Upregulation of cardiomyocyte ribonucleotide reductase increases intracellular 2 deoxy-ATP, contractility, and relaxation

F.S Korte,Jin Dai,Kate Buckley,Erik R Feest,Nancy Adamek,Michael A Geeves,Charles E Murry,Michael Regnier

doi:10.1016/j.yjmcc.2011.08.026

F.S Korte, Jin Dai + Show 6 more

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https://doi.org/10.1016/j.yjmcc.2011.08.026

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Abstract

We have previously demonstrated that substitution of ATP with 2 deoxy-ATP (dATP) increased the magnitude and rate of force production at all levels of Ca2+-mediated activation in demembranated cardiac muscle. In the current study we hypothesized that cellular [dATP] could be increased by viral-mediated overexpression of the ribonucleotide reductase (Rrm1 and Rrm2) complex, which would increase contractility of adult rat cardiomyocytes. Cell length and ratiometric (Fura2) Ca2+ fluorescence were monitored by video microscopy. At 0.5Hz stimulation, the extent of shortening was increased ~40% and maximal rate of shortening was increased ~80% in cardiomyocytes overexpressing Rrm1+Rrm2 as compared to non-transduced cardiomyocytes. The maximal rate of relaxation was also increased ~150% with Rrm1+Rrm2 overexpression, resulting in decreased time to 50% relaxation over non-transduced cardiomyocytes. These differences were even more dramatic when compared to cardiomyocytes expressing GFP-only. Interestingly, Rrm1+Rrm2 overexpression had no effect on minimal or maximal intracellular [Ca2+], indicating increased contractility is primarily due to increased myofilament activity without altering Ca2+ release from the sarcoplasmic reticulum. Additionally, functional potentiation was maintained with Rrm1+Rrm2 overexpression as stimulation frequency was increased (1Hz and 2Hz). HPLC analysis indicated cellular [dATP] was increased by approximately 10-fold following transduction, becoming ~1.5% of the adenine nucleotide pool. Furthermore, 2% dATP was sufficient to significantly increase crossbridge binding and contractile force during sub-maximal Ca2+ activation in demembranated cardiac muscle. These experiments demonstrate the feasibility of directly targeting the actin–myosin chemomechanical crossbridge cycle to enhance cardiac contractility and relaxation without affecting minimal or maximal Ca2+. This article is part of a Special issue entitled "Possible Editorial".

Highlights

Systolic and/or diastolic cardiac function is compromised in a number of cardiovascular diseases including myocardial infarction, ischemia/reperfusion injury, diabetes, high blood pressure and hypertrophic and dilated cardiomyopathy
Transudation with recombinant adenovirus containing appropriate coda constructs driven by the CMV promoter was used to induce over expression of muscle rib nucleotide reeducates 1 (Rrm1) and 2 (Rrm2) in cultured adult and neonatal rat cardiomyocytes
For our current study in intact cardiomyocytes we did not expect over expression of Rrm1+Rrm2 to result in high levels of dATP

Summary

Introduction

Systolic and/or diastolic cardiac function is compromised in a number of cardiovascular diseases including myocardial infarction, ischemia/reperfusion injury, diabetes, high blood pressure and hypertrophic and dilated cardiomyopathy. These pathophysiological conditions often involve alterations in the Ca2+ cycle[1], β-adrenergic responsiveness[2], and/or the contractile apparatus of cardiomyocytes[3, 4]. We have previously shown that replacing ATP with 2 deoxy-ATP (dATP) as the substrate for contraction of demembranated cardiac muscle increased isometric force and the rate of force development and shortening at all levels of Ca2+ activation, including saturating [Ca2+] (pCa 4.0) [6,7,8,9]. Replacement of ATP with dATP offers the potential to improve contraction independent of changes in [Ca2+]i or adrenergic signaling

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