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. 2012 Mar;143(3):720-726.e3.
doi: 10.1016/j.jtcvs.2011.08.048. Epub 2011 Dec 3.

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

Michael G Katz  1 , Anthony S Fargnoli, JaBaris D Swain, Catherine E Tomasulo, Michele Ciccarelli, Z Maggie Huang, Joseph E Rabinowitz, Charles R Bridges
Affiliations

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

Michael G Katz et al. J Thorac Cardiovasc Surg. 2012 Mar.

Abstract

Objective: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

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Figures

FIGURE 1
FIGURE 1
Effects of MCARD-βARKct gene transfer on LV mechanics. A, dP/dtmax. B, dP/dtmin. *P <.05; iso = 0.5 μg/min (dark bars) versus iso = 0 (light bars); †P<.05, 4 weeks versus control.
FIGURE 2
FIGURE 2
A, Effect of MCARD-βARKct gene transfer on potential energy. *P <.05; iso = 0.5 μg/min (dark bars) versus iso = 0 (light bars). B, Effect of MCARD-βARKct gene transfer on arterial elastance *P<.05; iso = 0.5 μg/min (dark bars) versus iso = 0 (light bars).
FIGURE 3
FIGURE 3
RT-qPCR: MCARD-βARKct left ventricle and collateral expression.
FIGURE 4
FIGURE 4
A, MCARD-βARKct quantitative Western blot analysis: left and right ventricles versus lung and liver. B, MCARD-βARKct quantitative Western blot analysis: LV regional expression. C, MCARD-βARKct Western blot: left ventricle, lung, and liver. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; LV, left ventricular; RV, right ventricular.
FIGURE 5
FIGURE 5
A, Cardiac cAMP level: MCARD-βARKct versus MCARD-GFP. B, Cardiac βAR density: MCARD-βARKct versus MCARD-GFP.
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