Translation of Cardiac Myosin Activation with 2-deoxy-ATP to Treat Heart Failure via an Experimental Ribonucleotide Reductase-Based Gene Therapy

Abstract

Despite recent advances, chronic heart failure remains a significant and growing unmet medical need, reaching epidemic proportions carrying substantial morbidity, mortality, and costs. A safe and convenient therapeutic agent that produces sustained inotropic effects could ameliorate symptoms, and improve functional capacity and quality of life. We discovered small amounts of 2-deoxy-ATP (dATP) activate cardiac myosin leading to enhanced contractility in normal and failing heart muscle. Cardiac myosin activation triggers faster myosin crossbridge cycling with greater force generation during each contraction. We describe the rationale and results of a translational medicine effort to increase dATP levels using a gene therapy strategy that upregulates ribonucleotide reductase, the rate-limiting enzyme for dATP synthesis, selectively in cardiomyocytes. In small and large animal models of heart failure, a single dose of this gene therapy has led to sustained inotropic effects with no toxicity or safety concerns identified to-date. Further animal studies are being conducted with the goal of testing this agent in patients with heart failure.

Keywords: Heart failure; dATP; gene therapy; myosin activation; ribonucleotide reductase.

Figure 1

Schematic Illustrations of Selected Inotropic Agents and Gene Therapies for Treatment of HF Schematic illustrations depicting targets and mechanisms of action of selected inotropic agents and gene therapies for treatment of heart failure (HF) highlighting cardiac myosin activators BB-R12 and omecamtiv. (A) 2-deoxy-ATP (dATP) and omecamtiv are cardiac myosin activators that act directly on the contractile apparatus. Gene therapies that act on calcium cycling and regulation work “upstream” of myosin. Drugs and gene therapies that act on the β-adrenergic system or inhibit phosphodiesterase and cyclic AMP work further upstream of the contractile apparatus and calcium regulation. (B) Myosin heads form transient cross-bridges with actin. ATP fuels a conformational change (“power stroke”) causing actin to slide past myosin, shortening sarcomere length and causing contraction. Cardiac myosin activators enable more myosin heads (independent force generators) to interact with actin per cardiac cycle (i.e., more active cross-bridges). This has been characterized as “more hands pulling on the rope” and increases the maximum force of contraction. (C) Molecular modeling indicates dATP detaches from myosin more rapidly than ATP, enabling faster cross-bridge cycling and more rapid force generation (i.e., increase in maximum rate of pressure rise [+dP/dt]). Replicating cells normally have a small supply of dATP for DNA synthesis and repair. Ribonucleotide reductase (R1R2) converts ATP to dATP under tight allosteric regulation but is down-regulated in nonreplicating cardiomyocytes. BB-R12 provides the gene for R1R2 under control of a cardiac troponin promoter, restricting synthesis to cardiomyocytes. dATP increases the maximum force of contraction, increases +dP/dt and −dP/dt, and does not prolong systole. Omecamtiv activates myosin by increasing the transition rate from weak to strong binding states between myosin and actin, increases the maximum force of contraction without a change in +dP/dt or −dP/dt, and prolongs systole. Omecamtiv figure adapted with permission from Malik et al. . AC6 = adenylyl-cyclase type 6; β-AR = beta-adrenergic receptor; BB-R12 = AAV6-cTnT-R1R2; cAMP = cyclic AMP; CK = creatine kinase; GRK2 = G-protein-coupled receptor kinase-2; PDE = phosphodiesterase; PLN = phospholamban; R1R2 = ribonucleotide reductase; SERCA2a = sarcoplasmic/endoplasmic reticulum calcium ATPase; SUMO1 = small ubiquitin-related modifier 1.

Figure 2

Effects on Force Production and Stiffness Effects on force production and stiffness with [2% dATP/98% ATP] compared with 100% ATP (5 mmol/l total) in rat cardiac muscle. (A) Example force trace (pCa 5.6) showing increased force production with transfer from 100% ATP to [2% dATP/98% ATP] and reduction of force after transfer back to 100% ATP. (B) % increases in force production and stiffness of cardiac trabeculae with [2% dATP/98% ATP] at submaximal (pCa 5.6) and maximal (pCa 4.0) Ca²⁺ levels.

Figure 3

Effects of ATP Versus dATP Effects of ATP versus dATP in ex vivo human cardiac tissue from end-stage HF patients demonstrating dATP increases force. (A) Representative isometric force trace of demembranated tissue at pCa 5.6 with ATP or dATP. (B) Force measured at pCa 5.6 with 10%, 25%, 50%, and 100% dATP compared with 100% ATP (n = 4 at each dATP concentration). Measurements are mean ± SEM. *p < 0.05 compared with 100% ATP by paired Student t test.

Figure 4

dATP Mechanism of Action (A) Molecular simulation results show the loss of O2′ on dADP disrupts contacts in the nucleotide binding pocket on myosin. Representative structures showing conformational changes within the nucleotide binding pocket at 50 ns with ADP and dADP. Phe129 (magenta) and the primary contacts it makes are shown (dotted black lines). (B) Representative acting binding surface structures on myosin (circled area on ribbon structure) with ADP and dADP simulations at 50 ns, showing conformational changes in myosin resulting in increased exposure of polar residues (green regions) in the actin binding surface with dADP binding. Modeling figures adapted with permission from S.G. Nowakowski, M. Regnier, V. Daggett, unpublished data, October 2016. (C) Schematic illustrating the chemomechanical cycle of muscle contraction. Transitions between actin (A) and myosin (M) binding states are labeled. Transition states in boxes are where dATP alters the cycle, with the magnitude of the dATP effect indicated by the number of plus symbols.

Figure 5

Effects of AV-R1 + AV-R2 Treatment on ARCs (A) Contractile response of adult rat cardiomyocytes (ARCs) at different stimulation frequencies. AV-R1 + AV-R2–treated cells (triangles) showed significantly greater response to Ca²⁺ at all frequencies. AV-GFP–treated cells (open circles); nontransduced cells (solid circles). *p < 0.05 compared with nontransduced; †p < 0.05 compared with AV-GFP–treated. (B to D) R1R2 protein expression in ARCs after AV-R1 + AV-R2 treatment. Increased R1 (B) and R2 (C) protein expression in AV-R1 + AV-R2–treated neonatal rat ventricular myocytes (NRVMs). (D) Increased intracellular dATP in AV-R1 + AV-R2–treated NRVMs. *p < 0.05 compared with AV-GFP–treated NRVMs. GFP = green fluorescent protein.

Figure 6

R1R2 Overexpression Increases hESC-CM Contractility and Enhances Contractility of Coupled Cardiomyocytes (A) Example of coupled green fluorescent protein (GFP)-expressing cell and WT cell. Contractile measurements were made individually on each cell in a doublet, as well as on uncoupled WT cells in culture. (B) Representative traces showing increased contractility of AV-R1 + AV-R2–treated cells and coupled WT cells compared with control cells. AV-R1 + AV-R2–treated hESC-CMs showed (C) significantly increased magnitude of contraction compared with AV-GFP–treated cells and (D) significantly increased maximum contraction velocity without affecting relaxation velocity. *p < 0.05; N.S. = not significant. (E) Schematic showing dATP generation in transplanted R1R2-overexpressing cells (dATP “Donor” cells) and the gap junction-mediated transfer of dATP through coupled host cardiomyocytes. hESC-CM = human embryonic stem cell-derived cardiomyocyte; WT = wild-type.

Figure 7

BB-R12 Vector in Healthy Mouse Tissues (A) Schematic diagram detailing the BB-R12 (AAV6-R1.2) vector construct containing a cardiac-specific promoter (cTnT455) and human sequences of the R1 (RRM1) and R2 (RRM2) subunits separated by a self-cleaving P2A peptide linker. (B) BB-R12 vector genome biodistribution in healthy mice tissues. Quantitative polymerase chain reaction results quantifying BB-R12 vector genomes/nuclei of liver, heart (ventricle), and gastrocnemius tissues. Bars represent mean ± SD. (C) R1R2 is selectively overexpressed in cardiac tissue of healthy mice treated with BB-R12. Western blot analysis of control and BB-R12–treated mouse ventricle and liver tissue, probing for R1, R2, and a loading control (GAPDH).

Figure 8

BB-R12 Effect on LVEF in Swine Model of MI/HF Left ventricular ejection fraction (LVEF) at each time point in the 2-month study of swine myocardial infarction (MI)/heart failure (HF) for sham (MI + saline) versus BB-R12 therapy at low, medium, and high doses (n = 4 to 5 per group). *p < 0.05, **p < 0.01 compared with sham, mixed effects regression model; data are represented as mean ± SEM.

Figure 9

BB-R12 Effect on Hemodynamic Parameters in Swine Model of MI/HF Day 0 (pre-treatment) to Day 56 changes in (A) LVEF, (B) LVEDP, (C) +dP/dt, and (D) −dP/dt. Data are mean change ± SEM. *p < 0.05. Adapted with permission from Kadota et al. . LVEDP = left ventricular end-diastolic pressure; other abbreviations as in Figures 1 and 8.