Methods for reversing heart failure using gene therapy - Patents.com

Abstract

Described herein are compositions comprising viral vectors. The viral vectors can encode T-tubule organizing proteins or peptides, such as cardiac isoform of bridging integrator 1 (cBIN1). Also disclosed herein are methods for treating or preventing heart failure in a subject in need thereof. The treatment or prevention methods can include administering to a subject a vector comprising cBIN1 to restore or increase contractile (systolic) function or relaxation (diastolic) function in the heart of a subject experiencing heart failure or with chronic myocardial stress.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 007,229, filed April 8, 2020, and No. 63 / 088,123, filed October 6, 2020, each of which is incorporated by reference in its entirety herein.

[0002] Federally Funded Research This invention was made with United States Government support under Grant Nos. HL133286, HL094414, and HL138577 from the National Institutes of Health. The United States Government has certain rights in the invention.

[0003] Technical Field Described herein are compositions comprising a viral vector. The viral vector may encode a t-tubule organizing protein or peptide, such as cardiac isoform of bridging integrator1 (cBIN1). Also disclosed herein are methods for treating or preventing heart failure in a subject in need thereof. The methods of treatment or prevention may include administering to a subject a vector comprising cBIN1 to restore or increase contractile (systolic) or relaxing (diastolic) function in the heart of a subject experiencing heart failure or having chronic myocardial stress. [Background technology]

[0004] Heart failure (HF) is the fastest growing cardiovascular disease, affecting more than 20 million people worldwide and 6.2 million in the United States [1-2]. The majority of HF-related mortality is associated with cardiac pump failure due to impaired inotropic and diastolic function of the myocardium, and sudden cardiac death due to increased arrhythmia burden from the failing heart. Furthermore, nearly 50% of patients with HF with preserved ejection fraction (HFpEF) [2] develop severe diastolic dysfunction with an even higher risk of arrhythmias, which is associated with poorer clinical outcomes and for which no effective pharmacological therapy exists. Thus, there is an urgent need to develop new therapeutic strategies that can limit and reverse the progression of HF.

[0005] The pathophysiological cellular hallmark of failing ventricular myocytes during HF development is abnormal calcium transients with impaired intracellular calcium homeostasis [3], which disrupts excitation-contraction (EC) coupling [4], impairs electrical stability [5], and disrupts mitochondrial metabolism [6]. Normal interbeat calcium transients depend on a series of intracellular events known as calcium-induced calcium release (CICR) [7], in which an initial calcium influx through t-tubule L-type calcium channels (LTCCs) subsequently induces massive calcium release from sarcoplasmic reticulum (SR) stores via ryanodine receptors (RyRs). During relaxation, the accumulated calcium is then released primarily into SR Ca2+, which is then released into SR Ca2+. +Calcium is removed from the cytoplasm by reabsorption into the SR via SR-ATPase (SERCA) and calcium export into the extracellular space [7]. In HF, abnormal remodeling of t-tubules [8-10] impairs LTCC-RyR coupling and synchronous CICR [3, 11], which reduces systolic release and leads to EC uncoupling and thus reduced contractility. Meanwhile, HF-associated leaky RyR

[12] and SERCA2a dysfunction

[13] leads to SR depletion and elevated diastolic calcium

[14] , which results in severe diastolic dysfunction and electrical instability

[15] . Furthermore, calcium homeostasis abnormalities cause loss of mitochondrial membrane potential

[16] and increased permeability

[17] , which promotes the risk of mitochondrial-driven cell death [18-19] and HF progression [18, 20]. In summary, calcium dyshomeostasis is important in controlling normal cardiac pump function, electrical stability and metabolism, and its disturbance can lead to pump failure, fatal arrhythmias and severe metabolic disorders.

[0006] Myocardial transverse tubules (T-tubules) are important for initiating calcium transients and maintaining efficient excitation-contraction (EC) coupling. Pathological T-tubule remodeling is a consequence of β-adrenergic stimulation in HF [21-23]. Furthermore, impairment of T-tubule microdomains has been implicated in HF progression [24-27]. Indeed, T-tubule remodeling may be a turning point from hypertrophy to failure

[10] . Normal calcium transients

[28] require the presence of L-type calcium channels (LTCCs) in T-tubule microdomains, which are crucial for cardiac contraction and relaxation. The T-tubule membrane scaffolding protein cardiac bridging integrator 1 (cBIN1)

[29] promotes LTCC trafficking

[30] and clustering for bimolecular organization and is also under the control of β-adrenergic receptor (β-AR) signaling

[31] . Furthermore, cBIN1 is reduced in HF [31-33], and consequently, disruption of cBIN1-microdomains impairs normal stress responses, limits contractile force, and promotes arrhythmias. Therapeutic approaches that preserve cBIN1-microdomains may benefit the heart under stress by protecting calcium handling machinery and slow the progression of HF.

[0007] Therefore, there remains a need to prevent remodeling within individual ventricular myocytes to improve global cardiac remodeling and have therapeutic benefit in the failing heart. Summary of the Invention

[0008] One embodiment described herein is a method for restoring cardiac tissue or ameliorating symptoms of cardiac failure in a subject experiencing cardiac failure or under chronic stress, comprising diagnosing cardiac failure or myocardial stress in a subject, and administering a transgene encoding Cardiac Bridging Integrator 1 (cBIN1) to cardiac tissue of the subject experiencing cardiac failure. In one aspect, diagnosing cardiac failure or myocardial stress comprises measuring reduced cBIN1 blood levels.

[0009] Other embodiments described herein are methods for restoring or increasing contractile (systolic) or relaxing (diastolic) function in the heart of a subject experiencing heart failure, comprising administering to the subject's cardiac tissue a transgene encoding Cardiac Bridging Integrator 1 (cBIN1), where after the transgene is delivered to and expressed in the cardiac tissue, the contractile function of the heart is restored or increased. In one aspect, the transgene is administered after the subject is diagnosed with heart failure. In another aspect, the diagnosis of heart failure comprises measuring reduced cBIN1 blood levels. In another aspect, the method comprises administering the transgene to the myocardium. In another aspect, the transgene is administered by injection. In another aspect, the transgene comprises a vector comprising a transgene encoding cBIN1. In another aspect, the transgene is administered in a dose of about 1×10 10 ~Approx. 5×10 10 In another aspect, the expression of cBIN1 remodels damaged myocardium. In another aspect, the expression of cBIN1 stabilizes the intracellular distribution of calcium handling machinery in myocardium. In another aspect, the expression of cBIN1 reduces concentric hypertrophy in myocardium. In another aspect, the expression of cBIN1 restores or increases t-tubule microfolds or microdomains in myocardium. In another aspect, the expression of cBIN1 restores or reduces hyperphosphorylation of ryanodine receptor 2 (RyR2) in myocardium. In another aspect, the expression of cBIN1 restores or improves cardiac contractility and lucitropy. In another aspect, the expression of cBIN1 restores or improves cardiac relaxation and diastolic function. In another aspect, the expression of cBIN1 is preventative against further damage to myocardium. In another aspect, the transgene is administered at least once. In another aspect, the subject is a mammal. In another aspect, the subject is a mouse or a dog. In another aspect, the subject is a human. In other embodiments, the subject experiences reduced ejection fraction (HFrEF).

[0010] Another embodiment described herein is the use of cBIN1 in medicine to restore myocardial tissue or repair myocardial damage in subjects experiencing heart failure or with chronic myocardial stress.

[0011] Another embodiment described herein is the use of cBIN1 in medicine to restore or increase contractile (systolic) or relaxing (diastolic) function in the heart of a subject experiencing heart failure or having chronic myocardial stress.

[0012] The patent or application file contains at least one color drawing. Copies of the patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0013] [Figure 1] Figure 1A-B show the experimental protocol for cardiac bridging integrator 1 (cBIN1) post-treatment in mice subjected to transaortic constriction (TAC). Figure 1A shows the schematic protocol; 47 mice were randomized into three groups: sham (N=12) or post-treated TAC mice administered AAV9-GFP (N=17) and AAV9-cBIN1 (N=18) 5 weeks after TAC. Figure 1B is the echocardiographic analysis of the transaortic pressure gradient (TAP) in the three groups. [Diagram 2] Figures 2A-B show that post-treatment with exogenous cBIN1 improves survival of mice after TAC. Figure 2A shows Kaplan-Meier survival curves for mice after TAC treated with AAV9-CMV virus transduced with all three groups of mice: sham control (N=12), GFP (N=17) or cBIN1 (N=18). For comparison between the three groups, the log-rank test was used. Figure 2B shows Kaplan-Meier survival curves for mice after post-TAC treatment with AAV9-GFP (N=16) or AAV9-cBIN1 (N=15) that were not in the end stage of disease (TAC EF ≥ 30% at 5 weeks after TAC) before AAV9 injection. For comparison of survival between the AAV9-GFP and AAV9-cBIN1 groups, the log-rank test was used. [Diagram 3]Figures 3A-C show that exogenous cBIN1 reduces TAC-induced hypertrophy and pulmonary edema. Figure 3A shows longitudinal heart sections stained with H&E (scale bar, 1 mm). Figure 3B shows the ratio of heart weight to tibia length (HW / TL) and Figure 3C shows the ratio of lung weight to tibia length (LW / TL) at 20 weeks after TAC. Data are presented as mean ± SEM, and statistical analysis was performed using two-way ANOVA with Fisher's LSD test. *, ***, p<0.05, 0.001 vs. sham; †, p<0.05 comparing between GFP and cBIN1 groups. [Figure 4-1] Figure 4A shows that cBin1 gene transfer maintains myocardial systolic and diastolic function in pressure-overloaded hearts. (A) Representative left ventricular (LV) short-axis M-mode images of each group (sham, AAV9-GFP, AAV9-cBIN1) at 5 weeks (before AAV9 injection) and 20 weeks (15 weeks after AAV9 injection) after TAC. Figures 4B-D show echocardiographically measured (Figure 4B) left ventricular ejection fraction (EF), (Figure 4C) end-diastolic volume, and (Figure 4D) left ventricular mass at 5 weeks (before AAV9) and 20 weeks (after AAV9) after TAC. Figure 4E shows representative mitral inflow pulsed wave Doppler images (top) and tissue Doppler images (bottom) of the septal mitral annulus (e') at 20 weeks (15 weeks after AAV9 injection) after TAC. Figure 4F shows quantification of E / e' from each group at 5 weeks (pre-AAV9) and 20 weeks (post-AAV9) after TAC. Figure 4G-H shows the delta change from 5 to 20 weeks (ΔSV=SV20w-SV5w; ΔCO=CO20w-CO5w) in (Figure 4G) stroke volume (SV) and (Figure 4H) cardiac output (CO) for each mouse. Data are shown as mean ± SEM, and statistical analysis was performed using two-way ANOVA with Fisher's LSD test. *, **, ***, p<0.05, 0.01, 0.001 versus sham; †, ††, †††, p<0.05, 0.01, 0.001 comparing AAV9-GFP and -cBIN1 groups at each time point. ‡‡, p<0.01 comparing pre- and post-AAV9 treatment for each group. [Figure 4-2]Figure 4A shows that cBin1 gene transfer maintains myocardial systolic and diastolic function in pressure-overloaded hearts. (A) Representative left ventricular (LV) short-axis M-mode images of each group (sham, AAV9-GFP, AAV9-cBIN1) at 5 weeks (before AAV9 injection) and 20 weeks (15 weeks after AAV9 injection) after TAC. Figures 4B-D show echocardiographically measured (Figure 4B) left ventricular ejection fraction (EF), (Figure 4C) end-diastolic volume, and (Figure 4D) left ventricular mass at 5 weeks (before AAV9) and 20 weeks (after AAV9) after TAC. Figure 4E shows representative mitral inflow pulsed wave Doppler images (top) and tissue Doppler images (bottom) of the septal mitral annulus (e') at 20 weeks (15 weeks after AAV9 injection) after TAC. Figure 4F shows quantification of E / e' from each group at 5 weeks (pre-AAV9) and 20 weeks (post-AAV9) after TAC. Figure 4G-H shows the delta change from 5 to 20 weeks (ΔSV=SV20w-SV5w; ΔCO=CO20w-CO5w) in (Figure 4G) stroke volume (SV) and (Figure 4H) cardiac output (CO) for each mouse. Data are shown as mean ± SEM, and statistical analysis was performed using two-way ANOVA with Fisher's LSD test. *, **, ***, p<0.05, 0.01, 0.001 versus sham; †, ††, †††, p<0.05, 0.01, 0.001 comparing AAV9-GFP and -cBIN1 groups at each time point. ‡‡, p<0.01 comparing pre- and post-AAV9 treatment for each group. [Figure 4-3]Figure 4A shows that cBin1 gene transfer maintains myocardial systolic and diastolic function in pressure-overloaded hearts. (A) Representative left ventricular (LV) short-axis M-mode images of each group (sham, AAV9-GFP, AAV9-cBIN1) at 5 weeks (before AAV9 injection) and 20 weeks (15 weeks after AAV9 injection) after TAC. Figures 4B-D show echocardiographically measured (Figure 4B) left ventricular ejection fraction (EF), (Figure 4C) end-diastolic volume, and (Figure 4D) left ventricular mass at 5 weeks (before AAV9) and 20 weeks (after AAV9) after TAC. Figure 4E shows representative mitral inflow pulsed wave Doppler images (top) and tissue Doppler images (bottom) of the septal mitral annulus (e') at 20 weeks (15 weeks after AAV9 injection) after TAC. Figure 4F shows quantification of E / e' from each group at 5 weeks (pre-AAV9) and 20 weeks (post-AAV9) after TAC. Figure 4G-H shows the delta change from 5 to 20 weeks (ΔSV=SV20w-SV5w; ΔCO=CO20w-CO5w) in (Figure 4G) stroke volume (SV) and (Figure 4H) cardiac output (CO) for each mouse. Data are shown as mean ± SEM, and statistical analysis was performed using two-way ANOVA with Fisher's LSD test. *, **, ***, p<0.05, 0.01, 0.001 versus sham; †, ††, †††, p<0.05, 0.01, 0.001 comparing AAV9-GFP and -cBIN1 groups at each time point. ‡‡, p<0.01 comparing pre- and post-AAV9 treatment for each group. [Figure 5-1] Figures 5A-C show that AAV9-cBIN1 post-treatment rescues EF in mouse hearts after TAC. Figure 5A shows the ΔEF changes (ΔEF) by echocardiographic monitoring from pre-AAV to 3, 6, 8, 10, and 15 weeks after AAV9 injection (correspondingly, 8, 11, 13, 15, and 20 weeks after TAC) in the AAV9-CMV-GFP and -cBIN1 treatment groups. Figures 5B-C show the histogram distribution of ΔEF at 6 weeks (Figure 5B) and 8 weeks (Figure 5C) after AAV9 treatment with Gaussian distribution fit curves. Data are shown as mean ± SEM. [Figure 5-2]Figures 5A-C show that AAV9-cBIN1 post-treatment rescues EF in mouse hearts after TAC. Figure 5A shows the ΔEF changes (ΔEF) by echocardiographic monitoring from pre-AAV to 3, 6, 8, 10, and 15 weeks after AAV9 injection (correspondingly, 8, 11, 13, 15, and 20 weeks after TAC) in the AAV9-CMV-GFP and -cBIN1 treatment groups. Figures 5B-C show the histogram distribution of ΔEF at 6 weeks (Figure 5B) and 8 weeks (Figure 5C) after AAV9 treatment with Gaussian distribution fit curves. Data are shown as mean ± SEM. [Figure 6-1] Figures 6A-F show that exogenous cBIN1 pretreatment improves myocardial pressure-volume (PV) loops. Figure 6A shows a schematic protocol: sham (N=5) or TAC with pretreatment of AAV9-GFP (N=10) or cBIN1 (N=10) administered 3 weeks prior to TAC. Figures 6B-F show representative PV loops (Figure 6B), EF (Figure 6C), dp / dt max (Figure 6D), dp / dt min (Figure 6E) and Tau (Figure 6F) of sham, AAV9-GFP and -cBIN1 hearts at 8 weeks after TAC surgery. Data are presented as mean ± SEM and one-way ANOVA with Fisher's LSD test was used for statistical analysis. **, ***: p<0.01, 0.001 versus sham; †, ††: p<0.05, 0.01 compared between the AAV9-GFP and AAV9-cBIN1 groups. [Figure 6-2]Figures 6A-F show that exogenous cBIN1 pretreatment improves myocardial pressure-volume (PV) loops. Figure 6A shows a schematic protocol: sham (N=5) or TAC with pretreatment of AAV9-GFP (N=10) or cBIN1 (N=10) administered 3 weeks prior to TAC. Figures 6B-F show representative PV loops (Figure 6B), EF (Figure 6C), dp / dt max (Figure 6D), dp / dt min (Figure 6E) and Tau (Figure 6F) of sham, AAV9-GFP and -cBIN1 hearts at 8 weeks after TAC surgery. Data are presented as mean ± SEM and one-way ANOVA with Fisher's LSD test was used for statistical analysis. **, ***: p<0.01, 0.001 versus sham; †, ††: p<0.05, 0.01 compared between the AAV9-GFP and AAV9-cBIN1 groups. [Figure 7-1] Figures 7A-C show that the reduction of cBIN1-microdomains in post-TAC hearts can be normalized by AAV9-cBIN1 pretreatment. Figures 7A-B show western blots of (Figure 7A) cBIN1, (Figure 7B) ryanodine receptor (RyR), and Cav1.2 from sham, AAV9-GFP, and AAV9-cBIN1 pretreated post-TAC heart lysates. Quantification is included in the bottom bar graphs (for cBIN1, n = 8 hearts per group; for cBIN1, n = 6 hearts per group). Figure 7C shows representative myocardial immunofluorescence spinning disk confocal images of BIN1 labeling (anti-BAR domain; top panel), RyR (middle panel), and Cav1.2 (bottom panel) from sham, AAV9-GFP, and AAV9-cBIN1 pretreated post-TAC hearts. Insets include magnified images of the corresponding boxed regions. Bottom row (left to right): Peak power density of BIN1, RyR and Cav1.2 distribution in sham, AAV9-GFP and cBIN1 pretreated hearts 8 weeks after TAC surgery (n=15-20 images from 5 hearts per group). Data are shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA with Fisher's LSD test. *, **, ***, p<0.05, 0.01, 0.001 vs. sham; †, ††, p<0.05, 0.01 compared between AAV9-GFP and -cBIN1 groups. [Figure 7-2] Figures 7A-C show that the reduction of cBIN1-microdomains in post-TAC hearts can be normalized by AAV9-cBIN1 pretreatment. Figures 7A-B show western blots of (Figure 7A) cBIN1, (Figure 7B) ryanodine receptor (RyR), and Cav1.2 from sham, AAV9-GFP, and AAV9-cBIN1 pretreated post-TAC heart lysates. Quantification is included in the bottom bar graphs (for cBIN1, n = 8 hearts per group; for cBIN1, n = 6 hearts per group). Figure 7C shows representative myocardial immunofluorescence spinning disk confocal images of BIN1 labeling (anti-BAR domain; top panel), RyR (middle panel), and Cav1.2 (bottom panel) from sham, AAV9-GFP, and AAV9-cBIN1 pretreated post-TAC hearts. Insets include magnified images of the corresponding boxed regions. Bottom row (left to right): Peak power density of BIN1, RyR and Cav1.2 distribution in sham, AAV9-GFP and cBIN1 pretreated hearts 8 weeks after TAC surgery (n=15-20 images from 5 hearts per group). Data are shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA with Fisher's LSD test. *, **, ***, p<0.05, 0.01, 0.001 vs. sham; †, ††, p<0.05, 0.01 compared between AAV9-GFP and -cBIN1 groups. [Figure 8-1]Figures 8A-H show that exogenous cBIN1 reduces concentric hypertrophy of mouse hearts after isoproterenol (ISO). Figure 8A shows the experimental protocol: 56 mice were randomized into four experimental groups: AAV9-GFP+PBS, AAV9-GFP+ISO, AAV9-cBIN1+PBS, AAV9-cBIN1+ISO (n=14 / group). Figure 8B shows mouse heart weight / body weight ratio (HW / BW) in the four groups. Figure 8C shows representative images of the longitudinal view of the left ventricle at both end diastole and end systole 4 weeks after PBS or ISO infusion. Figures 8D-G show echocardiographic analysis of end diastolic volume, LV mass, and relative wall thickness (Figure 8D), ejection fraction (Figure 8E), E / e' (Figure 8F), stroke volume (Figure 8G), and cardiac output (Figure 8H). Data are presented as mean ± SEM. Two-way ANOVA followed by Fisher's LSD test was used for multiple comparisons. *, **, *** indicate p<0.05, 0.01, 0.001 for PBS vs. ISO comparison within each AAV9 treatment group; ##, ### indicate p<0.01, 0.001 for GFP vs. cBIN1 comparison within each drug infusion group. [Figure 8-2]Figures 8A-H show that exogenous cBIN1 reduces concentric hypertrophy of mouse hearts after isoproterenol (ISO). Figure 8A shows the experimental protocol: 56 mice were randomized into four experimental groups: AAV9-GFP+PBS, AAV9-GFP+ISO, AAV9-cBIN1+PBS, AAV9-cBIN1+ISO (n=14 / group). Figure 8B shows mouse heart weight / body weight ratio (HW / BW) in the four groups. Figure 8C shows representative images of the longitudinal view of the left ventricle at both end diastole and end systole 4 weeks after PBS or ISO infusion. Figures 8D-G show echocardiographic analysis of end diastolic volume, LV mass, and relative wall thickness (Figure 8D), ejection fraction (Figure 8E), E / e' (Figure 8F), stroke volume (Figure 8G), and cardiac output (Figure 8H). Data are presented as mean ± SEM. Two-way ANOVA followed by Fisher's LSD test was used for multiple comparisons. *, **, *** indicate p<0.05, 0.01, 0.001 for PBS vs. ISO comparison within each AAV9 treatment group; ##, ### indicate p<0.01, 0.001 for GFP vs. cBIN1 comparison within each drug infusion group. [Figure 9-1]Figure 9A-C shows that isoproterenol reduces cBIN1 and disrupts the cBIN1-microfold, which is normalized by AAV9-cBIN1. Figure 9A shows western blots of cBIN1 and GAPDH from cardiac lysates and immunoprecipitated cardiac lysates from GFP+PBS, GFP+ISO, cBIN1+PBS, and cBIN1+ISO hearts. Quantification is shown in the bar graph on the right (N=6-7 hearts / group). Figure 9B shows representative cardiomyocyte images of Di-8-ANNEPs labeling (upper panel) (scale bar, 10 µm) and power spectra of the corresponding boxed regions of interest above (lower panel). Quantification of peak power density is included in the bar graph on the left. (N=26-31 cells from 3-4 hearts per group). Data presented as mean ± SEM. Two-way ANOVA followed by Fisher's LSD test for multiple comparisons was used. *, ** indicate p<0.05, 0.01 for comparison of PBS vs. ISO within each AAV9 treatment group; and #, ## indicate p<0.05, 0.01 for comparison of GFP vs. cBIN1 within each drug infusion group. Figure 9C shows transmission electron microscopy imaging of t-tube microfolds from myocardial tissue from all four groups (scale bar, 1 μm). Quantification of the extent of t-tube (TT) contours from each group is included in the bar graph on the left (N=232-305 TTs from 5-6 myocardial sections and 60-100 images of 2-3 hearts from each group). Chi-square test was used for comparison of TT contours between groups, p<0.001 for comparison of GFP+PBS vs. GFP+ISO, GFP+ISO vs. cBIN1+ISO, and cBIN1+PBS vs. other groups. [Figure 9-2]Figure 9A-C shows that isoproterenol reduces cBIN1 and disrupts the cBIN1-microfold, which is normalized by AAV9-cBIN1. Figure 9A shows western blots of cBIN1 and GAPDH from cardiac lysates and immunoprecipitated cardiac lysates from GFP+PBS, GFP+ISO, cBIN1+PBS, and cBIN1+ISO hearts. Quantification is shown in the bar graph on the right (N=6-7 hearts / group). Figure 9B shows representative cardiomyocyte images of Di-8-ANNEPs labeling (upper panel) (scale bar, 10 µm) and power spectra of the corresponding boxed regions of interest above (lower panel). Quantification of peak power density is included in the bar graph on the left. (N=26-31 cells from 3-4 hearts per group). Data presented as mean ± SEM. Two-way ANOVA followed by Fisher's LSD test for multiple comparisons was used. *, ** indicate p<0.05, 0.01 for comparison of PBS vs. ISO within each AAV9 treatment group; and #, ## indicate p<0.05, 0.01 for comparison of GFP vs. cBIN1 within each drug infusion group. Figure 9C shows transmission electron microscopy imaging of t-tube microfolds from myocardial tissue from all four groups (scale bar, 1 μm). Quantification of the extent of t-tube (TT) contours from each group is included in the bar graph on the left (N=232-305 TTs from 5-6 myocardial sections and 60-100 images of 2-3 hearts from each group). Chi-square test was used for comparison of TT contours between groups, p<0.001 for comparison of GFP+PBS vs. GFP+ISO, GFP+ISO vs. cBIN1+ISO, and cBIN1+PBS vs. other groups. [Figure 10-1]Figure 10A-D shows that cBIN1 increases Cav1.2 localization to t-tubules. Figure 10A shows western blots of Cav1.2 in cardiac lysates from GFP+PBS, GFP+ISO, cBIN1+PBS, and cBIN1+ISO hearts. Quantification (Cav1.2 / GAPDH and Cav1.2 / Troponin) is included in the bar graphs on the right (n=5-7 hearts per group). Figure 10B shows representative confocal images (100x) of anti-Cav1.2 labeling in mouse myocardium from each group (top, two panels) (scale bar, 10 µm). The third panel contains the power spectrum, and the fourth panel contains the fluorescence intensity profile within the boxed region along the cardiomyocyte longitudinal axis. Figure 10C shows quantification of Cav1.2 peak power density and immunofluorescence intensity in t-tubules in each group (n=15-32 cell images from 3-4 hearts per group). Scale bar: 10 μm. Figure 10D shows representative calcium transient traces and quantification of peak amplitude (ΔF / F0) from each group (n = 61-88 cells from 6 hearts per group). Data are presented as mean ± SEM. Two-way ANOVA followed by Fisher's LSD test for multiple comparisons was used. *** indicates p<0.001 for comparison of PBS vs. ISO within each AAV9 treatment group; ### indicates p<0.001 for comparison of GFP vs. cBIN1 within each drug infusion group. [Figure 10-2]Figure 10A-D shows that cBIN1 increases Cav1.2 localization to t-tubules. Figure 10A shows western blots of Cav1.2 in cardiac lysates from GFP+PBS, GFP+ISO, cBIN1+PBS, and cBIN1+ISO hearts. Quantification (Cav1.2 / GAPDH and Cav1.2 / Troponin) is included in the bar graphs on the right (n=5-7 hearts per group). Figure 10B shows representative confocal images (100x) of anti-Cav1.2 labeling in mouse myocardium from each group (top, two panels) (scale bar, 10 µm). The third panel contains the power spectrum, and the fourth panel contains the fluorescence intensity profile within the boxed region along the cardiomyocyte longitudinal axis. Figure 10C shows quantification of Cav1.2 peak power density and immunofluorescence intensity in t-tubules in each group (n=15-32 cell images from 3-4 hearts per group). Scale bar: 10 μm. Figure 10D shows representative calcium transient traces and quantification of peak amplitude (ΔF / F0) from each group (n = 61-88 cells from 6 hearts per group). Data are presented as mean ± SEM. Two-way ANOVA followed by Fisher's LSD test for multiple comparisons was used. *** indicates p<0.001 for comparison of PBS vs. ISO within each AAV9 treatment group; ### indicates p<0.001 for comparison of GFP vs. cBIN1 within each drug infusion group. [Figure 11-1]Figure 11A-D show that cBIN1 orchestrates the subcellular distribution of SERCA2a in the heart after isoproterenol. Figure 11A shows western blots of SERCA2a in cardiac lysates from GFP+PBS, GFP+ISO, cBIN1+PBS, and cBIN1+ISO hearts. Figure 11B shows quantification (SERCA2a / actin) included in the bar graph (n=6-8 hearts per group). Figure 11C shows representative confocal images of anti-SERCA2a labeling in mouse myocardium from each group (top, 2 panels). Scale bar: 10 μm. The third panel contains the power spectrum of SERCA2a in the boxed region above. Figure 11D shows quantification of peak power density of SERCA2a (n=11-15 cell images from 3-4 hearts per group). Data are presented as mean ± SEM. Two-way ANOVA followed by Fisher's LSD test for multiple comparisons was used. **,*** indicates p<0.01, 0.001 for comparison of PBS vs. ISO within each AAV9 treatment group; ### indicates p<0.001 for comparison of GFP vs. cBIN1 within each drug infusion group. [Figure 11-2]Figure 11A-D show that cBIN1 orchestrates the subcellular distribution of SERCA2a in the heart after isoproterenol. Figure 11A shows western blots of SERCA2a in cardiac lysates from GFP+PBS, GFP+ISO, cBIN1+PBS, and cBIN1+ISO hearts. Figure 11B shows quantification (SERCA2a / actin) included in the bar graph (n=6-8 hearts per group). Figure 11C shows representative confocal images of anti-SERCA2a labeling in mouse myocardium from each group (top, 2 panels). Scale bar: 10 μm. The third panel contains the power spectrum of SERCA2a in the boxed region above. Figure 11D shows quantification of peak power density of SERCA2a (n=11-15 cell images from 3-4 hearts per group). Data are presented as mean ± SEM. Two-way ANOVA followed by Fisher's LSD test for multiple comparisons was used. **,*** indicates p<0.01, 0.001 for comparison of PBS vs. ISO within each AAV9 treatment group; ### indicates p<0.001 for comparison of GFP vs. cBIN1 within each drug infusion group. [Figure 12]Figure 12A-B show sucrose gradient fractionation of myocardial microsomes. Figure 12A shows representative Western blots of Cav1.2 and cBIN1 in the F4 (TT) fraction of cardiac microsomes from GFP+PBS, GFP+ISO, cBIN1+PBS, and cBIN1+ISO hearts (2.5 μg protein loading per lane). Quantification is included in the bar graph (n=3 hearts per group). Figure 12B shows representative Western blots of RyR, phospholamban (PLN), and SERCA2a in the F2 (longitudinal SR enriched) and F3 (jSR enriched) fractions of cardiac microsomes from GFP+PBS, GFP+ISO, cBIN1+PBS, and cBIN1+ISO hearts (25 μg protein loading per lane). Quantification of SERCA2a in F2 and F3 is included in the bar graph on the right (n=3 hearts per group). Data are presented as mean ± SD. Two-way ANOVA followed by Fisher's LSD test was used for multiple comparisons. *, **, *** indicate p<0.05, 0.01, 0.001 for PBS vs. ISO comparison within each AAV9 treatment group; #, ## indicate p<0.05, 0.01 for GFP vs. cBIN1 comparison within each drug infusion group. [Figure 13-1]Figure 13A-D shows that exogenous cBIN1 brings together Cav1.2-RyR and SERCA2a-cBIN1 molecules in cardiomyocytes. Super-resolution STORM imaging and nearest neighbor analysis of Cav1.2-RyR (Figure 13A-B) and SERCA2a-cBIN1 (Figure 13C-D) molecules in cardiomyocytes isolated from GFP+PBS, cBIN1+PBS, GFP+ISO, and cBIN1+ISO hearts. Figure 13A,C shows, from top to bottom, a representative 2D-STORM cell image; a representative 3D-STORM image of a cupron; and a nearest neighbor distance distribution histogram obtained from a full-cell 3D-STORM image. Figure 13B,D shows quantification of the first peak of the nearest neighbor distance distribution histogram using full-cell image analysis (N=7-17 cells from 2-3 animals per group). Data are presented as mean ± SD. Two-way ANOVA followed by Fisher's LSD test was used for multiple comparisons. * indicates p<0.05 for comparison of PBS vs. ISO within each AAV9 treatment group; #, ### indicates p<0.05, 0.001 for comparison of GFP vs. cBIN1 within each drug infusion group. [Figure 13-2]Figure 13A-D shows that exogenous cBIN1 brings together Cav1.2-RyR and SERCA2a-cBIN1 molecules in cardiomyocytes. Super-resolution STORM imaging and nearest neighbor analysis of Cav1.2-RyR (Figure 13A-B) and SERCA2a-cBIN1 (Figure 13C-D) molecules in cardiomyocytes isolated from GFP+PBS, cBIN1+PBS, GFP+ISO, and cBIN1+ISO hearts. Figure 13A,C shows, from top to bottom, a representative 2D-STORM cell image; a representative 3D-STORM image of a cupron; and a nearest neighbor distance distribution histogram obtained from a full-cell 3D-STORM image. Figure 13B,D shows quantification of the first peak of the nearest neighbor distance distribution histogram using full-cell image analysis (N=7-17 cells from 2-3 animals per group). Data are presented as mean ± SD. Two-way ANOVA followed by Fisher's LSD test was used for multiple comparisons. * indicates p<0.05 for comparison of PBS vs. ISO within each AAV9 treatment group; #, ### indicates p<0.05, 0.001 for comparison of GFP vs. cBIN1 within each drug infusion group. [Figure 14-1]Figures 14A-H show echocardiograms of post-isoproterenol hearts receiving AAV9-GFP, cBIN1, BIN1, BIN1+17, and BIN1+13. Figure 14A shows representative LV short-axis M-mode images from each group at baseline (top) and 4 weeks after isoproterenol treatment (bottom). In all 4-week post-ISO images, papillary muscles are indicated with arrows. Figures 14B-D show quantitative analysis of LV mass (Figure 14B), relative wall thickness (Figure 14C), and ejection fraction (Figure 14D) from each group (N=10 mice per group). Figure 14E shows representative mitral inflow pulsed wave Doppler images (top) and tissue Doppler images of the septal mitral annulus 4 weeks after isoproterenol treatment (bottom). Figures 14F-H show quantitative analysis of E / e' (Figure 14F), stroke volume (Figure 14G), and cardiac output (Figure 14H) from each group (N=10 mice per group). Data are presented as mean ± SEM. Two-way ANOVA followed by Fisher's LSD test for multiple comparisons was used. *, **, *** indicate p<0.05, 0.01, 0.001 vs. baseline; #, ##, ### indicate p<0.05, 0.01, 0.001 vs. GFP group at 4 weeks post-ISO. [Figure 14-2]Figures 14A-H show echocardiograms of post-isoproterenol hearts receiving AAV9-GFP, cBIN1, BIN1, BIN1+17, and BIN1+13. Figure 14A shows representative LV short-axis M-mode images from each group at baseline (top) and 4 weeks after isoproterenol treatment (bottom). In all 4-week post-ISO images, papillary muscles are indicated with arrows. Figures 14B-D show quantitative analysis of LV mass (Figure 14B), relative wall thickness (Figure 14C), and ejection fraction (Figure 14D) from each group (N=10 mice per group). Figure 14E shows representative mitral inflow pulsed wave Doppler images (top) and tissue Doppler images of the septal mitral annulus 4 weeks after isoproterenol treatment (bottom). Figures 14F-H show quantitative analysis of E / e' (Figure 14F), stroke volume (Figure 14G), and cardiac output (Figure 14H) from each group (N=10 mice per group). Data are presented as mean ± SEM. Two-way ANOVA followed by Fisher's LSD test for multiple comparisons was used. *, **, *** indicate p<0.05, 0.01, 0.001 vs. baseline; #, ##, ### indicate p<0.05, 0.01, 0.001 vs. GFP group at 4 weeks post-ISO. [Figure 14-3]Figures 14A-H show echocardiograms of post-isoproterenol hearts receiving AAV9-GFP, cBIN1, BIN1, BIN1+17, and BIN1+13. Figure 14A shows representative LV short-axis M-mode images from each group at baseline (top) and 4 weeks after isoproterenol treatment (bottom). In all 4-week post-ISO images, papillary muscles are indicated with arrows. Figures 14B-D show quantitative analysis of LV mass (Figure 14B), relative wall thickness (Figure 14C), and ejection fraction (Figure 14D) from each group (N=10 mice per group). Figure 14E shows representative mitral inflow pulsed wave Doppler images (top) and tissue Doppler images of the septal mitral annulus 4 weeks after isoproterenol treatment (bottom). Figures 14F-H show quantitative analysis of E / e' (Figure 14F), stroke volume (Figure 14G), and cardiac output (Figure 14H) from each group (N=10 mice per group). Data are presented as mean ± SEM. Two-way ANOVA followed by Fisher's LSD test for multiple comparisons was used. *, **, *** indicate p<0.05, 0.01, 0.001 vs. baseline; #, ##, ### indicate p<0.05, 0.01, 0.001 vs. GFP group at 4 weeks post-ISO. [Figure 15-1]Figures 15A-J show that cBIN1 gene transfer improves heart failure-free survival in mice after TAC. Figure 15A shows a schematic protocol for the TAC study. Figure 15B shows trans-aortic pressure gradient measurements in all mice 5 days after surgery. Figure 15C shows Kaplan-Meier survival curves for heart failure-free survival (non-survival is death or EF < 35%) in WT vs. Bin1 HT mice (left), and AAV9-GFP or cBIN1 pre-treated mice (right). Log-rank test was used to compare survival rates. HW / BW (Figure 15D) and LW / BW (Figure 15E) of all mice at 8 weeks after TAC. (Figure 15F) Representative M-mode echocardiographic images of all mice 8 weeks after surgery. Echocardiographic measurements of left ventricular ejection fraction (FIG. 15G), end-diastolic volume (FIG. 15H), LV mass (FIG. 15I), and E / e' (FIG. 15J) for all mice 8 weeks after TAC. Data are shown as mean±SEM. Representative E and e' images in the AAV9-treated group are included in the right panel of (FIG. 15J). Unpaired T-test (or nonparametric Mann-Whitney test) used for comparison between WT and Bin1 HT. One-way ANOVA or Kruskal-Wallis test followed by Fisher's LSD test for multiple comparisons was used for comparison between Sham, AAV9-GFP, and AAV9-cBIN1. *, **, *** indicate p<0.05, 0.01, 0.001 for comparison with WT or Sham, and #, ## indicate p<0.05, 0.01 for comparison with AAV9-GFP and AAV9-cBIN1. [Figure 15-2]Figures 15A-J show that cBIN1 gene transfer improves heart failure-free survival in mice after TAC. Figure 15A shows a schematic protocol for the TAC study. Figure 15B shows trans-aortic pressure gradient measurements in all mice 5 days after surgery. Figure 15C shows Kaplan-Meier survival curves for heart failure-free survival (non-survival is death or EF < 35%) in WT vs. Bin1 HT mice (left), and AAV9-GFP or cBIN1 pre-treated mice (right). Log-rank test was used to compare survival rates. HW / BW (Figure 15D) and LW / BW (Figure 15E) of all mice at 8 weeks after TAC. (Figure 15F) Representative M-mode echocardiographic images of all mice 8 weeks after surgery. Echocardiographic measurements of left ventricular ejection fraction (FIG. 15G), end-diastolic volume (FIG. 15H), LV mass (FIG. 15I), and E / e' (FIG. 15J) for all mice 8 weeks after TAC. Data are shown as mean±SEM. Representative E and e' images in the AAV9-treated group are included in the right panel of (FIG. 15J). Unpaired T-test (or nonparametric Mann-Whitney test) used for comparison between WT and Bin1 HT. One-way ANOVA or Kruskal-Wallis test followed by Fisher's LSD test for multiple comparisons was used for comparison between Sham, AAV9-GFP, and AAV9-cBIN1. *, **, *** indicate p<0.05, 0.01, 0.001 for comparison with WT or Sham, and #, ## indicate p<0.05, 0.01 for comparison with AAV9-GFP and AAV9-cBIN1. [Figure 15-3]Figures 15A-J show that cBIN1 gene transfer improves heart failure-free survival in mice after TAC. Figure 15A shows a schematic protocol for the TAC study. Figure 15B shows trans-aortic pressure gradient measurements in all mice 5 days after surgery. Figure 15C shows Kaplan-Meier survival curves for heart failure-free survival (non-survival is death or EF < 35%) in WT vs. Bin1 HT mice (left), and AAV9-GFP or cBIN1 pre-treated mice (right). Log-rank test was used to compare survival rates. HW / BW (Figure 15D) and LW / BW (Figure 15E) of all mice at 8 weeks after TAC. (Figure 15F) Representative M-mode echocardiographic images of all mice 8 weeks after surgery. Echocardiographic measurements of left ventricular ejection fraction (FIG. 15G), end-diastolic volume (FIG. 15H), LV mass (FIG. 15I), and E / e' (FIG. 15J) for all mice 8 weeks after TAC. Data are shown as mean±SEM. Representative E and e' images in the AAV9-treated group are included in the right panel of (FIG. 15J). Unpaired T-test (or nonparametric Mann-Whitney test) used for comparison between WT and Bin1 HT. One-way ANOVA or Kruskal-Wallis test followed by Fisher's LSD test for multiple comparisons was used for comparison between Sham, AAV9-GFP, and AAV9-cBIN1. *, **, *** indicate p<0.05, 0.01, 0.001 for comparison with WT or Sham, and #, ## indicate p<0.05, 0.01 for comparison with AAV9-GFP and AAV9-cBIN1. [Figure 15-4]Figures 15A-J show that cBIN1 gene transfer improves heart failure-free survival in mice after TAC. Figure 15A shows a schematic protocol for the TAC study. Figure 15B shows trans-aortic pressure gradient measurements in all mice 5 days after surgery. Figure 15C shows Kaplan-Meier survival curves for heart failure-free survival (non-survival is death or EF < 35%) in WT vs. Bin1 HT mice (left), and AAV9-GFP or cBIN1 pre-treated mice (right). Log-rank test was used to compare survival rates. HW / BW (Figure 15D) and LW / BW (Figure 15E) of all mice at 8 weeks after TAC. (Figure 15F) Representative M-mode echocardiographic images of all mice 8 weeks after surgery. Echocardiographic measurements of left ventricular ejection fraction (FIG. 15G), end-diastolic volume (FIG. 15H), LV mass (FIG. 15I), and E / e' (FIG. 15J) for all mice 8 weeks after TAC. Data are shown as mean±SEM. Representative E and e' images in the AAV9-treated group are included in the right panel of (FIG. 15J). Unpaired T-test (or nonparametric Mann-Whitney test) used for comparison between WT and Bin1 HT. One-way ANOVA or Kruskal-Wallis test followed by Fisher's LSD test for multiple comparisons was used for comparison between Sham, AAV9-GFP, and AAV9-cBIN1. *, **, *** indicate p<0.05, 0.01, 0.001 for comparison with WT or Sham, and #, ## indicate p<0.05, 0.01 for comparison with AAV9-GFP and AAV9-cBIN1. [Figure 16] Figure 16 shows AAV9-transduced exogenous GFP-V5 and cBIN1-V5 protein expression in mouse cardiomyocytes. Representative images of adult mouse ventricular cardiomyocytes under transmitted light (top) or wide-field fluorescent light (rabbit anti-V5 labeling, bottom) from control (left), AAV9-GFP-V5 (middle), or AAV9-cBIN1-V5 (right) treated mice. [Figure 17]FIG. 17 shows that AAV9-delivered exogenous cBIN1 normalizes cardiomyocyte t-tubule microfolds in post-TAC hearts. Representative live cell membrane labeling (Di-8-ANNEPs) images of freshly isolated cardiomyocytes from sham, AAv9-GFP, and AAV9-cBIN1 treated post-TAC hearts. Quantification of t-tubule Di-8-ANNEPs intensity is included in the bar graph on the right (n=10 images from 5 hearts per group). All data are presented as mean±SEM. Statistical analysis used Kruskal-Wallis with LSD post-test. ***, p<0.001 compared to sham group; †, p<0.05 compared between AAV9-GFP and AAV9-cBIN1 groups. [Figure 18] FIG. 18A is a representative fluorescent confocal image (20×) of V5 and WGA labeling in myocardial cryosections obtained from mice 7 weeks after injection with AAV9 transducing GFP-V5 or cBIN1-V5, or from control hearts not injected with AAV9 (negative control). Positive V5 signal is detected in 63% and 57% of cells, respectively, from hearts 7 weeks after retroorbital injection of AAV9-GFP-V5 or AAV9-cBIN1-V5 (3×1010 vg). Scale bar, 100 μm. FIG. 18B shows quantification of the percent of myocardial area with detectable V5 signal. N=4-6 myocardial sections from 2-3 animals from each group. Data are presented as mean±SEM. Kruskal-Wallis test followed by Dunn's test for multiple comparisons was used. * indicates p<0.05 vs. no AAV9 negative control. [Figure 19] Figure 19A shows echocardiographic-based classification of LV remodeling in GFP+PBS, cBIN1+PBS, GFP+ISO, and cBIN1+ISO hearts. Figure 19B shows representative Western blots and quantification of α-smooth muscle actin in hearts from each group. Data are presented as mean ± SEM. Two-way ANOVA followed by Fisher's LSD test for multiple comparisons was used. * indicates p<0.05 for comparison of PBS vs. ISO within each AAV9 treatment group; ## indicates p<0.01 for comparison of GFP vs. cBIN1 within each drug infusion group. [Figure 20] Figure 20A shows a representative Western blot of total RyR2 protein expression in mice, along with quantification. Figure 20B shows representative confocal images (100x) of RyR2 in mouse cardiomyocytes from each group, followed by power spectrum analysis (n = 33-36 cells from 3-4 hearts per group). Scale bar: 10 μm. [Figure 21-1] FIG. 21 shows representative Western blots for RyR2 (total and phosphorylated pS2814, pS2808), Cav1.2, CAMKIIδ (total and phosphorylated pT287), and phospholamban (PLN, total and phosphorylated pS16, pT17). Quantification is included in the bar graphs on the right. Data are presented as mean ± SEM. N=4-7 hearts per group. Two-way ANOVA followed by Fisher's LSD test for multiple comparisons was used. * indicates p<0.05 for comparison of PBS vs. ISO within each AAV9 treatment group; # indicates p<0.05 for comparison of GFP vs. cBIN1 within each drug infusion group. [Figure 21-2] FIG. 21 shows representative Western blots for RyR2 (total and phosphorylated pS2814, pS2808), Cav1.2, CAMKIIδ (total and phosphorylated pT287), and phospholamban (PLN, total and phosphorylated pS16, pT17). Quantification is included in the bar graphs on the right. Data are presented as mean ± SEM. N=4-7 hearts per group. Two-way ANOVA followed by Fisher's LSD test for multiple comparisons was used. * indicates p<0.05 for comparison of PBS vs. ISO within each AAV9 treatment group; # indicates p<0.05 for comparison of GFP vs. cBIN1 within each drug infusion group. [Figure 22]Figure 22A shows a schematic protocol for sucrose gradient fractionation of cardiac microsomes (3-6 mg per heart). The total protein yields recovered from each fraction F1, F2, F3, F4 are between 0.001-0.02, 0.4-0.8, 0.04-0.06, and <0.008 mg per heart prep, respectively. Figure 22B shows representative Western blots of Cav1.2, Na+ / K+-ATPase, SERCA2a, cBIN1, and caveolin-3 from microsomal input (M) and recovered fractions from F1, F2, F3, F4, and pellets from GFP+PBS, GFP+ISO, cBIN1+PBS, and cBIN1+ISO hearts. [Figure 23-1] Figures 23A-E show that cBIN1 organizes LTCCs and SERCA2a in post-isoproterenol mouse hearts. Figure 23A shows representative western blots of Cav1.2 and SERCA2a in post-isoproterenol mouse hearts treated with AAV9-GFP, cBIN1, BIN1, BIN1+17, and BIN1+13. Quantification is included in the bar graphs on the right (n=3 hearts per group). Figures 23B-C show representative confocal images (100x) of anti-Cav1.2 (Figure 23B) and anti-SERCA2a (Figure 23C) labeling in mouse myocardium from each group. Scale bar: 5 μm. Figure 23D shows quantification of t-tubule Cav1.2 fluorescence intensity from each group. N=16-22 cells from 3 hearts from each group. Figure 23E shows quantification of SERCA2a peak power density from each group. N = 20 cell images from 2-3 hearts from each group. Data are shown as mean ± SEM. One-way ANOVA followed by Fisher's LSD test for multiple comparisons was used. *, **, *** indicate p<0.05, 0.01, 0.001 compared to the control GFP group. [Figure 23-2]Figures 23A-E show that cBIN1 organizes LTCCs and SERCA2a in post-isoproterenol mouse hearts. Figure 23A shows representative western blots of Cav1.2 and SERCA2a in post-isoproterenol mouse hearts treated with AAV9-GFP, cBIN1, BIN1, BIN1+17, and BIN1+13. Quantification is included in the bar graphs on the right (n=3 hearts per group). Figures 23B-C show representative confocal images (100x) of anti-Cav1.2 (Figure 23B) and anti-SERCA2a (Figure 23C) labeling in mouse myocardium from each group. Scale bar: 5 μm. Figure 23D shows quantification of t-tubule Cav1.2 fluorescence intensity from each group. N=16-22 cells from 3 hearts from each group. Figure 23E shows quantification of SERCA2a peak power density from each group. N = 20 cell images from 2-3 hearts from each group. Data are shown as mean ± SEM. One-way ANOVA followed by Fisher's LSD test for multiple comparisons was used. *, **, *** indicate p<0.05, 0.01, 0.001 compared to the control GFP group. [Figure 24] Figures 24A-D show that AAV9-cBIN1 rescues diabetic HFpEF in db / db mice. Maximum running distance on a mouse treadmill was measured by echocardiography in E / A (Figure 24A), E / e' (Figure 24B), and SV (Figure 24C); and (Figure 24D) in control db / m mice (db / m+GFP) and diabetic db / db mice treated with AAV9-GFP or cBIN1 (db / db+GFP or db / db+cBIN1). N=10 animals per group. Differences between groups were compared using one-way ANOVA followed by LSD Fisher's exact test. *, *** indicate p<0.05 or 0.001, respectively, compared to the db / m+GFP group. †, ††† indicate p<0.05 or 0.001 for the comparison of the db / db+GFP group vs. the db / db+cBIN1 group. [Diagram 25]Figure 25 shows that AAV9-cBIN1 rescues ischemic HFrEF in dogs. Left ventricular ejection fraction (LVEF) versus study week was measured in two tested dogs (dogs #1 and #2) by echocardiography. Time 0 corresponds to the time of LAD ligation. Arrows indicate the time of cBIN1 therapy. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Reorganization of intracellular calcium handling machinery can be achieved by targeting t-tubule membrane microdomains organized by its cardiac isoform of bridging integrator 1 (cBIN1)

[34] . cBIN1-microdomains were previously shown to promote intracellular trafficking

[13] and surface clustering of LTCCs [14,35], affect the electrochemical gradient between LTCCs by forming a protective slow-diffusion region for extracellular ions in the t-tubule lumen

[12] , and organize LTCC-RyR dyads [12,14] by recruiting RyR to the jSR to bind with LTCCs

[14] . More recently, cBIN1-microdomains were also shown to be important in organizing the intracellular distribution of SERCA2a for diastolic calcium regulation

[34] . In HF, cBIN1-microdomains are disrupted by decreased transcription of cBIN1 [16,36,37], impairing dyad formation, calcium transient control, and cardiac contractility. A decrease in myocardial cBIN1 can be detected in human blood as a result of cBIN1 membrane turnover and microparticle release

[38] . In humans, plasma CS (cBIN1 score) is an indicator of cardiomyocyte cBIN1 levels, which identifies myocardial structural remodeling and facilitates HF diagnosis and prognosis

[39] . In chronically stressed mouse hearts, pretreatment with exogenous cBIN1 maintains the microdomain-organized distribution of Cav1.2 and SERCA2a and preserves normal inotropy and lucitropy. These data indicate that cBIN1 supplementation could be an effective HF treatment with the potential to restore myocardial function in hearts with existing HF.

[0015] Because increased afterload is an important primary and secondary cause of HF

[40] , this study uses a mouse model of elevated afterload induced by transaortic constriction (TAC). It has been reported that cBIN1 pretreatment prevents the onset of HF in TAC mice. Furthermore, AAV9-mediated gene transfer was used to introduce exogenous cBIN1 into post-TAC mouse hearts with pre-existing HF. cBIN1 posttreatment reduces TAC-induced pathological remodeling as well as HF incidence and mortality. cBIN1 induces functional recovery in mouse hearts with pre-existing TAC-induced HF. This disclosure also explored whether overexpressing exogenous cBIN1 in vivo could limit myocardial remodeling and dysfunction. Continuous infusion of isoproterenol causes myocardial cBIN1 downregulation and perturbed intracellular distribution of calcium-handling proteins, and also induces pathological concentric hypertrophy with diastolic dysfunction. We found that normalization of cBIN1 by adeno-associated virus 9 (AAV9)-mediated gene transfer increased inotropy and maintained lucitropy, and suppressed pathological hypertrophy. In cardiomyocytes, we found that exogenous cBIN1 preserved the subcellular distribution of LTCCs and the localization of sarcoplasmic reticulum (SR) calcium ATPase2a (SERCA2a) in t-tubules. The protective effect of cBIN1 was confirmed to be isoform-specific and effective in a second model of aortic contraction (TAC)-induced cardiac hypertrophy and HF, indicating that exogenous cBIN1-mediated preservation of t-tubule microdomains may be a therapeutic approach to improve myocardial function in chronically stressed hearts.

[0016] Intramyocardial AAV9 viral delivery of exogenous cBIN1, when administered after reduced ejection fraction, could rescue cardiac contractile function in chronic pressure overload stressed mice and further limit ventricular dilation and HF development.

[0017] Under continued pressure overload, myocardial remodeling begins with an adaptive hypertrophic response, which then transitions to maladaptive cardiac dilatation, leading to the worsening of HF [41-43]. Previous studies have demonstrated that administration of AAV9-cBIN1 before TAC surgery preserves myocardial systolic and diastolic function, indicating the efficacy of cBin1 gene therapy in preventing HF. In this disclosure, we found that exogenous cBIN1 administration not only limits but also rescues the development of further HF in TAC-stressed hearts, attenuating cardiac hypertrophy, reducing pulmonary edema, and improving overall survival in mice. Furthermore, exogenous cBIN1 introduced by gene transfer improves myocardial remodeling and cardiac function measured by echocardiography. Most strikingly, mice with pre-existing severe HF showed recovery of EF after cBin1 gene therapy, indicating that the protective effect of exogenous cBIN1 may serve as a transformative therapy for patients with pre-existing structural remodeling and a diagnosis of HF.

[0018] Recently, AAV-mediated gene therapy has been shown to be a promising modality for the treatment of HF [44-45]. Currently, the β-adrenergic system, Ca 2+Several clinical trials of HF gene therapy targeting various pathways, such as circulating proteins and cell death pathways, as well as stem cell homing, are completed or underway

[46] . We recently found that targeting calcium-regulated microdomains of t-tubules can be effectively achieved by introducing cBIN1, an essential microdomain-organizing protein

[34] . By stabilizing t-tubule microdomains, cBIN1 potentially restores cytosolic calcium homeostasis and contributes to increased systolic calcium release, improved diastolic reabsorption, limited SR leakage to maintain electrical stability, and preserved mitochondrial function to limit mitochondria-associated cell death. This result indicates that this microdomain-targeting approach may serve as a novel therapeutic strategy to improve the efficiency of functional preservation and improve overall HF survival. Furthermore, the observed cBIN1-mediated improvement in overall survival is likely a combined effect of improved pump function and reduced arrhythmias, both of which are controlled by cBIN1-microdomains [7, 12, 36]. How cBIN1 therapy affects arrhythmia burden in failing hearts requires further analysis using in vivo telemetry monitoring in future studies. Furthermore, because TAC-induced HF is accompanied by myocyte death associated with mitochondrial dysfunction

[47] , it remains interesting in future studies to examine whether cBIN1 replacement therapy can preserve mitochondrial function and limit mitochondrial-associated cell death in failing hearts.

[0019] Regarding functional recovery, EF changes monitored from the beginning of AAV9-cBIN1 treatment showed a high peak of recovery at 6 weeks after AAV9, after which the therapeutic efficiency declined, although the rescue effect was maintained at 15 weeks after AAV9 injection. These data support the conclusion that a single administration of a relatively low dose of AAV9-cBIN1 (3 × 10 10vg) is sufficient to preserve cardiac function. Whether multiple administrations of exogenous cBIN1 at increasing doses are required to maximize therapeutic efficacy remains to be verified. However, the current rescue data indicate that for patients with existing HF, cBin1 gene therapy could potentially break the worsening cycle of HF progression and result in restoration of failing cardiac function.

[0020] This study reveals a protective role of exogenous cBIN1 in mouse hearts with pre-existing HF after pressure overload. In this first proof-of-concept study, we used a CMV promoter-driven AAV9 vector for gene delivery due to its consistent transduction efficiency and established cardiac tropism. Further testing in mice and larger mammals with cBin1 packaged in AAV9 with a more efficient cardiac-specific promoter will be required before clinical trials to validate the efficacy and efficiency of cBin1 gene therapy in HF patients. Future studies are also needed to understand the intracellular mechanisms by which cBIN1 balances calcium homeostasis among t-tubules, SR, and mitochondrial cytoplasmic microdomains. Further understanding of the downstream target molecules and signaling pathways of cBIN1 will also be required to better understand the interplay between cBIN1 gene therapy and HF pathophysiology.

[0021] The present disclosure also shows the beneficial effect of exogenous cBIN1 in preventing LV hypertrophy and cardiac dysfunction in stressed hearts. In mice receiving continuous infusion of isoproterenol, exogenous cBIN1 results in isoform-specific improvement of cardiac inotropy and lucitropy, limiting the development of LV hypertrophy. The cardioprotective effect of exogenous cBIN1 was further confirmed in mouse hearts subjected to pressure overload-induced HF.

[0022] Chronically elevated catecholamine levels and cardiac β-adrenergic receptor (β-AR) activation have important roles in the pathogenesis of HF. Impaired myocardial structure and function have been observed in animals with persistent sympathetic activation [48–49]. Isoproterenol, a synthetic catecholamine and nonselective β-AR agonist, has been used in studies to induce models of LV hypertrophy and dysfunction

[50] . Here, high doses of isoproterenol were used to induce LV concentric hypertrophy while preserving systolic function. LV hypertrophy due to chronic excessive cardiac workload is associated with a high risk of cardiovascular events

[51] , and preventing or reversing ventricular hypertrophy while preserving cardiac diastolic function is crucial to prevent the progression of a stressed heart to a failing heart. Here, we found that cBIN1 attenuates chronic isoproterenol-induced hypertrophy while simultaneously leading to isoform-specific improvements in stroke volume and cardiac output in hypertrophied hearts with preserved systolic function. The increased LV volumes in cBIN1 hearts are not due to pump failure and dilated cardiomyopathy, but rather reflect improved myocardial lucitropy (E / e') in parallel with increased intrinsic myocardial contractility (inotropy). This phenotype of cBIN1 hearts is typical of the athletes' hearts in adaptive endurance training, characterized by chamber enlargement and increased LV volumes, stroke volume and cardiac output [52-54]. Aerobic exercise training has been reported to improve myocardial function and inotropic and lucitropic responses in both animal models [55-56] and patients with hypertension

[57] and diastolic dysfunction

[58] . Thus, exogenous cBIN1 may provide additional exercise-like benefits to heart failure patients, improving exercise capacity and quality of life.

[0023] These post-isoproterenol hearts are in the hypertrophic stage with preserved contractile function, and exogenous cBIN1 can effectively convert the increased demands on the heart into functional benefits. As a result, these functional and efficient cBIN1 hearts are likely to be limited in the development of hypertrophy and prevent the next step of disease progression and HF development as occurs in clinical conditions. Next, the functional protective effect of exogenous cBIN1 in already decompressed hearts is also observed in a mouse model of TAC-induced hypertrophy and HF. Under pressure overload, compensatory hypertrophy is an adaptive response. The adaptive response transitions over time to cardiac dilation, and the associated remodeling becomes maladaptive, leading to the worsening of HF. We found that the fate of dilated cardiomyopathy development in hearts under pressure overload stress is inevitably determined by the amount of myocardial cBIN1 protein. Reduction of cardiac BIN1 in genetically deleted Bin1HT-TAC hearts after pressure overload is associated with more severe dilated cardiomyopathy, whereas increase of cBIN1 by gene transfer improves cardiac systolic and diastolic function, limits HF, and improves HF-free survival. It remains unclear whether exogenous cBIN1 reduces cardiomyocyte death, which also contributes to LV dilation in hearts after TAC. Future studies investigating the effect of cBIN1 on cardiomyocyte survival in stressed hearts will be necessary. However, our data show that exogenous cBIN1 not only limits the development of hypertrophy in stressed hearts, but also prevents myocardial transition from hypertrophy to dilated cardiomyopathy and HF in TAC mice.

[0024] The mechanism by which cBIN1 improves myocardial inotropic function is related to the known action of cBIN1 to organize t-tubule microdomains, which are required for bimolecular organization and efficient EC coupling. cBIN1 forms t-tubule microfolds, organizes slow-diffusion regions that capture extracellular t-tubule luminal ions, attracts LTCCs that traffic forward to t-tubules

[30] , clusters LTCCs already delivered to the cell surface

[35] , and recruits RyRs that bimolecularly couple with LTCCs

[31] . Here, we confirmed that exogenous cBIN1, but not other BIN1 isoforms, increases Cav1.2 localization to t-tubules in vivo. These results support that cBIN1-microdomains with organized LTCC distribution are involved in the positive inotropic effects observed in sympathetically overdriven cBIN1 hearts. Whether cBIN1-microdomains control phosphorylation of LTCCs and their functional responses to sympathetic stress, including the well-established β-subunit-regulated Cav1.2 channel response [59-60], remains to be explored experimentally. Furthermore, RyRs are important for inotropy, and hyperphosphorylated leaky RyRs are involved in HF progression

[14] . Consistent with previous reports in the isoproterenol model and in human HF [14, 61], we found that chronic isoproterenol activates PKA- and CAMKII-induced hyperphosphorylation of RyRs. AAV9-cBIN1 blunts these pathways, normalizing RyR phosphorylation and preventing SR leakage after chronic sympathetic activation.

[0025] A further novel finding of this disclosure is that exogenous cBIN1 increases SERCA2a function by organizing its subcellular distribution. Chronic isoproterenol-induced concentric hypertrophy is associated with disturbed subcellular distribution of SERCA2a, with increased global protein expression, while contractile function is preserved. It is well known that SERCA2a activity is decreased in end-stage HF. Our data indicate that in addition to decreased expression and impaired regulation by PLN, the subcellular distribution of SERCA2a may also contribute to abnormal SR calcium reuptake activity in HF. Furthermore, an adaptive increase in SERCA2a protein expression may occur by diastolic calcium-induced elevation of calcineurin / NFAT activation, as reported in adult rat ventricular cardiomyocytes with α-receptor agonist phenylephrine-induced hypertrophy

[62] . Thus, the increase in SERCA2a protein expression here may be an adaptive response induced by elevated diastolic calcium concentration, as shown by increased calcium-dependent phosphorylation at T287 of CAMKII. Thus, a transient increase in SERCA2a may occur in the early stages of all LV hypertrophy while preserving function. During disease progression, this adaptive increase in total SERCA2a protein expression plateaus or even decreases in levels, as occurs in end-stage HF, which results in severe diastolic and systolic dysfunction. In cBIN1 hearts, organized SERCA2a along the SR exhibits better calcium reabsorption and therefore less diastolic calcium overload in hearts still in the compensated phase. These results are consistent with previous studies in a rat model of HF that identified increased BIN1 expression as associated with SERCA2a expression

[63] . Future studies exploring cBIN1 regulation of diastolic calcium concentration and the calcineurin / NTAT pathway will be necessary to further understand its role in regulating SERCA2a expression and activity during disease progression. Note that the effect on SERCA2a organization is not cBIN1 specific and can be partially induced by other BIN1 isoforms, especially BIN1+17.This is consistent with the partial in vivo protective effects of BIN1+17 on cardiac hypertrophy and diastolic function. Whether and how BIN1 isoforms cooperate to orchestrate the distribution of SERCA2a in normal and diseased cardiomyocytes requires further exploration in future studies. Furthermore, through regulating calcium handling mechanisms in the SR, including SERCA2a distribution and RyR phosphorylation, cBIN1 may help maintain normal SR calcium load. As a limitation of the present study, future experiments are needed to quantify the effects of cBIN1 on SR calcium load, calcium release and reuptake kinetics, and arrhythmogenic spontaneous calcium release in chronically stressed hearts.

[0026] However, the most robust protection of both inotropy and lucitropy in sympathetically overdriven hearts was observed only in the cBIN1 group, suggesting that additional beneficial effects on lucitropy may result from cBIN1-dependent improvements in LTCC localization and bimolecular organization. Isoform-specific improvements in bimolecular organization reduce orphan leaky RyRs that accumulate outside of bimolecules

[31] , limiting calcium leakage from the SR and reducing cytoplasmic calcium concentrations during diastole. Together with the identified novel role for SERCA2a organization, our data indicate that cBIN1-microdomain-associated regulation provides a unique benefit in protecting cardiac lucitropy in addition to the inotropic effect. Meanwhile, cBIN1 overexpression may also suppress the pathological effects of isoproterenol stimulation by enhancing regulation of β-AR signaling and compartmentalization of second messenger and calcium-handling channels and pumps. Thus, cBIN1 stabilizes t-tubule microdomains and controls all aspects of calcium handling, thereby generating a positive feed-forward mechanism for efficient intracellular interbeat calcium cycling. In future studies, it will be interesting to determine whether exogenous cBIN1 alters β-AR expression, subcellular distribution, and functional regulation following chronic sympathetic activation.

[0027] In conclusion, we found that overexpression of exogenous cBIN1 was protective in mouse hearts exposed to chronic β-AR activation-induced concentric hypertrophy, as well as pressure overload-induced hypertrophy and HF. Future experiments in large mammals with common spontaneous heart failures such as hypertension and diabetes are needed. Improving viral infectivity in cardiomyocytes may further help to limit or prevent isoproterenol-induced membrane destruction in all cardiomyocytes and enhance the protective effect on the whole heart. Further experiments with cBin1 packaged in AAV9 with an efficient and cardiac-specific promoter will be needed to induce sufficient exogenous protein expression in all cardiomyocytes before clinical trials testing the efficacy and efficiency of cBin1 gene therapy. Future studies are needed to establish whether cBIN1 affects systemic hemodynamics and blood pressure. Finally, future studies are needed to explore how cBIN1-microdomains regulate the organization of intracellular calcium handling machinery, EC coupling, SR calcium loading and release, diastolic calcium concentration and downstream calcium signaling pathways, the interplay between signaling pathways of pathological and physiological hypertrophic remodeling, and the molecular transition from compensated hypertrophy to compensated cardiomyopathy.

[0028] One embodiment described herein is a method for restoring cardiac tissue or ameliorating symptoms of cardiac failure in a subject experiencing cardiac failure or under chronic stress, comprising diagnosing cardiac failure or myocardial stress in a subject, and administering a transgene encoding Cardiac Bridging Integrator 1 (cBIN1) to cardiac tissue of the subject experiencing cardiac failure. In one aspect, diagnosing cardiac failure or myocardial stress comprises measuring reduced cBIN1 blood levels.

[0029] Other embodiments described herein are methods for restoring or increasing contractile (systolic) or relaxing (diastolic) function in the heart of a subject experiencing heart failure, comprising administering to the subject's cardiac tissue a transgene encoding Cardiac Bridging Integrator 1 (cBIN1), where after the transgene is delivered to and expressed in the cardiac tissue, the contractile function of the heart is restored or increased. In one aspect, the transgene is administered after the subject is diagnosed with heart failure. In another aspect, the diagnosis of heart failure comprises measuring reduced cBIN1 blood levels. In another aspect, the method comprises administering the transgene to the myocardium. In another aspect, the transgene is administered by injection. In another aspect, the transgene comprises a vector comprising a transgene encoding cBIN1. In another aspect, the transgene is administered in a dose of about 1×10 10 ~Approx. 5×10 10 In another aspect, the expression of cBIN1 remodels damaged myocardium. In another aspect, the expression of cBIN1 stabilizes the intracellular distribution of calcium processing equipment in myocardium. In another aspect, the expression of cBIN1 reduces concentric hypertrophy in myocardium. In another aspect, the expression of cBIN1 restores or increases t-tubule microfolds or microdomains in myocardium. In another aspect, the expression of cBIN1 restores or reduces hyperphosphorylation of ryanodine receptor 2 (RyR2) in myocardium. In another aspect, the expression of cBIN1 restores or improves cardiac contractility and lucitropy. In another aspect, the expression of cBIN1 restores or improves cardiac relaxation and diastolic function. In another aspect, the expression of cBIN1 is preventative against further damage to myocardium. In another aspect, the transgene is administered at least once. In another aspect, the subject is a mammal. In another aspect, the subject is a mouse or a dog. In another aspect, the subject is a human. In other embodiments, the subject experiences reduced ejection fraction (HFrEF).

[0030] Another embodiment described herein is the use of cBIN1 in medicine to restore myocardial tissue or repair myocardial damage in subjects experiencing heart failure or with chronic myocardial stress.

[0031] Another embodiment described herein is the use of cBIN1 in medicine to restore or increase contractile (systolic) or relaxing (diastolic) function in the heart of a subject experiencing heart failure or having chronic myocardial stress.

[0032] It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, devices, assemblies, and uses described herein may be made without departing from the scope of any embodiment or aspect thereof. The compositions, devices, assemblies, and methods provided are exemplary and are not intended to limit the scope of the disclosed embodiments. All of the various embodiments, aspects, and options disclosed herein may be combined in any variation or iteration. The scope of the compositions, formulations, methods, devices, assemblies, and processes described herein includes all actual or potential combinations of the embodiments, aspects, options, examples, and preferences described herein. The compositions, formulations, devices, assemblies, or methods described herein may omit any component or step disclosed herein, may substitute any component or step disclosed herein, or may include any component or step disclosed elsewhere herein. The ratio of the mass of any component of a composition or formulation disclosed herein to the mass of the other components in the formulation or the total mass of the other components in the formulation is disclosed herein as if expressly disclosed. If the meaning of a term in any patent or publication incorporated by reference conflicts with the meaning of the term used in this disclosure, the meaning of the term or phrase in this disclosure shall control. All patents and publications cited herein are incorporated by reference herein for their specific teachings.

[0033] Various embodiments and aspects of the invention described herein are summarized by the following subsections: Item 1. A method for restoring cardiac tissue or ameliorating symptoms of heart failure in a subject experiencing heart failure or under chronic stress, the method comprising diagnosing heart failure or myocardial stress in a subject, and administering a transgene encoding Cardiac Bridging Integrator 1 (cBIN1) to cardiac tissue of the subject experiencing heart failure. Item 2. The method according to item 1, wherein the diagnosis of heart failure or myocardial stress comprises measuring a decreased cBIN1 blood level. Item 3. A method for restoring or increasing contractile function in the heart of a subject experiencing heart failure, comprising administering to the subject's cardiac tissue a transgene encoding Cardiac Bridging Integrator 1 (cBIN1), wherein after the transgene is delivered to and expressed in the cardiac tissue, the cardiac contractile function is restored or increased. Item 4. The method of Item 3, wherein the transgene is administered after the subject is diagnosed with heart failure. Item 5. The method according to Item 4, wherein diagnosing heart failure comprises measuring a decreased cBIN1 blood level. Item 6. The method according to any one of Items 1 to 5, which comprises administering a transgene to the myocardium. Item 7. The method of any one of items 1 to 6, wherein the transgene is administered by injection. Item 8. The method according to any one of Items 1 to 7, wherein the transgene comprises a vector containing a transgene encoding cBIN1. Item 9. The introduced gene is about 1 × 10 10 ~Approx. 5×10 10 The method according to any one of items 1 to 8, comprising a vector genome comprising: Item 10. The method according to any one of items 1 to 9, wherein expression of cBIN1 reconstructs damaged myocardium. Item 11. The method according to any one of Items 1 to 10, wherein expression of cBIN1 stabilizes the intracellular distribution of a calcium handling mechanism in cardiac muscle. Item 12. The method according to any one of Items 1 to 11, wherein expression of cBIN1 reduces concentric hypertrophy in the myocardium. Item 13. The method according to any one of items 1 to 12, wherein expression of cBIN1 restores or increases t-tubule folds or microdomains in cardiac muscle. Item 14. The method according to any one of items 1 to 13, wherein expression of cBIN1 restores or reduces hyperphosphorylation of ryanodine receptor 2 (RyR2) in cardiac muscle. Item 15. The method according to any one of items 1 to 14, wherein expression of cBIN1 restores or improves cardiac contractility and lucitropy. Item 16. The method according to any one of items 1 to 15, wherein expression of cBIN1 restores or improves cardiac relaxation and diastolic function. Item 17. The method according to any one of items 1 to 16, wherein expression of cBIN1 is protective against further damage to the myocardium. Item 18. The method according to any one of items 1 to 17, wherein the transgene is administered at least once. Item 19. The method according to any one of items 1 to 18, wherein the subject is a mammal. Item 20. The method according to any one of items 1 to 19, wherein the subject is a mouse or a dog. Item 21. The method according to any one of items 1 to 20, wherein the subject is a human. Item 22. The method according to any one of items 1 to 21, wherein the subject experiences reduced ejection fraction (HFrEF). Item 23. Use of cBIN1 in medicine to restore myocardial tissue or repair myocardial damage in subjects experiencing heart failure or with chronic myocardial stress. Item 24. Use of cBIN1 in medicine to restore or increase contractile (systolic) or relaxing (diastolic) function in the heart of a subject experiencing heart failure or with chronic myocardial stress. EXAMPLES

[0034] Example 1 Materials and Methods Animal procedures for functional rescue studies. Adult male C57BL / 6 mice (The Jackson Laboratory) were used. All mice were anesthetized at 8–10 weeks of age and subjected to open-chest sham or transcatheter aortic constriction (TAC) surgery. TAC was performed by tying a 7-0 silk thread to a 27-gauge needle between the first and second branches of the aortic arch. As sham controls, age-matched mice were subjected to open-chest mock surgery without TAC. For gene therapy, 5 weeks after the start of TAC, 3 × 10 of AAV9 virus (Welgen.Inc) transducing cBIN1-V5 or GFP-V5 was administered. 10 Mice were injected retroorbitally with 100 μL of vector genome (vg)

[64] .

[0035] Animal procedures for isoproterenol studies. For isoproterenol studies, adult male C57BL / 6 mice were injected with 3 × 10 6 immunized mice of AAV9 transduced with GFP or BIN1 isoforms. 10 Vector genome (vg) (Welgen, Inc.) was administered by retroorbital injection

[64] . Three weeks after vg administration, mice were subcutaneously implanted with osmotic minipumps releasing PBS or isoproterenol (30 mg / kg / day). Fifty-six mice were randomized to receive GFP+PBS, GFP+ISO, cBIN1+PBS, or cBIN1+ISO (N=14 / group). The other 50 mice were randomized to receive AAV9-GFP, cBIN1, BIN1, BIN1+17, or BIN1+13 (N=10 / group) prior to isoproterenol. AAV9 was used because it is a promising gene therapy vehicle and shows the highest cardiac tropism

[65] . The CMV promoter was used due to its efficiency and safety in cardiac gene transfer

[66] . AAV9-CMV-GFP was used as a negative control virus because it does not induce myocardial cytotoxicity and has been used successfully as a negative control virus in many gene therapy studies in animal models of cardiovascular disease

[67] . In the TAC study, adult male cardiac-specific Bin1 heterozygotes (Bin1 HT; Bin1 flox / + , Myh6-cre + ) and its wild type (WT; Bin1 flox / + , Myh6-cre -Either 100- or 100-week-old male C57BL / 6 mice (Jackson Laboratory) or 100- or 100-month-old littermates

[29] or adult male C57BL / 6 mice (Jackson Laboratory) were used. All mice were anesthetized at 8–10 weeks of age and subjected to open-chest TAC or mock surgery (Sham). For gene therapy, similar to the isoproterenol study, 3 × 10 AAV9 viruses transduced with cBIN1-V5 or GFP-V5 were administered 3 weeks before the start of TAC. 10 vg was injected retroorbitally into mice.

[0036] Isoproterenol minipump study. Fifty-six mice were injected with 3 × 10 of AAV9 transduced with V5-tagged GFP or cBIN1 via retroorbital injection while the mice were anesthetized with 1% isoflurane in oxygen. 10 Mice were randomized to receive a dose of vector genome (vg)

[68] . Three weeks after virus injection, mice were implanted with osmotic minipumps releasing isoproterenol or PBS (N=14 per group for each of the four test groups: AAV9-GFP+PBS, AAV9-GFP+ISO, AAV9-cBIN1+PBS, and AAV9-cBIN1+ISO). AAV9 was used in this study because AAV is the most promising gene therapy vehicle [21, 69] and AAV9 shows the highest cardiac tropism in mice (4–6). The CMV promoter was used because it has been established that AAV9-CMV can efficiently and safely deliver gene to the heart

[25] . AAV9-CMVGFP was used as a negative control virus because it did not induce cardiac injury or myocardial cytotoxicity [25-26] and GFP AAV9 has been successfully used as a negative control virus in numerous gene therapy studies using animal models of cardiovascular disease, including mouse models of hypertrophy and cardiomyopathy [26-29]. This protocol was repeated in a second set of animals. Similarly, 3 weeks prior to isoproterenol minipump implantation, 50 mice were injected with 3 × 10 of AAV9 transducing V5-tagged GFP, BIN1, BIN1+13, BIN1+17, or cBIN1 by retroorbital injection. 10Mice were randomized to receive vector genome (vg) (n=10 per group). Three weeks after AAV9 injection, mice were implanted with subcutaneous ALZET osmotic minipumps (Model 1004, Durect, Cupertino, CA, USA) that continuously release isoproterenol according to previously established procedures

[30] . Briefly, under light anesthesia with isoflurane inhalation, mice were implanted subcutaneously on the back with osmotic minipumps that continuously release 30 mg / kg / day of isoproterenol.

[0037] For cBIN1 deficiency studies, 8- to 10-week-old cardiac-specific Bin1 heterozygous deletion male mice (Bin1 HT; Bin1 flox / + , Myh6-Cre + ) and its wild type (WT; Bin1 flox / + , Myh6-Cre - TAC was performed on Bin1 HT and WT mice, which were generated as previously described. Specifically, heterozygous loxP-flanked Bin1 mice (loxP sites around exon 3 of the Bin1 gene) were transfected with Myh6-cre + Mice were bred to generate cardiomyocyte-specific Bin1 HT (N = 10) and WT littermate controls (n = 14). Genotypes were determined according to previously established methods: Bin1 + , Bin1 flox , and Cre + PCR was used to distinguish alleles. In the AAV9-mediated overexpression study, 5- to 7-week-old male C57BL / 6J mice (Jackson Laboratory) were transfected with AAV9 virus (3 × 10 10vg) was injected retroorbitally. Three weeks later, 8- to 10-week-old mice were anesthetized and subjected to open-chest TAC surgery. Age-matched mice subjected to open-chest mock surgery without TAC were used as sham controls (N=10). TAC was performed to induce pressure overload as previously described. Briefly, 8- to 12-week-old male mice were anesthetized by facemask administration of 3% isoflurane, intubated, and placed on a ventilator (Harvard Apparatus) with supplemental O2 and 1.5% isoflurane at a tidal volume of 0.2 mL and a respiratory rate of 120 breaths / min. The thoracic cavity was entered through a small incision in the second intercostal space at the superior border of the sternum, and aortic constriction was performed by tying a 7-0 nylon suture ligature against a 27-gauge needle between the first and second branches distal to the aortic arch. Buprenorphine (0.8 mg / kg) was administered subcutaneously for analgesia, and the mice were allowed to recover in a heated chamber with 100% O. Eight weeks after TAC, the animals were euthanized and tissues were harvested for analysis.

[0038] Adeno-associated virus 9 (AAV9) generation and administration. Five AAV9 vectors expressing GFP-V5, BIN1-V5, BIN1+13-V5, BIN1+17-V5, and cBIN1-V5 (BIN1+13+17-V5) driven by the CMV promoter were all custom-made and produced at Welgen, Inc. (Worcester, MA, USA). Previously published Gateway expression clones of V5-tagged GFP and mouse BIN1 isoforms

[31] were used and sequenced, then sent to Welgen for subsequent cloning into AAV vectors and virus preparation. These gene inserts (GFP-V5 or BIN1-V5) were then subcloned into the pAAV-CMV vector (Welgen, Inc., Worcester, MA, USA), and positive clones were selected by restriction enzyme digestion. pAAV-CMV-(GFP / BIN1)-V5 plasmid DNA was purified and sequenced. All AAV viruses were produced in HEK293 cells. Three plasmids, pAAV-CMV-(GFP / BIN1)-V5, pAAV-rep / cap9, and pHelper vectors, were transfected into 293 cells using polyethylenimine. After transfection, the supernatant and cells were harvested. AAV viruses were released from HEK293 cells by three freeze-thaw cycles. Virus in the medium was precipitated with PEG8000 (Sigma-Aldrich, St. Louis, MO, USA). The cell lysate and pelleted supernatant precipitate were combined and treated with Benzonase (Merck, Kenilworth, NJ, USA) for 1 h at 37°C. Viruses were purified by iodixanol gradient centrifugation and concentrated using Amicon Ultra-15 centrifugal filters (Sigma-Aldrich, St. Louis, MO, USA).

[0039] Echocardiography for functional rescue testing. In vivo systolic and diastolic left ventricular (LV) function was monitored by echocardiography in anesthetized mice using a Vevo 7700 at baseline, pre-surgery, and then biweekly until the end of the study protocol. Transaortic pressure gradients were measured 2 weeks after surgery using a modified Bernoulli equation (Δ pressure gradient (mmHg) = 4 × peak velocity 2 (m / s) 2 All mice surviving 5 weeks after TAC were included in the study.

[0040] Echocardiography for isoproterenol testing. Echocardiograms were recorded using a Vevo-3100 ultrasound system (Visual Sonics) equipped with a 70 MHz transducer. Protein interactions were analyzed by immunofluorescence imaging and biochemical coprecipitation. Peak intensities of Cav1.2 in t-tubules were quantified with Image J as previously described

[30] . Power spectrum analysis was performed using FFT transformation in Matlab [10, 30]. Intracellular protein distribution was analyzed by sucrose gradient fractionation as previously established

[70] . For calcium transient measurements, Cal-520-AM (AAT Bioquest) was used as previously described

[31] . For nearest-neighbor analysis between LTCC-RyR and SERCA2a-cBIN1 molecules, 3D super-resolution stochastic optical reconstruction microscopy (STORM) images were obtained

[31] .

[0041] Primary endpoints of severe HF-free survival vs. non-survival, and HF classification. Overall survival was analyzed in all groups. In addition, non-severe heart failure (HF)-survival was analyzed and compared between the AAV9-GFP and AAV9-cBIN1 groups. For severe HF-free survival, the primary endpoint was survival with an ejection fraction (EF) ≥ 35% as measured by echocardiography. Non-survival was death within 20 weeks after TAC or EF < 35%. At the end of the protocol, surviving TAC mice had tibial length (TL), lung weight (LW), and heart weight (HW) measured.

[0042] Immunofluorescence labeling and confocal imaging. For fluorescent labeling of cardiomyocyte membranes, freshly isolated ventricular cardiomyocytes from GFP-TAC and cBIN1-TAC mice were incubated with Di-8-ANNEPs for 20 min at room temperature (RT). Cells were then washed with HBSS to remove residual dye before live cell imaging. For fixed cell V5 imaging (10x), isolated cardiomyocytes were fixed in methanol at -20 °C for 5 min, permeabilized, and blocked with 0.5% Triton X-100 and 5% normal goat serum (NGS) in PBS for 1 h at RT. Cells were incubated with rabbit anti-V5 (Sigma) overnight at 4 °C and detected with Alexa555-conjugated goat anti-rabbit IgG. For tissue immunofluorescence imaging, frozen sections of myocardium were fixed in ice-cold acetone for 5 min. Primary antibodies used were mouse anti-BIN1-BAR (2F11, Rockland), mouse anti-RyR (Abcam), or rabbit anti-Cav1.2 (Alomone). After incubation with primary antibodies and several washes with 1× PBS, cells and tissue sections were incubated with Alexa488- or Alexa555-conjugated goat anti-mouse or rabbit secondary antibodies (Life Technologies) and mounted with ProLong gold containing DAPI. All confocal imaging was performed on a Nikon Eclipse Ti microscope equipped with a 100×1.49 numerical aperture (NA) and a 60×1.1 or 10× objective. High-resolution cardiomyocyte images were obtained using a spinning disk confocal unit (Yokogawa CSU10) using diode-pumped solid-state (DPSS) lasers (486 nm, 561 nm, 647 nm) generated from a Laser Merge Module 5 (Spectral applied research, CA). T-tubule membrane labeling fluorescence intensity profiles were generated using ImageJ, and peak intensities at T-tubules were quantified as previously described

[29] . Power spectrum analysis was performed using FFT transformation in Matlab, and normalized peak power densities at T-tubules were compared between groups [10, 30].

[0043] Immunofluorescence labeling and imaging by spinning disk confocal microscopy. Myocardial tissue sections were embedded in 100% OCT medium, flash frozen on dry ice with ethanol, stored in a −80°C freezer, and then sectioned at 10 μm as previously described

[32] . After fixation with acetone, tissue cryosections were permeabilized with 0.1% Triton X-100 and 5% normal goat serum (NGS, Life Technology) in PBS for 1 h at RT. For staining of V5, CaV1.2, and SERCA2a, tissue sections were incubated overnight at 4°C with rabbit anti-V5 (1:500, Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-CaV1.2 (1:250, Alomone Labs, Jerusalem, Israel), or mouse anti-SERCA2a (1:250, Abcam, Cambridge, MA, USA) primary antibodies. After several washes with 1× PBS, tissue sections were incubated with goat anti-mouse and anti-rabbit IgG conjugated to Alexa 4#88 and 555, respectively. Tissue sections were mounted with Prolong® Gold medium containing DAPI. All imaging was performed on a Nikon Eclipse Ti microscope equipped with a total internal reflection fluorescence objective with a numerical aperture of 40×1.1 or 100×1.49 and NIS Elements software (Nikon, Los Angeles, CA, USA). Z-step stacks of 0.5 μm were collected on a spinning disk confocal unit (Yokogawa CSU10, Sugar Land, Taxas, USA) connected to the same Ti microscope using diode-pumped solid-state lasers (486 nm, 561 nm) generated by a Laser Merge Module 5 (Spectral Applied Research, Richmond Hill, Ontario, Canada) and captured with a high-resolution ORCA-Flash 4.0 digital CMOS camera. T-tubule Cav1.2 fluorescence intensity profiles were generated using ImageJ, and peak intensities in t-tubules were quantified as previously described

[30] . Calcium transients were performed according to a previously described protocol

[31] .Briefly, freshly isolated cardiomyocytes were loaded with 10 μmol / L Cal-520-AM (AAT Bioquest) in 0.4% Pluronic F-127 in normal Tyrode's buffer for 30 min. After three washes with buffer containing 1 mmol / L probenecid, cells were placed in an imaging chamber and paced at 1 Hz with a field stimulator (Ionflux). Images were collected at 67 fps using a spinning disk confocal microscope and analyzed with Nikon Element Software. F0 (baseline fluorescence) and F. max The fluorescence signal of (maximal fluorescence at the peak of the calcium transient) was first background corrected and then ΔF / F = (F max The ratio (-F0) / F0 was calculated and compared between groups.

[0044] Power spectrum analysis. Frequency domain power spectra of cardiomyocyte immunofluorescence subsections were generated in Matlab using FFT transformation [10, 30]. Power spectra normalized to the maximum component were generated and plotted over distance (1 / frequency, μm). Normalized peak power density

[71] was quantified and compared between groups.

[0045] Super-resolution stochastic optical reconstruction microscopy (STORM) imaging and nearest neighbor analysis. For STORM imaging, cardiomyocytes were prepared as previously reported

[31] . On the day of imaging, fresh STORM imaging buffer (0.5 mg / mL glucose oxidase, 40 μg / mL catalase, 10% glucose with mercaptoethylamine) was added to the dish. STORM images were collected on a Nikon Eclipse Ti microscope equipped with lasers (488 nm, 561 nm from a self-contained 4-line laser module with acousto-optic tunable filters) and captured with a high-speed iXon DU897 Ultra EMCCD camera. Images were acquired and analyzed using the STORM module to generate three-dimensional (3D) projections of Cav1.2 / RyR and cBIN1 / SERCA2a images with nanoscale resolution. For nearest neighbor analysis, native 3D STORM images were displayed with the Gaussian rendering algorithm available in Nikon Elements software, and 3D stacks of 3D STORM images (two channels in one acquisition, Cav1.2 / RyR or cBIN1 / SERCA2a) were acquired at a depth of 500 nm with a z-spacing of 10 nm in molecule list text file format. The molecule list text file was loaded in ImageJ, and the nearest neighbor distance between molecules from the two channels (nearest neighbor distance) was calculated. The nearest neighbor distance was constructed and displayed as a frequency distribution histogram with a user-defined range and bin width, and the first peak value was detected and fitted with a 15th order polynomial curve. The distance between Cav1.2-RyR and SERCA2a-cBIN1 molecules at the corresponding first peak positions was quantified and compared between groups.

[0046] Transmission electron microscopy. All transmission electron microscopy (TEM) work was performed by the core facility of the Electron Imaging Center at The California NanoSystems Institute at UCLA. Tissue preparation was performed using a previously reported method

[72] . Briefly, mouse hearts were perfused with 20 mL of fresh fixative solution (2% glutaraldehyde and 2% paraformaldehyde in 1x PBS). Left ventricular tissue (1 mm 3) were post-fixed in 1% osmium tetroxide and incubated in 3% uranyl acetate. After dehydration in ethanol, the specimens were processed in propylene oxide, embedded in Spurr resin (Electron Microscopy Services), and sectioned using an ultramicrotome (Leica). Sections were mounted on grids and stained with uranyl acetate and lead citrate, after which images were acquired using a JEM1200-EX, JEOL microscope (Gatan). The degree of contoured t-tubules was quantified using a modified scoring system established previously

[29] .

[0047] Western blotting for functional rescue studies. Tissue lysates were made from hearts flash frozen in liquid nitrogen. Frozen tissues were homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer as previously described

[41] . Lysates were rotated head-to-toe for 40 min at 4 °C, sonicated, and then centrifuged (16,000 × g, 25 min at 4 °C) to remove cellular debris. Protein lysates were then prepared in 2 × sample buffer containing 5% β-mercaptoethanol (Bio-Rad, Hercules, CA), incubated for 30 min at RT, and resolved on an 8–12% gradient sodium dodecyl sulfate (SDS) polyacrylamide electrophoresis gel. Proteins were electrophoresed onto polyvinylidene difluoride (PVDF) membranes. After transfer, membranes were fixed with methanol, blocked with 5% BSA in 1× Tris-buffered saline (TBS) for 1 h at RT, and incubated with primary antibodies in 5% BSA in 1× TBS overnight at 4°C, followed by incubation with Alexa 647-conjugated secondary antibodies (Life Technology) for 1 h at RT. Primary antibodies consisted of custom-made polyclonal rabbit anti-BIN1 exon 13 (Anaspec)

[29] , mouse anti-RyR (Abcam), rabbit Cav1.2 antibody (Alomone), and mouse anti-GAPDH (Millipore).

[0048] Western blotting for isoproterenol testing. Frozen cardiac tissue was homogenized using RIPA lysis buffer with protease inhibitors, and protein concentration was determined using the Bradford assay. Samples were separated on NuPAGE™ Novex™ 4–12% Bis-Tris Protein Gels and then transferred to polyvinylidene difluoride membranes. After blocking with 5% bovine serum albumin (BSA) in 1× TNT buffer for 1 h, membranes were incubated with primary antibodies including rabbit anti-GAPDH or actin (Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-CaV1.2 (Alomone Labs, Jerusalem, Israel), or mouse anti-SERCA2a (Abcam, Cambridge, MA, USA) overnight at 4°C, followed by incubation with secondary antibodies (goat anti-rabbit or mouse IgG-Alexa 647) for 1.5 h at room temperature (RT). Immunoreactive bands were imaged with a Molecular Imager® Gel Doc™ XR+ System (Bio-Rad Laboratories, Irvine, CA, USA) and band intensity was quantified with Image Lab software (Bio-Rad Laboratories, Irvine, CA, USA).

[0049] Preparation of cardiac microsomes and sucrose gradient fractionation. Sucrose gradient fractionation of microsomes was prepared according to an established protocol with modifications

[70] . Myocardial membrane microsomes were prepared from one starting heart for each study group. Frozen cardiac tissue was homogenized using a Polytron handheld homogenizer in 2 mL of homogenization buffer (20 mM Tris pH 7.4, 250 mM sucrose, 1 mM EDTA supplemented with HALT protease inhibitors). The homogenate was then centrifuged at 12,000 × g (Beckman) for 20 min at 4 °C, and the supernatant (S1) was collected in a pre-weighed tube and stored on ice. The pellet was suspended in 1 mL of the same buffer, homogenized, and centrifuged at 12,000 × g for 20 min at 4 °C. The supernatant (S2) was collected and combined with S1 from the previous step. The combined microsomal supernatants (S1+S2) were then subjected to ultracentrifugation at 110,000 × g for 2 h at 4 °C. After ultracentrifugation, the supernatant was discarded, the pellet was weighed, and an appropriate amount of buffer (~1 mL) was added to ensure a final concentration of microsomes of ~25 mg / mL. The total protein concentration in the resuspended microsomes was measured using a Nanodrop 2000 for each sample and normalized among the four groups. Equal amounts of total microsomes from each sample (3–6 mg in 0.5 mL) were carefully placed on top of a discontinuous sucrose gradient [52, 58, 73] and 45%, v / w homogenizer buffer, 2 mL each) and ultracentrifuged at 150,000 × g for 16 h in a fixed-angle MLA-55 rotor using a Beckman Coulter Optima Max XP Benchtop Ultracentrifuge. Samples were then collected from the following fractions: F1, 27%; F2, 27 / 32%; F3, 32 / 38%; and F4, 38 / 45%; as well as the pellet from the bottom of the tube (P). For each fraction, approximately 1 mL was taken, diluted 4-fold with homogenization buffer, and ultracentrifuged at 120,000 × g, 4 °C for 2 h. The pellet was resuspended in 100 μL of homogenization buffer, and protein concentration was measured with a Nanodrop 2000. The yield of total protein collected from each fraction, F1, F2, F3, and F4, was 0.001-0.02, 0.4-0.8, 0.04-0.06, and <0.008 mg per heart, respectively.Sample buffer was added and samples were frozen and stored at -20°C prior to subsequent Western blot analysis.

[0050] Statistical analysis. All data are expressed as mean ± standard error of the mean (SEM) or standard deviation (SD) as specified. Normality was assessed using the Shapiro-Wilk test. Kaplan-Meier survival analysis was used, comparisons were made between two groups using the log-rank test and between three groups using the log-rank trend test. Continuous variables were compared using t-tests / Mann-Whitney U and one-way analysis of variance (ANOVA) / Kruskal-Wallis tests. Two-way ANOVA was used to determine differences between two groups at two different time points. Two-way ANOVA was used to determine differences between two AAV9 groups with different drug infusions, followed by Fisher's least significant difference (LSD) post-hoc adjustment for multiple pairwise comparisons. Categorical variables were analyzed using Fisher's exact test or chi-square test. Data were analyzed using GraphPad Prism (version 7.0; GraphPad Software, La Jolla, CA, USA). Two-tailed p values ​​were used, with p<0.05 considered statistically significant.

[0051] Example 2 Functional rescue by exogenous cBIN1 in mouse hearts with pressure overload-induced heart failure To investigate whether targeting cBIN1-microdomains could be a novel therapeutic approach for HF, we investigated how cardiac cBIN1 affects HF development in mice subjected to pressure overload stress. We performed transaortic aortic constriction (TAC) or mock surgery (sham) in 8-10 week-old adult male mice, after which we measured overall survival as well as severe systolic HF survival (death or non-survival as ejection fraction, EF < 35%) by echocardiographic monitoring. As shown in the experimental protocol in Figure 1A, mice were first subjected to TAC for 5 weeks before retro-orbital injection of AAV9 transduced with cBIN1-V5 or control GFP-V5, followed by echocardiographic monitoring for an additional 15 weeks after virus injection (20 weeks after TAC). In addition to the group of mice that underwent open-chest mock surgery (sham control, N = 10), 36 mice were subjected to TAC surgery. One mouse died before reaching time point 0 (5 weeks after TAC), and the remaining 35 surviving mice were 3 × 10 10 vg AAV9-GFP (N=17) or cBIN1 (N=18). Anti-V5 labeling of cardiomyocytes isolated from mice 15 weeks after AAV9 injection identified a positive V5 signal, indicating successful transfer of exogenous protein in cardiomyocytes (Figure 16). Comparable transaortic pressure gradients at 2 weeks after TAC (Figure 1B) and myocardial dysfunction at 5 weeks after TAC were observed in these mice, as evidenced by decreased left ventricular (LV) EF with increased LV end-diastolic volume (EDV) before AAV9 injection (Table 1).

[0052] [Table 1]

[0053] Next, we investigated the overall survival rate (non-survival as death) of all groups. As shown in the Kaplan-Meier curves in Figure 2, the overall survival of AAV9-cBIN1-treated TAC mice (survival rate 77.8%, 14 / 18) is located in the middle of that of sham (survival rate 100%, 10 / 10) and AAV9-GFP-treated TAC mice (survival rate 58.8%, 10 / 17) (p=0.0202 by log-rank test for trend when comparing among the three groups) (Figure 2A). Next, we further analyzed asystolic HF survival (death or non-survival as EF<35%) between the two virus groups during follow-up echocardiographic monitoring in all surviving TAC mice with EF≥35% at time point 0 (5 weeks after TAC, before virus injection). Asystolic HF survival is significantly improved by AAV9-cBIN1 (p=0.0225 by log-rank test when compared to the AAV9-GFP group) (Figure 2B). Of the 11 AAV9-GFP mice with EF ≥ 35% at time 0, 9 developed EF < 35% within 20 weeks after TAC, 2 died early, and 7 had progressive EF loss. Meanwhile, of the 13 AAV9-cBIN1 mice with EF ≥ 35% at time 0, only 5 developed EF < 35% within 20 weeks after TAC, and 1 died early. Furthermore, of these 5 mice counted as non-survivors in Kaplan-Meier curve analysis, 3 of the remaining 4 non-survivor AAV9-cBIN1-treated animals, excluding the 1 that died early, recovered EF ≥ 35% at 20 weeks after TAC. Surviving mice were then sacrificed and the ratios of HW / TL and LW / TL were evaluated. AAV9-cBIN1 mice did not have a significant increase in HW / TL and LW / TL as occurred in AAV9-GFP mice when compared with sham control mice (Figure 3A-B). These data indicate that cBin1 gene therapy protects cardiac function, effectively increases overall and HF-free survival, and prevents, postpones, or even reverses the worsening cycle of HF progression.

[0054] Among all mice that survived at 20 weeks after TAC, myocardial function and physiological parameters measured by echocardiography were further compared between groups both before and after AAV9 treatment (Table 1). At 20 weeks after TAC, AAV9-GFP mice developed significant LV systolic dysfunction (reduced EF) and chamber dilation (elevated EDV, Figure 4A-C, Table 1), which were normalized by AAV9-cBIN1 treatment. The increase in LV mass at 20 weeks after TAC was significantly reduced in AAV9-cBIN1-treated mice compared to the AAV9-GFP group (Figure 4D). Furthermore, the Δ decrease in stroke volume and cardiac output observed in the AAV9-GFP group was also reduced in the AAV9-cBIN1-treated group (Figure 4E-F). These results indicate that cBin1 gene therapy preserves myocardial function when administered to failing hearts.

[0055] To further examine the progression of contractile dysfunction in the post-TAC heart following virus injection at time point 0 (pre-AAV9, 5 weeks post-TAC), the ΔEF change (ΔEF) from pre-AAV to 3, 6, 8, 10, and 15 weeks post-AAV9 injection (corresponding to 8, 11, 13, 15, and 20 weeks post-TAC) was monitored by echocardiography (Figure 5A). Exogenous cBIN1-induced EF recovery peaked at 6–8 weeks post-AAV9 injection, with continued improvement in EF in the following weeks, whereas a progressive EF decrease was observed in the AAV9-GFP group. The observed EF recovery was illustrated on the histogram distribution of ΔEF by Gaussian fitting (Figure 5B–C). The AAV9-cBIN1 had a right-shifted histogram distribution of ΔEF compared to the AAV9-GFP group. For example, at 6 weeks after AAV9, a moderate decrease in EF (%) of -15.0 was observed in the AAV9-GFP group, whereas a moderate recovery in EF (%) of +6.9 was observed in the AAV9-cBIN1 group. These data indicate that exogenous cBIN1 can be administered 5 weeks after TAC to rescue myocardial contractile function in hearts with TAC-induced HF.

[0056] We recently reported that AAV9-cBIN1 pretreatment (3 × 10 10reported that the incidence of TAC-induced HF was significantly reduced in mice receiving exogenous AAV9 (vg; Figure 6A), resulting in improved HF-free survival at 8 weeks after TAC

[34] . These data are consistent with the myocardial protection observed when AAV9 was administered after TAC surgery. To further establish the cardioprotective effect of exogenous-cBIN1 in TAC mice, intracardiac hemodynamics were obtained in AAV9-pretreated mice using invasive PV loop recordings. Figure 6B contains representative PV loops of sham, AAV9-GFP, and AAV9-cBIN1 pretreated hearts 8 weeks after TAC surgery (Figure 6B). Systolic EF and maximum velocity of pressure change (dp / dt max) were decreased in the AAV9-GFP group, which were normalized by exogenous cBIN1 (Figure 6C-D). When investigating the effect on relaxation kinetics, it was found that the maximum rate of pressure decay (dp / dt min) was decreased in the AAV9-GFP group but normalized by AAV9-cBIN1 (Figure 6E). The increase in the time constant of isovolumic relaxation (Tau) in the AAV9-GFP group was also rescued by cBin1 gene transfer (Figure 6F), indicating that cardiac relaxation was improved. Together, these data indicate that exogenous cBIN1 improves cardiac inotropy and lucitropy in pressure-overloaded hearts.

[0057] We previously demonstrated that cBIN1 forms t-tubule microdomains, organizes LTCC-RyR dyads, and efficiently and dynamically regulates cardiac function and EC coupling

[25] . More recently, we found that cBIN1-microdomains are disrupted in mouse hearts with sympathetic overdrive diastolic dysfunction and are rescued by AAV9-cBIN1. Here, we also investigated the changes in cardiac t-tubule cBIN1-microdomains after TAC and the effects of exogenous cBIN1. Western blotting (Figure 7A) identified that myocardial cBIN1 protein was significantly decreased in mouse hearts 8 weeks after TAC (27% decrease from sham control, p<0.05), which was normalized by AAV9-cBIN1 pretreatment. In conjunction with cBIN1 rescue, membrane labeling with Di-8-ANNEPs (Figure 17) identifies that compared to sham cardiomyocytes, 8 weeks post-TAC cardiomyocytes have significantly reduced t-tubule cBIN1-microdomain intensity, which is normalized in cardiomyocytes from mice with AAV9-cBIN1 pretreatment. We next used immunofluorescence imaging to analyze the organization of cBIN1-microdomains and bimolecules at the cardiomyocyte level. Power spectrum analysis of BIN1 signals identified that the well-organized t-tubule distribution of cBIN1 in sham cardiomyocytes was disrupted in hearts after TAC, which was preserved by AAV9-cBIN1 pretreatment (Figure 7C). Although total LTCC and RyR protein levels are not significantly altered between groups by Western blotting (Figure 7B), myocardial distribution of LTCC and RyR (Figure 7C) is disorganized in post-TAC hearts, which also significantly improves in AAV9-cBIN1 pretreated hearts (p<0.05). These data indicate that exogenous cBIN1 normalizes TAC-induced myocardial cBIN1 reduction and leads to preservation of microdomains created by cBIN1 in t-tubules. Thus, cBIN1 replacement therapy reorganizes cardiac LTCC-RyR coupling required for calcium cycling and efficient EC coupling for heart rate sensing by normalizing t-tubule microdomains in pressure-loaded hearts.

[0058] Example 3 Exogenous cBIN1 reduces concentric hypertrophy in mouse hearts following isoproterenol infusion We investigated the effect of cBIN1 on myocardial function in animals that received 4 weeks of isoproterenol infusion (Figure 8A). AAV9 was used to introduce myocardial expression of exogenous V5-tagged GFP or cBIN1

[74] 3 weeks prior to the start of isoproterenol. Anti-V5 labeling identified similar percentages of myocardial areas with detectable V5 signal at 7 weeks after AAV9 injection (GFP, 62.4 + 10.5%; cBIN1, 57.9.2 + 7.8%), indicating successful introduction of exogenous protein in more than half of the cardiomyocytes (Figure 18). The remaining approximately 40% of negatively stained cardiomyocytes may express exogenous protein at low levels below the detection threshold of immunofluorescence. In all mice, isoproterenol significantly increased heart weight to body weight ratio (HW / BW), indicative of cardiac hypertrophy (Figure 8B). Cardiac geometry and function were assessed by echocardiography (Figure 8C-H). In AAV9-GFP pretreated animals, isoproterenol induced a significant increase in LV mass and relative wall thickness (RWT) without changing end-diastolic volume (EDV), consistent with echocardiographic classification of concentric hypertrophy

[75] . In AAV9-cBIN1 pretreated animals, isoproterenol increase in LV mass was attenuated with normal RWT and increased EDV, similar to the echocardiographic classification of "physiologic hypertrophy-like LV remodeling" using a previously reported method

[75] (Figure 19A). Furthermore, α-smooth muscle actin was increased in GFP+ISO hearts, but not in cBIN1+ISO hearts (Figure 19B), indicating that AAV9-cBIN1 limits isoproterenol-induced LV hypertrophy. In AAV9-GFP pretreated mice, isoproterenol led to a small increase in EF (p=0.050 vs. GFP+PBS and p=0.007 vs. cBIN1+PBS groups), but a strong increase in the onset of LV passive filling (E) to myocardial relaxation (e') ratio (E / e'), suggesting the onset of diastolic dysfunction. In AAV9-cBIN1 pretreated mice, isoproterenol still increased systolic function and, importantly, E / e' remained normal, indicating positive inotropy while maintaining lucitropy.Furthermore, although blood pressure measurements were not performed, isoproterenol significantly increased heart rate (HR) in all animals, indicating the effectiveness of isoproterenol in causing hemodynamic stress. However, HR after isoproterenol did not differ between GFP+ISO and cBIN1+ISO mice (Table 2), confirming that the further improvement in cardiac output after isoproterenol in AAV9-cBIN1 mice was due to muscle efficiency and not due to an increase in heart rate.

[0059] [Table 2]

[0060] Example 4 cBIN1-microdomains disrupted by chronic isoproterenol can be normalized by AAV9-cBIN1 It is well known that both myocardial inotropy and lucitropy are linked to calcium cycling in cardiomyocytes

[76] . cBIN1, a structural organizer of bilayer microdomains

[31] , mediates the regulation of extracellular Ca 2+It creates t-tubule microfolds that limit diffusion

[29] , promote microtubule-dependent forward trafficking of L-type calcium channels (LTCCs)

[30] and clustering of LTCCs already delivered to the t-tubule membrane. We therefore investigated how cBIN1-microdomains might remodel in the hypertrophied heart after chronic isoproterenol infusion. Western blots of cardiac lysates show that isoproterenol induces a marked decrease in cBIN1 protein, which is normalized by AAV9-cBIN1 (Figure 9A). Note that immunoprecipitation with anti-BIN1-exon 17 antibody followed by western blot detection with anti-BIN1-exon 13 antibody confirms that the protein band examined is the cBIN1 (BIN1+13+17) isoform. The overall t-tubule network organization was also examined in isolated cardiomyocytes labeled with the membrane dye Di-8-ANNEPs (Figure 9B). Live cell imaging followed by power spectrum analysis showed that overall t-tubule organization (normalized peak power density) was similar in GFP+ISO mice and increased by AAV9-cBIN1. Using transmission electron microscopy (TEM) imaging, it was noted that even though the total t-tubule network remained organized, there was a reduction in t-tubule microfolds in GFP+ISO hearts, which was retained in cBIN1+ISO hearts (Figure 9C). Quantification of the extent of t-tubule contour using a previously established modified scoring system

[29] (1, circular or dilated t-tubule lumen without folds; 2, t-tubule lumen with non-circular contour without folds; 3, t-tubules with 2-3 layers of folds; or 4, t-tubules with >3 layers of folds) confirmed a significant reduction in t-tubule contour in GFP+ISO hearts, which was normalized in cBIN1+ISO hearts (p<0.001, chi-square test). Note that in cBIN1+PBS hearts, microfolds are exaggerated (>3 layers of folds, score 4) as a result of the presence of cBIN1 at greater than physiological levels.These data indicate that cBIN1 is important for the formation of t-tubule microfolds, that folding is downregulated under chronic sympathetic overdrive, and that exogenous cBIN1 therapy restores folding.

[0061] We subsequently explored the expression and subcellular distribution of Cav1.2 in cardiomyocytes. In post-isoproterenol hearts, net myocardial protein expression of Cav1.2 was comparable (Figure 10A). However, myocardial tissue immunofluorescence labeling of Cav1.2 revealed that channel density along t-tubules was significantly reduced in GFP+ISO cardiomyocytes, which was normalized by AAV9-cBIN1 (Figure 10B-C), power spectrum and fluorescence profile analysis). These data are consistent with previous observations of altered Cav1.2 protein distribution despite similar total protein levels

[32] . Concomitant with the reduced Cav1.2 localization to t-tubules, the peak amplitude of calcium transients (ΔF / F0) was significantly reduced in GFP+ISO cardiomyocytes when compared to those from control GFP+PBS cardiomyocytes (Figure 10D), which was normalized by exogenous cBIN1. Rhinodin receptor 2 (RyR) total protein expression and subcellular distribution did not differ between all groups (Figure 20). However, RyR became hyperphosphorylated at both PKA-dependent S2808 and CAMKII-dependent S2814, consistent with previous reports

[61] . Together with increased phosphorylation of CAMKIIδ at T287, these data suggest that PKA- and CAMKII-activation-induced RyR hyperphosphorylation occurs after chronic isoproterenol infusion. Importantly, AAV9-cBIN1 pretreatment successfully blunted these pathways and reduced RyR hyperphosphorylation (Figure 21).

[0062] Example 5 Exogenous cBIN1 improves the distribution of SERCA2a along the SR Cardiac lucitropy is most directly linked to calcium reabsorption via SERCA2a. Surprisingly, despite impaired diastolic dysfunction in GFP+ISO hearts, total protein expression of SERCA2a was significantly increased after isoproterenol infusion (Figure 11A-B). Total protein levels of phospholamban (PLN) and its phosphorylated forms (pS16 and pT17) were unchanged (Figure 21). Previous studies have shown that acute isoproterenol-induced PLN phosphorylation is normalized after chronic isoproterenol infusion and that PLN dephosphorylation can also occur through activation of serine / threonine phosphatases PP1 and PP2A [73, 77]. Consistent with these reports, our results demonstrated that the lack of change in PLN phosphorylation after 4 weeks of isoproterenol infusion may be the net result of balanced local activation of both kinases and phosphatases. These data indicate that both SERCA2a protein and activity are not decreased in the post-isoproterenol hearts. Given the effect of cBIN1 on Cav1.2 localization, we examined the localization of SERCA2a. SERCA2a-labeled myocardial tissue sections were imaged by spinning disk confocal microscopy and compared between groups (Figure 11C). In GFP+PBS hearts, a subpopulation of SERCA2a was concentrated in the t-tubule / jSR region, giving rise to a systematic distribution with a major power spectrum peak at 1.8-2 µm, corresponding to the total length of the sarcomere. Overexpressing cBIN1 in cBIN1+PBS hearts further increased the SERCA2a signal near the t-tubule / jSR. In GFP+ISO hearts, the subcellular distribution of SERCA2a was disrupted with a marked decrease in peak power density, which was normalized in cBIN1+ISO hearts (quantification in Figure 11D).

[0063] The intracellular distribution of Cav1.2 and SERCA2a was further explored using biochemical sucrose gradient-based fractionation of cardiac microsomes

[70] . As shown in Figure 22A, fraction F4 has the lowest recovery yield compared to other fractions. However, even with the low yield, Cav1.2 and cBIN1 were able to utilize the limited Na + / K +SR protein was detectable in F4 along with -ATPase and depleted SERCA2a, indicating that F4 is enriched in t-tubule-derived microsomes (Figure 22B). When normalizing t-tubule protein concentration for F4 across all samples (2.5 μg protein loaded per lane), GFP+ISO hearts had a significant decrease in both cBIN1 and Cav1.2 protein per unit t-tubule compared to control GFP+PBS hearts, which was normalized by AAV9-cBIN1 pretreatment (Figure 12A). These data are consistent with immunofluorescence imaging, which identified a decrease in t-tubule localization of Cav1.2 channels after isoproterenol infusion and restoration by AAV9-cBIN1. On the other hand, SR protein was only detected in fractions F2 and F3. Normalization of SR protein concentration for F2 and F3 (25 μg protein loaded per lane) showed that F3 was relatively richer in RyR and less PLN than F2 (Figure 12B), with jSR enrichment toward the heavier F3 fraction. Quantification of SERCA2a expression in F2 and F3 showed that, when compared to AAV9-GFP, AAV9-cBIN1 caused a significant increase in SERCA2a distribution toward the heavier, more jSR-rich F3, but not the longitudinal SR-rich F2 fraction (Figure 12B). Of note, isoproterenol alone increased SERCA2a expression in F3 of AAV9-GFP mouse hearts, likely due to an overall increase in total protein expression of SERCA2a in the heart after isoproterenol (Figure 11A). These data indicate that in the heart after isoproterenol infusion, exogenous cBIN1 can maintain t-tubule microdomains to localize Cav1.2 and SERCA2a to their functional sites.

[0064] Given the reduction of Cav1.2 and SERCA2a in the t-tubule / jSR region, we analyzed the nanoscale protein-protein colocalization of Cav1.2-RyR and SERCA2a-cBIN1 using STORM imaging (Figure 13). Nearest-neighbor analysis quantified the distance between individual Cav1.2 molecules and their nearest RyR molecules. Histogram distribution of the distance between Cav1.2-RyR molecules from whole-cell images identified the first peak at around 40 nm in GFP+PBS, GFP+ISO, and cBIN1+ISO cardiomyocytes, which corresponds to a bimolecular couplet. In GFP+ISO hearts with preserved contractile function, the distribution histogram tended to shift to the right, but the position of the first peak remained maintained (Figure 13B). Interestingly, cBIN1+PBS myocytes had a left-shifted histogram distribution and a significantly decreased Cav1.2-RyR peak distance, indicating a closer coupling that is likely brought closer by the exaggeration of the cBIN1-microfold as observed by TEM imaging. Meanwhile, the distance between SERCA2a and its nearest neighbor cBIN1 in the t-tubules showed a tendency to increase after isoproterenol in AAV9-GFP pretreated animals (p=0.063, GFP+PBS vs. GFP+ISO), which was significantly decreased in AAV9-cBIN1 pretreated animals (p<0.001, GFP+ISO vs. cBIN1+ISO) (Figure 13C-D). These data indicate that the cBIN1-microfold may regulate the colocalization and interaction between EC coupling and calcium handling proteins.

[0065] Example 6 The cBIN1+ISO cardiac phenotype is isoform-specific and unique to cBIN1 To further investigate whether the observed phenotype of cBIN1+ISO hearts was an isoform-specific effect, we repeated the isoproterenol protocol in over 50 mice randomized to receive AAV9 transduced with GFP and cBIN1, as well as three other mouse cardiomyocytes expressing BIN1 isoforms, including small BIN1, BIN1+17, and BIN1+13. Similarly, three weeks after viral administration, mice were continuously subcutaneously infused with isoproterenol at 30 mg / kg / day for four weeks. Protein expression of Cav1.2 and SERCA2a in the heart after isoproterenol was not significantly different when compared among the five groups of mice transduced with GFP or BIN1 isoforms (Figure 23A). Myocardial tissue immunofluorescence labeling of Cav1.2 channels revealed that channel density along t-tubules was significantly increased only in cBIN1-expressing hearts, but not in other BIN1 isoforms (Figure 23B, quantification shown in Figure 23D). Immunofluorescence imaging revealed that exogenous cBIN1 introduced by AAV9 organized SERCA2a distribution (FIG. 23C, quantification shown in FIG. 23E), consistent with the data from FIG.

[0066] Next, the functional consequences of the different AAV9-BIN1 isoform pretreatment were explored using echocardiography. cBIN1-expressing mice reduced isoproterenol-induced increases in LV wall thickness, LV mass, and RWT when compared to the GFP group (Figure 14A-D, Table 3). In all animals, cardiac output after isoproterenol was significantly increased from baseline levels, which was a consequence of the isoproterenol-induced increase in cardiac RWT (Table 3). However, only cBIN1 hearts also had improved contractile function, normalized E / e', increased stroke volume, and further increased cardiac output when compared to GFP hearts after isoproterenol (Figure 14E-H). Of note, a partial cBIN1-like effect occurred in mice pretreated with BIN1+17, which significantly reduced LV mass, E / e', and reduced the isoproterenol-increased RWT. The partial diastolic function rescue observed from BIN1+17 was consistent with the partial rescue of SERCA2a subcellular distribution by immunofluorescence imaging (Figure 23C). However, no positive inotropic effect was observed in AAV9-BIN1+17 pretreated hearts after isoproterenol infusion, due to the inability of BIN1+17 to increase Cav1.2 distribution in t-tubules.

[0067] [Table 3]

[0068] Example 7 The myocardial protective effect of AAV9-cBIN1 in TAC-induced HF is confirmed The myocardial protective effect of cBIN1 was further investigated in another mouse model of pressure overload induced by TAC. Mice with either genetic deficiency of cBIN1 or AAV9-transduced cBIN1 overexpression were tested in this study (Figure 15A). Deficiency studies included cardiac-specific Bin1 HT mice and WT littermate controls

[29] , both of which were subjected to TAC for 8 weeks. Overexpression studies included mice subjected to 8 weeks of TAC pre-injected with AAV9 transduced with cBIN1-V5 or AAV9-GFP-V5, and mice subjected to open-chest mock surgery (sham). Mice were monitored and terminated 8 weeks after surgery. Virus (AAV9-GFP / cBIN1-V5), dose (3 × 10 10 vg), time of administration (3 weeks prior to surgery), and route (retroorbital injection) were the same as those used in the isoproterenol study. Aortic constriction in all TAC mice was confirmed by an elevated transaortic pressure gradient (Figure 15B), establishing a similar increase in hemodynamic afterload in all mice undergoing TAC. Kaplan-Meier curves summarizing the severe HF-free (EF ≥ 35%) survival rate were included in Figure 15C. The survival rate of Bin1 HT mice was 20.0% (8 of 10, 2 dead, 6 EF < 35%), reduced from 71.4% (4 of 14 non-survivors, 1 dead, 3 EF < 35%) in WT mice (p = 0.038 by log-rank test). As expected, all sham mice survived throughout the entire experimental protocol (10 of 10, black dotted line). Survival decreased to 64.3% in AAV9-GFP mice (5 of 14 non-survivors, 5 EF < 35%), which significantly improved to 93.7% in the AAV9-cBIN1 group (1 of 16 non-survivors, 1 EF < 35%) (p = 0.020 by log-rank test). These data indicate that higher cBIN1 protein content in the heart is associated with better survival without developing systolic HF after pressure overload.

[0069] At 8 weeks after TAC, surviving mice were sacrificed and evaluated for HW / BW and LW / BW ratios (Table 4, Figure 15D-E). Bin1 HT mice had significantly higher HW / BW and LW / BW than WT mice, suggesting that BIN1 deficiency exacerbated LV hypertrophy and pulmonary edema. Regarding gene therapy, AAV9-cBIN1 significantly reduced LW / BW from the control GFP-TAC group to the level of sham hearts, representing a marked reduction in TAC-induced pulmonary edema. AAV9-cBIN1 hearts still experienced hypertrophy, albeit to a lesser extent. These data indicated that exogenous cBIN1 reduced TAC-induced hypertrophy and prevented progression to HF. Furthermore, echocardiographic analysis (Figure 15F-J, Table 5) revealed that Bin1 HT-TAC hearts had a significant decrease in EF and an increase in EDV compared with WT-TAC mice, suggesting that BIN1 deficiency aggravated dilated cardiomyopathy. Meanwhile, AAV9-cBIN1 significantly reduced TAC-induced LV dilation (EDV) and systolic dysfunction (EF), limiting the development of dilated cardiomyopathy. As a result, pretreatment with AAV9-cBIN1 maintained stroke volume and cardiac output without dilating the heart after TAC. Furthermore, tissue Doppler revealed that the diastolic parameter E / e' values ​​of both the lateral and septal walls were significantly improved in AAV9-cBIN1 pretreated hearts, indicating that diastolic function is improved in exogenous cBIN1 mice. These data indicate that cBin1 gene therapy preserves myocardial systolic and diastolic function in stressed hearts and effectively prevents the development of dilated cardiomyopathy in mouse hearts exposed to pressure overload.

[0070] [Table 4]

[0071] [Table 5]

[0072] Example 8 Mouse diabetic cardiomyopathy (HFpEF) study We investigated myocardial function and the therapeutic potential of AAV9-cBIN1 in db / db mice with diabetic cardiomyopathy. The Db / db mouse strain (Leprdb homozygous Dock7m, Jackson Laboratories), which has been established as a mouse model of type 2 diabetes, has been used as a model of diabetic cardiomyopathy and heart failure with preserved ejection fraction (HFpEF)

[78] . Nine-week-old male and female db / db mice and their control littermate db / m mice were administered one dose (1 × 10 11 vg) of AAV9-cBIN1 or control GFP was administered by retro-orbital injection [79, 80]. Cardiac function and physiological parameters were measured both before AAV injection and 8 weeks after injection, when the animals were 17 weeks of age

[79] . Cardiac function was assessed by systolic parameters (ejection fraction and fractional shortening), diastolic parameters (E / A, E / e'), and stroke volume (SV) measured by echocardiography. We also evaluated the performance of these animals in an exercise test by measuring the maximum running distance on a mouse treadmill. Exercise tolerance is an important physiological parameter in the decline of cardiac functional reserve and HFpEF.

[0073] Myocardial function parameters measured by echocardiography indicate that db / db mice successfully develop diastolic dysfunction as early as 9 weeks of age. In 17-week-old db / db mice, where the left ventricular ejection fraction is still preserved, significant changes in diastolic parameters are seen, including decreased E / A (Figure 23A) and increased E / e' (Figure 24B). All these abnormal diastolic parameters can be rescued and normalized by treatment with AAV9-cBIN1. The improved diastolic function of AAV9-cBIN1 normalizes the decreased left ventricular stroke volume of db / db mice (Figure 24C). Along with the improved echocardiographic parameters, exercise intolerance (reduced maximum running distance on a treadmill) in these diseased db / db mice is also significantly improved by AAV9-cBIN1 (Figure 24D). Note that the body weight of all db / db mice was similar across both groups with either AAV9-GFP or cBIN1 treatment, indicating similar levels of obesity and type 2 diabetes. Taken together with the rescue of diastolic parameters measured by echocardiography, these data indicate that the AAV9-cBIN1-mediated rescue effect on exercise tolerance is due to improved myocardial functional reserve.

[0074] Example 9 Summary of canine ischemic cardiomyopathy (HFrEF) studies We investigated the effect of cBIN1 gene therapy to rescue impaired cardiac function in hearts with ischemic cardiomyopathy. Adult hound dogs (25–30 kg) underwent thoracotomy and permanent ligation of the proximal left anterior descending (LAD) coronary artery. The dogs were followed for 8–9 weeks after ligation with echocardiograms, hemodynamic parameters, and physiological parameters. The animals were anesthetized and injected with cBIN1 packaged with AAV9 virus into the left ventricular endocardium. Three-dimensional electroanatomical mapping of the LV endocardial chamber was performed using a NOGA XP (Biosense Webster / Johnson and Johnson). The same NOGA XP system was used to inject into the myocardium with a Myostar catheter with a 27-gauge nitinol needle. Twenty injection sites throughout the left ventricular endocardial chamber were injected in each heart. Each injection consisted of 2.5 × 1011 vg, totaling 5 × 10 per animal heart 12 vg. Animals were then monitored by echocardiogram, hemodynamic, and physiological parameters.

[0075] Paired study week measurements of left ventricular ejection fraction (LVEF) are reported as shown in Figure 25. Data for two animals were included. Time point 0 corresponds to the time of LAD ligation. Arrows indicate the time of cBIN1 therapy. Note that LVEF was reduced by half before cBIN1 treatment and then recovered to only mild dysfunction within 1-2 weeks after injection. The first dog continues to recover. The second dog ingested a foreign body at week 12, resulting in an ischemic colon, and treatment was discontinued.

[0076] In both animals, cBIN1 gene therapy led to a dramatic rescue of myocardial function in hearts with ischemic cardiomyopathy (HFrEF). Rescue lasted for at least 5 weeks. This study is currently ongoing, and the duration of treatment following a single episode of cBIN1 infusion remains to be determined.

[0077] References

[0078] [Table 6-1]

[0079] [Table 6-2]

[0080] [Table 6-3]

[0081] [Table 6-4]

[0082] [Table 6-5]

[0083]

Table 6-6

[0084]

Table 6-7

Claims

1. 1. A pharmaceutical comprising a transgene encoding cardiac isoform of Bridging integrator 1 (cBIN1) for use in a method for increasing contractile (systolic) or relaxing (diastolic) function in the heart or treating heart failure in a subject with existing heart failure, said method comprising administering to a subject with existing heart failure a transgene encoding cBIN1.

2. The pharmaceutical of claim 1 , wherein the introduced gene is administered after the subject is diagnosed with heart failure.

3. The pharmaceutical of claim 1 , wherein the transgene is administered by injection.

4. The method of claim 1 , wherein the medicament comprises an adeno-associated virus (AAV) vector comprising a transgene encoding cBIN1.

5. The method of claim 1 , wherein the transgene comprises about 3×10 10 vector genome.

6. The pharmaceutical composition of claim 1, wherein expression of cBIN1 remodels damaged myocardium.

7. The pharmaceutical composition of claim 1, wherein expression of cBIN1 stabilizes the intracellular distribution of calcium handling mechanisms in cardiac muscle.

8. The pharmaceutical composition of claim 1, wherein expression of cBIN1 reduces concentric hypertrophy in the myocardium.

9. The pharmaceutical of claim 1, wherein expression of cBIN1 restores or increases t-tubule microfolds or microdomains in the myocardium.

10. The pharmaceutical composition of claim 1, wherein expression of cBIN1 restores or reduces hyperphosphorylation of ryanodine receptor 2 (RyR2) in the myocardium.

11. The method of claim 1, wherein expression of cBIN1 restores or improves cardiac contractility and lucitropy.

12. The method of claim 1, wherein expression of cBIN1 restores or improves cardiac relaxation and diastolic function.

13. The method of claim 1, wherein expression of cBIN1 is protective against further damage to the myocardium.

14. The pharmaceutical composition of claim 1 , wherein the transgene is administered at least once.

15. The pharmaceutical composition of claim 1 , wherein the subject is a mouse.

16. The pharmaceutical composition of claim 1 , wherein the subject is a human.

17. The method of claim 1, wherein the subject is experiencing reduced ejection fraction (HFrEF).

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