Istaroxime Metabolite PST3093 Selectively Stimulates SERCA2a and Reverses Disease-Induced Changes in Cardiac Function.

Heart failure (HF) therapeutic toolkit would strongly benefit from the availability of ino-lusitropic agents with a favorable pharmacodynamics and safety profile. Istaroxime is a promising agent, which combines Na+/K+ pump inhibition with sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) stimulation; however, it has a very short half-life and extensive metabolism to a molecule named PST3093. The present work aims to investigate whether PST3093 still retains the pharmacodynamic and pharmacokinetic properties of its parent compound. We studied PST3093 for its effects on SERCA2a and Na+/K+ ATPase activities, Ca2+ dynamics in isolated myocytes, and hemodynamic effects in an in vivo rat model of diabetic [streptozotocin (STZ)-induced] cardiomyopathy. Istaroxime infusion in HF patients led to accumulation of PST3093 in the plasma; clearance was substantially slower for PST3093 than for istaroxime. In cardiac rat preparations, PST3093 did not inhibit the Na+/K+ ATPase activity but retained SERCA2a stimulatory activity. In in vivo echocardiographic assessment, PST3093 improved overall cardiac performance and reversed most STZ-induced abnormalities. PST3093 intravenous toxicity was considerably lower than that of istaroxime, and it failed to significantly interact with 50 off-targets. Overall, PST3093 is a "selective" SERCA2a activator, the prototype of a novel pharmacodynamic category with a potential in the ino-lusitropic approach to HF with prevailing diastolic dysfunction. Its pharmacodynamics are peculiar, and its pharmacokinetics are suitable to prolong the cardiac beneficial effect of istaroxime infusion. SIGNIFICANCE STATEMENT: Heart failure (HF) treatment would benefit from the availability of ino-lusitropic agents with favourable profiles. PST3093 is the main metabolite of istaroxime, a promising agent combining Na+/K+ pump inhibition and sarcoplasmic reticulum Ca2+ ATPase2a (SERCA2a) stimulation. PST3093 shows a longer half-life in human circulation compared to istaroxime, selectively activates SERCA2a, and improves cardiac performance in a model of diabetic cardiomyopathy. Overall, PST3093 as a selective SERCA2a activator can be considered the prototype of a novel pharmacodynamic category for HF treatment.

Endobronchial Aerosolized AAV1.SERCA2a Gene Therapy in a Pulmonary Hypertension Pig Model: Addressing the Lung Delivery Bottleneck.

A disappointing number of new therapies for pulmonary hypertension (PH) have been successfully translated to the clinic. Adeno-associated viral (AAV) gene therapy has the potential to treat the underlying pathology of PH, but the challenge remains in efficient and safe delivery. The aims of this study were (1) to test the efficacy of endobronchial aerosolization delivery for AAV1-mediated sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) gene therapy in a PH pig model and (2) to identify the most efficient airway administration modality for in-lung gene therapy in PH. We hypothesized that delivery to the distal bronchi increases lung viral uptake and avoids virus loss in off-target compartments. In part 1 of the study, PH was induced in pigs by surgically banding the pulmonary veins. Two months postsurgery, 1 × 1013 viral genomes (vg) of AAV1.SERCA2a or saline was endobronchially aerosolized using a bronchoscope. Two months after aerosolization, high vg copies (vgc) were detected in the lungs, accompanied by functional and morphometrical amelioration of PH. In part 2 of the study, we directly compared the endobronchial aerosolization gene delivery to the intratracheal aerosolization in PH pigs. Endobronchial delivery demonstrated higher viral expression (6,719 ± 927 vs. 1,444 ± 402 vgc/100 ng DNA, p = 0.0017), suggesting this delivery modality is a promising method for clinical AAV gene therapy for PH.

Intra-tracheal gene delivery of aerosolized SERCA2a to the lung suppresses ventricular arrhythmias in a model of pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) results in right ventricular (RV) failure, electro-mechanical dysfunction and heightened risk of sudden cardiac death (SCD), although exact mechanisms and predisposing factors remain unclear. Because impaired chronotropic response to exercise is a strong predictor of early mortality in patients with PAH, we hypothesized that progressive elevation in heart rate can unmask ventricular tachyarrhythmias (VT) in a rodent model of monocrotaline (MCT)-induced PAH. We further hypothesized that intra-tracheal gene delivery of aerosolized AAV1.SERCA2a (AAV1.S2a), an approach which improves pulmonary vascular remodeling in PAH, can suppress VT in this model. OBJECTIVE: To determine the efficacy of pulmonary AAV1.S2a in reversing electrophysiological (EP) remodeling and suppressing VT in PAH. METHODS: Male rats received subcutaneous injection of MCT (60 mg/kg) leading to advanced PAH. Three weeks following MCT, rats underwent intra-tracheal delivery of aerosolized AAV1.S2a (MCT + S2a, N = 8) or saline (MCT, N = 9). Age-matched rats served as controls (CTRL, N = 7). The EP substrate and risk of VT were determined using high-resolution optical action potential (AP) mapping ex vivo. The expression levels of key ion channel subunits, fibrosis markers and hypertrophy indices were measured by RT-PCR and histochemical analyses. RESULTS: Over 80% of MCT but none of the CTRL hearts were prone to sustained VT by rapid pacing (P < .01). Aerosolized gene delivery of AAV1.S2a to the lung suppressed the incidence of VT to <15% (P < .05). Investigation of the EP substrate revealed marked prolongation of AP duration (APD), increased APD heterogeneity, a reversal in the trans-epicardial APD gradient, and marked conduction slowing in untreated MCT compared to CTRL hearts. These myocardial EP changes coincided with major remodeling in the expression of K and Ca channel subunits, decreased expression of Cx43 and increased expression of pro-fibrotic and pro-hypertrophic markers. Intra-tracheal gene delivery of aerosolized AAV1 carrying S2a but not luciferase resulted in selective upregulation of the human isoform of SERCA2a in the lung but not the heart. This pulmonary intervention, in turn, ameliorated MCT-induced APD prolongation, reversed spatial APD heterogeneity, normalized myocardial conduction, and suppressed the incidence of pacing-induced VT. Comparison of the minimal conduction velocity (CV) generated at the fastest pacing rate before onset of VT or at the end of the protocol revealed significantly lower values in untreated compared to AAV1.S2a treated PAH and CTRL hearts. Reversal of EP remodeling by pulmonary AAV1.S2a gene delivery was accompanied by restored expression of key ion channel transcripts. Restored expression of Cx43 and collagen but not the pore-forming Na channel subunit Nav1.5 likely ameliorated VT by improving CV at rapid rates in PAH. CONCLUSION: Aerosolized AAV1.S2a gene delivery selectively to the lungs ameliorates myocardial EP remodeling and VT susceptibility at rapid heart rates. Our findings highlight for the first time the utility of a non-cardiac gene therapy approach for arrhythmia suppression.

New insights into SERCA2a gene therapy in heart failure: pay attention to the negative effects of B-type natriuretic peptides.

Sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA2a) is a target of interest in gene therapy for heart failure with reduced ejection fraction (HFrEF). However, the results of an important clinical study, the Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID) trial, were controversial. Promising results were observed in the CUPID 1 trial, but the results of the CUPID 2 trial were negative. The factors that caused the controversial results remain unclear. Importantly, enrolled patients were required to have a higher plasma level of B-type natriuretic peptide (BNP) in the CUPID 2 trial. Moreover, BNP was shown to inhibit SERCA2a expression. Therefore, it is possible that high BNP levels interact with treatment effects of SERCA2a gene transfer and accordingly lead to negative results of CUPID 2 trial. From this point of view, effects of SERCA2a gene therapy should be explored in heart failure with preserved ejection fraction, which is characterised by lower BNP levels compared with HFrEF. In this review, we summarise the current knowledge of SERCA2a gene therapy for heart failure, analyse potential interaction between BNP levels and therapeutic effects of SERCA2a gene transfer and provide directions for future research to solve the identified problems.

Randomized Clinical Trials of Gene Transfer for Heart Failure with Reduced Ejection Fraction.

Despite improvements in drug and device therapy for heart failure, hospitalization rates and mortality have changed little in the past decade. Randomized clinical trials using gene transfer to improve function of the failing heart are the focus of this review. Four randomized clinical trials of gene transfer in heart failure with reduced ejection fraction (HFrEF) have been published. Each enrolled patients with stable symptomatic HFrEF and used either intracoronary delivery of a virus vector or endocardial injection of a plasmid. The initial CUPID trial randomized 14 subjects to placebo and 25 subjects to escalating doses of adeno-associated virus type 1 encoding sarcoplasmic reticulum calcium ATPase (AAV1.SERCA2a). AAV1.SERCA2a was well tolerated, and the high-dose group met a 6 month composite endpoint. In the subsequent CUPID-2 study, 243 subjects received either placebo or the high dose of AAV1.SERCA2a. AAV1.SERCA2a administration, while safe, failed to meet the primary or any secondary endpoints. STOP-HF used plasmid endocardial injection of stromal cell-derived factor-1 to promote stem-cell recruitment. In a 93-subject trial of patients with ischemic etiology heart failure, the primary endpoint (symptoms and 6 min walk distance) failed, but subgroup analyses showed improvements in subjects with the lowest ejection fractions. A fourth trial randomized 14 subjects to placebo and 42 subjects to escalating doses of adenovirus-5 encoding adenylyl cyclase 6 (Ad5.hAC6). There were no safety concerns, and patients in the two highest dose groups (combined) showed improvements in left ventricular function (left ventricular ejection fraction and -dP/dt). The safety data from four randomized clinical trials of gene transfer in patients with symptomatic HFrEF suggest that this approach can be conducted with acceptable risk, despite invasive delivery techniques in a high-risk population. Additional trials are necessary before the approach can be endorsed for clinical practice.

Therapeutic efficacy of AAV1.SERCA2a in monocrotaline-induced pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by dysregulated proliferation of pulmonary artery smooth muscle cells leading to (mal)adaptive vascular remodeling. In the systemic circulation, vascular injury is associated with downregulation of sarcoplasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) and alterations in Ca(2+) homeostasis in vascular smooth muscle cells that stimulate proliferation. We, therefore, hypothesized that downregulation of SERCA2a is permissive for pulmonary vascular remodeling and the development of PAH. METHODS AND RESULTS: SERCA2a expression was decreased significantly in remodeled pulmonary arteries from patients with PAH and the rat monocrotaline model of PAH in comparison with controls. In human pulmonary artery smooth muscle cells in vitro, SERCA2a overexpression by gene transfer decreased proliferation and migration significantly by inhibiting NFAT/STAT3. Overexpresion of SERCA2a in human pulmonary artery endothelial cells in vitro increased endothelial nitric oxide synthase expression and activation. In monocrotaline rats with established PAH, gene transfer of SERCA2a via intratracheal delivery of aerosolized adeno-associated virus serotype 1 (AAV1) carrying the human SERCA2a gene (AAV1.SERCA2a) decreased pulmonary artery pressure, vascular remodeling, right ventricular hypertrophy, and fibrosis in comparison with monocrotaline-PAH rats treated with a control AAV1 carrying ß-galactosidase or saline. In a prevention protocol, aerosolized AAV1.SERCA2a delivered at the time of monocrotaline administration limited adverse hemodynamic profiles and indices of pulmonary and cardiac remodeling in comparison with rats administered AAV1 carrying ß-galactosidase or saline. CONCLUSIONS: Downregulation of SERCA2a plays a critical role in modulating the vascular and right ventricular pathophenotype associated with PAH. Selective pulmonary SERCA2a gene transfer may offer benefit as a therapeutic intervention in PAH.

SERCA2a gene transfer improves electrocardiographic performance in aged mdx mice.

BACKGROUND: Cardiomyocyte calcium overloading has been implicated in the pathogenesis of Duchenne muscular dystrophy (DMD) heart disease. The cardiac isoform of sarcoplasmic reticulum calcium ATPase (SERCA2a) plays a major role in removing cytosolic calcium during heart muscle relaxation. Here, we tested the hypothesis that SERCA2a over-expression may mitigate electrocardiography (ECG) abnormalities in old female mdx mice, a murine model of DMD cardiomyopathy. METHODS: 1 × 10(12) viral genome particles/mouse of adeno-associated virus serotype-9 (AAV-9) SERCA2a vector was delivered to 12-m-old female mdx mice (N = 5) via a single bolus tail vein injection. AAV transduction and the ECG profile were examined eight months later. RESULTS: The vector genome was detected in the hearts of all AAV-injected mdx mice. Immunofluorescence staining and western blot confirmed SERCA2a over-expression in the mdx heart. Untreated mdx mice showed characteristic tachycardia, PR interval reduction and QT interval prolongation. AAV-9 SERCA2a treatment corrected these ECG abnormalities. CONCLUSIONS: Our results suggest that AAV SERCA2a therapy may hold great promise in treating dystrophin-deficient heart disease.

SERCA2a gene transfer decreases sarcoplasmic reticulum calcium leak and reduces ventricular arrhythmias in a model of chronic heart failure.

BACKGROUND: Sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) gene therapy improves mechanical function in heart failure and is under evaluation in a clinical trial. A critical question is whether SERCA2a gene therapy predisposes to increased sarcoplasmic reticulum calcium (SR Ca(2+)) leak, cellular triggered activity, and ventricular arrhythmias in the failing heart. METHODS AND RESULTS: We studied the influence of SERCA2a gene therapy on ventricular arrhythmogenesis in a rat chronic heart failure model. ECG telemetry studies revealed a significant antiarrhythmic effect of SERCA2a gene therapy with reduction of both spontaneous and catecholamine-induced arrhythmias in vivo. SERCA2a gene therapy also reduced susceptibility to reentry arrhythmias in ex vivo programmed electrical stimulation studies. Subcellular Ca(2+) homeostasis and spontaneous SR Ca(2+) leak characteristics were measured in failing cardiomyocytes transfected in vivo with a novel AAV9.SERCA2a vector. SR Ca(2+) leak was reduced after SERCA2a gene therapy, with reversal of the greater spark mass observed in the failing myocytes, despite normalization of SR Ca(2+) load. SERCA2a reduced ryanodine receptor phosphorylation, thereby resetting SR Ca(2+) leak threshold, leading to reduced triggered activity in vitro. Both indirect effects of reverse remodeling and direct SERCA2a effects appear to underlie the antiarrhythmic action. CONCLUSIONS: SERCA2a gene therapy stabilizes SR Ca(2+) load, reduces ryanodine receptor phosphorylation and decreases SR Ca(2+) leak, and reduces cellular triggered activity in vitro and spontaneous and catecholamine-induced ventricular arrhythmias in vivo in failing hearts. SERCA2a gene therapy did not therefore predispose to arrhythmias and may represent a novel antiarrhythmic strategy in heart failure.

Augmentation of left ventricular mechanics by recirculation-mediated AAV2/1-SERCA2a gene delivery in experimental heart failure.

AIMS: Down-regulation of sarcoplasmic reticulum calcium ATPase (SERCA2a) is a key molecular abnormality in heart failure (HF), which is not currently addressed by specific pharmacotherapy. We sought to evaluate, in detail, the impact of augmented SERCA2a expression on left ventricular (LV) mechanics in a large animal model of HF. METHODS AND RESULTS: Heart failure was induced in adult sheep by rapid pacing (180 b.p.m.) for 1 month, followed by delivery of adeno-associated virus (AAV) 2/1-SERCA, using a percutaneous, recirculating system for gene delivery over a 10 min period. Left ventricular mechanics was investigated by echocardiography and conductance catheter measurements in sheep receiving AAV2/1-SERCA2a after a further 4 weeks of pacing in comparison with untreated HF controls. Left ventricular function was significantly improved in the AAV2/1-SERCA2a-treated group, despite continued pacing, as measured by fractional shortening (delta absolute FS, control -4.2 ± 1.5% vs. treatment 4.4 ± 1.5%; P < 0.01) and conductance catheterization (delta Ees, control -1.22 ± 0.60 vs. treatment 0.65 ± 0.51; P < 0.05). Western blots showed an increase in SERCA protein in AAV2/1-SERCA2a-treated animals, and an analysis of gene delivery showed no evidence of regional myocardial heterogeneity in the distribution of AAV2/1-SERCA. CONCLUSION: In a large animal model, AAV2/1-mediated SERCA2a gene delivery using percutaneous, recirculating cardiac delivery leads to improved LV function.

Neonatal gene transfer of Serca2a delays onset of hypertrophic remodeling and improves function in familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant genetic disorder linked to numerous mutations in the sarcomeric proteins. The clinical presentation of FHC is highly variable, but it is a major cause of sudden cardiac death in young adults with no specific treatments. We tested the hypothesis that early intervention in Ca(2+) regulation may prevent pathological hypertrophy and improve cardiac function in a FHC displaying increased myofilament sensitivity to Ca(2+) and diastolic dysfunction. A transgenic (TG) mouse model of FHC with a mutation in tropomyosin at position 180 was employed. Adenoviral-Serca2a (Ad.Ser) was injected into the left ventricle of 1-day-old non-transgenic (NTG) and TG mice. Ad.LacZ was injected as a control. Serca2a protein expression was significantly increased in NTG and TG hearts injected with Ad.Ser for up to 6 weeks. Compared to TG-Ad.LacZ hearts, the TG-Ad.Ser hearts showed improved whole heart morphology. Moreover, there was a significant decline in ANF and ß-MHC expression. Developed force in isolated papillary muscle from 2- to 3-week-old TG-Ad.Ser hearts was higher and the response to isoproterenol (ISO) improved compared to TG-Ad.LacZ muscles. In situ hemodynamic measurements showed that by 3 months the TG-Ad.Ser hearts also had a significantly improved response to ISO compared to TG-Ad.LacZ hearts. The present study strongly suggests that Serca2a expression should be considered as a potential target for gene therapy in FHC. Moreover, our data imply that development of FHC can be successfully delayed if therapies are started shortly after birth.