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 overexpression 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 0.5Hz 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 [Ca(2+)], 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 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 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+). This article is part of a Special issue entitled "Possible Editorial".

Figure 1. Representative traces and data summary

Representative cell length traces (a) and Ca²⁺ transients (b, Fura-2 fluorescence) of non-transducer (black), GFP-only (green), and Rrm1+Rrm2+GFP (red) transducer cardiomyocytes. Percentage change in contractile (c) and Ca²⁺ transient (d) properties of GFP-only and Rrm1+Rrm2+GFP transducer myocytes, stimulated at 0.5 Hz, as compared to non-transducer myocytes. V_short = velocity of shortening; FS = fractional shortening; V_rel = maximal relaxation velocity; RT_50,90 = time to 50% and 90% relaxation, respectively; FL = fluorescence; DT_50,90 = time to 50% and 90% Ca²⁺ decay, respectively *p<0.05 as compared to Non-Transducer.

Figure 2. Effect of stimulation frequency on contractile properties

Rrm1+Rrm2 transducer myocytes (open triangles) respond similarly to stimulation frequency as GFP-only transduce open circles) and non-transducer myocytes (closed circles) but show elevated fractional shortening (a) and shortening velocity (b) at all frequencies. Relaxation velocity (c) and time to 90% relaxation (d) are also similar between groups, with time to relaxation shortening as stimulation frequency increases. * = p<0.05 as compared to Non-Transducer, † = p<0.05 as compared to GFP, ‡ = p<0.05 as compared to 0.5 Hz for all groups.

Figure 3. Effect of stimulation frequency on Ca²⁺ handling properties

Rrm1+Rrm2 transducer myocytes (open triangles) respond similarly to stimulation frequency as non-transducer myocytes (closed circles) in minimal (a) and maximal (b) fluorescence, while GFP-only transducer myocytes (closed circles) showed a greater increase in both as frequency increased. As with cardiomyocyte relaxation, Ca²⁺ transient decay time (DT) to 50% (c) and 90% (d) is shortened with increased stimulation frequency, but both are dramatically shortened in R1R2 transducer cardiomyocytes. * = p<0.05 as compared to Non-Transducer, † = p<0.05 as compared to GFP, ‡ = p<0.05 as compared to 0.5 Hz for all groups.

Figure 4. Contractile Responsiveness

Contractile response as assessed as fractional shortening divided by maximal fura fluorescence (peak Ca²⁺) indicates Rrm1+Rrm2 transducer cardiomyocytes (open triangles) are significantly more responsive to Ca²⁺ at all stimulation frequencies, while GFP-only transducer cardiomyocytes (open circles) are less responsive to Ca²⁺ only at 2Hz stimulation frequency as compared to non-transducer cardiomyocytes (closed circles). * = p<0.05 as compared to Non-transducer, † = p<0.05 as compared to GFP.

Figure 5. Increased Rrm and dATP

(a) Western blot of Rrm1 transducer neonatal rat cardiomyocytes probed with anti-Rrm1 antibody indicates a >24-fold increase in Rrm1. (b) Western blot of Rrm2 transducer neonatal rat cardiomyocytes probed with anti-Rrm2 antibody indicates a > 46-fold increase in Rrm2. (c) Rrm1+Rrm2 over expression significantly increased intracellular [dATP] by >10-fold in neonatal rat cardiomyocytes as assessed by HPLC analysis. * = p<0.05 as compared to GFP transducer cardiomyocytes.

Figure 6. Nucleotide binding and actin-myosin dissociation

Rapid kinetic measurements of nucleotide binding and actin-myosin dissociation of mouse cardiac myosin taken at 10°C (top) and 20°C (bottom). There was no difference in k_obs between ATP and dATP at any [NTP] at either temperature.

Figure 7. Isometric force increases with 2% dATP

Isometric force development of demembranated cardiac trabeculae activated with 2% dATP, 98% ATP vs. 100% ATP (5 mM [NTP] total). (a) Force trace with pica 5.6 activation. (b) Summary of % increase in force with 2% dATP in activation solutions for sub-maximal (pica 5.6), but not maximal (pica 4.0) Ca²⁺ activation.