Sildenafil and cardiomyocyte-specific cGMP signaling prevent cardiomyopathic changes associated with dystrophin deficiency

Abstract

We recently demonstrated early metabolic alterations in the dystrophin-deficient mdx heart that precede overt cardiomyopathy and may represent an early "subclinical" signature of a defective nitric oxide (NO)/cGMP pathway. In this study, we used genetic and pharmacological approaches to test the hypothesis that enhancing cGMP, downstream of NO formation, improves the contractile function, energy metabolism, and sarcolemmal integrity of the mdx heart. We first generated mdx mice overexpressing, in a cardiomyocyte-specific manner, guanylyl cyclase (GC) (mdx/GC(+/0)). When perfused ex vivo in the working mode, 12- and 20-week-old hearts maintained their contractile performance, as opposed to the severe deterioration observed in age-matched mdx hearts, which also displayed two to three times more lactate dehydrogenase release than mdx/GC(+/0). At the metabolic level, mdx/GC(+/0) displayed a pattern of substrate selection for energy production that was similar to that of their mdx counterparts, but levels of citric acid cycle intermediates were significantly higher (36 +/- 8%), suggesting improved mitochondrial function. Finally, the ability of dystrophin-deficient hearts to resist sarcolemmal damage induced in vivo by increasing the cardiac workload acutely with isoproterenol was enhanced by the presence of the transgene and even more so by inhibiting cGMP breakdown using the phosphodiesterase inhibitor sildenafil (44.4 +/- 1.0% reduction in cardiomyocyte damage). Overall, these findings demonstrate that enhancing cGMP signaling, specifically downstream and independent of NO formation, in the dystrophin-deficient heart improves contractile performance, myocardial metabolic status, and sarcolemmal integrity and thus constitutes a potential clinical avenue for the treatment of the dystrophin-related cardiomyopathies.

Fig. 1.

Functional and physiological parameters of 12- and 20-week-old isolated working mdx and mdx/GC^+/0 mouse hearts. Data are means ± SEM of mdx (solid bars) and mdx/GC^+/0 (open bars) heart perfusion experiments (n = 4 mice per group). Values shown represent averages for the 20- to 30-min perfusion period. *, P < 0.05; **, P < 0.001 mdx vs. mdx/GC^+/0 mouse hearts. LVEDP, left ventricular end-diastolic pressure; RPP, rate-pressure product.

Fig. 2.

Flux parameters relevant to citrate synthesis and mitochondrial citric acid cycle (CAC) intermediates in isolated working mdx and mdx/GC^+/0 mouse hearts. Data are means ± SEM of perfusion experiments performed in isolated working mdx (solid bars) and mdx/GC^+/0 (open bars) mouse hearts (n = 4 per group). Procedures for the determination of (A) the contribution of carbohydrates (lactate, pyruvate, and glucose) and exogenous fatty acids (oleate) to acetyl-CoA formation for citrate synthesis (CS) via pyruvate decarboxylation (PDC) and β-oxidation (OLE), respectively referred to as PDC/CS and OLE/CS and (B) tissue levels of CAC intermediates are described in Materials and Methods. *, P < 0.05 mdx vs. mdx/GC^+/0 mouse hearts.

Fig. 3.

Heart rate responses and quantitation of in vivo cardiomyocyte sarcolemmal injury induced by an acute increase in cardiac mechanical stress after isoproterenol infusion in mdx and mdx/GC^+/0 mice. Values are means ± SEM of four to six experiments in mdx (solid bars) or mdx/GC^+/0 (open bars) mouse hearts. Heart rate was continuously monitored by a single-lead electrocardiogram (A), and cardiomyocyte sarcolemmal injury was evaluated by using the Evans blue vital dye (B). ***, P < 0.001 for mdx vs. mdx/GC^+/0 mouse hearts.

Fig. 4.

Changes in gene expression levels for atrial natriuretic factor (anf) and the α₁ subunit of soluble guanylyl cyclase (sgcα₁) in placebo vs. sildenafil-treated mdx mice. Data are means ± SEM of eight freeze–clamped placebo (solid bars) and sildenafil-treated (open bars) mdx mouse hearts. mRNA levels were normalized to total myocardial RNA. Statistical analyses were performed on raw data. *, P < 0.05 for placebo vs. sildenafil-treated mdx mouse hearts.

Fig. 5.

Heart rate responses and in vivo cardiomyocyte sarcolemmal injury induced by an acute increase in cardiac mechanical stress in placebo vs. sildenafil-treated mdx mice. Values are means ± SEM of four to six experiment in placebo (solid bars) or sildenafil-treated (open bars) mdx mouse hearts, as described in Fig. 3. *, P < 0.05; **, P < 0.01, #, P < 0.001 for placebo vs. sildenafil-treated mdx mouse hearts.