Biological Heart Rate Reduction Through Genetic Suppression of Gα(s) Protein in the Sinoatrial Node

Abstract

Background: Elevated heart rate represents an independent risk factor for cardiovascular outcome in patients with heart disease. In the sinoatrial node, rate increase is mediated by β(1) adrenoceptor mediated activation of the Gα(s) pathway. We hypothesized that genetic inactivation of the stimulatory Gα(s) protein in the sinoatrial node would provide sinus rate control and would prevent inappropriate heart rate acceleration during β-adrenergic activation.

Methods and results: Domestic pigs (n=10) were evenly assigned to receive either Ad-small interfering RNA (siRNA)-Gα(s) gene therapy to inactivate Gα(s) or adenovirus encoding for green fluorescent protein (Ad-GFP) as control. Adenoviruses were applied through virus injection into the sinoatrial node followed by epicardial electroporation, and heart rates were evaluated for 7 days. Genetic inhibition of Gα(s) protein significantly reduced mean heart rates on day 7 by 16.5% compared with control animals (110±8.8 vs 131±9.4 beats per minute; P<0.01). On β-adrenergic stimulation with isoproterenol, we observed a tendency toward diminished rate response in the Ad-siRNA-Gα(s) group (Ad-siRNA-Gα(s), +79.3%; Ad-GFP, +61.7%; n=3 animals per group; P= 0.294). Adverse effects of gene transfer on left ventricular ejection fraction (LVEF) were not detected following treatment (LVEF(Ad-siRNA-Gαs), 66%; LVEF(Ad-GFP), 60%).

Conclusions: In this preclinical proof-of-concept study targeted Ad-siRNA-Gα(s) gene therapy reduced heart rates during normal sinus rhythm compared with Ad-GFP treatment and prevented inappropriate rate increase after β-adrenergic stimulation. Gene therapy may provide an additional therapeutic option for heart rate reduction in cardiac disease. (J Am Heart Assoc. 2012;1:jah3-e000372 doi: 10.1161/JAHA.111.000372).

Keywords: electrophysiology; gene therapy; heart failure; heart rate; sinoatrial node.

Figure 1.

In vitro efficacy of Ad-siRNA-Gα_s gene transfer. Gα_s protein expression was analyzed using Western blot in HL-1 mouse atrial cardiac myocytes. (A) Gα_s protein levels evaluated in untreated control cells and following application of Ad-GFP (GFP) or Ad-siRNA-Gα_s (siRNA). (B) Quantification of optical density normalized to GAPDH protein. Gα_s expression was suppressed by 51% in HL-1 cells infected with Ad-siRNA-Gα_s (n=3) compared with controls (n=3), whereas Ad-GFP did not significantly alter Gα_s protein levels (n=3). Data are provided as mean±SEM; ***P<0.001 versus control HL-1 cells. GAPDH indicates glyceraldehyde-3-phosphate-dehydrogenase; GFP, green fluorescent protein.

Figure 2.

Heart rate reduction following Ad-siRNA-Gα_s gene therapy. (A) Representative ECG recordings obtained from pigs before gene therapy (day 1) and after application of Ad-siRNA-Gα_s or Ad-GFP (day 7), respectively. (B) Mean heart rates (± SEM) assessed by daily ECG recordings in control animals (n=5) and in pigs treated with Ad-siRNA-Gα_s (n=5). Statistical significances among groups were analyzed on days 1, 4, and 7, respectively (**P<0.01). (C) Relative changes in heart rates recorded on day 7 compared with the day of gene transfer (day 1). (D-G) Three animals from each group were subjected to isoproterenol challenge to assess adrenergic response. Administration of isoproterenol on day 7 significantly increased heart rates in Ad-GFP control animals (D, E). Heart rate acceleration by isoproterenol was attenuated in animals infected with Ad-siRNA-Gα_s (D, E). (F, G) Comparison of heart rates obtained from individual animals in the Ad-siRNA-Gα_s group (F) and in the Ad-GFP group (G) before and after isoproterenol challenge, respectively. Data represent mean values±SEM; *P<0.05, ***P<0.001 compared with respective drug-free control conditions. ECG indicates electrocardiogram; GFP, green fluorescent protein.

Figure 3.

Efficacy and cardiac distribution of transgene expression. (A) Representative microphotographs depicting SAN, RA, and LV after application of Ad-GFP (day 7). GFP reporter gene expression was analyzed via direct fluorescence measurements (scale bar, 100 μm). (B) The relation of GFP positive cells compared with the total number of cardiac cells (in %) is presented for SAN, RA, and LV tissue obtained from 5 animals. Data are given as mean±SEM; **P<0.01 versus sinoatrial node. GFP indicates green fluorescent protein; LV, left ventricle; RV, right atrium; SAN, sinoatrial node.

Figure 4.

Gα_s protein knockdown in the sinoatrial node. Expression of Gα_s protein was assessed by immunohistochemistry. (A) Representative microscopic findings after treatment with Ad-siRNA-Gα_s and Ad-GFP (scale bar, 50 μm). (B) Quantification of Gα_s protein levels in n=5 animals per group. Data are expressed as mean±SEM (***P<0.001 vs Ad-GFP). GFP indicates green fluorescent protein.

Figure 5.

Expression of proteins involved in β-adrenergic signaling after gene therapy. Representative Western blots (A, C, E) and mean optical density (OD) values (B, D, F) are presented for study animals treated with Ad-siRNA-Gα_s (siRNA) and Ad-GFP (GFP), respectively (n=5 animals per group). Ad-siRNA-Gα_s treatment did not significantly affect expression of β₁ adrenoceptors (A, B), adenylyl cyclase VI (C, D), and activated protein kinase A (E, F) in the sinoatrial node. GFP indicates green fluorescent protein.