Effects of PDE5 inhibition on dystrophic muscle following an acute bout of downhill running and endurance training

Abstract

Lack of sarcolemma-localized neuronal nitric oxide synthase mu (nNOSμ) contributes to muscle damage and fatigue in dystrophic muscle. In this study, we examined the effects of compensating for lack of nNOSμ with a phosphodiesterase type 5 (PDE5) inhibitor in mdx mice following downhill running and endurance training. Dystrophic mice (mdx) were treated with sildenafil citrate and compared with untreated mdx and wild-type mice after an acute bout of downhill running and during a progressive low-intensity treadmill running program (5 days/wk, 4 wk). Magnetic resonance imaging (MRI) and spectroscopy (MRS) transverse relaxation time constant (T2) of hindlimb and forelimb muscles were measured as a marker of muscle damage after downhill running and throughout training. The MRI blood oxygenation level dependence (BOLD) response and 31phosphorus MRS (31P-MRS) data were acquired after stimulated muscle contractions. After downhill running, the increase in T2 was attenuated (P < 0.05) in treated mdx and wild-type mice compared with untreated mdx. During training, resting T2 values did not change in wild-type and mdx mice from baseline values; however, the running distance completed during training was greater (P < 0.05) in treated mdx (>90% of target distance) and wild-type (100%) than untreated mdx (60%). The post-contractile BOLD response was greater (P < 0.05) in treated mdx that trained than untreated mdx, with no differences in muscle oxidative capacity, as measured by 31P-MRS. Our findings indicate that PDE5 inhibition reduces muscle damage after a single bout of downhill running and improves performance during endurance training in dystrophic mice, possibly because of enhanced microvascular function. NEW & NOTEWORTHY This study examined the combined effects of PDE5 inhibition and exercise in dystrophic muscle using high-resolution magnetic resonance imaging and spectroscopy. Our findings demonstrated that sildenafil citrate reduces muscle damage after a single bout of downhill running, improves endurance-training performance, and enhances microvascular function in dystrophic muscle. Collectively, the results support the combination of exercise and PDE5 inhibition as a therapeutic approach in muscular dystrophies lacking nNOSμ.

Keywords: Duchenne muscular dystrophy; phosphodiesterase type 5 (PDE5) inhibitor; sildenafil citrate; skeletal muscle damage.

Figures

Fig. 1.

Experimental timeline used to examine the effect of PDE5 inhibitor on downhill running and during progressive low-intensity treadmill training. MRI, magnetic resonance imaging; MRS, magnetic resonance spectroscopy; PDE5, phosphodiesterase type 5.

Fig. 2.

A: representative transverse relaxation time constant (T2)-weighted axial images (TR: 2 s; TE: 40 ms) of the upper forelimbs and lower hindlimbs of a wild-type and an untreated mdx mouse following downhill running. B: example single voxel 1H-MRS spectra acquired from the deep medial region of the lower hindlimb. C: MR T2 was calculated using a mono-exponential equation. MR, magnetic resonance; MRS, magnetic resonance spectroscopy; TE, echo time; TR, repetition time.

Fig. 3.

A: 1H-MRS 1H2O transverse relaxation time constant (T2) in upper anterior forelimb muscles before and following an acute bout of downhill running in wild-type (n = 10), mdx (n = 14), and treated mdx (mdxsil; n = 14) mice. B: 1H-MRS 1H2O T2 of the medial deep region of hindlimb muscles before and following downhill running in wild-type (n = 10), mdx (n = 14), and treated mdx (mdxsil; n = 14) mice. Paired t-test with Bonferroni correction were used to examine differences in T2 pre- and post-downhill running. *Significantly different from pre-downhill running at P < 0.05. MRS, magnetic resonance spectroscopy.

Fig. 4.

A: MRS 1H2O transverse relaxation time constant (T2) of the mdx mice measured weekly during training showed no changes in 1H2O T2 from baseline. B: percentage of prescribed distance covered by wild-type and by mdx mice that trained without treatment (mdxtrain) and with sildenafil citrate treatment (mdxtrain+sil; n = 5/group). Note that running volume during training was reduced (P < 0.05) in untreated mdx mice compared with treated mdx and wild type. Two-way analysis of variance (treatment group vs. time) with Tukey post hoc analysis were performed. *Significantly different (P < 0.05) from wild type. Values are mean (SD). MRS, magnetic resonance spectroscopy.

Fig. 5.

Total distance run by mice during the downhill running protocol performed before and after training in wild-type and mdx mice without treatment (mdxtrain) and that trained with sildenafil citrate treatment (mdxtrain+sil) and in mdx mice treated with sildenafil citrate without training (mdxsil; n = 5/group). Note that the mice in the treatment groups (mdxtrain+sil and mdxsil) were administered sildenafil citrate before the pretraining downhill run. Paired t-tests with Bonferroni correction were used for comparisons of before and after training. Values are expressed as percent of maximum distance. *Significantly different from pretraining at P < 0.05.

Fig. 6.

A: example post-contractile MRI blood oxygenation level dependence (BOLD) response following brief (2 s) tetanic stimulated contractions in an mdx mouse (timing of the stimulated contractions is depicted with arrow). B: MRI peak BOLD response in wild-type mice that trained, in mdx mice that trained without treatment (mdxtrain) and with sildenafil citrate treatment (mdxtrain+sil), and in mdx mice treated with sildenafil citrate without training (mdxsil; n = 5/group). One-way analysis of variance with Tukey multiple comparison test were used for comparisons. Values are mean (SD). *Significantly different from wild type; #significantly different from mdxtrain. MRI, magnetic resonance imaging; SI, signal intensity.

Fig. 7.

The time constant (τ) for phosphocreatine (PCr) recovery after electrically stimulated contractions (2 Hz) in wild-type mice that trained, in mdx mice that trained without treatment (mdxtrain) and with sildenafil citrate treatment (mdxtrain+sil), and in mdx mice treated with sildenafil citrate without training (mdxsil; n = 5/group). One-way analysis of variance with Tukey multiple comparison test were used for comparisons. *Significantly different from wild type.

Fig. 8.

A: representative images after Masson’s trichrome staining of the gastrocnemius in wild-type mice that trained, in mdx mice that trained without treatment (mdxtrain) and with sildenafil citrate treatment (mdxtrain+sil), and in mdx mice treated with sildenafil citrate without training (mdxsil; n = 5/group). mdx mice had significantly greater fibrosis in comparison to wild type, and no differences were observed among mdx groups. B: CD45+ cells were greater in all mdx groups compared with wild type. One-way analysis of variance with Tukey multiple comparison test were used for comparisons. *Significantly different from wild type (P < 0.05).