The goal of this project is to determine whether increased cardiomyocyte 2-deoxy ATP content [dATP], via over-expression of Ribonucleotide Reductase (R1R2), can be beneficial in potentiating cardiac function and treating heart failure. A multi-disciplinary team of investigators with considerable experience in the areas of cardiac contractile function, metabolism, electrophysiology and viral-mediated gene delivery has been assembled. Dr. Regnier (PI) previously demonstrated that dATP enhances contraction in demembranated cardiac muscle by increasing myosin binding to actin (crossbridge formation) and crossbridge cycling. In a recent paper (2011, JMCC. 51:894-901;Appendix 1), we demonstrated that increasing [dATP] in cultured adult rat cardiomyocytes by over-expression of R1R2 enhances contraction, speeds relaxation and has no effect on intracellular Ca2+ transient amplitude but speeds it's decay. In the same JMCC issue an editorial (2011, JMCC 51;883-4) urges that this novel approach be tested in animals to determine its potential for treating heart failure. This is the purpose of our proposal. Importantly, we present preliminary data demonstrating increasing [dATP] rescues depressed contractility and Ca2+ transients of cardiomyocytes from infarcted hearts. Additionally, we demonstrate that transgenic R1R2 over-expression mice (TG-R1R2) have elevated left ventricular (LV) function, measured by echocardiography and Langendorff perfusion. Here we propose a translational approach, i.e. delivery of an adeno-associated viral vector with a cardiac specific promoter (AAV6- R1R2cTnT455). We demonstrate that it results in R1R2 over-expression in the heart, but not skeletal muscle or lung, and that the AAV6 vector has sustained activity for at least 20 months in mice. We also report that a ~10x dose range of AAV6-R1R2cTnT455 injection is effective in increasing LV function (out to 6 weeks thus far). We will study AAV6-R1R2cTnT455 injected mice and TG-R1R2 mice under normal conditions (Aim 1) and in an acute (Aim 2) and chronic (Aim 3) infarct model. In vivo and in vitro whole heart studies will be complimented by trabeculae, intact cardiomyocyte and myofibril mechanical assessments during Ca2+ activated contraction. Because dATP increases crossbridge cycling and may affect other cellular ATPases, we will measure cardiac ATP synthesis and mitochondrial respiration, as well as high energy phosphate utilization, energetic reserve and oxygen consumption under basal conditions and with ?-adrenergic stress using NMR spectroscopy. We will also study action potential and Ca2+ transient behavior and assess hearts and mice for potential pathological condition. Mechanistic interpretations will be aided by proteomic analysis of myofilament and membrane proteins to assess isoform and phosphorylation profiles. We will also use computational models (in collaboration with Dr. Andrew McCulloch, UCSD) to integrate the multi-scale data and provide mechanistic insight. Results from these studies will provide valuable insight on whether sustained enhancement of myofilament contractility (with dATP) has potential for treatment of heart failure in animal models and humans.
Experiments proposed in this application will study a novel gene therapy based approach to improving cardiac function following myocardial infarct (heart attack). Our approach will be to use an adeno-associated (AAV6) viral vector with a cardiac specific promoter to increase cellular 2-deoxy ATP (dATP), which we have shown increase heart cell contraction. We will study the effectiveness of this approach in normal heart function and following coronary occlusion heart attack models to improve cardiac pump function via enhanced myofilament contractility.
|Teichman, Sam L; Thomson, Kassandra S; Regnier, Michael (2017) Cardiac Myosin Activation with Gene Therapy Produces Sustained Inotropic Effects and May Treat Heart Failure with Reduced Ejection Fraction. Handb Exp Pharmacol 243:447-464|
|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|
|Nowakowski, Sarah G; Regnier, Michael; Daggett, Valerie (2017) Molecular mechanisms underlying deoxy-ADP.Pi activation of pre-powerstroke myosin. Protein Sci 26:749-762|
|Ruan, Jia-Ling; Tulloch, Nathaniel L; Razumova, Maria V et al. (2016) Mechanical Stress Conditioning and Electrical Stimulation Promote Contractility and Force Maturation of Induced Pluripotent Stem Cell-Derived Human Cardiac Tissue. Circulation 134:1557-1567|
|Cheng, Yuanhua; Lindert, Steffen; Oxenford, Lucas et al. (2016) Effects of Cardiac Troponin I Mutation P83S on Contractile Properties and the Modulation by PKA-Mediated Phosphorylation. J Phys Chem B 120:8238-53|
|Zaunbrecher, Rebecca; Regnier, Michael (2016) Connecting Sarcomere Protein Mutations to Pathogenesis in Cardiomyopathies: The Development of ""Disease in a Dish"" Models. Front Physiol 7:566|
|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|
|Cheng, Yuanhua; Hogarth, Kaley A; O'Sullivan, M Lynne et al. (2016) 2-Deoxyadenosine triphosphate restores the contractile function of cardiac myofibril from adult dogs with naturally occurring dilated cardiomyopathy. Am J Physiol Heart Circ Physiol 310:H80-91|
|Cheng, Yuanhua; Regnier, Michael (2016) Cardiac troponin structure-function and the influence of hypertrophic cardiomyopathy associated mutations on modulation of contractility. Arch Biochem Biophys 601:11-21|
|Kolwicz Jr, Stephen C; Odom, Guy L; Nowakowski, Sarah G et al. (2016) AAV6-mediated Cardiac-specific Overexpression of Ribonucleotide Reductase Enhances Myocardial Contractility. Mol Ther 24:240-250|
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