Effect of elevated dATP on contractile function and the Frank-Starling relationship in models of dilated cardiomyopathy

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Abstract Dilated cardiomyopathy (DCM) is the most common form of systolic heart failure with poor prognosis and no specific treatment to address the underlying contractile deficit. In addition to the loss of contractility, there is also impairment in the Frank-Starling relationship, an adaptive process that is described as the increase in contractile force in response to increased preload. We are proposing to investigate a novel therapy for DCM that addresses both loss of contractility and impairment in the Frank-Starling relationship. This novel therapy is achieved by increasing intracellular levels of 2-deoxy ATP (dATP) in cardiomyocytes via increasing the expression of the enzyme ribonucleotide reductase (R1R2), the rate-limiting step in de novo dNTP biosynthesis. Increased intracellular levels dATP in cardiomyocytes increases contraction by enhancing cross-bridge binding and cycling kinetics and improving allosteric activation of contraction. Recent data suggests that in addition to improved contraction, increased dATP level also enhances the Frank Starling relationship. I will propose specific aims to assess the effect of increased dATP on the Frank Starling relationship and contraction in two animal models of DCM. In the first aim, I will use a genetic model of DCM with a D230N mutation in alpha-tropomyosin (D230N Tm). Using recombinant D230N Tm, I will test the hypothesis that this mutation reduces thin filament activation and, as a consequence, the kinetics of contractile activation and relaxation. I will then determine the effect of increasing dATP content on these deficits. Using tissue from transgenic D230N Tm mice, I will test the hypothesis that this mutation decreases calcium sensitivity of force and length-dependent activation. I will correct these functional deficits by increasing dATP content. Using AAV6-cR1R2, an adeno-associated viral vector that restricts R1R2 over-expression to cardiac myocytes, I will test the hypothesis that increasing dATP levels improves contractility of isolated cardiomyocytes, and improves systolic function in both adult and young D230N Tm mice. Similarly, I will use post-infarct model of DCM in rats to evaluate the effect of cross-bridge activation on length dependent activation (LDA)-the Frank Starling relationship at the sarcomere level. Next, I will use alternative agents to increase (dextran, EMD 50733) or decrease (BDM, beryllium fluoride, high inorganic phosphate) cross-bridge recruitment and evaluate their effect on LDA in demembranated post- infarct DCM rat trabecula. I will complete this aim by testing the hypothesis that cross-bridge activation augments LDA in human myocardium from patients with DCM. Extension Project: Use data from above studies in D230N Tm DCM rodent model to simulate realistic cardiac twitches and use machine-learning methods for parameter optimization.