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

Abstract

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.

Figure 1.. Incidence, characteristics, mode-of-induction, and reversibility of VT.

A. Bar graph showing the incidence of pacing-induced sustained VT in CTRL, MCT, and MCT + S2a hearts. B. Mode of induction of VT by progressive elevation of pacing rate. Representative action potential traces recorded from CTRL (black) and MCT (red) hearts during steady-state pacing at progressively shorter pacing cycle lengths leading up to the initiation of sustained VT in MCT but not CTRL. C. A contour map showing the distribution of the frequency of ventricular activation during VT in a representative MCT heart. D. Bar graph representing the average dominant frequency in the RV and LV of MCT hearts during VT.

Figure 2.. Action potential remodeling in untreated and S2a-treated MCT hearts relative to control.

A. Superimposed AP traces from representative CTRL (black), MCT (red), and MCT+S2a (blue) treated hearts during baseline steady-state pacing. Average epicardial APD measured at 75% of repolarization (APD₇₅) in CTRL (black), MCT (red), and MCT+S2a (blue).B. Contour maps representing APD₇₅ across the anterior epicardial surface. C. Average RV and LV APD₇₅ in CTRL (black), MCT (red), and MCT+S2a (blue). D. Average APD₇₅ gradient from the RV to the LV. N =4 CTRL, 9 MCT, and 5 MCT+S2a. APD = action potential duration; APD₇₅ = APD measured at 75% repolarization; PCL = pacing cycle length. E. APD heterogeneity indexed by the standard deviation of APD₇₅ values measured across all pixels in CTRL (black), MCT (red), and MCT+S2a (blue). N=4 control, 9 MCT, and 5 MCT+S2a. *P <0.05, **P <0.005, ***P <0.0005, ****P <0.0001 by one-way ANOVA and Tukey post-test. † P MCT vs MCT+S2a <0.05, †† P MCT vs MCT+S2a <0.005. ‡ P MCT+S2a vs CTRL <0.05, ‡‡ P MCT+S2a vs CTRL <0.005.

Figure 3.. mRNA expression of key K channel subunits in the RV and LV of CTRL, MCT and MCT+S2a hearts.

Top row mRNA expression of the Kv4.2 (N= 6 for all groups), Kv4.3 (N= 5 CTRL, 4 MCT, 7 MCT+S2a), and Kir2.1 (N=10 CTRL, 8 MCT, 7 MCT+S2a) K channels in the RV; Bottom row mRNA expression of Kv4.2, Kv4.3, and Kir2.1 in the LV of each group. *P <0.05, **P <0.005, ***P <0.0005 by one-way ANOVA and Tukey post-test.

Figure 4.. Conduction slowing and its Restoration by intra-tracheal delivery of aerosolized AAV1.S2a.

A. Representative isochrone maps for Ctrl, MCT and MCT+S2a hearts. B. Rate-dependence of CV in CTRL (black, N=4), MCT (red, N=8), and MCT+S2a (blue, N=6). C. Comparison of the mean critical conduction velocity (CV_c) across groups (left, N=4 CTRL, 8 MCT, 6 MCT+S2a), and between VT (+) and VT (−) hearts (right) (N=8 and N=10, respectively). *P <0.05, **P <0.005, ***P <0.0005, one-way ANOVA and Tukey post-test, † P MCT vs MCT+S2a <0.05, †† P MCT vs MCT+S2a <0.005.

Figure 5.. Changes in the expression of the molecular correlates of myocardial conduction.

A. mRNA expression of fibrosis markers Col1A1 (N=5 CTRL, 4 MCT, 7 MCT+S2a), Col1A2 (N= 5 CTRL, 4 MCT, 5 MCT+S2a), and Col3A1 (N= 4 CTRL, 4 MCT, 7 MCT+S2a), measured by RT-PCR in CTRL, MCT, and MCT+S2a hearts. B. mRNA expression of myosin heavy chain α (MHC-α) (N= 4 CTRL, 4 MCT, 7 MCT+S2a), atrial natriuretic peptide (ANP) (N= 4 CTRL, 4 MCT, 7 MCT+Sa), and brain natriuretic peptide (BNP) (N= 6 CTRL, 6 MCT, 7 MCT+S2a). C. mRNA expression of the pore-forming L- and T-type calcium channels, Cav1.2 and Cav3.1 in CTRL, MCT, and MCT+S2a RV samples. *P <0.05, **P <0.005, ***P <0.0005 by one-way ANOVA and Tukey post-test.

Figure 6.. Cx43 expression in the RV of CTRL, MCT and MCT+S2a rats.

A. Cx43 expression (N=8 CTRL, 8 MCT, 5 MCT+S2a) measured by RT-PCR in CTRL, MCT, and MCT+S2a hearts. B. Representative images of Cx43 (red) and α-actinin (green) immunostaining in RV tissue sections from CTRL, MCT, and MCT+S2a hearts. C. Representative western blot showing the expression of Nav1.5 in CTRL, MCT, and MCT+S2a hearts. D. Average Nav1.5 expression in the 3 groups. **P <0.005 by one-way ANOVA and Tukey post-test.

Figure 7:. Efficiency and selectivity of AAV1.S2a gene delivery to the lung.

A. Transgene copies were assessed by RT-PCR in RV and lung tissue samples to determine the efficiency and specificity of AAV1.S2a gene transfer to the lung. N=5-6 rats per group. ** P<0.01. B. Human and rat SERCA2a expression measured by RT-PCR in RV samples from CTRL, MCT, and MCT+S2a hearts. C. Representative immunofluorescence images and expression of SERCA2a (red) relative to α-actinin (green) in RV tissue sections from CTRL, MCT, and MCT+S2a hearts. D. Representative western blots of SERCA2a and GAPDH as a loading control, and quantification of relative SERCA2a protein expression in the RV of CTRL, MCT, and MCT+S2a hearts.