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. DCM has various causes including coronary artery disease, toxins, metabolites and genetic mutations with all leading to a common pathway of dilation and reduced contractility. In addition to the loss of contractility, there is also impairment in the Frank-Staring 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. Our previous published work has shown that that 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 (Tm). This allows us to test whether augmenting cross-bridge binding improves the contractile deficit caused by a thin filament mutation and suggest that our therapy can be used a wide variety of conditions. 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 demembranated trabecula 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-R1R2cTnT455, 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 adult D230N Tm mice and retard or prevent progression of heart failure in young mice with DCM. In the first aim, I will use post-infarct model of DCM in rats in addition to the D230N Tm mice to evaluate the effect of cross-bridge activation on length dependent activation (LDA)-the Frank Starling relationship at the sarcomere level. I will first tes the hypothesis that dATP enhances LDA in the two models of DCM. 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. The long-term goal of this project is provide a safe and effective treatment for DCM regardless of its cause. I am proposing the necessary preclinical experiments necessary to take this novel therapy to the clinic to improve morbidity and mortality in DCM.
Heart failure, the inability for the heart to keep up with its work load, is a growing epidemic in worldwide, with high mortality despite current available medical treatments. In the most common form, the heart is dilated with reduced contractile function. We are proposing a novel mechanism to treat this problem by altering the fuel used for contraction employing the naturally occurring molecule, deoxyadenosine triphosphate (dATP).
|Farris, Stephen D; Moussavi-Harami, Farid; Stempien-Otero, April (2017) Heart failure with preserved ejection fraction and skeletal muscle physiology. Heart Fail Rev 22:141-148|
|Olafsson, Sigurast; Whittington, Dale; Murray, Jason et al. (2017) Fast and sensitive HPLC-MS/MS method for direct quantification of intracellular deoxyribonucleoside triphosphates from tissue and cells. J Chromatogr B Analyt Technol Biomed Life Sci 1068-1069:90-97|
|Thomson, Kassandra S; Odom, Guy L; Murry, Charles E et al. (2016) Translation of Cardiac Myosin Activation with 2-deoxy-ATP to Treat Heart Failure via an Experimental Ribonucleotide Reductase-Based Gene Therapy. JACC Basic Transl Sci 1:666-679|