Cardiac troponin t peptide composition and methods of use

Abstract

A composition comprising at least one peptide derived from the C-terminal segment of troponin T, the at least one peptide optionally fused to at least one cardiac-targeting peptide and / or at least one cell-penetrating peptide, polynucleotides and vectors encoding the composition, and methods of using the composition and vector in a method of treating heart failure, e.g., diastolic heart failure.

Description

UIC0117WO PATENT CARDIAC TROPONIN T PEPTIDE COMPOSITION AND METHODS OF USEREFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority from U. S . Provisional Application Serial Number 63 / 760, 179, filed February 19, 2025, the content of which is incorporated herein by reference in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (name : UIC0117WO_ST26 .xml; size : 74, 334 bytes; and date of creation: January 17, 2026) is herein incorporated by reference in its entirety .FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0003] This invention was made with government support under grant no . HL138007 awarded by the National Institutes of Health. The government has certain rights in this invention.BACKGROUND OF THE INVENTION

[0004] Cardiac muscle contraction and relaxation are essential for the pumping function of the heart . The contractile apparatus of striated muscles is regulated by Ca2+via the troponin complex associated with the sarcomeric thin filament . The troponin complex is composed of three protein subunits : the Ca2+-binding subunit troponin C (TnC) , the inhibitory subunit troponin I (Tnl ) , and the tropomyosin-binding subunit troponin T (TnT) (Tobacman (2021 ) Biophys J. 120 : 1-9) . During muscle activation, the rise of cytosolic Ca2+binds TnC and induces a series of conformational changes in troponin and tropomyosin, which in turn allow the myosin head to form a strong cross bridge with the actin filament to activate myosin ATPase and a conformational power stroke that forces the sliding of the thinUIC0117WO PATENT filament toward the center of the sarcomere and contract the muscle .

[0005] TnT is at a central position in the thin filament regulation of striated muscle contraction and relaxation cycles . Extensive structure-function relationship studies have demonstrated that TnT binds TnT, TnC, tropomyosin and possibly F-actin (Wei & Jin (2016) Gene 582 : 1-13) . Two tropomyosinbinding sites were previously identified in the middle region and at the C-terminal region of the TnT protein (Pearlstone & Smillie ( 1977 ) Can. J. Biochem. 55 ( 10) : 1032-8 ; Heeley et al . ( 1987 ) J. Biol . Chem. 262 : 9971-9978 ; Jin & Chong (2010) Archives Biochem. Biophys. 500 : 144-150) as essential sites for the thin filament anchoring of the troponin complex . A recent study has revealed a third tropomyosin-binding site located in the C-terminal end segment of TnT encoded by the last exon of vertebrate TnT genes (Cao et al . (2023) bioRxiv 2023.02.20.529301 ) .

[0006] The C-terminal segment of TnT is conserved among cardiac, fast, and slow skeletal muscle TnT isoforms and across vertebrate species . The 14 amino acid C-terminal end segment of cardiac TnT encoded by exon 17 of the TNNT2 gene has an identical sequence in a number of mammalian species including marsupials and human (Cao et al . (2023) bioRxiv 2023.02.20.529301 ) . Several mutations in this segment including R278C, K280N, and R286C have been linked to hypertrophic cardiomyopathy (HCM) (Watkins et al . ( 1995) N. Engl . J. Med.332 : 1058-64 ; Morimoto et al . ( 1999) Biochem. Biophys. Res. Commun. 261 : 79-82 ; Sequeira et al . (2013) Circ Res. 112 : 1491-505) . The C-terminal 14 amino acids of cardiac TnT are required for full inhibition of myofilament and substitution of positively charged Arg and Lys residues in this segment with Ala results in increased activation of contraction (Johnson et al . (2019) J. Biol . Chem. 294 ( 51 ) : 19535-19545 ) .UIC0117WO PATENT

[0007] Deletion of the evolutionarily added N-terminal hypervariable region of TnT (cTnT-ND) has been shown to restore a Tnl-like ancestral molecular conformation (Chong & Jin (2009) J. Mol . Evol . 68 : 448-60) detected by an anti-Tnl monoclonal antibody that was generated against an epitope in the C-terminal segment of Tnl (Jin et al . (2001 ) Biochemistry 40 : 2623-2631 ) . A physiologically occurring restrictive proteolytic truncation of the N-terminal variable region of cardiac TnT in adaption to acute myocardial ischemia with contractility-af terload mismatch produces an adaptive decrease in the systolic velocity of cardiac muscle to elongate the rapid ventricular ej ection time and sustain stroke volume against afterload (Feng et al . (2008 ) J. Physiol . (London) 586 : 3537-3550) . The C-terminal segment of Tnl has been characterized as a tropomyosin-binding site with a function in the inhibitory and regulatory roles of Tnl, which is critical to muscle relaxation (Wong et al . (2019) J. Mol . Cell . Cardiol .136 : 42-52 ; Hornos et al . (2021 ) J. Biol . Chem. 296 : 100228 ) . The Tnl C-terminal segment-like molecular conformation restored in cardiac TnT by the restrictive N-terminal truncation is localized in the 14 amino acids C-terminal segment (Cao et al . (2023) bioRxiv 2023.02.20.529301 ) .SUMMARY OF THE INVENTION

[0008] This invention provides a composition comprising a troponin T peptide fused to at least one cardiac-targeting peptide and / or at least one cell-penetrating peptide, wherein the troponin T peptide has the amino acid sequence : SKTRGKAKVTGRWK (SEQ ID NO : 1 ) , SKTCGKAKVTGRWK (SEQ ID NO : 2 ) , SKTRGNAKVTGRWK (SEQ ID NO : 3 ) , SKTRGKAKVTGCWK (SEQ ID NO : 4 ) , SKKAGTPAKGKVGGRWK (SEQ ID NO: 5) , RKGAGKGRVGGRWK (SEQ ID NO : 6 ) , RKGAPKGRLGGRWK (SEQ ID NO: 9) , KKGAPKARLGGRWK (SEQ ID NO: 11 ) ,UIC0117WO PATENT or KKGAPKGRLGGRWK (SEQ ID NO: 12 ) , or a functional fragment, modified peptide, and / or multimer thereof .

[0009] The invention also provides a composition comprising a troponin T peptide having the amino acid sequence of any one Of SKTRGKAKVTGRWK (SEQ ID NO : 1 ) , SKTCGKAKVTGRWK (SEQ ID NO : 2 ) , SKTRGNAKVTGRWK (SEQ ID NO : 3 ) , SKTRGKAKVTGCWK (SEQ ID NO : 4 ) , SKKAGTPAKGKVGGRWK (SEQ ID NO: 5) , RKGAGKGRVGGRWK (SEQ ID NO : 6 ) , RKGAPKGRLGGRWK (SEQ ID NO: 9) , KKGAPKARLGGRWK (SEQ ID NO: 11 ) , or KKGAPKGRLGGRWK (SEQ ID NO: 12) , or a functional fragment thereof, wherein the troponin T peptide or functional fragment is a modified peptide .

[0010] Al so provided is a composition comprising a multimeric troponin T peptide comprising a troponin T peptide of any one Of SKTRGKAKVTGRWK (SEQ ID NO : 1 ) , SKTCGKAKVTGRWK (SEQ ID NO : 2 ) , SKTRGNAKVTGRWK (SEQ ID NO : 3 ) , SKTRGKAKVTGCWK (SEQ ID NO : 4 ) , SKKAGTPAKGKVGGRWK (SEQ ID NO: 5) , RKGAGKGRVGGRWK (SEQ ID NO : 6 ) , RKGAPKGRLGGRWK (SEQ ID NO: 9) , KKGAPKARLGGRWK (SEQ ID NO: 11 ) , or KKGAPKGRLGGRWK (SEQ ID NO: 12) , or a functional fragment thereof, optionally wherein the multimeric troponin T peptide is a dimer .

[0011] Further provided are methods of using a composition of the invention for treating a cardiomyopathy and / or heart failure .BRIEF DESCRIPTION OF DRAWINGS

[0012] FIG. 1 shows Localized Surface Plasmon Resonance (LSPR) analysis of the binding of peptides comprising the C-terminal 14 amino acids of cardiac TnT (cTnT-C14 ) and tropomyosin. Wildtype human cTnT-C14 peptide (HcTnT-C14 WT) or HcTnT-C14 peptides containing the HCM mutation R278C, K280N, or R286C were analyzed via a LSPR sensor chip with saturated coating of tropomyosin (a flow of 250 ,g / mL) . A control peptide, cTnT-E16 ( INVLRNRINDNQKV, SEQ ID NO: 77 ) was used as negative control .UIC0117WO PATENT The representative LSPR traces from three independent experiments demonstrate that HcTnTC-14 WT at 10 M concentration produced saturable binding to immobilized tropomyosin with fast association and dissociation rates . In contrast, the three HCM mutant peptides all showed weakened tropomyosin-binding. The results demonstrate that the C-terminal end segment of cardiac TnT functions as a tropomyosinbinding site in the form of isolated free peptide at physiologically relevant affinity, which is significantly impaired by the HCM mutations .

[0013] FIG. 2 shows LSPR analysis of the binding of cTnT-C14 peptides to tropomyosin-actin thin filaments . The sensor chip was coated with a low density of F-action (a flow of 30 pg / mL) . After saturable decoration of the immobilized F-actin with tropomyosin (a flow at 10 pM) and a brief wash to remove free tropomyosin, WT or HCM mutant HcTnT-C14 peptides were inj ected at 10 pM concentration to measure association to and dissociation from the tropomyosin-F-actin filaments . The cTnT-E16 peptide was used as negative control . The LSPR traces showed an initial peak representing the binding of tropomyosin to F-actin, followed by the binding of HcTnT-C14 peptides . The representative LSPR traces from at least three independent experiments demonstrated that HcTnT-C14 WT produced a rapid association and saturable binding to tropomyosin-F-actin filaments followed by a rapid dissociation, similar to its interaction with tropomyosin alone (FIG. 1 ) . Also similar to the binding patterns to tropomyosin alone, HcTnT-C14 R278C, HcTnT-C14 K280N and HcTnT-C14 R286C showed diminished binding affinity for tropomyosin. The results confirm that the C-terminal peptide of cardiac TnT binds tropomyosin in actin thin filaments with physiological relevance, which is impaired by the HCM mutations .UIC0117WO PATENT

[0014] FIG. 3 shows LSPR analysis of the direct binding of cTnT-C14 peptides to F-actin. LSPR sensor chip was coated with F-action (at flow of 30 pg / mL) to test the interaction of WT and mutant HcTnT-C14 peptides with F-actin. The representative LSPR traces show that 10 pM HcTnT-C14 WT produced strong binding to F-actin while the HcTnT-C14 R278C, HcTnT-C14 K280N and HcTnT-C14 R286C exhibited significantly weaker binding to F-actin .

[0015] FIGS . 4A-4B show the effect of HcTnT-C14 peptides on Ca2+activated contractility of permeabilized cardiac muscle sections . The force-pCa curves show that addition of HcTnT-C14 WT peptide to skinned sections of wild-type mouse left ventricular papillary muscle decreased Ca2+-sensitivity (FIG.4A) . At sarcomere length 2.0 m, the treatment of 20 pM HcTnT-C14 had a notable Ca2+-desensitization effect on cardiac muscle strips . At sarcomere length 2.3 pm, HcTnT-C14 WT had a more notable Ca2+-desensitization effect on cardiac muscle (FIG. 4A) . Adding the HcTnT-C14 WT peptide to skinned sections of cTnT-ND mouse left ventricular papillary muscle in force-pCa measurements did not produce a significant change in contractility (FIG. 4B) . The lack of additive effect suggests that free HcTnT-C14 peptide targets the same Ca2+-desensitization mechanism as that of cTnT-ND. HcTnI-C14 did not decrease maximum force development of cardiac muscle .

[0016] FIGS . 5A-5C show loss of function caused by HCM mutations in HcTnT-C14 peptides . Consistent with their diminished tropomyosin- and thin filament-binding capacities, the force-pCa curves show that addition of HcTnT-C14 R278C (FIG. 5A) , HcTnT-C14 K280N (FIG. 5B) or HcTnT-C14 R286C (FIG.5C) peptide to skinned sections of wild-type mouse left ventricular papillary muscle did not produce a significant effect on Ca2+-activated contraction, indicating a similar lossUIC0117WO PATENT of function underlying the mechanism for these point mutations' pathological impacts .

[0017] FIG. 6 shows a map of an Adeno-associated virus (AAV) shuttle vector for cardiomyocyte-specific expression of a peptide of the disclosure . The shuttle vector plasmid DNA may be used for recombinant packing and preparation of high titer stocks of AAV viruses for cardiomyocyte-specific expression of a peptide of the disclosure .

[0018] FIGS . 7A-7B show that treatment of cardiomyocytes with a fusion protein of the invention decreases contractile amplitude . FIG. 7A shows that treatment of isolated WT mouse cardiomyocytes with various concentrations of cardiac targeting and membrane penetrating cTnT-C14 fusion peptide produced significant functional effects on decreasing the contractile amplitude in a dose-dependent manner which plateau at 0.5 pM. FIG. 7B shows that treatment of Tm-E180G cardiomyocytes with 0.5 pM CTP-TAT-cTnT-C14 fusion peptide markedly reduced the contraction amplitude, effectively correcting the hypercontractility .DETAILED DESCRIPTION OF THE INVENTION

[0019] This invention is based, in part, on the finding that the C-terminal 14 amino acid segment of cardiac TnT in the form of isolated free peptide (cTnT-C14 ) modulates the contractile kinetics of cardiac muscle . The interaction of cTnT-C14 with tropomyosin and F-actin-tropomyosin composite thin filaments were analyzed using localized surface plasmon resonance (LSPR) spectroscopy together with functional studies of the effect of cTnT-C14 peptide on Ca2+activation of force production in skinned cardiac muscle strips . The physiological function of the conf ormationally modulated TnT C-terminal tropomyosinbinding site also led to the development of novel peptides and compositions that modulate the kinetics of cardiac muscleUIC0117WO PATENT contraction. In particular, the binding affinity and association and dissociation rates of a C-terminal TnT peptide of the disclosure for tropomyosin shows that the C-terminal TnT peptide finds use in modulating the kinetics of cardiac muscle and in the treatment of heart conditions such as a cardiomyopathy and / or heart failure . Accordingly, provided herein is a composition comprising a C-terminal TnT peptide and methods of using the same to decrease calcium sensitivity of activated myofilaments, modulate the kinetics of cardiac muscle, and in the treatment of heart failure, e. g. , diastolic heart failure .

[0020] In some aspects, a composition of the disclosure comprises a C-terminal troponin T peptide (referred to generally herein as a "troponin T peptide" or "TnT peptide") having the amino acid sequence of any one of SKTRGKAKVTGRWK (SEQ ID N0: l ) , SKTCGKAKVTGRWK (SEQ ID NO : 2 ) , SKTRGNAKVTGRWK (SEQ ID N0: 3) , SKTRGKAKVTGCWK (SEQ ID NO : 4 ) , SKKAGTPAKGKVGGRWK (SEQ ID NO: 5) , RKGAGKGRVGGRWK (SEQ ID NO: 6) , RKGAPKGRLGGRWK (SEQ ID NO: 9) , KKGAPKARLGGRWK (SEQ ID NO: 11 ) , or KKGAPKGRLGGRWK (SEQ ID NO: 12 ) , or a functional fragment thereof, wherein the troponin T peptide or functional fragment is a modified peptide .

[0021] In some aspects, a composition of the disclosure comprises a multimeric troponin T peptide comprising a troponin T peptide of any one of SKTRGKAKVTGRWK (SEQ ID N0: l ) , SKTCGKAKVTGRWK (SEQ ID NO : 2 ) , SKTRGNAKVTGRWK (SEQ ID NO : 3 ) , SKTRGKAKVTGCWK (SEQ ID NO : 4 ) , SKKAGTPAKGKVGGRWK (SEQ ID NO : 5 ) , RKGAGKGRVGGRWK (SEQ ID NO : 6 ) , RKGAPKGRLGGRWK (SEQ ID NO: 9) , KKGAPKARLGGRWK (SEQ ID NO: 11 ) , or KKGAPKGRLGGRWK (SEQ ID NO: 12 ) , or a functional fragment thereof, optionally wherein the multimeric troponin T peptide is a dimer .

[0022] In some aspects, a composition of the disclosure comprises at least one TnT peptide conjugated or fused to at least one cardiac-targeting peptide and / or at least one cellpenetrating peptide . In some aspects, the composition of theUIC0117WO PATENT disclosure comprises one, two, three, four, five, six, seven, eight or more troponin T peptides conjugated or fused to one, two, three, four, five, six, seven, eight or more cardiactargeting peptides and / or one, two, three, four, five, six, seven, eight or more cell-penetrating peptides . In some aspects, a cardiac-targeting peptide and / or cell-penetrating peptide is conjugated or fused to the N-terminus of the troponin T peptide . In other aspects, a cardiac-targeting peptide and / or cell-penetrating peptide is conjugated or fused to the C-terminus of the troponin T peptide . In some aspects, the troponin T peptide is flanked by a cardiac-targeting peptide on one end ( e. g. , the C-terminus or N-terminus) and the cellpenetrating peptide is on the other end. In some aspects, a troponin T peptide is conjugated to both a cardiac-targeting peptide and a cell-penetrating peptide, wherein the cardiactargeting peptide and cell-penetrating peptide are both conjugated or fused to the N-terminus of the troponin T peptide, e. g. , the fusion has the structure [ cardiac-targeting peptide] - [cell-penetrating peptide ] - [ troponin T peptide] .

[0023] "Troponin T" or "TnT" refers to a tropomyosin-binding subunit of the troponin complex that plays a central role in regulating striated muscle contraction and relaxation. The amino acid sequences of troponin T proteins are known in the art and available, e. g. , under GENBANK Accession Nos . NM_001276347 . 2 or NM_001291774 . 2 (human TnT proteins) ; or XM_065420704 . 1 XM_065570861 . 1 , XM_044988613 . 1 , XM_048833492 . 1 , or XM_037888443 . 2 (turtle TnT proteins) . As used herein, a "troponin T peptide, " "TnT peptide, " or "C-terminal troponin T peptide" refers to a peptide derived from or based on the amino acid sequence of the C-terminal end segment of troponin T . In some aspects, a troponin T peptide is derived from or based upon 14 to 30 ( e. g. , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) consecutive C-terminal amino acid residues of troponin T . In some aspects, a troponin TUIC0117WO PATENT peptide is derived from or based upon 14 to 30 consecutive C-terminal amino acid residues of human troponin T protein. In some aspects, a troponin T peptide is derived from or based upon 14 to 30 consecutive C-terminal amino acid residues of a turtle troponin T protein. In some aspects, a troponin T peptide is derived from or based upon 14 to 20, in particular 14 to 17, consecutive C-terminal amino acid residues of human troponin T protein. In some aspects, a troponin T peptide is derived from or based upon 14 to 20, in particular 14 to 17, consecutive C-terminal amino acid residues of turtle troponin T protein.

[0024] In some aspects, a troponin T peptide of the disclosure is not less than about 14 amino acid residues, 15 amino acid residues, 16 amino acid residues, 17 amino acid residues, 18 amino acid residues, 19, amino acid residues, or 20 amino acid residues in length. In other aspects, a troponin T peptide of the disclosure is not less than about 14 amino acid residues in length. In some aspects, a peptide of the disclosure is 14 to 20 amino acid residues in length, 14 to 19 amino acids in length, 14 to 18 amino acids in length, 14 to 17 amino acids in length, 15 to 20 amino acids in length, 15 to 19 amino acids in length, 15 to 18 amino acids in length, or 15 to 17 amino acids in length. In other aspects, a troponin T peptide of the disclosure is 14 to 17 amino acid residues in length.

[0025] In some aspects, a troponin T peptide of the disclosure exhibits tropomyosin-binding activity. In some aspects, a troponin T peptide exhibits a binding association, dissociation, and / or overall affinity for tropomyosin comparable to the binding of a full-length troponin T protein. In some aspects, a troponin T peptide exhibits a slower binding association, faster dissociation, and / or weaker overall affinity for tropomyosin compared to the binding of a full-length troponin T protein. In other aspects, a troponin T peptide exhibits binding to tropomyosin-F-actin composite filaments .UIC0117WO PATENT

[0026] In some aspects, a troponin T peptide of the disclosure has the amino acid sequence : SKTRGKAKVTGRWK (SEQ ID NO: 1 ) , SKTCGKAKVTGRWK (SEQ ID NO : 2 ) , SKTRGNAKVTGRWK (SEQ ID NO : 3 ) , SKTRGKAKVTGCWK (SEQ ID NO : 4 ) , SKKAGTPAKGKVGGRWK (SEQ ID NO : 5 ) , RKGAGKGRVGGRWK (SEQ ID NO : 6 ) , RKGAPKGRLGGRWK (SEQ ID NO: 9) , KKGAPKARLGGRWK (SEQ ID NO: 11 ) , or KKGAPKGRLGGRWK (SEQ ID NO: 12 ) , or a functional fragment, modified peptide, and / or multimer thereof .

[0027] As used herein, the term "active fragment" or "functional fragment" refers to a portion of a reference peptide that mediates the same effect as the reference peptide, e. g. , a functional fragment of troponin T peptide exhibits tropomyosin-binding activity and / or binding to tropomyosin-F-actin composite filaments . In some aspects, an active fragment or functional fragment of a troponin T peptide having the amino acid sequence of any one of SEQ ID NOs : l-6, 9, 11 or 12 is at least about 12 amino acid residues in length. In some aspects, an active fragment or functional fragment of a troponin T peptide having the amino acid sequence of any one of SEQ ID NOs : l-6, 9, 11 or 12 is no less than 12 or 13 consecutive amino acid residues of SEQ ID NO: 1-6, 9, 11 or 12. Exemplary functional fragments include C-terminal truncations, N-terminal truncations, or truncations of both the C-terminus and N-terminus of a reference peptide ( e. g. , deletions of at least 1, at least 2, at least 3, at least 4, or at least 5 amino acids from the N-terminus, the C-terminus, or both) . In some aspects, by sequentially deleting N- and / or C-terminal amino acids from any one of SEQ ID NOs : l-6, 9, 11 or 12 and assessing the function of the resulting peptide fragment, alone or as fusion, can identify a functional fragment of troponin T peptide for use in a composition and method of the disclosure .

[0028] In some aspects, a functional fragment of a troponin T peptide may have the amino acid sequence of any one of SKTRGKAKVTGR (SEQ ID NO : 7 ) , SKTCGKAKVTGR (SEQ ID N0: 19) ,UIC0117WO PATENT SKTRGNAKVTGR (SEQ ID NO: 20) , SKTRGKAKVTGC (SEQ ID N0: 21 ) , SKKAGTPAKGKVGGR (SEQ ID NO: 22 ) , RKGAGKGRVGGR (SEQ ID NO: 23) , RKGAPKGRLGGR (SEQ ID NO: 24 ) , KKGAPKARLGGR (SEQ ID NO: 25) , or KKGAPKGRLGGR (SEQ ID NO: 26) .

[0029] A troponin T peptide of the disclosure may be composed of the 20 "standard" L-amino acids, D-amino acids, or nonstandard, modified, or unusual amino acids well-defined in the art . Accordingly, in some aspects, a troponin T peptide of the disclosure may be a "modified troponin T peptide, " "modified TnT peptide, " or "modified peptide ." In some aspects, a modified peptide may include one or more non-standard, modified, or unusual amino acids such as p-alanine ( p-Ala) and other a-amino acids such as 3-aminopropionic acid, 2, 3-diaminopropionic acid (Dpr) , 4-aminobutyric acid and so forth; a-aminoisobutyric acid (Aib) ; s-aminohexanoic acid (Aha) ; 5-aminovaleric acid (Ava) ; N-methylglycine or sarcosine (MeGly) ; ornithine (Orn) ; citrulline (Git) ; t-butylalanine (t-BuA) ; t-butylglycine (t-BuG) ; N-methylisoleucine (Me-Ile) ; phenylglycine (Phg) ; norleucine (Nle) ; 4-chlorophenylalanine (Phe (4-Cl) ) ; 2-f luorophenylalanine (Phe (2-F) ) ; 3-f luorophenylalanine (Phe (3-F) ) ; 4-f luorophenylalanine (Phe (4-F) ) ; penicillamine (Pen) ; 1 , 2 , 3 , 4-tetrahydroisoquinoline-3-carboxylic acid (Tic) ; homoarginine (hArg) ; N-acetyl lysine (AcLys) ; 2 , 4-diaminobutyric acid (Dbu) ; 2 , 4-diaminobutyric acid (Dab) ; p-aminophenylalanine (Phe (pNH2) ) ; N-methyl valine (MeVal) ; homocysteine (hCys) , homophenylalanine (hPhe) and homoserine (hSer) ; hydroxyproline (Hyp) , homoproline (hPro) , N-methylated amino acids and peptoids (N-substituted glycines) . In some aspects, an amino acid residue of any one of SEQ ID NOs : l-6, 9, 11 or 12, or any one of SEQ ID NOs : 7 or 19-26 may be replaced with a non-standard, modified, or unusual amino acid residue of the same . For example, a valine residue may be replaced with MeVal, a lysine residue may be replaced with AcLys, and / or alanine may be replaced with MeGly.UIC0117WO PATENT

[0030] In some aspects, a modified peptide may include one or more amino acid residues that are chemically modified by post-translational modification. Any modification known in the art may be made to an amino acid of the troponin T peptide or functional fragment or multimer using any method known in the art . In some aspects, a troponin T peptide or functional fragment or multimer of the disclosure may be glycosylated, phosphorylated, sulfated, amidated, carboxylated, acetylated PEGylated, palmitoylated and / or myristoylated . For example, the C-terminus of a peptide may be modified with amidation, addition of peptide alcohols and aldehydes, addition of esters, or addition of p-nitroaniline and thioesters . The N-terminal and / or side chains of a peptide may be modified by PEGylation, acetylation, formylation, addition of a fatty acid, addition of benzoyl, addition of bromoacetyl, addition of pyroglutamyl, succinylation, addition of tetrabutyoxycarbonyl and addition of 3-mercaptopropyl , acylation ( e. g. , lipopeptides ) , biotinylation, phosphorylation, sulfation, glycosylation, introduction of a maleimido group, chelating moiety, chromophore and / or fluorophore . In some aspects, a troponin T peptide or functional fragment or multimer is phosphorylated, e. g. , a threonine of the troponin T peptide or functional fragment or multimer is phosphorylated. In some aspects, a modified troponin T peptide may have the amino acid sequence SKTRGKAKV (pT) GRWK (SEQ ID NO : 8 ) .

[0031] In some aspects, a composition of the disclosure includes an oligomer or multimer of a troponin T peptide having the amino acid sequence of any one of SEQ ID N0s : l-6, 9, 11 or 12 or a functional fragment ( e. g. , SEQ ID Nos : 7 or 19-26) or modified peptide thereof . In some aspects, a multimer may be a polymer comprising two or more troponin T peptide sequences of the disclosure . In some aspects, a multimer may be a polymer comprising two, three, four, five, six, seven, eight or more troponin T peptides . In some aspects, each troponin T peptide,UIC0117WO PATENT fragment or modified peptide of a multimer may have the same sequence . In some aspects, each troponin T peptide, fragment or modified peptide of a multimer may have a different sequence . In multimers comprising three or more troponin T peptide, fragment or modified peptide, said multimer may be composed of two or more different sequences . By way of illustration a trimer may be composed of two troponin T peptides having identical sequences and a third peptide having a sequence that is different from the other two . In some aspects, a multimer of the disclosure is a dimer comprising two troponin T peptides, fragments and / or modified peptides of the disclosure, said two peptides being identical or non-identical with respect to each other . In another aspect, a multimer of the disclosure is a trimer comprising three troponin T peptides, fragments and / or modified peptides of the disclosure, said peptides being identical or non-identical with respect to each other . In another aspect, a multimer of the disclosure is a tetramer comprising four troponin T peptides, fragments and / or modified peptides of the disclosure, said peptides being identical or non-identical with respect to each other . In some aspects, a multimer comprises two identical amino acid sequences of the disclosure (i . e. , dimer) .

[0032] In some aspects, a multimer is a dendrimer comprising a plurality of troponin T peptides, fragments and / or modified peptides . Dendrimers are repeatedly branched, roughly spherical large molecules, typically symmetric around the core, and often adopting a spherical three-dimensional morphology. Dendrimers may comprise four peptides, eight peptides, 16 peptides, or 32 peptides . In some aspects, a dendrimer comprises four peptides (i . e. , a tetrameric dendrimer) or eight peptides (i . e. , octameric dendrimer) .

[0033] A multimer of the disclosure may be made by linking two or more peptide monomers directly to one another via a peptide bond (i . e. , a peptide fusion) or by linking two or more peptideUIC0117WO PATENT monomers via a linker group . In one aspect, a linker may be composed of glycine and / or serine residues, e. g. , Gly-Gly-Ser . In another aspect, peptides of a multimer may be linked to a lysine backbone, such as a lysine residue (each peptide chain is linked to a single lysine residue) , or coupled to a polymer carrier, for example a protein carrier . Said linker group in one aspect comprises a plurality of lysine residues, such as a core moiety having a plurality of lysine residues, such as seen in a lysine-based dendromeric structure containing three, seven, fifteen and more lysine residues . Any other linking of peptide monomers known to the skilled person may be envisioned.

[0034] In some aspects, a multimer of a troponin T peptide may have the amino acid sequence of any one of S KT RG KAKVT GRWKS KT RG KAKVT GRWK (SEQ ID NO: 27 ) , SKTCGKAKVTGRWKSKTCGKAKVTGRWK (SEQ ID NO: 28 ) , SKTRGNAKVTGRWKSKTRGNAKVTGRWK (SEQ ID NO: 29) , SKTRGKAKVTGCWKSKTRGKAKVTGCWK (SEQ ID NO: 30) , SKKAGTPAKGKVGGRWKSKKAGTPAKGKVGGRWK (SEQ ID NO: 31 ) , RKG AG KG RVGG RW KRKG AG KG RVGG RW K ( SEQ ID NO : 32 ) , RKGAPKGRLGGRWKRKGAPKGRLGGRWK (SEQ ID NO: 10) , SKTRGKAKV (pT) GRWKSKTRGKAKV ((SEQ ID NO: 33) , KKGAPKARLGGRWKKKGAPKARLGGRWK (SEQ ID NO: 34 ) , SKTRGKAKVTGRSKTRGKAKVTGR (SEQ ID NO: 35) , or KKGAPKGRLGGRWKKKGAPKGRLGGRWK (SEQ ID NO: 36) .

[0035] In some aspects, a troponin T peptide, functional fragment, modified peptide, or multimer of the disclosure consists essentially of the amino acid sequence of any one of SEQ ID NOs : l-12 or 15-36. In some aspects, a troponin T peptide, functional fragment, modified peptide, or multimer of the disclosure consists of the amino acid sequence of any one of SEQ ID NOs : l-12 or 15-36. As used herein, the phrase "consisting essentially of" is to be interpreted to encompass the specified features, steps, operations, elements, components, nucleic acids and / or amino acid residues and additional features,UIC0117WO PATENT steps, operations, elements, components, nucleic acids and / or amino acid residues that do not materially affect the basic and novel characteristic ( s ) of the claimed invention. Thus, the term "consisting essentially of" when used herein is not intended to be interpreted to be equivalent to "comprising." The phrase "consisting of" is intended to exclude any additional features, steps, operations, elements, components, nucleic acids and / or amino acid residues other than those subsequently following the phrase . When "consisting of" is used to define a composition, the phrase does not exclude impurities ordinarily associated therewith composition.

[0036] In accordance with the composition of the disclosure, a troponin T peptide is fused, conjugated, or linked to at least one cardiac-targeting peptide and / or at least one cellpenetrating peptide . "Con ugated, " "linked, " "fused, " and "fusion" are used interchangeably herein to refer to the joining of two or more chemical elements or components by any means including chemical conjugation or recombinant means . In some aspects, a troponin T peptide is fused, conjugated, or linked to at least one cardiac-targeting peptide and / or at least one cell-penetrating peptide by a peptide bond. Thus, in one aspect, a troponin T peptide fused to a cardiac-targeting peptide and / or a cell-penetrating peptide results in a single protein comprising the troponin T peptide, cardiac-targeting peptide and / or a cell-penetrating peptide . In other aspects, a troponin T peptide is fused, conjugated, or linked to at least one cardiac-targeting peptide and / or at least one cellpenetrating peptide by a linker or non-peptidic bond.

[0037] A "cardiac-targeting peptide" or "CTP" refers to peptide that specifically targets cardiac tissue . "Specifically targets cardiac tissue" means that when said CTP is administered to a mammal, the CTP is targeted to cardiac tissue at much higher levels than it is targeted to other tissues, such as, for example, liver, kidney, lung, skeletal muscle, orUIC0117WO PATENT brain. This functional property of a CTP is not abolished by its inclusion in a composition of the disclosure . Accordingly, the entire composition is delivered to cardiac tissue . In some aspects, the ratio of targeting of a CTP that "specifically targets cardiac tissue" into cardiac tissue relative to liver, kidney, lung, skeletal muscle, or brain is at least 2 : 1 or is at least 3 : 1.

[0038] In some aspects, a cardiac-targeting peptide may be a peptide that is between 6 and 25 amino acids in length or between 8 and 20 amino acids in length. In some aspects, a cardiac-targeting peptide of a composition of the disclosure comprises the amino acid sequence WLSEAGPWTVRALRGTGSW (SEQ ID NO: 37 ; see Sahagun et al . (2023) Biomolecules 13 ( 12 ) : 1690 ) , CSTSMLKAC (SEQ ID NO: 38 ; see Kanki et al . (2011 ) J. Mol . Cell Cardiol . 50 ( 5 ) : 841-8 ) , APWHLSSQYSRT (SEQ ID NO: 13, see CAS No .1052692-86-0) , SQYSRT (SEQ ID NO: 39, see US 2021 / 0206805) , or a variation thereof in which one amino acid is either deleted, substituted by another amino acid, or one amino acid is added.

[0039] As used herein, a "cell-penetrating peptide, " "CPP, " or "membrane translocating peptide" refers to a class of short peptides that is capable of targeting to and / or penetrating the membrane of a cell, permitting delivery to the interior of said cell . CPPs are typically used to facilitate the cellular uptake of various molecules ( from nanosize particles to small chemical molecules and large fragments of DNA) , which are attached as cargo to a CPP by covalent or non-covalent linkage . A CPP may thus be used for the in vitro and in vivo cellular delivery of a cargo molecule attached to the CPP . A number of defined classes of CPP are recognized in the art . These are described in more detail below and are encompassed in the definition of CPP used herein. However, it will be recognized that said definition also encompasses any peptide that exhibits the stated cell delivery properties . In some aspects, a CPP of the composition of the disclosure has the functional property ofUIC0117WO PATENT being able to target and / or penetrate into the interior of a mammalian cell, in particular a human cell, preferably a heart cell . This functional property of a CPP is not abolished by its inclusion in a composition of the disclosure . Accordingly, the entire composition is delivered to the interior of the cell .

[0040] In some aspects, a CPP of use in a composition of the disclosure may be a Tat peptide having the amino acid sequence GRKKRRQRRRPPQ (SEQ ID NO: 14 ) ; a Transportan having the amino acid sequence GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO : 40 ) ; a Penetratin having the amino acid sequence RQIKIWFQNRRMKWKK (SEQ ID N0: 41 ) ; a R6-Penetratin having the amino acid sequence RRRRRRRQIKIWFQNRRMKWKK (SEQ ID NO: 42 ) ; pVEC having the amino acid sequence LLI ILRRRIRKQAHAHSK (SEQ ID NO: 43) ; MPG having the amino acid sequence GALFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 44 ) , Pepl having the amino acid sequence KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 45) , MAP having the amino acid sequence KLALKLALKALKAALKLA (SEQ ID NO: 46) ; or R6W3 having the amino acid sequence RRWWRRWRR (SEQ ID NO: 47 ) . In other aspects, a CPP of use in a composition of the disclosure may be a PIP series CPP, e. g. , as described in detail in WO 2009 / 147368 and WO 2013 / 030569.

[0041] The functional properties of a CTP and / or CPP, or of any composition of the disclosure comprising a CTP and / or CPP, may be assessed by any suitable method. Suitable methods are well-known and include, for example, directly visualizing targeting and / or delivery into the cytoplasm of a cell by attachment of a suitable label, such as a fluorescent label, to the molecule to be tested. The labeled molecule is then incubated with cells in vitro and targeting / internalization is quantified, e. g. , with a fluorescence microscope . Alternatively, cell delivery may be assessed by a secondary indicator, such as the activity in a cultured cell of a cargo attached to the CTP and / or CPP or the molecule comprising the CTP and / or CPP . The level of cargo activity will indicate theUIC0117WO PATENT level of cell delivery. In some aspects, the functional property of a CTP and / or GPP for targeting / internalization of a troponin T peptide may be assessed by measuring, e. g. , Ca2+-activated force as described herein.

[0042] In some aspects, a composition comprising a troponin T peptide fused to at least one cardiac-targeting peptide and / or at least one cell-penetrating peptide may have the amino acid sequence of any one of GRKKRRQRRRPPQSKTRGKAKVTGRWK (SEQ ID NO: 48 ) , GRKKRRQRRRPPQSKTCGKAKVTGRWK (SEQ ID NO 49) , GRKKRRQRRRPPQSKTRGNAKVTGRWK (SEQ ID NO 50) , GRKKRRQRRRPPQSKTRGKAKVTGCWK (SEQ ID NO 51 ) , GRKKRRQRRRPPQSKTRGKAKVTGR (SEQ ID NO 52 ) , GRKKRRQRRRPPQSKTRGKAKV (pT) GRWK (SEQ ID NO 53) , GRKKRRQRRRPPQRKGAGKGRVGGRWK (SEQ ID NO 54 ) , GRKKRRQRRRPPQRKGAPKGRLGGRWK (SEQ ID NO 55) , GRKKRRQRRRPPQRKGAPKGRLGGRWKRKGAPKGRLGGRWK (SEQ ID NO 56) , GRKKRRQRRRPPQKKGAPKARLGGRWK (SEQ ID NO 57 ) , GRKKRRQRRRPPQKKGAPKGRLGGRWK (SEQ ID NO 58 ) , APWHLSSQYSRTSKTRGKAKVTGRWK (SEQ ID NO 59) , APWHLSSQYSRTSKTCGKAKVTGRWK (SEQ ID NO 60) , APWHLSSQYSRTSKTRGNAKVTGRWK (SEQ ID NO 61 ) , APWHLSSQYSRTSKTRGKAKVTGCWK (SEQ ID NO 62 ) , APWHLSSQYSRTSKTRGKAKVTGR (SEQ ID NO 63) , APWHLSSQYSRTSKTRGKAKV (pT) GRWK (SEQ ID NO 64 ) , APWHLSSQYSRTRKGAGKGRVGGRWK (SEQ ID NO 65) , APWHLSSQYSRTRKGAPKGRLGGRWK (SEQ ID NO 66) , APWHLSSQYSRTRKGAPKGRLGGRWKRKGAPKGRLGGRWK (SEQ ID NO 67 ) , APWHLSSQYSRTGRKKRRQRRRPPQKKGAPKARLGGRWK (SEQ ID NO 68 ) , APWHLSSQYSRTGRKKRRQRRRPPQKKGAPKGRLGGRWK (SEQ ID NO 69) , APWHLSSQYSRTGRKKRRQRRRPPQSKTRGKAKVTGRWK (SEQ ID NO 15) , APWHLSSQYSRTGRKKRRQRRRPPQSKTCGKAKVTGRWK (SEQ ID NO 70) , APWHLSSQYSRTGRKKRRQRRRPPQSKTRGNAKVTGRWK (SEQ ID NO 71 ) , APWHLSSQYSRTGRKKRRQRRRPPQSKTRGKAKVTGCWK (SEQ ID NO 72 ) , APWHLSSQYSRTGRKKRRQRRRPPQSKTRGKAKVTGR (SEQ ID NO 73) ,UIC0117WO PATENT APWHLSSQYSRTGRKKRRQRRRPPQSKTRGKAKV (pT) GRWK (SEQ ID NO: 74 ) , APWHLSSQYSRTGRKKRRQRRRPPQRKGAGKGRVGGRWK (SEQ ID NO: 16) , APWHLSSQYSRTGRKKRRQRRRPPQRKGAPKGRLGGRWK (SEQ ID NO: 17 ) , APWHLSSQYSRTGRKKRRQRRRPPQRKGAPKGRLGGRWKRKGAPKGRLGGRWK (SEQ ID NO: 18 ) , APWHLSSQYSRTGRKKRRQRRRPPQKKGAPKARLGGRWK (SEQ ID NO: 75) , or APWHLSSQYSRTGRKKRRQRRRPPQKKGAPKGRLGGRWK (SEQ ID NO : 76 ) .

[0043] In some aspects, a composition of the disclosure may be further labeled with a heavy isotope, e. g. ,15N or13C, FITC, conjugated to an imaging agent, FRET substrate with a f luorophore / quencher pair, DNA molecule, RNA molecule, chemotherapeutic agent, or antibody. In some aspects, a composition of the disclosure may be tagged, e. g. , with a 6XHis tag, Myc tag, FLAG tag, or an epitope tag and the like to facilitate detection and / or isolation of the composition. In some aspects, a tag is fused to a composition of the disclosure via cleavable linker, e. g. , protease cleavable linker .

[0044] In some aspects, a composition of the invention does not include a full-length troponin T protein ( e. g. , a troponin T protein under GENBANK Accession No . NM_001276347 . 2 , NM_001291774 .2 XM_065420704 . 1 , XM_065570861 . 1 , XM_044988613 . 1 , XM_048833492 . 1, or XM_037888443 . 2 . In some aspects, a composition of the invention is not composed solely of any one of SEQ ID NOs : l-4, i . e. , a composition comprising any one of SEQ ID NOs : l-4 also includes either a cardiac-targeting peptide and / or a cell-penetrating peptide, which is exogenous to SEQ ID NOs : l-4, or is modified. A cardiac-targeting peptide and cell-penetrating peptide that is "exogenous" to any one of SEQ ID NOs : l-4 refers to a cardiac-targeting peptide and cellpenetrating peptide this is not derived from troponin T, i . e. , the cardiac-targeting peptide and cell-penetrating peptide does not have an amino acid sequence of a troponin T protein.

[0045] A composition of the disclosure may be prepared synthetically, recombinantly, or a combination thereof . In oneUIC0117WO PATENT aspect, a composition of the disclosure may be synthesized recombinantly using recombinant DNA techniques . Thus, in another aspect, the invention provides a polynucleotide encoding a composition comprising at least one troponin T peptide alone or fused to at least one cardiac-targeting peptide and / or at least one cell-penetrating peptide . In a related aspect, the invention provides vectors, particularly expression vectors that comprise a polynucleotide encoding a composition comprising at least one troponin T peptide alone or fused to at least one cardiac-targeting peptide and / or at least one cell-penetrating peptide . In some aspects, a vector provides replication, transcription, and / or translation regulatory sequences that facilitate recombinant synthesis of the desired composition in a eukaryotic cell or prokaryotic cell . Accordingly, the invention also provides host cells for recombinant expression of a composition of the disclosure and methods of harvesting and purifying the composition produced by the host cells . Production and purification of a recombinant composition is routinely practiced by one of skill in the art . The composition may be purified by any suitable method known in the art including, without limitation, gel filtration and / or affinity purification. When a composition of the invention is produced in the form of a fusion protein comprising, e. g. , a tag, the tag may optionally be cleaved off using a protease before further analysis and / or use .

[0046] Alternatively, a composition of the disclosure may be synthesized by any of the chemical synthesis techniques known in the art, particularly solid-phase synthesis techniques, for example, using commercially available automated peptide synthesizers . See, for example, Stewart & Young ( 1984 ) Solid Phase Peptide Synthesis, 2nded. , Pierce Chemical Co . ; Tam et al . ( 1983) J. Am. Chem. Soc. 105 : 6442 ; Merrifield ( 1986) Science 232 : 341-347 ; Barany et al . ( 1987 ) Int . J. Peptide Protein Res. 30 : 705-739; and U. S . Patent No . 5, 424, 398.UIC0117WO PATENT

[0047] A composition of the disclosure comprising at least one troponin T peptide or troponin T peptide fused to at least one cardiac-targeting peptide and / or at least one cell-penetrating peptide exhibits binding to tropomyosin and F-actin-tropomyosin composite thin filaments thereby modulating the kinetics of cardiac muscle . Accordingly, a composition of the disclosure finds use in a method of treating heart failure . In one aspect, the method of the invention comprises the step of administering to a subj ect in need of treatment an effective amount of a composition comprising at least one troponin T peptide or troponin T peptide or fused to at least one cardiac-targeting peptide and / or at least one cell-penetrating peptide . In another aspect, the method of the invention comprises the step of administering to a subj ect in need of treatment an effective amount of a vector comprising a polynucleotide encoding a composition comprising at least one troponin T peptide or troponin T peptide or fused to at least one cardiac-targeting peptide and / or at least one cell-penetrating peptide .

[0048] As used herein, "treatment" or "treating" means any treatment of a disease in a patient, including: (a) reducing the severity of the disease; (b) arresting the development of the disease or disorder; (c) inhibiting worsening of the disease or disorder; (d) limiting or preventing recurrence of the disease or disorder in patients that have previously had the disease or disorder; (e) causing regression of the disease or disorder; ( f ) improving or eliminating the symptoms of the disease or disorder; and / or (g) improving survival . Treatment of diseases and disorders herein is intended to also include the prophylactic administration of a composition described herein to a subj ect (i . e. , an animal, preferably a mammal, most preferably a human) believed to be in need of preventative treatment, such as, for example, heart failure . Accordingly, the invention provides for the treating, preventing, orUIC0117WO PATENT reducing the severity of heart failure or one or more complications of heart failure .

[0049] As used herein, "heart failure" refers to an abnormality in cardiac function in which the heart of a subj ect does not pump blood at a rate required by metabolic tissues . A subj ect that may benefit from treatment in accordance with the methods herein include those having, suspected of having, at risk of having a cardiomyopathy and / or heart failure, or predisposed to developing a cardiomyopathy and / or heart failure . Such a cardiomyopathy and / or heart failure may include congestive heart failure, myocardial infarction, tachyarrhythmia, familial hypertrophic cardiomyopathy, ischemic heart disease, idiopathic dilated cardiomyopathy, or myocarditis . Heart failure can result from many causes, including, but not limited to, ischemic, congenital, rheumatic, viral, toxic, or idiopathic forms . Chronic cardiac hypertrophy is a serious disease state that is a precursor to congestive heart failure and cardiac arrest . In some aspects, a composition of the invention provides benefit in the treatment of conditions such as hypertrophic cardiomyopathy and diastolic heart failure . Hypertrophic cardiomyopathy refers to a heart disease in which the walls of the left ventricle thicken in the absence of other signs such as aortic valve stenosis or high blood pressure sufficient to induce left ventricular hypertrophy. Diastolic heart failure, also known as heart failure with preserved ej ection fraction (HFpEF) , occurs when the heart ' s left ventricle becomes stiff and thick, preventing it from fully relaxing and filling with enough blood between beats, even though its pumping strength (ej ection fraction) remains normal . Symptoms include breathlessness, fatigue, and swelling, often linked to high blood pressure, aging, diabetes, or obesity.

[0050] As used herein, the terms "amount effective, " "effective amount" or "therapeutically effective amount" refer to an amount of a composition or polynucleotide ( e. g. , vector)UIC0117WO PATENT of the disclosure or a pharmaceutical composition comprising the composition or polynucleotide sufficient to achieve the stated desired result, for example, treating heart failure . The amount of the composition or polynucleotide which constitutes an "effective amount" or "therapeutically effective amount" may vary depending on the severity of the disease, the condition, weight, or age of the subj ect to be treated, the frequency of dosing, and / or the route of administration, but may be determined routinely by one of ordinary skill in the art . A clinician may titer the dosage or route of administration to obtain the optimal therapeutic effect . Typical dosages of a composition comprising at least one troponin T peptide fused to at least one cardiac-targeting peptide and / or at least one cell-penetrating peptide may range from about 0. 1 pg / kg to up to about 100 mg / kg or more, depending on the factors mentioned above . In some aspects, the dosage may range from 0.1 pg / kg up to about 100 mg / kg, or 1 pg / kg up to about 100 mg / kg, or 5 pg / kg up to about 100 mg / kg.

[0051] In some aspects, a composition or polynucleotide ( e. g. , vector) of the disclosure is provided in a pharmaceutical composition. In some aspects, the pharmaceutical composition may be specially formulated for oral administration in solid or liquid form or for intraperitoneal, intravenous, intramuscular, intracoronary or intramyocardial inj ection. Optimal pharmaceutical compositions may be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage . See, for example, Remington's Pharmaceutical Sciences , latest edition .

[0052] A composition or polynucleotide ( e. g. , vector) of the disclosure may be incorporated in a conventional systemic dosage form, such as a tablet, capsule, soft gelatin capsule, elixir or inj ectable formulation. The dosage forms may also include the necessary physiologically acceptable carrier,UIC0117WO PATENT diluent, or excipient, including, e. g. , lubricant, buffer, surfactant, antibacterial, bulking agent (such as mannitol) , antioxidant (ascorbic acid or sodium bisulfite) and the like .

[0053] Acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. The pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine) ; antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulf ite) ; buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids) ; bulking agents (such as mannitol or glycine) ; chelating agents (such as ethylenediamine tetraacetic acid (EDTA) ) ; complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin) ; fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose or dextrins) ; proteins (such as serum albumin, gelatin or immunoglobulins) ; coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone) ; low molecular weight polypeptides; salt-forming counterions (such as sodium) ; preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide) ; solvents (such as glycerin, propylene glycol or polyethylene glycol) ; sugar alcohols (such as mannitol or sorbitol) ; suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20 and polysorbate 80, Triton, trimethamine, lecithin, cholesterol, or tyloxapal) ; stabilityUIC0117WO PATENT enhancing agents (such as sucrose or sorbitol) ; tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol, or sorbitol) ; delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants . See, for example, Remington's Pharmaceutical Sciences , Id.

[0054] The primary carrier, diluent, or excipient in a pharmaceutical composition may be either aqueous or non-aqueous in nature . For example, a suitable carrier, diluent, or excipient may be water for inj ection, physiological saline solution, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles . Pharmaceutical compositions may comprise Tris buffer of about pH 7. 0-8. 5, or acetate buffer of about pH 4. 0-5. 5, which may further include sorbitol or a suitable substitute thereof . Pharmaceutical compositions may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences , Id. ) in the form of a lyophilized cake or an aqueous solution. Further, a composition or polynucleotide of the disclosure may be formulated as a lyophilizate using appropriate excipients such as sucrose .

[0055] Administration routes for the pharmaceutical compositions herein include orally, through inj ection by intravenous, intraperitoneal, intracerebral (intra-parenchymal ) , intracerebroventricular , intramuscular, intramyocardial , intracoronary, intra-ocular , intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices . A pharmaceutical composition may be administered by bolus inj ection or continuously by infusion, or by implantation device . A pharmaceutical composition also may be administered locally via implantation of a membrane, sponge or another appropriateUIC0117WO PATENT material onto which the desired molecule has been absorbed or encapsulated. Where an implantation device is used, the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed-release bolus, or continuous administration.

[0056] A pharmaceutical composition herein may be delivered parenterally. When parenteral administration is contemplated, a composition or polynucleotide ( e. g. , vector) for use herein may be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired composition or polynucleotide ( e. g. , vector) in a pharmaceutically acceptable carrier . A particularly suitable carrier for parenteral inj ection is sterile distilled water in which a composition or polynucleotide is formulated as a sterile, isotonic solution, appropriately preserved. Preparation may involve the formulation of the desired composition or polynucleotide with an agent, such as inj ectable microspheres, nanoparticles, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid) , beads, or liposomes, that may provide controlled or sustained release of the composition or polynucleotide which may then be delivered via a depot inj ection. Formulation with hyaluronic acid has the effect of promoting sustained duration in the circulation. Implantable drug delivery devices may be used to introduce the desired molecule . In some aspects, the invention provides a liposome encapsulating a composition comprising at least one troponin T peptide alone or fused to at least one cardiac-targeting peptide and / or at least one cell-penetrating peptide . In some aspects, the invention provides a nanoparticle encapsulating a composition comprising at least one troponin T peptide alone or fused to at least one cardiac-targeting peptide and / or at least one cell-penetrating peptide . In some aspects, the liposome or nanoparticle is modified with a cardiac-targeting peptide and / or cell-penetrating peptide as described herein.UIC0117WO PATENT In some aspects, a liposome or nanoparticle modified with a cardiac-targeting peptide encapsulates a composition comprising at least one troponin T peptide fused to at least one cell-penetrating peptide so that the liposome or nanoparticle delivers the composition to cardiac tissue .

[0057] Pharmaceutical compositions may also be formulated for inhalation. For inhalation, a composition or polynucleotide of the disclosure may be formulated as a dry powder for inhalation, or inhalation solutions may also be formulated with a propellant for aerosol delivery, such as by nebulization . Pulmonary administration is further described in PCT / US94 / 001875, which describes pulmonary delivery of chemically modified proteins .

[0058] Pharmaceutical compositions may be delivered through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art . A composition or polynucleotide of the disclosure that is administered in this fashion may be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules . A capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents may be included to facilitate absorption of a peptide disclosed herein. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed.

[0059] Pharmaceutical compositions may also contain adjuvants such as preservative, wetting agents, emulsifying agents and dispersing agents . Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid and the like . It may also be desirable to include isotonic agents such as sugars, sodium chloride and the like .UIC0117WO PATENT Prolonged absorption of the inj ectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

[0060] The invention further provides a means for delivering polynucleotides encoding a composition comprising at least one troponin T peptide alone or fused to at least one cardiactargeting peptide and / or at least one cell-penetrating peptide . Such delivery means may include vector (s) harboring polynucleotide ( s ) encoding the composition. Vectors may include a DNA plasmid, a bacterial artificial chromosome (BAG) , a yeast artificial chromosome (YAC) , a viral vector, or a nucleic acid complexed with a delivery vehicle such as a nanoparticle, liposome or poloxamer . In some aspects, the vector may be a plasmid or a viral vector such as an adeno-associated viral (AW) or lentiviral vector .

[0061] In some aspects, a vector described herein comprises a cardiac-specific promoter operatively linked to a polynucleotide that encodes a composition comprising at least one troponin T peptide alone or fused to at least one cardiactargeting peptide and / or at least one cell-penetrating peptide, wherein expression of the composition treats a subj ect in need thereof ( e. g. , a subj ect having or at risk of having heart failure) . For example, in some aspects, the vector is an AAV-based vector comprising a cardiac TnT promoter operatively linked to a polynucleotide that encodes a composition comprising at least one troponin T peptide alone or fused to at least one cardiac-targeting peptide and / or at least one cell-penetrating peptide for the treatment of heart failure . An AW vector may be from the group consisting of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 or chimeric AAV derived thereof . In some aspects, an AAV expression vector is pseudotyped to enhance targeting. A pseudotyping strategy can promote gene transfer and sustain expression in a target cell type . For example, the AAV2 genome can be packaged into the capsid ofUIC0117WO PATENT another AAV serotype such as AAV5, AAV7, or AAV8, producing pseudotyped vectors such as AAV2 / 5, AAV2 / 7, and AAV2 / 8 respectively, as described in Balaj i et al . ( (2013) J. Surg. Res. 184 ( 1 ) : 691-698 ) . In some aspects, an AAV9 vector may be used and engineered as described, e. g. , in Asokari et al . ( (2013) Hum. Gene Then. 24 ( 11 ) : 906-913 ; Pozsgai et al . (2017 ) Mol . Then. 25 ( 4 ) : 855-869 ) ; Kotterman & Schaffer ( (2014 ) Nature Rev. Genet . 15 : 445-451 ) ; and US 2016 / 0340393 Al . In some aspects, the viral vector is AAV engineered to increase target cell infectivity as described in US 2018 / 0066285 Al or a naturally evolved variant that has cardiospecificity or cardiotropism as described in Zeissler et al . (2025) Circulation 152 ( 6) : 416-419 .

[0062] In some aspects, the disclosure provides a vector comprising one or more regulatory elements operatively linked to a polynucleotide encoding a composition comprising at least one troponin T peptide alone or fused to at least one cardiactargeting peptide and / or at least one cell-penetrating peptide . In some aspects, the regulatory element is a cardiac-specific promoter that is operatively linked to a polynucleotide encoding the composition for the treatment of heart disease . As used herein, the term "regulatory element" refers those nontranslated regions of the vector (e. g. , origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno sequence or Kozak sequence) introns, a polyadenylation sequence, 5 ' and 3 ' untranslated regions) the interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. The transcriptional regulatory element may be functional in either a eukaryotic cell ( e. g. , a mammalian cell) or a prokaryotic cell ( e. g. , bacterial or archaeal cell) . In some aspects, a polynucleotide sequence encoding the composition described herein is operably linkedUIC0117WO PATENT to multiple control elements that allow expression of the polynucleotide in both prokaryotic and eukaryotic cells .

[0063] In some aspects, expression of a composition may be controlled by a muscle-specific promoter . In some aspects, the promoter is a cardiac cell-specific promoter . In some aspects, the promoter is a cardiomyocyte-specific promoter . A "cardiomyocyte-specific promoter, " as used herein, specifies a promoter whose activity in cardiomyocytes is at least 2-fold higher than in any other non-cardiac cell type or cardiac cell which is not a cardiomyocyte . In some aspects, a cardiomyocytespecific promoter suitable for being used in a vector of the present disclosure has an activity in cardiomyocytes that is at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 50-fold higher compared to its activity in a non-cardiac cell type or a cardiac cell type which is not a cardiomyocyte . In some aspects, the cardiacspecific or cardiomyocyte-specific promoter is a human promoter . Examples of cardiac-specific or cardiomyocytespecific promoter include, but are not limited to, the alpha myosin heavy chain promoter, the myosin light chain 2v promoter, the alpha-cardiac actin promoter, the alphatropomyosin promoter, the cardiac troponin C promoter, the cardiac troponin I promoter, the cardiac troponin T promoter, the cardiac myosin-binding protein C promoter, and the sarco / endoplasmic reticulum Ca2+ATPase (SERCA) promoter ( e. g. , isoform 2 of SERCA2 ) . In some aspects, the cardiac-specific promoter is the cardiac TnT promoter . Exemplary TnT promoters of use in a vector of the disclosure may have the nucleotide sequence :cagctggctggcttgtgtcagcccctcgggcactcacgtatctccgtccgacgggtt taaaatagcaaaactctgaggccacacaatagcttgggcttatatgggctcctgtggggga agggggagcacggagggggccggggccgctgctgccaaaatagcagctcacaagtgttgca ttcctctctgggcgccgggcacattcctgctggctctgcccgccccggggtgggcgccgggUIC0117WO PATENT gggaccttaaagcctctgccccccaaggagcccttcccagacagccgccggcacccaccgc tccgtgggacgatccccgaagc (SEQ ID NO: 78 ) or caggttttgctattttaaacccgtcggacggagatacgtgagtgcccgaggggctga cacaagccagccagtccgctgctgccaaaatagcagctcacaagtgttgcattcctctctg ggcgccgggcacattcctgctggctctgcccgccccggggtgggcgccggggggaccttaa agcctctgccccccaaggagcccttcccagacagccgccggcacccaccgctccgtgggac gatccccgaagc (SEQ ID NO: 79) .

[0064] In some aspects, a vector of the disclosure further includes an enhancer . The term "enhancer" refers to a segment of DNA that contains sequences capable of providing enhanced transcription and in some instances can function independent of their orientation relative to another control sequence . An enhancer may function cooperatively or additively with promoters and / or other enhancer elements . An enhancer may overlap with a promoter or be upstream or downstream of the promoter . In some aspects, a vector of the invention comprises a cardiac TnT promoter and one or more enhancers . An exemplary enhancer may have the nucleotide sequence :agtagaaaaacagccaagctagggaggctgggaggccaagccccagataccttacat agctctgctcagcctctgtctcattaggaactccatttttaggatgcagttgtttcaggct aaaaataaatcatgcaatgaataaaaaagttagatacgacactgtagagggattcgctgat acagtctgtccga (SEQ ID NO: 80) .

[0065] The following non-limiting examples are provided to further illustrate the present invention.Example 1 : Materials and Methods

[0066] cTnT-C14 Peptides . Wild-type (WT) human cTnT-C14 peptide and derivatives containing HCM mutations R278C, R286C or K280N were commercially synthesized by Peptide 2.0 Inc (Chantilly, VA) with mass spectrometry certification for the anticipated molecular mass and purities >95% in HPLC analysis . High concentration stocks of the peptides were made by dissolving them in a buffer containing 0.1 M KC1, 3 mN MgC12,UIC0117WO PATENT 10 mN imidazole-HCl , pH 7.0 and stored at -20°C until use in experiments . A non-tropomyosin-binding and non-actin-binding peptide of the same length was synthesized for use as control .

[0067] SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) . To assess the purity of proteins prepared for the present study, SDS-PAGE was carried out as described previously (Feng & Jin (2020) J. Exp. Physiol . 28 : 10 . 1113 / EP088521 ; Cao et al . (2023) bioRxiv 2023.02.20.529301 ) . The resolving gel was 14% with an acrylamide / bis-acrylamide ratio of 29 : 1. The protein samples were prepared in an SDS-gel sample buffer containing 2% SDS and 1% p-mercaptoethanol . The samples were heated at 80°C for 5 minutes and clarified by centrifugation in a microcentrifuge at top speed (20, 000 x g) for 5 minutes before loading to the gel . Electrophoresis was run at a constant current of 22 mA per 0.75 mm thick BioRad mini-gel . The gel was then stained with Coomassie Brilliant Blue R-250, distained, and scanned for image documentation.

[0068] Preparation of Tropomyosin and F-actin. Rabbit cardiac and back muscle acetone powders were prepared from frozen tissue stocks . After being minced with a precooled stainless-steel grinder and washed in cold water with stirring for 2-3 minutes and standing at 4 °C for 20 minutes, the solubilized material was removed by squeezing out through two layers of cheesecloth. The tissue residue was sequentially washed at 4 °C with stirring followed by filtered through cheesecloth with 50% ethanol three times, 95% ethanol twice, and 100% acetone twice . All equipment and reagents were precooled at 4 °C . The resulting grayish-white acetone powder was spread thinly over filter paper in a fume hood and air-dried at room temperature . The final dried powder was stored at -20°C in aliquots until use .

[0069] As previously described (Smillie ( 1982 ) Methods Enzymol . 85 Pt B : 234-41 ) , a-tropomyosin was purified from the cardiac muscle acetone powder by extraction with an extraction buffer containing 1.0 M KC1 and 0.5 mM dithiothreitol (DTT) ,UIC0117WO PATENT 10 mM imidazole-HCl, pH 7.0 for 16 hours at room temperature with gentle stirring. The solubilized material was collected by squeezing out through two layers of cheesecloth, and the residue was re-extracted with the same buffer for 2 hours . The extracts were combined, adjusted to pH 4. 6 using HC1, and stirred at 4 °C for 30 minutes . The precipitate was collected by centrifugation at 6, OOOxg at 4 °C for 20 minutes and dissolved in the extraction buffer with stirring for 20 minutes . The insoluble materials were removed by centrifugation at 6, OOOxg at 4 °C for 10 minutes . The isoelectric precipitation at pH 4. 6 of tropomyosin and dissolution at pH 7.0 were repeated two more times . The final precipitate was dissolved in 0.5 mM DTT, 10 mM imidazole-HCl, pH 7.0. and precipitated by slowly adding solid ammonium sulfate to 53% saturation while maintaining pH at 7.0 at 0°C with stirring. After standing for 30 minutes, the precipitate was removed by centrifugation at 11, 000 x g at 4 °C for 30 minutes . More solid ammonium sulfate was added to the supernatant to 65% saturation at 0°C while keeping the pH at 7.0 with NaOH, and the precipitate was collected by centrifugation at 11, OOOxg at 4 °C for 30 minutes . The purified tropomyosin in the final precipitate was redissolved in deionized water containing 0.5 mM DTT and dialyzed against cold water containing 2 mM p-mercaptoethanol for three changes and lyophilized.

[0070] As described previously (Jin ( 1995) J. Biol . Chem.270 : 6908-6916) , muscle actin was extracted from rabbit back muscle acetone powder with a pre-cooled buffer containing 2 mM Tris-HCl, pH 8.0, 2 mM CaCl2, 0.2 mM NaATP and 0.5 mM DTT by stirring gently in an ice bath for 30 minutes . The mixture was then filtered and centrifuged in a Beckman Coulter Optima XPN-100 Ultracentrifuge at 10, 250 rpm in a TYPE 50.2 Ti rotor at 4 °C for 25 minutes to remove residual materials . KC1 and MgCl2were then gradually added to the supernatant to finalUIC0117WO PATENT concentrations of 50 mM and 2 mM, respectively, to initiate actin polymerization. This solution was gently stirred at room temperature for 1 hour . More KC1 was added to reach a final concentration of 0.9 M followed by stirring at room temperature for 2 hours to separate tropomyosin from F-actin. The mixture was then ultracentrifuged as above at 45, 000 rpm at 4 °C for 3 hours to pellet the polymerized F-actin. The F-actin pellet was completely dissolved in a minimal volume of the buffer containing 2 mM Tris-HCl, pH 8.0, 2 mM CaCl2, 0.2 mM NaATP and 0.5 mM DTT and to ensure a uniform suspension to depolymerize and dialyzed against the same buffer at 4 °C with twice changes . The solution of depolymerized actin was ultracentrifuged as above at 45, 000 rpm at 4 °C for 2 hours to remove any insoluble materials . The clarified supernatant containing purified G- actin was repolymerized by adding KC1 and MgCl2to reach final concentrations of 50 mM and 2 mM, respectively. The final product of F-actin was pelleted by ultracentrifuged as above at 45, 000 rpm at 4 °C for 3 hours, resuspended in a small volume of the polymerization buffer and stored at 4 °C for use within 7-10 days . The protein concentration of tropomyosin and F-actin stocks was determined with UV absorbance at 280 nm.

[0071] Preparation of Recombinant AAV9 Viruses . Recombinant AAV9 viruses are constructed to express a C-terminal TnT peptide, or functional fragment, multimer, or modified peptide thereof in cardiomyocytes . The AAV shuttle vector design is shown in FIG. 6. An enhanced cardiac troponin T promoter (SEQ ID NO: 78 or SEQ ID NO: 79) using a human calsequestrin cis-enhancer (SEQ ID NO: 80) Chamberlain et al . (2018 ) Hum. Gene Ther. 29 ( 8 ) : 927-937 ) is used to drive high level expression of a C-terminal TnT peptide, or functional fragment, multimer, or modified peptide thereof in cardiac muscle . Aiming for a higher level expression, two versions of the cardiac troponin T promotor containing a 5' -segment in its forward or reverseUIC0117WO PATENT orientation ( lannello et al , ( 1991 ) J. Biol . Chem. 266 (5) : 3309-16) have been constructed. The shuttle vectors are used to package AAV9 viruses and produce high titer viral stocks for therapeutic delivery of C-terminal TnT peptide, or functional fragment, multimer, or modified peptide thereof .

[0072] Localized Surface Plasmon Resonance (LSPR) Assay. LSPR spectroscopy was carried out using a Nicoya Lifesciences LSPR system to study the interaction between cTnT-C14 peptides and tropomyosin or tropomyosin-actin composite thin filaments . The Nicoya High Sensitivity Carboxyl LSPR sensor chip was precoated with carboxyl-modif led gold nanoparticles . The LSPR system was first flushed with 80% isopropanol at the maximum flow rate of 150 pL / min to remove any air bubbles . To activate the carboxyl groups on the gold nanoparticle surface before the coating of tropomyosin or F-actin, l-ethyl-3- ( 3-dimethylaminopropyl ) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were dissolved separately in deionized water both at the concentration of 0.1 M. The EDC / NHS activation solution stocks were made fresh by 1 : 1 mixing of the stocks immediately prior to use to ensure optimal activation efficiency. The EDC / NHS activation solution was inj ected at a flow rate of 20 pL / min for 4 minutes . The protein to be studied (tropomyosin or F-actin) was immobilized on the chip surface by flowing the protein solution at 20 pL / min for 4 minutes for dehydration condensation of carboxyl groups on the chip with the amino groups on the protein.

[0073] For cTnT-C14 peptide-tropomyosin binding assays, purified tropomyosin was dissolved at 250 pg / mL in 10 mM sodium acetate, pH 4.0. The pH was at least 0.5 units below the isoelectric point of tropomyosin (4. 6) to ensure its positive overall charge . After tropomyosin immobilization and equilibration in LSPR assay buffer consisting of 0.1 M KC1, 3 mM MgC12 and 10 mM imidazole-HCl , pH 7.0, I M ethanolamine was inj ected at 20 pL / min for 4 minutes to block the unreactedUIC0117WO PATENT carboxyl groups on the gold surface of the chip . After reequilibration with LSPR assay buffer, 1 mM 6-mercapto-l-hexanol (MCH) in 30% ethanol was inj ected at 20 pL / min for 12 min to block exposed gold nanoparticles and prevent nonspecific interaction with free cysteine on protein samples . cTnT-C14 or control peptide diluted at 10 pM in the LSPR assay buffer was flowed over the chip at a rate of 20 pL / min for 4 minutes, allowing for binding (association) to occur . The flow was then switched to the LSPR assay buffer at the same rate to elute the bound cTnT-C14 peptide and the course of dissociation was measured until the signal curve back to baseline . When needed, the LSPR buffer flow rate was increased to remove residual peptide in the inj ection loop . For peptides with a strong binding affinity, a regeneration with 1 M KC1 and 3 mM MgC12 at high flow rate was employed to return the signal to baseline level .

[0074] For cTnT-C14-tropomyosin-F-actin binding assays, F-actin was coated to the chip same as for tropomyosin except at the protein concentration 30 pg / mL . To study the binding of cTnT-C14 peptides to tropomyosin-F-actin composite thin filament, tropomyosin was diluted in the LSPR buffer at 10 pM and added to the F-actin-coated sensor chip by flowing in at the rate of 20 pL / min for 4 minutes, allowing tropomyosin to bind the immobilized F-actin at saturation. After a 2-minute brief washing with the LSPR buffer to remove free tropomyosin, cTnT-C14 peptide was inj ected at 10 pM in the flow rate of 20 pL / min for 4 minutes to test the association to the immobilized tropomyosin-F-actin complex . The flow was then switched to LSPR buffer and the dissociation of cTnT-C14 peptide and likely some tropomyosin from the immobilized F-actin was measured. To achieve complete regeneration of the F-actin coated chip, a high-speed flow of the regeneration buffer containing 1 M KC1 and 3 mM MgC12 was applied, which was essential to remove anyUIC0117WO PATENT residual tropomyosin and TnT-C14 peptides to recondition the system for repeating analysis . The direct binding of cTnT-C14 peptides to chip-immobilized F-actin was measured similarly to the cTnT-C14-tropomyosin binding assay.

[0075] Two of the cTnT-C14 mutations, R278C and R286C, contain a cysteine residue, which can form dimers via disulfide bonding. To prevent dimerization that could affect the binding properties for tropomyosin or F-actin, 1 mM tris (2-carboxyethyl ) phosphine (TCEP) was added in the R278C and R286C stock solution at least 1 hour before testing to reduce the disulfide bonds . After being diluted to 10 pM peptides in the LSPR working solution, the final TCEP concentration was 100 pM, which did not generate any background signal while ensuring the disulfide bonds were fully reduced.

[0076] Measurements of Ca2+-activated Force of Skinned Cardiac Muscle Strips. Permeabilized left ventricular papillary muscles from wild-type (WT) C57B / L6 mice or transgenic mice expressing N-terminal truncated cardiac TnT (cTnT-ND) (Feng et al . (2008 ) J. Physiol . (London) 586 : 3537-3550) at 3-4 months of age were prepared using cryosection for contractility studies ( Feng & Jin (2020) J. Exp. Physiol . 28 : 10 . 1113 / EP088521 ) . 120-150 pm wide and 35 pm thick cardiac muscle strips were mounted between two aluminum T-clips and transferred to a chamber of a thermocontrolled stage ( 802D, Aurora Scientific) at 6-8 °C in a relaxation buffer (BES 40 mM, EGTA 10 mM, MgC12 6.86 mM, ATP 5.96 mM, DTT 1 mM, creatine phosphate 33 mM, creatine kinase 200 U / mL, K-propionate 3.28 mM, pH 7.0, plus protease inhibitor cocktail) . The muscle preparation was connected to a force transducer (403A, Aurora Scientific) and a length controller (322C, Aurora Scientific) . The buffer was then switched to a skinning solution (relaxation buffer containing 1% Triton™ X-100) for 20 minutes . After a wash with relaxation buffer, the permeabilized muscle strip was placed in pCa 9.0 buffer and theUIC0117WO PATENT sarcomere length was measured through a digital camera attached to the microscope and adjusted to 2 . 0 pm and / or 2.3 pm. Calcium activated force was examined at pCa 6.5, 6.3, 6.0, 5.8, 5.5, 5.0, and 4.5 at 15°C . WT or HCM mutant HcTnT-C14 peptides or the control peptide was then added at 20 pM and the force-pCa measurements repeated.

[0077] Contractility Measurements of Isolated Adult Mouse Cardiomyocytes . Cardiomyocytes were enzymatically isolated from the hearts of 2 to 5 months old wild-type and a-tropomyosin E180G HCM mutation transgenic mice (Wei & Jin (2015) Am. J. Physiol . Cell Physiol . 308 : C397-C404 ) . The isolated cardiomyocytes were incubated with various concentrations of CTP-TAT-cTnT-C14 fusion peptide at room temperature for 30 minutes . After the peptide treatment, the cardiomyocytes were loaded into a perfusion chamber of 0.25 mL volume mounted on the stage of an inverted microscope using a heating adapter with feedback temperature control and superfused at 1 mL / min flow rate with oxygenated buffer containing 132 mM NaCl, 4.8 mM KC1, 1.2 mM MgC12, 10 mM HEPES, 15 mM glucose, 2 mM sodium pyruvate, 1.8 mM CaCl2, pH 7.4, at 36.5-37 °C . Contractions were induced by field electrical pacing with a Myopacer stimulator ( lonOptix, Milton, MA) using 10 volts 4 ms pulses at 1, 2 and 5 Hz . Cell shortening and re-lengthening were recorded using a CCD video camera ( lonOptix) . Sarcomere length detection data were acquired and analyzed using SoftEdge computer program ( lonOptix) and the Origin 8 software .

[0078] Data Analysis and Statistics . Statistical analysis of LSPR results were analyzed for Ka, Kd, and KD and compared using Student' s t test . All values are presented as mean ± SD. The force-pCa curves were plotted and fitted using a Hill exponential equation (y = START + (END-START) *xAn / (kAn+xAn) ) for data analysis and statistical analysis was performed using paired Student' s t-test .UIC0117WO PATENTExample 2 . Binding of cTnT-C14 Peptides to Tropomyosin

[0079] The functions of WT and three HCM mutant cTnT-C14 peptides were investigated. The amino acid sequences and physical properties of the cTnT-C14 peptides are shown in Table 1 .TABLE 1&

[0080] The amino acid sequence alignment of the cTnT-C14 segment in representative mammalian species shows a striking feature that this segment is 100% conserved in mammalian hearts, indicating functional importance . The C-terminal end segments of human fast and slow skeletal muscle TnT are aligned as an example to show isoform variations . The three cTnT-C14 peptides containing hypertrophic cardiomyopathy (HCM) mutations R278C, K280N or R286C investigated herein are also shown .UIC0117WO PATENT

[0081] Representative LSPR traces (FIG. 1 ) showed that WT HcTnT-C14 peptide at 10 M concentration binds immobilized tropomyosin with rapid association to reach saturation. Once saturation was reached and the flow was switched to LSPR buffer washing, the dissociation of WT HcTnT-C14 peptide occurred rapidly. The physiologically relevant binding affinity and kinetics demonstrate that the C-terminal end segment corresponding to the third tropomyosin-binding site of TnT functions in isolation in the form of free peptide (Table 2 ) . The high binding affinity indicates a potent physiological role, and the fast association and dissociation kinetics indicate a dynamic binding mechanism where transitions can occur readily between bound and unbound states .TABLE 2*p < 0.05; **p < 0.01 vs WT in Student' s t test .

[0082] As shown in Table 2, binding kinetic analysis depicted the association (Kon) and dissociation (KOff) rates and affinity (KD) of WT and three HCM mutant cTnT-C14 peptides with tropomyosin. In comparison with HcTnT-C14 WT, HcTnT-C14 K278C, HcTnT-C14 K280N and HcTnT-C14 K286C exhibit significantly weaker binding affinity.

[0083] The LSPR analysis and results in FIG. 1 and Table 2 further show that HcTnT-C14 peptides containing one of the three HCM mutations (R278C, K280N or R286C) have significantly decreased binding to tropomyosin . Compared to WT HcTnT-C14 under the same concentration and experimental conditions andUIC0117WO PATENT time parameters, the maximum tropomyosin-binding signal of the mutant peptides were significantly lower .Example 3 : Binding of cTnT-C14 Peptides to Tropomyosin-F-actin Composite Thin Filaments

[0084] The LSPR results in Table 3 and FIG. 2 show that WT HcTnT-C14 peptide also binds to tropomyosin-F-actin composite filaments with high affinity, similar to its binding to tropomyosin alone with saturable binding and fast association and dissociation rates . Also similar to the results of tropomyosin-binding assays, HCM mutants HcTnT-C14 R278C, HcTnT- C14 K280N and HcTnT-C14 R286C exhibited significantly weakened binding to tropomyosin-F-actin composite filaments .TABLE 3*p < 0.05 vs WT in Student' s t test .

[0085] The results further demonstrate the physiological relevance of the binding of cTnT-C14 peptide to tropomyosin. The similar patterns of WT and HCM mutant HcTnT-C14 peptides interactions with the assembly of tropomyosin and F-actin filaments to that with tropomyosin alone indicate that the binding to tropomyosin is the primary functional mechanism that is preserved for the tropomyosin-F-actin composite thin filament . The weakened binding caused by the HCM mutations demonstrate the pathogenic and pathophysiological mechanisms through disrupting the interaction of the tropomyosin-binding site 3 with tropomyosin-F-actin thin filament . The similar changes caused by the HCM mutations in the binding affinity forUIC0117WO PATENT tropomyosin and tropomyosin-F-actin thin filament also support that the functional changes impair the interaction of cTnT-C14 with tropomyosin to result in an abnormal regulation of cardiac muscle contraction and relaxation.Example 4 : cTnT-C14 Peptide Directly Binds F-actin

[0086] The C-terminal domain of TnT has previously been shown to exhibit F-actin binding activity (Pearlstone & Smillie ( 1985) Can. J. Biochem. Cell Biol . 63 (3) : 212-8 ; Schaertl et al . ( 1995) Biochemistry 34 : 15890-15894 ) . The HcTnT-C14 peptides bind tropomyosin-F-actin composite thin filament similar to their binding patterns for tropomyosin alone (FIGS . 1-2 ) indicating a primary interaction with tropomyosin. Therefore, their direct binding to F-actin was investigated in LSPR under the same conditions . The results in FIG. 3 and Table 4 show that WT TnT-C14 peptide binds to F-actin with a physiologically relevant affinity. The HCM mutation also decreased the binding of HcTnT-C14 peptides to F-actin, similar to that with tropomyosin or tropomyosin-F-actin thin filaments .TABLE 4*p < 0.05; **p < 0.01 vs WT in Student' s t test .

[0087] Binding kinetic analysis depicted the association (Kon) and dissociation (KOff) rates and affinity (KD) of WT and three HCM mutant cTnT-C14 peptides with F-action.

[0088] The results demonstrate that cTnT-C14 peptide is involved in the direct binding of TnT to F-actin. The similar patterns of HcTnT-C14 peptides' binding to tropomyosin-F-actinUIC0117WO PATENT thin filaments and tropomyosin alone support the functionality of the C-terminal end segment of TnT in regulating muscle contractility via its tropomyosin-binding activity.Example 5 : Measurement of cTnT-C14 Peptide on the Contractility of Skinned Cardiac Muscle

[0089] Force-pCa studies showed that the addition of HcTnT-C14 WT peptide to skinned sections of WT mouse left ventricular papillary muscle decreased Ca2+-sensitivity (FIG. 4A) . At sarcomere length 2.0 m, the treatment of 20 pM HcTnT-C14 had a notable Ca2+-desensitization effect on cardiac muscle strips . At sarcomere length 2.3 pm, HcTnT-C14 had higher Ca2+-desensitization effects cardiac muscle . (Table 5) . HcTnT-C14 did not decrease maximum force development of cardiac muscle .TABLE 5* p<0.05, **p<0.01, vs control in Student' s t test , n, Hill coefficient .

[0090] When adding the HcTnT-C14 WT peptide to skinned sections of cTnT-ND mouse left ventricular papillary muscle in Force-pCa measurements, no significant change was detected (FIG. 4B and Table 6) . This result indicates that the same mechanism has been utilized by the conformational configuration by restrictive truncation of the N-terminal variable region (Feng et al . (2008 ) J. Physiol . (London) 586 : 3537-3550) that restored a Tnl-like inhibitory function corresponding to an epitope structure in the C-terminal end segment of cardiac TnT (Cao et al . (2025) Proc Natl Acad Sci, 122 (27 ) : e2507107122 ) which is detected by an anti-Tnl monoclonal antibody (Chong & Jin (2009) J. Mol . Evol . 68 : 448-60) . Subsequently, the exogenous cTnT-C14 peptide did not produce an additive effect .UIC0117WO PATENT TABLE 6*p<0.05 vs SL 2.0 pm in Student' s t test , n, Hill coefficient .

[0091] Consistent with their diminished tropomyosin and thin filament binding capacities, addition of the three HCM mutant HcTnT-C14 peptides (R278C, K280N or R286C) to skinned sections of WT mouse left ventricular papillary muscle for contractility analysis did not produce notable a functional effect (FIGS . 5A-5C and Table 7 ) . The results demonstrate the loss of function as the underlying mechanism for these cardiac TnT point mutations' pathological impacts .TABLE 7n, Hill coefficient .The loss of tropomyosin- and F-actin-binding functions of the three HcTnT-C14 peptides containing HCM-associated mutations (FIGS . 1-3) confirms the functional importance of this regulatory structure . The loss of binding affinity of the HCM mutant HcTnT-C14 peptides for tropomyosin-F-actin thin filament abolished their Ca2+-desensitization effect in contractility studies (FIGS . 5A-5C) , which likely impairs the coordinated transition between muscle contraction and relaxation to impair relaxation and cause hypercontractile phenotypes . The LSPR kinetic studies show that the HCM mutations in the C-terminalUIC0117WO PATENT end segment weaken the association to tropomyosin and tropomyosin-F-actin filament (FIGS . 1, 2, and 5A-5B) . The results herein demonstrate the use of the TnT-C14 peptide in therapeutic applications for the treatment of hypercontractile cardiomyopathies and diastolic heart failure .Example 6 : Additional Peptides for Modulating Contractile Function

[0092] Screened peptides . Additional peptides were generated (Table 8 ) and tested for use in modulating contractile function .TABLE 8"p, " phosphorylated residue .

[0093] Design of Modified Human Slow TnT-C14 . Turtle muscles are rich in slow-tonic fibers with unique contraction properties including prolonged contractions, stable contractions, precisely controlled contractions and fatigue resistance . The C14 segment of turtle slow TnTs has two unique residues that may contribute to the functional features by increasing the function in elongating the duration of peak force .UIC0117WO PATENT

[0094] Creation of a peptide of TnT-C14 repeats. It was observed that in the absence of reducing agent, both human Cardiac TnT-C14-R278C and R286C mutants, but not the K280N mutant, showed significantly higher than WT binding to tropomyosin. Such effect was abolished in the presence of reducing agents, indicating an underlying mechanism of disulfide bond dimerization. This mechanism has been demonstrated by the novel design of a peptide with multiple C14 repeats .

[0095] Additional TnT sequences of use in the compositions and methods herein (Table 9) .TABLE 9

[0096] Heart homing and membrane penetrating C14 fusion peptides . Peptides of the disclosure are fused to heart targeting (CTP, APWHLSSQYSRT ; SEQ ID NO: 13) ) and cellpenetrating (TAT, GRKKRRQRRRPPQ; SEQ ID NO: 14 ) peptides, CTPUIC0117WO PATENT and TAT, operably linked to the N-terminus of a peptide of the disclosure (Table 10) . Nucleic acids encoding a peptide of the disclosure ( e. g. , as described in Tables 1, 8, 9 and / or 10) may be inserted in a bacterial, eukaryotic or viral (e . g. , AAV-9) expression vector by standard methods and used to transduce cardiac cells in the treatment of hypercontractile cardiomyopathies and diastolic heart failure . AAV-9 expression vector by standard methods and used to transduce cardiac cells in the treatment of hypercontractile cardiomyopathies and diastolic heart failure . See, e. g. , FIG. 6.TABLE 10Example 7 : Effective Delivery of cTnT-C14 into Cardiomyocytes to Therapeutically Modulate Contractility

[0097] Treatment of isolated wild-type mouse cardiomyocytes with various concentrations of cardiac targeting and membrane penetrating cTnT-C14 fusion peptide produced significant functional effects on decreasing the contractile amplitude in a dose-dependent manner which plateau at 0.5 pM (FIG. 7A, ***P<0.001 in Student' s test) . The results demonstrate that cTnT-C14 peptide has a potent inhibitory effect on contractility through its troponin I-like inhibitory function. The plateaued reduction in contraction amplitude indicates a self-restrictive modulation plausible for use as a therapeutic reagent .UIC0117WO PATENT

[0098] Cardiomyocytes isolated from Tm-E180G transgenic mice exhibited a significantly greater sarcomere shortening amplitude compared with wild-type control, demonstrating a hypercontractile phenotype consistent with hypertrophic cardiomyopathy phenotype . The treatment of Tm-E180G cardiomyocytes with 0.5 pM CTP-TAT-cTnT-C14 fusion peptide markedly reduced the contraction amplitude, effectively correcting the hypercontractility (FIG. 7B, ***P<0.001 in Student t test) . The results demonstrate that cTnT-C14 peptide can selectively attenuate abnormal hypercontractility caused by the Tm-E180G HCM mutation, supporting its potential use as a thin-f ilament kinetics-targeted modulator to treat HCM and other myocardial hypercontractility-caused heart failure, such as diastolic heart failure .

Claims

UIC0117WO PATENT WHAT IS CLAIMED IS :1 . A composition comprising at least one troponin T peptide fused to at least one cardiac-targeting peptide and / or at least one cell-penetrating peptide , wherein the troponin T peptide has the amino acid sequence of any one of SK RGKAKVTGRWK ( SEQ ID NO : 1 ) , SKTCGKAKVTGRWK ( SEQ ID NO : 2 ) , SKTRGNAKVTGRWK ( SEQ ID NO : 3 ) , SKTRGKAKVTGCWK ( SEQ ID NO : 4 ) , SKKAGTPAKGKVGGRWK ( SEQ ID NO : 5 ) , RKGAGKGRVGGRWK ( SEQ ID NO : 6 ) , RKGAPKGRLGGRWK ( SEQ ID NO : 9 ) , KKGAPKARLGGRWK ( SEQ ID NO : 11 ) , or KKGAPKGRLGGRWK ( SEQ ID NO : 12 ) , or a functional fragment , modi fied peptide , and / or multimer thereof .2 . The composition of claim 1 , wherein the functional fragment of troponin T peptide is at least about 12 amino acid residues in length .3 . The composition of claim 1 , wherein the functional fragment of the troponin T peptide has the amino acid sequence of any one of SKTRGKAKVTGR ( SEQ ID NO : 7 ) , SKTCGKAKVTGR ( SEQ ID NO : 19 ) , SKTRGNAKVTGR ( SEQ ID NQ : 20 ) , SKTRGKAKVTGC ( SEQ ID NO : 21 ) , SKKAGTPAKGKVGGR ( SEQ ID NO : 22 ) , RKGAGKGRVGGR ( SEQ ID NO : 23 ) , RKGAPKGRLGGR ( SEQ ID NO : 24 ) , KKGAPKARLGGR ( SEQ ID NO : 25 ) , or KKGAPKGRLGGR ( SEQ ID NO : 26 ) .4 . The composition of claim 1 , wherein the modi fied troponin T peptide is glycosylated, phosphorylated, sul fated, amidated, carboxylated, acetylated, PEGylated, palmitoylated and / or myristoylated .5 . The composition of claim 1 , wherein the multimer of the troponin T peptide is a dimer .UIC0117WO PATENT 6 . The composition of claim 1 , wherein each troponin T peptide of the multimer is the same or di f ferent .7 . The composition of claim 1 , wherein the multimer of the troponin T peptide has the amino acid sequence of any one of SKTRGKAKVTGRWKSKTRGKAKVTGRWK ( SEQ ID NO : 27 ) , SKTCGKAKVTGRWKSKTCGKAKVTGRWK ( SEQ ID NO : 28 ) , SKTRGNAKVTGRWKSKTRGNAKVTGRWK ( SEQ ID NO : 29 ) , SKTRGKAKVTGCWKSKTRGKAKVTGCWK ( SEQ ID NO : 30 ) , SKKAGTPAKGKVGGRWKSKKAGTPAKGKVGGRWK ( SEQ ID NO : 31 ) , RKGAGKGRVGGRWKRKGAGKGRVGGRWK ( SEQ ID NO : 32 ) , RKGAPKGRLGGRWKRKGAPKGRLGGRWK ( SEQ ID NO : 10 ) ,SKTRGKAKV (pT ) GRWKSKTRGKAKV (pT ) GRWK ( SEQ ID NO : 33 ) , KKGAPKARLGGRWKKKGAPKARLGGRWK ( SEQ ID NO : 34 ) , SKTRGKAKVTGRSKTRGKAKVTGR ( SEQ ID NO : 35 ) , or KKGAPKGRLGGRWKKKGAPKGRLGGRWK ( SEQ ID NO : 36 ) .8 . The composition of claim 1 , wherein the at least one cardiac-targeting peptide has the amino acid sequence of any one of WLSEAGPWTVRALRGTGSW ( SEQ ID NO : 37 ) , CSTSMLKAC ( SEQ ID NO : 38 ) , APWHLSSQYSRT ( SEQ ID NO : 13 ) , or SQYSRT ( SEQ ID NO : 39 ) .9 . The composition of claim 1 , wherein the at least one cell-penetrating peptide has the amino acid sequence of any one of GRKKRRQRRRPPQ ( SEQ ID NO : 14 ) ; GWTLNSAGYLLGKINLKALAALAKKIL ( SEQ ID NO : 40 ) ; RQIKIWFQNRRMKWKK ( SEQ ID NO : 41 ) ; RRRRRRRQIKIWFQNRRMKWKK ( SEQ ID NO : 42 ) ; LLI ILRRRIRKQAHAHSK ( SEQ ID NO : 43 ) ; GALFLGFLGAAGSTMGAWSQPKKKRKV ( SEQ ID NO : 44 ) , KETWWETWWTEWSQPKKKRKV ( SEQ ID NO : 45 ) , KLALKLALKALKAALKLA ( SEQ ID NO : 46 ) ; or RRWWRRWRR ( SEQ ID NO : 47 ) .10 . The composition of claim 1 , wherein the composition has the amino acid sequence of any one of GRKKRRQRRRPPQSKTRGKAKVTGRWK ( SEQ ID NO : 48 ) ,UIC0117WO PATENT GRKKRRQRRRPPQSKTCGKAKVTGRWK ( SEQ ID NO : 49 ) , GRKKRRQRRRPPQSKTRGNAKVTGRWK ( SEQ ID NO : 50 ) , GRKKRRQRRRPPQSKTRGKAKVTGCWK ( SEQ ID NO : 51 ) , GRKKRRQRRRPPQSKTRGKAKVTGR ( SEQ ID NO : 52 ) , GRKKRRQRRRPPQSKTRGKAKV (pT ) GRWK ( SEQ ID NO : 53 ) , GRKKRRQRRRPPQRKGAGKGRVGGRWK ( SEQ ID NO : 54 ) , GRKKRRQRRRPPQRKGAPKGRLGGRWK ( SEQ ID NO : 55 ) , GRKKRRQRRRPPQRKGAPKGRLGGRWKRKGAPKGRLGGRWK ( SEQ ID NO : 56 ) , GRKKRRQRRRPPQKKGAPKARLGGRWK ( SEQ ID NO : 57 ) , GRKKRRQRRRPPQKKGAPKGRLGGRWK ( SEQ ID NO : 58 ) , APWHLSSQYSRTSKTRGKAKVTGRWK ( SEQ ID NO : 59 ) , APWHLSSQYSRTSKTCGKAKVTGRWK ( SEQ ID NO : 60 ) , APWHLSSQYSRTSKTRGNAKVTGRWK ( SEQ ID NO : 61 ) , APWHLSSQYSRTSKTRGKAKVTGCWK ( SEQ ID NO : 62 ) , APWHLSSQYSRTSKTRGKAKVTGR ( SEQ ID NO : 63 ) , APWHLSSQYSRTSKTRGKAKV (pT ) GRWK ( SEQ ID NO : 64 ) , APWHLSSQYSRTRKGAGKGRVGGRWK ( SEQ ID NO : 65 ) , APWHLSSQYSRTRKGAPKGRLGGRWK ( SEQ ID NO : 66 ) , APWHLSSQYSRTRKGAPKGRLGGRWKRKGAPKGRLGGRWK ( SEQ ID NO : 67 ) , APWHLSSQYSRTGRKKRRQRRRPPQKKGAPKARLGGRWK ( SEQ ID NO : 68 ) , APWHLSSQYSRTGRKKRRQRRRPPQKKGAPKGRLGGRWK ( SEQ ID NO : 69 ) , APWHLSSQYSRTGRKKRRQRRRPPQSKTRGKAKVTGRWK ( SEQ ID NO : 15 ) , APWHLSSQYSRTGRKKRRQRRRPPQSKTCGKAKVTGRWK ( SEQ ID NO : 70 ) , APWHLSSQYSRTGRKKRRQRRRPPQSKTRGNAKVTGRWK ( SEQ ID NO : 71 ) , APWHLSSQYSRTGRKKRRQRRRPPQSKTRGKAKVTGCWK ( SEQ ID NO : 72 ) , APWHLSSQYSRTGRKKRRQRRRPPQSKTRGKAKVTGR ( SEQ ID NO : 73 ) , APWHLSSQYSRTGRKKRRQRRRPPQSKTRGKAKV (pT ) GRWK ( SEQ ID NO : 74 ) ,APWHLSSQYSRTGRKKRRQRRRPPQRKGAGKGRVGGRWK ( SEQ ID NO : 16 ) , APWHLSSQYSRTGRKKRRQRRRPPQRKGAPKGRLGGRWK ( SEQ ID NO : 17 ) , APWHLSSQYSRTGRKKRRQRRRPPQRKGAPKGRLGGRWKRKGAPKGRLGGRWK( SEQ ID NO : 18 ) ,APWHLSSQYSRTGRKKRRQRRRPPQKKGAPKARLGGRWK ( SEQ ID NO : 75 ) orUIC0117WO PATENT APWHLSSQYSRTGRKKRRQRRRPPQKKGAPKGRLGGRWK (SEQ ID NO: 76) .

11. A liposome or nanoparticle encapsulating the composition of claim 1.

12. A polynucleotide encoding the composition of claim 1.

13. A vector comprising the polynucleotide of claim 12, optionally wherein the vector is an AAV vector .

14. A composition comprising the composition of claim 1 and a pharmaceutically acceptable carrier, diluent, or excipient .

15. A method of treating heart failure comprising administering to a subj ect in need of treatment an effective amount of the composition of claim 1 thereby treating the subj ect' s heart failure .

16. The method of claim 15, wherein the heart failure comprises diastolic heart failure .

17. A method of treating heart failure comprising administering to a subj ect in need of treatment an effective amount of a vector comprising a polynucleotide encoding the composition of claim 1 thereby treating the subj ect' s heart failure .

18. A composition comprising a troponin T peptide having the amino acid sequence of any one of SKTRGKAKVTGRWK (SEQ ID NO: 1 ) , SKTCGKAKVTGRWK (SEQ ID NO : 2 ) , SKTRGNAKVTGRWK (SEQ ID N0: 3) , SKTRGKAKVTGCWK (SEQ ID NO : 4 ) , SKKAGTPAKGKVGGRWK (SEQ ID NO: 5) , RKGAGKGRVGGRWK (SEQ ID NO: 6) , RKGAPKGRLGGRWK (SEQ ID NO: 9) , KKGAPKARLGGRWK (SEQ ID NO: 11 ) , or KKGAPKGRLGGRWK (SEQ IDUIC0117WO PATENT NO: 12 ) , or a functional fragment thereof, wherein the troponin T peptide or functional fragment is a modified peptide .

19. The composition of claim 18, wherein the modified troponin T peptide is glycosylated, phosphorylated, sulfated, amidated, carboxylated, acetylated, PEGylated, palmitoylated and / or myristoylated .

20. A composition comprising a multimeric troponin T peptide comprising a troponin T peptide of any one of SKTRGKAKVTGRWK (SEQ ID NO : 1 ) , SKTCGKAKVTGRWK (SEQ ID NO : 2 ) , SKTRGNAKVTGRWK (SEQ ID NO : 3 ) , SKTRGKAKVTGCWK (SEQ ID NO : 4 ) , SKKAGTPAKGKVGGRWK (SEQ ID NO: 5) , RKGAGKGRVGGRWK (SEQ ID NO : 6 ) , RKGAPKGRLGGRWK (SEQ ID NO: 9) , KKGAPKARLGGRWK (SEQ ID NO: 11 ) , or KKGAPKGRLGGRWK (SEQ ID NO: 12 ) , or a functional fragment thereof, optionally wherein the multimeric troponin T peptide is a dimer .

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