Broad Transgenic, and Cardiac-Specific Viral Mediated, Over-Expression of Ribonucleotide Reductase Increases In Vivo Cardiac Contractility

Abstract

We have previously demonstrated that substitution of ATP with 2 deoxy-ATP (dATP) increased the magnitude and rate of force production at all activating [Ca2+] in demembranated cardiac muscle via increased crossbridge binding and cycling rates. Furthermore, we demonstrated that cellular [dATP] is increased by adenoviral-driven overexpression of the ribonucleotide reductase (Rrm) complex, which increased contractility with no decrement of relaxation in adult rat cardiomyocytes from normal or infarcted hearts. In this study we hypothesized that in vivo overexpression of Rrm would increase cardiac contractility. Elevated cardiac Rrm was accomplished by 1) transgenic (Tg) overexpression in mice via the chicken beta-actin promoter and 2) intravenous delivery of recombinant adeno-associated virus (rAAV6) to drive Rrm production using a cardiac specific promoter (cTnT455). Echocardiographic fractional shortening was significantly increased in 3-month old Tg animals (43.5 ± 1.4%) as compared to wild-type (WT) littermates (34.5 ± 2.2%), and this increase persisted at 6 months of age (42.8 ± 2.5% vs 28.8 ± 2.7%). Similar increases in FS were seen in rAAV6-Rrm transduced animals (43.0 ± 2.1%) as compared to sham-injected control animals (32.1 ± 2.9%). LV pressure development (balloon catheterization) was elevated in Tg-Rrm vs. WT Langendorff perfused hearts, and concurrent NMR spectra indicated no difference in PCr:ATP ratio during high workload challenge. Upregulation of Rrm was confirmed via RT-PCR, western blot analysis, and immunohistochemistry. Preliminary western blot results also suggest no difference in SERCA expression, but increased Ser-16 phosphorylation of phospholamban, in Tg-Rrm vs WT hearts. This may, in part, explain our previously reported acceleration of Ca2+ transient decay seen in Rrm-overexpressing cardiomyocytes in vitro. Overall, these experiments demonstrate the feasibility of directly targeting the actin-myosin crossbridge to enhance cardiac contractility without impairing relaxation. HL65497, HL091368, AHA2310117.