Chloroquine Decreases Cardiomyocyte Autophagy and Improves Cardiac Function in a Mouse Model of Duchenne Muscular Dystrophy

Background: Duchenne muscular dystrophy (DMD), a severe degenerative skeletal and cardiac muscle disease, has a poor prognosis, and no curative treatments are available. Because autophagy has been reported to contribute to skeletal muscle degeneration, therapies targeting autophagy are expected to improve skeletal muscle hypofunction. However, the role of this regulatory mechanism has not been evaluated clearly in DMD cardiomyocytes. Methods: In the present study, we demonstrated that autophagy was enhanced in the cardiomyocytes of mdx mice, a model of DMD, and that increased autophagy contributed to the development of cardiomyopathy in this context. Results: As assessed by GFP-mRFP-LC3 transfection, autophagosomes were more abundant in cardiomyocytes of mdx mice compared with control wild-type (WT) mice. The number of autophagosomes was signicantly enhanced by isoproterenol-induced cardiac stress (4 weeks) in cardiomyocytes of mdx but not WT mice. Simultaneously, isoproterenol increased cardiomyocyte brosis in mdx but not WT mice. Administration of chloroquine, an autophagy inhibitor, signicantly decreased cardiomyocyte autophagy and brosis in mdx mice, even after isoproterenol treatment. Left ventricle size and function were evaluated by echocardiography. Left ventricular contraction was decreased in mdx mice after isoproterenol treatment compared with control mice, which was alleviated by chloroquine administration. Conclusions: These ndings suggested that heart failure of DMD could be associated with autophagy. Therefore, autophagy inhibitors, such as chloroquine, are a potential therapeutic modality for heart failure in DMD patients.


Background
Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy, and is caused by mutations in the gene encoding dystrophin located on chromosome Xp21 [1,2,3]. DMD is inherited in an autosomal recessive manner and is relatively common, with an incidence of approximately 1 per 3,500 male births [4,5].
Muscle degeneration and subsequent brosis occur at early ages in DMD patients. Muscle weakness results in walking di culties, and ultimately in the development of respiratory muscle failure and heart failure. Respiratory and heart failure are common lethal complications of DMD, and frequently affect patients in their late teens and early twenties [1]. Recently, ventilator support devices, such as home ventilators, have been developed, prolonging the mean lifespan of ventilated DMD patients to over 35 years [6,7,8,9,10,11]. Therefore, cardiomyopathy is now the leading cause of death in DMD patients.
The percentage of DMD patients that died from cardiac complications increased from 8-44% after the development of home ventilation devices in the 1990s [12].
Fibrosis-associated cardiomyopathy generally leads to dilated cardiomyopathy. More than 80% of DMD patients older than 18 years have reduced cardiac function, and 90% of DMD patients develop dilated cardiomyopathy [13,14]. General therapeutic protocols for dilated cardiomyopathy secondary to DMD are not curative, e.g. combinations of diuretics, vasodilators, and beta-blockers [15,16]. To develop more targeted approaches to treatment, it is crucial to delineate the regulatory mechanisms of cardiomyopathy in DMD. A recent report suggested that cardiomyocyte apoptosis contributes to DMD-induced cardiomyopathy in an in vitro study using induced pluripotent stem cells (iPSCs) [17]. In the present study, we focused on another type of cell death, autophagy, to determine if this regulatory mechanism contributed to DMD-induced cardiomyopathy using the mdx mouse model of DMD.

Materials And Methods
Experimental animals C57BL/6 wild-type (WT) mice were purchased from CLEA Japan, Inc. (Japan). Mdx mice, a model of DMD, were bred at Kyoto University. All animal procedures were conducted in accordance with the guidelines of the Kyoto University Animal Committee and with prior approval from the Institutional Ethical Committee and an ARRIVE guideline. Animals were handled in accordance with the Declaration of Helsinki.
To increase cardiac load, capsulated isoproterenol was inserted subcutaneously from 12 to 16 weeks in both WT and mdx mice for a dosage of 0.5 mg/kg/day. For chloroquine treatment, capsulated chloroquine was subcutaneously inserted simultaneously with isoproterenol for a dosage of 0.4 mg/kg/day. Clinical observations to monitor the side effects of all experiments were recorded every other day. Echocardiography was performed at 16 weeks of age, as was histological evaluation. A summarized animal protocol is shown in Fig. 1.

Electron microscopy
For transmission electron microscopy analysis, left ventricular sections from 16 week non-treated WT and mdx mice were dissected after sacri ce and xed for 1 h in a pH 7.4 solution containing 4% paraformaldehyde (PFA) and 0.5% glutaraldehyde in 0.1 M cacodylate buffer. Ultra-thin sections were cut at a thickness of 7 µm and embedded on glass slides for observation with an H-7650 electron microscope (Hitachi, Ltd., Japan).

Statistics
All experiments were performed at least three times, and statistical signi cance was evaluated with a one-way ANOVA followed by the Tukey-Kramer test using JMP® Pro156 (11.0.0) software. p-values < 0.05 were considered statistically signi cant.

Increased autophagy in mdx mouse cardiomyocytes
To determine if autophagy occurred in DMD cardiomyocytes, we rst observed autophagosomes in cardiomyocytes of non-treated 16-week-old WT mice and mdx mice using an electron microscope. ( Fig. 2A) Abundant GFP-mRFP-LC3 positive dots, representing autophagosomes, were present in mdx mouse cardiomyocytes. By contrast, sparse GFP-mRFP-LC3 positive dots were detected in WT mouse cardiomyocytes. (Fig. 2B (a and c)) After enhancing cardiac stress by administration of isoproterenol for 1 month, the number of GFP-mRFP-LC3 positive dots increased in cardiomyocytes of 16 week WT and mdx mice. (Fig. 2B (b and d)) However, the upregulation ratio of GFP-mRFP-LC3 positive dots was dramatically enhanced by isoproterenol in cardiomyocytes of mdx mice compared with those of WT mice. Subsequently, chloroquine, an autophagy inhibitor, was administered to mdx mice simultaneously with isoproterenol for 1 month to verify that the GFP-mRFP-LC3 positive dots were autophagosomes. Administration of chloroquine diminished nearly all GFP-mRFP-LC3 positive dots in 16 week mdx mouse cardiomyocytes. (Fig. 2B (e)) As previous papers mentioned that autophagy was over activated in mdx mouse, we performed western blotting assessing LC1 and LC3. LC1 and LC3 were not clearly detected in heart sections of both BL/7 control mice and mdx mice without cardiac stress by isoproterenol [18]. (Supplementary Fig. 1) These ndings indicated that autophagy occurred constitutively in mdx mouse cardiomyocytes, and that mdx cardiomyocytes were more sensitive to cardiac stress than WT cardiomyocytes.
Isoproterenol-induced cardiac stress increased cardiomyocyte brosis signi cantly in only mdx mice and was inhibited by chloroquine To further evaluate the downstream effects of cell death, the brotic area in the left ventricle (LV) was measured after 1 month of isoproterenol-induced cardiac stress. The LV brotic area in 16 week mdx mice was remarkably larger than that of WT mice. (Fig. 3A (a and b)) Interestingly, simultaneous administration of chloroquine with isoproterenol signi cantly inhibited LV brosis in 16 week mdx mice. (Fig. 3A (c)) The calculated brosis area was also signi cantly larger in the LV of mdx mice. No mice treated with chloroquine exhibited side effects during the study. These ndings indicated that cardiac stress signi cantly induced cardiac regeneration in mdx mice by accelerated autophagy.

Chloroquine alleviated impaired cardiac contraction in mdx mice
To explore the role of autophagy in mdx mouse cardiac function, we measured the key echocardiographic markers lLVDd, LVDs, LVFS, LVEF, IVSd, and LVPWd after treatment or non-treatment with isoproterenol with or without chloroquine, as shown in Fig. 1. LVDd, LVDs, IVSd, and LVPWd were not signi cantly affected by isoproterenol or chloroquine in WT or mdx mice. (Fig. 4A, B, E, F) However, LVFS and LVEF, markers of cardiac contraction, were signi cantly decreased by isoproterenol treatment in mdx mice. Reductions of LVFS and LVEF in mdx mice were alleviated by simultaneous administration of chloroquine with isoproterenol. (Fig. 4C, D) Major well-characterized side effects of chloroquine, including vomiting and weight-loss, were not observed in WT or mdx mice. These results indicated that inhibiting autophagy with chloroquine protected cardiac function in isoproterenol-stressed mdx mice.

Discussion
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 identi ed that cardiomyocyte autophagy was accelerated in mdx mice, and that the number of autophagosomes was increased by cardiac stress with isoproterenol. In addition, cardiac brosis was increased by autophagy in this context. These data con icted with prior reports of skeletal muscle autophagy in DMD. Thus, we administered the autophagy inhibitor chloroquine to mdx mice to con rm our ndings. Chloroquine dramatically decreased cardiomyocyte autophagosome formation and cardiac brosis under isoproterenol cardiac stress in mdx mice. These ndings suggest that enhanced autophagy is a potential contributor to cardiomyopathy in DMD patients, accelerating cardiomyocyte cell death. These ndings were consistent with a recent study demonstrating enhanced cardiac autophagy in DMD, which supports the validity of our ndings [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 signi cantly 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 ndings of the present study, and suggest a tissue-and cell typespeci c 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 speci cally 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 ndings suggested that cardiomyocyte apoptosis did not signi cantly 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 ndings 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.

Declarations
Ethics approval and consent to participate: All animal procedures were conducted in accordance with the guidelines of the Kyoto University Animal Committee and with prior approval from the Institutional Ethical