Transgenic overexpression of ribonucleotide reductase improves cardiac performance

Abstract

We previously demonstrated that cardiac myosin can use 2-deoxy-ATP (dATP) as an energy substrate, that it enhances contraction and relaxation with minimal effect on calcium-handling properties in vitro, and that contractile enhancement occurs with only minor elevation of cellular [dATP]. Here, we report the effect of chronically enhanced dATP concentration on cardiac function using a transgenic mouse that overexpresses the enzyme ribonucleotide reductase (TgRR), which catalyzes the rate-limiting step in de novo deoxyribonucleotide biosynthesis. Hearts from TgRR mice had elevated left ventricular systolic function compared with wild-type (WT) mice, both in vivo and in vitro, without signs of hypertrophy or altered diastolic function. Isolated cardiomyocytes from TgRR mice had enhanced contraction and relaxation, with no change in Ca(2+) transients, suggesting targeted improvement of myofilament function. TgRR hearts had normal ATP and only slightly decreased phosphocreatine levels by (31)P NMR spectroscopy, and they maintained rate responsiveness to dobutamine challenge. These data demonstrate long-term (at least 5-mo) elevation of cardiac [dATP] results in sustained elevation of basal left ventricular performance, with maintained β-adrenergic responsiveness and energetic reserves. Combined with results from previous studies, we conclude that this occurs primarily via enhanced myofilament activation and contraction, with similar or faster ability to relax. The data are sufficiently compelling to consider elevated cardiac [dATP] as a therapeutic option to treat systolic dysfunction.

Fig. 1.

Scheme illustrating a four-step cross-bridge model of contraction. Major transitions are labeled. Dark arrows indicate transitions that we hypothesize to be enhanced when dATP is used as a substrate for contraction rather than ATP.

Fig. 2.

Response of LV function during 20 min of acute physiological demand. (A–C) LVDevP, HR, and RPP, the product of LVDevP and HR, measured in isolated hearts perfused at baseline (BL) and dobutamine infusion (n = 5 each group). (D) Coronary flow, estimated by collecting the perfusate effluent over a 2-min period, in Langendorff heart preparations during normal workload and dobutamine infusion (n = 5 each group). (E and F) Rate of pressure change calculated by the first derivative of the LV pressure wave (dP/dt) in Langendorff heart preparations at baseline and during dobutamine infusion. The positive maximum (+dP/dt) is an index of the rate of LV pressure development. The negative maximum (−dP/dt) is an index of the rate of ventricular relaxation. *P < 0.05 vs. WT at baseline (n = 5 each group).

Fig. 3.

Energetic response to dobutamine challenge. Ratio of PCr and ATP, as measured by ³¹P NMR spectroscopy in isolated perfused hearts at baseline (BL) and at the end of 300 nM dobutamine infusion (DOB). *P < 0.05 vs. WT at baseline (n = 5 each group).

Fig. 4.

Smooth muscle contraction is not disrupted in WT mice with full exchange of dATP for ATP. An example trace from smooth muscle contraction assay is shown. Maximum force was not different between two groups: WT mouse smooth muscle strips with either 5 mM ATP or 5 mM dATP. Vertical lines before force development and immediately following plateau are noise resulting from switching calcium solutions.

Fig. 5.

Representative sarcomere length (A) and calcium (B) traces from TgRR and WT single isolated ventricular myocytes. (A) Representative sarcomere length traces obtained using video microscopy (IonOptix). WT shortening is shown in gray and TgRR is shown in black. (B) Representative Fura-2 fluorescence traces obtained during shortening. No observable difference in calcium handling was noted. WT is shown in gray and TgRR in black. Quantification of these traces (and others) can be found in Table 2.