Methods of using gene therapy to repair heart failure

Abstract

Described herein are compositions comprising viral vectors. The viral vectors can encode a tubulin organizing protein or peptide, such as a cardiac isoform of bridging integrator 1 (cBIN1). Also disclosed herein are methods of treating or preventing heart failure in a subject in need thereof. The methods of treatment or prevention can comprise administering a vector comprising cBIN1 to a subject who has experienced heart failure or who has chronic myocardial stress, to repair or enhance contractile (systolic) function or relaxation (diastolic) function in the subject's heart.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 007,229, filed on April 8, 2020, and U.S. Provisional Patent Application No. 63 / 088,123, filed on October 6, 2020, each of which is incorporated herein by reference in its entirety.

[0003] Federally funded research

[0004] This invention was made with U.S. Government support under National Institutes of Health Grant Nos. HL133286, HL094414, and HL138577. The U.S. Government has certain rights in this invention. Technical Field

[0005] Compositions comprising viral vectors are described herein. The viral vectors may encode a t-tubule organizing protein or peptide, such as the cardiac isoform of bridging integrin 1 (cBIN1). Also disclosed herein are methods for treating or preventing heart failure in subjects in need thereof. The treatment or prevention methods may include administering a vector comprising cBIN1 to a subject who has experienced heart failure or has chronic myocardial stress to repair or enhance contractile (systolic) function or relaxation (diastolic) function in the subject's heart. Background Art

[0006] Heart failure (HF) is the fastest growing cardiovascular disorder, affecting more than 20 million people worldwide and 6.2 million Americans [1-2]. HF-related mortality is largely related to cardiac pump failure due to myocardial systolic and diastolic dysfunction and sudden cardiac death due to the increased arrhythmia burden of the failing heart. In addition, severe diastolic failure with a further increased risk of arrhythmias occurs in nearly 50% of patients with HF with preserved ejection fraction (HFpEF) [2], who have even worse clinical outcomes and lack effective pharmacological treatments. Therefore, there is an urgent need to develop new therapeutic strategies that can limit and reverse the progression of heart failure.

[0007] During the development of HF, the pathophysiological cellular hallmarks of ventricular myocyte failure are abnormal calcium transients and impaired intracellular calcium homeostasis [3], which disrupt excitation-contraction (EC) coupling [4], impair electrical stability [5], and perturb mitochondrial metabolism [6]. Normal beat-to-beat calcium transients rely on a series of intracellular events called calcium-induced calcium release (CICR) [7], in which an initial calcium influx mediated by t-tubule L-type calcium channels (LTCCs) subsequently induces a massive calcium release from sarcoplasmic reticulum (SR) stores via ryanodine receptors (RyRs). Then, during relaxation, calcium is released primarily via SR Ca 2+ The β-ATPase (SERCA) removes accumulated calcium from the cytoplasm by reuptake of calcium into the SR and expulsion of calcium into the extracellular space [7]. In HF, abnormal t-tubule remodeling [8-10] impairs LTCC-RyR coupling and synchronized CICR [3,11], leading to reduced contractile release, EC uncoupling, and thus reduced contractility. On the other hand, HF-associated RyR leakage

[12] and abnormal SERCA2a function

[13] lead to SR depletion and increased diastolic calcium

[14] , resulting in severe diastolic failure and electrical instability

[15] . In addition, impaired calcium homeostasis triggers loss of mitochondrial membrane potential

[16] and increased permeability

[17] , which increases the risk of mitochondrial-induced cell death [18-19] and HF progression [18,20]. In summary, abnormal calcium homeostasis is crucial for controlling normal cardiac pump function, electrical stability, and metabolism, and when disturbed, it can lead to pump failure, lethal arrhythmias, and severe metabolic disorders.

[0008] The cardiac transverse tubules (t-tubules) are essential for initiating calcium transients and maintaining effective excitation-contraction (EC) coupling. Pathological t-tubule remodeling is a consequence of β-adrenergic stimulation in HF [21-23]. Furthermore, impaired t-tubule microdomains are associated with HF progression [24-27]. Indeed, t-tubule remodeling may be the turning point from hypertrophy to failure

[10] . Normal calcium transients

[28] require L-type calcium channels (LTCCs) to reside in t-tubule microdomains, which are essential for cardiac contraction and relaxation. The t-tubule membrane scaffold protein cardiac bridging integrin 1 (cBIN1)

[29] , which promotes LTCC trafficking

[30] and aggregation for dyad organization, is also regulated by β-adrenergic receptor (β-AR) signaling

[31] . Furthermore, cBIN1 is decreased in HF [31-33], and the resulting disruption of cBIN1 microdomains impairs the normal stress response, thereby limiting contractility and promoting arrhythmias. Therapeutic approaches that preserve the cBIN1 microdomain may benefit the stressed heart by protecting calcium handling mechanisms, thereby slowing HF progression.

[0009] Therefore, there remains a need to prevent remodeling within individual ventricular myocytes in order to improve overall cardiac remodeling and have therapeutic benefit in the failing heart. Summary of the Invention

[0010] One embodiment described herein is a method for repairing cardiac tissue or ameliorating symptoms of heart failure in a subject who has experienced heart failure or is under chronic stress, the method comprising diagnosing heart failure or myocardial stress in the subject; and administering a transgene encoding cardiac bridging integrator 1 (cBIN1) to the cardiac tissue of the subject who has experienced heart failure. In one aspect, diagnosing heart failure or myocardial stress comprises measuring decreased blood levels of cBIN1.

[0011] Another embodiment described herein is a method of repairing or enhancing contractile (systolic) function or relaxation (diastolic) function in the heart of a subject who has experienced heart failure, the method comprising administering a transgene encoding cardiac bridging integrator 1 (cBIN1) to the subject's cardiac tissue, wherein after the transgene is delivered to the cardiac tissue and expressed, the contractile function of the heart is repaired or enhanced. In one aspect, the transgene is administered after the subject is diagnosed with heart failure. In another aspect, diagnosing heart failure comprises measuring decreased 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 comprises approximately 1×10 10 to about 5×10 10 vector genomes. In another aspect, expression of cBIN1 reconstructs damaged myocardium. In another aspect, expression of cBIN1 stabilizes the intracellular distribution of calcium handling machinery in the myocardium. In another aspect, expression of cBIN1 reduces centripetal hypertrophy in the myocardium. In another aspect, expression of cBIN1 repairs or increases t-tubule microfolding or microdomains in the myocardium. In another aspect, expression of cBIN1 repairs or reduces hyperphosphorylation of ryanodine receptor 2 (RyR2) in the myocardium. In another aspect, expression of cBIN1 repairs or improves cardiac contractility and diastolic force. In another aspect, expression of cBIN1 repairs or improves cardiac relaxation and diastolic function. In another aspect, expression of cBIN1 prevents further damage to the 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 another aspect, the subject experiences reduced ejection fraction (HFrEF).

[0012] Another embodiment described herein is the use of cBIN1 in a medicament for repairing myocardial tissue or repairing myocardial damage in a subject that has experienced heart failure or suffers from chronic myocardial stress.

[0013] Another embodiment described herein is the use of cBIN1 in a medicine for restoring or enhancing contractile (systolic) function or relaxing (diastolic) function in the heart of a subject that has experienced heart failure or suffers from chronic myocardial stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] This patent or application file contains at least one drawing printed in color. Copies of this patent or patent application publication in color will be provided by the Office upon request and payment of the necessary fee.

[0015] Figure 1A to Figure 1B Shown is the experimental protocol for cardiac bridging integrin 1 (cBIN1) post-treatment in mice subjected to transverse aortic constriction (TAC). Figure 1A Schematic scheme is shown: 47 mice were randomly divided into three groups: sham-operated (N=12), or TAC mice treated with AAV9-GFP (N=17) and AAV9-cBIN1 (N=18) 5 weeks after TAC. Figure 1B Shown are echocardiographic analyses of transaortic pressure gradient (TAP) in the three groups.

[0016] Figures 2A to 2B It was shown that post-treatment with exogenous cBIN1 improved the survival rate of mice after TAC. Figure 2A Kaplan-Meier survival curves are shown for all three groups of mice: sham-operated control group (N=12), post-TAC mice transduced with AAV9-CMV virus for GFP (N=17), or treated with cBIN1 (N=18). The log-rank test was used for comparisons among the three groups. Figure 2B Figure 2 shows Kaplan-Meier survival curves for post-TAC mice that were not in the end-stage of disease before AAV9 injection (EF ≥ 30% 5 weeks after TAC) after treatment with AAV9-GFP (N = 16) or AAV9-cBIN1 (N = 15). The log-rank test was used to compare survival between the AAV9-GFP and AAV9-cBIN1 groups.

[0017] Figures 3A to 3C It was shown that exogenous cBIN1 alleviated TAC-induced hypertrophy and pulmonary edema. Figure 3A Longitudinal heart sections stained with H&E are shown (scale bar, 1 mm). Figure 3B Shown are the heart weight to tibia length ratio (HW / TL) at 20 weeks after TAC. Figure 3C Lung weight relative to tibia length (LW / TL) at 20 weeks after TAC is shown. Data are expressed as mean ± SEM and statistically analyzed using two-way ANOVA with Fisher's LSD test. *, ***p < 0.05, 0.001 vs. sham group; Comparison between GFP and cBIN1 groups.

[0018] Figure 4A cBin1 gene transfer preserves myocardial systolic and diastolic function in pressure-overloaded hearts. (A) Representative left ventricular (LV) short-axis M-mode images from each group (sham, AAV9-GFP, AAV9-cBIN1) at 5 weeks after TAC (before AAV9 injection) and 20 weeks after TAC (15 weeks after AAV9 injection). Figures 4B to 4D Echocardiographic measurements at 5 weeks (before AAV9) and 20 weeks (after AAV9) after TAC are shown ( Figure 4B )Left ventricular ejection fraction (EF), ( Figure 4C ) end-diastolic volume and ( Figure 4D )Left ventricular mass. Figure 4E Representative pulsed-wave Doppler images of mitral inflow (upper panels) and tissue Doppler images of the septal mitral annulus (e') (lower panels) are shown 20 weeks after TAC (15 weeks after AAV9 injection). Figure 4F Shown are the quantifications of E / e' for each group at 5 weeks (pre-AAV9) and 20 weeks (post-AAV9) post-TAC. Figures 4G to 4H The results show that each mouse Figure 4G ) stroke volume (SV) and ( Figure 4H ) Δ change in cardiac output (CO) from 5 to 20 weeks (ΔSV = SV 20周 -SV 5周 ; ΔCO=CO 20周 -CO 5周 Data are expressed as mean ± SEM and were statistically analyzed using two-way ANOVA with Fisher's LSD test. *, **, *** p < 0.05, 0.01, 0.001, compared with the sham group; Comparison of the AAV9-GFP group and the AAV9-cBIN1 group at each time point. When comparing before AAV9 treatment with after AAV9 treatment in each group.

[0019] Figures 5A to 5C Shown that AAV9-cBIN1 post-treatment rescued EF in mouse hearts after TAC. Figure 5A Shown are echocardiographically monitored ΔEF changes (ΔEF) from pre-AAV to 3, 6, 8, 10, and 15 weeks after AAV9 injection (8, 11, 13, 15, and 20 weeks after TAC, respectively) in the AAV9-CMV-GFP and AAV9-CMV-cBIN1 treated groups. Figures 5B to 5C Shown are the 6 weeks after AAV9 treatment ( Figure 5B ) and 8 weeks ( Figure 5CThe ΔEF of α was fitted with a Gaussian distribution curve. Data are presented as mean ± SEM.

[0020] Figures 6A to 6F It was shown that exogenous cBIN1 pretreatment improved myocardial pressure-volume (PV) loops. Figure 6A Schematic scheme is shown: sham operation (N=5) or TAC, pre-treatment with AAV9-GFP (N=10) or cBIN1 (N=10) administered 3 weeks before TAC. Figures 6B to 6F Representative PV loops in AAV9-GFP and AAV9-cBIN1 hearts at 8 weeks after sham, TAC surgery are shown ( Figure 6B )、EF( Figure 6C )、dp / dt maximum value( Figure 6D )、dp / dt minimum value( Figure 6E ) and τ( Figure 6F Data are expressed as mean ± SEM and statistically analyzed using one-way ANOVA with Fisher's LSD test. **, ***p < 0.01, 0.001, compared with sham-operated group; Comparison between the AAV9-GFP group and the AAV9-cBIN1 group.

[0021] 7A to 7C It was shown that the reduction of cBIN1 microdomains in the heart after TAC could be normalized with AAV9-cBIN1 pretreatment. 7A to 7B ( ) of heart lysates after TAC of sham, AAV9-GFP and AAV9-cBIN1 pre-treated hearts are shown. Figure 7A )cBIN1、( Figure 7B ) Western blot for ryanodine receptor (RyR) and Cav1.2. Quantification is included in the bar graph in the lower panel (n = 8 hearts per group for cBIN1, n = 6 hearts per group for cBIN1). Figure 7C Representative myocardial immunofluorescence spinning disk confocal images of BIN1 labeling (anti-BAR domain; upper panel), RyR (middle panel), and Cav1.2 (lower panel) in sham-operated, AAV9-GFP, and AAV9-cBIN1-pretreated hearts after TAC are shown. The insets include magnified images of the corresponding boxed areas. Bottom row (from left to right): Peak power density of BIN1, RyR, and Cav1.2 distribution in sham-operated, AAV9-GFP, and cBIN1-pretreated hearts 8 weeks after TAC surgery (n = 15-20 images of five hearts per group). Data are expressed as mean ± SEM and statistically analyzed using one-way ANOVA with Fisher's LSD test. *, **, *** p < 0.05, 0.01, 0.001 vs. sham-operated group; Comparison between the AAV9-GFP group and the AAV9-cBIN1 group.

[0022] Figures 8A to 8H It was shown that exogenous cBIN1 attenuated the centripetal hypertrophy of mouse hearts after isoproterenol (ISO). Figure 8A The experimental scheme is shown: 56 mice were randomly divided into four experimental groups: AAV9-GFP+PBS, AAV9-GFP+ISO, AAV9-cBIN1+PBS, AAV9-cBIN1+ISO (n=14 / group). Figure 8B Shown are the heart weight to body weight ratios (HW / BW) of mice in the four groups. Figure 8C Shown are representative images of longitudinal views of the left ventricle at end-diastole and end-systole 4 weeks after PBS or ISO infusion. Figures 8D to 8G End-diastolic volume, LV mass and relative wall thickness are shown ( Figure 8D ), ejection fraction ( Figure 8E )、E / e'( Figure 8F ), stroke volume ( Figure 8G ) and cardiac output ( Figure 8H ) echocardiographic analysis. Data are expressed as mean ± SEM. Multiple comparisons were performed using two-way ANOVA followed by Fisher's LSD test. *, **, *** indicate p < 0.05, 0.01, and 0.001 for the PBS vs. ISO comparison within each AAV9-treated group; ## , ### Indicates p<0.01, 0.001 for the comparison of GFP vs. cBIN1 within each drug infusion group.

[0023] Figures 9A to 9C It was shown that isoproterenol decreased cBIN1 and disrupted cBIN1 microfolding, which could be normalized by AAV9-cBIN1. Figure 9A Western blots of cBIN1 and GAPDH from cardiac lysates and immunoprecipitated cardiac lysates from GFP+PBS, GFP+ISO, cBIN1+PBS and cBIN1+ISO hearts are shown. Quantification in bar graphs is on the right (N=6-7 hearts per group). Figure 9B Representative images of cardiomyocytes labeled with di-8-ANNEP (upper panel) (scale bar, 10 μm) and power spectra (lower panel) of the corresponding framed regions of interest are shown. Quantification of peak power density is included to the left of the bar graph. (N = 26-31 cells from 3-4 hearts per group). Data are presented as mean ± SEM. Multiple comparisons were performed using a two-way ANOVA followed by Fisher's LSD test. *, ** indicates p < 0.05, 0.01 for PBS vs. ISO within each AAV9-treated group; # ,## Indicates p<0.05, 0.01 for the comparison of GFP vs. cBIN1 within each drug infusion group. Figure 9C Transmission electron microscopy images of t-tubule microfolding of myocardial tissue from all four groups are shown (scale bar, 1 μm). Quantification of the extent of t-tubule (TT) outlines for each group is included in the bar graph on the left (N = 232-305 TTs, 60-100 images from 5-6 myocardial sections and 2-3 hearts for each group). TT outlines were compared between groups using the chi-square test, with p < 0.001 for comparisons of GFP + PBS vs. GFP + ISO, GFP + ISO vs. cBIN1 + ISO, and cBIN1 + PBS vs. other groups.

[0024] 10A to 10D cBIN1 is shown to increase Cav1.2 localization to t-tubules. Figure 10A Western blots of Cav1.2 in cardiac lysates from GFP+PBS, GFP+ISO, cBIN1+PBS, and cBIN1+ISO hearts are shown. Quantification (Cav1.2 / GAPDH and Cav1.2 / troponin) is included in the bar graph on the right (n=5-7 hearts per group). Figure 10B Representative confocal images (100×) of anti-Cav1.2 labeling in mouse myocardium from each group are shown (top two panels) (scale bar, 10 μm). The third panel includes the power spectrum, and the fourth panel includes the fluorescence intensity spectrum along the longitudinal axis of the cardiomyocyte within the boxed area. Figure 10C Quantification of Cav1.2 peak power density and immunofluorescence intensity at the t-tubules in each group is shown (n=15-32 cell images from 3-4 hearts per group). Scale bar: 10 μm. Figure 10D Representative calcium transient tracings and peak amplitude quantification (ΔF / F0) for each group are shown (n = 61-88 cells from 6 hearts per group). Data are presented as mean ± SEM. Multiple comparisons were performed using a two-way ANOVA followed by Fisher's LSD test. *** indicates p < 0.001 for PBS vs. ISO comparison within each AAV9-treated group; ### Indicates p < 0.001 for the comparison of GFP vs. cBIN1 within each drug infusion group.

[0025] 11A to 11D Shown is the intracellular distribution of cBIN1 organizing SERCA2a in the heart following isoproterenol. Figure 11A Shown are Western blots of SERCA2a in cardiac lysates from GFP+PBS, GFP+ISO, cBIN1+PBS, and cBIN1+ISO hearts. Figure 11B Quantification (SERCA2a / Actin) is shown and included in the bar graph (n=6-8 hearts per group). Figure 11C Representative confocal images of anti-SERCA2a labeling in the myocardium of mice from each group are shown (top two panels). Scale bar: 10 μm. The third panel includes the power spectrum of SERCA2a in the top boxed area. Figure 11D Quantification of the peak power density of SERCA2a is shown (n = 11-15 cell images from 3-4 hearts per group). Data are presented as mean ± SEM. Multiple comparisons were performed using a two-way ANOVA followed by Fisher's LSD test. **, *** indicate p < 0.01, 0.001 for PBS vs. ISO within each AAV9-treated group; ### Indicates p < 0.001 for the comparison of GFP vs. cBIN1 within each drug infusion group.

[0026] FIG. 12A to FIG. 12B Sucrose gradient fractionation of cardiac microsomes is shown. Figure 12A Representative Western blots of Cav1.2 and cBIN1 in the F4(TT) fraction of cardiac microsomes from GFP+PBS, GFP+ISO, cBIN1+PBS, cBIN1+ISO hearts are shown (2.5 μg protein loaded per lane). Quantification is included in the bar graph (n=3 hearts per group). Figure 12B Representative Western blots of RyR, phospholamban (PLN), and SERCA2a in the F2 (longitudinal SR enrichment) and F3 (jSR enrichment) fractions of cardiac microsomes from GFP+PBS, GFP+ISO, cBIN1+PBS, and cBIN1+ISO hearts are shown (25 μg protein loaded 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 expressed as mean ± SD. Multiple comparisons were performed using a two-way ANOVA followed by Fisher's LSD test. *, **, *** indicate p < 0.05, 0.01, 0.001 for the PBS vs. ISO comparison within each AAV9-treated group; #,## Indicates p<0.05, 0.01 for the comparison of GFP vs. cBIN1 within each drug infusion group.

[0027] 13A to 13D Figure 3 shows that exogenous cBIN1 brings together Cav1.2-RyR and SERCA2a-cBIN1 molecules in cardiomyocytes. Cav1.2-RyR ( 13A to 13B ) and SERCA2a-cBIN1( FIG. 13C to FIG. 13D )Super-resolution STORM imaging and nearest neighbor analysis of molecules. Figure 13A 、 Figure 13CShown from top to bottom are representative 2D-STORM cell images; representative 3D-STORM images of couplers; and histograms of nearest neighbor distance distributions obtained from whole-cell 3D-STORM images. Figure 13B 、 Figure 13D Quantification of the first peak of the nearest neighbor distance distribution histogram using whole-cell image analysis is shown (N = 7-17 cells from 2-3 animals per group). Data are expressed as mean ± SD. Multiple comparisons were performed using a two-way ANOVA followed by Fisher's LSD test. * indicates p < 0.05 for PBS vs. ISO comparison within each AAV9-treated group; #,### Indicates p<0.05, 0.001 for the comparison of GFP vs. cBIN1 within each drug infusion group.

[0028] 14A to 14H Shown are echocardiograms of hearts after isoproterenol administration of AAV9-GFP, cBIN1, BIN1, BIN1+17, and BIN1+13. Figure 14A Representative LV short-axis M-mode images of each group at baseline (upper panel) and 4 weeks after isoproterenol treatment (lower panel) are shown. In all 4-week post-ISO images, the papillary muscles are marked with arrows. 14B to 14D The LV mass of each group is shown ( Figure 14B ), relative wall thickness ( Figure 14C ) and ejection fraction ( Figure 14D ) were quantitatively analyzed (N = 10 mice per group). Figure 14E Shown are representative mitral inflow pulsed-wave Doppler images (upper) and tissue Doppler images (lower) of the septal mitral annulus 4 weeks after isoproterenol treatment. Figures 14F to 14H The E / e'( Figure 14F ), stroke volume ( Figure 14G ) and cardiac output ( Figure 14H ) (N = 10 mice per group). Data are expressed as mean ± SEM. Multiple comparisons were performed using a two-way ANOVA followed by Fisher's LSD test. *, **, *** indicate p < 0.05, 0.01, 0.001 compared to baseline; # , ## , ### Indicates p < 0.05, 0.01, 0.001 compared with the GFP group 4 weeks after ISO.

[0029] Figures 15A to 15J It was shown that cBIN1 gene transfer improved heart failure-free survival of mice after TAC. Figure 15A A schematic scheme of the TAC study is shown. Figure 15B Transaortic pressure gradient measurements in all mice 5 days after surgery are shown. Figure 15CKaplan-Meier survival curves for heart failure-free survival (non-survival defined as death or EF < 35%) of WT versus Bin1 HT mice (left) and mice pretreated with AAV9-GFP or cBIN1 (right). The log-rank test was used for survival comparison. HW / BW ( Figure 15D ) and LW / BW( Figure 15E ). ( Figure 15F ) Representative M-mode echocardiographic images of all mice 8 weeks after surgery. Left ventricular ejection fraction (LVEF) measured by echocardiography of all mice 8 weeks after TAC Figure 15G ), end-diastolic volume ( Figure 15H ), LV quality ( Figure 15I ) and E / e'( Figure 15J ). Data are expressed as mean ± SEM. Representative E and e' images of the AAV9-treated group are included in ( Figure 15J ) in the right panel. Unpaired T-test (or nonparametric Mann-Whitney test) was used to compare WT and Bin1 HT. Comparisons between sham, AAV9-GFP, and AAV9-cBIN1 were performed using one-way ANOVA or Kruskal-Wallis test followed by Fisher's LSD test for multiple comparisons. *, **, *** indicate p < 0.05, 0.01, and 0.001 compared with WT or sham; # , ## Indicates p<0.05, 0.01 when comparing AAV9-GFP vs. AAV9-cBIN1.

[0030] Figure 16 Figure 2. Expression of exogenous GFP-V5 and cBIN1-V5 proteins in mouse cardiomyocytes following AAV9 transduction. Representative images of adult mouse ventricular cardiomyocytes from control (left), AAV9-GFP-V5 (center), or AAV9-cBIN1-V5 (right)-treated mice under transmitted light (top) or widefield fluorescence (rabbit anti-V5 labeling, bottom).

[0031] Figure 17 Figure 3 shows that exogenous cBIN1 transduced by AAV9 normalizes the microfolding of cardiomyocyte t-tubules in post-TAC hearts. Representative live cell membrane marker (di-8-ANNEP) images of freshly isolated cardiomyocytes from sham-operated, AAv9-GFP, and AAV9-cBIN1-treated post-TAC hearts. Quantification of t-tubule di-8-ANNEP intensity is included in the bar graph on the right (n = 10 images of 5 hearts per group). All data are expressed as mean ± SEM. Statistical analysis was performed using the Kruskal-Wallis test with LSD post-test. ***, p < 0.001 when compared to the sham group; p < 0.05 when compared between the AAV9-GFP group and the AAV9-cBIN1 group.

[0032] Figure 18A Representative fluorescent confocal images (20×) of V5 and WGA labeling in myocardial cryosections obtained from mice 7 weeks after injection of AAV9-transduced GFP-V5 or cBIN1-V5 or control hearts without AAV9 injection (negative control) are shown. 10 7 weeks after 4 vg, positive V5 signals were detected in 63% and 57% of cardiac cells, respectively. Scale bar, 100 μm. Figure 18B Quantification of the percentage of myocardial area with detectable V5 signal is shown. N = 4-6 myocardial sections from 2-3 animals per group. Data are presented as mean ± SEM. Multiple comparisons were performed using the Kruskal-Wallis test followed by the Dunn test. * indicates p < 0.05 compared to the no AAV9 negative control.

[0033] Figure 19A Echocardiography-based classification of LV remodeling in GFP+PBS, cBIN1+PBS, GFP+ISO, and cBIN1+ISO hearts is shown. Figure 19B Representative Western blots and quantification of α-smooth muscle actin in hearts from each group are shown. Data are presented as mean ± SEM. Multiple comparisons were performed using a two-way ANOVA followed by Fisher's LSD test. * indicates p < 0.05 for PBS vs. ISO within each AAV9-treated group; ## Indicates p < 0.01 for the comparison of GFP vs. cBIN1 within each drug infusion group.

[0034] Figure 20A Shown are representative Western blots and quantification of total RyR2 protein expression in mice. Figure 20B Representative confocal images (100×) of RyR2 in mouse cardiomyocytes from each group are shown, followed by power spectrum analysis (n=33-36 cells from 3-4 hearts per group). Scale bar: 10 μm.

[0035] Figure 21 shows representative Western blots for RyR2 (total and phosphorylated pS2814 and pS2808), Cav1.2, CAMKIIδ (total and phosphorylated pT287), and phospholamban (PLN, total and phosphorylated pS16 and pT17). Quantification is included in the bar graph on the right. Data are presented as mean ± SEM. N = 4-7 hearts per group. Multiple comparisons were performed using a two-way ANOVA followed by Fisher's LSD test. * indicates p < 0.05 for PBS vs. ISO within each AAV9-treated group;# p<0.05 for GFP vs. cBIN1 in each drug infusion group.

[0036] Figure 22A Schematic protocol showing sucrose gradient fractionation of cardiac microsomes (3-6 mg per heart). Total protein yield recovered from fractions F1, F2, F3, F4 was between 0.001-0.02, 0.4-0.8, 0.04-0.06 and <0.008 mg per heart preparation, respectively. Figure 22B Representative Western blots showing Cav1.2, Na + / K + -ATPase, SERCA2a, cBIN1 and caveolin 3, as well as bulk from GFP+PBS, GFP+ISO, cBIN1 +PBS and cBIN1 +ISO hearts, and recovered fractions from F1, F2, F3, F4.

[0037] Figures 23A to 23E Representative confocal images (100x) showing cBIN1 organization of LTCC and SERCA2a in isoproterenol post mouse hearts. Figure 23A Representative Western blots showing Cav1.2 and SERCA2a in isoproterenol post mouse hearts treated with AAV9-GFP, cBIN1, BIN1, BIN1 +17 and BIN1 +13. Quantification is included in the right bar graph (n=3 hearts per group). Figures 23B to 23C Representative confocal images (100x) showing anti-Cav1.2 Figure 23B ) and anti-SERCA2a Figure 23C ) labeling in mouse myocardium of each group. Scale bar: 5 pm. Figure 23D Quantification of t-tubule Cav1.2 fluorescence intensity of each group. N=16-22 cells per 3 hearts per group. Figure 23E Quantification of SERCA2a peak power density of each group. N=20 cell images per 2-3 hearts per group. Data are expressed as mean ± SEM. One-way ANOVA followed by Fisher’s LSD test for multiple comparisons. *, **, *** indicate p<0.05, 0.01, 0.001 when compared to GFP control group.

[0038] 24A to 24D AAV9-cBIN1 rescues diabetic HFpEF in db / db mice. Echocardiographic measurements of E / A Figure 24A ), E / e' Figure 24B ) and SV( Figure 24C ); and ( Figure 24D ) maximum running distance on a mouse treadmill. N = 10 animals per group. One-way ANOVA followed by LSD Fisher's exact test was used to compare differences between groups. *, *** means p < 0.05 or 0.001 when compared to db / m + GFP group, respectively. means p < 0.05 or 0.001 when comparing db / db + GFP vs. db / db + cBINl groups.

[0039] Figure 25 shows that AAV9-cBINl rescued ischemic HFrEF in dogs. Echocardiography measured left ventricular ejection fraction (LVEF) in two study dogs (Dog 1 and Dog 2) vs. study week number. Time 0 corresponds to the time of LAD ligation. Arrows indicate the time of cBINl treatment. DETAILED DESCRIPTION

[0040] Reorganization of intracellular calcium handling machinery can be achieved by targeting the t-tubule membrane microdomain organization by cardiac isoform of bridging integrator factor 1 (cBINl)

[34] . It was previously discovered that the cBINl microdomain organizes the LTCC-RyR dyad [12, 14] by facilitating intracellular trafficking

[13] and surface clustering [14, 35] of the LTCC, influencing the electrochemical gradient of extracellular ions across the LTCC by generating a protective slow-diffusion zone within the t-tubule lumen

[12] , and recruiting RyR to the jSR for coupling with the LTCC

[14] . The cBINl microdomain was also recently found to be critical in organizing the intracellular distribution of SERCA2a for diastolic phase calcium regulation

[34] . In HF, the cBINl microdomain is disrupted due to reduced cBINl transcription [16, 36, 37], impairing dyad formation, calcium transient regulation, and cardiac contractility. Myocardial cBINl reduction can be detected in human blood, as a result of cBINl membrane turnover and microparticle release

[38] . In humans, plasma CS (cBINl score) is an indicator of myocyte cBINl levels, identifies myocardial structural remodeling, and thus aids in HF diagnosis and prognosis

[39] . In mouse hearts subjected to chronic stress, pretreatment with exogenous cBINl preserved the microdomain organization distribution of Cavl.2 and SERCA2a, maintaining normal inotropy and lusitropy. These data suggest that cBINl replacement can be an effective HF therapy, with the potential to restore myocardial function in previously existing HF hearts.

[0041] Since increased afterload is an important primary and secondary cause of HF

[40] , the current study used a mouse model of elevated afterload induced by transverse aortic constriction (TAC). In TAC mice, cBIN1 pretreatment has been reported to protect against the development of HF. Here, we also used AAV9-mediated gene transfer to introduce exogenous cBIN1 into the hearts of mice with pre-existing HF after TAC. cBIN1 posttreatment reduced TAC-induced pathological remodeling, as well as HF episodes and mortality. cBIN1 induced functional recovery in the hearts of mice with pre-existing TAC-induced HF. Furthermore, in this disclosure, we explored whether in vivo overexpression of exogenous cBIN1 could limit myocardial remodeling and dysfunction. Continuous isoproterenol infusion resulted in decreased myocardial cBIN1 expression and disturbed intracellular distribution of calcium handling proteins, and also induced pathological concentric hypertrophy with diastolic dysfunction. We found that normalizing cBIN1 through adeno-associated virus 9 (AAV9)-mediated gene transfer both increased contractility and preserved relaxation, thereby reducing pathological hypertrophy. Within cardiomyocytes, we found that exogenous cBIN1 preserved the intracellular distribution of LTCC at the t-tubule level and the localization of sarcoplasmic reticulum (SR) calcium-ATPase 2a (SERCA2a). The protective effects of cBIN1 were both isoform-specific and validated in a secondary model of transverse aortic constriction (TAC)-induced cardiac hypertrophy and heart failure, suggesting that exogenous cBIN1-mediated preservation of t-tubule microdomains is a potential therapeutic approach to improve myocardial function under chronic stress.

[0042] Administration of exogenous cBIN1 into AAV9-transduced myocardium after ejection fraction reduction rescued cardiac contractile function and limited the development of ventricular dilatation and HF in mice subjected to chronic pressure overload stress.

[0043] Under sustained pressure overload, myocardial remodeling begins with an adaptive hypertrophic response that subsequently transitions to maladaptive cardiac dilation, leading to worsening HF [41-43]. In previous studies, we demonstrated that administration of AAV9-cBIN1 prior to TAC surgery preserved myocardial systolic and diastolic function, indicating the efficacy of cBIN1 gene therapy in HF prevention. In the current disclosure, we found that in mice, exogenous cBIN1 administration not only limited but also rescued TAC-stressed hearts from further HF development, improved overall survival, reduced cardiac hypertrophy, and alleviated pulmonary edema. Furthermore, exogenous cBIN1 introduced by gene transfer improved myocardial remodeling and cardiac function, as measured by echocardiography. Most strikingly, mice with pre-existing severe HF exhibited restored EF after cBIN1 gene therapy, suggesting that the protective effects of exogenous cBIN1 could be used as a translatable treatment for patients diagnosed with pre-existing structural remodeling and HF.

[0044] Recently, AAV-mediated gene therapy has been shown to be a promising therapeutic modality for HF [44-45]. Currently, various pathways such as the β-adrenergic system, Ca 2+ Several clinical trials targeting circulating proteins and cell death pathways, as well as homing stem cells for HF gene therapy, have been completed or are ongoing

[46] . We recently showed that targeting calcium regulatory microdomains at the t-tubules can be effectively achieved by transducing the essential microdomain organizing protein cBIN1

[34] . By stabilizing t-tubule microdomains, cBIN1 may restore cytosolic calcium homeostasis and contribute to increased systolic calcium release, improved diastolic reuptake, limited S leak to maintain electrical stability, and preserved mitochondrial function to limit mitochondrial-associated cell death. The results suggest that this microdomain-targeted approach could be used as a novel therapeutic strategy with improved efficiency in functional preservation, thereby improving overall HF survival. Furthermore, the observed cBIN1-mediated improvement in overall survival may be a combined effect of improved pump function and arrhythmia reduction, as both are regulated by cBIN1 microdomains [7, 12, 36]. How cBIN1 treatment affects the arrhythmia burden in the failing heart requires further analysis in future studies using in vivo telemetry monitoring. Furthermore, since TAC-induced HF is associated with myocyte death associated with mitochondrial dysfunction

[47] , it would be interesting to explore in future studies whether cBIN1 replacement therapy could preserve mitochondrial function and limit mitochondrial-associated cell death in failing hearts.

[0045] In terms of functional recovery, although the EF changes monitored from the start of AAV9-cBIN1 treatment showed that the peak of recovery occurred at 6 weeks after AAV9 treatment and the therapeutic efficiency subsequently decreased, the rescue effect was still maintained at 15 weeks after AAV9 injection. These data indicate that even with a relatively low dose (3×10 10 vg) A single administration of AAV9-cBIN1 was also sufficient to preserve cardiac function. Whether multiple administrations of exogenous cBIN1 in increasing doses are necessary to maximize its therapeutic effect remains to be determined. However, our current rescue data suggest that, in patients with established HF, cBIN1 gene therapy may break the cycle of HF progression and induce functional recovery in failing hearts.

[0046] This study revealed the protective effect of exogenous cBIN1 in the hearts of mice with HF after experiencing pressure overload. For this first proof-of-concept study, we used AAV9 vectors driven by the CMV promoter for gene delivery because of its consistent transduction efficiency and defined cardiotropism. Further experiments using cBinl packaged in AAV9 with more efficient cardi-specific promoters are needed in mice and large mammals before clinical trials testing the efficacy and efficiency of cBinl gene therapy in HF patients. Future studies also need to explore the intracellular mechanisms of cBIN1 in calcium homeostasis among the cytosolic microdomains of t-tubules, SR, and nearby mitochondria. Further understanding of the downstream targeted molecules and signaling pathways of cBIN1 is also needed to better understand the interaction between cBinl gene therapy and HF pathophysiology.

[0047] The present disclosure also demonstrates the beneficial effects of exogenous cBIN1 in preventing LV hypertrophy and cardiac dysfunction in stressed hearts. In mice receiving continuous isoproterenol infusion, exogenous cBIN1 provided isoform-specific improvements in cardiac contractility and relaxation, limiting the development of LV hypertrophy. The cardioprotective effects of exogenous cBIN1 were further confirmed in the hearts of mice with HF induced by pressure overload.

[0048] Chronic elevation of catecholamine levels and activation of cardiac β-adrenergic receptors (β-ARs) play a key role in the pathogenesis of HF. Impaired myocardial structure and function have been observed in animals subjected to sustained sympathetic activation [48-49]. Isoproterenol, a synthetic catecholamine and nonselective β-AR agonist, has been used to induce a model of LV hypertrophy and dysfunction in studies

[50] . Here, a high dose of isoproterenol was used to induce LV concentric hypertrophy while preserving systolic function. Chronic excessive cardiac load-induced LV hypertrophy is associated with an increased risk of cardiovascular events

[51] , and prevention or reversal of ventricular hypertrophy while preserving diastolic function is critical to prevent a stressed heart from progressing to a failing heart. Here, we found that cBIN1 attenuated chronic isoproterenol-induced hypertrophy, while showing isoform-specific improvements in stroke volume and cardiac output in the hypertrophied heart while preserving systolic function. The increased LV volume in cBIN1 hearts was not secondary to pump failure and dilated cardiomyopathy, but rather reflected an improvement in myocardial relaxation (E / e') and a parallel increase in intrinsic myocardial contractility (systolic force). This phenotype in cBIN1 hearts is typical of athletic hearts in adaptive endurance training, which is characterized by enlarged cardiac chambers and increased LV volume, stroke volume, and cardiac output [52-54]. Aerobic exercise training has been reported to improve myocardial function and systolic and diastolic responses in animal models [55-56] as well as in patients with hypertension

[57] and diastolic failure

[58] . Thus, exogenous cBIN1 can provide additional exercise-like benefits to heart failure patients, resulting in improved exercise capacity and quality of life.

[0049] These post-isoproterenol hearts are in a hypertrophic phase that preserves systolic function, where exogenous cBIN1 can effectively translate increased cardiac demands into functional effects. Consequently, these functionally competent cBIN1 hearts exhibit limited hypertrophy, potentially preventing the next steps in disease progression and the development of heart failure (HF) that occur in clinical settings. Next, the functional protective effects of exogenous cBIN1 in already decompensated hearts were also observed in a mouse model of TAC-induced hypertrophy and HF. Under pressure overload, compensatory hypertrophy is an adaptive response. Over time, this adaptive response gives way to cardiac dilation, and the subsequent remodeling process becomes maladaptive, leading to worsening HF. We found that the fate of pressure-overload-stressed hearts to develop dilated cardiomyopathy is determined by the myocardial content of cBIN1 protein. Following pressure overload, lower cardiac BIN1 levels in HT-TAC hearts with genetic deletion of Bin1 were associated with more severe dilated cardiomyopathy, whereas higher cBIN1 levels concomitant with gene transfer improved both systolic and diastolic function, limited HF, and improved HF-free survival. It remains unclear whether exogenous cBIN1 reduces myocyte death, which also contributes to LV dilation in hearts after TAC. Future studies will be necessary to explore the effects of cBIN1 on myocyte survival in stressed hearts. However, our data suggest that exogenous cBIN1 not only limits the development of hypertrophy in stressed hearts but also prevents the transition from hypertrophy to dilated cardiomyopathy and HF in TAC mice.

[0050] The mechanism by which cBIN1 improves cardiac contractile function is related to its known effects on organizing t-tubule microdomains required for bigram organization and efficient EC coupling. cBIN1 generates t-tubule microfolds to organize slow-diffusion zones that capture extracellular t-tubule luminal ions, attract LTCCs for forward transport to the t-tubules

[30] , aggregate LTCCs that have already been delivered to the cell surface

[35] , and recruit RyRs to couple with LTCCs at bigrams

[31] . Here, we demonstrate in vivo that exogenous cBIN1, but not any other BIN1 isoform, increases Cav1.2 localization to the t-tubules. These results support that the preservation of cBIN1 microdomains with organized LTCC distribution is responsible for the positive inotropic effects observed in cBIN1 hearts subjected to sympathetic overdrive. Whether cBIN1 microdomains regulate LTCC phosphorylation and its functional responses to sympathetic stress, including the well-established β-subunit-regulated Cav1.2 channel responses [59-60], awaits future experimental investigation. Furthermore, RyRs are crucial for contractility, and hyperphosphorylated, leaky RyRs play a role in HF progression

[14] . Consistent with previous reports in the isoproterenol model and in humans with HF [14, 61], we found that chronic isoproterenol activated PKA and CAMKII-induced RyR hyperphosphorylation. AAV9-cBIN1 attenuated these pathways, thereby normalizing RyR phosphorylation after chronic sympathetic activation and preventing SR leakiness.

[0051] An additional novel finding of the present disclosure is that exogenous cBIN1 enhances SERCA2a function by organizing its intracellular distribution. Chronic isoproterenol-induced concentric hypertrophy with preserved contractile function is associated with a disturbed intracellular distribution of SERCA2a but increased overall protein expression. It is generally accepted that SERCA2a activity is reduced in the end stage of HF. Our data suggest that, in addition to reduced PLN expression and impaired regulation, the intracellular distribution of SERCA2a may also contribute to the abnormal SR calcium reuptake activity in HF. Furthermore, it has been reported that in adult rat ventricular cardiomyocytes with α-agonist phenylephrine-induced hypertrophy, SERCA2a protein expression is adaptively increased due to increased diastolic calcium-induced calcineurin / NFAT activation

[62] . Therefore, the increased SERCA2a protein expression here may be an adaptive response induced by elevated diastolic calcium concentrations, as indicated by increased calcium-dependent phosphorylation at T287 of CAMKII. Therefore, a transient increase in SERCA2a may occur in the early stages of all function-preserving LV hypertrophy. During disease progression, this adaptive increase in total SERCA2a protein expression levels off and even decreases in end-stage HF, leading to severe diastolic and systolic failure. In cBIN1 hearts, organized SERCA2a along the SR indicates better calcium reuptake, resulting in less diastolic calcium overload in hearts that remain in the compensatory phase. These results are consistent with previous studies in a rat model of HF that found that increased BIN1 expression correlated with SERCA2a expression

[63] . Future studies exploring the regulation of diastolic calcium concentrations and the calcineurin / NTAT pathway by cBIN1 are needed to further understand its role in regulating SERCA2a expression and activity during disease progression. Of note, the effects on SERCA2a organization are not cBIN1 specific and can be partially induced by other BIN1 isoforms, particularly 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 organize the distribution of SERCA2a in normal and diseased cardiomyocytes needs to be further explored in future studies. Furthermore, by regulating calcium-handling mechanisms at the SR, including SERCA2a distribution and RyR phosphorylation, cBIN1 may contribute to maintaining normal SR calcium loading. As a limitation of the current study, future experiments are needed to quantify the effects of cBIN1 on SR calcium loading, calcium release and reuptake kinetics, and arrhythmogenic spontaneous calcium release in chronically stressed hearts.

[0052] However, the most robust protection of contractility and diastolic tone in sympathetically overdriven hearts was observed only in the cBIN1 group, suggesting possible further beneficial effects of cBIN1-dependent LTCC localization and improvements in dyad organization on diastolic tone. With the isoform-specific improvement of dyad organization, fewer isolated leaky RyRs accumulate outside the dyad

[31] , limiting calcium leakage from the SR and reducing diastolic cytosolic calcium concentrations. Together with the newly identified effects on SERCA2a organization, our data suggest that microdomain-associated regulation of cBIN1 provides unique benefits in protecting cardiac diastolic tone beyond its inotropic effects. On the other hand, cBIN1 overexpression can also inhibit the pathological effects of isoproterenol stimulation by enhancing control of β-AR signaling and compartmentalization of secondary messengers and calcium handling channels and pumps. Thus, by stabilizing t-tubule microdomains to regulate all aspects of calcium handling, cBIN1 generates a positive feed-forward mechanism for efficient intracellular beat-to-beat calcium cycling. In future studies, it will be interesting to determine whether exogenous cBIN1 alters β-AR expression, intracellular distribution, and functional regulation following chronic sympathetic nerve activation.

[0053] In summary, we found that overexpression of exogenous cBIN1 has a protective effect on mouse hearts subjected to chronic β-AR activation-induced concentric hypertrophy, as well as pressure overload-induced hypertrophy and heart failure. Future experiments will need to be conducted in larger mammals with common comorbidities of natural heart failure, such as hypertension and diabetes. Improving viral infectivity in cardiomyocytes could also help limit or prevent isoproterenol-induced membrane disruption in all cardiomyocytes, thereby increasing protection to the entire heart. Before clinical trials testing the efficacy and efficiency of cBIN1 gene therapy, further experiments using cBIN1 packaged in AAV9 with an effective cardiac-specific promoter to induce sufficient exogenous protein expression in all cardiomyocytes are needed. Future studies are needed to determine whether cBIN1 affects systemic hemodynamics and blood pressure. Finally, future studies are needed to explore how cBIN1 microdomains regulate the organization of the intracellular calcium handling machinery, EC coupling, SR calcium loading and release, diastolic calcium concentrations and their downstream calcium signaling pathways, the interplay between pathological and physiological hypertrophic remodeling signaling pathways, and the molecular transition from compensated hypertrophy to decompensated cardiomyopathy.

[0054] One embodiment described herein is a method for repairing cardiac tissue or ameliorating symptoms of heart failure in a subject who has experienced heart failure or is under chronic stress, the method comprising diagnosing heart failure or myocardial stress in the subject; and administering a transgene encoding cardiac bridging integrator 1 (cBIN1) to the cardiac tissue of the subject who has experienced heart failure. In one aspect, diagnosing heart failure or myocardial stress comprises measuring decreased blood levels of cBIN1.

[0055] Another embodiment described herein is a method of repairing or enhancing contractile (systolic) function or relaxation (diastolic) function in the heart of a subject who has experienced heart failure, the method comprising administering a transgene encoding cardiac bridging integrator 1 (cBIN1) to the subject's cardiac tissue, wherein after the transgene is delivered to the cardiac tissue and expressed, the contractile function of the heart is repaired or enhanced. In one aspect, the transgene is administered after the subject is diagnosed with heart failure. In another aspect, diagnosing heart failure comprises measuring decreased 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 comprises approximately 1×10 10 to about 5×10 10 vector genomes. In another aspect, expression of cBIN1 reconstructs damaged myocardium. In another aspect, expression of cBIN1 stabilizes the intracellular distribution of calcium handling machinery in the myocardium. In another aspect, expression of cBIN1 reduces centripetal hypertrophy in the myocardium. In another aspect, expression of cBIN1 repairs or increases t-tubule microfolding or microdomains in the myocardium. In another aspect, expression of cBIN1 repairs or reduces hyperphosphorylation of ryanodine receptor 2 (RyR2) in the myocardium. In another aspect, expression of cBIN1 repairs or improves cardiac contractility and diastolic force. In another aspect, expression of cBIN1 repairs or improves cardiac relaxation and diastolic function. In another aspect, expression of cBIN1 prevents further damage to the 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 another aspect, the subject experiences reduced ejection fraction (HFrEF).

[0056] Another embodiment described herein is the use of cBIN1 in a medicament for repairing myocardial tissue or repairing myocardial damage in a subject that has experienced heart failure or suffers from chronic myocardial stress.

[0057] Another embodiment described herein is the use of cBIN1 in a medicine for restoring or enhancing contractile (systolic) function or relaxing (diastolic) function in the heart of a subject that has experienced heart failure or suffers from chronic myocardial stress.

[0058] It will be apparent to those skilled in the art that suitable modifications and adjustments may be made to the compositions, formulations, methods, processes, equipment, components, and applications described herein without departing from the scope of any embodiment or aspect thereof. The compositions, equipment, components, and methods provided are exemplary and are not intended to limit the scope of any disclosed embodiment. All embodiments, aspects, and options disclosed herein may be combined in any variation or iteration. The scope of the compositions, formulations, methods, equipment, components, and processes described herein includes all actual or potential combinations of the embodiments, aspects, options, examples, and preferences described herein. The compositions, formulations, equipment, components, or methods described herein may omit any component or step, replace any component or step disclosed herein, or include any component or step disclosed elsewhere herein. The mass of any component of any composition or formulation disclosed herein is disclosed herein to the mass of any other component in the formulation or to the ratio of the total mass of the other components in the formulation, as if they were explicitly disclosed. If the meaning of any term in any patent or disclosure 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 prevail. The specific teachings of all patents and publications cited herein are incorporated herein by reference.

[0059] The following clauses summarize various embodiments and aspects of the invention described herein:

[0060] Item 1. A method for repairing cardiac tissue or ameliorating symptoms of heart failure in a subject who has experienced heart failure or is under chronic stress, the method comprising diagnosing heart failure or myocardial stress in the subject; and administering a transgene encoding cardiac bridging integrator 1 (cBIN1) to the cardiac tissue of the subject who has experienced heart failure.

[0061] Clause 2. The method of Clause 1, wherein the diagnosis of heart failure or myocardial stress comprises measuring decreased blood levels of cBIN1.

[0062] Item 3. A method for repairing or enhancing contractile function in the heart of a subject that has experienced heart failure, the method comprising administering a transgene encoding cardiac bridging integrator 1 (cBIN1) to the cardiac tissue of the subject, wherein after the transgene is delivered to the cardiac tissue and expressed, the contractile function of the heart is repaired or enhanced.

[0063] Clause 4. The method of Clause 3, wherein the transgene is administered after the subject is diagnosed with heart failure.

[0064] Clause 5. The method of Clause 4, wherein the diagnosis of heart failure comprises measuring decreased blood levels of cBIN1.

[0065] Clause 6. The method of any one of Clauses 1 to 5, wherein the method comprises administering the transgene to the myocardium.

[0066] Clause 7. The method of any one of Clauses 1 to 6, wherein the transgene is administered by injection.

[0067] Clause 8. The method of any one of Clauses 1 to 7, wherein the transgene comprises a vector comprising a transgene encoding cBIN1.

[0068] Clause 9. The method according to any one of clauses 1 to 8, wherein the transgene comprises about 1×10 10 to about 5×10 10 vector genomes.

[0069] Clause 10. The method according to any one of Clauses 1 to 9, wherein expression of cBIN1 remodels damaged myocardium.

[0070] Clause 11. The method of any one of Clauses 1 to 10, wherein expression of cBIN1 stabilizes the intracellular distribution of calcium handling machinery in the myocardium.

[0071] Clause 12. The method of any one of Clauses 1 to 11, wherein expression of cBIN1 reduces centripetal hypertrophy in the myocardium.

[0072] Clause 13. The method according to any one of clauses 1 to 12, wherein expression of cBIN1 repairs or increases t-tubule microfolds or microdomains in the myocardium.

[0073] Clause 14. The method according to any one of clauses 1 to 13, wherein expression of cBIN1 restores or reduces hyperphosphorylation of ryanodine receptor 2 (RyR2) in the myocardium.

[0074] Clause 15. The method according to any one of Clauses 1 to 14, wherein expression of cBIN1 restores or improves cardiac contractility and diastolic force.

[0075] Clause 16. The method according to any one of clauses 1 to 15, wherein expression of cBIN1 restores or improves cardiac relaxation and diastolic function.

[0076] Clause 17. The method of any one of Clauses 1 to 16, wherein expression of cBIN1 prevents further damage to the myocardium.

[0077] Clause 18. The method according to any one of clauses 1 to 17, wherein the transgene is administered at least once.

[0078] Clause 19. The method of any one of clauses 1 to 18, wherein the subject is a mammal.

[0079] Clause 20. The method of any one of Clauses 1 to 19, wherein the subject is a mouse or a dog.

[0080] Clause 21. The method according to any one of clauses 1 to 20, wherein the subject is a human.

[0081] Clause 22. The method of any one of clauses 1 to 21, wherein the subject experiences heart failure with reduced ejection fraction (HFrEF).

[0082] Clause 23. Use of cBIN1 in medicine for repairing myocardial tissue or repairing myocardial damage in a subject who has experienced heart failure or suffers from chronic myocardial stress.

[0083] Clause 24. Use of cBIN1 in medicine for restoring or enhancing contractile (systolic) function or relaxing (diastolic) function in the heart of a subject that has experienced heart failure or suffers from chronic myocardial stress.

[0084] Example

[0085] Example 1

[0086] Materials and methods

[0087] Animal procedures for functional rescue studies. Adult male C57BL / 6 mice (Jackson Laboratory) were used. All 8- to 10-week-old mice were anesthetized and underwent thoracotomy sham surgery or transverse aortic constriction (TAC) surgery. TAC was performed by tying a 7-0 silk suture to a 27-gauge needle between the first and second branches of the aortic arch. For the sham control group, age-matched mice underwent thoracotomy mock surgery without TAC. For gene therapy, 5 weeks after the start of TAC, mice received a retroorbital injection of 100 μL of AAV9 virus (Welgen, Inc.) transducing cBIN1-V5 or GFP-V5 3×10 10 vector genome (vg)

[64] .

[0088] Animal Procedures for Isoproterenol Studies For isoproterenol studies, adult male C57BL / 6 mice were administered 3 × 10 10AAV9 vector genomes (vg) transducing GFP or BIN1 isoforms (Wilgen)

[64] . Three weeks after vg administration, osmotic minipumps releasing PBS or isoproterenol (30 mg / kg / day) were subcutaneously implanted into mice. 56 mice were randomly divided into GFP+PBS, GFP+ISO, cBIN1+PBS, or cBIN1+ISO groups (N=14 / group). Another 50 mice were randomized to receive AAV9-GFP, cBIN1, BIN1, BIN1+17, or BIN1+13 (N=10 / group) before isoproterenol. AAV9 was used because it is a promising gene therapy vector and shows the highest cardiac tropism

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

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

[67] . For TAC studies, adult male cardiac-specific Bin1 heterozygotes (Bin1 HT; Bin1 flox / + , Myh6-cre + ) and its wild type (WT; Bin1 flox / + , Myh6-cre - ) littermates

[29] ; or adult male C57BL / 6 mice (Jackson Laboratory). All 8- to 10-week-old mice were anesthetized and underwent open-chest TAC or mock surgery (sham surgery). For gene therapy, as in the isoproterenol study, mice received a retroorbital injection of 3 × 10 AAV9 viruses transduced with cBIN1-V5 or GFP-V5 3 weeks before TAC. 10 A vg.

[0089] Isoproterenol minipump studies. Fifty-six mice were randomly assigned to receive 3 × 10 AAV9 transduced with V5-tagged GFP or cBIN1 via retroorbital injection while the mice were anesthetized with 1% isoflurane in oxygen. 10Vector genomes (vg)

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

[25] . AAV9-CMV-GFP was used as a negative control virus because AAV9-CMVGFP does not induce cardiac injury and cardiomyocyte toxicity [25-26] and GFP AAV9 has been successfully used as a negative control virus in many gene therapy studies using animal models of cardiovascular disease, including mouse models of hypertrophy and cardiomyopathy [26-29]. The protocol was also repeated in a second group of animals. Likewise, three weeks before isoproterenol minipump implantation, fifty mice were randomized to receive 3×10 AAV9 transduced with V5-tagged GFP, BIN1, BIN1+13, BIN1+17, or cBIN1 via retroorbital injection. 10 Vector genomes (vg) (n = 10 / group). Three weeks after AAV9 injection, mice were implanted with subcutaneous ALZET osmotic minipumps (model 1004, Duret, Cupertino, CA, USA) that continuously release isoproterenol according to previously described procedures

[30] . Briefly, under light anesthesia by inhalation of isoflurane, a mini-osmotic pump that continuously releases isoproterenol at 30 mg / kg / day was subcutaneously implanted in the back of the mice.

[0090] Transverse aortic constriction (TAC) studies. For cBIN1 deficiency studies, cardiac-specific heterozygous deletion of Bin1 (Bin1 HT; Bin1 flox / + , Myh6-Cre + ) and its wild type (WT; Bin1 flox / + , Myh6-Cre - ) littermate (WT) male mice were subjected to TAC at 8-10 weeks of age. Bin1 HT and WT mice were generated as previously described. Specifically, heterozygous loxP-flanked Bin1 (loxP sites around exon 3 of the Bin1 gene) mice were crossed with Myh6-cre + Mice were crossed to generate cardiomyocyte-specific Bin1 HT (n=10) and WT littermate controls (n=14). Genotypes were confirmed by PCR to distinguish Bin1 according to previously established methods. + 、Bin1flox and Cre + For AAV9-mediated overexpression studies, 5- to 7-week-old male C57BL / 6J mice (Jackson Laboratory) were retroorbitally injected with AAV9 virus (3 × 10 10 vg). Three weeks later, 8-10 week old mice were anesthetized and underwent open-chest TAC surgery. Age-matched mice that underwent open-chest mock surgery without TAC were used as sham controls (N=10). TAC was performed as previously described to induce pressure overload. Briefly, 8-12 week old male mice were anesthetized by administering 3% isoflurane via a face mask, then intubated and placed on a ventilator (Harvard Apparatus) supplemented with O2 and 1.5% isoflurane, with a tidal volume of 0.2 mL and a respiratory rate of 120 breaths / minute. The thoracic cavity was entered through a small incision in the second intercostal space above the sternum, and aortic constriction was performed by tying a 7-0 nylon suture to a 27-gauge needle between the first and second branches of the aortic arch. Subcutaneous buprenorphine (0.8 mg / kg) was administered to relieve pain, and the mice were allowed to recover in a heated chamber with 100% O2. After 8 weeks of TAC, the animals were euthanized, and tissues were collected for analysis.

[0091] Adeno-associated virus 9 (AAV9) production and administration. All five AAV9 vectors (BIN1+13+17-V5) expressing GFP-V5, BIN1-V5, BIN1+13-V5, BIN1+17-V5, and cBIN1-V5 driven by the CMV promoter were custom-made and produced at Wilgen (Worcester, MA, USA). We used previously reported gated expression clones of V5-tagged GFP and mouse BIN1 isoforms

[31] , which were sequenced and sent to Wilgen for subsequent cloning into AAV vectors and virus preparations. Next, these gene inserts (GFP-V5 or BIN1-V5) were subcloned into the pAAV-CMV vector (Wilgen, 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 polyethyleneimine. After transfection, the supernatant and cells were collected. AAV virus was released from HEK293 cells by three freeze-thaw cycles. Virus in the culture medium was precipitated using PEG8000 (Sigma-Aldrich, St. Louis, MA, USA). The cell lysate and pelleted supernatant precipitate were combined and treated with universal nuclease (Merck, Kenilworth, NJ, USA) for 1 hour at 37°C. The virus was purified by iodixanol gradient centrifugation and concentrated using Amicon Ultra-15 centrifugal filters (Sigma-Aldrich, St. Louis, MO, USA).

[0092] Echocardiography for Functional Rescue Studies. Systolic and diastolic left ventricular (LV) function in anesthetized mice was monitored in vivo by echocardiography using a Vevo 7700 at baseline, before surgery, and every other week thereafter until the end of the experimental protocol. At 2 weeks postoperatively, the modified Bernoulli equation (Δpressure gradient (mm Hg) = 4 × peak velocity) was used. 2 (m / s) 2 All surviving mice 5 weeks after TAC were included in the study.

[0093] Echocardiography for isoproterenol studies. 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 coimmunoprecipitation. The peak intensity of Cav1.2 at the t-tubules was quantified by Image J as previously reported

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

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

[31] . Three-dimensional super-resolution stochastic optical reconstruction microscopy (STORM) images

[31] were obtained for nearest neighbor analysis between LTCC-RyR and SERCA2a-cBIN1 molecules.

[0094] The primary endpoint of severe HF-free survival versus non-survival, and HF classification. Overall survival was analyzed for all groups. In addition, severe heart failure (HF)-free 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 defined as death or EF < 35% within 20 weeks after TAC. At the end of the protocol, tibia length (TL), lung weight (LW), and heart weight (HW) were measured in surviving TAC mice.

[0095] Immunofluorescence labeling and confocal imaging. For fluorescent labeling of myocyte membranes, freshly isolated ventricular cardiomyocytes from GFP-TAC and cBIN1-TAC mice were incubated with di-8-ANNEP for 20 minutes at room temperature (RT). The cells were then washed with HBSS to remove excess dye before live cell imaging. For fixed cell V5 imaging (10x), isolated cardiomyocytes were fixed in methanol at -20°C for 5 minutes, then permeabilized and blocked with PBS containing 0.5% Triton X-100 and 5% normal goat serum (NGS) for 1 hour at room temperature. Cells were incubated with rabbit anti-V5 (Sigma) at 4°C overnight and detected with Alexa555-conjugated goat anti-rabbit IgG. For tissue immunofluorescence imaging, myocardial cryosections were fixed with ice-cold acetone for 5 minutes. The 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 DAPI containing ProLong gold. All confocal imaging was performed on a Nikon Eclipse Ti microscope 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), with diode-pumped solid-state (DPSS) lasers (486 nm, 561 nm, 647 nm) generated by a Laser Merger Module 5 (Spectral Applied Research, CA). Fluorescence intensity spectra of T-tubule membrane markers were generated by ImageJ, and the peak intensity at the T-tubule was quantified as previously reported

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

[0096] Immunofluorescence labeling and imaging were performed using a spinning disk confocal microscope. Myocardial tissue sections were embedded in 100% OCT medium, snap-frozen on dry ice with ethanol, sectioned at 10 μm as previously reported

[32] , and then stored in a −80°C freezer. After fixation with acetone, tissue cryosections were permeabilized with PBS containing 0.1% Triton X-100 and 5% normal goat serum (NGS, Life Technology) for 1 hour at room temperature. For V5, CaV1.2, and SERCA2a staining, tissue sections were incubated with primary antibodies against 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) at 4°C overnight. After washing several times with 1×PBS, the tissue sections were incubated with goat anti-mouse and anti-rabbit IgG conjugated with Alexa 4#88 and 555, respectively. Gold medium DAPI fixation. All images were acquired using a Nikon Eclipse Ti microscope with a 40×1.1 or 100×1.49 numerical aperture total internal reflection fluorescence objective and NIS Elements software (Nikon, Los Angeles, CA, USA). Confocal Z stacks with a Z step increment of 0.5 μm were collected using a spinning disk confocal unit (Yokogawa CSU10, Sugar Land, TX, USA) connected to the same titanium microscope with a diode-pumped solid-state laser (486 nm, 561 nm) generated by a Laser Merger Module 5 (Spectra Applied Research, Richmond Hill, ON, Canada) and captured by a high-resolution ORCA-Flash 4.0 digital CMOS camera. T-tubule Cav1.2 fluorescence intensity spectra were generated by ImageJ and the peak intensity at the t-tubule was quantified as previously reported

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

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

[0097] Power Spectrum Analysis. Frequency-domain power spectra of cardiomyocyte immunofluorescence subsections were generated in Matlab using FFT transformation [10, 30]. Power spectra were generated normalized to the maximum component and plotted against distance (1 / frequency, μm). Normalized peak power density

[71] was quantified and compared between groups.

[0098] 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, and 10% glucose with mercaptoethylamine) was added to the culture dish. STORM images were collected using a Nikon Eclipse Ti microscope with lasers (488 nm, 561 nm, from a separate 4-line laser module with an acousto-optic tunable filter) and captured by a high-speed iXon DU897 Ultra EMCCD camera. The STORM module was used to acquire and analyze images to generate 3-dimensional (3D) projections of Cav1.2 / RyR and cBIN1 / SERCA2a images with nanometer resolution. For nearest neighbor analysis, the original 3D STORM images were displayed using the Gaussian rendering algorithm available in Nikon Elements software, and 3D stacks of 3D STORM images in the molecule list text file (two channels per acquisition, Cav1.2 / RyR or cBIN1 / SERCA2a) were acquired at a z-spacing of 10 nm and a depth of 500 nm. The molecule list text file was imported into ImageJ and the closest distances (nearest neighbor distances) between the molecules of the two channels were calculated. The nearest neighbor distances were constructed and displayed as a frequency distribution histogram in a user-defined range and bin width and fitted to a 15th-degree polynomial curve with the first peak detected. The distances between the Cav1.2-RyR and SERCA2a-cBIN1 molecules at the corresponding first peak positions were quantified and compared between groups.

[0099] Transmission electron microscopy. All transmission electron microscopy (TEM) work was performed at the core facility of the Electron Imaging Center at the California NanoSystems Institute at UCLA. Tissue preparation was performed using previously reported methods

[72] . Briefly, mouse hearts were perfused with 20 mL of fresh fixative (2% glutaraldehyde and 2% paraformaldehyde in 1× PBS). Left ventricular tissue (1 mm 3) were postfixed with 1% osmium tetroxide and incubated in 3% uranyl acetate. After dehydration in ethanol, the samples were treated with propylene oxide, embedded in Spurr resin (Electron Microscopy Services), and sectioned using an ultramicrotome (Leica Microsystems). Sections were mounted on grids and stained with uranyl acetate and lead citrate before image acquisition using a JEM1200-EX, JEOL microscope (Gatan). The extent of the outlined t-tubules was quantified using a modified scoring system previously established

[29] .

[0100] Western blot for functional rescue studies. Tissue lysates were prepared from hearts that had been snap-frozen in liquid nitrogen. Frozen tissue was homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer as previously described

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

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

[0101] Western blot for isoproterenol studies. Frozen heart tissue was homogenized using RIPA lysis buffer containing protease inhibitors and protein concentration was determined using the Bradford assay. TM Novex TMThe samples were separated on 4-12% Bis-Tris protein gels and then transferred to polyvinylidene difluoride membranes. After blocking with 1× TNT buffer containing 5% bovine serum albumin (BSA) for 1 hour, the 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) at 4°C overnight, followed by incubation with secondary antibodies (goat anti-rabbit or mouse IgG-Alexa 647) at room temperature (RT) for 1.5 hours. The membranes were then incubated with Molecular ELISA kits. Gel Doc TM Immunoreactive bands were imaged using the XR+ system (Bio-Rad Laboratories, Irvine, CA, USA), and band intensities were quantified using Image Lab software (Bio-Rad Laboratories, Irvine, CA, USA).

[0102] Preparation of cardiac microsomes and sucrose gradient fractionation. Preparation of microsomal sucrose gradient fractionation was modified according to an established protocol

[70] . For each experimental group, myocardial membrane microsomes were prepared from starting material from one heart. 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 minutes at 4°C, and the supernatant (S1) was collected in a pre-weighed tube and kept on ice. The pellet was resuspended in 1 mL of the same buffer, homogenized, and centrifuged at 12,000 × g for 20 minutes at 4°C. The supernatant (S2) was collected and combined with S1 from the previous step. The combined microsomal supernatant (S1 + S2) was then ultracentrifuged at 110,000 × g for 2 hours at 4°C. After ultracentrifugation, the supernatant was discarded, the pellet was weighed, and an appropriate amount of buffer (~1 mL) was added to achieve a final microsomal concentration of ~25 mg / mL. The total protein concentration of the resuspended microsomes was measured for each sample using a Nanodrop 2000 and normalized across the four groups. The same amount of total microsomes from each sample (3-6 mg, 0.5 mL) was carefully overlaid on top of a discontinuous sucrose gradient (52, 58, 73) and 45% v / w in homogenization buffer, 2 mL each) and ultracentrifuged at 150,000 × g for 16 hours using a Beckman Coulter Optima Max XP benchtop ultracentrifuge in a fixed-angle MLA-55 rotor. Samples were then collected from the following fractions: F1, 27%; F2, 27 / 32%; F3, 32 / 38%; F4, 38 / 45%; and the pellet (P) from the bottom of the tube. For each fraction, approximately 1 mL was collected, diluted 4-fold in homogenization buffer, and ultracentrifuged at 120,000 × g for 2 hours at 4°C. The pellet was resuspended in 100 μL of homogenization buffer, and the protein concentration was measured by Nanodrop2000. The total protein yield recovered 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 / heart, respectively. Sample buffer was added before the sample was frozen and stored at -20°C before subsequent Western blot analysis.

[0103] 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 used the log-rank test to compare two groups and the log-rank trend test to compare three groups. Continuous variables were compared using T-test / Mann-Whitney U test and one-way analysis of variance (ANOVA) / Kruskal-Wallis test. Two-way ANOVA analysis was used to determine the differences between the two groups at two different time points. Two-way ANOVA was used to determine the differences between the 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 or chi-square test. Data were analyzed using GraphPad Prism (version 7.0; GraphPad Software, La Jolla, CA, USA). Two-sided p values ​​were used, and p < 0.05 was considered statistically significant.

[0104] Example 2

[0105] Exogenous cBIN1 functionally rescues mouse hearts from pressure-overload-induced heart failure

[0106] To explore whether targeting the cBIN1 microdomain could be a novel therapy for HF, we investigated how cardiac cBIN1 affects the development of HF in mice subjected to pressure overload. Transverse aortic constriction (TAC) or a mock procedure (sham surgery) was performed in 8-10 week old adult male mice, followed by echocardiographic monitoring to determine overall survival and survival without severe systolic HF (non-survival was defined as death or ejection fraction < 35%). Figure 1A As indicated in the experimental protocol, mice were first treated with TAC for 5 weeks, followed by retroorbital injection of AAV9 transduced with cBIN1-V5 or control GFP-V5, and then monitored by echocardiography for 15 weeks after viral injection (20 weeks after TAC). In addition to a group of mice that underwent thoracotomy (sham control group, N = 10), 36 mice underwent TAC. One mouse died before reaching time 0 (5 weeks after TAC), and the remaining 35 surviving mice were randomly assigned to receive 3 × 10 10 Vg of AAV9-GFP (N = 17) or cBIN1 (N = 18). Anti-V5 labeling of cardiomyocytes isolated from mice 15 weeks after AAV9 injection identified positive V5 signals, indicating that the exogenous protein was successfully transduced into cardiomyocytes ( Figure 16 Comparable transaortic pressure gradients were observed in these mice 2 weeks after TAC ( Figure 1B ) and 5 weeks after TAC, as evidenced by decreased left ventricular (LV) EF and increased LV end-diastolic volume (EDV) before AAV9 injection ( Table 1 ).

[0107]

[0108]

[0109] Then, the overall survival rate of all groups was explored (death was counted as non-survival). As shown by the Kaplan-Meier curve in Figure 2, the overall survival rate of AAV9-cBIN1-treated TAC mice (survival rate 77.8%, 14 / 18) was between the sham-operated group (survival rate 100%, 10 / 10) and AAV9-GFP-treated TAC mice (survival rate 58.8%, 10 / 17) (when compared among the three groups, p = 0.0202, by log-rank test for trend) ( Figure 2A Next, among all surviving TAC mice with EF ≥ 35% at time 0 (5 weeks after TAC and before virus injection), the survival rate without contractile HF between the two virus groups during follow-up echocardiographic monitoring was further analyzed (death or EF < 35% was counted as non-survival). AAV9-cBIN1 significantly improved the survival rate without contractile HF (p = 0.0225 when compared with the AAV9-GFP group, by log-rank test) ( Figure 2B ). Of the 11 AAV9-GFP mice with an EF ≥ 35% at time 0, 9 developed an EF < 35% within 20 weeks after TAC, of ​​which 2 died prematurely and 7 developed progressive EF decrease. In contrast, of the 13 AAV9-cBIN1 mice with an EF ≥ 35% at time 0, only 5 developed an EF < 35% within 20 weeks after TAC, and 1 died prematurely. In addition, of these 5 mice counted as non-survivors in the Kaplan-Meier curve analysis, except for one mouse that died prematurely, the remaining 3 of the 4 non-surviving AAV9-cBIN1-treated animals had an EF recovery rate of more than 35% at 20 weeks after TAC. The surviving mice were then sacrificed and the HW / TL and LW / TL ratios were evaluated. When compared with sham-operated control mice, the HW / TL and LW / TL of AAV9-cBIN1 mice did not increase significantly as they did in AAV9-GFP mice ( Figures 3A to 3B These data suggest that cBin1 gene therapy protects cardiac function and effectively improves overall and HF-free survival, thereby interrupting, delaying, and even reversing the degenerative cycle of HF progression.

[0110] In all mice that survived 20 weeks after TAC, echocardiographic measurements of myocardial function and physiological parameters were further compared between the groups before and after AAV9 treatment (Table 1). At 20 weeks after TAC, AAV9-GFP mice developed significant LV systolic dysfunction (decreased EF) and ventricular dilatation (increased EDV). Figures 4A to 4C, Table 1), which were normalized by AAV9-cBIN1 treatment. AAV9-cBIN1-treated mice had significantly reduced LV mass gain at 20 weeks after TAC when compared with the AAV9-GFP group ( Figure 4D Furthermore, the observed Δ reduction in stroke volume and cardiac output in the AAV9-GFP group was also abolished in the AAV9-cBIN1-treated group ( Figures 4E to 4F These results suggest that cBin1 gene therapy preserves myocardial function when administered to failing hearts.

[0111] To further explore the progression of systolic dysfunction in the heart after TAC at time 0 after virus injection (before AAV9 and 5 weeks after TAC), the incremental EF changes (ΔEF) from before AAV to 3, 6, 8, 10, and 15 weeks after AAV9 injection (8, 11, 13, 15, and 20 weeks after TAC, respectively) were monitored by echocardiography ( Figure 5A ). Exogenous cBIN1-induced EF recovery peaked at 6 to 8 weeks after AAV9 injection, and EF continued to improve in the following weeks, while progressive EF reduction was noted in the AAV9-GFP group. The observed EF recovery was demonstrated by the Gaussian fit of the ΔEF histogram distribution ( Figures 5B to 5C ). The ΔEF histogram distribution of AAV9-cBIN1 was right-shifted when compared to the AAV9-GFP group. For example, at 6 weeks after AAV9, there was a moderate EF (%) decrease of -15.0 in the AAV9-GFP group, while a moderate EF (%) recovery of +6.9 was observed in the AAV9-cBIN1 group. These data suggest that exogenous cBIN1 can rescue myocardial contractile function in TAC-induced HF hearts when administered 5 weeks after TAC.

[0112] We recently reported that in mice pretreated with AAV9-cBIN1 (3 × 10 10 vg; Figure 6A ), the incidence of TAC-induced HF was significantly reduced, resulting in a higher HF-free survival rate at 8 weeks after TAC

[34] . These data are consistent with the cardioprotective effect observed when AAV9 was administered after TAC surgery. To further determine the cardioprotective effect of exogenous cBIN1 in TAC mice, intracardiac hemodynamics were obtained in AAV9-pretreated mice using invasive PV loop recordings. Figure 6B Representative PV loops containing sham-operated, AAV9-GFP, and AAV9-cBIN1-pretreated hearts 8 weeks after TAC surgery ( Figure 6B The AAV9-GFP group showed reduced EF and maximum rate of pressure change (dp / dt max) during contraction, which were normalized by exogenous cBIN1 ( Figures 6C to 6D). In exploring the effects on relaxation kinetics, we found that the rate of decline in maximum pressure in the AAV9-GFP group (dp / dt min) was reduced, but normalized by AAV9-cBINl ( Figure 6E ). The increase in isovolumic relaxation time constant (τ) in the AAV9-GFP group was also rescued by cBinl gene transfer ( Figure 6F ), indicating improved cardiac relaxation. Taken together, these data suggest that exogenous cBINl improves both systolic and diastolic function in pressure-overloaded hearts.

[0113] We previously found that cBINl generates t-tubule microdomains and organizes LTCC-RyR dyads for efficient dynamic regulation of cardiac function and EC coupling

[25] . Recently, we found that cBINl microdomains were disrupted in sympathetically overdriven mouse hearts developing diastolic dysfunction and rescued by AAV9-cBINl. Here, we also explored alterations in cardiac t-tubule cBINl microdomains and the role of exogenous cBINl in post-TAC hearts. Western blotting ( Figure 7A ) identified a significant reduction in myocardial cBINl protein in post-TAC 8-week mice (27% less than sham control, p<0.05), which was normalized by AAV9-cBINl pretreatment. Along with cBINl rescue, membrane labeling with di-8- ANNEP ( Figure 17 ) determined a significant reduction in t-tubule cBINl microdomain intensity in post-TAC 8-week cardiomyocytes compared to sham cardiomyocytes, which was normalized in AAV9-cBINl pretreated mouse cardiomyocytes. Next, immunofluorescence imaging was used to analyze cBINl microdomain and dyad organization at the myocyte level. Power spectral analysis of BINl signal determined that the well-organized t-tubule distribution of cBINl in sham myocardium was disrupted in post-TAC hearts, which was preserved with AAV9-cBINl pretreatment ( Figure 7C ). Although there were no significant changes in total LTCC and RyR protein levels ( Figure 7B ) among groups by Western blotting, myocardial distribution of LTCC and RyR ( Figure 7C ) became disorganized in post-TAC hearts, which was also significantly improved in AAV9-cBINl pretreated hearts (p<0.05). These data suggest that exogenous cBINl normalizes post-TAC myocardial cBINl reduction, resulting in the preservation of cBINl microdomains at t-tubules. Via the normalization of t-tubule microdomains in pressure-overloaded hearts, cBINl replacement therapy thus reconstitutes the cardiac LTCC-RyR coupling subunits required for beat-to-beat calcium cycling and efficient EC coupling.

[0114] Example 3

[0115] Exogenous cBIN1 reduces concentric hypertrophy in mouse hearts after isoproterenol infusion

[0116] studied the effects of cBIN1 on myocardial function in animals receiving 4 weeks of isoproterenol infusion ( Figure 8A ). AAV9 was used to introduce myocardial expression of exogenous V5-tagged GFP or cBIN1

[74] 3 weeks before the start of isoproterenol. Anti-V5 labeling identified a similar percentage of myocardial area with detectable V5 signal 7 weeks after AAV9 injection (GFP, 62.4+10.5%; cBIN1, 57.9.2+7.8%), indicating that the exogenous protein was successfully transduced in more than half of the cardiomyocytes (Figure 18). The remaining nearly 40% of negatively stained cardiomyocytes probably expressed the exogenous protein at low levels below the threshold of immunofluorescence detection. In all mice, isoproterenol significantly increased the ratio of heart weight to body weight (HW / BW), indicating cardiac hypertrophy ( Figure 8B ). Echocardiography to assess cardiac geometry and function ( Figures 8C to 8H In AAV9-GFP pretreated animals, isoproterenol induced a significant increase in LV mass and relative wall thickness (RWT) without altering end-diastolic volume (EDV), consistent with echocardiographic classification of concentric hypertrophy

[75] . In AAV9-cBIN1 pretreated animals, the isoproterenol increase in LV mass was attenuated with normal RWT and increased EDV, similar to the results using previously reported methods

[75] ( Figure 19A ) echocardiographic classification of “physiological hypertrophic-like remodeling”. In addition, α-smooth muscle actin was increased in GFP+ISO hearts but not in cBIN1+ISO hearts ( Figure 19B ), indicating that AAV9-cBIN1 limited isoproterenol-induced LV hypertrophy. In mice pretreated with AAV9-GFP, isoproterenol caused a small increase in EF (p = 0.050 vs. GFP + PBS and p = 0.007 vs. cBIN1 + PBS groups), but the ratio of LV passive filling (E) to myocardial relaxation (e') (E / e') began to increase strongly, indicating the onset of diastolic dysfunction. In mice pretreated with AAV9-cBIN1, isoproterenol still increased systolic function and, importantly, maintained normal E / e', indicating positive inotropy and preserved diastolic tension. In addition, although blood pressure was not measured, isoproterenol significantly increased heart rate (HR) in all animals, indicating the effectiveness of isoproterenol in causing hemodynamic stress. However, HR after isoproterenol was not different 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 rather than increased heart rate.

[0117]

[0118] Example 4

[0119] Chronic isoproterenol-induced disruption of cBIN1 microdomains can be normalized by AAV9-cBIN1

[0120] It is well known that myocardial contractility and relaxation are both related to myocardial calcium cycling

[76] . The structural organizing factor cBIN1 of the dimeric microdomain

[31] generates t-tubule microfolding to limit extracellular Ca 2+ Diffusion

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

[30] and LTCC clusters delivered to the t-tubule membrane. Therefore, we explored how cBIN1 microdomains are remodeled in hypertrophic hearts after chronic isoproterenol infusion. Western blotting of cardiac lysates showed that isoproterenol induced a significant decrease in cBIN1 protein, which was 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 confirmed that the protein band examined was the cBIN1 (BIN1+13+17) isoform. The total t-tubule network structure was also examined in isolated cardiomyocytes labeled with the membrane dye di-8-ANNEP ( Figure 9B Live cell imaging and power spectrum analysis showed that overall t-tubule organization (normalized peak power density) remained similar in GFP+ISO mice but was enhanced by AAV9-cBIN1. Although the overall t-tubule network remained organized when imaged using transmission electron microscopy (TEM), a reduction in t-tubule microfolding was noted in GFP+ISO hearts, which was preserved in cBIN1+ISO hearts ( Figure 9C ). Quantification of the extent of outlined t-tubules using a previously established modified scoring system

[29] (1, round or dilated t-tubules with no luminal folds; 2, non-circular outlined t-tubules with no luminal folds; 3, t-tubules with 2-3 layers of folds; or 4, t-tubules with >3 layers of folds) identified a significant reduction in t-tubule outlines in GFP+ISO hearts, which was normalized in cBIN1+ISO hearts (p<0.001, chi-square test). Note that enlarged microfolds (more than 3 layers of folds, score 4) were found in cBIN1+PBS hearts, a result above the physiological level of cBIN1. These data suggest that cBIN1 is critical for the formation of t-tubule microfolds, that the folds are downregulated under chronic sympathetic overdrive, and that the folds can be restored by exogenous cBIN1 treatment.

[0121] Subsequently, we explored the expression and intracellular distribution of Cav1.2 in cardiomyocytes. In hearts treated with isoproterenol, the net myocardial protein expression of Cav1.2 was similar ( Figure 10A However, immunofluorescence labeling of myocardial tissues with Cav1.2 revealed a significant decrease in channel density along t-tubules in GFP+ ISO cardiomyocytes, which was reversed by AAV9-cBIN1 ( FIG. 10B to FIG. 10C ) were normalized (power spectrum and fluorescence spectrum analysis). These data are consistent with previous observations of altered Cav1.2 protein distribution, but with similar total protein levels

[32] . With reduced localization of Cav1.2 to t-tubules, the peak amplitude of calcium transients (ΔF / F0) was significantly reduced in GFP+ISO cardiomyocytes when compared to control GFP+PBS cardiomyocytes ( Figure 10D ), which was normalized by exogenous cBIN1. Total protein expression and intracellular distribution of ryanodine receptor 2 (RyR) were not different in all groups (Figure 20). However, RyR was hyperphosphorylated at PKA-dependent S2808 and CAMKII-dependent S2814, consistent with previous reports

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

[0122] Example 5

[0123] Exogenous cBIN1 improves SERCA2a distribution along the SR

[0124] Cardiac diastolic function is most directly related to calcium reuptake via SERCA2a. Surprisingly, despite impaired diastolic function in GFP+ISO hearts, total SERCA2a protein expression was significantly increased after isoproterenol infusion ( Figures 11A to 11B). 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 can normalize after chronic isoproterenol infusion, and even possible dephosphorylation of PLN due to activation of serine / threonine phosphatases PP1 and PP2A [73, 77]. Consistent with these reports, the results of the present invention showed that the lack of change in PLN phosphorylation after 4 weeks of isoproterenol infusion can be the net result of localized activation of both kinases and phosphatases in balance. These data show that SERCA2a protein and activity are not reduced in the isoproterenol post-heart. Given the effects of cBIN1 on Cav1.2 localization, SERCA2a localization was examined. Myocardial tissue sections with SERCA2a labeling were imaged with spinning-disk confocal microscopy and compared between groups Figure 11C ). In GFP+PBS hearts, SERCA2a subpopulation localized to the t-tubule / jSR region, resulting in an organized distribution with a major power spectral peak at 1.8-2 pm, corresponding to the full length of sarcomere. cBIN1 overexpression in cBIN1+PBS hearts further increased SERCA2a signal near the t-tubule / jSR. In GFP+ISO hearts, the intracellular distribution of SERCA2a was disorganized with a significantly reduced peak power density, which was normalized in cBIN1+ISO hearts Figure 11D ).

[0125] Biochemical sucrose gradient fractionation of cardiac microsomes was used to further explore the intracellular distribution of Cav1.2 and SERCA2a

[70] . As shown in Figure 22A Fraction F4 had the lowest recovery yield when compared to other fractions. However, even with the low yield, Cav1.2 and cBIN1 were detectable in F4 with limited Na + / K + -ATPase and depleted SERCA2a, indicating that F4 is enriched in t-tubule-derived microsomes Figure 22B ). When normalizing for t-tubule protein concentration in F4 (2.5 pg protein loaded per lane) in all samples, cBIN1 and Cav1.2 protein per unit of t-tubule were significantly reduced in GFP+ISO hearts compared to control GFP+PBS hearts, which was normalized by AAV9-cBIN1 pretreatment Figure 12AThese data are consistent with immunofluorescence imaging, which confirmed that the t-tubule localization of Cav1.2 channels was reduced after isoproterenol infusion and restored with AAV9-cBIN1. On the other hand, SR proteins were only detected in fractions F2 and F3. When the SR protein concentrations of F2 and F3 were normalized (25 μg protein loaded per lane), F3 had relatively more RyRs and fewer PLNs compared with F2 ( Figure 12B ), indicating that jSR was more enriched in the heavier F3 fraction. Quantification of SERCA2a expression in F2 and F3 confirmed that AAV9-cBIN1 resulted in a significant increase in SERCA2a distribution to the heavier and more jSR-enriched F3, rather than the longitudinal SR-enriched F2 fraction, when compared with AAV9-GFP ( Figure 12B Notably, isoproterenol alone did increase SERCA2a expression in F3 hearts of AAV9-GFP mice, likely due to an overall increase in total SERCA2a protein expression in the heart following isoproterenol ( Figure 11A These data suggest that exogenous cBIN1 can maintain t-tubule microdomains to localize Cav1.2 and SERCA2a to their functional sites in isoproterenol-infused hearts.

[0126] Given the reduction of Cav1.2 and SERCA2a in the t-tubule / jSR region, STORM imaging was used to analyze the nanoscale protein-protein colocalization of Cav1.2-RyR and SERCA2a-cBIN1 (Figure 13). Using nearest neighbor analysis, the distance between each Cav1.2 molecule and its nearest RyR molecule was quantified. The histogram distribution of the distance between Cav1.2-RyR molecules from whole-cell images identified the first peak around 40 nm corresponding to the dyad coupler in GFP+PBS, GFP+ISO, and cBIN1+ISO cardiomyocytes. In GFP+ISO hearts with preserved contractile function, the distribution histogram tended to shift to the right, but the first peak position was retained (Figure 14). Figure 13B Interestingly, the histogram distribution of cBIN1+PBS myocytes shifted to the left and the distance between Cav1.2-RyR peaks was significantly reduced, suggesting that the magnified cBIN1 microfolding observed in TEM imaging may bring the tightened couplers closer. On the other hand, in animals pretreated with AAV9-GFP, the distance between SERCA2a and its nearest neighbor cBIN1 at the t-tubule tended to increase after isoproterenol (p=0.063, GFP+PBS vs. GFP+ISO), which was significantly reduced in animals pretreated with AAV9-cBIN1 (p<0.001, GFP+ISO vs. cBIN1+ISO) ( FIG. 13C to FIG. 13D These data suggest that cBIN1-microfolds can regulate colocalization and interactions between EC coupling and calcium handling proteins.

[0127] Example 6

[0128] The cBIN1+ISO cardiac phenotype is isoform-specific and unique to cBIN1.

[0129] To further explore whether the observed cBIN1+ISO cardiac phenotype was an isoform-specific effect, the isoproterenol protocol was repeated in more than 50 additional mice, randomized to receive AAV9 transduction with GFP and cBIN1, as well as three other BIN1 isoforms expressed in mouse cardiomyocytes, including mini-BIN1, BIN1+17, and BIN1+13. Similarly, three weeks after viral administration, mice received continuous subcutaneous isoproterenol infusion at 30 mg / kg / day for 4 weeks. When compared between the five groups of mice transduced with GFP or BIN1 isoforms, protein expression of Cav1.2 and SERCA2a in the heart after isoproterenol was not significantly different ( Figure 23A Immunofluorescence labeling of myocardial tissue for Cav1.2 channels revealed that channel density along the t-tubules was significantly increased only in hearts expressing cBIN1, but not in other BIN1 isoforms ( Figure 23B , quantified in Figure 23D Immunofluorescence imaging revealed that exogenous cBIN1 introduced by AAV9 organized SERCA2a distribution ( Figure 23C , Figure, quantification in 23E), consistent with the data in Figure 11 .

[0130] Next, echocardiography was used to explore the functional consequences of pretreatment with different AAV9-BIN1 isoforms. Mice expressing cBIN1 had reduced isoproterenol-induced increases in LV wall thickness, LV mass, and RWT compared to the GFP group ( 14A to 14D , Table 3). In all animals, cardiac output increased significantly from its baseline level after isoproterenol, as a result of the isoproterenol-induced increase in heart rate RWT (Table 3). However, only cBIN1 hearts had improved contractile function, normalized E / e', increased stroke volume, and further increased cardiac output when compared to GFP hearts after isoproterenol ( Figures 14E to 14H Notably, a partial cBIN1-like effect occurred in mice pretreated with BIN1+17, which significantly reduced LV mass, E / e', and attenuated the isoproterenol-induced RWT. The partial rescue of diastolic function observed with BIN1+17 was consistent with the partial rescue of the intracellular distribution of SERCA2a by immunofluorescence imaging ( Figure 23CHowever, because BIN1+17 fails to increase Cav1.2 distribution at the t-tubules, there is no positive inotropic effect after isoproterenol infusion in AAV9-BIN1+17-pretreated hearts.

[0131]

[0132] Example 7

[0133] The cardioprotective effect of AAV9-cBIN1 was confirmed in TAC-induced HF

[0134] The cardioprotective effects of cBIN1 were further explored in an additional mouse model of TAC-induced pressure overload. In this study, mice with either genetic deficiency of cBIN1 or AAV9-transduced cBIN1 overexpression were tested. Figure 15A ). Deficiency studies involved cardiac-specific Bin1 HT mice and WT littermate controls

[29] , both of which received TAC for 8 weeks. Overexpression studies involved mice that received 8 weeks of TAC and had been previously injected with AAV9 transduced with cBIN1-V5 or AAV9-GFP-V5, as well as mice that underwent open-chest mock surgery (sham surgery). Mice were monitored and terminated 8 weeks after surgery. Virus (AAV9-GFP / cBIN1-V5), dose (3 × 10 10 vg), administration time (3 weeks before surgery), and route (retroorbital injection) were the same as those used in the isoproterenol study. Aortic constriction was confirmed in all TAC mice by an increase in the transaortic pressure gradient ( Figure 15B ), thereby establishing a similar increase in hemodynamic afterload in all mice receiving TAC. Kaplan-Meier curves summarizing survival without severe HF (EF ≥ 35%) are included in Figure 15C The survival rate of Bin1 HT mice was 20.0% (8 of 10, 2 deaths and 6 EF <35%), which was reduced compared to 71.4% of WT mice (4 of 14, 1 death and 3 EF <35%) (log-rank test p = 0.038). As expected, all sham-operated mice survived throughout the experimental protocol (10 of 10, black dashed line). The survival rate of AAV9-GFP mice decreased to 64.3% (5 of 14 did not survive, 5 EF <35%), which was significantly improved to 93.7% in the AAV9-cBIN1 group (1 of 16 did not survive, 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 systolic HF after pressure overload.

[0135] At 8 weeks after TAC, surviving mice were sacrificed and the HW / BW and LW / BW ratios were evaluated (Table 4, Figures 15D to 15E ). Both HW / BW and LW / BW in Bin1 HT mice were significantly higher than those in WT mice, indicating that BIN1 deficiency leads to worsening of LV hypertrophy and pulmonary edema. Regarding gene therapy, AAV9-cBIN1 significantly reduced LW / BW from the levels of the control GFP-TAC group to the levels of sham-operated hearts, indicating that TAC-induced pulmonary edema was significantly reduced. Hypertrophy still existed in AAV9-cBIN1 hearts, but to a lesser extent. These data indicate that exogenous cBIN1 alleviates TAC-induced hypertrophy and prevents deterioration to HF. Echocardiographic analysis ( Figures 15F to 15J , Table 5) also showed that there was a significant decrease in EF and an increase in EDV in Bin1 HT-TAC hearts when compared to WT-TAC mice, indicating that BIN1 deficiency leads to worsening of dilated cardiomyopathy. On the other hand, AAV9-cBIN1 significantly reduced TAC-induced LV dilation (EDV) and systolic dysfunction (EF), thereby limiting the development of dilated cardiomyopathy. Therefore, AAV9-cBIN1 pretreatment maintained the stroke volume and cardiac output of the heart after TAC without dilation of the heart. In addition, tissue Doppler determined that the diastolic parameter E / e' values ​​of the lateral and septal walls in the AAV9-cBIN1 pretreated hearts were significantly improved, indicating that the diastolic function of mice with exogenous cBIN1 was better. 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 the hearts of pressure-overloaded mice.

[0136]

[0137]

[0138]

[0139] Example 8

[0140] Study on diabetic cardiomyopathy (HFpEF) in mice

[0141] Myocardial function and the therapeutic potential of AAV9-cBIN1 were explored in db / db mice with diabetic cardiomyopathy. The db / db mouse line (Leprdb homozygous Dock7m, Jackson Laboratory) is an established mouse model of type 2 diabetes that 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 littermate control db / m mice were given a single dose (1 × 10 11vg) AAV9-cBIN1 or control GFP [79,80]. When the animals were 17 weeks old, cardiac function and physiological parameters were measured before and 8 weeks after AAV injection

[79] . Myocardial function was assessed by echocardiographic measurements of systolic parameters (ejection fraction and fractional shortening), diastolic parameters (E / A, E / e'), and stroke volume (SV). We also assessed the performance of these animals in an exercise exhaustion test by measuring their maximum running distance on a mouse treadmill. Exercise intolerance is an important physiological parameter of impaired cardiac reserve and HFpEF.

[0142] Echocardiographic measurements of myocardial function parameters showed that diastolic failure developed as early as 9 weeks of age in db / db mice. In 17-week-old db / db mice with preserved left ventricular ejection fraction, significant changes in diastolic parameters were observed, including a decrease in E / A ( Figure 23A ) and E / e' increased ( Figure 24B All of these abnormal diastolic parameters could be rescued and normalized by AAV9-cBIN1 treatment. As AAV9-cBIN1 improved diastolic function, the reduced left ventricular stroke volume in db / db mice was normalized ( Figure 24C Along with the improvement in echocardiographic parameters, treatment with AAV9-cBIN1 also significantly improved exercise intolerance (reduced maximum running distance on a treadmill) in these diseased db / db mice ( Figure 24D ). Note that the two groups of db / db mice treated with AAV9-GFP or cBIN1 had similar body weights, indicating similar levels of obesity and type 2 diabetes. Together with the rescue of diastolic parameters measured by echocardiography, these data suggest that the AAV9-cBIN1-mediated rescue of exercise tolerance is due to improved myocardial functional reserve.

[0143] Example 9

[0144] Overview of Canine Ischemic Cardiomyopathy (HFrEF) Research

[0145] We explored the effect of cBIN1 gene therapy on rescuing diminished cardiac function in hearts with ischemic cardiomyopathy. Adult beagle dogs (25-30 kg) underwent thoracotomy and permanent ligation of the proximal left anterior descending (LAD) coronary artery. Dogs were followed 8 to 9 weeks after ligation by echocardiography, hemodynamic parameters, and physiological parameters. The animals were anesthetized and injected into the left ventricular endocardium with cBIN1 packaged in AAV9 virus. The NOGA XP (Bernsons Webster / Johnson & Johnson) was used for 3D electroanatomical mapping of the LV endocardial cavity. Using the same NOGA XP system, we injected the myocardium with a Myostar catheter with a 27-gauge nitinol needle. Each heart was injected at 20 injection sites throughout the left ventricular endocardium. Each injection consisted of 2.5 × 10 11 Each animal heart consists of 5×10 12 The animals were then monitored continuously by echocardiography, hemodynamics, and physiological parameters.

[0146] like Figure 25 As shown, we report the changes in measured left ventricular ejection fraction (LVEF) over the course of the study week. Data from two animals are included. Time 0 corresponds to the time of LAD ligation. Arrows indicate the time of cBIN1 treatment. 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 continued to recover. The second dog was terminated at 12 weeks due to colonic ischemia caused by ingestion of a foreign body.

[0147] In both animals, cBIN1 gene therapy significantly rescued myocardial function in hearts with ischemic cardiomyopathy (HFrEF). The rescue occurred for at least five weeks. Trials are ongoing, and the duration of treatment after a single cBIN1 injection event remains to be determined.

[0148] References

[0149] 1. Roger, “Epidemiology of heart failure,” Circulation Research 113:646–659 (2013).

[0150] 2. Virani et al., “Heart Disease and Stroke Statistics-2020 Update: A Report from the American Heart Association,” Circulation 141:e139–e596 (2020).

[0151] 3. Gomez et al., “Defective excitation-contraction coupling in experimental cardiac hypertrophy and heart failure,” Science 276: 800-806 (1997).

[0152] 4. Perreault et al., “Abnormalities in intracellular calcium regulation and contractile function in myocardium from dogs with pacing-induced heart failure,” J Clin Exam 89:932-938 (1992).

[0153] 5. Landstrom et al., “Calcium Signaling and Cardiac Arrhythmias,” Circulation Research 120:1969–1993 (2017).

[0154] 6. Lopez-Crisosto et al., “Sarcoplasmic reticulum-mitochondria communication in cardiovascular pathophysiology,” Nature Reviews Cardiology 14:342–360 (2017).

[0155] 7. Bers, “Calcium cycling and signaling in cardiac myocytes,” Annual Review of Physiology 70, 23-49 (2008).

[0156] 8. Lyon et al., “Loss of T-tubules and other changes to surface topography in ventricular myocytes from failing human and rat heart,” Proceedings of the National Academy of Sciences of the United States of America, 106, 6854–6859 (2009).

[0157] 9. Louch et al., “There goes the neighborhood: pathological alterations in T-tubule morphology and consequences for cardiomyocyte Ca2+ handling,” Journal of Biomedicine & Biotechnology 503906 (2010).

[0158] 10. Wei et al. T-tubule remodeling during transition from hypertrophy to heart failure. Circulation Research 107:520-531 (2010).

[0159] 11. Litwin et al., "Asynchronous Ca( 2+ )Spark(Dyssynchronous Ca( 2 + )sparks in myocytes from infarcted hearts),” Circulation Research 87:1040-1047 (2000).

[0160] 12. Erkasap, “SERCA in genesis of arrhythmias: what we already know and what is new?” Anatolian Journal of Cardiology 7 Suppl 1: 43-46 (2007).

[0161] 13. Houser et al., "Functional properties of failing human ventricular myocytes", Trends Cardiovasc Med 10: 101-107 (2000).

[0162] 14. Marx et al., "PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts", Cell 101 : 365-376 (2000).

[0163] 15. Periasamy and Huke, "SERCA pump level is a critical determinant of Ca(2+) homeostasis and cardiac contractility", J Mol Cell Cardiol 33: 1053-1063 (2001).

[0164] 16. Santulli et al., "Mitochondrial calcium overload is a key determinant in heart failure", Proc Natl Acad Sci U S A 112: 11389-11394 (2015).

[0165] 17. Kinnally et al., "Is mPTP the gatekeeper for necrosis, apoptosis, or both?" Biochim Biophys Acta 1813: 616-622 (2011).

[0166] 18. Nakayama et al., “Ca2+- and mitochondrial-dependent cardiomyocyte necrosis as a primary mediator of heart failure,” Journal of Clinical Invest 117:2431-2444 (2007).

[0167] 19. Odagiri et al., “Local control of mitochondrial membrane potential, permeability transition pore and reactive oxygen species by calcium and calmodulin in rat ventricular myocytes,” Journal of Molecular and Cellular Cardiology 46:989–997 (2009).

[0168] 20. Zhou et al., “Mitochondrial dysfunction in pathophysiology of heart failure,” Journal of Clinical Investigation 128:3716–3726 (2018).

[0169] 21. Nikolaev et al., “Beta2-adrenergic receptor redistribution in heart failure changes cAMP compartmentation,” Science, 327:1653–1657 (2010).

[0170] 22. Ibrahim et al., “The structure and function of cardiac t-tubules in health and disease,” Acta Biol Sci 278: 2714-2723 (2011).

[0171] 23. Schobesberger et al., “T-tubule remodelling disturbs localized beta2-adrenergic signalling in rat ventricular myocytes during the progression of heart failure,” Cardiovasc Res 113:770–782 (2017).

[0172] 24. Balycheva et al., “Microdomain-specific localization of functional ion channels in cardiomyocytes: an emerging concept of local regulation and remodelling,” Biophysical Reviews 7: 43–62 (2015).

[0173] 25. Hong and Shaw, “Cardiac T-Tubule Microanatomy and Function,” Physiological Reviews 97: 227–252 (2017).

[0174] 26. Glukhov et al., “Direct Evidence for Microdomain-Specific Localization and Remodeling of Functional L-Type Calcium Channels in Rat and Human Atrial Myocytes,” Circulation 132: 2372–2384 (2015).

[0175] 27. Sanchez-Alonso et al., “Microdomain-Specific Modulation of L-Type Calcium Channels Leads to Triggered Ventricular Arrhythmia in Heart Failure,” Circulation Research 119:944–955 (2016).

[0176] 28. Bers, “Cardiac excitation-contraction coupling,” Nature 415: 198-205 (2002).

[0177] 29. Hong et al., “CardiacBIN1 folds T-tubule membrane, controlling ion flux and limiting arrhythmia,” Nature Medicine 20: 624–632 (2014).

[0178] 30. Hong et al., “BIN1 localizes the L-type calcium channel to cardiac T-tubules,” PLoS Biol 8: e1000312 (2010).

[0179] 31. Fu et al., “Isoproterenol Promotes Rapid Ryanodine Receptor Movement to Bridging Integrator 1 (BIN1)-Organized Dyads,” Circulation 133:388–397 (2016).

[0180] 32. Hong et al., “BIN1 is reduced and Cav1.2 trafficking is impaired in human failing cardiomyocytes,” Heart Rhythm 9:812–820 (2012).

[0181] 33. Caldwell et al., “Dependence of cardiac transverse tubules on the BAR domain protein amphiphysin II (BIN-1),” Circulation Research 115: 986–996 (2014).

[0182] 34. Liu et al., “In Mice Subjected to Chronic Stress, Exogenous cBIN1 Preserves CalciumHandling Machinery and Cardiac Function,” Journal of the American College of Cardiology, Basic & Translational Science, 5:561–578 (2020).

[0183] 35. Fu and Hong, “BIN1 regulates dynamic t-tubule membrane,” Biochimica et Biophysica Sinica 1863:1839–1847 (2016).

[0184] 36. Hong et al., “BIN1 is reduced and Cav1.2 trafficking is impaired in human failing cardiomyocytes,” Heart Rhythm 9:812–820 (2012).

[0185] 37. Zhou and Hong, “Cardiac BIN1 (cBIN1) is a regulator of cardiac contractile function and an emerging biomarker of heart muscle health,” Science China: Life Sciences, 60:257–263 (2017).

[0186] 38. Xu et al., “The ESCRT-III pathway facilitates cardiomyocyte release of cBIN1-containing microparticles,” PLoS Biol 15: e2002354 (2017).

[0187] 39. Nikolova et al., “Association of the plasma cardiac bridging integrator 1 score, a novel diagnostic biomarker, with heart failure with preserved ejection fraction and cardiovascular hospitalizations,” JAMA Cardiology 3:1206–1210 (2018).

[0188] 40. Blaufarb and Sonnenblick, “Therenin-angiotensin system in left ventricular remodeling,” Am J Cardiol 77:8C-16C (1996).

[0189] 41. Liu et al., “Peroxisome proliferator-activated receptor beta / delta activation in adult hearts facilitates mitochondrial function and cardiac performance under pressure-overload condition,” Hypertension 57, 223–230 (2011).

[0190] 42. Zhang et al., “Critical Roles of STAT3 in beta-Adrenergic Functions in the Heart,” Circulation 133:48–61 (2016).

[0191] 43. Zhou et al., “The Relationship between Galectin-3 and Different Patterns of Ventricular Geometry Remodelling in Aortic Valve Stenosis,” Heart Lung & Circulation 25:371–377 (2016).

[0192] 44. Rincon et al., “Gene therapy for cardiovascular disease: advances in vector development, targeting, and delivery for clinical translation,” Cardiovasc Res 108:4-20 (2015).

[0193] 45. Bass-Stringer et al., “Adeno-Associated Virus Gene Therapy: Translational Progress and Future Prospects in the Treatment of Heart Failure,” Heart Lung & Circulation 27:1285-1300 (2018)

[0194] 46. ​​Tilemann et al., “Gene therapy for heart failure,” Circulation Research 110:777-793 (2012).

[0195] 47. Thai et al., “Cardiac-specific Conditional Knockout of the 18-kDa Mitochondrial Translocator Protein Protects from Pressure Overload Induced Heart Failure,” Scientific Reports 8:16213 (2018).

[0196] 48. Osadchii, “Cardiac hypertrophy induced by sustained beta-adrenoreceptor activation: pathophysiological aspects,” Heart Failure Reviews 12: 66-86 (2007).

[0197] 49. Soltysinska et al., “Myocardial structural, contractile and electrophysiological changes in the guinea-pig heart failure model induced by chronic sympathetic activation,” Exp Physiol 96:647–63 (2011).

[0198] 50. Balakumar et al., “Rodent models of heart failure,” Journal of Pharmacological and Toxicological Methods 56: 1-10 (2007).

[0199] 51. Frey et al., “Hypertrophy of the heart: a new therapeutic target?” Circulation 109: 1580-1589 (2004).

[0200] 52. Oliveira et al., “Cardiac anti-remodelling effect of aerobic training is associated with a reduction in the calcineurin / NFAT signalling pathway in heart failure mice,” J Physiol 587:3899–3910 (2009).

[0201] 53. Neves et al., “Exercise training in hypertension: Role of microRNAs,” World Journal of Cardiology 6:713-27 (2014).

[0202] 54. Ooi et al., “The therapeutic potential of miRNAs regulated in settings of physiological cardiac hypertrophy,” Future Med Chem 6:205-222 (2014).

[0203] 55. MacDonnell et al., “Improved myocardial beta-adrenergic responsiveness and signaling with exercise training in hypertension,” Circulation 111: 3420-3428 (2005).

[0204] 56. Hoydal et al., “Exercise training restores Ca 2+ Exercise training reverses myocardial dysfunction induced byCaMKIIdeltaC overexpression by restoring Ca 2+ homeostasis),” Journal of Applied Physiology (1985) 121:212-220 (2016).

[0205] 57. Pitsavos et al., “The impact of moderate aerobicphysical training on left ventricular mass, exercise capacity and blood pressure response during treadmill testing in borderline and mildly hypertensive males,” Hellenic Journal of Cardiology 52:6–14 (2011).

[0206] 58. Dieberg et al., “Clinical outcomes and cardiovascular responses to exercise training in heart failure patients with preserved ejection fraction: a systematic review and meta-analysis,” Journal of Applied Physiology (1985) 119:726–733 (2015).

[0207] 59. Yang et al., “Cardiac CaV1.2 channels require beta subunits for beta-adrenergic-mediated modulation but not trafficking,” Journal of Clinical Investigation 129:647–658 (2019).

[0208] 60. Miriyala et al., “Role of CaVbeta subunits, and lack of functional reserve, in proteinkinase A modulation of cardiac CaV1.2 channels,” Circulation Research 102:e54-64 (2008).

[0209] 61. Shan et al., "Phosphorylation of the ryanodine receptor mediates the cardiac fight or flight response in mice", J. Clin. Invest. 120:4388-4398 (2010).

[0210] 62. Anwar et al., "PKC-independent signal transduction pathways increase SERCA2 expression in adult rat cardiomyocytes", J. Mol. Cell. Cardiol. 39:911-919 (2005).

[0211] 63. Lyon et al., "Plasticity of surface structures and beta(2)-adrenergic receptor localization in failing ventricular cardiomyocytes during recovery from heart failure", Circ. Heart Fail. 5:357-365 (2012).

[0212] 64. Basheer et al., "GJA1-20k Arranges Actin to Guide Cx43 Delivery to Cardiac Intercalated Discs", Circ. Res. 121 : 1069-1080 (2017).

[0213] 65. Zincarelli et al., “Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection,” Mol Therapy 16: 1073-1080 (2008).

[0214] 66. Chen et al., “Targeting transgene to the heart and liver with AAV9 by different promoters,” Clin Exp Pharmacol Physiol 42: 1108–1117 (2015).

[0215] 68. Denegri et al., “Single delievery of an adeno-associated viral construct to transfer the CASQ2 gene to knock-in mice affected by catecholaminergic polymorphic ventriculartachycardia is able to cure the disease from birth to advanced age,” Circulation 129:2673–2681 (2014).

[0216] 68. Writing Group et al., “Heart Disease and Stroke Statistics-2016 Update: A Report from the American Heart Association,” Circulation 133:e38-360 (2016).

[0217] 69. Verloop et al., “A systematic review concerning the relation between the sympathetic nervous system and heart failure with preserved left ventricular ejection fraction,” PLoS One 10:e0117332 (2015).

[0218] 70. Amoasii et al., “Myotubularin and PtdIns3P remodel the sarcoplasmic reticulum in muscle in vivo,” Journal of Cell Science, 126: 1806–1819 (2013).

[0219] 71. Guo and Song, AutoTT: automated detection and analysis of T-tubule architecture in cardiomyocytes”, Biophysical Journal 106(12): 2729–2736 (2014).

[0220] 72. Basheer et al., “Stress response protein GJA1-20k promotes mitochondrial biogenesis, metabolic quiescence, and cardioprotection against ischemia / reperfusion injury,” Joint Commission International Insight 3(20):e121900 (2018).

[0221] 73. Kirchhefer et al., “Chronic beta-adrenergic stimulation reverses depressed Cahandling in mice overexpressing inhibitor-2 of protein phosphatase 1,” Journal of Molecular and Cellular Cardiology 125:195–204 (2018).

[0222] 74. Fu and Shaw, “CASAAV Technology to Examine Regulators of Heart Failure: Cause or Effect.” Circulation Research 120:1846–1848 (2017).

[0223] 75. Gaasch and Zile, “Left ventricular structural remodeling in health and disease: with special emphasis on volume, mass, and geometry,” Journal of the American Heart Association 58:1733–1740 (2011).

[0224] 76. Periasamy and Janssen, “Molecular basis of diastolic dysfunction,” Heart Failure Clin 4:13-21 (2008).

[0225] 77. Boknik et al., “Protein phosphatase activity is increased in a rat model of long-term beta-adrenergic stimulation,” Nauern-Schmidtberg Archives of Pharmacology 362: 222-231 (2000).

[0226] 78. Valero-Munoz, et al., “Murine Models of Heart Failure with Preserved Ejection Fraction: a 'Fishing Expedition',” Journal of the American College of Cardiology, Basic & Translational Science 2(6):770-789 (2017).

[0227] 79. Liu et al., “In Mice Subjected to Chronic Stress, Exogenous cBIN1 Preserves Calcium-Handling Machinery and Cardiac Function,” Journal of the American College of Cardiology, Basic & Translational Science 5(6):561–578 (2020).

[0228] 80. Li et al., “Exogenous Cardiac Bridging Integrator 1 Benefits Mouse Hearts with Pre-existing Pressure Overload-Induced Heart Failure,” Frontiers in Physiology 11:708 (2020).

Claims

1. Use of a transgene encoding cBIN1 in the preparation of a medicament for use in a method for enhancing contractile function or relaxation function in the heart in a human or mouse subject with pre-existing heart failure or treating heart failure, the method comprising administering a transgene encoding cBIN1 to the human or mouse subject with pre-existing heart failure.

2. The use according to claim 1, wherein the transgene is administered after the subject is diagnosed with heart failure.

3. The use according to claim 1, wherein the method comprises administering the transgene by injection. The use according to claim 1 , wherein the medicament comprises an adeno-associated viral vector comprising a transgene encoding cBIN1.

5. The use according to claim 1, wherein the transgene comprises 3×10 10 vector genomes. The use according to claim 1 , wherein the expression of cBIN1 remodels damaged myocardium.

7. The use according to claim 1, wherein expression of cBIN1 stabilizes the intracellular distribution of calcium handling machinery in the myocardium.

8. The use according to claim 1, wherein expression of cBIN1 reduces centripetal hypertrophy in the myocardium.

9. The use according to claim 1, wherein the expression of cBIN1 repairs or increases t-tubule microfolds or microdomains in the myocardium.

10. The use according to claim 1, wherein expression of cBIN1 restores or reduces hyperphosphorylation of ryanodine receptor 2 in myocardium. The use according to claim 1 , wherein the expression of cBIN1 restores or improves cardiac contractility and diastolic force.

12. The use according to claim 1, wherein the expression of cBIN1 restores or improves cardiac relaxation and diastolic function.

13. The use according to claim 1, wherein expression of cBIN1 prevents further damage to the myocardium.

14. The use according to claim 1, wherein the transgene is administered at least once.

15. The use according to claim 1, wherein the subject is a mouse.

16. The use according to claim 1, wherein the subject is a human.

17. The use of claim 1, wherein the subject experiences improved heart failure with reduced ejection fraction.

Images