Treatment of heart disease by disruption of anchoring of pp2a

Abstract

To provide a method of treating heart failure with reduced ejection fraction.SOLUTION: The present invention provides a method that involves administering a pharmaceutically effective amount of a composition which inhibits the anchoring of PP2A to mAKAPβ to a patient at risk of such damage. This composition is preferably in the form of a viral based gene therapy vector that encodes a fragment of mAKAPβ to which PP2A binds.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application is a continuation of U.S. Provisional Patent Application No. 62 / 848,156, filed May 15, 2019. No. 2015 / 010999, filed on Oct. 1, 2015, which claims priority to US Provisional Patent Application Publication No. 2015 / 0109999, filed on Oct. 1, 2015, which application is incorporated by reference in its entirety. The U.S. patent application, Ser. No. 14 / 821,082, was filed on August 7, 2018, and is currently pending. U.S. Patent No. 9,937,228 issued on March 10, 2014, and U.S. Patent No. 9,937,228 filed on March 14, 2014. The patent application is U.S. Patent Application No. 14 / 213,583, now published September 15, 2015. U.S. Patent No. 9,132,174 filed July 5, 2018, and U.S. Patent Application No. 1 No. 6 / 028,004, U.S. Provisional Patent Application No. 61 / 798 filed March 15, 2013 ,268, and U.S. Provisional Patent Application No. 62 / 529,224, filed July 6, 2017 No. 6,299,523, filed on Oct. 1, 2004, incorporated by reference in

[0002] [Statement regarding government support] This invention was made possible through the award of the National Institutes of Health under Contract No. RO1 HL 07539 This invention was made possible with government support under Patent Application HL126825 and Patent Application HL126825. The government has certain rights in this invention. do. [Background technology]

[0003] During chronic stress, the primary compensatory mechanism of the heart is a nonmitotic increase in contractile cell volume. This results in muscle cell hypertrophy, which is an additional factor in adult mammals (Hill and Olson 2008). Cardiac muscle cells are roughly cylindrical and can grow either widthwise or lengthwise. contributes to the majority of the cardiac muscle mass in the lung (Jugdutt 2003), and Eccentric hypertrophy and eccentric hypertrophy result in thickening of the ventricular wall and dilation of the ventricle, respectively. The widthwise "centripetal" myocyte growth involved in the structure reduces ventricular wall stress ( Law of LaPlace), an "eccentric" longitudinal muscle fiber that contributes to the continuous structure of the sarcomere. Cell growth occurs at greater speeds without stretching individual sarcomeres beyond their optimal length for contraction. The cardiac muscle can adapt to various ventricular volumes (length-tension relationship) (Grossman, Jones s, and McLaurin 1975). The left ventricle is affected by physiological changes such as pregnancy or exercise training. Concentric ventricular hypertrophy occurs in the ventricles due to hypertension or aortic stenosis. It is the primary early response to increased systolic wall stress present in pressure overload diseases such as stenosis. Eccentric ventricular hypertrophy occurs in conditions of volume overload, such as that which occurs after myocardial infarction, and Certain forms of cardiovascular disease, including those characterized primarily by pressure overload from concentric hypertrophy Transition to a dilated heart in heart failure with reduced ejection fraction (HFrEF) Eccentric and concentric hypertrophy are also predominant in hereditary hypertrophy and dilated cardiomyopathy, respectively. exists in.

[0004] At the cellular level, cardiomyocyte hypertrophy increases protein synthesis within individual myocytes as well as the sarcomere. This occurs as a result of increased size and organization. For discussion, see Kehat (2010) and Hill (2008). Each of these is incorporated by reference in its entirety herein. The prevailing view is that cardiac hypertrophy is a cause of cardiac failure. The conventional method of treating heart failure is to Afterload reduction, beta-adrenergic receptor (β-AR) blockade, and mechanical support in patients with However, this technique does not prevent or treat pathological cardiac hypertrophy. Further mechanisms are needed to

[0005] In this study, the mechanism that induces "compensatory" concentric hypertrophy early in pressure overload-related heart disease was It has been suggested that vasopressin predisposes the liver to subsequent systolic dysfunction and eventual failure (Sch iattarella and Hill 2015). In this regard, RSK3-mA Targeting the KAPβ complex suppresses cardiac remodeling caused by pressure overload and prevents heart failure. The results show that this is possible (Kritzer et al. 201 4; Li, Kritzer, et al. 2013). This includes concentric hypertrophy. Inhibition of signaling pathways that induce remodeling is desirable early in pressure overload disease. However, if the heart is in the later stages of pressure overload-related disease or in the advanced stages of volume overload-related disease, Even in the early stages, it is a disease characterized by eccentric growth and ventricular enlargement that leads to HFrEF. During the progression of the disease, signals that promote concentric hypertrophy and oppose eccentric hypertrophy are maintained. The aim of this study was to determine whether efforts to maintain cardiac volume and contraction during induction could be achieved. Furthermore, the increased and / or polarized concentric myocyte hypertrophy in familial dilated cardiomyopathy is unclear. It is unknown whether inhibition of cardiac myocyte hypertrophy could be beneficial.

[0006] AKAP and cardiac remodeling Ventricular myocyte hypertrophy is a condition in which the myocardium is hypertrophied due to myocardial infarction, hypertension, and congenital heart disease, or due to neurohumoral activation. This is the primary compensatory mechanism that reduces ventricular wall tension when stressed. Non-dividing growth of muscle cells, myofibril organization, and the expression of "fetal" genes normally expressed during embryonic development. associated with upregulation of specific subsets (Frey 2004, Hill 2008). Synchronous abnormal cardiac contractions, Ca 2+ Handling and myocardial energy are interstitial fibrosis and Inappropriate use of steroids, including steroids and cardiomyocyte death, increases the risk of developing heart failure and malignant arrhythmias. This adaptation is related to changes in responsiveness (Cappola 2008, Hill 2008). Both involve both systolic and diastolic dysfunction, which are present in varying proportions depending on the underlying disease. contribute to the damage caused by the pathological remodeling of muscle cells (Sharma and Kass 2014). The enzymes involved in the activation of mitogen-activated protein kinases (MAPKs), cyclic nucleotides, and Ca 2+ Complex intracellular signaling pathways, including hypoxia, hypoxia, and phosphoinositide-dependent signaling pathways, Regulated by a ring network (Heineke and Molkent in 2006).

[0007] Heart failure is estimated to be 6% in the United States due to increased prevalence caused by risk factors such as smoking and obesity. It affects 200,000 adults, with approximately 1,000,000 new cases diagnosed in adults each year. (Benjamin et al. 2019). Prevalence and incidence of heart failure Rates are increasing, primarily due to increased life expectancy, but also due to risk factors (high blood pressure, diabetes, etc.). Increased prevalence of diabetes, dyslipidemia, and obesity, as well as other types of cardiovascular disease (myocardial infarction [MI] and arrhythmias) is also due to increased survival (Heidenreich et al. 2 013). First-line therapy for patients with heart failure is associated with increased survival and quality of life for these patients. Improved longevity and reduced mortality in patients with left ventricular dysfunction Angiotensin-converting enzyme (ACE) inhibitors and β-adrenergic receptor blockers (β- Subsequent or alternative therapies include aldosterone and angiotensin II receptor blockers, neprilysin inhibitors, loop diuretics and thiazolinone. Diuretics, vasodilators, and I f Current blocking agents and device-based therapy (Po Nikowski et al. 2016). Nevertheless, 5% of symptomatic heart failure The annual mortality rate remains approximately 50%, including a mortality rate of over 40% after MI (Heid enreich et al. 2013; Gerber et al. 2016) .

[0008] Cardiac hypertrophy is mediated by G protein-coupled receptors, cytokine receptors, and growth factor tyrosine kinases. Various neurohumoral and paracrine receptors activate several receptor families, including the ATPase receptor. It can be induced by both inflammatory and autocrine stimuli (Brown 2006, Fr In this context, A-kinase anchoring proteins (AKAPs) These receptors can assemble into multiprotein complexes that integrate the hypertrophic pathways emanating from these receptors. In particular, recent research has revealed that scaffold proteins It functions as a protein and orchestrates hypertrophic pathways activated by stress signals. mAKAP, AKAP-Lbc, and D-AKAP play central roles in regulating Anchor proteins, including 1, have been identified so far.

[0009] Scaffolding proteins act as organizers of "nodes" in intracellular signaling networks ment of interest as a potential therapeutic target (Negro, Dodge- Kafka, and Kapiloff 2008). In cells, scaffolding proteins These signal transduction pathways constitute a key mechanism responsible for the specificity and efficacy of intracellular signal transduction. They can assemble into multimolecular complexes called “glucosomes” (Scott and First, many signaling enzymes have broad substrate specificity. Scaffolding proteins localize these pleiotropic enzymes with their respective substrates, allowing them to bind to the substrates. Selectively improves catalytic activity while providing a degree of specificity that is not inherent to the active site of the enzyme. (Scott and Pawson 2009). Second, some signaling Scaffolding proteins can colocalize rare enzymes with their substrates. Third, many scaffolds are multivalent, allowing for more efficient signaling. Field coupling can orchestrate the co-regulation of individual substrate effectors by multiple enzymes. Muscle A-kinase anchor protein (mAKAP, also known as AKAP6) Protein kinase A (PKA) and Ca with broad substrate specificity 2+ / Calmodulin signaling enzymes such as the calcium-dependent phosphatase calcineurin (CaN) and particularly low- Both signaling enzymes, such as p90 ribosomal S6 kinase 3 (RSK3), which is abundant in It is a large scaffold expressed in cardiac and skeletal muscle cells and neurons that binds to Figure 1) (Wang et al. 2015;Pare, Easlick, et a l. 2005;Michel et al. 2005a;Kapiloff et al. mAKAPβ is an alternative splicing enzyme expressed in muscle cells. It is a phosphodiesterase (PDS) form that binds the integral membrane protein nesprin-1α in cells. and localizes to the outer nuclear envelope (Pare, Easlick, et al. 2005).

[0010] Its role as a scaffolding protein for stress-related signaling molecules in cardiomyocytes Consistently, mAKAPβ deletion in neonatal rat ventricular myocytes in vitro Deficiency of α-adrenergic receptors, β-adrenergic receptors, endothelin-1, and angiotensin II, and hypertrophy induced by leucine inhibitor / gp130 receptor signaling. inhibited (Zhang et al. 2011; Pare, Bauman, et al. 2005;Dodge-Kafka et al. 2005;Guo et al. al. 2015). In vivo mAKAP gene targeting in mice suppresses hypertrophy induced by short-term pressure overload and chronic β-adrenergic stimulation, , inhibiting the development of heart failure after prolonged pressure overload and providing a survival benefit (Kritzer et al. al. 2014). Specifically, mAKAP fl / fl , Tg(Myh6-cre / E sr1*), tamoxifen-induced, conditional knockout mice AKAP gene deletion suppresses myocyte apoptosis and interstitial apoptosis following 16-week aortic coarctation. Left ventricular hypertrophy was suppressed while significantly inhibiting cardiac fibrosis, left atrial hypertrophy, and pulmonary edema (edema lung weight). decreased (Kritzer et al. 2014).

[0011] Also, mAKAP gene targeting is beneficial after myocardial infarction (Kapiloff, unpublished). Permanent ligation of the left anterior descending artery (LAD) in mice results in extensive myocyte remodeling. This results in myocardial infarction, including death, scar formation, and subsequent left ventricular (LV) remodeling. Four weeks after AD ligation, mAKAP conditional knockout mice showed a significant reduction in infarction. Compared with the control cohort, LV dimensions and function were preserved. Knockout mice showed a significant reduction in infarct size compared to controls. The LV ejection fraction and index atrial mass were maintained.

[0012] mAKAP and cardiac remodeling explanation mAKAP was originally a novel cAMP-dependent protein kinase (PKA) regulatory subunit. nit (R subunit) binding protein, i.e., A-kinase anchor protein or AKAP cDNA library screening (Mccartn mAKAP was initially isolated from the original cDNA fragment. Based on the size of the protein encoded by this gene, it was named "AKAP100" (Mc Cartney et al. 1995). Subsequently, mAKA expressed in neurons Defined the full-length mRNA sequence of mAKAPα, an alternative splicing isoform of P and revealed that wild-type mAKAPα is a 255 kDA scaffold (Kapilo ff et al. 1999b). The 230 kDa mAKAP expressed in striated muscle cells The sequence of mAKAPβ, an alternative splicing isoform of mAb, was later obtained. KAP corresponds to mAKAPα residue Met-245 when expressed in cardiac or skeletal muscle. showed that it migrates from an internal initiation site (Michel et al. 2005 a).

[0013] mAKAP is expressed in three cell types: neurons and It is localized to the nuclear envelope of both cardiac and skeletal striated muscle cells (Fig. 6) (Kapiloff et al. l. 1999b;Pare, Easlick, et al. 2005;Mich mAKAP is not a transmembrane domain protein. , which contains three spectrin-like repeat regions (residues 772–1187) that mediate localization (Kapiloff et al. 1999b). Binding of the third spectrin repeat (residues 1074-1187) of mAKAP by ATP-1α inhibits nuclear membrane localization of mAKAP at least in muscle cells and when expressed in heterologous cells. necessary and sufficient for the transformation (Pare, Easlick, et al. 2005 Nesprin-1α is also present in the inner nuclear membrane, where it can bind A-type lamins and emerin. Interestingly, mutations in lamin A / C, emerin, and nesprin-1α The mutation is associated with Emery-Dreifuss muscular dystrophy and other forms of cardiomyopathy. (Bonne et al. 1999;Fatkin et al. 1999;Mu Chir et al. 2000;Bione et al. 1994;Zhang However, disease-causing mutations in human mAK mAKAPβ in mouse hearts during early development, yet to be identified in AP genes Knockout does not induce cardiomyopathy (Kritzer et al. 2014). In addition to binding nesprin-1α, mAKAPβ also binds the first spectrophotometric signal of mAKAP. Binds phospholipase Cε (PLCε) via the trine repeats, enhancing its association with the nuclear membrane This may be due to the fact that mAKAPβ is transported to the sarcoplasmic reticulum (Zhang et al. 2011). There are early reports that it exists (Mccartney et al. 1995; rx et al. 2000; Yang et al. 1998) have confirmed these findings. has been called into question due to technical issues, including antibody specificity (Kapiloff, Jac kson, and Airhart 2001;Kapiloff et al. 1 999b).

[0014] mAKAPβ interacts with adenylyl cyclin in addition to PKA, PLCε, and nesprin-1α. Enzyme type 5 (AC5), exchange protein activated by cAMP-1 (Epac 1), cAMP-specific phosphodiesterase type 4D3 (PDE4D3), MEK5 and and ERK5 MAP kinase, 3-phosphoinositide-dependent protein kinase-1 ( PDK1), p90 ribosomal S6 kinase 3 (RSK3), protein kinase Cε ( PKCε), protein kinase D (PKD1, PKCμ), protein phosphatase Calcineurin (CaN), Aβ and PP2A, type 2 ryanodine receptor (RyR2) , sodium / calcium exchanger NCX1, ubiquitin E3- involved in HIF1α regulation ligase, and myopodin (Pare, Bauman, et al. t al. 2005;Pare, Easlick, et al. 2005;Do dge-Kafka et al. 2005;Marx et al. 2000;K apiloff, Jackson, and Airhart 2001;Miche l et al. 2005a;Li et al.;Wong et al. 20 08;Zhang et al. 2011;Dodge-Kafka and Kap iloff 2006;Vargas et al. 2012;Faul et al. . 2007;Schulze et al. 2003;Kapiloff et a l. 2009; Zhang et al. 2013), which is a muscle cell stress response These signaling molecules bind a wide variety of important proteins. It binds to the transcription factors hypoxia-inducible factor 1α (HIF1α) and myocyte enhancer factor-2 (MEF2), and nuclear factor of activated T cells (NFATc) transcription factors, and type II histone deacetylases (Fig. 7) (Kritzer et al. 2014; Li, Vargas, et al. 2013;Li et al. 2010;Wong et al. 2008;Li et al. 2019;Dodge-Kafka et al. 2018). Some of these molecules directly and some indirectly. mAKAP binds to the ATP-dependent ATPases, some in a constitutive and some in a regulated manner. The composition of the β-signalosome is thought to correspond to the underlying state of the muscle cell. As research continues on Pβ, the list of binding partners grows, and remodeling This supports the postulated role of ATP as a key coordinator of signaling pathways required for ATP production. Most of what is known about mAKAPβ is derived from cultured neonatal rat ventricular myocytes. This is based on studies using cytoplasmic vesicles, in which mAKAPβ binds to both α- and β-adrenergic receptors. Required for hypertrophy induction by a variety of upstream receptors, including nalin and cytokine receptors It was early recognized that Dodge-Kafka et al. 2005). However, in recent years, cardiomyocytes The phenotype of specific mAKAPβ conditional knockout mice was published, and remodeling This study confirmed the central role of mAKAPβ in the regulation of ATP synthesis (Kritzer et al. 2014). The mAKAPβ signalosome influences pathological cardiac remodeling. There are various upstream inputs, downstream effectors (outputs), and integrating circuits within the do.

[0015] mAKAPβ - Prototype A-kinase anchor protein Like most AKAPs, mAKAPs contain an amphipathic helix that is responsible for binding PKA. containing the nucleotide sequence (residues 2055–2072) (Kapiloff et al. 1999 b; Kritzer et al. 2012). PKA plays a key role in the formation of the CRRC. It is a heterotetramer of two R subunits and two catalytic C subunits. Within the enzyme, the N-terminal docking and dimerization domain of the R subunit of PKA It forms an X-shaped antiparallel four-helix bundle (Newlon et al. 1999 This bundle contains a hydrophobic groove that corresponds to the hydrophobic face of the AKAP amphipathic helix. KAPβ is a high-affinity (K D =119 nM) and selectively (including the RII subunit) It binds type II PKA (Zakhary et al. 2000). In contrast, PKA-mAKAPβ binding increased 16-fold after autophosphorylation of RIIα (Zak hary et al. 2000), in altered states of β-adrenergic signaling. In addition to mAKAPβ, more than 12 Other AKAPs are expressed in muscle cells, each with its own specific localization and binding properties. Toner groups (Kritzer et al. 2014). AKAP is one of the rarest AKAPs in muscle cells, and loss of mAKAP results in perinuclear It does not even affect the localization of peripheral PKA (Kapiloff, unpublished observations). ) Despite the low level of scaffold expression, endogenous mAKAPβ was expressed in muscle cells. Substitution of a full-length mAKAPβ mutant that cannot bind to PKA inhibits myocyte hypertrophy induction. This is sufficient for inhibition (Pare, Bauman, et al. 2005). Furthermore, the mAKAPβ signalosome demonstrates that PKA signaling is precisely regulated even in individual organelles. and that the expression level of a protein or protein complex can be determined by its This is an example of both the fact that the functional significance of proteins does not necessarily indicate their role.

[0016] mAKAPβ is not only an effector of cAMP signaling but also a regulator of cAMP synthesis and This is important because it also binds enzymes responsible for degradation (Kapiloff et al. 2009; Dodge et al. 2001). The synthesis of cAMP from ATP is The metabolism of cAMP to 5'AMP, while catalyzed by adenylyl cyclase (AC) is catalyzed by phosphodiesterases (PDEs). AC and PDE AKAP The differential association of cAMP with ATP contributes to the compartmentalization of cAMP in cells and regulates the local activity of cAMP effectors. Local cAMP activation and specific regulatory feedback and feedforward loops. (Scott, Dessauer, and Tasken 2013). mAKAP can bind to both AC2 and AC5, but not AC 5 is recognized as a relevant mAKAPβ binding partner in the heart (Kapilof f et al. 2009). The N-terminal, C1 and C2 domains of AC5 are involved in the synthesis of mAKAP. AC5 directly binds to a unique N-terminal site (residues 275-340) of β. by PKA feedback phosphorylation promoted by mAKAPβ complex formation This negative feedback is inhibited (Kapiloff et al. 2009). , which is considered physiologically relevant for maintaining basal cAMP signaling. The link to KAPβ was inhibited by a competitor peptide containing the mAKAP AC5 binding domain. When inhibited by ATP, both the cAMP content and size of myocytes increase without the presence of a hypertrophic stimulus. increased (Kapiloff et al. 2009).

[0017] mAKAP was the first AKAP shown to bind to PDEs (Dodge et al. 2001). The sites 1286–1831 in mAKAP are involved in the regulation of PDE4D3. Binds to a specific N-terminal domain. Binds to phosphorylation of serine residues 13 and 54 of PDE4D3. These results in increased scaffold binding and increased PDE catalytic activity, respectively (Dodge et al. al. 2001;Sette and Conti 1996;Carlisle Michel et al. 2004). Increased PDE4D3 activity accelerates cAMP degradation. Thus, PKA and PDE4D3 can regulate local cAMP levels and PKA activity. This creates a significant negative feedback loop (Dodge et al. 2001). PDE4D3 bound to KAP acts not only as a PDE but also as a MAPK, MEK5 and E RK5 and the cAMP-dependent Rap1-guanine nucleotide exchange factor Epac1. It also functions as an adaptor protein recruiting to the scaffold (Dodge-Kafka et al. al. 2005). Activation of MEK5 and ERK5 by upstream signals is mediated by Ser- 579, resulting in phosphorylation of PDE4D3, inhibiting PDE and increasing cAMP accumulation and and PKA activation (Dodge-Kafka et al. 2005; Hof fmann et al. 1999;Mackenzie et al. 2008) Epac1 is not as sensitive to cAMP as PKA, and is inhibited by extremely high cAMP levels. This leads to further activation of mAKAP-associated Epac1. c1 inhibits ERK5 activity via Rap1, thus inhibiting MAPK signaling. Prevents PDE4D3 inhibition and potentially prevents maximal PDE4D3 activity due to concomitant PKA phosphorylation. (Dodge-Kafka et al. 2005). As such, Epac1, ERK5, and PDE4D3 are involved in the regulation of cAMP uptake when cAMP levels are elevated. A third load may suppress cAMP levels in the vicinity of the mAKAP complex, counteracting the increase in This forms a feedback loop.

[0018] Further complexity is due to the C-terminal domain of the serine-threonine phosphatase PP2A, mAKAP. The binding to the end (residues 2083-2319) is given by Dodge-Kafka e t al. 2010). PP2A catalyzes the dephosphorylation of Ser-54 of PDE4D3. By integrating the mAKAP complex, PDEs can be inhibited without the presence of upstream stimuli. PP2A associated with PKA contains the B56δB subunit, a PKA substrate. ion enhances PP2A catalytic activity (Ahn et al. 2007) and inhibits mAKAP-bound P Phosphorylation of B56δ by KA increases PDE4D3 dephosphorylation and inhibits PDE This likely increases cAMP levels and stimulates cAMP signaling. This forms a positive feedforward loop that initiates the activation of PKA and ERK5. This negative feedback loop, based on AC5 phosphorylation and PDE4D3 regulation by m cAMP levels in the AKAPβ signalosome are regulated by upstream β-adrenergic and MA It can be predicted that AC5 and E may be tightly controlled by PK signaling. The upstream signaling of RK5 is initially via PP2A feedforward signaling. It can promote cAMP signaling, while negatively filtering PKA and Epac1. PDE4D3 activation by feedback and AC5 inhibition can suppress signaling. Interestingly, Rababa'h et al. found that mAKAP proteins containing nonsynonymous polymorphisms We showed how differently PKA and PDE4D3 bind to each other (Raba ba'h et al. 2013). cAMP signaling mediates the upstream signaling pathway. The possibility that they are differentially regulated by crosstalk between tracts or by human polymorphisms is unclear. We look forward to further studies in muscle cells that demonstrate the relevance of this complex signaling network. It shall be worthy of

[0019] mAKAPβ and MAP-kinase-RSK3 signaling The recruitment of ERK5 to the mAKAPβ complex by PDE4D3 was initially initiated by the aforementioned feed It has been shown to be involved in the local regulation of cAMP by the back loop (Dodge-Kaf However, ERK5 is also important for muscle cell hypertrophy. It has also been shown to be a potent inducer of longitudinal growth (eccentric growth) in cultured muscle cells. While it induces concentric hypertrophy, it is also important for pressure overload-induced concentric hypertrophy in vivo. (aortic coarctation in mice) (Nicol et al. 2001; Kimura In particular, RNA interference of mAKAPβ expression in cultured muscle cells RNAi-mediated inhibition of interleukin-6-type cytokine leukemia inhibitors inhibited eccentric growth induced by inhibitory factor (LIF) (Dodge-Kafka et al. 2005). Possible effectors of mAKAPβ-bound ERK5 are However, in both the heart and brain, In this study, mAKAP, together with ERK (ERK1, 2, or 5), acts as a MAPK effector p It binds PDK1, a kinase that can activate 90RSK, and activates the MAPK effector p 90RSK is also a kinase that associates with mAKAP (Ranganathan et al. Michel et al. 2005a). Importantly, PDK Binding of 1 to mAKAP abolished the requirement for membrane association for RSK activation (Mi chel et al. 2005a). Both of these data suggest that mAKAPβ acts upstream We demonstrate that RSK activation can be coordinated in muscle cells in response to MAPK signaling. It was something like that.

[0020] p90RSK regulates many cellular processes, including cell proliferation, survival, migration, and invasion. RSK activity is mediated by most hypertrophic stimuli in muscle cells. (Anjum and Blenis 2008; Sadoshima et al. 1995). Furthermore, RSK activity is significantly increased in cardiac tissues of human end-stage dilated cardiomyopathy. It was found that the RSK activity increased in the Family members consist of two distinct domains: an N-terminal kinase domain and a C-terminal kinase domain. It contains two catalytic domains (Anjum and Blenis 2008). The enzyme domain phosphorylates the RSK substrate, resulting in the C-terminal and C-terminal cleavage by ERK and PDK1, respectively. It is activated by sequential phosphorylation in the activation loop of the N-terminal kinase domain PDK1 phosphorylation of the N-terminal domain at Ser-218 indicates full activation of the enzyme. There are four ubiquitously expressed mammalian RSK family members. However, only RSK3 binds mAKAPβ (Li, Kritzer, et al. 2013). The unique N-terminal domain (1–30) in RSK3 is involved in the regulation of mAKAPβ Direct binding of residues 1694–1833 explains the selective association of this isoform with the scaffold. (Li, Kritzer, et al. 2013). RSK3 is a ubiquitous nucleotide analogue of other R Although it is expressed in muscle cells at lower levels than SK family members, it is involved in neonatal muscle cell hypertrophy. The main findings were RNAi of RSK3, inactivation of the N-terminal kinase domain of RSK3, and the anchoring of RSK3. Inhibition of RSK3 binding to mAKAP using a RING disruptor peptide It was found that this could be achieved (Li, Kritzer, et al. 2013). Notably, RSK3 expression in vivo was significantly affected by pressure overload and catecholamine infusion. Induction of cardiac hypertrophy by both α- and β-glucanase and associated heart failure in a mouse model of familial hypertrophic cardiomyopathy (α-tropomyosin Glu180Gly) (Li, Kr itzer, et al. 2013;Passariello et al. 20 13) Furthermore, ERK1 / 2 MAP-kinase has been reported to selectively induce concentric hypertrophy. Consistent with its proposed role (Kehat et al. 2011), RSK3 gene deletion Loss of Raf1 in a mouse model of Noonan syndrome L613V Due to mutation The concentric hypertrophy induced by the steroid was inhibited (Passariello et al. 201 6) The recognition that this particular RSK isoform is required for cardiac remodeling This makes it a compelling candidate for therapeutic targeting.

[0021] mAKAPβ and phosphatidylinositide signaling The cAMP effector Epac1 mediates the mAKAPβ complex, which influences ERK5 signaling. activating Rap1 in coalescence (Dodge-Kafka et al. 2005 Furthermore, Epac1-Rap1, whose Ras-association domain is involved in the first step of mAKAPβ, Activates PLCε, a phospholipase that directly binds pectin repeat-like domains (Zhang et al. 2011). PLCε, like mAKAPβ, is an RNA by removing mAKAPβ from the target protein by i or by expression of a competitive binding peptide Even though it was found to be a nutrient-rich compound, it was required for neonatal muscle cell hypertrophy. In an excellent paper by Ory, mAKAPβ-bound PLCε was found to be a novel phosphatidyl Regulating PKCε and PKD activation via the inositol-4-phosphate (PI4P) pathway In this pathway, PLCε converts perinuclear PI4P into diacylglycerol. and inositol-1,4-bisphosphate (Zhang et al. l. 2013). PKD1 is a type II histone deacetylase (HDAC 4 / 5 / 7 / 9), inducing their nuclear export and derepressing hypertrophic gene expression (M onovich et al. 2010;Xie and Hill 2013). S Mrcka et al. reported that PLCε inhibits pressure overload-induced PKD activation, type II HDAC phosphorylation, and found that ATP is required for hypertrophy in vivo (Zhang et al. 2013). Subsequently, mAKAPβ was also shown to increase the vascular wall thickness in response to pressure overload in vivo. It was found to be required for PKD activation and HDAC4 phosphorylation (Kritzer et al. 2014). Notably, mAKAPβ mediates PKD and HDAC These results together suggest that local cAMP signaling is involved in the formation of a ternary complex with 4. This demonstrates how this can affect the regulation of cardiac gene expression.

[0022] Recently, it has been reported that mAKAPβ acts as a scaffold for HDAC5 in cardiomyocytes, and that HDAC5, PK It was reported that the signalosome containing D and PKA was formed (Dodge-Kaf Inhibition of mAKAPβ expression was associated with the inhibition of α- and β- Suppressed HDAC5 phosphorylation by PKD and PKA in response to adrenergic receptor stimulation Importantly, inhibition of mAKAPβ-HDAC5 anchoring inhibits α-adrenergic receptor agonism. Impeded induction of HDAC5 nuclear export by phosphoreceptor signaling and PKD phosphorylation Furthermore, inhibition of mAKAPβ-PKA anchoring abolished α-adrenergic-induced HD. These data suggest that AC5 inhibits the nuclear export of AC5 by β-adrenergic receptor stimulation. Both studies demonstrated that the mAKAPβ signalosome mediates the nucleoplasmic localization of class IIa HDACs. Thus, it has been revealed that mAKAPβ functions to regulate the direction of The scaffolds both suppress and activate pathological gene expression in health and disease, respectively. It functions as a node in the muscle cell regulatory network that controls

[0023] mAKAPβ and calcium signaling mAKAPβ mediates cAMP, phosphoinositide, and MAP-kinase signaling In addition, Ca 2+ They contribute to the regulation of neurotransmitter signaling. The second binding partner of β is Ca from intracellular stores. 2+ induced Ca 2+ The cause of the release Ryanodine receptor Ca 2+ release channel (RyR2) (Kapiloff, Jackson, and Airhart 2001;Marx et al. 20 00). RyR2 is a stimulator of bulk Ca 2+ In excitation-contraction coupling, PKA phosphorylation enhances RyR2 currents (Valdiv et al., 2013). ia et al. 1995;Dulhunty et al. 2007;Bers 2006), the importance of PKA-catalyzed RyR2 phosphorylation for excitation-contraction coupling remains unclear. This is open to debate (Houser 2014; Dobrev and Wehre ns 2014). A portion of RyR2 is probably located in perinuclear dyads (Escoba r et al. 2011) immunoprecipitated with mAKAPβ and nesprin-1α antibodies. (Pare, Easlick, et al. 2005; Kap iloff, Jackson, and Airhart 2001). mAKAPβ is important for regulating the nuclear events involved in pathological remodeling. The signaling molecular components are seen to be integrated together. Thus, the mAKAPβ complex This may provide a mechanism by which contraction responds to hypertrophy induction. Stimulation leads to increased PKA phosphorylation of mAKAPβ-associated RyR2 (Pare, B PKA-catalyzed RyR2 phosphorylation is associated with increased exchange stimulation. Local Ca concentration in the vicinity of the mAKAPβ signalosome during stress 2+ The release may be enhanced.

[0024] Although it is unlikely that a small number of mAKAPβ-associated RyR2 molecules could affect overall contraction, the perikarya Ca of the enclosure 2+ Possible targets for increase are scaffold-binding Ca 2+ / Calmodulin-dependent The catalytic subunit of CaN may be the dependent phosphatase calcineurin (CaN). There are three isoforms (α, β, γ) of the CaNAβ-mAKAPβ complex. Only ATP has been detected in muscle cells (Li et al. 2010). CaNAβ is important for the induction of cardiac hypertrophy and postischemic myocyte survival in vivo. It is a CaNA isoform (Bueno et al. 2002; Bueno et al. 2004). CaNAβ binds to a unique site within mAKAPβ (residue 128 6-1345) (Pare, Bauman, et al. 2005 ; Li et al. 2010). CaNAβ binding to mAKAPβ was regulated by adrenaline. by stimulation and Ca 2+ / Calmodulin directly enhances in cells (Li et al. 2010). In particular, CaNAβ-mAKAPβ binding is It is required for α-adrenergic-induced neonatal myocyte hypertrophy in vitro. (Li et al. 2010).

[0025] mAKAPβ and gene expression CaN is responsible for the activation of NFATc and MEF2 transcription factors, among other substrates. The NFATc transcription factor family is expressed in muscle cells and mediates muscle cell hypertrophy. Contains four CaN-dependent isoforms that may contribute to induction (Wilkins et al. al. 2004). In general, NFATc family members contain a regulatory domain within the N-terminal When it is heavily phosphorylated at multiple serine-rich motifs, it is retained in the cytoplasm. When this motif is dephosphorylated by CaN, NFATc translocates into the nucleus. NFATc family members can bind mAKAPβ, and the binding of mAKAPβ was required for CaN-dependent dephosphorylation of NFATc3 in muscle cells. (Li et al. 2010). Therefore, mAKAPβ expression also It was also required for NFAT nuclear translocation and transcriptional activity in tro (Li et al. 2010; Pare, Bauman, et al. 2005). These results In vivo, NFAT-dependent gene expression was observed in mAKAPβ myocytes after aortic coarctation surgery. This correlates with recent findings that the gene expression was suppressed by cell-specific knockout (Krit zer et al. 2014).

[0026] Like NFATc2 and NFATc3, MEF2D regulates cardiac hypertrophy in vivo. It is a transcription factor that is highly required for transcription (Kim et al. 2008; Wilkins et al. 2010). t al. 2002; Bourajjaj et al. 2008). MEF2fa Milli members contain conserved DNA fragments containing both MADS boxes and MEF2 homology domains. Contains the binding domain (Potthoff and Olson 2007). MEF2D The DNA-binding domain of mAKAP directly binds to the N-terminal domain of mAKAP (Vargas et al. 2012; Kim et al. 2008). CaN and MEF2D , which is important not only in the heart but also in skeletal muscles (Naya et al. 1999;N aya and Olson 1999;Black and Olson 1998; Friday et al. 2003;Wu et al. 2001). MEF2- Interference with mAKAPβ binding inhibits MEF2 transcriptional activity and endogenous expression in C2C12 skeletal myoblasts. blunted MEF2 target gene expression (Vargas et al. 2 012). Furthermore, as evidenced by a decrease in cell fusion and differentiation marker expression, Inhibition of the MEF2-mAKAP complex suppresses the differentiation of C2C12 myoblasts into myotubes. (Vargas et al. 2012). Notably, the CaN-MEF2 In cardiomyocytes, this is mAKAPβ-dependent (Li, Vargas, et al. Therefore, inhibition of CaN-mAKAPβ binding inhibits C2C12 cell proliferation. inhibited both MEF2 transcriptional activity in cells and cardiomyocyte hypertrophy (Li, Vargas , et al. 2013). Like NFATc2, it is also involved in the in vivo response to pressure overload. MEF2D dephosphorylation in o after conditional knockout of mAKAPβ This correlates with decreased expression of MEF2 target genes, including atrial natriuretic factor (ANF) expression. (Kritzer et al. 2014).

[0027] In vivo mAKAPβ-mediated NFATc, MEF2, and Regulation of HDAC4 indicates the importance of mAKAPβ for stress-regulated gene expression. (Kritzer et al. 2014). , NFATc and MEF2 are regulated by CaN, while HDAC4 and It has been shown how HDAC5 is regulated by PKD and PKA (Li, Vargas, et al. 2013;Zhang et al. 2013;L i et al. 2010; Dodge-Kafka et al. 2018). m AKAPβ facilitates the regulation of these gene regulatory proteins by other signaling enzymes For example, mAKAPβ-associated ERK5 phosphorylates MEF2, which acts as a transcription factor. Furthermore, PKA can activate MEFs (Kato et al. 2000). 2 can be phosphorylated to affect its DNA binding affinity (Wang et al. On the other hand, Olson et al. reported that PKA phosphorylation of HDAC4 is a novel Generation of a novel HDAC4 proteolytic fragment can inhibit MEF2 activity It has been proposed that mAKAPβ binds to the ATP-dependent ATPase (Backs et al. 2011). How the activities of the partners are ultimately integrated to control gene expression is a key issue in v It can be studied both in vitro and in vivo.

[0028] Other mAKAPβ binding partners The significance of docking to scaffolds, including myopodin and NCX1, has been There are other binding partners of mAKAPβ that are not well characterized (Faul et al. 2007; Schulze et al. 2003) and hypoxia. HIF-1α, a transcription factor that regulates systemic responses to inflammatory cytokines, also binds mAKAPβ (W ong et al. 2008). Under normoxia, HIF-1α in cells The abundance of HIF-1α is kept low by ubiquitin-mediated proteasomal degradation. prolyl hydroxylases (PHD1, PHD2, and PHD3) are hydroxylated by a family of hydroxylase dioxygenases (Ohh et al. 2000). Hydroxylated HIF-1α then binds to elongin C ubiquitin ligands. Recruiting the gauze complex to ubiquitinate HIF-1α and its proteasome-dependent It is recognized by the von Hippel-Lindau protein (pVHL), which promotes its degradation ( Maxwell et al. 1999). Under hypoxic conditions, PHD is inactivated. This results in decreased HIF-1α degradation and accumulation of HIF-1α in the nucleus, where it co-expresses with HIF-1β. mAKAPβ can cleave and promote the transcription of target genes. In the cells, HIF-1α, PHD, pVHL, and E3 ligase Siah2 (seven It can assemble a signaling complex containing the in absentia homolog 2 (Wong et al. 2008). Under normoxia, the mAKAPβ anchor -PHD and pVHL promote HIF-1α ubiquitination and degradation (Wong et al. However, under hypoxic conditions, Siah2 activation results in Induce proteasomal degradation of bound PHDs and promote HIF-1α accumulation (Wong mAKAPβ knockout inhibits cardiomyocyte survival after ischemia-reperfusion. may affect.

[0029] mAKAPβ - the orchestrator of remodeling The above description is based on several factors known to be important in cardiac hypertrophy and pathological remodeling. How signaling pathways are regulated by binding of key signaling intermediates to the mAKAPβ scaffold This shows how cardiomyocyte-specific mAKAP conditional knockdown is regulated. The mAKAPβ signalosome is involved in the development of AKAPβ signaling in vivo. It has been characterized as a AKAPβ (Kritzer et al. 2014). In cardiac myocytes, the ATPase inhibitors are required for the induction of cardiac hypertrophy by aortic coarctation and isoproterenol injection. However, most notably, myocardial apoptosis and interstitial Prevention of pathological remodeling, including stromal fibrosis, and preservation of cardiac function in the face of prolonged pressure overload Both of these compounds significantly prolonged the survival of mice (Kritzer et al. 2014). 014). These results suggest that its loss may confer a survival benefit in heart disease. As a first scaffold, mAKAPβ was identified. Importantly, Nkx2-5-targeted The use of the cre deletion strain did not result in any obvious phenotype at 6 months of age. mAKAPβ is not known to be required for either the development or maintenance of normal adult cardiac function. (Kritzer et al. 2014). Although it suppresses physiological hypertrophy induced by forced exercise (swimming), it does not cause any disease-related effects. Targeting the mAKAPβ complex in the inflammatory process remains relevant.

[0030] Targeting the mAKAPβ complex in humans, including siRNA knockdown of the scaffold Various methods can be envisioned. However, the structure and function of the mAKAPβ signalosome A relatively detailed understanding of these pathways will provide further approaches to target these pathways. For example, mAKAPβ-CaNAβ, mAKAPβ-MEF2D, and mAKAPβ-P Anchoring disruptor peptides targeting LCε and mAKAPβ-RSK3 binding Targets key protein-protein interactions involving mAKAPβ, including thiol Expression of the peptide has already been shown to be effective in vitro. (Li, Vargas, et al. 2013;Li, Kritzer, et al. al. 2013;Vargas et al. 2012;Zhang et al. Heart failure, a leading cause of death, costs over $30 billion per year in the United States alone. Even with modern treatment, this disease has a 50% mortality rate within 5 years of diagnosis. (G Many potential targets in cardiac disease are multifaceted. This complicates the development of drugs with sufficient specificity in vivo. Specific targeting of the AKAPβ signalosome contributes to pathological cardiac remodeling A relatively rare protein whose elimination can be promoted without significant side effects. This provides an opportunity to target the interactions between proteins and the target molecules to treat patients with heart failure. Developing new and effective treatments for heart disease, as well as other cardiovascular diseases such as coronary artery disease, hypertension, and valvular disease There is a real need for prevention of the onset of associated vascular disease. Summary of the Invention

[0031] The following brief summary is not intended to be inclusive of all features and aspects of the present invention, nor is it intended to be exhaustive of all aspects of the present invention. It is implied that all features and aspects described in this Summary must be included. isn't it.

[0032] The inventors have used drugs that target specific protein-protein interactions. By inhibiting the signaling properties of individual mAKAP signaling complexes, we aim to improve cardiac disease. The researchers discovered a method for treating a specific process. Such a treatment involves selective inhibition of a given cellular response. This offers advantages over conventional treatment approaches because it allows for harm.

[0033] In particular, the present inventors have developed a method for inhibiting mAKAP-mediated protein-protein interactions. This results in the expression of key transcription factors that initiate the cellular processes that lead to pathological cardiac remodeling. Inhibits the ability of mAKAP to regulate the activation of enzymes that play a central role in activation It was found that it can be used for this purpose.

[0034] Specifically, the inventors have demonstrated that by inhibiting the binding interaction between PP2A and mAKAPβ, For example, after a myocardial infarction, it can protect the heart from damage that can lead to heart failure. discovered.

[0035] Thus, the present invention, in certain aspects, includes a method for protecting the heart from damage, The method comprises administering a pharmaceutically effective amount of a composition that inhibits the interaction between PP2A and mAKAPβ to a subject. This is done by administering the drug to patients at risk of the disorder.

[0036] The present invention also provides a pharmaceutical composition for inhibiting the interaction between PP2A and mAKAPβ. The present invention relates to a method for treating cardiac disease by administering a substance to a patient.

[0037] The present invention also relates to a composition that inhibits the interaction between PP2A and mAKAPβ.

[0038] In yet another embodiment, the inhibitor inhibits the expression or activity of PP2A and mAKAPβ. This includes any molecule that inhibits

[0039] The above and other objects, features and advantages of the present invention are set forth in the accompanying drawings, in which like reference characters identify the same parts throughout the various drawings. As shown in the accompanying drawings, which refer to the same parts, the following more specific embodiments of the present invention will now be described. The drawings are not necessarily to scale, but rather are illustrative of the principles of the present invention. The emphasis is on demonstrating the principles of Ming.

[0040] This patent or application contains at least one color drawing. Copies of the patent or patent application publication will be provided by the Patent Office upon request and payment of the necessary fees. will be done. [Brief explanation of the drawings]

[0041] [Figure 1]Figure 1 shows a model of mAKAPβ-regulated, SRF-dependent gene expression. Anchored RSK3 is a Gq protein-coupled receptor-ERK effector that phosphorylates SRF associated with the perinuclear mAKAPβ complex. mAKAPβ-anchored PP2A, which can be activated by cAMP-dependent protein kinase A (PKA), counteracts SRF phosphorylation. Phosphorylated SRF induces gene expression that promotes concentric hypertrophy. [Figure 2] FIG. 2 shows the amino acid sequence of human RSK3 (SEQ ID NO: 1). [Figure 3] Figure 3 shows the amino acid sequence of rat mAKAP (SEQ ID NO: 2). It is noted that within this document, references to the mAKAP sequence, whether written as "mAKAPβ" or "mAKAP," are by numbering of the mAKAPα alternative splice form, which includes the entire mAKAPβ and is identical to the originally described mAKAP sequence as shown in this figure (Kapiloff 1999, Michel 2005). "mAKAP" is also referred to as "AKAP6" in reference databases and literature. mAKAPβ begins at residue 245, while mAKAPα begins at residue 1. The PP2A-binding domain begins at residue 2134. [Figure 4] 4 shows the amino acid sequence of the PBD of rat mAKAP when expressed in an AAV vector, including the N-terminal myc tag (SEQ ID NO: 12). [Figure 5] FIG. 5 shows the plasmid of the PBD of pscA-TnT-myc-rat mAKAP used to generate the AAV9sc.rat PBD (SEQ ID NOs: 13 and 14). [Figure 6]Figure 6 shows the perinuclear scaffolding of mAKAPβ. The top panel shows mouse heart sections (left ventricle) stained with mAKAP antibody (grayscale panels and green), Hoechst nuclear stain (blue), and wheat germ agglutinin (red, shown only in the enlarged control image). The bottom left panel is from a control, mAKAP knockout mouse. Horizontal bar = 20 μm. The middle panel shows an adult rat myocyte stained with antibodies against mAKAP (green) and actinin (red). The bottom panel shows the mAKAP domain structure. Direct binding partners, whose sites have been fine-mapped in mAKAPβ, are shown. mAKAPβ begins at residue 245 of mAKAPα; therefore, all binding sites are numbered relative to mAKAPα. Image from Kritzer et al. (Kritzer et al. 2014). [Figure 7] Figure 7 shows the signaling modules of mAKAPβ. mAKAPβ binds multiple signaling enzymes and gene regulatory proteins. Modules involved in cAMP, Ca2+, hypoxia, phosphatidylinositide, and MAPK signaling can be defined. See above for details. In this figure, the mAKAPβ scaffold is shown as a yellow sphere positioned on a gray base representing nesprin-1α, associating various signaling molecules. The gold cylinder represents the nuclear pore complex inserted into the nuclear membrane. [Figure 8]Figure 8 shows that okadaic acid-sensitive phosphatase regulates mAKAP-associated PDE4D3. A. Transfected HEK293 cells expressing both mAKAP and PDE4D3 were treated with either 300 μM okadaic acid (OA) or 500 μM cyclosporine A (CsA) for 30 minutes, followed by stimulation with 5 μM forskolin (Fsk) for 10 minutes. The phosphorylation status of PDE4D3 present in the mAKAP antibody immunoprecipitates was measured using an antibody specific for Ser-54 of phosphorylated PDE4D3 (top panel). Total PDE4D3 (middle panel) and mAKAP (bottom panel) present in the mAKAP antibody immunoprecipitates were detected using a non-phospho-specific antibody. Note that in these experiments, mAKAP was GFP-tagged, and PDE4D3 was VSV- and GFP-tagged, resulting in increased molecular weight. n=3. B, PDE activity associated with mAKAP antibody immunoprecipitates prepared as in A was assayed using [H]cAMP substrate. *p<0.05 compared to untreated cells (bar 1). C, Endogenous protein complexes were isolated from clarified adult rat heart extracts (500 μg total protein) using control (IgG) or mAKAP-specific antibodies. PDE activity associated with the immunoprecipitates was assayed in the presence of 10 nM OA or 50 nM PKI. n=3, *p<0.05. [Figure 9]Figure 9 shows that protein phosphatase PP2A associates with the mAKAP scaffold in adult rat heart. A. Phosphatase activity associated with protein complexes immunoprecipitated with mAKAP antibodies from adult rat heart extracts (500 μg total protein) was assayed using 32P-labeled histone substrate in the presence or absence of 30 nM PP2A inhibitor I (Li, Makkinje, and Damuni 1996) and 100 nM PKA-phosphorylated PP1 inhibitor-1 (Endo et al. 1996). n = 3. *p < 0.05. B and C. Protein complexes were isolated from clarified adult rat heart extracts (2 mg total protein) using control (IgG) or mAKAP-specific antibodies. PP2A (panel B) and PP1 (panel C) catalytic subunits were detected by immunoblotting in extracts (80 μg) and immunoprecipitates (25% loading). n = 3. [Figure 10]Figure 10 shows that PP2A binds the C-terminal mAKAP domain. A, Schematic diagram of the mAKAP domain and GFP- and myc-tagged mAKAP proteins used herein. mAKAP fragments containing the rat and human proteins are shown in black and gray, respectively. The hatched bars indicate the three spectrin repeat domains responsible for nuclear membrane targeting in muscle cells (Kapiloff et al. 1999a). Binding sites are shown for proteins known to directly bind mAKAP, including 3-phosphoinositide-dependent kinase-1 (PDK1, mAKAP residues 227-232) (Michel et al. 2005b), nesprin-1α (1074-1187) (Pare, Easlick, et al. 2005), ryanodine receptor (RyR2, 1217-1242) (Marx et al. 2000), PP2B (1286-1345) (Li et al. 2009), PDE4D3 (1285-1833) (Dodge et al. 2001), and PKA (2055-2072) (Kapiloff et al. 1999a). The shaded bars indicate the PP2A binding site. The first and last residues of each fragment are indicated. B. Purified GST-PP2A A subunit fusion proteins were incubated with extracts prepared from HEK293 cells expressing the indicated GFP-mAKAP fusion proteins and detected by pulldown using glutathione resin. GFP-mAKAP fragments were detected in pulldowns (25% load, upper panel) and extracts (5% load, lower panel) using GFP antibodies. n = 3. C. Myc-tagged mAKAP fragments were expressed in HEK293 cells, and phosphatase binding was detected by immunoprecipitation with control (IgG) or myc-tagged antibodies, followed by a phosphatase assay using a 32P-labeled histone substrate. n = 3. *p < 0.05 compared with other samples. It is noted that the C-terminal homology domains of both rat and human mAKAP bind PP2A. [Figure 11]Figure 11 shows that PP2A association with the mAKAP-PDE4D3 complex is required for the inhibition of PDE4D3 phosphorylation. A. HEK293 cells expressing PDE4D3 (VSV- and GFP-tagged) and myc-tagged mAKAP1286-2312 or 1286-2083, which lacks the PP2A binding site, were treated with 300 μM OA for 30 minutes, followed by stimulation with 5 μM Fsk for 10 minutes. The protein complex was immunoprecipitated using a myc-tagged antibody in the presence of phosphatase inhibitors. The phosphorylation status of co-immunoprecipitated PDE4D3 was measured using an antibody specific for Ser-54 of phosphorylated PDE4D3 (P-PDE4D3, top panel). Total PDE4D3, myc-mAKAP, and PP2A C-subunit present in the immunoprecipitates were detected using non-phospho-specific antibodies (bottom three panels). n=3. B, PDE activity associated with myc antibody immunoprecipitates isolated from additional cells treated as in A was assayed using [3H]cAMP. n=3. *p<0.05 compared to bar 1. [Figure 12]Figure 12 shows that mAKAP-associated PP2A contains the B56δ subunit and is cAMP-activated. A, Protein complexes were immunoprecipitated from adult rat heart extracts (500 μg of total protein) using control (IgG) or mAKAP-specific antibodies as in Figure 2B and assayed for associated phosphatase activity. Immunoprecipitates were preincubated for 5 min without or with 50 μM CPT-cAMP, 10 nM OA, or 50 nM PKI, as indicated, followed by the addition of [32P]histone substrate. n = 3. *p < 0.05. B, Endogenous protein complexes were immunoprecipitated from adult heart extracts (2 mg of total protein) with B56δ and control (IgG) antibodies. mAKAP was detected by immunoblotting in 80 μg of extract and immunoprecipitates (25% loading). n = 3. C, Flag-tagged B56δ and / or GFP-tagged mAKAP were expressed in HEK293 cells. Protein complexes were immunoprecipitated using mAKAP antibody. B56δ in immunoprecipitates (25% load) and total extracts (5% load) was detected with Flag antibody by immunoblotting. n = 3. D, Phosphatase activity associated with mAKAP antibody immunoprecipitates prepared as in C was assayed using 32P-labeled histone substrate. n = 3. E, HEK293 cells expressing mAKAP and B56δ were treated with 5 μM Fsk and 10 μM IBMX (Fsk / IBMX) for 10 min, after which protein complexes were immunoprecipitated with mAKAP antibody. Phosphatase activity associated with the immunoprecipitates was assayed using [32P]histone substrate. n = 3. It is noted that PP2A B56δ and C subunit binding to mAKAP was not affected by Fsk / IBMX (see Figure 13 below). [Figure 13]Figure 13 shows that phosphorylation of B56δ by PKA increases mAKAP-associated PP2A activity. A, B56δ is phosphorylated by PKA at serine residues 53, 68, 81, and 566 (Ahn et al. 2007). Wild-type or alanine-substituted (S4A) B56δ at all four PKA sites was coexpressed in HEK293 cells with wild-type mAKAP or a full-length mAKAP mutant lacking the PKA binding site (ΔPKA, see Figure 3A). After stimulation with 5 μM Fsk and 50 μM IBMX, the protein complex was immunoprecipitated with mAKAP antibody, and associated proteins were detected by immunoblotting with B56δ, mAKAP, and PP2A-C antibodies (bottom three panels). PKA phosphorylation of B56δ was detected by immunoblotting with a B56δ phospho-Ser-566 specific antibody (P-B56δ, upper panel). n=3. B, Immunoprecipitates prepared in B were assayed for associated phosphatase activity. n=3. *p<0.05. [Figure 14] Figure 14 shows that PKA phosphorylation of B56δ enhances the dephosphorylation of mAKAP-associated PDE3D3. A. HEK293 cells expressing mAKAP (GFP-tagged), PDE4D3 (VSV- and GFP-tagged), and either wild-type B56δ or the B56δ S4A mutant of the PKA phosphorylation site were treated with 300 μM OA for 30 minutes as indicated, followed by stimulation with 5 μM Fsk for 10 minutes. Protein complexes were immunoprecipitated with mAKAP antibody in the presence of phosphatase inhibitors. The phosphorylation status of PDE4D3 present in the immunoprecipitates was measured using an antibody specific for Ser-54 of phosphorylated PDE4D3 (top panel). Total PDE4D3, mAKAP, B56δ, and PP2A-C proteins present in the immunoprecipitates were detected using non-phospho-specific antibodies (bottom four panels). n=3. B, PDE activity associated with protein complexes isolated from additional cells treated as in A was assayed using [3H]cAMP. n=3. *p<0.05 compared to bar 1. [Figure 15]Figure 15 shows that PKA and PP2A associated with the mAKAP complex cooperate to regulate PDE4D3 activity and cAMP degradation. PKA is composed of two regulatory subunits and two catalytic subunits. mAKAP-bound PP2A contains the A, B56δ, and C (catalytic) subunits. A, In unstimulated cells, basal PP2A activity maintains PDE4D3 dephosphorylation, presumably resulting in a more rapid rise in cAMP levels in response to subsequent agonists than would occur if PDE4D3 were phosphorylated and activated. At the same time, basal PDE4D3 activity should maintain low local cAMP levels, preventing spurious signaling. B, Gs-coupled receptor stimulation induces cAMP synthesis, exceeding the rate of cAMP degradation by PDE4D3 and activating mAKAP-bound PKA. PKA phosphorylates and activates both PDE4D3 and PP2A. PDE4D3 activity should limit peak cAMP levels and accelerate the rate of cAMP clearance after GPCR downregulation, whereas PP2A activation opposes PDE4D3 phosphorylation by PKA, reducing cAMP degradation and contributing to a larger and longer-lasting cAMP signal. [Figure 16] Figure 16 shows confirmation of PKA-phosphorylated PP1 inhibitory activity. Protein complexes were immunoprecipitated from rat heart extracts with PP1 or control IgG antibodies, and the associated phosphatase activity was assayed using [P]histone substrate in the presence or absence of 100 nM PKA-phosphorylated PP1 inhibitor-1 (Endo et al. 1996). n=3. [Figure 17]Figure 17 shows the distribution of mAKAP and PP2A catalytic subunits in neonatal rat cardiomyocytes. Neonatal rat ventricular myocytes were isolated as previously described (Pare, Easlick, et al. 2005). After treatment with 50 μM phenylephrine for 1 week to induce myofibril organization and mAKAP expression, cells were fixed and stained with 0.25 μg / ml mouse anti-PP2A-C (green), 0.1 μg / ml OR010 rabbit anti-mAKAP (red) affinity-purified antibodies, and rhodamine phalloidin (blue in the composite image) to reveal actin myofibrils, as previously described. Four-color images were acquired at 400x on a Zeiss LSM510 / UV confocal microscope. For clarity, separate images of the PP2A-C subunit and mAKAP are shown. The PP2A-C subunit is present in a diffuse, punctuated pattern in the cytosol, while mAKAP is restricted to the nuclear membrane. The presence of PP2A-C subunit staining at the nuclear membrane is consistent with the presence of PP2A-mAKAP complexes (yellow in the composite image). Control IgG staining is shown in the right panel. n=3. [Figure 18] Figure 18 shows that mAKAP fragments do not bind to PP1 in HEK293 cells. mAKAP-GFP fusion protein was expressed in HEK293 cells, and the protein complex was immunoprecipitated with PP1 antibody. Despite strong expression (lower panel), the mAKAP fusion protein was not precipitated with PP1 antibody. n=3. [Figure 19]Figure 19 shows that SRF phosphorylation is regulated by the mAKAPβ signalosome in cardiomyocytes. (A) SRF domain structure. Known phosphorylation residues are indicated (Li et al. 2014; Mack 2011; Janknecht et al. 1992). (B) Neonatal rat ventricular myocytes (NRVMs) were transiently transfected with siRNA and SRE-luciferase and control Renilla luciferase plasmids. Normalized luc:rluc ratios are shown. n=3. (C) Co-immunoprecipitation of endogenous complexes from mouse heart extracts. n=3. (D) HA-tagged RSK3WT or S218A inactive mutant (Li, Kritzer, et al. 2013) and / or myc-mAKAPβ were expressed in COS-7 cells for co-immunoprecipitation assays. n=3. (E) NRVM extracts were obtained 2 days after siRNA transfection with or without 10 μM PE. See Figure S1B. n=3. * for control siRNA + PE, † for control siRNA + no drug. (F) Adult rat ventricular myocytes (ARVM) were infected with adenovirus expressing myc-GFP or myc-GFP-RBD and treated with 20 μM PE for 1 day. n=3. * for myc-GFP + PE, † for myc-GFP + no drug. (G) NRVM in minimal maintenance medium were treated with 1 μM okadaic acid (OA) or 1 μg / ml cyclosporine A (CsA) for 1 hour. n=4. * for no drug control. (H) NRVM transfected with control or mAKAP siRNA were used for co-immunoprecipitation assays. PP2A holoenzyme contains a homodimeric core of A and C subunits and a scaffolding B subunit (Dodge-Kafka et al. 2010). PP2A C subunit (PP2A-C) was detected by immunoblotting. n=3. (I) NRVM were infected with adenovirus expressing myc-PBD or β-gal, followed by co-immunoprecipitation assay. n=3. (J) ARVM were infected with myc-PBD or β-gal adenovirus and treated with 10 μM Iso for 1 day. n=4. * for β-gal + Iso, † for β-gal + no drug. [Figure 20] Figure 20 shows that SRF S103 phosphorylation is a determinant of myocyte centripetal growth. Adult rat ventricular myocytes (ARVMs) were infected with adenovirus and cultured with or without 20 μM PE or 10 μM Iso for 24 hours, followed by immunocytochemical staining and cell width and length measurements (maximum dimension parallel to or perpendicular to the striations; horizontal bar = 25 μm). (A, B) Myocytes were infected with adenoviruses expressing β-gal (control) or HA-tagged RSK3 and maintained in minimal medium. At the top, α-actinin is red, nuclei are blue, and HA-RSK3 is green. At the bottom, HA-RSK3 is grayscale. n = 4. (C-F) Myocytes were infected with adenoviruses expressing SRF WT, S103D, or S103A, or with control virus. Flag-SRF is green, α-actinin is red, and nuclei are blue. * vs. no drug with the same virus, † vs. control under the same treatment conditions, ‡ vs. SRF WT under the same treatment conditions. D: n=3, F: n=5. (G, H) Myocytes were infected with adenovirus expressing myc-GFP or myc-GFP-RBD (green). (I, J) Myocytes were infected with adenovirus expressing myc-PBD or β-gal control. (G-J) α-actinin—red, nuclei—blue. * vs. no drug control with the same protein, † vs. control protein under the same treatment conditions. n=4. The relative frequency distribution of cells measured in these experiments is shown in Figure S4. [Figure 21] Figure 21 shows that PP2A dephosphorylates SRF S103. GST-SRF fusion protein purified from bacterial extracts and plated on glutathione beads was incubated with 0.5 μg of purified RSK3 (Millipore) for 30 minutes, washed twice with PP2A reaction buffer, and then incubated with 50 ng of purified PP2A with or without 10 nM okadaic acid for 30 minutes. [Figure 22]Figure 22 shows the AAV9sc.myc-PBD. A. The AAV9sc.myc-PBD contains a minigene expressing a myc-tagged rat PBD peptide (rat mAKAP aa 2134-2314) and a deleted right ITR, conferring self-complementarity and presumably reducing delay and increasing expression efficiency (Andino et al., 2007). The AAV harbors the cardiotrophic serotype 9 capsid protein and expresses the encoded protein under the control of the cardiomyocyte-specific tritroponin T promoter (cTnT) (Prasad et al., 2011). B. The shuttle plasmid for the AAV9sc.myc-PBD is shown. [Figure 23] Figure 23 shows PBD anchoring disruptor treatment. (A) Myc-tagged rat mAKAP PBD (AAV9sc.myc-PBD) and myc-GFP (AAV9sc.GFP) were expressed in mice using self-complementary AAV9 and the cardiomyocyte-specific tritroponin T promoter (Prasad et al., 2011). (B) The time course of the AAV9sc.myc-PBD treatment study is shown in C-H. Mice were 8 weeks old at the start of the study. (C) Representative whole-heart photographs at the endpoint are shown. Horizontal bar = 5 μm. (D-H) Sequential M-mode echocardiography is shown. n = AAV9sc.myc-PBD-8 (green), AAV9sc.GFP-5 (black). *P value for the difference between cohorts at a given time point. LV remodeling index = mass / end-diastolic volume. LVAW; d = left ventricular anterior wall thickness in diastole. [Figure 24] FIG. 24 shows the nucleotide sequence of human RSK3 (SEQ ID NO: 15). [Figure 25] FIG. 25 shows the nucleotide sequence of the rat mAKAPα mRNA translated from the open reading frame (SEQ ID NOs: 2 and 16). [Figure 26] FIG. 26 shows the nucleotide sequence of the human mAKAPβ mRNA translated from the open reading frame (SEQ ID NOs: 17 and 18). [Figure 27]FIG. 27 shows the nucleotide sequence of the human mAKAPα mRNA translated from the open reading frame (SEQ ID NOs: 19 and 20). [Figure 28] Figure 28 shows the amino acid sequence of human mAKAP (SEQ ID NO: 8). mAKAPα starts at residue 1 and mAKAPβ starts at residue 243. The PBD is shown in bold. [Figure 29] FIG. 29 shows the amino acid sequence of the human PBD expressed in AAV (SEQ ID NO: 9). [Figure 30] Figure 30 shows an alignment of the amino acid sequences of human and rat PBDs expressed in AAV species (SEQ ID NOs: 9 and 12). The rat PBD has an N-terminal Myc-tag [EQKLISEEDL] (SEQ ID NO: 21) (Figure 4). [Figure 31] FIG. 31 shows a map of the human PBD shuttle plasmid. [Figure 32] FIG. 32 shows the nucleotide sequence of the pscAAV-hmAKAP PBD plasmid (SEQ ID NOs: 10 and 11). [Figure 33]Figure 33 shows that SRF phosphorylation is decreased in dilated hearts. (A-E) Protein extracts from mouse ventricles were assayed for phosphorylated and total SRF 5 minutes (short-term pressure overload, n = 4, 4) or 16 weeks (heart failure, n = 15, 19) after TAC or sham surgery. (A) Representative Western blots are shown. (B) Densitometry of the upper panel in A is shown. (C) Five minutes after pressure overload, RSK3 was immunoprecipitated using the N-16 RSK3-specific antibody and detected using the OR43 RSK3 antibody and the RSK3 S218 phospho-specific antibody, demonstrating RSK3 activity. The immunoprecipitation-Western assay was validated using RSK3- / - mice (not shown). n = 3 for each condition. (D) Heart failure was induced by 16 weeks of pressure overload. M-mode echocardiography for left ventricular (LV) volumes in diastole and systole and ejection fraction demonstrated that TAC hearts were dilated and dysfunctional. Measurement of edematous lung weight (linked to tibia length), indicating the presence of pulmonary edema, demonstrated that TAC mice had heart failure. (E) Densitometry of the lower panel in A is shown. (F-H) Left ventricular tissue from human patients (including nonischemic and ischemic cardiomyopathies, nondilated congenital heart disease, and controls) was assayed for SRF S103 phosphorylation and separated by normal (<5.3 cm, n = 7) or elevated (>5.3 cm, n = 8) left ventricular internal diameter (LVID; d) in diastole. Equal loading on blots was confirmed using Ponceau S staining of the major protein bands (not shown). DETAILED DESCRIPTION OF THE INVENTION

[0042] As mentioned above, AKAP-derived signaling complexes are involved in physiological and pathological cardiac signaling. Therefore, the present inventors have demonstrated that mitochondrial vasculopathy plays a central role in the regulation of cardiac pathological processes. Targeting specific protein-protein interactions as an approach to suppress the By using drugs that inhibit the signaling properties of individual AKAP signaling complexes, Such a therapeutic approach would allow for the selective inhibition of a given cellular response, making it possible to This offers advantages over previous treatment approaches.

[0043] Anchoring proteins, including mAKAP, are therapeutic targets for the treatment of cardiac hypertrophy and heart failure. In particular, the present inventors have demonstrated that AKAP-mediated protein-protein interactions can be inhibited. This is due to the activation of key transcription factors that initiate the remodeling process that leads to cardiac hypertrophy. can be used to inhibit the ability of mAKAPs to regulate the activation of enzymes that play a central role in I discovered that it was possible.

[0044] One aspect of the present invention is the improvement of ventricular geometry, i.e., the reduction of LV volume by less elongated myocytes. Reduced diameter and / or increased LV wall thickness due to wider myocytes By reducing wall stress (Laplace's law), the systolic flow in HFrEF-prone hearts Prevention of systolic dysfunction may improve function. Protein phosphatase 2A (PP2 A) Muscle A-kinase anchor protein (mAKAP, also known as AKAP6) A new gene therapy vector based on expression of an anchoring disruptor peptide derived from It is shown in the figure.

[0045] As described below, the present inventors have demonstrated that the transcription factor serum response factor (SRF) mediates myocardial In cells, RSK3 mediates the mAKAPβ signalosome, activating Ser 103 Phosphorylated by SRF is then catalyzed by scaffold-bound protein phosphatase 2A (PP2A). Recently, we have discovered that HFrEF can be dephosphorylated by ATP. Methods for inhibiting eccentric changes in ventricular morphology are the subject of the present invention.

[0046] Protein phosphatase 2A (PP2A) has been considered a constitutive housekeeping enzyme. It was previously thought to be a protein, but has been shown to contribute to the regulation of many phosphorylation events. For example, in cardiomyocytes, PP2A regulates calcium and MAPK signaling. Involved in regulating nulling (duBell, Lederer, and Rogers 1996; duBell et al. 2002; Liu and Hofman n 2004). PP2A is a stable, ubiquitously expressed catalytic (PP2A-C) and scaffold (PP2A-A) subunit heterodimer, as well as 21 known diverse B subunits. Serine / threonine phosphatase inhibitors exist as heterotrimeric complexes consisting of one of (Lechward et al. 2001; Wera and Hemmi ngs 1995). PP2A B subunits are B (or PR55), B' (or B 56) and B'' (or PR72), It has been proposed to regulate both the catalytic activity and the intracellular targeting of phosphatases (V The present inventors have identified a protein phosphatase (PPH) that associates with the mAKAP complex. Phosphatase 2A (PP2A) catalyzes the dephosphorylation of PDE4D3 serine residue 54 It is possible to reverse PDE4D3 activation by mAKAP complexes in heterologous cells. (Dodge-Kafka et al. 2017) 10) Mapping studies have revealed that the C-terminal mAKAP domain (residues 2085–2319) revealed that PP2A binds to the ATP-dependent ATPase (Dodge-Kafka et al. 2 010). Binding to mAKAP is required for PP2A function in PDE4D3, and C Deletion of the terminal domain inhibits both baseline and forskolin-stimulated PDE4D3 activity. Interestingly, the mAKAP complex in the heart The PP2A holoenzyme contains the PP2A targeting subunit B56δ (Dodge-Kafk a et al. 2010). Like PDE4D3, B56δ is a PKA substrate, PKA phosphorylation of mAKAP-bound B56δ doubles the phosphatase activity of the complex. Therefore, the activity of PKA phosphorylated mAKAP in heterologous cells was increased. Expression of the B56δ mutant, which is unable to inhibit PDE4D3, resulted in increased PDE4D3 phosphorylation. These findings together suggest that PP2A associated with the mAKAP complex mediates PDE4D3 dephosphorylation. stimulates PDE4D3 in unstimulated cells and inhibits adenylyl cyclase activity mediating a cAMP-induced positive feedback loop following activation and B56δ phosphorylation Thus, we demonstrated that PKA-PDE4D3-PP2A- The mAKAP complex is a complex of protein kinases and phosphatases that mediates how molecular signals This example illustrates how the complexes involved in the regulation of localized intracellular signaling can be dynamically regulated. The relationship to cell function and any potential therapeutic implications remains to be defined in previous studies. (Dodge-Kafka et al. 2010).

[0047] The present inventors have identified a novel mechanism of action of mAKAP-bound PP2A in cardiomyocytes and the mechanism of this action. The therapeutic implications of this mechanism are disclosed here. The inventors demonstrate that the transcription factor SRF mediates the binding of mAKAPβ to R Ser by SK3 103(Figure 19) and Ser 103 SRF in We demonstrate that phosphorylation constitutes an epigenetic switch that promotes concentric cardiomyocyte hypertrophy. Importantly, the Ser of SRF 103 However, mAKAPβ scaffold It is disclosed that PP2A can dephosphorylate ATP-dependent ATPases (Fig. 19 and Fig. 21). SRF Ser 103 Phosphorylation of α-glucan has been shown to induce afferent muscle cell hypertrophy. These findings provide a novel mechanism for regulating cardiomyocyte morphology and mAKAPβ In particular, the inventors discovered an unexpected function of mAKAPβ-bound SRF Consistent with the role of PP2A as a phosphatase for mAK in vitro Removal of PP2A from APβ inhibits Ser-mediated ischemia in cardiomyocytes. 103 phosphorylation of (Fig. 1 9) and can promote concentric cardiomyocyte hypertrophy (Figure 20), It may protect against the development of systolic dysfunction after myocardial infarction (Figure 23).

[0048] Inhibition of PP2A binding to mAKAPβ is due to the PP2A binding site mapping in mAKAPβ. We demonstrate novel improvements in pharmacokinetics and in vivo mAKAP-PP2A in cardiac disease. mAKAPβ2134-2314 (Figure 19) or human mAKA This can be achieved by expressing a competitive peptide containing residues 2132-2319 of Pβ. The C-terminal domain of human mAKAP, which is homologous to that of other mAKAPs, also binds PP2A. It should be noted that the results shown in Fig. 10 are as follows. Human sequence for mouse mAKAP2134-2314 (human mAKAP amino acid residue homology) 2132-2319) are also predicted to bind PP2A and are involved in PP2A-mAKAP binding competition. Constitutes a synthetic peptide.

[0049] PP2A anchoring disruptor peptide delivered via virus-based gene therapy Effective delivery is demonstrated by efficacy in a mouse infarction model (Figure 23). Alternatively, delivery of such peptides that can inhibit PP2A-mAKAPβ interaction may be Cell-penetrating sequences such as transactivators of transcription peptides and polyarginine tails or conjugation with fatty-derived groups such as stearates. It is also possible to create peptide mimetics (i.e., peptides with structural variations in the original sequence). The peptides are resistant to exoproteases and endoproteases without affecting their structural and functional properties. Stability can be improved by the use of a hydroxybenzoate (which provides protection against protease enzymes).

[0050] The inventors also demonstrated that small molecule disruptors disrupt specific interactions within AKAP-derived complexes. We found that small molecule disruptors can be used to target Such compounds can be identified through a combination of chemical design and screening methods. For the treatment of chronic heart failure, a specific binding site of AKAP is targeted to induce AKAP activation in cardiomyocytes. can be designed to inhibit the interaction of PP2A with ATP and improve normal cardiac contractility. .

[0051] The present invention relates to any cardiac disease caused by the interaction between PP2A and mAKAPβ. Such cardiac dysfunction is manifested by signs and symptoms such as shortness of breath and fatigue. These conditions can include high blood pressure, coronary artery disease, myocardial infarction, valvular disease, primary cardiomyopathy, and congenital heart disease. These include diseases such as heart disease, cardiac arrhythmias, pulmonary disease, diabetes, anemia, hyperthyroidism, and other systemic diseases. There may be various causes, including but not limited to these.

[0052] In accordance with the present invention, conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art may be employed. Such techniques are well explained in the literature. rook et al., “Molecular Cloning: A Laborat ory Manual” (4th Ed., 2012); otocols in Molecular Biology” Volumes I- III [Ausubel, RM, ed. (1994)]; Biology: A Laboratory Handbook” Volumes I-III [JE Celis, 3rd ed. (2005))]; Current Protocols in Immunology” Volumes I-III [Coligan, JE, ed. (2005)]; Gonucleotide Synthesis” (MJ Gait ed. 1 984); “Nucleic Acid Hybridization” [BD Hames & SJ Higgins eds. (1985)]; nscription And Translation” [BD Hames & SJ Higgins, eds. (1984)]; ll Culture” [RI Freshney, ed. (1986)]; “Immobilized Cells And Enzymes” [IRL Pr ess, (1986)]; B. Perbal, “A Practical Gui C. Mach ida, “Viral Vectors for Gene Therapy: Met hods and protocols” (2010); J. Reidhaar- Olson and C. Rondinone, “Therapeutic App Applications of RNAi: Methods and Protocols ” (2009) is referred to.

[0053] The following definitions and abbreviations are used herein: AC5 - Adenylyl cyclase type 5 ACE - Angiotensin-converting enzyme ANF- atrial natriuretic factor ARVM-adult rat ventricular myocytes CaN-calcineurin CArG Box- (CC9AT)6GG CPT - cAMP-8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate phosphoric acid CsA- cyclosporine A CTKD - C-terminal kinase domain ERK - extracellular signal-regulated kinase FBS - Fetal Bovine Serum Fsk- Forskolin GFP - Green Fluorescent Protein GPCR - G-protein coupled receptor HDAC - histone deacetylase Gs-stimulatory G protein GST-glutathione-S-transferase HIF1α-hypoxia-inducible factor 1α HFrEF - Heart failure with reduced ejection fraction IBMX- 3-Isobutyl-1-methylxanthine Iso-Isoproterenol LIF- leukemia inhibitory factor MADS- (MCM1, agamous, deficiens, SRF) domain- CArG box (CC9AT) 6GG mediates DNA binding to serum response element (SRE) The MADS-box gene family consists of the first four members, ignoring ARG80. It was later named as an acronym for MCM1 from the budding yeast Saccharomyces cerevisiae AGAMOUS from Arabidopsis thaliana DEFICIENS from Antirrhinum majus SRF from humans Homo sapiens mAKAP - Muscle A-kinase anchoring protein mAKAPα - alternatively spliced ​​isoform expressed in neurons, 255 kDa mAKAPβ - alternatively spliced ​​isoform expressed in striated muscle cells, 230 kDa MAPK - mitogen-activated protein kinase MEF2 - Myocyte enhancer factor-2 MgAc - Magnesium acetate MI - Myocardial infarction NCX1 - sodium / calcium exchanger NFATc - Nuclear factor of activated T cells NRVM- neonatal rat ventricular myocytes NTKD - N-terminal kinase domain OA- Okadaic acid PBD-PP2A- Anchoring disruptor-Attenuates eccentric hypertrophy PDE4D3 - cAMP-specific phosphodiesterase type 4D3 PDK1- 3´ phosphoinositide-dependent kinase 1 PE- Phenylephrine PHD - prolyl hydroxylase PI4P - phosphatidylinositol-4-phosphate PKA - Protein Kinase A PKD - Protein kinase D PKI - Protein Kinase Inhibitors PLCε-phospholipase Cε PKA- cAMP-dependent protein kinase PP2A-protein (serine-threonine) phosphatase-SRF Ser 10 3 Dephosphorylates PP2B - calcium / calmodulin-dependent protein phosphatase 2B RBD-isoform-specific N-terminal RSK3 domains are distinct within mAKAPβ The "RSK3 binding domain" binds at residues 1694-1833 (RBD) RSK- p90 ribosomal S6 kinase RyR2 - type 2 ryanodine receptor siRNA - small interfering RNA oligonucleotides shRNA - short hairpin RNA SRE - serum response element SRF - Serum response factor - transcription factor (SRF Ser 103 Phosphorylation of Induce myocyte and cardiac hypertrophy. Inhibition of phosphorylation improves cardiac structure and function. (It is.) siRNA - small interfering RNA TAC- Aortic coarctation TCA - Trichloroacetic acid VSV - vesicular stomatitis virus

[0054] Unless otherwise defined, all technical and scientific terms used herein are defined by the present invention. The meanings used herein are the same as those commonly understood by those skilled in the art. Any methods and materials similar or equivalent to those described may be used in the practice or testing of the present invention. Generally, cell and molecular biology is used, but preferred methods and materials are described. The terminology and techniques used in the field of biochemistry and molecular chemistry are well known and understood. Those commonly used in the art. Specific experimental techniques not specifically specified. are generally well known in the art and are cited and described herein. This is done by conventional methods as described in various general and more specific references available in the literature. For clarity, the following terms are defined below:

[0055] The present invention relates to various intracellular signals and pathways that lead to cardiomyocyte hypertrophy and / or dysfunction. We therefore acknowledge that this is mediated by the interaction between PP2A and mAKAPβ. The inventors have utilized this interaction to prevent and / or treat cardiomyocyte hypertrophy and / or dysfunction. Various methods for inhibiting its action have been discovered.

[0056] Thus, the present invention includes a method for protecting the heart from damage, the method comprising administering to the heart a PP2A and mAb. A pharmaceutically effective amount of a composition that inhibits interaction with KAPβ is administered to a subject at risk of such a disorder. The "pharmacologically effective amount" is determined empirically based on the method of delivery. It should be understood that the dosage can be determined and may vary depending on the method of delivery.

[0057] The present invention also provides a pharmaceutical composition for inhibiting the interaction between PP2A and mAKAPβ. It also relates to a method of treating cardiac disease by administering the substance to a patient.

[0058] The present invention also relates to compositions that inhibit the interaction between PP2A and mAKAPβ. In certain embodiments, these inhibitory compositions or "inhibitors" include gene therapy delivery. In another embodiment, the inhibitor is a peptide inhibitor that can be administered by any known method. It may be a small molecule inhibitor.

[0059] Specifically, the present invention relates to methods and compositions for treating or protecting the heart from injury, The methods include (1) inhibiting the interaction between PP2A and mAKAPβ, (2) inhibiting the interaction between PP2A and mA (3) a drug that inhibits the activity of PP2A and mAKAPβ; or (4) a drug that inhibits the expression of PP2A and mAKAPβ. The therapeutic effect is achieved by administering a therapeutically effective amount of the composition to a patient at risk for such a disorder.

[0060] The present invention also relates to a method of treating or protecting the heart from damage, the method comprising administering PP a pharmaceutically effective amount of a composition that inhibits a cellular process mediated by anchoring of 2A. This is done by administering it to patients at risk of such disorders.

[0061] In one embodiment, the composition comprises mAKAPβ and a peptide. The mAKAPβ peptide is derived from the carboxy terminus of the mAKAPβ amino acid sequence. In a particularly preferred embodiment, the mAKAPβ peptide comprises at least mAKAPβ It is a fragment of amino acids 2083 to 2319 of the amino acid sequence.

[0062] In a preferred embodiment, the mAKAPβ peptide comprises at least mAKAPβ amino acid sequence. It is a fragment of amino acids 2133 to 2319 of the amino acid sequence.

[0063] In another embodiment, the composition comprises a compound that inhibits expression of either or both of PP2A and mAKAPβ. In a preferred embodiment, the siRNA comprises a small interfering RNA that inhibits the expression of mAK. siRNAs that inhibit APβ expression are expressed using short hairpin RNA expression vectors or biological agents. It is produced in vivo after administration of shRNA.

[0064] The compositions of the present invention can be administered directly or can be administered using a viral vector. In a preferred embodiment, the vector is an adeno-associated virus (AAV).

[0065] In other embodiments, the composition comprises a small molecule inhibitor. The molecule is a PP2A inhibitor.

[0066] In other embodiments, the composition comprises a molecule that inhibits the binding, expression, or activity of mAKAPβ. In a preferred embodiment, the molecule is a mAKAPβ peptide. Expression can be achieved using viral vectors, including avian-associated viruses (AAV).

[0067] In yet other embodiments, the composition comprises a molecule that interferes with a mAKAPβ-mediated cellular process. In a preferred embodiment, the molecule interferes with the anchoring of PP2A.

[0068] The present invention also provides a method for determining whether the binding interaction between PP2A and mAKAPβ occurs directly or via a PP2 The potential for cardiac disease, as measured by measuring downstream effects of binding of A to mAKAPβ The present invention also relates to diagnostic assays for determining disease propensity. We provide test kits for this purpose.

[0069] In yet other embodiments, the inhibitor is an antisense RNA, including shRNA. Expression of PP2A and mAKAPβ, including ribozymes and small interfering RNA (siRNA), This includes any molecule that inhibits the expression of

[0070] The present invention also provides a compound useful for inhibiting the expression and / or binding of PP2A and mAKAPβ. In one embodiment, the present invention includes an assay system for screening for potentially effective drugs. The test drug showed no significant effect on the binding activity of PP2A and mAKAPβ compared with the control. To measure its effect on the The present invention also provides an extract containing 2A and mAKAPβ. A test kit for the assay is provided.

[0071] In preparing the peptide compositions of the present invention, the amino acid sequence of PP2A or mAKAP (Figure 3) All or part of the sequence can be used. The carboxy-terminal region of the protein is used as an inhibitor. At least 10 amino acids of the mAKAP sequence are used. 25 amino acids are used. Most preferably, amino acids 2133 to 231 of mAKAP are used. The peptide portion of 9 is used.

[0072] Various amino acid substitutions, deletions, or insertions also inhibit the interaction between PP2A and mAKAPβ It should be understood that the ability of an inhibitory peptide to inhibit the activity of such a compound may be improved. Substitution mutations are made in a non-conservative manner (i.e., by substituting amino acids with particular sizes or characteristics). (by changing an amino acid belonging to one group to an amino acid belonging to another group) or in a conservative manner (i.e., by identifying amino acids belonging to a group of amino acids with a particular size or property) , by changing to an amino acid belonging to the same group), the amino acid of the resulting protein This conservative change can generally be done to The structure and function of the protein are less affected by non-conservative changes. The present invention provides a method for producing a protein having a structure, activity, or function that is more easily altered than conventional methods. It is considered to include sequences containing conservative changes that do not significantly alter the activity or binding characteristics of the molecule. It is necessary to

[0073] Below are some examples of different amino acid classifications: Nonpolar R-group amino acids: alanine, valine, leucine, isoleucine, proline, phenyl Nilauranine, tryptophan, and methionine. Amino acids with polar uncharged R groups: glycine, serine, threonine, cysteine, tyrosine, Asparagine, glutamine. Amino acids with polar R groups (negatively charged at pH 6.0): aspartic acid, glutamic acid. Basic amino acids (positively charged at pH 6.0): lysine, arginine, histidine (pH 6 (The one in .0).

[0074] Another classification could be amino acids with a phenyl group: phenylalanine, tryptophan Ah, tyrosine.

[0075] Another classification can be by molecular weight (i.e., size of the R group): glycine (75 ), alanine (89), serine (105), proline (115), valine (117), leonine (119), cysteine ​​(121), leucine (131), isoleucine (13 1), asparagine (132), aspartic acid (133), glutamine (146), dextrin (146), glutamic acid (147), methionine (149), histidine (pH 6 (155), phenylalanine (165), arginine (174), Tyrosine (181), tryptophan (204).

[0076] Particularly preferred substitutions are Lys to Arg and vice versa, so as to be able to maintain a positive charge; - Glu to Asp and vice versa, so as to maintain a negative charge; - Ser to Thr, so as to maintain a free -OH, and - Gln versus Asn, so as to be able to maintain free NH2.

[0077] Amino acid substitutions may also be introduced to replace amino acids with particularly desirable properties. For example, Cys can be a candidate site for disulfide bridges with other Cys. His can be introduced specifically as a "catalytic" site. (i.e., His can act as an acid or a base, the most common in biochemical catalysis. Pro is a particularly important amino acid that induces β-turns in protein structures. Due to the surface structure, it is possible to introduce at least about 70% (preferably less and (most preferably) at least about 80%, and most preferably at least about 90% or 95% of the amino acid residues are identical. or represent conservative substitutions, two amino acid sequences are "substantially homologous" .

[0078] Similarly, the nucleotide sequences used in the present invention may also contain substitutions, deletions, or insertions. When the codons encoding a particular amino acid are degenerate, Any codon that encodes one amino acid can be used. If it is desired to replace the nucleotide sequence according to the known genetic code, It is possible.

[0079] Nucleotides and oligonucleotides can also be modified. For a description of nucleotides and oligonucleotides, see U.S. Pat. No. 6,223,629, which is incorporated by reference in its entirety. No. 7,807,816 describes example modifications.

[0080] At least about 70% (preferably at least about 80%, most preferably at least Two nucleotide sequences are considered "real" when they share approximately 90% or 95% (or more) of their nucleotides. "Qualitatively homologous" or "substantially identical."

[0081] At least about 70% (preferably at least about 80%, most preferably at least When two nucleotides (approximately 90% or 95%) are able to hydrogen bond to the target sequence, The sequences of the primers are "substantially complementary."

[0082] The term "standard hybridization conditions" refers to hybridization and washing conditions. In both cases, the salt conditions and temperature conditions are substantially equivalent to 5x SSC and 60°C. However, those skilled in the art will appreciate that such "standard hybridization conditions" include: The sodium and magnesium concentrations in the nucleotide sequence, the length and concentration of the nucleotide sequence, and the mismatch It will be understood that the results will depend on certain conditions, including the rate of polymerization, and the rate of formamide. In addition, the important thing in determining the "standard hybridization conditions" is The two sequences to be transduced are RNA-RNA, DNA-DNA, or RNA-DNA. Such standard hybridization conditions are Typically, the wash is performed at a higher stringency than the predicted or determined T m Yo This is 10 to 20°C lower than the above range and can be easily determined by those skilled in the art according to well-known procedures.

[0083] The term "pharmaceutically acceptable" means a substance that is physiologically acceptable and suitable for administration to humans. When administered, it usually produces allergic or similar adverse reactions such as stomach upset and dizziness. refers to molecular entities and compositions that cannot be identified.

[0084] The term "therapeutically effective amount" refers to an amount sufficient to prevent clinically significant changes in cardiomyocyte characteristics. Preferably, at least about 30 percent, more preferably at least 50 percent by an amount sufficient to reduce the As used herein,

[0085] Preparation of therapeutic compositions containing polypeptides, analogs, or active fragments as active ingredients The preparation of such compositions is well understood in the art. Typically, such compositions are prepared as liquid formulations. They are prepared as injections or suspensions, but are suitable for dissolution or suspension in liquid prior to injection. Solid forms containing the active therapeutic ingredient can also be prepared. The preparation can also be emulsified. The ingredients are often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, or ethanol. and combinations thereof. Further, if desired, the composition may contain Minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like, may be present to enhance effectiveness.

[0086] The polypeptides, analogs, or active fragments, and small molecule inhibitors are pharmaceutically acceptable. The compound can be formulated into a therapeutic composition as a neutral salt form. , inorganic acids such as hydrochloric acid or phosphoric acid, or acetic acid, oxalic acid, tartaric acid, and mandelate. formed with organic acids such as hydroxybenzoates (with free amino groups of polypeptide or antibody molecules) Also included are acid addition salts formed from free carboxyl groups, e.g. Inorganic salts such as sodium, potassium, ammonium, calcium, or ferric hydroxide and isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine The bases may be derived from organic bases such as ginseng and procaine.

[0087] The therapeutic compositions of the present invention are conventionally administered intravenously, for example, by injection of a unit dose. The term "unit dose" when used in reference to the therapeutic compositions of the present invention means a dose for a human With respect to physically discrete units suitable as unit dosages, each unit is a dose of the dilution required. A compound calculated to produce a desired therapeutic effect in association with a diluent, i.e., a carrier or vehicle. The formulation contains a predetermined amount of active substance.

[0088] The compositions are administered in a manner compatible with the dosage formulation, and in such amount as is therapeutically effective. The subject to be treated, the capacity of the subject's immune system to use the active ingredient, and the desired PP2A- The exact amount of active ingredient required for administration will depend on the degree of inhibition of mAKAPβ binding. However, appropriate dosages may vary depending on the individual and are determined by the physician's judgment. Approximately 0.1 to 20, preferably about 0.5 to about 10, per kilogram of individual body weight per day and more preferably in the range of 1 to several milligrams of active ingredient, depending on the route of administration. The appropriate dosage regimen for initial administration and booster injections may vary, but initial administration, and subsequent re-administration at intervals of 1 hour or more by subsequent injection or other administration is typical. Alternatively, sufficient to maintain a concentration of 10 nanomolar to 10 micromolar in the blood. Consideration should be given to continuous intravenous infusion.

[0089] Since the inhibitor needs to reach the cytosol, the peptides of the present invention It may be necessary to modify the peptide to allow its translocation in the membrane, or to coat it with a peptide inhibitor. Similarly, nucleic acid inhibitors (siRNA, s RNAs (including hRNA and antisense RNA) can be expressed by the vector. Any vector that is capable of entering the cells to be transfected can be used in the present invention. In particular, viral vectors "infect" cells and express a desired RNA or peptide. Any viral vector that can "infect" cells can be used. A particularly preferred viral vector is the adeno-associated virus (AAV).

[0090] siRNAs inhibit the translation of target mRNA through a process called RNA interference. When an siRNA is perfectly complementary to a target mRNA, the siRNA binds to the mRNA. shRNAs act by promoting the degradation of ribosomal protein A. , have certain advantages over siRNA in that they are produced as oligonucleotides. siRNA oligonucleotides are typically synthesized in the laboratory and then delivered to the cytoplasm. In contrast, shRNAs are delivered to cells using a vector-based delivery system. The shRNA is expressed as a minigene delivered to the cell nucleus via transcription. siRNAs are processed into mature siRNA species by cellular enzymes such as ribosomal and Dicer. While typically 99% decomposed after 48 hours, shRNA can be expressed for up to 3 years. shRNA can be delivered at much lower copy numbers than siRNA (5 copies) (vs. low nM), are much less likely to cause off-target effects, immune activation, inflammation, and toxicity siRNA is suitable for acute disease conditions that can tolerate high doses, whereas shRNA , and is appropriate for chronic serious diseases or disorders where low doses are desired (http: / / www.b enitec.com / technology / sirna-vs-shrna).

[0091] Guidelines for designing siRNA and shRNA are provided in the text of Elbashir (2001). and https: / / www.thermofisher.com / us / en / home / references / ambion-tech-support / rnai -sirna / general-articles / -sirna-design-gu idelines.html and http: / / www.invivogen.com / On various websites, including review-sirna-shrna-design all of which are incorporated herein by reference in their entirety. 1 nucleotide is A or G. 25-29 nucleotide siRNAs are shorter. shRNAs with double strand lengths of 19 to 21 are more effective than those with double strand lengths of 19 to 21. siRNA and shRNA are preferably 19 to 29 nucleotides. The loop sequence in the shRNA can be 3 to 9 nucleotides in length, and can be 5, 7, or 9 nucleotides. Nucleotides are preferred.

[0092] Regarding small molecule inhibitors, any small molecule that inhibits the interaction between PP2A and mAKAPβ Furthermore, any compound that inhibits the activity of PP2A and / or mAKAPβ can be used. Any small molecule can be used.

[0093] Small molecules of similar structure and function can be determined similarly by rational and screening approaches. It is Noh.

[0094] Similarly, any small molecule that inhibits the expression of PP2A and / or mAKAPβ can be used. This can be done.

[0095] More particularly, the present invention is described by the following paragraphs which set out preferred embodiments thereof. can be. 1. A method for treating or preventing heart failure with reduced ejection fraction, comprising administering to cardiac cells of a patient a vasopressin-containing compound. a method for treating a patient with rhesus malabsorption, comprising administering a composition that maintains phosphorylation levels in serum response factor (SRF) Law. 2.SRF is Ser 103 The method according to item 1, wherein the protein is phosphorylated at 3. Dephosphorylation activity of protein (serine-threonine) phosphatase 2A (PP2A) Item 1, the method of inhibiting sex. 4. Anchoring of PP2A to muscle A-kinase anchoring protein (mAKAPβ) The method according to item 3, wherein the activating agent inhibits the activation of the activating agent. 5. The method according to item 4, wherein the composition comprises a fragment of mAKAPβ. 6. The composition has at least 90% sequence identity with a fragment of mAKAPβ. The method of claim 5, comprising an amino acid sequence. 7. The composition according to item 5, wherein the composition comprises a fragment of amino acids 2132 to 2319 of mAKAP. The method described below. 8. The method according to item 5, wherein the composition comprises amino acids 2132 to 2319 of mAKAP. 9. The method according to paragraph 4, wherein the composition comprises a fragment of PP2A. 10. The composition according to paragraph 4, wherein the composition comprises a vector encoding a fragment of mAKAP. method. 11. The composition has at least 90% sequence identity with a fragment of mAKAP. The method of claim 4, comprising a vector encoding the amino acid sequence. 12. The vector encodes a fragment of amino acids 2132 to 2319 of mAKAP. Item 11. The method according to Item 10. 13. The vector encodes amino acids 2132 to 2319 of mAKAP. The method described. 14. The method of paragraph 10, wherein the vector is an adeno-associated virus (AAV). 15. A composition encoding a molecule that inhibits anchoring of PP2A to mAKAP. 16. The composition of paragraph 15, wherein the molecule comprises a fragment of mAKAP. 17. Amino acid sequences with at least 90% sequence identity to a fragment of mAKAP Item 16. The composition according to item 15, comprising a sequence. 18. The fragment of amino acids 2132 to 2319 of mAKAP described in item 16. Composition of. 19. The composition according to item 16, comprising amino acids 2132 to 2319 of mAKAPβ. 20. The composition according to item 15, comprising a fragment of PP2A. 21. A vector encoding a molecule that inhibits the anchoring of PP2A to mAKAP is prepared. A composition comprising: 22. The composition of paragraph 21, wherein the vector encodes a fragment of mAKAP. 23. The vector has at least 90% sequence identity with a fragment of mAKAP. 22. The composition of claim 21, wherein the composition encodes an amino acid sequence comprising: 24. The vector encodes a fragment of amino acids 2132 to 2319 of mAKAP. Item 22. The composition according to Item 21. 25. The vector encodes amino acids 2132 to 2319 of mAKAP. The composition described. 26. The composition of paragraph 21, wherein the vector encodes a fragment of PP2A. 27. The composition of paragraph 21, wherein the vector is an adeno-associated virus (AAV).

[0096] The following examples are provided to aid in understanding the present invention and to discern its true scope. The scope of the present invention is set forth in the appended claims. It is understood that variations can be made without [Example]

[0097] The compositions and processes of the present invention are intended to be illustrative only and not to limit the scope of the present invention. This will be better understood with reference to the following examples, which are not intended to be limiting. Various modifications to the disclosed embodiments are possible. Changes and modifications will be apparent to those skilled in the art. However, the present invention is not limited to the process, formulation and / or method of the present invention. This can be done without departing from the scope of the appended claims.

[0098] [Example 1] SRF regulation by the mAKAPβ signalosome Materials and Methods Culture of neonatal rat ventricular myocytes: Sprague-Dawley rats, 1 to 3 days old, were decapitated, and the excised hearts were placed in 1x ADS buffer. The solution was placed in a 16 mM NaCl, 20 mM HEPES, 1 mM NaHPO 4, 5.5 mM glucose, 5.4 mM KCl, 0.8 mM MgSO4, pH 7. 35) The atria were carefully removed and washed to remove blood. The ventricles were minced and gently shaken. While stirring, 3.3 mg of type II collagenase (Worthington, 230 U 15 mL of 1x ADS buffer containing 0.01 mg / mg and 9 mg of pancreatin (Sigma) After 15 minutes, the dissociated cardiomyocytes were stirred at 50 g for 1 minute. The cells were separated by centrifugation for 1 hour, resuspended in 4 mL of horse serum, and incubated at 37°C with occasional stirring. To maximize yield, the enzymatic digestion and isolation steps of myocytes were This was repeated 10–12 times. The myocytes were pooled and centrifuged again at 50 g for 2 minutes, and then resuspended in 10% water. Maintenance medium (DMEM:M199, 4:1) supplemented with 5% WT and 5% fetal bovine serum ) and resuspended in gelatin-coated tissue culture plastic to remove any contaminating fibroblasts. The cells were pre-plated for 1 hour before plating into the container. The following day, the medium was changed to 0.1 mM bromodeoxygenase (BMD) and 0.1 mM bromodeoxygenase (BMD) were added. The maintenance medium was changed to one containing oxyuridine to suppress fibroblast growth.

[0099] Isolation and culture of adult rat ventricular myocytes: Two- to three-month-old rats were treated with 1000 U of heparin for cardiac resection and then administered ketamine (8 Anesthetize using acetaminophen (0-100 mg / kg) and xylazine (5-10 mg / kg) intraperitoneally. The heart was perfused with cold perfusion buffer (NaCl) pre-equilibrated with 95% O and 5% CO. l 120mM, KCl 5.4mM, Na2HPO4 7H2O 1.2mM, NaH CO3 20.0mM, MgCl2.6H2O 1.6mM, taurine 5mM, gluco Immediately transferred into the 2,3-butanedione monoxime (5.6 mM, 10 mM). After removal of extraneous tissue, the heart was cannulated with a Harvard Langendorff apparatus. Attached via the aorta. 2+ Use non-perfused perfusion to remove 8-10 mL of remaining blood. The hearts were then perfused at a constant rate of 1 / min at 37°C. Collagenase I (Worthington, 245 U / mg), 0.1 mg of protease 50 mL perfusion buffer containing Sigma type XIV and 0.1% BSA After perfusion, the atria were removed and the ventricular myocytes were sliced. The cell debris was separated by repeated pipetting. The solution was filtered and the myocytes were harvested by centrifugation at 50 g for 1 min. 2 + The concentration was gradually restored to 1.8 mM, and myocytes were resuspended in ACCT medium (M199 medium (In Vitrogen 11150-059), Creatine 5mM, L-Carnitine 2mM M, taurine 5mM, HEPES 25mM, 2,3-butanedione monoxime 1 0 mM, BSA 0.2% and 1x insulin-transferrin-selenium The cells were resuspended and plated onto dishes pre-coated with 10 μg / ml laminin. The cells were washed with ACCT medium 1.5 hours after plating and then diluted with 100-200 ml of each Adenovirus and Dharmafect1 (Dharmac) at a multiplicity of infection (MOI) of 100 nmol / L of siRNA mixed with 100 nmol / L of siRNA was used for adenovirus infection or The next day, adrenergic agents were added and biochemical assays were performed. and morphological measurements were performed 24 hours after stimulation.

[0100] Other cell cultures: HEK293 and COS-7 cells were cultured in DMEM containing 10% FBS and 1% P / S. These cells were maintained in Lipofect® as recommended by the manufacturer. Transiently transfected with amine2000 (Invitrogen) or adenosine and Adeno-X Tet-Off virus (Clontech). .

[0101] Luciferase assay: 225,000 neonatal rat ventricular myocytes in a 24-well dish were cultured with control RSK3- or RSK3-specific siRNA oligonucleotides (10 nM) and Dharmafe The cells were transfected with ct1 reagent (Thermofisher). The next day, the cells were washed with medium. Afterwards, muscle cells were treated with 100 ng of SRE-luc (firefly luciferase) and 100 ng of -36Prl-rluc (Renilla luciferase) reporter plasmid and T The cells were transfected with transfast reagent for 1 hour, and then incubated in a medium containing 4% horse serum. After overnight incubation, the cells were washed with medium and incubated with or without 10 μM PE for 1 day. The samples were collected in 100 μl of PLB and incubated with the Promega Dual Luciferase Kit. Assays were performed using a Berthold Centro X luminometer. .

[0102] Co-immunoprecipitation: Tissues were homogenized using a Polytron or cells were incubated with an inhibitor cocktail (1 μg / ml leupeptin, 1 μg / ml pepstatin, 1 mM benzamidine, 1 mM IP buffer containing AEBSF, 50 mM NaF, and 1 mM sodium orthovanadate buffer (50mM HEPES pH7.4, 150mM NaCl, 5mM EDT A, 10% glycerol, 1% Triton-X 100, 1mM DTT) The soluble proteins were separated by centrifugation at 3-10,000 g for 10 minutes. Antibodies and protein-G agarose beads (50% slurry, Upstate) were extracted. The beads were added to the IP buffer and incubated overnight at 4°C with rocking. The mixture was washed four times at 4°C. Bound proteins were analyzed by SDS-PAGE gel. The samples were sorted by size and then imaged using a Fujifilm LAS-3000 or GE-AI600 imaging system. The antibodies were developed by immunoblotting as previously described using the ELISA kit (4 6) Protein markers were Precision Plus Protein Stan dards (Bio-Rad, 1610373).

[0103] Immunocytochemistry: Myocytes on coverslips were fixed in 3.7% formaldehyde in PBS for 1 hour. Permeabilized with 0.3% TritonX-100 and containing 0.2% BSA and 1% horse serum The slides were then blocked in PBS. The slides were then stained with primary antibodies diluted in blocking buffer. Antibody and Alexa fluorochrome-conjugated specific secondary antibodies (Invitrogen, 1: The slips were then incubated continuously with 1000 mL of blocking buffer for 1 hour. The cells were washed three times with 1 μg / mL Hoechst 33258 in the final wash to label the nuclei. The cells were then stopped with SlowFade Gold anti-fade buffer ( The slides were sealed in a Leica DM4000 microscope (Invitrogen, S36938). A microscope was used to obtain wide-field images.

[0104] GST-SRF phosphorylation assay: GST-SRF protein was transformed into BL21 E. coli and and purified using glutathione-Sepharose (Vargas et al. 2014). 12) The GST-SRF on the beads was incubated with 0.5 μg of GST-SRF in ATP-containing kinase buffer. Active recombinant full-length His6-tagged human RSK3 (Millipore 14-462)+ The cells were incubated with 50 nM BI-D1870 for 30 min. -SRF beads were eluted with Laemmli buffer or PP2A phosphatase buffer. After washing with HCl, the cells were incubated for another 30 min in the presence of 50 ng of PP2A + / - 10 nM okadaic acid. After incubation for 1 min, the protein was eluted with Laemmli buffer. Protein loading was measured by Ponceau staining, and SRF phosphorylation was measured by phospho-SRF. S 103 Detection was carried out using specific antibodies.

[0105] Plasmid constructs SRE-luciferase reporter - SRE-luc was generated as previously described ( Kapiloff et al. 1991) -36bp rat promoter in firefly luciferase reporter plasmid c-fosSRF response element at the XhoI site upstream of the lactin promoter (TCGAC AGG ATG TCC ATA TTA GGA CAT CTG)( By subcloning two copies of the sequence shown in SEQ ID NO: 1 (Treisman 1985), It was produced by.

[0106] -36Prl-Renilla luciferase - Rat prolactin -36-+36 with BglII and HindIII affinity ends An oligonucleotide containing the gin promoter (GATCT CGA AGG TTT A TA AAG TCA ATG TCT GCA GAT GAG AAA GCA GTG GTT CTC TTA GGA CTT CTT GGG GAA GTG TGG TC) (SEQ ID NO: ) was subcloned into pRL-null (Promega). A control Renilla luciferase vector was obtained by cross-linking.

[0107] mAKAP fragment expression vector: pS-EGFPC1-mAKAP-1694-1833-mh adenovirus shuttle The vector was constructed using the myc, His6, and GF promoter sequences in pEGFPC1 (Clontech). Encoding the P-tagged mAKAP aa 1694-1833 fragment (RBD) cDNA (Li, Kritzer, et al. 2013) was used to Subcloning into pTRE shuttle vector modified to contain an immediate early promoter pS-EGFPC1-mh was prepared by removing the deletion of the mAKAP sequence. Myc-tagged mAKAP aa 2134-2314 (PBD) The pTRE-myc-mAKAP PBD encoding fragment was cloned into ApaI-Sc a pTR containing the full-length, N-terminally myc-tagged mAKAP cDNA in I It was produced by digesting and ligating E-myc-mAKAP. Cloning pTRE-βgal, encoding the lactosidase control protein pAcTnTS-EGFP-m, which was used to generate AAV-RBD, was obtained from The AKAP 1694-1833mh plasmid is pEGFPC1-rmAKAP-16 The NheI- BamHI fragment of 94-1833-mh (Li, Kritzer, et al. 2013) was generously provided by Dr. Brent French of the University of Virginia. Subcloned into pAcTnTs (Prasad et al. 2011), which was also provided by To generate the AAV-GFP control virus, The pAcTnTs-EGFP-mh plasmid was cloned into pA cTnTS-EGFP-mAKAP 1694-1833mh was digested, blunt-ended, and Other mAKAP plasmids were prepared by ligation. The results were similar to those published in the journal (Pare, Bauman, et al. 2005; Kapiloff, Jackson, and Airhart 2001).

[0108] SRF construct - pFlag-SRF expressing Flag-tagged SRF protein was cloned into pCGN-SR Human SRF cDNA from F (Addgene plasmid #11977) was cloned into pSH1 Subcloning into the XbaI / EcoRI site of the 60c NFATc1 expression plasmid pTRE-Flag-hS was constructed by the following procedure (Ho et al. 1995). RF was prepared by transfecting Flag-tagged SRF cDNA into the pTRE shuttle vector (Clontech). pTRE-3xHA-hSRF was generated by subcloning into pTRE-3xHA-hSRF. pTRE-Flag-hSRF, which replaces the Flag tag with three tandem HA tags. It was created by inserting a custom sequence into the fiI and SanDI sites. The A and S103D mutations were introduced into the pTRE plasmid by site-directed mutagenesis. and instead of GAGCCTGAGCGAG (SEQ ID NO: ), the sequence ATCGCTGG CAGAG (SEQ ID NO: ) and GAGCCTGGATGAA (SEQ ID NO: ) were introduced. pGEX-4T1-FLAG-hSRF for GST-SRF expression in bacteria was prepared using p The NcoI (blunt-ended)-EcoRI fragment of TRE-Flag-hSRF was transformed into p Subcloning into the BamHI (blunt-ended)-EcoRI site of GEX-4T1 It was produced by.

[0109] RSK3 expression vector: Protease activity of HA-tagged RSK3 wild-type and S218A mutant and RSK3 fragments Plasmids were as previously described (Li, Kritzer, et al. (2013). pS-HA-hRSK3 1-42 adenovirus shuttle vector The vector inserted the HA-tagged 1-42 cDNA into the BsaBI and pS-EGFPC1-mh fragments. and NheI sites of the tagged GFP cDNA was replaced.

[0110] Adenovirus was generated by PI-SceI and I-CeuI subcloning into pTR E shuttle vector and Adeno-X Tet-off system (Clontech) Prepared using the Vivapure AdenoPACK kit (Sartorius These adenoviruses were amplified and purified using a tetracycline (TetrA). When infected with a virus expressing a cullin transactivator, the recombinant protein expresses proteins (adeno-tTA for "tet-off" or reverse for "tet-on" Some adenoviruses were transfected with a modified pTRE vector containing a constitutive CMV promoter. It was produced using the vector (pS).

[0111] result Considering the role of RSK3 and mAKAPβ in determining concentric muscle cell growth, The focus was on identifying the RSK3 cardiomyocyte substrate. The transcription factor serum response factor (SRF) It regulates both cardiac development and adult function by regulating genes involved in growth and the actin cytoskeleton. SRF plays an important role in the treatment of cancer (Miano 2010). 103 Phosphorus It undergoes multiple post-translational modifications (Figure 1), including oxidation (Mack 2011). The prominent role of SRF in the RSK family and its role in the RSK-dependent phenotypes of other RSK family members have been shown. Due to SRF phosphorylation by ATP (Miano 2010; Rivera et al. 1 993;Janknecht et al. 1992;Hanlon, Sturgi ll, and Sealy 2001), SRF inhibits the expression of RSK3 in cardiomyocytes. It is thought that RSK3 is the vector responsible for the activation of SRF Ser. 103 Phosphorylation of purified Glutathione-S-transferase (GST)-SRF fusion protein was used to SRF is a nucleotide sequence encoding the CArG box [CC(A / T)6G G] DNA binding to serum response elements (SREs) and homo- or heterodimerization with other transcription factors Conserved MADS (MCM1, agamous, deficiens, The SRF domain is expressed in the neonatal mouse embryo by RNA interference (RNAi) (Fig. 19A). RSK3 small interfering nucleic acid for depletion from primary cultures of rat ventricular myocytes (NRVM) Using siRNA (ribonucleotide-linked immunosorbent assay), we demonstrated that deletion of RSK3 inhibits the α-adrenergic agonist phenylalanine. Inhibits SRE-dependent transient reporter activity, including that induced by leuconazole We determined that RSK3 binds the scaffold protein mAKAPβ (PE, Figure 19B). Li, Kritzer, et al. 2013), and SRF also regulates mAKAPβ signaling. We then tested whether endogenous mAKAP associates with the endotheliosome and promotes its phosphorylation. β is stably co-immunoprecipitated with SRF in adult mouse heart extracts using SRF antibodies Furthermore, when expressed in heterologous cells, SRF and RSK3 downregulated mAb. In the presence of KAPβ, they were able to associate to form a ternary complex (Fig. 19D). Therefore, inhibition of RSK3 and mAKAPβ expression in NRVMs suppressed PE-induced SRF Ser 103 inhibited the phosphorylation of RSK3 (Fig. 19E). The domain identifies a distinct "RSK3-binding domain" within mAKAPβ at residues 1694–183 3 (RBD) (Li, Kritzer, et al. 2013) Myc-tagged green fluorescent protein (G) was used to compete for mAKAPβ-RSK3 binding. FP) RBD fusion protein expression (Li, Kritzer, et al. 201 3) in primary cultures of NRVM and adult rat ventricular myocytes (ARVM, Fig. 19 and data PE-induced SRF Ser in both the α- and β-actin-dependent manner (data not shown). 103 Inhibited phosphorylation of N-terminal Similar results were obtained by anchoring inhibition using the RSK3 peptide (data not shown). These results were confirmed in vivo. 103 phosphoric acid The induction was achieved by the previously described RSK3 systemic and mAKAPβ myocyte-specific conditioning agents. in hearts from both IL-1 knockout and IL-1 knockout mice (Kritzer et al. 2 014;Li, Kritzer, et al. 2013) and in vivo In RBD-expressing mice (data not shown), the expression of β-glucan was reduced. SRF mediates its phosphorylation in response to catecholamine stimulation via the mAKAPβ signaling pathway. We found that it is a substrate of RSK3 in muscle cells, determined by its association with the IL-1 receptor.

[0112] mAKAPβ is a Ca 2+ / calmodulin-dependent phosphatase calcineurin ( PP2B, PPP3), and PP2A protein kinases containing the B56δ subunit A (PKA) isoenzyme binds two phosphatases (Dodge- Kafka et al. 2010; Li et al. 2010). Okadaic acid, a PP1 / PP2A inhibitor, is used instead of cyclosporine A (CsA), a phosphorus inhibitor. Treatment of NRVM with (OA) significantly reduced baseline SRF Ser 103 Phosphorylation Thus, purified PP2A stimulated the binding of SRF to Ser. 103 Easily remove SRF and PP2A were phosphorylated only in the presence of mAKAPβ (Fig. 21). Since RSK3 can be co-immunoprecipitated in the presence of SRF, PP2A, and m AKAPβ forms a ternary complex in NRVM (Figure 19H). PP2A binds to mAb. It binds the C-terminal domain of KAPβ (Dodge-Kafka et al. 2010 ), PP2A-binding domain (myc-PBD, Fig. 4), expression of endogenous myocytes. cAMP competed with mAKAPβ-PP2A association (Fig. 19I). Consistent with previously published findings that activate A (Dodge-Kafka et al. 2010), PBD expression was significantly increased by β-adrenergic isoproterenol (Is o, Fig. 19J) in ARVM stimulated with SRF Ser 103 Enhanced induction of phosphorylation Taken together, these results suggest that the mAKAPβ signalosome mediates various upstream SRF Ser in response to stimulation 103 Phosphorylation can be regulated Shows.

[0113] [Example 2] SRF Ser 103 Phosphorylation promotes concentric hypertrophy Both neonatal rat ventricular myocytes (NRVM) and adult rat ventricular myocytes (ARVM) to study molecular signaling pathways involving α-adrenergic and β-adrenergic-induced hypertrophy. While useful, the two cell samples differ in shape, ultrastructure, and in some cases, cell type. The sections differ significantly (Peter, Bjerke, and Leinwand 201 6) ARVMs, due to their roughly cylindrical shape, are subject to cardiac remodeling in vivo. It was developed as an in vitro model for the morphological hypertrophy associated with RSK3. The characteristics of the qout mice suggested that RSK3 is important for concentric hypertrophy (P assariello et al. 2016; Li, Kritzer, et al. (2013). High expression of RSK3 selectively increased the width of cultured ARVMs. The length / width ratio was significantly decreased (Fig. 20A, B). These results were similar to those obtained after 1 day of myocyte culture below (PE, Fig. 20C, D). PE induced an 8-10% increase in width and an 8-14% decrease in length / width ratio over 24 hours. This is approximately 1 / 2 the width of mouse myocytes in vivo 2 weeks after aortic coarctation. This compares favorably with a 7–21% increase and a 14–21% decrease in the length / width ratio (8, 1 6) Notably, expression of the SRF S103D phosphorylation mimetic mutant also inhibited A SRF increased the width of the RVM and induced concentric hypertrophy to the same extent as PE treatment. Expression of the S103A mutant did not affect basal myocyte size but increased PE-induced apoptosis. This result was consistent with the inhibition of SRF Ser. 103 Inhibits phosphorylation (Fig. 19F), and expression of the RBD RSK3 anchoring disruptor peptide In contrast to the high expression of PE and RSK3, the β-adduct Chronic stimulation with the renal agonist Iso increased both the length and width of the ARVM, resulting in a more This resulted in symmetric hypertrophy (Fig. 20I, J), which is consistent with the long-term Iso The effect was similar to that of injection (Li, Kritzer, et al. 201 3) Like RBD and SRF S103A expression, P from the mAKAPβ signalosome Eliminating P2A phosphatase had no effect on underlying ARVM morphology. Moreover, like SRF S103D expression, PBD anchoring disruptor expression In contrast, in the presence of Iso, PDB-derived The present study demonstrated that Isoproliferated concentric hypertrophy of ARVMs, and the Isoproliferative effect of Isoproliferative Modification on the width and length of ARVMs was significantly enhanced by P With the exception of P2A, there were trends toward larger and smaller values, respectively. Inducible SRF Ser 103 This was consistent with the PDB-dependent enhancement of phosphorylation (Figure 19J). These results suggest that mAKAPβ anchoring RSK3 and PP2A mediate the function of afferent myocardium. Ser of SRF promotes cell hypertrophy 103 This supports a model for regulating phosphorylation.

[0114] [Example 3] Regulation of PDE4D3 by mAKAPβ-bound PP2A Antibody - Mouse monoclonal anti-GFP (Santa Cruz; 1:500), mouse monoclonal Clonal anti-VSV tag (Sigma: 1:1000), mouse monoclonal anti- mAKAP (Covance, 1:1000), 9E10 mouse anti-myc (Santa Cruz, Inc., 1:500 dilution), polyclonal anti-PP2A-C (Santa Cruz, 1:500), and polyclonal anti-PP1 catalytic subunit (Sant a Cruz, Inc., 1:500) primary antibody was used for immunoblotting. We generated a phospho-specific antibody against phospho-PDE4D3 Ser-54 and identified a target protein containing residues 70–81. Phosphorylated and non-phosphorylated human PDE4D3 peptides (21st Century Bio The antibody was affinity purified using ochemicals and used at a dilution of 1:500. Both non-phosphospecific and phospho-Ser-566 specific polyclonal B56δ Antibodies were as previously described (Ahn et al. 2007).

[0115] Expression construct - Flag-tagged B56δ, glutathione-S-transferase (GST) PP2 AA fusion proteins, and myc- and green fluorescent protein (GFP)-tagged rat and the expression vector for human mAKAP was as previously described (Ahn et al. 2007;Pare, Bauman, et al. 2005;K apiloff et al. 1999a;Kapiloff, Jackson, and Airhart 2001). Myc-tagged mAb, which is deficient in PP2A binding, was The KAP construct was generated by PCR using rat mAKAP1286-2083. The cDNA fragment encoding the mAKAPα and mAKAPβ were prepared by cloning the AKAPα and AKAPβ proteins, respectively. and two alternatively spliced ​​isoforms of mAKAP expressed in the brain ( Michel et al. 2005b). mAKAPβ binds to mAKAPα residue 245. ~2314 and all recombinant mAKAP proteins shown in this specification The protein is based on mAKAPα. - a cDNA encoding VSV-tagged PDE4D3 (Dodge et al. 2001) was subcloned into a GFP expression vector (Clontech). Thus, a dual-tagged PDE4D3 protein was obtained.

[0116] Immunoprecipitation - HEK293 cells can readily express a variety of wild-type and mutant proteins, The cells cultured in 60 mm plates were used in this study as a heterologous line lacking KAP. Cells were incubated with calcium phosphate buffer using 6 μg of each DNA construct per plate. The cells were transfected at a confluency of 50% to 70% depending on the method. in buffer (HEPES, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100 and protease inhibitors) were used. The supernatant was mixed with 3 μg of antibody and 15 μl of pre-washed protein A or G agarose beads. After overnight incubation at 4°C, the immunoprecipitates were incubated with the same buffer. The plate was washed three times with buffer and the bound proteins were analyzed by immunoblotting.

[0117] For immunoprecipitation of endogenous native mAKAP complexes, adult rat hearts (Pel- The cells (freeze) were homogenized in 10 ml of HSE buffer at 15,000 × g. After centrifugation at RT for 25 min, the clarified extracts were immunoprecipitated as described above.

[0118] PDE assay - PDE activity associated with immunoprecipitated protein complexes was determined according to the method by Beavo et al. Assayed (Beavo, Bechtel, and Krebs 1974). The sample was diluted with 45 μl of PDE buffer A (100 mM MOPS, pH 7.5, 4 mM E GTA, 1.0 mg / ml bovine serum albumin) and 50 μl of PDE buffer B [ 100 mM MOPS, pH 7.5, 75 mM MgAc, 1 μM cAMP, and 10 0,000cpm[ 3 Inhibition was assayed in [H]cAMP (Dupont, NEN). The agents were included as indicated.

[0119] Phosphatase assay - Phosphatase activity as a substrate 32 Using P-labeled histones, the method of Ahn et al. Histones were measured in 250 mM MOP. S, pH 7.4, 2.5 mM MgAc, 100 mM β-mercaptoethanol, purified PKA catalytic subunit, 1 μM ATP, 20 μM histone, and 1 mCi [γ- 32 Radiolabeling was performed in reactions containing [P]ATP (6000 Ci / mmol). The reaction was stopped by adding 50% TCA. 32 [P] Histones released by centrifugation Purified from reactive nucleotides. 32 [P] Histone pellet was collected in 1 ml of ether / once with ethanol / HCl (4:1:0.1) and once with 1 ml of ether / ethanol (4: The plate was washed three times with 1) and the substrate was then diluted in 200 μl of PP2A assay buffer (25 1 mM Tris, pH 7.4, 1 mM DTT, and 10 mM MgCl After that, the mixture was precipitated with 50% TCA. After repeated washing, 32 200 μl of [P] histones The cells were suspended in PP2A buffer.

[0120] To measure phosphatase activity, immunoprecipitated protein complexes were incubated in HSE buffer. The immunoprecipitates were washed twice in PP2A reaction buffer and once in PP2A reaction buffer. Without any problems, 100,000 cpm 32 20 μl PP2A assay containing [P] histones The mixture was incubated in the buffer at 30°C for 30 minutes. Purified I-1 was phosphorylated by PKA. After cleavage, the reaction was incubated with 100 μl of 20% TCA, which was used as a specific PP1 inhibitor. The mixture was stopped by heating and then centrifuged for 10 minutes. 32 T containing PO4 CA supernatants were measured by scintillation counting.

[0121] GST Dropdown - PP2A-A subunit GST fusion protein or GST control protein The adsorbed glutathione resin was incubated with HEK293 cell extracts. After overnight incubation, the beads were washed three times. Bound proteins were analyzed by immunoblotting. The analysis was carried out by

[0122] Statistics - Each "n" refers to a completely independent experiment performed using a separate culture or heart sample. All p values ​​were calculated using Student's t test.

[0123] result Regulation of mAKAP-bound PDE4D3 by okadaic acid-sensitive phosphatase cAMP activation of PKA, PKA phosphorylation and activation of PDE4D3, and PDE4 The negative feedback loop inherent in the mAKAP complex, involving D3-catalyzed cAMP degradation, PDE4D3 phosphate has been previously described (Dodge et al. 2001). The activation of mAKAP was dependent on PKA binding to mAKAP. PP2A and Ca2+ may be responsible for PDE4D3 dephosphorylation. 2+ / Cal Both modulin-dependent protein phosphatase and calcineurin (PP2B) are involved in cardiac Associated with mAKAP scaffolds in muscle cells (Pare, Bauman, et al. 2005; Kapiloff, Jackson, and Airhart 20 01; Li et al. 2009). PP2B binds to Se, a residue in the upstream conserved region of PDE4D3 that is required for PKA activation. To test whether r-54 can dephosphorylate PDE4D3, a heterologous system was used. mAKAP and PDE4D3 were used (Sette and Conti 1996). Highly expressing HEK293 cells were treated with 300 μM okadaic acid (OA) to induce the expression of PP2A (and and protein phosphatase 1 [PP1]) activity, or 500 μM cyclosporin Treatment with porin A (CsA) inhibited PP2B activity (Fig. 8A). After immunoprecipitation of the protein complex using the antibody, phosphorylation of PDE4D3 was measured at the engineered residues. Assayed by immunoblotting with a phosphospecific antibody against Ser-54 OA treatment increased baseline PDE4D3 Ser-54 phosphorylation, whereas Inhibition of PP2B had no effect (Fig. 8A, top panel, second row). When PKA is activated by the addition of the adenylyl cyclase agonist forskolin, , and further increased by 1.8-fold (Fsk, Fig. 8A, top panel, fifth row). Skolin alone, without phosphatase inhibition, had no significant effect (Fig. 8A, column 4). A non-phospho-specific antibody against PDE4D3 and an antibody against mAKAP were used. Immunoblotting showed that the two proteins precipitated similarly under each condition ( Figure 8A, bottom panel).

[0124] Phosphorylation of Ser-54 in PDE4D3 increases phosphodiesterase activity by twofold. In (Sette and Conti 1996), OA treatment also inhibited mAKAP-binding PD We tested whether E4D3 activity could be enhanced by the mAKAP complex. Immunoprecipitated from K293 cells and assayed for associated phosphodiesterase activity. (Fig. 8B). The mAKAP-associated phosphodiesterase activity in untreated cells was significantly increased by mAb. KAP was detected only when coexpressed with PDE4D3 (Fig. 8B, bar 1, and data (not shown), PDE4D3 associates with mAKAP in cardiomyocytes as a phosphodiesterase inhibitor. This was consistent with previous findings that the enzyme activity corresponds to all of the enzyme activity (Dodge et al. 2001). In agreement with the results obtained with the phospho-Ser-54 antibody, Fs Treatment with kappa b alone was not sufficient to stimulate mAKAP-bound PDE4D3 activity in HEK293 cells. While Fsk and OA treatments could not stimulate PDE4D3 activity, both Fsk and OA treatments synergistically enhanced PDE4D3 activity (Fig. 8B, bars 3 and 6). CsA had no effect on either basal or stimulated PDE4D3 activity. There was no effect of PP2B on PDE4D binding to mAKAP in cells under these conditions. These results suggest that OA-sensitive IL-3 is not regulated in this heterologous system. Baseline and Fsk-stimulated PDE4D3 binding to mAKAP by soluble phosphatases Both demonstrate potent inhibition of phosphorylation and activity.

[0125] The enhancement of phosphodiesterase activity by OA was confirmed by the recombinant protein in HEK293 cells. Expression of proteins as well as native mAKAP complexes from adult rat heart extracts Both PDE4D3 and PKA were also observed in the purified mAb (Fig. 8C). It is active in the KAP complex (Dodge et al. 2001). PKA activity present in the AKAP complex was inhibited by the specific PKA inhibitor PKI. A two-fold increase in phosphodiesterase activity was evident in the inhibition of bound PKA. Importantly, OA inhibition inhibits the mAKAP function. 6 when phosphodiesterase activity is also inhibited by 30% (bars 2 and 3) and PKA is also inhibited. 0% (bars 4 and 5). These data together indicate that the AKAP complex is associated with OA-sensitive phosphatases inhibit the dephosphorylation and phosphodiesterase activity of PDE4D3 Indicates that it is a regulatory factor.

[0126] PP2A associates with the mAKAP scaffold in the heart. Since we observed that OA-sensitive phosphatases associate with the mAKAP complex, The mAKAP complex isolated from cardiac cell extracts was identified by co-immunoprecipitation experiments. The phosphatase activity associated with the body is 32[P] histones were measured using There was a three-fold increase in phosphatase activity compared to control IgG immunoprecipitates (Figure 9A). , bars 1 and 2). The mAKAP-associated phosphatase responsible for the immunoprecipitation activity is PP2A. The phosphatase activity was abolished by 30 nM of the PP2A inhibitor I. completely inhibited (Li, Makkinje, and Damuni 1996), This is because the addition of 100 nM of PKA phosphorylation PP1 inhibitor-1 did not inhibit the activity. (Endo et al. 1996). As a positive control, PKA-phosphorylated PP PP1 inhibitor-1 was isolated from HEK293 cell extracts by immunoprecipitation with PP1 antibody. PP1 was indeed inhibited (Fig. 16). This is not due to PP2B binding, but to Ca 2+ / Calmodulin is a phosphatase These results were supported by the fact that mAKAP The immunoprecipitates were analyzed by immunoblotting. Furthermore, the PP2A-C subunit was detected in the mAKAP-specific immunoprecipitates (Fig. 1 8B and C).

[0127] Like PKA, PP2A associates with many cellular substrates and is expressed in diverse intracellular compartments. It is predicted to be present in cultured primary myocardium of neonatal rats (Virshup 2000). Confocal fluorescence microscopy of cells revealed that PP2A-C subunits were expressed in a fine, punctuated pattern. We have revealed that the cytoplasmic distribution of the ATP-dependent ATPases is regulated by the ATP-dependent ATPases (Fig. 17, green). As previously reported, mAKAP is primarily localized to the nuclear membrane (Pare, Easlic k, et al. 2005). mAKAP and PP2 from adult rat heart extracts Consistent with co-immunoprecipitation in A, overlapping PP2A and mAKAP staining was detected at the nuclear membrane. (Figure 17, composite image), PP2A, PKA, and PDE4D3, as well as the scaffold mA A model in which localized signaling complexes consisting of distinct pools of KAP exist in cardiomyocytes This confirmed the theory.

[0128] mAKAP residues 2083–2319 contain the PP2A-binding domain. To map the PP2A binding site in mAKAP, bacterially expressed PP2A mAK expressed in HEK293 cells using a GST-A subunit fusion protein GFP-tagged fragments of AP were detected in the pulldown (Figures 10A and 10B). -PP2A-A is a fragment of mAKAP containing the domain at residue 2085 from the C-terminus. Only rat mAKAP1835-2312 and human 2 were stably detected by pull-down. GFP fusion proteins of human and rat mAKAP, including 085-2319, were identified as GS As a negative control, GFP-mAKAP fusion protein was used. Proteins did not bind PP1 in HEK293 cells and did not bind PP1 and mAb from heart extracts. This was consistent with the lack of co-immunoprecipitation of KAP (Figure 18). To confirm the mapping of the site, myc-tagged mAKA was expressed in HEK293 cells. The P fragment was immunoprecipitated with myc-tagged antibody and assayed for associated PP2A activity. The 1286-2083 region of mAKAP was not detected (Fig. 10C). 86-2312 co-immunoprecipitated with OA-sensitive phosphatase activity. These data Both PP2A and PKA bind to PKA, PDE4D3, and other known mAKAP-binding proteins. This indicates that the AKAP binds to a C-terminal site in mAKAP that is distant from the binding site of AKAP (Fig. 10A ).

[0129] mAKAP-anchoring PP2A in the complex regulates PDE4D3 phosphorylation. Data obtained using mAKAP complexes isolated from rat heart extracts showed that mA This suggests that KAP-bound PP2A regulates PDE4D3 in a complex (Fig. 8C). To test whether PP2A anchoring is required for PDE4D3 dephosphorylation, PDE4D3 was expressed in HEK293 cells, and the binding of PDE4D3, PKA, and PP2A was investigated. mAKAP construct containing the binding site (myc-mAKAP 1286-2312), or a similar mAKAP construct without the PP2A binding site (myc-mAKAP 1 The cells were stimulated with Fsk and OA to express the mAKAP complex. The phosphorylation of mAKAP-bound PDE4D3 was confirmed by immunoprecipitation. The full-length mAKAP was assayed by immunoblotting with a 4-phosphate-specific antibody. As seen in the expression of OA (Fig. 8A), phosphatase activity was suppressed by OA. The phosphorylation of PDE4D3 bound to myc-mAKAP1286-2312 was examined. Notably, myc-mAKAP lacked significant PP2A binding. Upon expression of 1286-2083 (Figure 11A, columns 4-6), baseline mAKAP results We detected an increase in PDE4D3 phosphorylation (0.49 ± 0.1 of the level obtained in OA). 9-fold, Figure 11A, column 4 vs. column 3). Furthermore, upon deletion of the PP2A-binding domain, Fs k alone is comparable to that associated with PP2A-containing complexes treated with both Fsk and OA. phosphodiesterase phosphorylation to levels of 3, 5, and 6 (Fig. 11A). (Column 3) Changes in Ser-54 phosphorylation of PDE4D3 result in changes in phosphodiesterase activity. PDE4D3 activity was reflected in the myc-mAKAP1 PP2A-free ATPase (Fig. 11B). 30% higher in 286-2083 immunoprecipitates than in complexes containing phosphatase (Bars 1 and 4) Importantly, no significant difference in PDE4D3 activity was observed in the absence of PP2A. No significant difference was observed between Fsk stimulation and Fsk stimulation in the presence of OA in the complex ( These data support the role of PP2 in regulating PDE4D3 phosphorylation and activity. Furthermore, these results suggest that PP2A is a PKA-activated phosphodiesterase (PKA)-dependent phosphodiesterase (PP2A)-dependent phosphodiesterase (PKA ... Not only does it suppress diesterase activity, but it also reduces the basal low PDE4D3 levels in unstimulated cells. This indicates that it functions to maintain activity levels.

[0130] The mAKAP-binding PP2A holoenzyme containing the B56δ subunit is regulated by PKA. can be. PP2A holoenzyme is a heterodimer of core A and C subunits, and the holoenzyme is identified. It consists of three subunits, including a B subunit that can target to intracellular organelles. (Virshup 2000). Three closely related B The subunits B56δ, B56γ1, and B56γ3 have been identified (Gigena et al. 2005; McCright et al. 1996). Our study revealed that the B56δ-containing PP2A holoenzyme is regulated by PKA phosphorylation. PP2A associated with the mAKAP complex also interacts with P We tested whether mAKAP activity could be regulated by the native mAKAP complex. Immunoprecipitated from adult rat heart extracts and assayed for associated phosphatase activity. The mAKAP-associated phosphatase activity catalyzed the non-hydrolytic decomposition of bound PKA (Fig. 12A). Stimulation with the cAMP analog CPT-cAMP increased the expression level by 2.5-fold (lines 2 and 3). As a control, all immunoprecipitated phosphatase activities were assayed in 10 mM OA. Therefore, it inhibits (column 4) the CPT-cAMP-stimulated increase in phosphatase activity by PK These data together show that the ATP-dependent ATPase activity in the heart was blocked by the addition of the A inhibitor PKI (column 5). PP2A activity associated with the mAKAP complex in the nucleus is upregulated by PKA-dependent cAMP signaling. This indicates that the effect is enhanced by

[0131] Since mAKAP-bound PP2A is regulated by PKA activity, mAKAP-bound PP2 We tested whether the A holoenzyme contains the B56δ subunit. Immunoprecipitation from adult rat heart extracts using 56δ and control (IgG) antibodies mAKAP was stably immunoprecipitated with B56δ antibody (Fig. 12B). Flag-tagged B56δ was expressed in HEK293 cells, and B56δ was expressed as a GFP-tagged ) showed that it was immunoprecipitated with mAKAP antibody only when coexpressed with mAKAP (Figure 12C). Finally, the binding of B56δ to mAKAP complexes the PP2A-C subunit. This indicates that mAKAP immunoprecipitated from HEK293 cell extracts is recruited to the endothelium. The complex was larger when GFP-mAKAP was coexpressed with Flag-B56δ. This was due to its association with phosphatase activity (Fig. 12D, columns 2 and 3). Based on these results, B56δ binds the PP2A-A / C core heterodimer to mAKA in the heart. It recruits the Fs to the P complex, conferring cAMP-dependent phosphatase activity. The increase in intracellular cAMP by the phosphodiesterase inhibitor IBMX and mAK mAKAP-associated phosphoproteins in HEK293 cells were detected only when AP was coexpressed with B56δ. increased the ATPase activity (Fig. 12E).

[0132] PKA binding is required for cAMP-dependent PP2A activity in the mAKAP complex. Previous studies have shown that PKA binds to four serine residues (53, 68, 81, and 566). It has been found that PP2A phosphorylates B56δ, and Ser-566 is involved in PP2A activity. It has been suggested that this corresponds to induction of mAKAP (Ahn et al. 2007). Since the complex contains both PKA and PP2A, PP2A binding to mAKAP is essential for PDE4 The association of these molecules into a complex mediates PP2A phosphorylation, as required for D3 dephosphorylation. To test this hypothesis, we transformed B56δ into the wild-type full-length A full-length mAb containing an internal deletion of residues 2053 to 2073, which contains the mAKAP or PKA binding site. KAP mutant (ΔPKA, Fig. 13A) was expressed in HEK293 cells (Pa re, Bauman, et al. 2005). After increasing intracellular cAMP, the mAKAP complex was isolated by immunoprecipitation and expressed as B5. The phosphorylation status of B56δ was determined using a phospho-specific antibody against Ser-566 of B56δ. The FSK / I was measured (Fig. 13A, upper panel) (Ahn et al. 2007). Only after BMX treatment and in combination with wild-type mAKAP but not with the ΔPKA mutant, Phosphorylation of B56δ was detected only when the control was expressed (Fig. 13A, lanes 2 and 6). Equivalent expression of mutant and wild-type mAKAP and B56δ proteins as a role was demonstrated by immunoblotting with a non-phospho-specific antibody (Fig. 13A, center and (Bottom panel). Furthermore, wild-type mAKAP was ligated to each of the four PKA substrate sites. This was co-expressed with a mutant form of B56δ containing the nin residue (S4A). As expected, Fs k / IBMX stimulation did not induce phosphorylation of S4A of B56δ (Fig. 13A, column 4). Since phosphorylation of B56δ increases PP2A catalytic activity, mAKAP antibody immunoprecipitates was assayed for phosphatase activity (Fig. 13B). Consistent with the results obtained using ATPase inhibitors, elevated cAMP activates phosphatases in the mAKAP complex. The enzyme activity increased 1.7-fold (Fig. 13B, lanes 2 and 3). Since the body did not show an increase in PP2A activity due to the increase in cAMP (column 5), this increase Similarly, the addition of B56δ to mAKAP required phosphorylation of B56δ. mAKAP, as no increase was obtained when coexpressed with the KA mutant scaffold (column 6). PKA binding to mAK was required to induce PP2A activity. Fsk / IBMX-induced increase in AP-associated PP2A activity is associated with the activation of the mAKAP complex. was not due to increased P2A-C subunit binding (Fig. 13A, lanes 1 and 2). This result suggests that phosphorylation of B56δ induces PP cleavage through a conformational change that does not affect holoenzyme formation. This is consistent with previous suggestions that it increases 2A catalytic activity (Ahn et al. al. 2007).

[0133] PP2A regulates PDE4D3 phosphorylation in a PKA-dependent manner. The above results suggest that PP2A dephosphorylation of PDE4D3 in the B56δ-mAKAP complex This suggests that activation should be enhanced by PKA-catalyzed phosphorylation of the phosphatase. To identify the role of B56δ phosphorylation in the regulation of PDE4D3, we investigated the role of PDE4D3 in the regulation of B56δ phosphorylation. 3 and mAKAP of wild-type B56δ or the B56δ S4A mutant, which does not respond to PKA. The cells were co-expressed with either Fsk or mAKAP. The cells were stimulated with Fsk and then the mAKAP complex was isolated. As detected by immunoblotting with phosphate-specific antibodies and enzymatic assays, P Fsk stimulation of DE4D3 Ser-54 phosphorylation and phosphodiesterase activity was observed in PP When 2A was inhibited by OA, only in the mAKAP complex containing wild-type B56δ This was observed (Fig. 14A and B, 1-3), which was consistent with the data previously described (Fig. 8). Expression of S4A in 6δ results in detectable fsk-stimulated PDE4D3 phosphorylation (0.39 ± 0.15-fold increase in Fsk / OA-stimulated cells, Fig. 4A, column 5), and phosphodiesterase PP2A activity was increased by OA (Fig. 14B, column 5). The inhibition was not as potent as when the control group was directly inhibited (Fig. 14A and B, columns 3 and 6). Together with the results shown in 13, PKA-stimulated PP2A holoenzyme anchoring was also associated with mAKAP. Factors that suppress both basal and PKA-stimulated PDE4D3 activity in the signaling complex This becomes:

[0134] Consider The results described herein demonstrate that the scaffold protein mAKAP binds to PKA phosphorylated P Define the biochemical mechanism of DE4D3 dephosphorylation and inactivation. A, C, and B56 PP2A heterotrimers composed of δ subunits are similar to other known mAKAP partners. It binds to a C-terminal site in mAKAP that is different from the binding site of PP2A (Fig. 10). The association with the mAKAP scaffold has functional significance in two important and novel ways. First, m AKAPs bind both PP2A and PDE4D3, thereby inhibiting phosphodiesterase activity. The enzyme traps the phosphatase in close proximity to the enzyme, allowing for efficient PDE4D3 dephosphorylation and dephosphorylation. Second, mAKAP interacts with PKA and PP2A, allowing for downregulation of ATP (Figure 11). By binding both PP2A and B56δ subunits, the cAMP-dependent phosphorylation of B56δ subunit of PP2A is suppressed. This promotes oxidation and the induction of PP2A activity (Fig. 13). The association was evident upon expression of mAKAP mutants lacking the binding sites for PP2A and PKA. It was.

[0135] The idea of ​​targeting phosphatases to obtain substrate specificity was first proposed by the first PP1 targeting subunit The first was in the mid-1980s with the identification of glycogen particle-associated proteins as units. Since this initial finding, , several other phosphatase target motifs have been identified (Virshup 20 00). AKAPs represent an important mechanism for linking phosphatases to their appropriate substrates. Some AKAPs bind protein phosphatases. Lucineurin) and that this interaction is PP2B-dependent N It has recently been shown to be important for FATc3 activation (Li et al. 2009). However, inhibition of PP2B inhibits Ser-54 phosphorylation or phosphodiesterase activity of PDE4D3. PP2B binding to mAKAP regulates PDE4D3, as it did not affect esterase activity. This data does not suggest that dephosphorylation of phosphodiesterases is involved in the ATP synthesis. P binding to mAKAP in and consequently in the regulation of local cAMP levels This supports the unique role of P2A.

[0136] The role of global phosphatases in regulating cellular cAMP concentrations has yet to be fully explored. In rat adipocytes, PP2A inhibits both PDE3B activity and phosphorylation. PDE4D3 has been shown to regulate S In addition to being phosphorylated by PKA at er-54, it is present in the mAKAP complex. It is phosphorylated at Ser-579 by MAP kinases, including ERK5, which are present in the Hoffmann et al. 1999;Dodge-Kafka et al. 2005). PP1 was not observed to bind mAKAP (Fig. 9 and Fig. 18), but PP 1 can dephosphorylate Ser-579 of PDE4D3 in other cytoplasmic domains, which By adding purified PP1 to isolated PDE4D3, phosphorylation at this site was observed. In addition, phosphatases bind to the phosphodiesterase at Ser-579 and PD mAKAP-binding PDE4D3 at Ser-16 of the second PKA site in E4D3 This is responsible for the dephosphorylation of (Carlisle Michel et al. 2004 ).

[0137] The anchoring hypothesis involves localizing both proteins to the same signaling complex. AKAPs function to target the action of PKA to specific substrates through Herein, we present a novel mechanism for PKA in the mAKAP complex. This suggests that the B56δ subunit of PP2A is a new target. Phosphorylation of B56δ stimulates PP2A activity and enhances dephosphorylation of DARPP-32 Consistent with these results, myocardial Stimulating cells with β-adrenergic receptor agonists increases PP2A activity (De Arcangelis, Soto, and Xiang 2008). mA The KAP scaffold can associate anchoring proteins with both PKA and PP2A. It may be important for the cAMP-induced increase in phosphatase activity and thus facilitate this event. (Figs. 11 and 13). Therefore, mAKAP plays a role in regulating phosphatase activity in the heart. It has a role to play.

[0138] Based on these results, it is possible that PP2A regulates cAMP levels near the mAKAP signaling complex. We present a model for the dual role of mAKAP in regulating signal transduction (Figure 15). First, mAKAP PP2A in the complex is dephosphorylated to a minimally active state without GPCR stimulation. E4D3 should maintain cAMP levels (Fig. 15A), presumably in response to agonists. Second, after GPCR stimulation induces activation of cAMP levels, PKA can phosphorylate both PDE4D3 and PP2A (Figure 15B). In contrast to the negative feedback on cAMP levels mediated by increased phosphorylation of 3 In turn, PKA phosphorylation of PP2A opposes PDE4D3 activation. By inhibiting PP2A, PP2A likely acts as part of a positive feedback loop. Thus, the previously described mAKA In conjunction with the possibility of inhibition of PDE4D3 by P-bound ERK5 (not shown) (Dodge- Kafka et al. 2005), the mAKAP signaling complex is Multiple feedback loops and crosstalk with upstream MAPK signaling pathways PP2A expression and cell proliferation are poised to finely regulate local cAMP levels through both It has been observed that intracellular localization is altered in heart failure (Reiken et al. al. 2001; Ai and Pogwizd 2005). PP2A-mediated positive The positive feedback or PDE4D3-mediated negative feedback is localized to the mAKAP complex. Whether PP2A primarily regulates the cAMP levels present in mAKAP in pathological conditions Stoichiometry of binding and PDE4D3 phosphorylation and dephosphorylation by PKA and PP2A The relative speed of the two forces can ultimately be determined by both the

[0139] This example demonstrates the association of the scaffold protein mAKAP with PDE4D3, PKA, and PP2A. This suggests a novel mechanism for maintaining dynamic regulation of anchored PDE4D3 activity. Each of these three enzymes regulates the temporal concentration of cAMP near the perinuclear mAKAP complex. The mAKAP signalosome plays a key role in regulating local cA This complex regulation of MPs may be due to the involvement of AKAPs and HO in the regulation of cAMP compartmentalization. This indicates the broad role of phospholipases.

[0140] [Example 4] Use of PBD as a treatment for HFrEF Heart failure, the common end stage of heart disease, is a syndrome of great public health importance. It affects 6.5 million Americans, with 960,000 new cases each year (Ben Patients with symptomatic heart failure have a reduced ejection fraction. Heart failure can be divided roughly equally into those with preserved ejection fraction (HFrEF) and those with preserved ejection fraction (HFrEF). First-line therapy has been shown to improve survival and quality of life, at least in HFrEF. angiotensin-converting enzyme (ACE) inhibitors, which can improve blood circulation and reduce mortality These include antihistamines and beta-adrenergic receptor blockers (beta-blockers) (Ponikowski et al. 2016). However, despite these and other adjuvant therapies, The 5-year mortality rate for heart failure is only about 50% (3% in a 2016 study after myocardial infarction). 9%)(Benjamin et al. 2017; Gerber et al. 2016), new therapeutic approaches are needed. Phosphorylation of SRF is essential for H A novel mechanism regulating the transition from compensatory hypertrophy to a dilated failing heart in FrEF Correct.

[0141] As mentioned above, SRF S103 both in vitro and in vivo Expression of PP2A anchoring disruptor D can promote afferent muscle cell hypertrophy. Expression of the -PBD inhibits eccentric hypertrophy induced by Iso treatment in cultured adult muscle cells. These results suggest that SRF S 103 Phosphorylation of promotes widthwise growth These results, along with the long-term results, suggest that it inhibits the elongation of cardiomyocytes. Association of SRF dephosphorylation with pressure overload-induced systolic dysfunction (data not shown) ), restoration of normal or increased SRF phosphorylation may be beneficial in chronic pressure overload and ischemia. This may prevent ventricular dilation that leads to HFrEF in chronic heart disease.

[0142] Mechanisms that induce "compensatory" concentric hypertrophy early in heart disease may predispose the heart to later systolic dysfunction and and eventual failure (Schiattarella and Hill 2018). 15) In this regard, targeting the RSK3-mAKAPβ complex may be effective in preventing cardiac dysfunction caused by pressure overload. may suppress remodeling of the heart and prevent heart failure (Kritzer et al. 2014 ; Li, Kritzer, et al. 2013). Remodeling, including concentric hypertrophy Although inhibition of signaling pathways that induce vasoconstriction may be desirable early in the disease, the heart , a disease process characterized by eccentric growth and ventricular enlargement that leads to HFrEF During this phase, signals that promote concentric muscle cell hypertrophy and suppress eccentric muscle cell hypertrophy are maintained. The question arises as to whether efforts to preserve cardiac volume and evoked contractility can be achieved by Therefore, maintaining SRF phosphorylation may be a key factor in end-stage disease and HFrEF. This provides a method to suppress the eccentric changes in ventricular morphology that characterize SRF phosphorylation. Maintaining oxygenation is a hallmark of end-stage disease and HFrEF, characterized by eccentric ventricular morphology. This means that SRF phosphorylation is suppressed in mice subjected to short-term pressure overload. The inventors have shown that vasoconstriction increases in mice and humans undergoing ventricular dilation and decreases in humans. This is further supported by new findings. 218RSK, as detected by phosphorylation Phosphorylated SRF was activated after pressure overload induction, as was the case with a 1.9-fold increase in SRF activation (Fig. 33C). Within 5 minutes, there was a 28% increase in the total left ventricular extract (containing approximately one-third of the myocytes by cell number) (Figures 33A and B). Notably, 16 weeks after aortic coarctation surgery, the heart was enlarged and When mice developed heart failure (Fig. 33D), phosphorylated SRF was significantly higher than in sham-operated controls. These results suggest that RSK3 catalytic activity is essential for the production of RSK3-dependent ATP. In contrast to phosphorylation, the phosphatase responsible for dephosphorylating SRF during eccentric hypertrophy induction The relevance of this finding to human disease was assessed using patient tissue samples. SRF Ser in left ventricular tissue of patients with abnormal left ventricular internal diameter 103 Compared to phosphorylation Therefore, SRF Ser in patients with dilated hearts 103 Phosphorylation decreased by 53% (p=0.005, Figures 33F-H).

[0143] Improved ventricular geometry, i.e., reduced LV internal diameter due to less elongated myocytes and / or wider myocytes increase LV wall thickness, reducing wall stress. (Laplace's law), may improve systolic function in HFrEF-prone hearts Prevention of systolic dysfunction is due to the expression of a novel AVV gene, mAKAPβ-derived PBD. It has been obtained in therapeutic vectors (Figure 22).

[0144] Treatment of myocardial infarction Coronary heart disease is the primary cause of HFrEF (Writing Group et Eight-week-old C57BL / 6WT mice were treated with either permanent LAD ligation or sham. Two days after surgery, cardiac function was evaluated by echocardiography, and the mice were evaluated for EF and The mice were randomized by age and weight (Figure 23B). Two cohorts of mice were treated with either AAVsc.GFP (n=5), Two days after LAD ligation, the mean ejection fraction was determined to be 34% (Figure 23D). , 5x10 on the mouse 11 vg was injected via the tail vein. In control GFP mice, In PBD mice, systolic function was significantly improved, whereas ejection fraction was progressively reduced (EF was 21%). Long-term recovery was achieved (EF = 43% 8 weeks after surgery, p < 0.0001). Myc-PBD-treated mice had reduced left ventricular volume (systolic contractility), consistent with improved cardiac function. Diastole - 69 μl for PBD vs 156 μl for GFP, p<0.001, Diastole - 11 8 μl vs 192 μl, p<0.001). Ventricular and atrial hypertrophy were reduced (p=0.053 and 0.024, respectively), and tibia length was associated with (Fig. 23C) and pulmonary edema, a sign of heart failure, tended to improve (p=0.078). PP2A can dephosphorylate SRF, excluding PP2A from mAKAPβ. Culling disruptor therapy prevents heart failure with reduced ejection fraction in ischemic heart disease These results indicate that this constitutes a novel therapeutic approach.

[0145] method Common method for left coronary artery ligation 5% isoflurane for induction and 2.5-3% isoflurane for maintenance thereafter The patient was anesthetized with flurane. Oral endotracheal intubation was performed using a 16G catheter, followed by myocardial intubation. Mice were mechanically ventilated using a Nivent ventilator. The skin is prepped and draped for sterility using 10% povidone-iodine solution. A heating pad was used to keep the mouse warm during the procedure to prevent heat loss. To prevent this, a surgically sterile non-medicated eye ointment was applied to the eyes before surgery. Once adequate sedation was achieved, the chest was opened via a left thoracotomy at the fourth intercostal space. If muscle bleeding occurs, use a thermal cautery device (e.g., Finetip, Bovie) Hemostasis was achieved. A 3 mm retractor was used to separate the ribs. After pericardiotomy, the anterior wall M The left coronary artery was ligated with a 7-0 prolene suture to obtain I. A 5-0 absorbable suture (muscle The chest was closed in three layers with 6-0 silk (two ligatures for the ribs and skin). A single dose of 0.5-1 mg / kg subcutaneous sustained-release buprenorphine was administered immediately after surgery to reduce the duration of surgery. (Bup-SR-LAB) was administered. Immediately after surgery, fluid replacement (e.g., sterile saline) was administered. The mice were allowed to recover until they were alert and active. Sham-operated mice performed all other procedures served as controls.

[0146] Echocardiography Minimally anesthetized mice with 1–2% isoflurane were placed on a high-resolution imaging system. The study was carried out using a Vevo2100® (VisualSonics) system. M-mode images were obtained in anesthetized mice at various time points. and systolic thickness and left ventricular cavity end-diastolic diameter (LVEDD) and end-systolic diameter (LV ESD) was measured, and LV volumes, shortening fraction, and ejection fraction were estimated.

[0147] The patent and scientific literature referred to herein represents knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are hereby incorporated by reference. All published foreign patents and patent applications cited herein are incorporated by reference. All other published references, documents cited herein are incorporated by reference. , manuscripts and scientific literature are incorporated herein by reference.

[0148] While the present invention has been particularly shown and described with reference to preferred embodiments thereof, it will be apparent to those skilled in the art that Various modifications may be made in form and detail without departing from the scope of the invention as encompassed by the appended claims. It is understood that variations may be made.

[0149] [References] Using siRNA for gene silencing is a rap idly evolving tools in molecular biology, ThermoFisher Scientific, retrieved June 16, 2017 <https: / / http: / / www.thermofish er.com / us / en / home / references / ambion-tech -support / rnai-sirna / general-articles / -si rna-design-guidelines.html>. Abrenica B, AlShaaban M, Czubryt MP. Th e A-kinase anchor protein AKAP121 is an egative regulator of cardiomyocyte hyperplasia trophy. J Mol Cell Cardiol 46: 674-681, 2009. Ahn JH, McAvoy T, Rakhilin SV, Nishi A, Greengard P, Nairn AC (2007) Protein ki nase A activates protein phosphatase 2A by phosphorylation of the B56delta subun it. Proc Natl Acad Sci USA 104:2979-2984 . Ai X, Pogwizd SM (2005) Connexin 43 dow nregulation and dephosphorylation in non ischemic heart failure is associated wit h enhanced colocalized protein phosphata se type 2A. Circ Res 96:54-63. Andino LM, Conlon TJ, Porvasnik SL, Boy e SL, Hauswirth WW, Lewin AS (2007) Rapi d, widespread transduction of the muri ne myocardium using self-complementary Adeno-associated virus. Genetic vaccine s and therapy 5:13. Anjum R, Blenis J. The RSK family of ki nases: emerging roles in cellular signal ling. Nat Rev Mol Cell Biol. 2008;9(10): 747-758. Appert-Collin A, Cotecchia S, Nenniger- Tosato M , Pedrazzini T , Diviani D. The A -kinase anchoring protein (AKAP)-Lbc-sig naling complex mediates alpha1 adrenergic c receptor-induced cardiomyocyte hypertr ophy. Proc Natl Acad Sci USA 104:10140–101. 10145 , Avkiran M, Cook AR, Neck F. Targeting Na+ / H+ exchanger regula- tion for cardi ac protection: a RSKy approach? Curr Opi n Pharmacol. 2008;8:133–140. Bain J , Plater L , Elliott M , Shpiro N , Hastie CJ, McLauchlan H, Klevernic I, Ar thur JS, Alessi DR, Cohen P. The selecti vity of protein kinase inhibitors: a fur ther update. Biochem J. 2007;408:297–315 . . . . Backs J, Worst BC, Lehmann LH, Patrick DM, Jebessa Z, Kreusser MM, Sun Q, Chen L, Heft C, Katus HA, Olson EN (2011) Sel ective repression of MEF2 activity by PK A-dependent proteolysis of HDAC4. J Cell Biol 195:403-415. Bauman AL, Scott JD (2002) Kinase- and phosphatase-anchoring proteins: harnessi ng the dynamic duo. Nat Cell Biol 4:E203 -206. Bauman AL, Michel JJ, Henson E, Dodge-K afka KL, Kapiloff MS, “The mAKAP signalo some and cardiac myocyte hypertrophy,” I UBMB Life. 2007 Mar;59(3):163-9. Review. Beavo JA, Bechtel PJ, Krebs EG (1974) P reparation of homogeneous cyclic AMP-dep endent protein kinase(s) and its subunit s from rabbit skeletal muscle. Methods E nzymol 38:299- 308. Beene DL, Scott JD. A-kinase anchoring proteins take shape. Curr Opin Cell Biol 19: 192-198, 2007. Benjamin EJ et al. (2017) Heart Disease and Stroke Statistics-2017 Update: A Re port From the American Heart Association . Circulation 135:e146-e603. Benjamin EJ et al. (2019) Heart Disease and Stroke Statistics-2019 Update: A Re port From the American Heart Association . Circulation 139: e56-e528. Bers DM (2006) Cardiac ryanodine· recep tor phosphorylation: target sites and fu nctional consequences. Biochem J 396:el- 3. Bers DM. Calcium cycling and signaling in cardiac myocytes. Annu Rev Physiol 70 : 23-49, 2008. Bione S, Maestrini E, Rivella S, Mancin i M, Regis S, Romeo G, Toniolo D (1994) Identification of a novel X-linked gene responsible for Emery-Dreifuss muscular dystrophy. Nat Genet 8:323-327. Black BL, Olson EN (1998) Transcription al control of muscle development by myoc yte enhancer factor-2 (MEF2) proteins. A Nu Rev Cell Dev Biol 14:167–196. Bonne G, DiBarletta MR, Vamous S, Beca and HM , Hammouda EH , Merlini L , Muntoni F , Greenberg CR , Gary F , Urtizberea JA , D uboc D , Fardeau M , Toniolo D , Schwartz K (1999) Mutations in the gene encoding l all NC cause autosomal dominant Emery- . Dreifuss muscular dystrophy. Nat Genet 2 1:285-2 Bourajjaj M, Armand AS, Martin Coast s PA, Weijts B, van der Nagel R, Heenema n S, Wehrens XH, De Windt LJ (2008) NFAT C2 is a necessary mediator of calcineuria n- dependent cardiac hypertrophy and hea rt failure. J Biol Chem 283:22295–22303. Brown JH, Del Re DP, Sussman MA. The Ra c and Rho Hall of Fame: A Decade of Hype rtrophic signaling hits. Circ Res 98: 73 0-742, 2006. Burns-Hamuro LL, Ma Y, Kammerer S, Rein eke U, Self C, Cook C, Designing isofor m-specific peptide disruptors of protein kinase A localization. Proc Natl Acad S ci U S A. 2003 Apr 1;100(7):4072-7. Brunton LL, Hayes JS, Mayer SE (1979) H ormonally specific phosphorylation of ca rdiac troponin I and activation of glyco gen phosphorylase. Nature 280:78-80. Buck M, Chojkier M. C / EBPbeta-Thr217 ph osphorylation signaling contributes to t he development of lung injury and fibros is in mice. PLoS One. 2011;6(10):e25497. Bueno OF, Wilkins BJ, Tymitz KM, Glasco ck BJ, Kimball TF, Lorenz JN, Molkentin JD (2002) Impaired cardiac hypertrophic response in Calcineurin Abeta -deficient mice. Proc Natl Acad Sci U S A 99:4586- 4591. Bueno OF, Lips DJ, Kaiser RA Wilkins BJ , Dai YS, Glascock BJ, Klevitsky R, Hewe tt TE, Kimball TR, Aronow BJ, Doevendans PA, Molkentin JD (2004) Calcineurin Abe ta gene targeting predisposes the myocar dium to acute ischemia-induced apoptosis and dysfunction. Circ Res 94:91-99. Burchfield JS, Xie M, Hill JA (2013) Pa thological ventricular remodeling: mecha nisms: part 1 of 2. Circulation 128:388- 400. Cappola TP. Molecular remodeling in hum an heart failure. J Am Coll Cardiol 51: 137-138, 2008. Cariolato L, Cavin S, Diviani D. A-kina se anchoring protein (AKAP)- Lbc anchors a PKN-based signaling complex involved in alpha1-adrenergic receptor-induced p3 8 activation. J Biol Chem 286: 7925-7937 , 2011. Carlisle Michel JJ, Dodge KL, Wong W, M ayer NC, Langeberg LK, Scott JD (2004) P KA- phosphorylation of PDE4D3 facilitate s recruitment of the mAKAP signalling co mplex. Biochem J 381:587-592. Carlucci A, Lignitto L, Feliciello A. C ontrol of mitochondria dynamics and oxid ative metabolism by cAMP, AKAPs and the proteasome. Trends Cell Biol 18: 604-613 , 2008. Carnegie GK, Smith FD, McConnachie G, L angeberg LK, Scott JD. AKAP-Lbc nucleate s a protein kinase D activation scaffold . Mol Cell 15: 889 -899, 2004. Carnegie GK, Soughayer J, Smith FD, Ped roja BS, Zhang F, Diviani D, Bristow MR, Kunkel MT, Newton AC, Langeberg LK, Sco tt JD. AKAP-Lbc mobilizes a cardiac hype rtrophy signaling pathway. Mol Cell 32: 169 -179, 2008. Chaturvedi D, Poppleton HM, Stringfield T, Barbier A, Patel TB. Subcellular loc alization and biological actions of acti vated RSK1 are determined by its interac tions with subunits of cyclic AMP-depend ent protein kinase. Mol Cell Biol. 2006; 26:4586-4600. Chen L, Kurokawa J, Kass RS. Phosphoryl ation of the A-kinase anchoring protein Yotiao contributes to protein kinase A r egulation of a heart potassium channel. J Biol Chem 280: 31347-31352, 2005. Chen L, Kurokawa J, Kass RS. Phosphoryl ation of the A-kinase- anchoring protein Yotiao contributes to protein kinase A regulation of a heart potassium channel. J Biol Chem 280: 31347-31352, 2005. Chen L, Marquardt ML, Tester DJ, Sampso n KJ, Ackerman MJ, Kass RS. Mutation of an A-kinase-anchoring protein causes lon g-QT syndrome. Proc Natl Acad Sci USA 10 4: 20990-20995, 2007. Chen PP, Patel JR, Rybakova IN, Walker JW, Moss RL. Protein kinase A-induced my ofilament desensitization to Ca2+ as a r esult of phosphorylation of cardiac myos in-binding protein C. J Gen Physiol 136: 615-627, 2010. Christian F, Szaszak M, Friedl S, Drewi anka S, Lorenz D, Goncalves A, Furkert J , Vargas C, Schmieder P, Gotz F, Zuhlke K, Moutty M, Gottert H, Joshi M, Reif B, Haase H, Morano I, Grossmann S, Klukovi ts A, Verli J, Gaspar R, Noack C, Bergma nn M, Kass R, Hampel K, Kashin D, Genies er HG, Herberg FW, Willoughby D, Cooper DM, Baillie GS, Houslay MD, von Kries JP , Zimmermann B, Rosenthal W, Klussmann E . Small molecule AKAP-protein kinase A ( PKA) interaction disruptors that activat e PKA interfere with compartmentalized c AMP signaling in cardiac myocytes. J Bio l Chem 286: 9079-9096, 2011. Clerk A, Cullingford TE, Fuller SJ, Gir aldo A, Markou T, Pikkarainen S, Sugden PH (2007) Signaling pathways mediating c ardiac myocyte gene expression in physio logical and stress responses. J Cell Phy siol 212:311-322. Consensus (1987). “Effects of enalapril on mortality in severe congestive heart failure. Results of the Cooperative Nor th Scandinavian Enalapril Survival Study (CONSENSUS).” N Engl J Med 316(23): 142 9-1435. Cuello F, Snabaitis AK, Cohen MS, Taunt on J, Avkiran M. Evidence for direct reg ulation of myocardial Na+ / H+ exchanger i soform 1 phosphorylation and activity by 90-kDa ribosomal S6 kinase (RSK): effec ts of the novel and specific RSK inhibit or fmk on responses to alpha1-adrenergic stimulation. Mol Pharmacol. 2007;71:799 -806. De Arcangelis V, Soto D, Xiang Y (2008) Phosphodiesterase 4 and phosphatase 2A differentially regulate cAMP / protein kin ase a signaling for cardiac myocyte cont raction under stimulation ofbetal adren ergicreceptor. Mol Pharmacol 74:1453-146 2. Diviani D, Abuin L, Cotecchia S, Pansie r L. Anchoring of both PKA and 14-3-3 in hibits the Rho-GEF activity of the AKAP- Lbc signaling complex. EMBO J 23: 2811-2 820, 2004. Diviani D, Dodge-Kafka KL, Li J, Kapilo ff MS. A-kinase anchoring proteins: scaf folding proteins in the heart,” Am J Phy siol Heart Circ Physiol. 2011 Nov;301(5) :H1742-53. Dobrev D, Wehrens XH (2014) Role of RyR 2 phosphorylation in heart failure and a rrhythmias: Controversies around ryanodi ne receptor phosphorylation in cardiac d isease. Circ Res 114:1311-1319; discussi on 1319. Dodge-Kafka, K. L., M. Gildart, J. Li, H. Thakur, and M. S. Kapiloff. 2018. Bid irectional regulation of HDAC5 by mAKAPb eta signalosomes in cardiac myocytes’, J ournal of Molecular and Cellular Cardiol ogy, 118: 13-25. Dodge-Kafka, K. L., A. Bauman, N. Mayer , E. Henson, L. Heredia, J. Ahn, T. McAv oy, A. C. Nairn and M. S. Kapiloff (2010 ). “cAMP-stimulated protein phosphatase 2A activity associated with muscle A kin ase-anchoring protein (mAKAP) signaling complexes inhibits the phosphorylation a nd activity of the cAMP-specific phospho diesterase PDE4D3.” J Biol Chem 285(15): 11078-11086. Dodge-Kafka, K. L. and M. S. Kapiloff ( 2006). “The mAKAP signaling complex: int egration of cAMP, calcium, and MAP kinas e signaling pathways.” Eur J Cell Biol 8 5(7): 593-602. Dodge-Kafka, K. L., J. Soughayer, G. C. Pare, J. J. Carlisle Michel, L. K. Lang eberg, M. S. Kapiloff and J. D. Scott (2 005). “The protein kinase A anchoring pr otein mAKAP coordinates two integrated c AMP effector pathways.” Nature 437(7058) : 574-578. Dodge, K. L., S. Khouangsathiene, M. S. Kapiloff, R. Mouton, E. V. Hill, M. D. Houslay, L. K. Langeberg and J. D. Scott (2001). “mAKAP assembles a protein kina se A / PDE4 phosphodiesterase cAMP signali ng module.” EMBO J 20(8): 1921-1930. Diviani D, Soderling J, Scott JD. AKAP- Lbc anchors protein kinase A and nucleat es Galpha 12-selective Rho-mediated stre ss fiber formation. J Biol Chem 276: 442 47-44257, 2001. Dodge KL, Khouangsathiene S, Kapiloff M S, Mouton R, Hill EV, Houslay MD, Langeb erg LK, Scott JD. mAKAP assembles a prot ein kinase A / PDE4 phosphodiesterase cAMP signaling module. EMBO J 20: 1921-1930, 2001. Dodge-Kafka KL, Bauman A, Kapiloff MS, A-kinase anchoring proteins as the basis for cAMP signaling,” Handb Exp Pharmaco l. 2008;(186):3-14. Dodge-Kafka KL, Bauman A, Mayer N, Hens on E, Heredia L, Ahn J, McAvoy T, Nairn AC, Kapiloff MS. cAMP-stimulated protein phosphatase 2A activity associated with muscle A kinase-anchoring protein (mAKA P) signaling complexes inhibits the phos phorylation and activity of the cAMP- sp ecific phosphodiesterase PDE4D3. J Biol Chem. 2010;285:11078-11086. Dodge-Kafka KL, Kapiloff MS, “The mAKAP signaling complex: integration of cAMP, calcium, and MAP kinase signaling pathw ays,” Eur J Cell Biol. 2006 Jul;85(7):59 3-602. Epub 2006 Feb 7. Review. Dodge-Kafka KL, Langeberg L, Scott JD ( 2006) Compartmentation of cyclic nucleot ide signaling in the heart: the role of A-kinase anchoring proteins. Circ Res 98 :993-1001. duBell WH, Lederer WJ, Rogers TB (1996) Dynamic modulation of excitation-contra ction coupling by protein phosphatases i n rat ventricular myocytes. J Physiol 49 3 ( Pt 3):793- 800. duBell WH, Gigena MS, Guatimosim S, Lon g X, Lederer WJ, Rogers TB (2002) Effect s of PP1 / PP2A inhibitor calyculin A on t he E-C coupling cascade in murine ventri cular myocytes. Am J Physiol Heart Circ Physiol 282:H38-48. Dulhunty AF, Beard NA, Pouliquin P, Cas arotto MG (2007) Agonists and antagonist s of the cardiac ryanodine receptor: pot ential therapeutic agents? Pharmacol The r 113:247-263. Dummler BA, Hauge C, Silber J, Yntema H G, Kruse LS, Kofoed B, Hemmings BA, Ales si DR, Frodin M. Functional characteriza tion of human RSK4, a new 90-kDa ribosom al S6 kinase, reveals constitutive activ ation in most cell types. J Biol Chem. 2 005;280:13304-13314 Edgley AJ, Krum H, Kelly DJ. Targeting fibrosis for the treatment of heart fail ure: a role for transforming growth fact or-beta. Cardiovasc Ther. 2012;30(1):e30 -40. Eide T, Coghlan V, Orstavik S, Holsve C , Solberg R, Skalhegg BS, Lamb NJ, Lange berg L, Fernandez A, Scott JD, Jahnsen T , Tasken K. Molecular cloning, chromosom al localization, and cell cycle-dependen t subcellular distribution of the A-kina se anchoring protein, AKAP95. Exp Cell R es 238: 305-316, 1998. Elbashir SM, Martinez J, Patkaniowska A , Lendeckel W, Tuschl T, Functional anat omy of SiRNAs for mediating efficient RN Ai in Drosophila melanogaster embryo lys ate, The EMBO Journal, Vol. 20, No. 23, pp. 6877-6888, 2001. Endo S, Zhou X, Connor J, Wang B, Sheno likar S (1996) Multiple structural eleme nts define the specificity of recombinan t human inhibitor-1 as a protein phospha tase-1 inhibitor. Biochemistry 35:5220- 5228. Escobar M, Cardenas C, Colavita K, Petr enko NB, Franzini- Armstrong C. Structur al evidence for perinuclear calcium micr odo- mains in cardiac myocytes. J Mol Ce ll Cardiol 50: 451-459, 2011. Fabiato A. Calcium-induced release of c alcium from the cardiac sarco- plasmic r eticulum. Am J Physiol Cell Physiol 245: C1-C14, 1983. Farah CS, Reinach FC. The troponin comp lex and regulation of muscle contraction . FASEB J 9: 755-767, 1995. Fatkin D, MacRae C, Sasaki T, Wolff MR, Porcu M, Frenneaux M, Atherton J, Vidai llet HJ, Jr., Spudich S, De Girolami U, Seidman JG, Seidman C, Muntoni F, Muehle G, Johnson W, McDonough B (1999) Missen se mutations in the rod domain of the la min NC gene as causes of dilated car diomyopathy and conduction-system d isease. N Engl J Med 341:1715-1724. Faul C, Dhume A, Schecter AD, Mundel P. Protein kinase A, Ca2+ / calmodulin-depen dent kinase II, and calcineurin regulate the intracellular trafficking of myopod in between the Z-disc and the nucleus of cardiac myocytes. Mol Cell Biol 27: 821 5-8227, 2007. Fink MA, Zakhary DR, Mackey JA, Desnoye r RW, Apperson- Hansen C, Damron DS, Bon d M. AKAP-mediated targeting of protein kinase a regulates contractility in card iac myocytes. Circ Res 88: 291- 297, 200 1. Fischmeister R, Castro LR, Abi-Gerge s A, Rochais F, Jurevicius J, Leroy J, V andecasteele G (2006) Compartmentation o f cyclic nucleotide signaling in the hea rt: the role of cyclic nucleotide phosph odiesterases. Circ Res 99:816-828. Fodstad H, Swan H, Laitinen P, Piippo K , Paavonen K, Viitasalo M, Toivonen L, K ontula K. Four potassium channel mutatio ns account for 73% of the genetic spectr um underlying long-QT syndrome (LQTS) an d provide evidence for a strong founder effect in Finland. Ann Med 36, Suppl 1: 53-63, 2004. Francis SH, Corbin JD. Structure and fu nction of cyclic nuleotide- dependent pr otein kinases. Annu Rev Physiol 56: 237- 272, 1994. Fraser ID, Tavalin SJ, Lester LB, Lange berg LK, Westphal AM, Dean RA, Marrion N V, Scott JD. A novel lipid-anchored A-ki nase anchoring protein facilitates cAMP- responsive membrane events. EMBO J 17: 2 261-2272, 1998. Frey N, Katus HA, Olson EN, Hill JA. Hy pertrophy of the heart: a new therapeutic c target? Circulation 109:1580–1589,2 004. Friday BB, Mitchell PO, Kegley KM, Pavl ath GK (2003) Calcineurin initiates sp lethal muscle differentiation by activation of MEF2 and MyoD. Differentiation 71:217 -227. Fuller MD, Emrick MA, Sadilek M, Scheue r T, Catterall WA. Molecular mechanism f calcium channel regulation in the figh t-or-flight response. Sci Signal 3 : ra70 , 2010. Gaffin RD, Pena JR, Alves MS, Dias FA. Chowdhury SA, Heinrich LS, Goldspink PH, 2005; Cranias EG, Wieczorek DF, Wolska BM. Lo ng-term rescue of a familial hypertrophy c cardiomyopathy caused by a mutation in the thin filament protein, tropomyosin, via modulation of a calcium cycling pro tein. J. Mol. Cell. Cardiol. 2011. Gao T, Yatani A, Dell’Acqua ML, Sako H, Green SA, Dascal N, Scott JD, Hosey MM. cAMP-dependent regulation of cardiac L- type Ca2+ channels requires membrane tar geting of PKA and phosphorylation of cha nnel subunits. Neuron 19: 185-196, 1997. Gao Y, Dickerson JB, Guo F, Zheng J, Zh eng Y. Rational design and characterizat ion of a Rac GTPase-specific small molec ule inhibitor. Proc Natl Acad Sci USA 10 1: 7618-7623, 2004. Gelb BD, Tartaglia M. RAS signaling pat hway mutations and hypertro- phic cardio myopathy: getting into and out of the th ick of it. J Clin Invest. 2011;121:844-8 47. GentilucciL,TolomelliA,SquassabiaF.Pept idesandpeptidomimetics in medicine, surg ery and biotechnology. Curr Med Chem 13: 2449- 2466, 2006. Gerber, Y., S. A. Weston, M. Enriquez-S arano, C. Berardi, A. M. Chamberlain, S. M. Manemann, R. Jiang, S. M. Dunlay and V. L. Roger (2016). “Mortality Associat ed With Heart Failure After Myocardial I nfarction: A Contemporary Community Pers pective.” Circ Heart Fail 9(1): e002460. Gigena MS, Ito A, Nojima H, Rogers TB ( 2005) A B56 regulatory subunit of protei n phosphatase 2A localizes to nuclear s peckles in cardiomyocytes. Am J Physiol Heart Circ Physiol 289:H285-294. Go AS et al. (2014) Heart disease and s troke statistics--2014 update: a report from the American Heart Association. Cir culation 129:e28-e292. Gold MG, Lygren B, Dokurno P, Hoshi N, McConnachie G, Tasken K, Carlson CR, Sco tt JD, Barford D. Molecular basis of AKA P specificity for PKA regulatory subunit s. Mol Cell 24: 383-395, 2006. Goldschmidt-Clermont PJ, Seo DM, Wang L , Beecham GW, Liu ZJ, Vazquez-Padron RI, Dong C, Hare JM, Kapiloff MS, Bishopric NH, Pericak-Vance M, Vance JM, Velazque z OC, “Inflammation, stem cells and athe rosclerosis genetics,” Curr Opin Mol The r. 2010 Dec;12(6):712-23. Review. Good MC, Zalatan JG, Lim WA. Scaffold p roteins: hubs for controlling the flow o f cellular information. Science. 2011;33 2:680-686. Gould KL, Bretscher A, Esch FS, Hunter T. cDNA cloning and sequencing of the pr otein-tyrosine kinase substrate, ezrin, reveals homol- ogy to band 4.1. EMBO J 8 : 4133-4142, 1989. Gray PC, Scott JD, Ca tterall WA. Regulation of ion channels b y cAMP-dependent protein kinase and A-ki nase anchoring proteins. Curr Opin Neuro biol 8: 330-334, 1998. Grossman W, Jones D, McLaurin LP (1975) Wall stress and patterns of hypertrophy in the human left ventricle. J Clin Inv est 56:56-64. Group, Consensus Trial Study. 1987. ‘Ef fects of enalapril on mortality in sever e congestive heart failure. Results of t he Cooperative North Scandinavian Enalap ril Survival Study (CONSENSUS)’, New Eng land Journal of Medicine, 316: 1429-35. Guo T, Cornea RL, Huke S, Camors E, Yan g Y, Picht E, Fruen BR, Bers DM. Kinetic s of FKBP12.6 binding to ryanodine recep tors in permeabilized cardiac myocytes a nd effects on Ca sparks. Circ Res 106: 1 743-1752, 2010. Guo, H., B. Liu, L. Hou, E. The, G. Li, D. Wang, Q. Jie, W. Che and Y. Wei (201 5). “The role of mAKAPbeta in the proces s of cardiomyocyte hypertrophy induced b y angiotensin II.” Int J Mol Med 35(5): 1159-1168. Hagemann D, Xiao RP. Dual site phosphol amban phosphorylation and its physiologi cal relevance in the heart. Trends Cardi ovasc Med 12: 51-56, 2002. Hanks SK, Quinn AM, Hunter T. The prote in kinase family: conserved features and deduced phylogeny of the catalytic doma ins. Science. 1988;241:42-52. Hanlon M, Sturgill TW, Sealy L (2001) E RK2- and p90(Rsk2)-dependent pathways re gulate the CCAAT / enhancer-binding protei n-beta interaction with serum response f actor. J Biol Chem 276:38449-38456. Harada H, Becknell B, Wilm M, Mann M, H uang LJ, Taylor SS, Scott JD, Korsmeyer SJ. Phosphorylation and inactivation of BAD by mitochondria-anchored protein kin ase A. Mol Cell 3: 413-422, 1999. Hayes JS, Brunton LL, Mayer SE (1980) S elective activation of particulate cAMP- dependent protein kinase by isoprotereno l and prostaglandin El. J Biol Chem 255 :5113-5119. Heidenreich, P. A., N. M. Albert, L. A. Allen, D. A. Bluemke, J. Butler, G. C. Fonarow, J. S. Ikonomidis, O. Khavjou, M . A. Konstam, T. M. Maddox, G. Nichol, M . Pham, I. L. Pina, J. G. Trogdon, C. Am erican Heart Association Advocacy Coordi nating, T. Council on Arteriosclerosis, B. Vascular, R. Council on Cardiovascula r, Intervention, C. Council on Clinical, E. Council on, Prevention and C. Stroke (2013). “Forecasting the impact of hear t failure in the United States: a policy statement from the American Heart Assoc iation.” Circ Heart Fail 6(3): 606-619. Heineke J, Molkentin JD (2006) Regulat ion of cardiac hypertrophy by intracel lular signaling pathways. Nat Rev Mol Cell Biol 7:589-600. Hell JW. Beta-adrenergic regulation of the L-type Ca2+ channel CaV1.2 by PKA re kindles excitement. Sci Signal 3: pe33, 2010. Henn V, Edemir B, Stefan E, Wiesner B, Lorenz D, Theilig F, Schmitt R, Vossebei n L, Tamma G, Beyermann M, Krause E, Her berg FW, Valenti G, Bachmann S, Rosentha l W, Klussmann E. Identification of a no vel A-kinase anchoring protein 18 isofor m and evidence for its role in the vasop ressin-induced aquaporin-2 shuttle in re nal principal cells. J Biol Chem 279: 26 654-26665, 2004. Hill JA, Olson EN. Cardiac plasticity. N Engl J Med 358: 1370-1380, 2008. Ho SN, Thomas DJ, Timmerman LA, Li X, F rancke U, Crabtree GR (1995) NFATc3, a l ymphoid-specific NFATc family member tha t is calcium-regulated and exhibits dist inct DNA binding specificity. J Biol Che m 270:19898-19907. Hoffmann R, Baillie GS, MacKenzie SJ, Y arwood SJ, Houslay MD (1999) The MAP kin ase ERK2 inhibits the cyclic AMP-specifi c phosphodiesterase HSPDE4D3 by phosphor ylating it at Ser579. EMBO J 18:893-903. Houser SR (2014) Role ofRyR2 phosphoryl ation in heart failure and arrhythmias: protein kinase A-mediated hyperphosphory lation of the ryanodine receptor at seri ne 2808 does not alter cardiac contracti lity or cause heart failure and arrhythm ias. Circ Res 114:1320-1327; discussion 1327. Huang LJ, Durick K, Weiner JA, Chun J, Taylor SS. D-AKAP2, a novel protein kina se A anchoring protein with a putative R GS domain. Proc Natl Acad Sci USA 94: 11 184-11189, 1997. Huang LJ, Durick K, Weiner JA, Chun J, Taylor SS. Identification of a novel dua l specificity protein kinase A anchoring protein, D-AKAP1. J Biol Chem 272: 8057 -8064, 1997. Huang LJ, Durick K, Weiner JA, Chun J, Taylor SS. Identification of a novel pro tein kinase A anchoring protein that bin ds both type I and type II regulatory su bunits. J Biol Chem. 1997;272:8057-8064. Hulme JT, Ahn M, Hauschka SD, Scheuer T , Catterall WA. A novel leucine zipper t argets AKAP15 and cyclic AMP-dependent p rotein ki- nase to the C terminus of the skeletal muscle Ca2 channel and modu- l ates its function. J Biol Chem 277: 4079 -4087, 2002. Hulme JT, Lin TW, Westenbroek RE, Scheu er T, Catterall WA. Beta-adrenergic regu lation requires direct anchoring of PKA to cardiac CaV1.2 channels via a leucine zipper interaction with A kinase-anchor - ing protein 15. Proc Natl Acad Sci USA 100: 13093-13098, 2003. Hulme JT, Westenbroek RE, Scheuer T, Ca tterall WA. Phosphory- lation of serine 1928 in the distal C-terminal domain of cardiac CaV1.2 channels during beta1-adr energic regulation. Proc Natl Acad Sci U SA 103: 16574 -16579, 2006. Hundsrucker C, Klussmann E. Direct AKAP -mediated protein-protein interactions a s potential drug targets. Hand Exp Pharm acol 186:483–503,2008. Hundsrucker C , Krause G , Beyermann M , P Rinz A, Zimmermann B, Diekmann O, Lorenz D, Stefan E, Nedvetsky P, Dathe M, Chri stian F , McSorley T , Krause E , McConnach ie G , Herberg FW , Scott JD , Rosenthal W , Klussmann E. The high-affinity AKAP7delta rotein kinase A interaction yields novel protein kinase A-anchoring disruptor ptides. Biochem J 396:297–306. [ PMC free article ] [ PubMed ] Jacola P, Mole DR, Tian YM, Wilson MI , Gielbert J , Gaskell SJ , Kriegsheim A , Hebestreit HF, Mukherji M, Schofield CJ, 2005; [ PubMed ] Maxwell PH, Pugh CW, Ratcliffe PJ. Targ eting of HIF-alpha to the von Hippel- Li O2-regulated ndau ubiquitylation complex given prolyl hydroxylation. Science 292 : 4 Rev. 68-472, Janknecht R, Hipskind RA, Houthaeve T, Nordheim A, Stunnenberg HG (1992) Identity fication of multiple SRF N-terminal phos phorylation sites affecting DNA binding properties. EMBO J 11:1045-1054. Jugdutt BI (2003) Remodeling of the myo cardium and potential targets in the col lagen degradation and synthesis pathways . Curr Drug Targets Cardiovasc Haematol Disord 3:1-30. Kamisago M, Sharma SD, DePalma SR, Solo mon S, Sharma P, McDonough B, Smoot L, M ullen MP, Woolf PK, Wigle ED, Seidman JG , Seidman CE. Mutations in sarcomere pro tein genes as a cause of dilated cardiom yopathy. N Engl J Med 343: 1688-1696, 20 00. Kammerer S, Burns-Hamuro LL, Ma Y, Hamo n SC, Canaves JM, Shi MM, Nelson MR, Sin g CF, Cantor CR, Taylor SS, Braun A. Ami no acid variant in the kinase binding do main of dual-specific A kinase-anchoring protein 2: a disease susceptibility pol ymorphism. Proc Natl Acad Sci USA 100: 4 066-4071, 2003. Kapiloff MS, Chandrasekhar KD, “A-kinas e anchoring proteins: temporal and spati al regulation of intracellular signal tr ansduction in the cardiovascular system, ” Journal Cardiovasc Pharmacol. 2011 Oct ;58(4):337-8. Kapiloff MS, Jackson N, Airhart N. mAKA P and the ryanodine receptor are part of a multi-component signaling complex on the cardiomyocyte nuclear envelope. J Ce ll Sci 114: 3167-3176, 2001. Kapiloff MS, Piggott LA, Sadana R, Li J , Heredia LA, Henson E, Efendiev R, Dess auer CW, “An adenylyl cyclase-mAKAPbeta signaling complex regulates cAMP levels in cardiac myocytes,” J Biol Chem. 2009 Aug 28;284(35):23540-6. Kapiloff MS, Mathis JM, Nelson CA, Lin CR, Rosenfeld MG (1991) Calcium / calmodul in- dependent protein kinase mediates a pathway for transcriptional regulati on. Proc Natl Acad Sci US A 88:3710-37 14. Kapiloff MS, Schillace RV, Westphal AM, Scott JD. mAKAP: an A-kinase anchoring protein targeted to the nuclear membrane of differentiated myocytes. J Cell Sci 112: 2725-2736, 1999. Kato Y, Zhao M, Morikawa A, Sugiyama T, Chakravortty D, Koide N, Yoshida T, Tap ping RI, Yang Y, Yokochi T, Lee JD (2000 ) Big mitogen-activated kinase regulates multiple members of the MEF2 protein fa mily. J Biol Chem 275:18534-18540. Keely SL (1977) Activation of cAMP-depe ndent protein kinase without a correspon ding increase in phosphorylase activity. Res Commun Chem Pathol Pharmacol 18:283 -290. Keely SL (1979) Prostaglandin El activ ation of heart cAMP-dependent protein ki nase: apparent dissociation of protein k inase activation from increases in phosp horylase activity and contractile force. Mol Pharmacol 15:235-245. Kehat I, Davis J, Tiburcy M, Accornero F, Saba-El-Leil MK, Maillet M, York AJ, Lorenz JN, Zimmermann WH, Meloche S, Mol kentin JD. Extracellular signal-regulate d kinases 1 and 2 regulate the balance b etween eccentric and concentric cardiac growth. Circ Res. 2011;108:176-183. Kehat I, Molkentin JD. Molecular pathwa ys underlying cardiac re- modeling durin g pathophysiological stimulation. Circul ation. 2010;122:2727-2735. Kentish JC, McCloskey DT, Layland J, Pa lmer S, Leiden JM, Martin AF, Solaro RJ. Phosphorylation of troponin I by proteins n kinase A accelerates relaxation and cr Ossbridge Cycle Kinetics in Mouse Ventri c- ular muscle. Circ Res 88:1059–1065; 2001. Kido M, Du L, Sullivan CC, Li X, Deutsc h R, Jamieson SW, Thistlethwaite PA. Hyp oxya-inducible factor 1-alpha reduces in farction and attenuates progression of c ardiac dysfunction after myocardial in- farction in the mouse. J Am Coll Cardiol 46: 2116–2124, Kim Y , Phan D , van Rooij E , Wang DZ , Mc Anally J , Qi X , Richardson JA , Hill JA . Bassel- Duby R, Olson EN (2008) The MEF2 D transcription factor mediates stre ss-dependent cardiac remodeling in mice. J Clin Invest 118:124–132. Kimura TE , Jin J , Zi M , Prehar S , Liu W , Oceandy D, Abe J, Neyses L, Weston AH, Cartwright EJ, Wang X. Targeted deletio n of the extracel- lular signal-regulate d protein kinase 5 attenuates hypertroph ic response and promotes pressure overlo ad-induced apoptosis in the heart. Circ Res. 2010;106:961-970. Kinderman FS, Kim C, von Daake S, Ma Y, Pham BQ, Spraggon G, Xuong NH, Jennings PA, Taylor SS. A dynamic mechanism for AKAP binding to RII isoforms of cAMP-dep endent protein kinase. Mol Cell 24: 397- 408, 2006. Klussmann E, Edemir B, Pepperle B, Tamm a G, Henn V, Klauschenz E, Hundsrucker C , Maric K, Rosenthal W. Ht31: the first protein kinase A anchoring protein to in tegrate protein kinase A and Rho signali ng. FEBS Lett 507: 264-268, 2001. Kodama H, Fukuda K, Pan J, Sano M, Taka hashi T, Kato T, Makino S, Manabe T, Mur ata M, Ogawa S. Significance of ERK casc ade compared with JAK / STAT and PI3-K pat hway in gp130-mediated cardiac hypertrop hy. Am J Physiol Heart Circ Physiol. 200 0;279(4):H1635-1644. Kontaridis MI, Yang W, Bence KK, Cullen D, Wang B, Bodyak N, Ke Q, Hinek A, Kan g PM, Liao R, Neel BG. Deletion of Ptpn1 1 (Shp2) in cardiomyocytes causes dilate d cardiomyopathy via effects on the extr acellular signal-regulated kinase / mitoge n-activated protein kinase and RhoA sign aling pathways. Circulation. 2008;117:14 23-1435. Kritzer MD, Li J, Dodge-Kafka K, Kapilo ff MS, “AKAPs: the architectural underpi nnings of local cAMP signaling,” J Mol C ell Cardiol. 2012 Feb;52(2):351-8. Kritzer, M. D., J. Li, C. L. Passariell o, M. Gayanilo, H. Thakur, J. Dayan, K. Dodge-Kafka and M. S. Kapiloff (2014). “ The scaffold protein muscle A-kinase anc horing protein beta orchestrates cardiac myocyte hypertrophic signaling required for the development of heart failure.” Circ Heart Fail 7(4): 663-672. Kumar, D., T. A. Hacker, J. Buck, L. F. Whitesell, E. H. Kaji, P. S. Douglas an d T. J. Kamp (2005). “Distinct mouse cor onary anatomy and myocardial infarction consequent to ligation.” Coron Artery Di s 16(1): 41-44. Lacana E, Maceyka M, Milstien S, Spiege l S. Cloning and character- ization of a protein kinase A anchoring protein (AKA P)-related protein that interacts with a nd regulates sphingosine kinase 1 activi ty. J Biol Chem 277: 32947-32953, 2002. Layland J, Solaro RJ, Shah AM. Regulati on of cardiac contractile function by tr oponin I phosphorylation. Cardiovasc Res 66: 12-21, 2005. Lechward K, Awotunde OS, Swiatek W, M uszynska G (2001) Protein phosphatase 2A: variety of forms and diversity of functions. Acta Biochim Pol 48:921-933. Lehnart, S. E., X. H. Wehrens, S. Reike n, S. Warrier, A. E. Belevych, R. D. Har vey, W. Richter, S. L. Jin, M. Conti and A. R. Marks (2005). “Phosphodiesterase 4D deficiency in the ryanodine-receptor complex promotes heart failure and arrhy thmias.” Cell 123(1): 25-35. Lester LB, Langeberg LK, Scott JD. Anch oring of protein kinase A facilitates ho rmone-mediated insulin secretion. Proc N atl Acad Sci USA 94: 14942-14947, 1997. Li CL, Sathyamurthy A, Oldenborg A, Tan k D, Ramanan N (2014) SRF phosphorylatio n by glycogen synthase kinase-3 promotes axon growth in hippocampal neurons. J N eurosci 34:4027-4042. Li H, Adamik R, Pacheco-Rodriguez G, Mo ss J, Vaughan M. Protein kinase A-anchor ing (AKAP) domains in brefeldin A-inhibi ted guanine nucleotide-exchange protein 2 (BIG2). Proc Natl Acad Sci USA 100: 16 27-1632, 2003. Li J, Kritzer MD, Michel JJ, Le A, Thak ur H, Gayanilo M, Passariello CL, Negro A, Danial JB, Oskouei B, Sanders M, Hare JM, Hanauer A, Dodge-Kafka K, Kapiloff MS, “Anchored p90 ribosomal S6 kinase 3 is required for cardiac myocyte hypertro phy,” Circ Res. 2013 Jan 4;112(1):128-39 . Li J, Negro A, Lopez J, Bauman AL, Hens on E, Dodge-Kafka K, Kapiloff MS. The mA KAPbeta scaffold regulates cardiac myocy te hypertrophy via recruitment of activa ted calcineurin. J Mol Cell Cardiol 48: 387-394, 2010. Li J, Negro A, Lopez J, Bauman AL, Hens on E, Dodge-Kafka K, Kapiloff MS, “The m AKAPbeta scaffold regulates cardiac myoc yte hypertrophy via recruitment of activ ated calcineurin,” J Mol Cell Cardiol. 2 010 Feb;48(2):387-94. Li J, Vargas MA, Kapiloff MS, Dodge-Kaf ka KL, Regulation of MEF2 transcriptiona l activity by calcineurin / mAKAP complexe s,” Exp Cell Res. 2013 Feb 15;319(4):447 -54. Li, J., S. Aponte Paris, H. Thakur, M. S. Kapiloff, and K. L. Dodge-Kafka. 2019 . ‘Muscle A-kinase-anchoring protein-bet a-bound calcineurin toggles active and r epressive transcriptional complexes of m yocyte enhancer factor 2D’, Journal of B iological Chemistry, 294: 2543-54. Li M, Makkinje A, Damuni Z (1996) Molec ular identification of I1PP2A, a novel p otent heat- stable inhibitor protein of protein phosphatase 2A. Biochemistry 35: 6998-7002. Liu Q, Hofmann PA (2004) Protein phosph atase 2A-mediated cross-talk between p 38 MAPK and ERK in apopfosis of cardia c myocytes. Am J Physiol Heart Circ Phys iol 286:H2204- 2212. Lohse MJ, Engelhardt S, Eschenhagen T. What is the role of beta- adrenergic sig naling in heart failure? Circ Res 93: 89 6-906, 2003. Lu JT, Kass RS. Recent progress in cong enital long QT syndrome. Curr Opin Cardi ol 25: 216-221, 2010. Lygren B, Carlson CR, Santamaria K, Lis sandron V, McSorley T, Lorenz D, Wiesner B, Rosenthal W, Zaccolo M, Tasken K, Kl ussmann E. AKAP-complex regulates the Ca 2+ reuptake into heart sarcoplasmic reti culum. EMBO Rep 8: 1061-1067, 2007. Lygren B, Tasken K. The potential use o f AKAP18delta as a drug target in heart failure patients. Expert Opin Biol Ther 8: 1099-1108, 2008. Mack CP (2011) Signaling mechanisms that regulate smooth muscle cell differentiation. Arterioscler Thromb Vase Biol 31:1495-1505. Mackenzie KF, Topping EC, Bugaj-Gaweda B, Deng C, Cheung YF, Olsen AE, Stockard CR, High Mitchell L, Baillie GS, Grizzl e WE, De Vivo M, Houslay MD, Wang D, Bol ger GB (2008) Human PDE4A8, a novel brai n-expressed PDE4 cAMP-specific phosphodi esterase that has undergone rapid evolut ionary change. Biochem J 411:361- 369. MacKenzie SJ, Baillie GS, McPhee I, Bol ger GB, Houslay MD (2000) ERK2 mitogen-a ctivated protein kinase binding, phospho rylation, and regulation of the PDE4D cA MP-specific phosphodiesterases. The invo lvement of COOH-terminal docking sites a nd NH2-terminal UCR regions. J Biol Chem 275:16609-16617. Maloney DJ, Hecht SM. Synthesis of a po tent and selective inhibitor of p90 Rsk. Org Lett. 2005;7:1097-1099. Maron BJ, Maron MS. Hypertrophic cardio myopathy. Lancet. 2013;381(9862):242-255 . Maruyama Y, Nishida M, Sugimoto Y, Tana be S, Turner JH, Kozasa T, Wada T, Nagao T, Kurose H. Galpha(12 / 13) mediates alp ha(1)- adrenergic receptor-induced cardi ac hypertrophy. Circ Res 91: 961-969, 20 02. Martinez, E. C., C. L. Passariello, J. Li, C. J. Matheson, K. Dodge-Kafka, P. R eigan and M. S. Kapiloff (2015). “RSK3: A regulator of pathological cardiac remo deling.” IUBMB Life 67(5): 331-337.Marx SO, Kurokawa J, Reiken S, Motoike H, D’A rmiento J, Marks AR, Kass RS. Requiremen t of a macromolecular signaling complex for beta adrenergic receptor modulation of the KCNQ1-KCNE1 potassium channel. Sc ience 295: 496 -499, 2002. Marx SO, Reiken S, Hisamatsu Y, Gaburja kova M, Gaburjakova J, Yang YM, Rosembli t N, Marks AR. Phosphorylation-dependent regulation of ryanodine receptors: a no vel role for leucine / isoleucine zippers. J Cell Biol. 2001;153:699-708. Marx SO, Reiken S, Hisamatsu Y, Jayaram an T, Burkhoff D, Rosemblit N, Marks AR. PKA phosphorylation dissociates FKBP12. 6 from the calcium release channel (ryan odine receptor): defective regula tion i n failing hearts. Cell 101: 365-376, 200 0. Maxwell PH, Wiesener MS, Chang GW, Clif ford SC, Vaux EC, Cockman ME, Wykoff CC, Pugh CW, Maher ER, Ratcliffe PJ. The tu mour suppressor protein VHL targets hypo xia-inducible factors for oxygen-depende nt proteolysis. Nature 399: 271-275, 199 9. Mayers CM, Wadell J, McLean K, Venere M , Malik M, Shibata T, Driggers PH, Kino T, Guo XC, Koide H, Gorivodsky M, Grinbe rg A, Mukhopadhyay M, Abu-Asab M, Westph al H, Segars JH. The Rho guanine nucleot ide exchange factor AKAP13 (BRX) is esse ntial for cardiac development in mice. J Biol Chem 285: 12344-12354, 2010. Mccartney S, Little BM, Langeberg LK, S cott JD (1995) Cloning and Characterizat ion of a- Kinase Anchor Protein-100 (Aka plOO) - a Protein That Targets a-Kinase to the Sarcoplasmic-Reticulum. J Biol Ch em 270:9327-9333. McConnell BK, Popovic Z, Mal N, Lee K, Bautista J, Forudi F, Schwartzman R, Jin JP, Penn M, Bond M. Disruption of prote in kinase A interaction with A-kinase-an choring proteins in the heart in vivo: e ffects on cardiac contractility, protein kinase A phosphorylation, and troponin I proteolysis. J Biol Chem 284: 1583-159 2, 2009. McCright B, Rivers AM, Audlin S, Virshu p DM (1996) The B56 family of protein ph osphatase 2A (PP2A) regulatory subunits encodes differentiation-induced phosphop roteins that target PP2A to both nucleus and cytoplasm. J Biol Chem 271:22081-22 089. McKinsey TA, Kass DA. Small-molecule th erapies for cardiac hypertrophy: moving beneath the cell surface. Nat Rev Drug D iscov. 2007;6:617-635. Miano JM (2010) Role of serum response factor in the pathogenesis of disease. L ab Invest 90:1274-1284. Michel JJ, Townley IK, Dodge-Kafka KL, Zhang F, Kapiloff MS, Scott JD, “Spatial restriction of PDK1 activation cascades by anchoring to mAKAPalpha,” Mol Cell. 2005 Dec 9;20(5):661-72. Michele DE, Gomez CA, Hong KE, Westfall MV, Metzger JM. Cardiac dysfunction in hypertrophic cardiomyopathy mutant tropo myosin mice is transgene-dependent, hype rtrophy-independent, and improved by bet a-blockade. Circ. Res. 2002;91(3):255-26 2. Monovich L, Vega RB, Meredith E, Mirand a K, Rao C, Capparelli M, Lemon DD, Phan D, Koch KA, Chapo JA, Hood DB, McKinsey TA (2010) A novel kinase inhibitor esta blishes a predominant role for protein k inase D as a cardiac class Ila histone d eacetylase kinase. FEBS Lett 584:631-637 . Morissette MR, Sah VP, Glembotski CC, B rown JH. The Rho effector, PKN, regulate s ANF gene transcription in cardiomyocyt es through a serum response element. Am J Physiol Heart Circ Physiol 278: H1769- H1774, 2000. Muchir A, Bonne G, van der Kooi AJ, van Meegen M, Baas F, Bolhuis PA, de Visser M, Schwartz K (2000) Identification of mutations in the gene encoding lamins A / C in autosomal dominant limb girdle musc ular dystrophy with atrioventricular con duction disturbances (LGMDlB). Hum Mol G enet 9:1453-1459. Naga Prasad SV, Barak LS, Rapacciuolo A , Caron MG, Rockman HA. Agonist-dependent t recruitment of phosphoinositide 3-kina is to the membrane by beta-adrenergic re receptor kinase 1. A role in receptor sequ extraction. J Biol Chem 276:18953–18959, 2001. Naga Prasad SV, Laporte SA, Chamberlain D, Caron MG, Barak L, Rockman HA. Phosp hoinositide 3-kinase regulates beta2-adr energetic receptor endocytosis by AP-2 rec ruitment to the receptor / beta-arrestin c complex. J Cell Biol 158:563–575, 2002. Nakagami H, Kikuchi Y, Katsuya T, Maurice we R, Akasaka H, ​​Saitoh S, Rakugi H, K friend Y, Shimamoto K, Ogihara T. Gene po lymor- phism of myospryn (cardiomyopathy -associated 5) is associated with left v enterricular wall thickness in patients wi th hypertension. Hypertens Res 30: 1239- 1246, 2007. Nakamura A, Rokosh DG, Paccanaro M, Yee RR, Simpson PC, Grossman W, Foster E. L V systolic performance improves with dev elopment of hypertrophy after transverse aortic constriction in mice. Am J Physi ol Heart Circ Physiol. 2001;281:H1104-11 12 Nakayama K, Frew IJ, Hagensen M, Skals M, Habelhah H, Bhoumik A, Kadoya T, Erdj ument-Bromage H, Tempst P, Frappell PB, Bowtell DD, Ronai Z. Siah2 regulates sta bility of prolyl-hydroxylases, controls HIF1alpha abundance, and modulates physi ological responses to hypoxia. Cell 117: 941-952, 2004. Nauert JB, Klauck TM, Langeberg LK, Sco tt JD. Gravin, an autoan- tigen recogniz ed by serum from myasthenia gravis patie nts, is a kinase scaffold protein. Curr Biol 7: 52-62., 1997. Naya FJ, Olson E (1999) MEF2: a transcr iptional target for signaling pathways c ontrolling skeletal muscle growth and di fferentiation. Curr Opin Cell Biol 11:68 3-688. Naya FJ, Wu C, Richardson JA, Overbeek P, Olson EN (1999) Transcriptional activ ity of MEF2 during mouse embryogenesis m onitored with a MEF2-dependent transgene . Development 126:2045-2052. Nerbonne JM, Kass RS. Molecular physiol ogy of cardiac repolariza-tion. Physiol Rev 85: 1205-1253, 2005. Negro A, Dodge-Kafka K, Kapiloff MS, “S ignalosomes as Therapeutic Targets,” Pro g Pediatr Cardiol. 2008 Apr; 25(1):51-56 . Nichols CB, Rossow CF, Navedo MF, Weste nbroek RE, Catterall WA, Santana LF, McK night GS. Sympathetic stimulation of adu lt cardiomyocytes requires association o f AKAP5 with a subpopulation of L-type c alcium channels. Circ Res 107: 747-756, 2010. Newlon MG, Roy M, Morikis D, Hausken ZE , Coghlan V, Scott JD, Jennings PA (1999 ) The molecular basis for protein kina se A anchoring revealed by solution N MR. Nat Struct Biol 6:222-227. Nicol RL, Frey N, Pearson G, Cobb M, Ri chardson J, Olson EN. Activated MEK5 ind uces serial assembly of sarcomeres and e ccentric cardiac hypertrophy. EMBO J. 20 01;20:2757-2767. Niggli E, Lederer WJ. Voltage-independe nt calcium release in heart muscle. Scie nce 250: 565-568, 1990. Papa S, Sardanel li AM, Scacco S, Petruzzella V, Techniko va- Dobrova Z, Vergari R, Signorile A. T he NADH: ubiquinone oxidoreductase (comp lex I) of the mammalian respiratory chai n and the cAMP cascade. J Bioenerg Biome mbr 34: 1-10, 2002. Ohh M, Park CW, Ivan M, Hoffman MA, Kim TY, Huang LE, Pavletich N, Chau V, Kael in WG (2000) Ubiquitination of hypoxia-i nducible factor requires direct binding to the beta- domain of the von Hippel-Li ndau protein. Nat Cell Biol 2:423-427. Oka T, Xu J, Kaiser RA, Melendez J, Ham bleton M, Sargent MA, Lorts A, Brunskill EW, Dorn GW, 2nd, Conway SJ, Aronow BJ, Robbins J, Molkentin JD. Genetic manipu lation of periostin expression reveals a role in cardiac hypertrophy and ventric ular remodeling. Circ. Res. 2007;101(3): 313-321. Okumura, S., G. Takagi, J. Kawabe, G. Y ang, M. C. Lee, C. Hong, J. Liu, D. E. V atner, J. Sadoshima, S. F. Vatner and Y. Ishikawa (2003). “Disruption of type 5 adenylyl cyclase gene preserves cardiac function against pressure overload.” Pro c Natl Acad Sci U S A 100(17): 9986-9990 . Olson GL, Cantor CR, Braun A, Taylor SS . Designing isoform-specific peptide dis ruptors of protein kinase A localization . Proc Natl Acad Sci USA 100: 4072-4077, 2003. Pare GC, Bauman AL, McHenry M, Michel J J, Dodge-Kafka KL, Kapiloff MS. The mAKA P complex participates in the induction of cardiac myocyte hypertrophy by adrene rgic receptor signaling. J Cell Sci 118: 5637-5646, 2005. Pare GC, Easlick JL, Mislow JM, McNally EM, Kapiloff MS. Nesprin-1alpha contrib utes to the targeting of mAKAP to the ca rdiac myocyte nuclear envelope. Exp Cell Res 303: 388-399, 2005. Passariello, C. L., J. Li, K. Dodge-Kaf ka and M. S. Kapiloff (2015). “mAKAP-a m aster scaffold for cardiac remodeling.” J Cardiovasc Pharmacol 65(3): 218-225. Passariello CL, Martinez EC, Thakur H, Cesareo M, Li J, Kapiloff MS (2016) RSK3 is required for concentric myocyte hype rtrophy in an activated Rafl model for N oonan syndrome. J Mol Cell Cardiol 93:98 -105. Passariello CL, Gayanilo M, Kritzer MD, Thakur H, Cozacov Z, Rusconi F, Wieczor ek D, Sanders M, Li J, Kapiloff MS (2013 ) p90 ribosomal S6 kinase 3 contributes to cardiac insufficiency in alpha-tropom yosin Glul 80Gly transgenic mice. Am J P hysiol Heart Circ Physiol 305:H1010-1019 . Patel HH, Hamuro LL, Chun BJ, Kawaraguc hi Y, Quick A, Re- bolledo B, Pennypacke r J, Thurston J, Rodriguez-Pinto N, Self C, Olson G, Insel PA, Giles WR, Taylor SS, Roth DM. Disruption of protein kinas e A localization using a trans-activator of transcription (TAT)-conjugated A-kin ase-anchoring peptide reduces cardiac fu nction. J Biol Chem 285: 27632-27640, 20 10. Pawson CT, Scott JD. Signal integration through blending, bolstering and bifurc ating of intracellular information. Nat Struct Mol Biol 17: 653-658, 2010. Pawson T, Nash P (2003) Assembly of cel l regulatory systems through protein int eraction domains. Science 300:445-452. Perino A, Ghigo A, Ferrero E, Morello F , Santulli G, Baillie GS, Damilano F, Du nlop AJ, Pawson C, Walser R, Levi R, Alt ruda F, Silengo L, Langeberg LK, Neubaue r G, SH, Lembo G, Wymann MP, Wetzker R, Houslay MD, Iaccarino G, Scott JD, Hirsc h E. Integrating cardiac PIP3 and cAMP s signaling through a PKA anchoring function n of p110gamma. Mol Cell 42: 84 -95, 201 1. Perrino C, Feliciello A, Schiattarella GG, Esposito G, Guerriero R, Zaccaro L, Del Gatto A, Saviano M, Garbi C, Carangi R, Di Lorenzo E, Donato G, Indolfi C, A VE provision, Chiariello M. AKAP121 dow nregulation impairs protective cAMP sign als, promotes mitochondrial dysfunctio n, and increases oxidative stress. Cardi ovasc Res 88: 101–110, 2010. Perrino C, Naga Prasad SV, Mao L, Noma T, Yan Z, Kim HS, Smithies O, Rockman HA . Intermittent pressure overload trigger s hypertrophy-independent cardiac dysfu nction and vascular rarefaction. J Clin Invest. 2006;116:1547-1560. Peter AK, Bjerke MA, Leinwand LA (2016) Biology of the cardiac myocyte in heart disease. Mol Biol Cell 27:2149-2160. Ponikowski, P., A. A. Voors, S. D. Anke r, H. Bueno, J. G. Cleland, A. J. Coats, V. Falk, J. R. Gonzalez-Juanatey, V. P. Harjola, E. A. Jankowska, M. Jessup, C. Linde, P. Nihoyannopoulos, J. T. Pariss is, B. Pieske, J. P. Riley, G. M. Rosano , L. M. Ruilope, F. Ruschitzka, F. H. Ru tten, P. van der Meer and M. Authors / Tas k Force (2016). “2016 ESC Guidelines for the diagnosis and treatment of acute an d chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the Europe an Society of Cardiology (ESC) Developed with the special contribution of the He art Failure Association (HFA) of the ESC .” Eur Heart J 37(27): 2129-2200. Potthoff MJ, Olson EN (2007) MEF2: a ce ntral regulator of diverse developmental programs. Development 134:4131-4140. Prabhakar R, Boivin GP, Grupp IL, Hoit B, Arteaga G, Solaro JR, Wieczorek DF. A familial hypertrophic cardiomyopathy al pha-tropomyosin mutation causes severe c ardiac hypertrophy and death in mice. J. Mol. Cell. Cardiol. 2001;33(10):1815-18 28. Prasad, K. M., Y. Xu, Z. Yang, S. T. Ac ton and B. A. French (2011). “Robust car diomyocyte-specific gene expression foll owing systemic injection of AAV: in vivo gene delivery follows a Poisson distrib ution.” Gene Ther 18(1): 43-52. Rababah A, Craft JW, Jr., Wijaya CS, At rooz F, Fan Q, Singh S, Guillory AN, Kat sonis P, Lichtarge 0, McConnell BK (2013 ) Protein kinase A and phosphodiesterase -4D3 binding to coding polymorphisms of cardiac muscle anchoring protein (mAKAP) . J Mol Biol 425:3277-3288, Ranganathan A, Pearson GW, Chrestensen CA, Sturgill TW, Cobb MH (2006) The MAP kinase ERK5 binds to and phosphorylates p90 RSK. Arch Biochem Biophys 449:8-16. Reiken S, Gaburjakova M, Gaburjakova J, He Kl KL, Prieto A, Becker E, Yi Gh GH, Wang J, Burkhoff D, Marks AR (2001) bet a-adrenergic receptor blockers restore c ardiac calcium release channel (ryanodin e receptor) structure and function in he art failure. Circulation 104:2843-2848. Resjo S, Oknianska A, Zolnierowicz S, M anganiello V, Degerman E (1999) Phosphor ylation and activation of phosphodiester ase type 38 (PDE3B) in adipocytes in res ponse to serine / threonine phosphatase in hibitors: deactivation of PDE3B in vitro by protein phosphatase type 2A. Biochem J 341 (Pt 3):839-845. Reynolds JG, McCalmon SA, Tomczyk T, Na ya FJ. Identification and mapping of pro tein kinase A binding sites in the costa meric protein myospryn. Biochim Biophys Acta 1773: 891-902, 2007. Richards SA, Dreisbach VC, Murphy LO, B lenis J. Characterization of regulatory events associated with membrane targetin g of p90 ribosomal S6 kinase 1. Mol Cell Biol. 2001;21:7470-7480. Rivera VM, Miranti CK, Misr;i RP, Ginty DD, Chen RH, Blenis J, Greenberg ME (19 93) A growth factor-induced kinase phosp horylates the serum response factor at a site that regulates its DNA-binding act ivity. Mol Cell Biol 13:6260-6273. Rockman HA, Koch WJ, Lefkowitz RJ. Seve n-transmembrane-span- ning receptors and heart function. Nature 415: 206-212, 20 02. Rockman HA, Ross RS, Harris AN, Knowlto n KU, Steinhelper ME, Field LJ, Ross J J r, Chien KR. Segregation of atrial-speci fic and in- ducible expression of an atr ial natriuretic factor transgene in an i n vivo murine model of cardiac hypertrop hy. Proc Natl Acad Sci USA. 1991;88:8277 -8281. Roger VL, Go AS, Lloyd-Jones DM, Adams RJ, Berry JD, Brown TM, Carnethon MR, Da i S, de Simone G, Ford ES, Fox CS, Fulle rton HJ, Gillespie C, Greenlund KJ, Hail pern SM, Heit JA, Ho PM, Howard VJ, Kiss ela BM, Kittner SJ, Lackland DT, Lichtma n JH, Lisabeth LD, Makuc DM, Marcus GM, Marelli A, Matchar DB, McDermott MM, Mei gs JB, Moy CS, Mozaffarian D, Mussolino ME, Nichol G, Paynter NP, Rosamond WD, S orlie PD, Stafford RS, Turan TN, Turner MB, Wong ND, Wylie-Rosett J; American He art Association Statistics Committee and Stroke Statistics Subcommittee. Heart d isease and stroke statistics-2011 update : a report from the American Heart Assoc iation. Circulation 123: e18-e209, 2011. Rose BA, Force T, Wang Y. Mitogen-activ ated protein kinase signaling in the hea rt: angels versus demons in a heart-brea king tale. Physiol Rev. 2010;90:1507-154 6. Russell MA, Lund LM, Haber R, McKeegan K, Cianciola N, Bond M. The intermediate filament protein, synemin, is an AKAP i n the heart. Arch Biochem Biophys 456: 2 04 -215, 2006. Sadoshima J, Qiu Z, Morgan JP, Izumo S. Angiotensin II and other hypertrophic s timuli mediated by G protein-coupled rec eptors activate tyrosine kinase, mitogen -activated protein kinase, and 90-kD S6 kinase in cardiac myocytes. The critical role of Ca(2+)-dependent signaling. Cir c. Res. 1995;76(1):1-15. Sapkota GP, Cummings L, Newell FS, Arms trong C, Bain J, Frodin M, Grauert M, Ho ffmann M, Schnapp G, Steegmaier M, Cohen P, Alessi DR. BI-D1870 is a specific in hibitor of the p90 RSK (ribosomal S6 kin ase) isoforms in vitro and in vivo. Bioc hem J. 2007;401:29-38. Schiattarella GG, Hill JA (2015) I nhibition of hypertrophy is a good th erapeutic strategy in ventricular pres sure overload. Circulation 131:1435-144 7. Scholten A, Poh MK, van Veen TA, van Br eukelen B, Vos MA, Heck AJ. Analysis of the cGMP / cAMP interactome using a chemic al proteom- ics approach in mammalian he art tissue validates sphingosine kinase type 1-interacting protein as a genuine and highly abundant AKAP. J Proteome Res 5: 1435-1447, 2006. Scholten A, van Veen TA, Vos MA, Heck A J. Diversity of cAMP- dependent protein kinase isoforms and their anchoring prot eins in mouse ventricular tissue. J Prot eome Res 6: 1705-1717, 2007. Schulze DH, Muqhal M, Lederer WJ, Ruknu din AM. Sodium / calcium exchanger (NCX1) macromolecular complex. J Biol Chem 278: 28849 -28855, 2003. Scott JD, Dessauer CW, Tasken K (2013) Creating order from chaos: cellular regu lation by kinase anchoring. Annu Rev Pha rmacol Toxicol 53:187-210. Scott, J. D. and T. Pawson (2009). “Cel l signaling in space and time: where pro teins come together and when they’re apa rt.” Science 326(5957): 1220-1224. Semenza GL. Hypoxia-inducible factor 1 (HIF-1) pathway. Sci STKE 2007: cm8, 200 7. Semenza GL. Regulation of oxygen homeos tasis by hypoxia-inducible factor 1. Phy siology 24: 97-106, 2009. Sette C, Conti M (1996) Phosphorylation and activation of a cAMP-specific phos phodiesterase by the cAMP-dependent prot ein kinase. Involvement of serine 54 in the enzyme activation. J Biol Chern 271: 16526-16534. Sfichi-Duke L, Garcia-Cazarin ML, Suman dea CA, Sievert GA, Balke CW, Zhan DY, M orimoto S, Sumandea MP. Cardiomyopathy- causing deletion K210 in cardiac troponi n T alters phosphorylation propensity of sarcomeric proteins. J Mol Cell Cardiol 48: 934-942, 2010. Shan J, Betzenhauser MJ, Kushnir A, Rei ken S, Meli AC, Wronska A, Dura M, Chen BX, Marks AR. Role of chronic ryanodine receptor phosphorylation in heart failur e and beta-adrenergic receptor blockade in mice. J Clin Invest 120: 4375-4387, 2 010. Shan J, Kushnir A, Betzenhauser MJ, Rei ken S, Li J, Lehnart SE, Lindegger N, Mo ngillo M, Mohler PJ, Marks AR. Phosphory lation of the ryanodine receptor mediate s the cardiac fight or flight response i n mice. J Clin Invest 120: 4388-4398, 20 10. Sharma K, Kass DA (2014) Heart failure with preserved ejection fraction: mechan isms, clinical features, and therapies. Circ Res 115:79-96. Shyu KG, Wang MT, Wang BW, Chang CC, Le u JG, Kuan P, Chang H. Intramyocardial i njection of naked DNA encoding HIF- 1alp ha / VP16 hybrid to enhance angiogenesis i n an acute myocardial infarction model i n the rat. Cardiovasc Res 54: 576-583, 2 002. Silva, J. M., M. Z. Li, K. Chang, W. Ge , M. C. Golding, R. J. Rickles, D. Siola s, G. Hu, P. J. Paddison, M. R. Schlabac h, N. Sheth, J. Bradshaw, J. Burchard, A . Kulkarni, G. Cavet, R. Sachidanandam, W. R. McCombie, M. A. Cleary, S. J. Elle dge and G. J. Hannon (2005). “Second-gen eration shRNA libraries covering the mou se and human genomes.” Nat Genet 37(11): 1281-1288. Singh A, Redden JM, Kapiloff MS, Dodge- Kafka KL, “The large isoforms of A-kinas e anchoring protein 18 mediate the phosp horylation of inhibitor-1 by protein kin ase A and the inhibition of protein phos phatase 1 activity,” Mol Pharmacol. 2011 Mar;79(3):533-40. Skroblin P, Grossmann S, Schafer G, Ros enthal W, Klussmann E. Mechanisms of pro tein kinase A anchoring. Int Rev Cell Mo l Biol 283: 235-330, 2010. Smith FD, Langeberg LK, Cellurale C, Pa wson T, Morrison DK, Davis RJ, Scott JD. AKAP-Lbc enhances cyclic AMP control of the ERK1 / 2 cascade. Nat Cell Biol 12: 1 242-1249, 2010. Smith JA, Poteet-Smith CE, Xu Y, Erring ton TM, Hecht SM, Lannigan DA. Identific ation of the first specific inhibitor of p90 ribosomal S6 ki- nase (RSK) reveals an unexpected role for RSK in cancer ce ll prolifera- tion. Cancer Res. 2005;65: 1027-1034. Spinale FG, Janicki JS, Zile MR. Membra ne-associated matrix proteolysis and hea rt failure. Circ. Res. 2013;112(1):195-2 08. Steinberg SF, Brunton LL (2001) Cornpar trnentation of G protein-coupled signali ng pathways in cardiac rnyocytes. Annu R ev Pharmacol Toxicol 41:751-773. Stelzer JE, Patel JR, Walker JW, Moss R L. Differential roles of cardiac myosin- binding protein C and cardiac troponin I in the myofi- brillar force responses t o protein kinase A phosphorylation. Circ Res 101: 503-511, 2007. Sumandea CA, Garcia-Cazarin ML, Bozio C H, Sievert GA, Balke CW, Sumandea MP. Ca rdiac troponin T, a sarcomeric AKAP, tet hers protein kinase A at the myofilament s. J Biol Chem 286: 530-541, 2011 Takeishi Y, Huang Q, Abe J, Che W, Lee JD, Kawakatsu H, Hoit BD, Berk BC, Walsh RA. Activation of mitogen-activated pro tein kinases and p90 ribosomal S6 kinase in failing human hearts with dilated ca rdiomy- opathy. Cardiovasc Res. 2002;53: 131-137. Terrenoire C, Houslay MD, Baillie GS, K ass RS. The cardiac IKs potassium channe l macromolecular complex includes the ph osphodies terase PDE4D3. J Biol Chem 284 : 9140-9146, 2009. Thomas GM, Rumbaugh GR, Harrar DB, Huga nir RL. Ribosomal S6 kinase 2 interacts with and phosphorylates PDZ domain-conta ining proteins and regulates AMPA recept or transmission. Proc Natl Acad Sci USA. 2005;102:15006-15011. Tingley WG, Pawlikowska L, Zaroff JG, K im T, Nguyen T, Young SG, Vranizan K, Kw ok PY, Whooley MA, Conklin BR. Gene-trap ped mouse embryonic stem cell-derived ca rdiac myocytes and human genet- ics impl icate AKAP10 in heart rhythm regulation. Proc Natl Acad Sci USA 104: 8461-8466, 2007. Treisrnan R (1985) Transient accumulati on of c-fos RNA following serum stimulat ion requires a conserved 5’ element and c-fos 3’ sequences. Cell 42:889-902. Uys GM, Ramburan A, Loos B, Kinnear CJ, Korkie LJ, Mouton J, Riedemann J, Moolm an-Smook J. Myomegalin is a novel A-kina se anchoring protein involved in the pho sphorylation of cardiac myosin binding p rotein C. BMC Cell Biol 12: 18, 2011. Valdivia HH, Kaplan JH, Ellis-Davies GC , Lederer WJ (1995) Rapid adaptation of cardiac ryanodine receptors: modulation by Mg2+ and phosphorylation. Science 267 :1997-2000. Vargas MA, Tirnauer JS, Glidden N, Kapi loff MS, Dodge-Kafka KL, “Myocyte enhanc er factor 2 (MEF2) tethering to muscle s elective A-kinase anchoring protein (mAK AP) is necessary for myogenic differenti ation,” Cell Signal. 2012 Aug;24(8):1496 -503. Virshup DM (2000) Protein phosphatase 2 A: a panoply of enzymes. Curr Opin Cell Biol 12:180- 185. Wang X, Tang X, Li M, Marshall J, Mao Z (2005) Regulation of neuroprotective ac tivity of myocyte-enhancer factor 2 by c AMP-protein kinase A signaling pathway i n neuronal survival. J Biol Chem 280:167 05-16713. Wang, Y., E. G. Cameron, J. Li, T. L. S tiles, M. D. Kritzer, R. Lodhavia, J. He rtz, T. Nguyen, M. S. Kapiloff and J. L. Goldberg (2015). “Muscle A-Kinase Ancho ring Protein-alpha is an Injury-Specific Signaling Scaffold Required for Neurotr ophic- and Cyclic Adenosine Monophosphat e-Mediated Survival.” EBioMedicine 2(12) : 1880-1887. Wang, Z., H. I. Ma, J. Li, L. Sun, J. Z hang and X. Xiao (2003). “Rapid and high ly efficient transduction by double-stra nded adeno-associated virus vectors in v itro and in vivo.” Gene Ther 10(26): 210 5-2111. Wera S, Hemmings BA (1995) Serine / threo nine protein phosphatases. Biochem J 311 ( Pt 1):17- 29. Wilkins BJ, De Windt LJ, Bueno OF, Braz JC, Glascock BJ, Kimball TF, Molkentin JD (2002) Targeted disruption of NFATc3, but not NFATc4, reveals an intrinsic de fect m calcineurin-mediated cardiac hype rtrophic growth. Mol Cell Biol 22:7603-7 613. Wilkins BJ, Dai YS, Bueno OF, Parsons S A, Xu J, Plank DM, Jones F, Kimball TR, Molkentin JD (2004) Calcineurin / NFAT cou pling participates in pathological, but not physiological, cardiac hypertrophy. Circ Res 94:110-118. Wong W, Goehring AS, Kapiloff MS, Lange berg LK, Scott JD, “mAKAP compartmentali zes oxygen-dependent control of HIF-1alp ha,” Sci Signal. 2008 Dec 23;1(51). Welch EJ, Jones BW, Scott JD. Networkin g with AKAPs: context- dependent regulat ion of anchored enzymes. Mol Interv 10: 86 -97, 2010. 114. Wu X, Simpson J, Hong JH, Kim KH, Thavarajah NK, Backx PH, Ne el BG, Araki T. MEK-ERK pathway modulati on ameliorates disease phe- notypes in a mouse model of Noonan syndrome associat ed with the Raf1(L613V) mutation. J Clin Invest. 2011;121:1009-1025. Wollert KC, Taga T, Saito M, Narazaki M , Kishimoto T, Glembotski CC, Vernallis AB, Heath JK, Pennica D, Wood WI, Chien KR. Cardiotrophin-1 activates a distinct form of cardiac muscle cell hypertrophy . Assembly of sarcomeric units in series VIA gp130 / leukemia inhibitory factor re receptor-dependent pathways. J Biol Chem. 1996;271:9535-9545. Writing Group, M., D. Mozaffarian, E.J . Benjamin, A. S. Go, D. K. Arnett, M. J . Blaha, M. Cushman, S. R. Das, S. de Fe rranti, JP Despres, HJ Fullerton, VJ Howard, MD Huffman, CR Isasi , MC Jimenez, SE Judd, BM Kisse la, JH Lichtman, LD Lisabeth, S. L iu, RH Mackey, DJ Magid, DK McG uire, E. R. Mohler, 3rd, C. S. Moy, P. M untner, M.E. Mussolino, K. Nasir, RW Neumar, G. Nichol, L. Palaniappan, D.K . Pandey, M. J. Reeves, C. J. Rodriguez, W. Rosamond, P. D. Sorlie, J. Stein, A. Towfighi, T. N. Turan, S. S. Virani, D. Woo, R. W. Yeh, M. B. Turner, C. Americ an Heart Association Statistics and S. S troke Statistics (2016). “Heart Disease and Stroke Statistics-2016 Update: A Rep ort From the American Heart Association. ” Circulation 133(4): e38-360. Wu H, Rothermel B, Kanatous S, Rosenber g P, Naya FJ, Shelton JM, Hutcheson KA, DiMaio JM, Olson EN, Bassel-Duby R, Will iams RS (2001) Activation of MEF2 by mus cle activity is mediated through a calci neurin-dependent pathway. EMBO J 20:6414 -6423. Xie M, Hill JA (2013) HDAC-dependent ve ntricular remodeling. Trends Cardiovasc Med 23:229-235. Xu J, Ismat FA, Wang T, Lu MM, Antonucc i N, Epstein JA. Cardiomyocyte-specific loss of neurofibromin promotes cardiac h ypertrophy and dysfunction. Circ Res. 20 09;105:304-311. Yang J, Drazba JA, Ferguson DG, Bond M (1998) A-kinase anchoring protein 100 (A KAPlOO) is localized in multiple subcell ular compartments in the adult rat heart . J Cell Biol 142:511-522. Yang KC, Jay PY, McMullen JR, Nerbonne JM. Enhanced car- diac PI3Ka signalling mitigates arrhythmogenic electrical remo del- ling in pathological hypertrophy an d heart failure. Cardiovasc Res. 2012;93 :252-262. Zakhary DR, Fink MA, Ruehr ML, Bond M ( 2000) Selectivity and regulation of A-ki nase anchoring proteins in the heart. Th e role of autophosphorylation of the typ e II regulatory subunit of cAMP-dependen t protein kinase. J Biol Chem 275:41389- 41395. Zhang L, Malik S, Kelley GG, Kapiloff M S, Smrcka AV, “Phospholipase Cepsilon sc affolds to muscle-specific A kinase anch oring protein (mAKAPbeta) and integrates multiple hypertrophic stimuli in cardia c myocytes,” J Biol Chem. 2011 Jul 1;286 (26):23012-21. Zhang, L., S. Malik, J. Pang, H. Wang, K. M. Park, D. I. Yule, B. C. Blaxall an d A. V. Smrcka (2013). “Phospholipase Ce psilon hydrolyzes perinuclear phosphatid ylinositol 4-phosphate to regulate cardi ac hypertrophy.” Cell 153(1): 216-227. Zhang Q, Bethmann C, Worth NF, Davies J D, Wasner C, Feuer A, Ragnauth CD, Yi Q, Mellad JA, Warren DT, Wheeler MA, Ellis JA, Skepper JN, Vorgerd M, Schlotter-We igel B, Weissberg PL, Roberts RG, Wehner t M, Shanahan CM (2007) Nesprin-1 and -2 are involved in the pathogenesis of Eme ry Dreifuss muscular dystrophy and are c ritical for nuclear envelope integrity. Hum Mol Genet 16:2816-2833. Zhao Y, Bjorbaek C, Moller DE. Regulati on and interaction of pp90(rsk) isoforms with mitogen-activated protein kinases. J Biol Chem. 1996;271:29773-29779. Zhao Y, Bjorbaek C, Weremowicz S, Morto n CC, Moller DE. RSK3 encodes a novel pp 90rsk isoform with a unique N-terminal s equence: growth factor-stimulated kinase function and nuclear translocation. Mol Cell Biol. 1995 Aug; 15(8): 4353-436.

Claims

1. comprising a nucleic acid encoding a molecule having at least 80% sequence identity to the sequence of SEQ ID NO:22 or SEQ ID NO:23 or a fragment thereof, The molecule inhibits the dephosphorylation activity of protein (serine-threonine) phosphatase 2A (PP2A) to maintain the phosphorylation level of serum response factor (SRF), thereby providing a composition for treating heart failure.

2. The composition of claim 1 , wherein the nucleic acid encodes a molecule having at least 90% sequence identity to the sequence of SEQ ID NO:22 or SEQ ID NO:23 or a fragment thereof.

3. The composition of claim 1 , wherein the nucleic acid encodes a molecule having at least 95% sequence identity to the sequence of SEQ ID NO:22 or SEQ ID NO:23 or a fragment thereof.

4. The composition of claim 1 , wherein the nucleic acid encodes a molecule having at least 80% sequence identity to the sequence of SEQ ID NO:22 or SEQ ID NO:

23.

5. The composition of claim 1 , wherein the nucleic acid encodes a molecule having at least 90% sequence identity to the sequence of SEQ ID NO:22 or SEQ ID NO:

23.

6. The composition of claim 1 , wherein the nucleic acid encodes a molecule having at least 95% sequence identity to the sequence of SEQ ID NO:22 or SEQ ID NO:

23.

7. The composition of claim 1 , wherein the nucleic acid encodes a molecule of sequence SEQ ID NO:22 or SEQ ID NO:

23.

8. 2. The composition of claim 1, wherein the nucleic acid encodes a molecule having at least 80% sequence identity to amino acids 2132-2319 of human muscle A-kinase anchor protein (mAKAP) or a fragment thereof.

9. The composition of claim 1, wherein the nucleic acid encodes a molecule having at least 90% sequence identity to amino acids 2132-2319 of mAKAP or a fragment thereof.

10. The composition of claim 1, wherein the nucleic acid encodes a molecule having at least 95% sequence identity to amino acids 2132-2319 of mAKAP or a fragment thereof.

11. The composition of claim 1, wherein the nucleic acid encodes a molecule having at least 80% sequence identity to amino acids 2132-2319 of mAKAP.

12. The composition of claim 1, wherein the nucleic acid encodes a molecule having at least 90% sequence identity to amino acids 2132-2319 of mAKAP.

13. The composition of claim 1, wherein the nucleic acid encodes a molecule having at least 95% sequence identity to amino acids 2132-2319 of mAKAP.

14. The composition of claim 1 , wherein the nucleic acid comprises an adeno-associated virus (AAV).

15. The composition of claim 14, wherein the AAV is AAV9.

16. The composition of claim 1 , wherein the molecule inhibits phosphorylation of B56δ (PPP2R5D) of PP2A.

17. The phosphorylation of SRF is １０３ The composition of claim 1, wherein the phosphorylation of

18. The composition of claim 1 , wherein the heart failure is heart failure with reduced ejection fraction.

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