Upregulation of Cardiomyocyte Ribonucleotide Reductase Increases Intracellular 2 deoxy-ATP, Contractility, and Relaxation

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 Ca ²⁺-mediated activation in demembranated cardiac muscle. In the current study we hypothesized that cellular [dATP] could be increased by viral-mediated over expression of the ribonucleotide reductase (Rrm1 and Rrm2) complex, which would increase contractility of adult rat cardiomyocytes. Cell length and ratiometric (fura2) Ca ²⁺ fluorescence were monitored by video microscopy. At 0.5 Hz 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 over expression, 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 over expression had no effect on minimal or maximal intracellular [Ca ²⁺] (Fura2 fluorescence), indicating increased contractility is primarily due to increased myofilament activity without altering Ca ²⁺ release from the sarcoplasmic reticulum. Additionally, functional potentiation was maintained with Rrm1+Rrm2 over expression as stimulation frequency was increased (1 Hz and 2 Hz). 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 Ca ²⁺ 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 Ca ²⁺.