The mechanisms and applications of a nucleotide-based heart failure therapy

Abstract

The naturally-occurring nucleotide 2-deoxy-ATP (dATP) is known to increase the amount of force produced by cardiac muscle when used as a substrate in place of ATP. Cytosolic concentration of dATP can be elevated by expressing both subunits of the enzyme ribonucleotide reductase (RNR), and overexpression of RNR in the heart has been previously shown to increase the magnitude of pressure development in the heart as well as increase the rates of contraction and relaxation in transgenic mice as well as virally-transduce rodent and large-animal models. The first aim of this dissertation utilizes Brownian dynamics simulations in concert with small-angle x-ray diffraction analysis of sarcomere structure to demonstrate that dATP induces structural changes in the conformation of myosin that increases its electrostatic affinity for actin and leads to an altered structural confirmation at rest that resembles changes caused by calcium-mediated activation. The second aim applies our therapeutic to a disease model and explores possible changes in skeletal muscle activation through a combinatorial gene therapy for a mouse model of Duchenne muscular dystrophy. Overexpression of RNR in concert with an artificial microdystrophin construct improved fractional shortening in cardiomyocytes but did not alter the overall force or kinetics of hindlimb muscle in either our disease model or in a mouse model of transgenic overexpression of ribonucleotide reductase. The third aim explores methods by which dATP can be elevated. We describe cardiac function after overexpression of Rrm1 and Rrm2B (RNRB), an isoform of the small subunit of RNR that has not been characterized in the heart. RNRB elevates cardiomyocyte dATP in a transgenic mouse model to the same degree as RNR, resulting in increases in cardiac function on multiple biophysical scales. Calcium handling was also altered in cardiomyocytes through an increase in the rate of calcium signal decay during contraction, a novel result which has not been previously described. These changes were reproducible in vitro in adult rat cardiomyocytes overexpressing RNRB through virally-mediated transduction in culture. Identifying a form of RNR that can be stably overexpressed in cardiomyocytes is an important step towards developing a therapeutic based on elevation of dATP.