Patent Application
20230108316
Described herein are compositions comprising viral vectors. The viral vectors may encode a t-tubule organizing protein or peptide such as cardiac isoform of bridging integrator 1 (cBIN1). Also disclosed herein are methods for treatment or prophylaxis of heart failure in a subject in need thereof. The method of treatment or prophylaxis may include administering a vector comprising cBIN1 to the subject for rehabilitating or increasing contractile (systolic) function or relaxation (diastolic) function in the heart of a subject having experienced heart failure or having chronic myocardial stress.
Heart failure (HF) is the fastest growing cardiovascular disorder affecting over 20 million people worldwide and 6.2 million Americans [1-2]. The majority of HF related mortality is associated with cardiac pump failure due to myocardial inotropic and lusitropic dysfunction, as well as sudden cardiac death due to increased arrhythmia burden of failing hearts. Furthermore, in nearly 50% of patients with HF with preserved ejection fraction (HFpEF) [2], severe diastolic failure with further increased arrhythmia risks occurs, which has even worse clinical outcomes and also lacks effective medical therapy. Thus, there is an urgent need to develop new therapeutic strategies that can limit and reverse heart failure progression.
During HF development, the pathophysiologic cellular hallmark of failing ventricular myocytes is abnormal calcium transients with impaired intracellular calcium homeostasis [3], which disrupts excitation-contraction (EC) coupling [4], impairs electrical stability [5], and disturbs mitochondrial metabolism [6]. Normal beat-to-beat calcium transient relies on a sequence of intracellular events known as calcium-induced-calcium-release (CICR) [7], where t-tubule L-type calcium channel (LTCC)-mediated initial calcium influx will subsequently induce a massive calcium release via ryanodine receptors (RyRs) from the sarcoplasmic reticulum (SR) store. During relaxation, the accumulated calcium will then be removed from the cytoplasm mainly by calcium reuptake to SR via SR Ca2+-ATPase (SERCA) together with calcium exclusion into the extracellular space [7]. In HF, abnormal t-tubule remodeling [8-10] impairs LTCC-RyR coupling and synchronous CICR [3, 11], resulting in diminished systolic release, EC uncoupling, and thus reduced contractility. On the other hand, HF-associated leaky RyRs [12] and abnormal SERCA2a function [13] will result in SR depletion and elevated 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 promotes the risk of mitochondrial-initiated cell death [18-19] and HF progression [18, 20]. Taken together, abnormal calcium homeostasis is critical in controlling normal cardiac pump function, electrical stability, and metabolism, which, when disturbed, will lead to pump failure, lethal arrhythmias, and severe metabolic disorder.
Cardiac transverse tubules (t-tubule) are critical for the initiation of calcium transients and maintenance of efficient excitation-contraction (EC) coupling. Pathological t-tubule remodeling is a consequence of β-adrenergic stimulation in HF [21-23]. Furthermore, impaired t-tubule microdomains have been implicated in HF progression [24-27]. In fact, t-tubule remodeling can be the tipping point from hypertrophy to failure [10]. Normal calcium transients [28], which require L-type calcium channels (LTCCs) to be at t-tubule microdomains, are crucial to cardiac contraction and relaxation. The t-tubule membrane scaffolding protein cardiac bridging integrator 1 (cBIN1) [29], which facilitates LTCC trafficking [30] and clustering for dyad organization, is also under the regulation of β-adrenergic receptor (β-AR) signaling [31]. Furthermore, cBIN1 is reduced in HF [31-33] and the resultant cBIN1-microdomain disruption impairs normal stress response, limiting contractility and promoting arrhythmias. Therapeutic approaches that preserve cBIN1-microdomains may benefit stressed hearts by protecting the calcium handling machinery, slowing HF progression.
Therefore, there remains a need for preventing remodeling within individual ventricular myocytes to improve overall cardiac remodeling and have therapeutic benefits for failing hearts.
One embodiment described herein is a method for rehabilitating heart tissue or ameliorating symptoms of heart failure in a subject having experienced heart failure or under chronic stress, the method comprising, diagnosing heart failure or myocardial stress in a subject; and administering a transgene encoding a Cardiac Bridging Integrator 1 (cBIN1) to heart tissue of the subject having experienced heart failure. In one aspect, the diagnosis of heart failure or myocardial stress comprises measuring reduced cBIN1 blood levels.
Another embodiment described herein is a method for rehabilitating or increasing contractile (systolic) function or relaxation (diastolic) function in the heart of a subject having experienced heart failure, the method comprising administering a transgene encoding Cardiac Bridging Integrator 1 (cBIN1) to heart tissue of the subject, wherein after the transgene is delivered to the heart tissue and expressed, contractile function of the heart is rehabilitated or increased. In one aspect, the transgene is administered after the subject is diagnosed with heart failure. In another aspect, the diagnosis of heart failure comprises measuring reduced cBIN1 blood levels. In another aspect, the method comprises administering the transgene to myocardium. In another aspect, the transgene is administered by injection. In another aspect, the transgene comprises a vector comprising the transgene encoding cBIN1. In another aspect, the transgene comprises about 1×1010 to about 5×1010 of vector genome. In another aspect, the expression of cBIN1 restructures damaged myocardium. In another aspect, the expression of cBIN1 stabilizes intracellular distribution of calcium handling machinery in the myocardium. In another aspect, the expression of cBIN1 reduces concentric hypertrophy in the myocardium. In another aspect, the expression of cBIN1 rehabilitates or increases t-tubule microfolds or microdomains in the myocardium. In another aspect, the expression of cBIN1 rehabilitates or decreases hyperphosphorylation of ryanodine receptor 2 (RyR2) in the myocardium. In another aspect, the expression of cBIN1 rehabilitates or improves cardiac contractility and lusitropy. In another aspect, the expression of cBIN1 rehabilitates or improves cardiac relaxation and diastolic function. In another aspect, the expression of cBIN1 is prophylactic for further damage to the myocardium. In another aspect, the transgene is administered at least once. In another aspect, the subject is mammal. In another aspect, the subject is a mouse or dog. In another aspect, the subject is a human. In another aspect, the subject experiences reduced ejection fraction (HFrEF).
Another embodiment described herein is the use of cBIN1 in a medicament for rehabilitation of myocardial tissue or repairing myocardial damage in a subject having experienced heart failure or having chronic myocardial stress.
Another embodiment described herein is the use of cBIN1 in a medicament for rehabilitating or increasing contractile (systolic) function or relaxation (diastolic) function in the heart of a subject having experienced heart failure or having chronic myocardial stress.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The reorganization of intracellular calcium handling machinery can be achieved by targeting t-tubule membrane microdomains organized by that cardiac isoform of bridging integrator 1 (cBIN1) [34]. It was previously found that cBIN1-microdomains organize LTCC-RyR dyads [12,14] by facilitating intracellular trafficking [13] and surface clustering of LTCCs [14, 35], affecting the electrochemical gradient across LTCCs via generating a protective slow diffusion zone within t-tubule lumen for extracellular ions [12], and recruiting RyRs to jSR for coupling with LTCCs [14]. More recently, it was found that cBIN1-microdomain is also critical in organizing the intracellular distribution of SERCA2a for diastolic calcium regulation [34]. In HF, cBIN1-microdomains are disrupted due to transcriptional reduction in cBIN1 [16, 36, 37], impairing dyad formation, calcium transient regulation, and cardiac contractility. Reduced myocardial cBIN1 can be detected in human blood, a result of cBIN1-membrane turnover and microparticle release [38]. In humans, plasma CS (cBIN1 score) is an index of myocyte cBIN1 level, which identifies myocardial structural remodeling, facilitating HF diagnosis and prognosis [39]. In mouse hearts subjected to chronic stress, pretreatment with exogenous cBIN1 preserves the microdomain-organized distribution of Cav1.2 and SERCA2a, maintaining normal inotropy and lusitropy. These data indicate that cBIN1 replacement can be an effective HF therapy with the potential to recover myocardial function in hearts with preexisting HF.
Since increased afterload is an important primary and secondary cause of HF [40], the current study uses a mouse model of elevated afterload induced by transverse-aortic constriction (TAC). In TAC mice, it was reported that cBIN1 pretreatment prevents HF development. Here we further used AAV9-mediated gene transfer to introduce exogenous cBIN1 in post-TAC mouse hearts with pre-existing HF. cBIN1 post-treatment reduces TAC-induced pathological remodeling, as well as the onset of HF and death. In mouse hearts with pre-existing TAC-induced HF, cBIN1 induces functional recovery. Also, in the present disclosure, we explored whether in vivo over-expression of exogenous cBIN1 can limit myocardial remodeling and dysfunction. Continuous isoproterenol infusion, which causes reduced myocardial cBIN1 expression and disorganized intracellular distribution of calcium handling proteins, also induces pathologic concentric hypertrophy with diastolic dysfunction. We found that normalization of cBIN1 through adeno-associated virus 9 (AAV9) mediated gene transfer both increases inotropy and preserves lusitropy, reducing pathologic hypertrophy. Within cardiomyocytes, we found that exogenous cBIN1 preserves the intracellular distribution of LTCCs at t-tubules, and the localization of the sarcoplasmic reticulum (SR) calcium-ATPase 2a (SERCA2a). The protective effects of cBIN1 are both isoform specific and confirmed effective in a second model of transverse aortic constriction (TAC) induced cardiac hypertrophy and HF, indicating that exogenous cBIN1 mediated preservation of t-tubule microdomains is a possible therapeutic approach to improve myocardial function in hearts under chronic stress.
AAV9 virus transduced-exogenous cBIN1 in myocardium, applied after a reduction in ejection fraction, can rescue cardiac systolic function and limit further development of ventricular chamber dilation and HF in mice subjected to chronic pressure overload stress.
Under continuous pressure overload, myocardial remodeling starts with an adaptive hypertrophic response followed by transitioning into maladaptive cardiac dilatation, leading to worsening HF [41-43]. In previous studies, we proved that the administration of AAV9-cBIN1 prior to TAC surgery preserves myocardial systolic and diastolic function, indicating the efficacy of cBin1 gene therapy in HF prevention. In the current disclosure, we found that exogenous cBIN1 administration not only limits but rescues the TAC-stressed hearts from further HF development and improves the overall survival with attenuated cardiac hypertrophy and lessened pulmonary edema in mice. Furthermore, exogenous cBIN1 introduced by gene transfer improves myocardial remodeling and cardiac function as measured by echocardiography. Most strikingly, mice with pre-existing severe HF exhibited recovered EF following cBin1 gene therapy, indicating the protective effect of exogenous cBIN1 may serve as a translatable treatment for patients with diagnosed pre-existing structural remodeling and HF.
Recently, AAV-mediated gene therapy has been shown as a promising modality for the treatment of HF [44-45]. There are currently several completed or ongoing clinical trials of HF gene therapies targeting various pathways such as the p-adrenergic system, Ca2+ cycling proteins, and cell death pathways, as well as homing stem cells [46]. We recently found that targeting the calcium regulating microdomains at t-tubules can be effectively achieved by transducing the essential microdomain-organizing protein cBIN1 [34]. By stabilizing t-tubule microdomains, cBIN1 potentially restores cytosolic calcium homeostasis and contributes to increasing systolic calcium release, improving diastolic reuptake, limiting SR leak for electrical stability maintenance, as well as preserving mitochondrial function to limit mitochondrial-associated cell death. The results indicate that this microdomain-targeting approach may serve as a new therapeutic strategy with improved efficiency in functional preservation, improving overall HF survival. Furthermore, the observed cBIN1-mediated improvement in overall survival is a possible combined effect from improved pump function and reduced arrhythmias, both of which are regulated by cBIN1-microdomains [7, 12, 36]. How cBIN1 therapy affects arrhythmia burden in failing hearts will need further analysis using in vivo telemetry monitoring in future studies. In addition, since TAC-induced HF is associated with mitochondrial disorder-associated myocyte death [47], it remains interesting in future studies to explore whether cBIN1 replacement therapy can preserve mitochondrial function and limit mitochondrial-related cell death in failing hearts.
With regard to functional recovery, although EF changes monitored from the beginning of AAV9-cBIN1 treatment shows a high peak of recovery at week 6 post-AAV9 followed by descending therapeutic efficiency, the rescue effect is maintained at 15-week post AAV9 injection. These data indicate even a single administration of AAV9-cBIN1 at a relatively low dose (3×1010 vg) is sufficient to preserve cardiac function. Whether multiple administrations of exogenous cBIN1 with increased dosage are needed to maximize its therapeutic effect remains to be tested. Nevertheless, our current rescue data indicate that, for patients with existing HF, cBin1 gene therapy could potentially break the worsening cycles of HF progression and result in functional recovery of failing hearts.
This study reveals a protective role of exogenous cBIN1 in mouse hearts with existing HF after subjected to pressure overload. For this first proof-of-concept study, we used the AAV9 vector driven by the CMV promoter for gene delivery due to its consistent transduction efficiency and established cardiac tropism. Further experiments using cBin1 packaged in AAV9 with a more efficient cardiac-specific promoter in mice and large mammals will be needed before clinical trials testing the efficacy and efficiency of cBin1 gene therapy in HF patients. Future studies are also needed to explore the intracellular mechanism for cBIN1 in balancing calcium homeostasis among cytosolic microdomains at t-tubules, SR, and nearby mitochondria. Further understanding of the downstream targeting molecules and signaling pathways of cBIN1 will be needed as well for a better understanding of the interplay between cBin1 gene therapy and HF pathophysiology.
This disclosure also indicates a beneficial effect of exogenous cBIN1 in preventing LV hypertrophy and cardiac dysfunction in stressed hearts. In mice subjected to continuous isoproterenol infusion, exogenous cBIN1 offers an isoform-specific improvement in cardiac inotropy and lusitropy, limiting the development of LV hypertrophy. The cardiac protective effect of exogenous cBIN1 is further confirmed in mouse hearts subjected to pressure overload induced HF.
Chronically elevated catecholamine levels and activation of cardiac β-adrenergic receptors (β-ARs) have a critical role in the pathogenesis of HF. Impaired myocardial structure and function have been observed in animals subjected to sustained sympathetic activation [48-49]. Isoproterenol, which is a synthetic catecholamine and non-selective β-AR agonist, has been used in research to induce the model of LV hypertrophy and dysfunction [50]. A high dose of isoproterenol was used here to induce LV concentric hypertrophy with preserved systolic function. Chronic excessive cardiac workload induced LV hypertrophy is associated with elevated risk of cardiovascular events [51] and preventing or reversing ventricular hypertrophy with preserved cardiac diastolic function is crucial to preventing the progression of stressed hearts to failing hearts. Here we found that cBIN1 attenuates chronic isoproterenol-induced hypertrophy and at the same time conveys an isoform-specific improvement in stroke volume and cardiac output in hypertrophic hearts with preserved systolic function. The increase of LV volume in the cBIN1 hearts is not secondary to pump failure and dilated cardiomyopathy, but rather it reflects improvement in myocardial lusitropy (E/e′) with a parallel increase of intrinsic myocardial contractility (inotropy). This phenotype of cBIN1 hearts is typical of athletic hearts in adaptation endurance training as characterized by chamber enlargement and increases of LV volume, stroke volume, and cardiac output [52-54]. Aerobic exercise training has been reported to improve myocardial function and inotropic and lusitropic responses in both animal models [55-56] and patients with hypertension [57] and diastolic failure [58]. Thus, exogenous cBIN1 may provide additional exercise like benefit to patients with heart failure, improving exercise capacity and quality of life.
These post-isoproterenol hearts are at a stage of hypertrophy with preserved systolic function, in which exogenous cBIN1 can effectively translate the increased demands on the heart into a functional effect. As a result, these functional and efficient cBIN1 hearts have limited hypertrophy development, which will likely prevent the next step of disease progression and HF development as occurs in the clinical setting. Next, the functional protective effect of exogenous cBIN1 in already decompensated hearts is also observed in a mouse model of TAC-induced hypertrophy and HF. Under pressure overload, compensated hypertrophy is an adaptive response. Over time, the adaptive response concedes to cardiac dilatation and the ensuing remodeling process becomes maladaptive, leading to worsening HF. We found that the fate of dilated cardiomyopathy development in pressure overload stressed hearts is causally determined by myocardial content of cBIN1 protein. Following pressure overload, less cardiac BIN1 in genetically deleted Bin1 HT-TAC hearts is associated with more severe dilated cardiomyopathy, whereas greater cBIN1 with gene transfer improves cardiac systolic and diastolic function, limits HF, and improves HF-free survival. It remains unclear whether exogenous cBIN1 reduces myocyte death, which also contributes to LV dilation in post-TAC hearts. Future studies will be necessary to explore the effect of cBIN1 on myocyte survival in stressed hearts. Nevertheless, our data indicate that exogenous cBIN1 not only limits hypertrophy development in stressed hearts but also prevents myocardial transition from hypertrophy to dilated cardiomyopathy and HF in TAC mice.
The mechanism of improvement in cardiac inotropic function by cBIN1 is linked to its known effect in organizing t-tubule microdomains required for dyad organization and efficient EC coupling. cBIN1 creates t-tubule microfolds to organize a slow diffusion zone trapping extracellular t-tubule lumen ions, attracts LTCCs forward trafficking to t-tubules [30], clusters LTCCs that are already delivered to cell surface [35], and recruits RyRs to couple with LTCCs at dyads [31]. Here we confirm in vivo that exogenous cBIN1, rather than any other BIN1 isoforms, increases Cav1.2 localization to t-tubules. These results support that preserved cBIN1-microdomain with organized LTCC distribution is responsible for the observed positive inotropic effect in sympathetically overdriven cBIN1 hearts. Whether cBIN1-microdomain regulates LTCC phosphorylation and its functional response to sympathetic stress including a well-established β-subunit-modulated Cav1.2 channel response [59-60] awaits future experimental explorations. Furthermore, RyR is critical to inotropy and hyper-phosphorylated leaky RyR plays a role in HF progression [14]. Consistent with previous reports in isoproterenol model and human HF [14, 61], we found that chronic isoproterenol activates PKA and CAMKII-induced RyR hyperphosphorylation. AAV9-cBIN1 blunts these pathways, normalizing RyR phosphorylation following chronic sympathetic activation and preventing SR leak.
An additional novel finding from the current disclosure is that exogenous cBIN1 increases SERCA2a function through organizing its intracellular distribution. Chronic isoproterenol-induced concentric hypertrophy with preserved systolic function is associated with disorganized intracellular distribution of SERCA2a yet increased overall protein expression. It is well accepted that SERCA2a activity is decreased in end stage HF. Our data indicate that in addition to reduced expression and impaired regulation by PLN, intracellular distribution of SERCA2a may also contribute to abnormal SR calcium reuptake activity in HF. Furthermore, as reported in adult rat ventricular cardiomyocytes with α-receptor agonist phenylephrine induced-hypertrophy, an adaptive increase in SERCA2a protein expression can occur due to elevated diastolic calcium-induced calcineurin/NFAT activation [62]. Thus, increased SERCA2a protein expression here is a possible adaptive response induced by elevated diastolic calcium concentration as indicated in elevated calcium-dependent phosphorylation at T287 of CAMKII. Thus, a transient increase in SERCA2a may occur at an early stage of all LV hypertrophy with preserved function. During disease progression, this adaptive increase in total SERCA2a protein expression will level off and even decrease as occurs in end stage HF, resulting in severe diastolic and systolic failure. In cBIN1 hearts, organized SERCA2a along SR indicates better calcium reuptake, therefore less diastolic calcium overload for hearts still at compensated stage. These results are consistent with a previous study in a rat model of HF which identified that increased BIN1 expression is associated with SERCA2a expression [63]. Future studies exploring cBIN1 regulation of diastolic calcium concentration and calcineurin/NTAT pathways will be needed to further understand its role in regulating SERCA2a expression and activity during disease progression. Note the effect on SERCA2a organization is 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 from BIN1+17 on cardiac hypertrophy and diastolic function. Whether and how BIN1 isoforms cooperate to organize SERCA2a distribution in normal and diseased cardiomyocytes require further exploration in future studies. Furthermore, through regulation of calcium handling machineries at SR including SERCA2a distribution and RyR phosphorylation, cBIN1 may help maintain normal SR calcium load. As a limitation of the current study, future experiments are needed to quantify the effect of cBIN1 on SR calcium load, calcium release and reuptake kinetics, and arrhythmogenic spontaneous calcium release in chronically stressed hearts.
Nevertheless, the most robust protection of both inotropy and lusitropy in sympathetic overdriven hearts is only observed in the cBIN1 group, indicating possible further beneficial effect on lusitropy from cBIN1-dependent improvement in LTCC localization and dyad organization. With isoform-specific improvement in dyad organization, less orphaned leaky RyRs accumulate outside of dyads [31], limiting calcium leak from SR and decreasing cytosolic calcium concentration during diastolic phase. Together with the newly identified role on SERCA2a organization, our data indicate that a cBIN1-microdomain related regulation offers a unique benefit in protecting cardiac lusitropy in addition to its inotropy effect. On the other hand, cBIN1 overexpression may also suppress the pathological effects of isoproterenol stimulation by enhancing the control of β-AR signaling and the compartmentalization of secondary messengers and calcium handling channels and pumps. Thus, by stabilizing t-tubule microdomains to regulate all aspects of calcium handling, cBIN1 produces a positive feedforward mechanism for efficient intracellular beat-to-beat calcium cycling. In future studies it will be interesting to identify whether exogenous cBIN1 alters β-AR expression, intracellular distribution, and functional regulation following chronic sympathetic activation.
In conclusion, we found that over-expression of exogenous cBIN1 is protective in mouse hearts subjected to chronic β-AR activation induced concentric hypertrophy as well as pressure overload induced hypertrophy and HF. Future experiments in large mammals with common natural heart failure comorbidities such as hypertension and diabetes will be needed. Improving the viral infectivity in cardiomyocytes can additionally help limit or prevent isoproterenol-induced membrane disruption in all cardiomyocytes, increasing the protective effect on the entire heart. Further experiments using cBin1 packaged in AAV9 with an efficient and cardiac specific promoter to induce sufficient exogenous protein expression in all cardiomyocytes will be needed before clinical trials testing the efficacy and efficiency of cBin1 gene therapy. Future studies are needed to establish whether cBIN1 will impact systemic hemodynamics and blood pressure. Finally, future studies are needed to explore how cBIN1-microdomain regulates the organization of intracellular calcium handling machineries, EC coupling, SR calcium load and release, diastolic calcium concentration and its downstream calcium signaling pathways, the interplay between signaling pathways of pathologic and physiologic hypertrophic remodeling, as well as the molecular transition from compensated hypertrophy to decompensated cardiomyopathy.
One embodiment described herein is a method for rehabilitating heart tissue or ameliorating symptoms of heart failure in a subject having experienced heart failure or under chronic stress, the method comprising, diagnosing heart failure or myocardial stress in a subject; and administering a transgene encoding a Cardiac Bridging Integrator 1 (cBIN1) to heart tissue of the subject having experienced heart failure. In one aspect, the diagnosis of heart failure or myocardial stress comprises measuring reduced cBIN1 blood levels.
Another embodiment described herein is a method for rehabilitating or increasing contractile (systolic) function or relaxation (diastolic) function in the heart of a subject having experienced heart failure, the method comprising administering a transgene encoding Cardiac Bridging Integrator 1 (cBIN1) to heart tissue of the subject, wherein after the transgene is delivered to the heart tissue and expressed, contractile function of the heart is rehabilitated or increased. In one aspect, the transgene is administered after the subject is diagnosed with heart failure. In another aspect, the diagnosis of heart failure comprises measuring reduced cBIN1 blood levels. In another aspect, the method comprises administering the transgene to myocardium. In another aspect, the transgene is administered by injection. In another aspect, the transgene comprises a vector comprising the transgene encoding cBIN1. In another aspect, the transgene comprises about 1×1010 to about 5×1010 of vector genome. In another aspect, the expression of cBIN1 restructures damaged myocardium. In another aspect, the expression of cBIN1 stabilizes intracellular distribution of calcium handling machinery in the myocardium. In another aspect, the expression of cBIN1 reduces concentric hypertrophy in the myocardium. In another aspect, the expression of cBIN1 rehabilitates or increases t-tubule microfolds or microdomains in the myocardium. In another aspect, the expression of cBIN1 rehabilitates or decreases hyperphosphorylation of ryanodine receptor 2 (RyR2) in the myocardium. In another aspect, the expression of cBIN1 rehabilitates or improves cardiac contractility and lusitropy. In another aspect, the expression of cBIN1 rehabilitates or improves cardiac relaxation and diastolic function. In another aspect, the expression of cBIN1 is prophylactic for further damage to the myocardium. In another aspect, the transgene is administered at least once. In another aspect, the subject is mammal. In another aspect, the subject is a mouse or dog. In another aspect, the subject is a human. In another aspect, the subject experiences reduced ejection fraction (HFrEF).
Another embodiment described herein is the use of cBIN1 in a medicament for rehabilitation of myocardial tissue or repairing myocardial damage in a subject having experienced heart failure or having chronic myocardial stress.
Another embodiment described herein is the use of cBIN1 in a medicament for rehabilitating or increasing contractile (systolic) function or relaxation (diastolic) function in the heart of a subject having experienced heart failure or having chronic myocardial stress.
It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, apparata, assemblies, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions, apparata, assemblies, and methods provided are exemplary and are not intended to limit the scope of any of the disclosed embodiments. All the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, apparata, assemblies, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences described herein. The compositions, formulations, apparata, assemblies, or methods described herein may omit any component or step, substitute any component or step disclosed herein, or include any component or step disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the other components in the formulation are hereby disclosed as if they were expressly disclosed.
Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof.
Various embodiments and aspects of the inventions described herein are summarized by the following clauses:
Materials and Methods
Animal procedures for functional rescue studies. Adult male C57BL/6 mice (The Jackson Laboratory) were used. All mice were anesthetized at the age of 8-10 weeks and subjected to open-chest sham or transverse aortic constriction (TAC) surgery. TAC was performed by tying a 7-0 silk suture against a 27-gauge needle between the first and second branch of the aortic arch. For sham controls, age-matched mice were subjected to open-chest mock surgery without TAC being performed. For gene therapy, at 5 weeks post the onset of TAC, mice received retro-orbital injection of 100 μL of 3×1010 vector genome (vg) of AAV9 virus (Welgen, Inc.) transducing cBIN1-V5 or GFP-V5 [64].
Animal procedures for isoproterenol studies. For the isoproterenol study, adult male C57BL/6 mice were administered 3×1010 vector genome (vg) of AAV9 transducing GFP or BIN1 isoforms (Welgen, Inc.) via retro-orbital injection [64]. Three weeks after vg administration, mice were implanted subcutaneously with osmotic mini pumps releasing PBS or isoproterenol (30 mg/kg/day). 56 mice were randomized into GFP+PBS, GFP+ISO, cBIN1+PBS, or cBIN1+ISO group (N=14/group). Another 50 mice were randomized into receiving AAV9-GFP, cBIN1, BIN1, BIN1+17, or BI N1+13 (N=10/group) before isoproterenol. AAV9 was used since it is a promising gene therapy vehicle and exhibits 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 the negative control virus since it does not induce cardiomyocyte toxicity and has been successfully used as a negative control virus in numerous gene therapy studies with animal models of cardiovascular diseases [67]. For TAC study, either adult male cardiac-specific Bin1 heterozygotes (Bin1 HT; Bin1flox/+, Myh6-cre+) with their wild type (WT; Bin1flox/+, Myh6-cre−) littermates [29]; or adult male C57BL/6 mice (Jackson Laboratory) were used. All mice were anesthetized at the age of 8-10 weeks and subjected to open-chest TAC or mock surgery (Sham). For gene therapy, same as the isoproterenol study, mice received retro-orbital injection of 3×1010 vg of AAV9 virus transducing cBIN1-V5 or GFP-V5 at 3 weeks prior to the onset of TAC.
Isoproterenol mini pump study. Fifty-six mice were randomized to receive a dose of 3×1010 vector genome (vg) of AAV9 transducing V5-tagged GFP or cBIN1 via retro-orbital injection while mice were anesthetized with 1% isoflurane in oxygen [68]. Three weeks after viral injection, mice were subjected to implantation of osmotic mini pump releasing isoproterenol or PBS (N=14 per 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 since AAV is the most promising gene therapy vehicle [21, 69] and AAV9 exhibits the highest cardiac tropism in mice (4-6). The CMV promoter was used since it has been established that AAV9-CMV can efficiently and safely direct cardiac gene transfer [25]. AAV9-CMV-GFP was used as the negative control virus since AAV9-CMVGFP does not induce cardiac damage and cardiomyocyte toxicity [25-26], and GFP AAV9 has been successfully used as a negative control virus in numerous gene therapy studies with animal models of cardiovascular diseases, including mouse models of hypertrophy and cardiomyopathy [26-29]. This protocol was also repeated in a second set of animals. Similarly, three weeks before isoproterenol mini pump implantation, fifty mice were randomized to receive a dose of 3×1010 vector genome (vg) of AAV9 transducing V5-tagged GFP, BIN1, BIN1+13, BI N1+17, or cBIN1 (n=10 per group) via retro-orbital injection. Three weeks after AAV9 injection, mice were implanted with subcutaneous ALZET osmotic minipump (Model 1004, Durect, Cupertino, Calif., USA) continuously releasing isoproterenol following previously established procedure [30]. In brief, under light anesthesia with inhalation of isoflurane, mice were implanted subcutaneously on the back with osmotic mini pumps, which continuously release isoproterenol at 30 mg/kg/day.
Transverse aortic constriction (TAC) study. For cBIN1 deficiency study, TAC was performed on male mice with cardiac-specific Bin1 heterozygote deletion (Bin1 HT; Bin1flox/+, Myh6-Cre+) and their wild type (WT; Bin1flox/+, Myh6-Cre−) littermates (WT) at the age of 8-10 weeks old. Bin1 HT and WT mice were generated as previously described. Specifically, heterozygote loxp site flanked Bin1 (loxP sites around exon 3 of the Bin1 gene) mice were interbred with Myh6-cre+ mice to generate cardiomyocyte specific Bin1 HT (N=10) and WT littermate controls (n=14). Genotypes were confirmed by PCR to differentiate Bin1+, Bin1flox, and Cre+ alleles according to a previously established method. For AAV9 mediated over-expression study, 5 to 7-week old male C57BL/6J mice (Jackson Laboratory) received retro-orbital injection of AAV9 virus (3×1010 vg) transducing cBIN1-V5 (N=18) or GFP-V5 (N=18). After three weeks, mice were anesthetized at the age of 8-10 weeks and subjected to open-chest TAC surgery. Age-matched mice subjected to open-chest mock surgery without TAC being performed were used as sham controls (N=10). TAC was performed to induce pressure overload as previously described. Briefly, 8-12 weeks old male mice were anesthetized by face mask administration of 3% isoflurane and then intubated and placed on a ventilator (Harvard Apparatus) with supplemental O2 and 1.5% isoflurane using a tidal volume of 0.2 mL and a respiratory rate of 120 breaths/min. The chest cavity was entered in the second intercostal space at the upper sternal border through a small incision, and aortic constriction was performed by tying a 7-0 nylon suture ligature against a 27-gauge needle between the first and second branch off the aortic arch. Subcutaneous buprenorphine (0.8 mg/kg) was administered for pain relief, and mice were allowed to recover in a heated chamber with 100% O2. Animals were euthanized, and tissues harvested for analysis after 8 weeks of TAC.
Generation and administration of adeno-associated virus 9 (AAV9). All five AAV9 vectors expressing GFP-V5, BIN1-V5, BIN1+13-V5, BIN1+17-V5, and cBIN1-V5 (BIN1+13+17-V5) driven by the CMV promoter were custom made and produced at Welgen, Inc. (Worcester, Mass., USA). We used previously reported gateway expression clones of V5-tagged GFP and mouse BIN1 isoforms [31], which were sequenced and then sent to Welgen for subsequent cloning into AAV vector and viral preparation. Next, these gene inserts (GFP-V5 or BIN1-V5) were subcloned into the pAAV-CMV vector (Welgen, Inc., Worcester, Mass., USA), and the positive clones were selected by restriction enzyme digestion. The pAAV-CMV-(GFP/BIN1)-V5 plasmid DNA were purified and sequenced. All AAV viruses were produced in HEK293 cells. Three plasmids, pAAV-CMV-(GFP/BIN1)-V5, pAAV-rep/cap9, and pHelper vectors were transfected into 293 cells using polyethylenimine. Following transfection, the supernatant and cells were harvested. The AAV viruses were released from HEK293 cells by 3 freeze-thaw cycles. The viruses in the medium were precipitated using PEG8000 (Sigma-Aldrich, St. Louis, MO, USA). The cell lysate and pelleted supernatant precipitate were combined and treated by Benzonase (Merck, Kenilworth, N.J., USA) at 37° C. for 1 h. The virus was purified by iodixanol gradient centrifugation and concentrated with Amicon Ultra-15 centrifugal filter (Sigma-Aldrich, St. Louis, Mo., USA).
Echocardiography for functional rescue studies. In vivo systolic and diastolic left ventricular (LV) functions were monitored by echocardiography in anesthetized mice using Vevo 7700 at baseline, pre-surgery, and every other week thereafter until the end of the experimental protocol. The trans-aortic pressure gradient was recorded using the modified Bernoulli equation (ΔPressure gradient (mm Hg)=4×peak velocity2 (m/s)2) at 2-weeks post-surgery. All surviving mice at 5 weeks post-TAC were included in the study.
Echocardiography for isoproterenol studies. Echocardiography were recorded using a Vevo-3100 ultrasound system (Visual Sonics) equipped with 70 MHz transducer. Protein interaction was analyzed by immunofluorescent imaging and biochemical coimmunoprecipitation. Peak intensity of Cav1.2 at t-tubules was quantified by Image J as previously reported [30]. Power spectrum analysis was analyzed in Matlab using FFT conversion [10, 30]. Intracellular protein distribution was analyzed by sucrose gradient fractionation using a previously established method [70]. For calcium transient measurement, Cal-520-AM (AAT Bioquest) was used as previously described [31]. Three-dimensional super-resolution stochastic optical reconstruction microscopy (STORM) images were obtained [31] for nearest neighbor analysis between LTCC-RyR and SERCA2a-cBIN1 molecules.
Primary endpoint of severe HF-free survival versus non-survival, and HF classification. Overall survival was analyzed in all groups. Furthermore, severe heart failure (HF)-free survival was also analyzed and compared between the AAV9-GFP and AAV9-cBIN1 groups. For severe HF-free survival, the primary endpoint is survival with ejection fraction (EF)≥35% measured by echocardiography. Non-survival is either death or EF<35% within 20-weeks post-TAC. At the end of the protocol, survived TAC mice were measured for tibial length (TL), lung weight (LW), and heart weight (HW).
Immunofluorescence Labeling and Confocal Imaging. For cardiomyocyte membrane fluorescent labeling, freshly isolated ventricular cardiomyocytes from GFP-TAC and cBIN1-TAC mice were incubated with Di-8-ANNEPs for 20 min at room temperature (RT). The cells were then washed with HBSS to remove the remaining dye before live-cell imaging. For fixed-cell V5 imaging (10×), isolated cardiomyocytes were fixed in methanol at −20° C. for 5 min and permeabilized and blocked with 0.5% Triton X-100 and 5% normal goat serum (NGS) in PBS for 1 h at RT. Cells were incubated with rabbit anti-V5 (Sigma) overnight at 4° C. and detected by Alexa555 conjugated goat anti rabbit IgG. For tissue immunofluorescent imaging, myocardial cryo-sections were fixed with ice-cold acetone for 5 min. The primary antibodies used were mouse anti-BIN1-BAR (2F11, Rockland), mouse anti-RyR (Abcam), or rabbit anti-Cav1.2 (Alomone). Following incubation with primary antibodies and several washes with 1×PBS, cells and tissue sections were then 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 60×1.1 or 10×objectives. High-resolution cardiomyocyte images were obtained using a spinning-disc confocal unit (Yokogawa CSU10) with diode-pumped solid state (DPSS) lasers (486 nm, 561 nm, 647 nm) generated from laser merge module 5 (Spectral applied research, Calif.). T-tubule membrane labeling fluorescent intensity profiles were generated by ImageJ, and peak intensity at t-tubules is quantified as previously reported [29]. Power spectrum analysis was analyzed in Matlab using FFT conversion and normalized peak power density at t-tubules was compared across groups [10, 30].
Immunofluorescence labeling and imaging with spinning disc confocal microscopy. Myocardial tissue sections were embedded in 100% OCT media and flash frozen on dry ice with ethanol and stored in a −80° C. freezer before being sectioned at 10 μm as previously reported [32]. After fixation by acetone, tissue cryosections were permeabilized with 0.1% Triton X-100 and 5% normal goat serum (NGS, Life Technology) in PBS for 1 h at RT. For 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, Mass., USA) overnight at 4° C. After several washes with 1×PBS, tissue sections were then incubated with goat anti-mouse and anti-rabbit IgG conjugated with Alexa 4#88 and 555, respectively. Tissue sections were mounted with DAPI containing Prolong® Gold medium. All imaging was obtained with 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, Calif., USA). Confocal Z stacks at Z-step increments of 0.5 μm were collected with a spinning-disk confocal unit (Yokogawa CSU10, Sugar Land, Tex., USA) connected to the same Ti microscope with diode-pumped solid state lasers (486 nm, 561 nm) generated from laser merge module 5 (Spectral Applied Research, Richmond Hill, Ontario, Canada), and captured by a high-resolution ORCA-Flash 4.0 digital CMOS camera. T-tubule Cav1.2 fluorescent intensity profiles were generated by ImageJ and peak intensity at t-tubules is quantified as previously reported [30]. Calcium transients were performed following previously described protocol [31]. Briefly, freshly isolated cardiomyocytes were loaded with 10 μmol/L Cal-520-AM (AAT Bioquest) in 0.4% Pluronic F-127 in normal Tyrode buffer for 30 minutes. After 3 washes in buffer containing 1 mmol/L probenecid cells were placed in imaging chamber and paced with field stimulator (lonflux) at 1 Hz. Images were collected using spinning disc confocal microscope at 67 fps and analyzed using Nikon Element Software. Fluorescent signals of F0 (baseline fluorescence) and Fmax (maximal fluorescence at the peak of calcium transient) were background corrected first followed by ratio calculation of ΔF/F0=(Fmax-F0)/F0 for comparison across groups.
Power Spectrum Analysis. The frequency domain power spectrum of cardiomyocyte immunofluorescent subsections was generated in Matlab using FFT conversion [10, 30]. Power spectrum normalized to maximal component was generated and plotted over distance (1/frequency, μm). Normalized peak power density [71] was quantified and compared among groups.
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 dish. The STORM images were collected with the Nikon Eclipse Ti microscope with lasers (488 nm, 561 nm from a self-contained 4-line laser module with acousto-optic tunable filters) and captured by a high-speed iXon DU897 Ultra EMCCD camera. The STORM module was used to obtained and analyze the images to generate 3-dimensional (3D) projections of Cav1.2/RyR and cBIN1/SERCA2a images at nanoscale resolution. For nearest neighbor analysis, the native 3D STORM images are displayed with the gaussian rendering algorithm available in Nikon Elements software, and 3D stacks of 3D STORM images (two channels per acquisition, either Cav1.2/RyR or cBIN1/SERCA2a) in molecule list text file format were obtained at a z-spacing of 10 nm for a depth of 500 nm. The molecule list text files were imported in ImageJ and the nearest distance between molecules from two channels (nearest neighbor distance) was calculated. The nearest neighbor distances were constructed and displayed in user-defined range and bin-width as frequency distribution histogram and fitted in 15th-degree polynomial curve with the first peak value detected. The distance between Cav1.2-RyR and SERCA2a-cBIN1 molecules at the corresponding first peak position were quantified and compared among groups.
Transmission Electron microscopy. All transmission electron microscopy (TEM) work was done by the core facility at the Electron Imaging Center of The California NanoSystems Institute, UCLA. Tissue preparation was performed using a previously reported method [72]. Briefly, mouse hearts were perfused with 20 mL of fresh fixative solution (2% glutaraldehyde and 2% paraformaldehyde in 1×PBS). Left ventricular tissue (1 mm3) were post-fixed with 1% osmium tetroxide and incubated in 3% uranyl acetate. After dehydration in ethanol, samples were treated with propylene oxide, embedded in Spurr resin (Electron Microscopy Services), and sectioned using an ultramicrotome (Leica). The sections were mounted on grid and stained with uranyl acetate and lead citrate before image acquisition using the JEM1200-EX, JEOL microscope (Gatan). The degree of contoured t-tubules was quantified using a modified scoring system established previously [29].
Western Blotting for functional rescue studies. Tissue lysates were made from hearts flash frozen in liquid nitrogen. Frozen tissue was homogenized in radio-immunoprecipitation assay (RIPA) lysis buffer as previously described [41]. Lysates were rotated head-to-toe in 4° C. for 40 min, sonicated, followed by centrifugation (16,000×g for 25 min at 4 ° C.) to clear cellular debris. Protein lysates were then prepared 2×sample buffer (Bio-Rad, Hercules, Calif.) containing 5% 8-mercaptoethanol, incubated in RT for 30 min, and separated on an 8-12% gradient sodium dodecyl sulfate (SDS) polyacrylamide electrophoresis gel. Proteins were electro-transferred to polyvinylidene difluoride (PVDF) membrane. After transfer, membranes were fixed in methanol and blocked with 5% BSA in 1×Tris-buffered saline (TBS) for 1 h at RT, and incubated with primary antibody in 5% BSA in 1×TBS overnight at 4° C., followed by incubation with Alexa 647 conjugated secondary antibody (Life Technology) for 1 h at RT. Primary antibodies consisted of a custom-made polyclonal rabbit anti-BIN1 exon 13 (Anaspec) [29], mouse anti-RyR (Abcam), rabbit Cav1.2 antibody (Alomone), and mouse anti-GAPDH (Millipore).
Western Blotting for isoproterenol studies. Frozen heart tissues were homogenized using RIPA lysis buffer with protease inhibitor, and a Bradford assay was used to determine the protein concentration. Samples were separated on NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gels and then transferred to polyvinylidene difluoride membranes. After blocking for 1 h with 5% Bovine Serum Albumin (BSA) in 1×TNT buffer, membranes were incubated overnight at 4° C. with primary antibody 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), followed by incubation with secondary antibody (goat anti rabbit or mouse IgG-Alexa 647) for 1.5 hour at room temperature (RT). Immunoreactive bands were imaged with the Molecular Imager® Gel Doc™ XR+System (Bio-Rad Laboratories, Irvine, Calif., USA) and band intensities quantified with Image Lab software (Bio-Rad Laboratories, Irvine, Calif., USA).
Cardiac microsome preparation and sucrose gradient fractionation. Microsome sucrose gradient fractionation was prepared according to an established protocol with modifications [70]. Myocardial membrane microsomes were prepared from starting material of one heart for each experimental group. Frozen heart tissue was homogenized with a Polytron Handheld homogenizer in 2 mL homogenization buffer (20 mM Tris pH 7.4, 250 mM sucrose, 1 mM EDTA supplemented with HALT protease inhibitor). 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 the S1 from previous step. The combined microsomal supernatant (S1+S2) was then subjected to ultracentrifugation at 110,000×g for 2 hours at 4° C. After ultracentrifugation, the supernatant was disposed, the pellet was weighted, and the appropriate amount of buffer (˜1 mL) was added to bring a final concentration of microsome ˜25 mg/mL. The total protein concentration in the resuspended microsome was measured using Nanodrop 2000 for each sample and normalized among the four groups. The same amount (3-6 mg in 0.5 mL) of total microsome from each sample was carefully laid over the top of a discontinuous sucrose gradient [52, 58, 73], and 45%, v/w in homogenization buffer, 2 mL each) and ultracentrifuged in a fixed angle MLA-55 rotor at 150,000×g for 16 hours with a Beckman Coulter Optima Max XP Benchtop Ultracentrifuge. Samples were then collected from the following fractions: F1, 27%; F2, 27/32%; F3, 32/38%; and F4, 38/45%; as well as the pellet (P) from the bottom of the tube. For each fraction, ˜1 mL was collected, diluted 4×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, followed by protein concentration measurement by Nanodrop 2000. The yield of total amount of protein recovered from each fraction F1, F2, F3, F4 is between 0.001-0.02, 0.4-0.8, 0.04-0.06, and <0.008 mg per heart, respectively. Sample buffer was added before samples were frozen and stored at −20° C. before subsequent Western Blot analysis.
Statistical analysis. All data are expressed as mean±standard error of the mean (SEM) or standard deviation (SD) as specified. Normality was assessed using the Shapiro-Wilk test. Kaplan-Meier survival analysis was used to compare across two groups using the log-rank test and across three groups using the log-rank trend test. Continuous variables were compared using T-test/Mann-Whitney U and one-way analysis of variance (ANOVA)/Kruskal-Wallis tests. Two-way ANOVA was used to determine differences between two groups at two different time points. Two-way ANOVA was used to determine differences between two AAV9 groups with different drug infusion, which was then 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 tests. Data were analyzed using GraphPad Prism (version 7.0; GraphPad Software, La Jolla, Calif., USA). Two-sided p values were used and p<0.05 is considered statistically significant.
Functional Rescue by Exogenous cBIN1 in Mouse Hearts with Pressure Overload-Induced Heart Failure
To explore whether targeting cBIN1-microdomain can be a new therapy for HF, how cardiac cBIN1 affects HF development in mice subjected to pressure overload stress was investigated. Either transverse aortic constriction (TAC) or a mock surgery (sham) was performed in adult male mice at the age of 8-10 weeks, followed by echocardiography monitoring to determine overall survival as well as severe systolic HF free survival (non-survival as death or ejection fraction, EF<35%). As indicated in the experimental protocol in
#Indicates p < 0.05.
##Indicates p < 0.01.
###Indicates p < 0.001 for AAV9-GFP vs. AAV9-cBIN1.
Then, the overall survival rate (non-survival is death) in all groups was explored. As indicated in the Kaplan-Meier curves in
Of all the mice that survived at 20 weeks post-TAC, echocardiography measured myocardial function and physiological parameters were further compared across groups both before and after AAV9 treatment (Table 1). At 20 weeks post-TAC, AAV9-GFP mice developed significant LV contractile dysfunction (EF reduction) and chamber dilation (EDV elevation,
To further explore the progression of systolic dysfunction in post-TAC hearts after viral injection at Time 0 (pre-AAV9, 5 weeks post-TAC), the delta EF changes (ΔEF) from pre-AAV to 3, 6, 8, 10, 15 weeks post-AAV9 injection (correspondingly 8, 11, 13, 15, 20 weeks post-TAC) were monitored by echocardiography (
We recently reported that, in mice receiving AAV9-cBIN1 pretreatment (3×1010 vg at 3 weeks prior to TAC surgery;
We previously identified that cBIN1 creates t-tubule microdomains and organizes LTCC-RyR dyads for efficient and dynamic regulation of cardiac function and EC coupling [25]. More recently, we found that in sympathetic overdriven mouse hearts developing diastolic dysfunction, cBIN1-microdomains are disrupted and are rescued by AAV9-cBIN1. Here, we also explored the alterations in cardiac t-tubule cBIN1-microdomains and the effect of exogenous cBIN1 in post-TAC hearts. Western blotting (
Exogenous cBIN1 Reduces Concentric Hypertrophy in Mouse Hearts after Isoproterenol Infusion
The effect of cBIN1 on myocardial function in animals subjected to 4 weeks of isoproterenol infusion was investigated (
#, ##, ### indicates P < 0.05, 0.01, 0.001 respectively for comparison of AAV8-GFP vs. AAV9-cBIN1 with isoproterenol (ISO) infusion.
Chronic Isoproterenol-Disrupted cBIN1-Microdomains can be Normalized by AAV9-cBIN1
It is well known that both myocardial inotropy and lusitropy are related to cardiomyocyte calcium cycling [76]. cBIN1, the structural organizer for dyad microdomains [31], creates t-tubule microfolds to limit extracellular Ca2+ diffusion [29], facilitates microtubule-dependent forward trafficking of L-type calcium channels (LTCCs) [30], and clusters of LTCCs that are already delivered to t-tubule membrane. Therefore, how cBIN1-microdomains may remodel in hypertrophic hearts after chronic isoproterenol infusion was explored. Western blots of heart lysates indicate that isoproterenol induced a significant reduction in cBIN1 protein, which is normalized by AAV9-cBIN1 (
Subsequently, Cav1.2 expression and intracellular distribution in cardiomyocytes was explored. In post-isoproterenol hearts, the net myocardial protein expression of Cav1.2 was similar (
Exogenous cBIN1 Improves SERCA2a Distribution Along SR
Cardiac lusitropy is most directly related to calcium reuptake via SERCA2a. Surprisingly, despite impaired diastolic dysfunction in GFP+ISO hearts, total protein expression of SERCA2a was significantly increased after isoproterenol infusion (
Intracellular distribution of Cav1.2 and SERCA2a was further explored using biochemical sucrose gradient-based fractionation of cardiac microsomes [70]. As indicated in
Given reduced Cav1.2 and SERCA2a at t-tubule/jSR region, STORM imaging was used to analyze nanoscale protein-protein colocalization for Cav1.2-RyR and SERCA2a-cBIN1 (
The Phenotype of cBIN1+ISO Hearts is Isoform Specific and Unique to cBIN1
To further explore whether the observed phenotype of cBIN1+ISO hearts was an isoform specific effect, the isoproterenol protocol was repeated in 50 more mice, which were randomized to receive AAV9 transducing GFP and cBIN1, as well as the other three mouse cardiomyocyte expressing BIN1 isoforms including the small BIN1, BIN1+17, and BIN1+13. Similarly, three weeks after viral administration, mice were subjected to continuous subcutaneous isoproterenol infusion at 30 mg/kg/day for 4 weeks. The protein expression of Cav1.2 and SERCA2a in post- isoproterenol hearts were not significantly different when compared across five groups of mice transduced with GFP or BIN1 isoforms (
Next, the functional consequence of different AAV9-BIN1 isoform pretreatment was explored using echocardiography. cBIN1-expressing mice, when compared to the GFP group, lessened the isoproterenol induced increase in LV wall thickness, LV mass, and RWT (
#,
##,
###indicates P < 0.05, 0.01, 0.001 vs. the GFP group of 4 w post-ISO.
The Cardiac Protective Effect of AAV9-cBIN1 is Confirmed in TAC-Induced HF
The myocardial protective effect of cBIN1 was further explored in a separate mouse model of pressure overload induced by TAC. Mice with either genetic deficiency of cBIN1 or AAV9- transuced cBIN1 over-expression were tested in this study (
At 8 weeks after TAC, surviving mice were sacrificed and evaluated for ratios of HW/BW and LW/BW (Table 4,
#indicates p < 0.05 for AAV9-GFP vs. AAV9-CBIN1.
#indicates p < 0.05 for AAV9-GFP vs. AAV9-cBIN1.
Mouse Diabetic Cardiomyopathy (HFpEF) Studies
Myocardial function was explored in db/db mice developing diabetic cardiomyopathy, as well as the therapeutic potential of AAV9-cBIN1. The Db/db mouse line (homozygous Dock7m for Leprdb, Jackson Laboratories), an established mouse model of type 2 diabetes, has been used as a model of diabetic cardiomyopathy and heart failure with preserved ejection fraction (HFpEF) [78]. Nine-weeks old male and female db/db mice and their littermate control db/m mice were administered with one dose (1×1011 vg) of AAV9-cBIN1 or control GFP through retro-orbital injection [79, 80]. Cardiac function and physiological parameters were measured both prior to AAV injection and 8 weeks after injection when animals are 17 weeks of age [79]. Myocardial function was evaluated by echocardiography-measured systolic parameters (ejection fraction and fractional shortening), diastolic parameters (E/A, E/e′), as well as stroke volume (SV). We also evaluated the performance on exercise exhaustion test of these animals by measuring their maximal running distance on a mouse treadmill. Exercise intolerance is an important physiological parameter of impaired cardiac functional reserve and HFpEF.
Echocardiography measured myocardial functional parameters indicate a successful development of diastolic failure as early as in 9 weeks of age in db/db mice. In 17-week old db/db mice with still preserved left ventricular ejection fraction, there is a significant alteration in diastolic parameters including reduced E/A (
Summary of Canine Ischemic Cardiomyopathy (HFrEF) Studies
We explored the effect of cBIN1 gene therapy to rescue diminished cardiac function in hearts with ischemic cardiomyopathy. Adult canine hound dogs (25-30 kg) were subjected to open thoracotomy and permanent ligation of the proximal Left Anterior Descending (LAD) coronary artery. Dogs were followed by echocardiography, hemodynamic parameters, and physiologic parameters 8 to 9 weeks post ligation, the animals were anesthetized, and their left ventricular endocardium was injected with cBIN1 packaged in AAV9 virus. The NOGA XP (Biosense Webster/Johnson and Johnson) was used to perform 3D electroanatomical mapping of the LV endocardial chamber. Using the same NOGA XP system, we inject the myocardium with a Myostar catheter that has a 27-gauge nitinol needle. Each heart was injected at 20 injections sites throughout left ventricular endocardium. Each injection consisted of 2.5×1011 vg mixed in 250 pL of PBS, for a total of 5×1012 vg per animal heart. Animals were then continued to be monitored by echocardiography, hemodynamic, and physiological parameters.
As shown in
In both animals, cBIN1 gene therapy provided a dramatic rescue of myocardial function in heart with ischemic cardiomyopathy (HFrEF). Rescue occurred for at least five weeks. The experiments are ongoing, and duration of therapy after a single episode of cBIN1 injection remains to be determined.
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This application claims priority to U.S. Provisional Patent Application N. 63/007,229, filed on Apr. 8, 2020, and 63/088,123, filed on Oct. 6, 2020, each of which is incorporated by reference herein in its entirety.
This invention was made with United States government support under National Institutes of Health grant numbers HL133286, HL094414, and HL138577. The United States government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/026131 | 4/7/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63088123 | Oct 2020 | US | |
| 63007229 | Apr 2020 | US |
