In the present study, we demonstrated that autophagy is a major contributing mechanism to cardiomyopathy in the mdx mouse model of DMD, and is accompanied by reduced cardiac contraction.
Although cardiomyopathy is a leading cause of death in DMD patients, effective therapies to prevent or slow the progression of disease are not available [15, 16]. Because the regulatory mechanisms of cardiomyopathy remain incompletely understood, current approaches to treatment of cardiomyopathy are palliative therapies, including diuretics, angiotensin-converting enzyme inhibitors, and beta-blockers.
Recent reports have demonstrated that the mean lifespan of DMD patients is as high as 35 years of age, and that more than half of DMD patients die from cardiomyopathy [12, 13, 14]. To improve the prognosis of DMD-related cardiomyopathy, mechanistic studies to yield more targeted approaches to treatment are necessary. In the present study, we focused on autophagic cardiomyocyte cell death using the mdx mouse model of DMD.
Previous reports suggested that skeletal muscle autophagy is decreased in DMD patients and mdx mice. As a result of this mechanism, waste products accumulate in the cytosol, impairing skeletal muscle function [19, 20, 21]. Unexpectedly, in the present study, we identified that cardiomyocyte autophagy was accelerated in mdx mice, and that the number of autophagosomes was increased by cardiac stress with isoproterenol. In addition, cardiac fibrosis was increased by autophagy in this context. These data conflicted with prior reports of skeletal muscle autophagy in DMD. Thus, we administered the autophagy inhibitor chloroquine to mdx mice to confirm our findings. Chloroquine dramatically decreased cardiomyocyte autophagosome formation and cardiac fibrosis under isoproterenol cardiac stress in mdx mice. These findings suggest that enhanced autophagy is a potential contributor to cardiomyopathy in DMD patients, accelerating cardiomyocyte cell death. These findings were consistent with a recent study demonstrating enhanced cardiac autophagy in DMD, which supports the validity of our findings [22]. Thus, our results, together with prior studies of skeletal muscle, presumably indicate that cellular stress responses are cell type-dependent, and that targeted therapies for DMD should be tailored to the affected organ or cell type.
In clinical settings, cardiac function data are the primary criteria for monitoring cardiomyopathy in DMD patients [23]. Therefore, an echocardiographic approach was used to evaluate cardiac function in mdx mice in the present study. In DMD patients, heart failure follows hypertrophic cardiomyopathy in the early stages, characterized by thinning of the ventricular wall and dilated cardiomyopathy in the late stages [24]. In accordance with clinical parameters were evaluated by echocardiography in mdx mice. Although left ventricular wall thinning and dilatation were not clearly observed in the present study, LVFS and LVEF, indicative of cardiac contractile function, were significantly decreased in mdx mice with isoproterenol cardiac stress. The lack of left ventricular wall thinning and dilatation was likely due to the relatively short duration of our studies, as one month exposure to isoproterenol-induced cardiac stress could be too short for cardiac morphological abnormalities to develop. Nevertheless, cardiac contractile function was impaired by cardiac stress, which was alleviated by chloroquine. These data demonstrated that inhibition of autophagy positively affected cardiac function in this context. These in vivo results suggest the use of chloroquine to improve cardiac function or inhibit cardiac deterioration in DMD patients. In the present study, isoproterenol was used to induce cardiac stress by increasing heart rate. Considering this effect of isoproterenol, combination therapy of chloroquine and beta-blockers could be more effective for treatment of cardiomyopathy in DMD patients.
The clinical application of chloroquine in DMD patients could potentially impair skeletal muscle function, as previous reports have demonstrated that skeletal muscle autophagy is suppressed in DMD [19, 20, 21]. These results are not consistent with the findings of the present study, and suggest a tissue- and cell type-specific role of autophagy in the context of DMD. Further studies are needed to evaluate the potential therapeutic application of chloroquine in DMD, but this modality could potentially reduce skeletal muscle function in DMD patients. Thus, side effects in organs dependent on skeletal muscle function, including the respiratory system, should be carefully evaluated in the clinical application of chloroquine for the treatment of DMD-associated cardiomyopathy. If chloroquine does not adversely affect skeletal muscle function, this modality could be used to treat cardiomyopathy in DMD patients. Alternatively, modalities that specifically inhibit cardiomyocyte autophagy could be developed.
In this study, we focused primarily on autophagy. Cardiomyocyte apoptosis was simultaneously evaluated by using WT and mdx mice. Our findings suggested that cardiomyocyte apoptosis did not significantly differ between these mice. Further studies should be conducted to evaluate the contribution of other cell death mechanisms, including necrosis and mitophagy, to cardiomyocyte dysfunction in DMD [25, 26, 27].
In conclusion, our findings demonstrated that accelerated autophagy was a potential contributing mechanism to cardiomyopathy in the context of DMD. The autophagy inhibitor chloroquine is a potential therapeutic modality for cardiomyopathy of DMD.