Abstract 618: Overexpression of Rrm2B Elevates dATP and Cardiac Function

Cardiac myosin can use 2-deoxy-ATP (dATP) instead of ATP as energy substrate, significantly enhancing the rate and magnitude of force development and relaxation. Our group has shown repeatedly that overexpression of the enzyme ribonucleotide reductase (RNR; the rate-limiting step in de novo dNTP synthesis) in cardiomyocytes, by either transgenic or viral vector approach, enhances cardiac function by significantly elevating cytosolic concentrations of dATP. We are exploring different promotor, gene constructs and engineered mutations of RNR to be able to vary the amount of dATP maintained in cardiomyocytes. RNR activity is partially governed by levels of the small subunit, Rrm2, whose concentration oscillates with the cell cycle via degradation by the ubiquitin-proteasome system. The p53-inducible small subunit Rrm2B is naturally present in quiescent cells and does not contain the amino acid sequence that is targeted by the ubiquitin-conjugating enzyme. We hypothesized that Rrm2B overexpression may be more stable than Rrm2 through its resistance to degradation by this pathway. Transgenic mice that overexpress Rrm1 and Rrm2B have a ~60 fold increase in myocardial [dATP] as measured by liquid chromatography-mass spectrometry compared to wild-type littermates. This translates to significantly elevated left-ventricular developed pressure as well as higher rates of contraction and relaxation in perfused isolated hearts, as well as enhanced shortening and relaxation in isolated cardiomyocytes. Isolated rat cells also have an increase in [dATP] and corresponding greater fractional shortening and rates of contraction and relaxation when transduced in vitro with adenovirus encoding Rrm1 and Rrm2B when compared to cells transduced with a control vector. We are currently in the process of comparing the effectiveness of adeno-associated viruses encoding Rrm2 and Rrm2B in vivo. Our ultimate goal is to develop an improved vector to enable stable and consistent overexpression of RNR and elevation of dATP in cardiomyocytes transduced in vivo , thereby improving both systolic and diastolic function for treatment of cardiomyopathies.