11. Gene Therapy Mediated Increase in dATP Improves Cardiac Performance in Models of Systolic Heart Failure

Abstract

Heart Failure results in more deaths and hospitalizations than almost any other single cause. We are exploring gene therapy treatment strategies that increase 2 deoxy-adenosine triphosphate (dATP) levels via cardiac specific expression of ribonucleotide reductase (RNR) in heart muscle. dATP is produced by RNR. In vitro studies show it increases the magnitude & rate of contraction in rodent, pig & failing human heart muscle without altering calcium handling or slowing relaxation. RNR expression in heart muscle increases intracellular dATP, cardiomyocyte contraction & cardiac performance in rodents. To do this we ligated a cDNA encoding both human RNR subunits to miniaturized cardiac specific enhancer/promoter portions of human cardiac troponin T and packaged the therapeutic “BB-R12” gene in AAV6. In the current study expanded the types of heart failure treated by BB-R12 by testing the effect of dATP on contractile function in a transgenic mouse model of familial dilated cardiomyopathy (DCM). These mice carry and phenocopy a missense mutation (D230N) in alpha-tropomyosin (Tm) that was identified in two DCM family cohorts that exhibit progressive ventricular dilation and loss of systolic function. We measured the effect of dATP on the Ca2+ sensitivity of force in demembranated trabeculae from young adult D230N & WT mice (3 mice & 8-10 trabeculae per group). pCa50 was decreased (lower Ca2+ sensitivity) for D230N Tm (5.47±0.01) compared to WT (5.59±0 0.03) mice, and substituting dATP for ATP rescued this defect (5.55±0.03). dATP also increased isometric force ~20% at pCa=5.8, the approximate level of intracellular Ca2+ seen in a cardiomyocyte twitch. We also measured force and the kinetics of activation & relaxation of isolated myofibrils. Force at pCa=5.6 decreased from 85±14 mN/mm2 in WT myofibrils to 52±7 mN/mm2 (p<0.05) in D230N Tm myofibrils. Importantly, the force deficit was improved to 72±14 mN/mm2 (p=0.47) with dATP. Similarly, the activation rate decreased in D230N Tm myofibrils (1.6±0.2 s-1) vs. WT mice (3.5±0.3 s-1), and this was partially corrected by dATP (2.5±0.4 s-1). dATP also corrected alterations in the early, slow relaxation phase of myofibrils from D230N Tm mice. In ongoing studies, we are systemically injecting 4-6 week old D230N Tm mice with AAV6-BB-R12 to determine how this affects cardiac function acutely, as well as the time course of reduced systolic function, progressive ventricular dilation & wall remodeling. In related studies, we found AAV6-BB-R12 rescues cardiac function in an infarcted pig heart failure model. These studies assess the ability of BB-R12 to acutely treat diseased hearts and act as a preventative agent in progressive heart failure.