Sildenafil reverses cardiac dysfunction in the mdx mouse model of Duchenne muscular dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is a progressive and fatal genetic disorder of muscle degeneration. Patients with DMD lack expression of the protein dystrophin as a result of mutations in the X-linked dystrophin gene. The loss of dystrophin leads to severe skeletal muscle pathologies as well as cardiomyopathy, which manifests as congestive heart failure and arrhythmias. Like humans, dystrophin-deficient mice (mdx mice) show cardiac dysfunction as evidenced by a decrease in diastolic function followed by systolic dysfunction later in life. We have investigated whether sildenafil citrate (Viagra), a phosphodiesterase 5 (PDE5) inhibitor, can be used to ameliorate the age-related cardiac dysfunction present in the mdx mice. By using echocardiography, we show that chronic sildenafil treatment reduces functional deficits in the cardiac performance of aged mdx mice, with no effect on normal cardiac function in WT controls. More importantly, when sildenafil treatment was started after cardiomyopathy had developed, the established symptoms were rapidly reversed within a few days. It is recognized that PDE5 inhibitors can have cardioprotective effects in other models of cardiac damage, but the present study reports a prevention and reversal of pathological cardiac dysfunction as measured by functional analysis in a mouse model of DMD. Overall, the data suggest that PDE5 inhibitors may be a useful treatment for the cardiomyopathy affecting patients with DMD at early and late stages of the disease.

Fig. 1.

Timelines of the different sildenafil treatment regimens. (A) Untreated control with echocardiographic measurements at approximately 4, 8, 12, and 15 mo of age; (B) chronic sildenafil treatment beginning at 1 mo of age for prevention analysis; (C) acute sildenafil treatment beginning at 12 mo of age for reversal analysis; and (D) 2-wk sildenafil treatment starting at 12 mo of age with successive echocardiographic measurements before, during, and after treatment (Inset, close-up of timeline with black lines representing echocardiographic time points). Sildenafil treatment started on day 0, with echocardiographic measurements on days 3, 7, and 14. Sildenafil was removed and echocardiograms acquired on days 18, 24, 31, and 40.

Fig. 2.

Left ventricular dysfunction is reduced and reversed in mdx mice. (A) Pulse-wave Doppler MPI timeline of mdx and WT mice. Sildenafil treatment prevents an increase in MPI to 15 mo and reverses the abnormal MPI when administration begins at 12 mo. (B) Tissue Doppler Ea/Aa ratio timeline. Values are means ± SE; *P < 0.05, mdx vehicle vs. WT vehicle; ^#P < 0.05, mdx after 3 mo of sildenafil treatment (15 mo old) vs. baseline at 12 mo; n = 4–12 mice/group; two-way ANOVA. (C) MPI of 12 mo-old mdx mice before, during, and after a 2-wk sildenafil treatment regimen. MPI is expressed as a percentage change from baseline before sildenafil administration. Sildenafil treatment started on day 0, with MPI acquisition on days 3, 7, and 14. Sildenafil was removed and MPI acquired on days 18, 24, 31, and 40. Values are means ± SE; *P < 0.05 for time points compared with baseline; n = 6 mice; one-way ANOVA. (D) MPI was obtained by pulse-wave Doppler imaging of mitral valve inflow (Right) from the apical four-chamber view (Left). RV, right ventricle; LV, left ventricle; RA, right atrium; LA, left atrium; IVCT, isovolumic contraction time; IVRT, isovolumic relaxation time; EV, left ventricular ejection time.

Fig. 3.

Sildenafil-mediated cardioprotection in 16-mo-old mdx hearts is associated with inactivation of GSK-3β. (A) Representative Western blots showing relative abundance of PKG, pGSK-3β, GSK-3β, and GAPDH. (B) PKG protein expression normalized to GAPDH and (C) pGSK-3β protein expression normalized to total GSK-3β; n = 7 mice/group; *P < 0.05 analyzed by unpaired t test.