Abstract 369: Engineering Rrm2 to Elevate 2-deoxy-ATP and Improve Cardiomyocyte Function

Abstract

Overexpression of ribonucleotide reductase (RNR) in cardiomyocytes increases the amount of cytosolic 2-deoxy-ATP (dATP), which can be used by myosin and significantly increases contraction of cardiac muscle at all levels of calcium activation. Our group is working to develop enhanced dATP as a therapeutic option for heart failure. We have demonstrated that virally-mediated overexpression of RNR elevates dATP and increases the rate and magnitude of contraction and increases left ventricular contraction in normal hearts as well as rodent models of myocardial infarction and dilated cardiomyopathy. RNR is a heterotetramer containing two subunits, Rrm1 and Rrm2. While cardiomyocyte-specific overexpression of Rrm1 is stable, we have observed high variability in expression levels of the Rrm2 subunit in multiple disease models. We hypothesized that this variability was largely due to protein degradation via the ubiquitin-proteasome complex (UPC). We found that pharmacological inhibition of proteasome activity leads to increased expression of Rrm2 in virally-transduced cardiomyocytes in vitro. To confirm the hypothesis that the overexpressed Rrm2 is degraded via UPC-mediated degradation, we engineered mutations in specific ubiquitin-binding degrons of the Rrm2 gene. Transfecting human induced pluripotent stem cell-derived cardiomyocytes resulted in higher levels of Rrm2 than those overexpressing wild-type protein and resulted in higher levels of cytosolic dATP as measured by Liquid chromatography-mass spectrometry. Ongoing and planned experiments will compare the effects of this engineered mutation on Rrm2 overexpression, dATP production, and contractility in cultured cardiomyocytes. Our goal is to develop an improved RNR vector that will be resistant to degradation through the ubiquitin-proteasome pathway and therefore enable more stable and consistent RNR enzyme activity and deoxynucleotide levels of cardiomyocytes transduced in vivo.