AAV6-βARKct gene delivery mediated by molecular cardiac surgery with recirculating delivery (MCARD) in sheep results in robust gene expression and increased adrenergic reserve

Abstract

Genetic modulation of heart function is a novel therapeutic strategy. We investigated the effect of molecular cardiac surgery with recirculating delivery (MCARD)-mediated carboxyl-terminus of the β-adrenergic receptor kinase (βARKct) gene transfer on cardiac mechanoenergetics and β-adrenoreceptor (βAR) signaling. After baseline measurements, sheep underwent MCARD-mediated delivery of 10(14) genome copies of self-complimentary adeno-associated virus (scAAV6)-βARKct. Four and 8 weeks after MCARD, mechanoenergetic studies using magnetic resonance imaging were performed. Tissues were analyzed with real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting. βAR density, cyclic adenosine monophosphate levels, and physiologic parameters were evaluated. There was a significant increase in dP/dt(max) at 4 weeks: 1384 ± 76 versus 1772 ± 182 mm Hg/s; and the increase persisted at 8 weeks in response to isoproterenol (P<.05). Similarly, the magnitude of dP/dt(min) increased at both 4 weeks and 8 weeks with isoproterenol stimulation (P<.05). At 8 weeks, potential energy was conserved, whereas in controls there was a decrease in potential energy (P<.05) in response to isoproterenol. RT-qPCR confirmed robustness of βARKct expression throughout the left ventricle and undetectable expression in extracardiac tissues. Quantitative Western blot data confirmed higher expression of βARKct in the left ventricle: 0.46 ± 0.05 versus 0.00 in lung and liver (P<.05). Survival was 100% and laboratory parameters of major organ function were within normal limits. MCARD-mediated βARKct delivery is safe, results in robust cardiac-specific gene expression, enhances cardiac contractility and lusitropy, increases adrenergic reserve, and improves energy utilization efficiency in a preclinical large animal model.