Chloroquine decreases cardiac fibrosis 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 decreased 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 this present study, we evaluated myocardial fibrosis and its mechanism in mdx mice, a model of DMD, and also evaluated changes in cardiac function. Results As assessed by LC3 immunohistochemistry, a small number of autophagosomes were detected in cardiomyocytes of both mdx mice and control wild-type (WT) mice. The number of autophagosomes was significantly enhanced by 4 weeks of isoproterenol-induced cardiac stress in cardiomyocytes of mdx but not WT mice. Simultaneously, isoproterenol increased cardiomyocyte fibrosis in mdx but not WT mice. Administration of chloroquine significantly decreased cardiomyocyte fibrosis 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 Heart failure in DMD patients is possibly treated with chloroquine, and the mechanism probably involves chloroquine’s anti-inflammatory effects.


Introduction
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 fibrosis occur at early ages in DMD patients.Muscle weakness results in walking difficulties, 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 who died from cardiac complications increased from 8% to 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, and fibrosis in cardiomyocytes to determine if this regulatory mechanism contributed to DMD-induced cardiomyopathy using the mdx mouse model of DMD.

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 following the guidelines of the Kyoto University Animal Committee and with prior approval from the Institutional Ethical Committee and an ARRIVE guideline.Animals were handled under the Declaration of Helsinki.When sacrificing mice for experiments, a high-concentration CO 2 container was used.All mouse experiments were performed using anesthesia to alleviate pain.
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, chloroquine was dissolved at 75 mg in 250 mL of clean water and given to mice using an automatic water dispenser.Water loss was checked every other day, and it was the same in all mouse groups.Clinical observations to monitor the side effects, activity, appetite (weight loss), and diarrhea, 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 nontreated WT and mdx mice were dissected after sacrifice and fixed 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).

Histology and immunohistochemistry
Mouse hearts were isolated, and 4%-PFA fixed-left ventricles were sliced at 7 μm and stained using a Hematoxylin-Eosin Stain Kit and Picro-Sirius Red Stain Kit (COSMO BIO, Japan).Fibrosis was imaged using a BZ-X-710 microscope (KEYENCE, Japan), and fibrotic areas in each cross-section of the left ventricle at the papillary muscle level were quantified using the automated calculation software of the BZ-X-Analyzer software (KEYENCE, Japan).Autophagosome was stained with LC3 (Santa Cruz, sc-28266) and automatically counted the number of LC3-positive dots by using a BZ-X-Analyzer software and Photoshop software (Adobe).

Statistics
All experiments used at least 3 mice in each group and were performed 3 or more times.In detail, electron microscopy evaluation, LC3 staining, and Western blotting were performed in three individual experiments from three different mice hearts.For the evaluation of fibrosis of the left ventricle of mice, we conducted experiments with five mice in each group.Regarding the echocardiogram study, experiments were conducted with five mice in each group.Statistical significance was evaluated with a one-way ANOVA followed by the Tukey-Kramer test using JMP1 Pro156 (11.0.0) software.p-values <0.05 were considered statistically significant.

Increased autophagosomes after cardiac stress in mdx mouse cardiomyocytes
To determine if autophagy occurred in DMD cardiomyocytes, we first observed autophagosomes in cardiomyocytes of non-treated 16-week-old WT mice and mdx mice using an electron microscope (Fig 2A).LC3 positive dots were detected in cardiomyocytes of both WT and mdx mice, but the numbers were small and at similar levels (Fig 2A (a) and (c)).After enhancing cardiac stress by administration of isoproterenol for 1 month, the number of LC3 positive dots increased in cardiomyocytes of both 16-week WT and mdx mice (Fig 2B (b) and (d)).However, the upregulation ratio of LC3 positive dots was dramatically enhanced by isoproterenol in cardiomyocytes of mdx mice compared with those of WT mice.Subsequently, chloroquine, an autophagy inhibitor as well as an anti-inflammatory and anti-immunosuppressive agent, was administered to mdx mice simultaneously with isoproterenol for 1 month to verify that the LC3-positive dots were autophagosomes.Administration of chloroquine diminished nearly all LC3 positive dots in 16-week mdx mouse cardiomyocytes (Fig 2B (e)).Quantitative evaluation of LC3 dots showed that the number of dots increased significantly in mdx mice cardiomyocytes after isoproterenol stress and decreased with chloroquine administration (Fig 2C).As previous papers mentioned that autophagy, assessed by LC3 positive dots, was over-activated in mdx mice [18], we performed western blotting assessing LC3-I and LC3-II to confirm the LC3 dots results.LC3-I expression was strong and LC3-II expression was weak in WT mice.The expression level ratio did not change even after the administration of isoproterenol with/without chloroquine.When LC3 expression was also evaluated in mdx mice, LC3-I expression was stronger than LC3-II expression under non-cardiac stress.After administration of isoproterenol, the expression of LC3-I and LC3-II became almost the same levels.In other words, cardiac stress reduced the expression level of LC3-I and increased the expression level of LC3-II.When an autophagy inhibitor, chloroquine, was administrated to mdx mice, the ratio of LC3-I and LC3-II expression levels did not change even after the administration of isoproterenol.We also evaluated the expression of LC3-I and LC3-II in 30-week-old mice, and the expression pattern was similar to that of WT mice (Fig 3A).Cardiomyocyte expression of P62/SQSTM-1 did not significantly change in WT mice after administration of isoproterenol or chloroquine(Fig 3A).The expression levels of LC3-I, LC3-II, and p62/SQSTM-1 were quantified.When the LC3-II/LC3-I ratio and the expression level of p62/SQSTM-1 in the cardiomyocytes of untreated WT mice were set as 1, and the ratios of other expression levels were At least the evaluation only in LC3-II, there were no significant differences between each group.These findings indicated that mdx mouse cardiomyocytes were more sensitive to cardiac stress than WT mouse cardiomyocytes.

Isoproterenol-induced cardiac stress increased cardiomyocyte fibrosis significantly in only mdx mice and was inhibited by chloroquine
To further evaluate the cardiac stress, the fibrotic area in the left ventricle (LV) was measured after 1 month of isoproterenol-induced cardiac stress.The LV fibrotic area in 16-week mdx mice was remarkably larger than that of WT mice (Fig 4A  investigate another cell death.No significant changes were observed in the expression of caspase 3 in cardiomyocytes in both WT and mdx mice before/after isoproterenol administration and after chloroquine administration (Fig 4C).These findings indicated that cardiac stress significantly induced cardiac fibrosis in mdx mice, and possibly chloroquine reduced the stressinduced cardiac fibrosis.

Chloroquine alleviated impaired cardiac contraction in mdx mice
To explore the effect of cardiac fibrosis and chloroquine on mdx mouse cardiac function, we measured the key echocardiographic markers LVEDd, LVESd, LVFS, LVEF, IVSd, and LVPWd after treatment or non-treatment with isoproterenol with or without chloroquine, as shown in Fig 1 .LVEDd, LVESd, IVSd, and LVPWd were not significantly affected by isoproterenol or chloroquine in WT and mdx mice.(Fig 5A , 5B, 5E and 5F).However, LVFS and LVEF, markers of cardiac contraction, were significantly decreased by isoproterenol treatment

Discussion/Conclusions
In the present study, we demonstrated that cardiac stress significantly increased cardiac fibrosis in the mdx mouse model of DMD and was accompanied by significantly reduced cardiac contraction.
Although cardiomyopathy is a leading cause of death in DMD patients, effective therapies to prevent or slow the progression of the disease are not available [15,16].Because the regulatory mechanisms of cardiomyopathy remain incompletely understood, current approaches to the 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 and therapeutic 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 as well as cardiac fibrosis.
Previous reports suggested that skeletal muscle autophagy is decreased in DMD patients and mdx mice [19][20][21].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 the number of autophagosomes was increased by cardiac stress with isoproterenol.In addition, cardiac fibrosis was increased by cardiac stress with isoproterenol.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.After administration of chloroquine autophagosomes dramatically decreased in cardiomyocytes of mdx mice under isoproterenol cardiac stress.If autophagy is assumed to be the main cause of cardiac fibrosis, chloroquine administration should further increase the number of autophagosomes.However, in this experiment, the number of autophagosomes decreased with chloroquine administration, suggesting the possibility that other effects of chloroquine, e.g.anti-inflammatory or antiimmunosuppressing effects, were thought to have suppressed cardiac fibrosis.Indeed, there is no solid evidence that autophagy suppresses cardiac fibrosis in DMD mice in our experiments, but considering that autophagosomes increase with cardiac stress, it seems possible that autophagy has an effect.In a recent study, there is a report regarding the association of myocardial autophagy with DMD [22].Thus, in further study, the concentration of chloroquine and the administration methods of chloroquine should be considered to elucidate the mechanisms of cardiomyopathy in DMD patients.
In clinical settings, cardiac function data are the primary criteria for monitoring cardiomyopathy in DMD patients [23].Therefore, the present study used an echocardiographic approach to evaluate cardiac function in mdx mice.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 stress impaired cardiac contractile function, which was alleviated by chloroquine.These data demonstrated that reducing cardiac fibrosis 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, a combination therapy of chloroquine and beta-blockers could be more effective for the 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].Further studies are needed to evaluate the potential therapeutic application of chloroquine in DMD cardiomyopathy.Moreover, 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 at a certain level of blood concentration, this modality could be used to treat cardiomyopathy in DMD patients.Alternatively, modalities that specifically inhibit cardiac fibrosis could be developed.
In this study, we focused primarily on autophagy.Cardiomyocyte autophagy may occur significantly in DMD model mice, but this was not clear in detailed studies using chloroquine.At the same time, it was difficult to say that apoptosis occurred significantly in DMD model mice.However, stress-induced cardiac fibrosis clearly occurred in DMD model mice, and chloroquine administration suppressed the fibrosis, suggesting that chloroquine's anti-inflammatory effects may have contributed to the fibrosis-suppressing effect.Further studies should be conducted to evaluate the contribution of other cell death mechanisms, including necrosis and mitophagy as well as the anti-inflammatory effect of chloroquine, to cardiomyocyte dysfunction in DMD [25][26][27].
In conclusion, our findings demonstrated that chloroquine effects have a potential contributing mechanism to cardiomyopathy in the context of DMD and chloroquine is a potential therapeutic modality for cardiomyopathy of DMD by reducing cardiac fibrosis.

Fig 2 .
Fig 2. Autophagosome detection in left ventricle (LV) sections, and LC3 expression in mdx mice cardiomyocytes.(A) A representative autophagosome in an mdx LV cardiomyocyte administrated isoproterenol.(Arrow), Scale bar, 100 nm.(B) LC3 positive autophagosomes in LV sections with and without isoproterenol treatment.a) WT untreated, b) WT isoproterenol-treated, c) mdx untreated, d) mdx isoproterenol-treated, e) mdx isoproterenol + chloroquinetreated.Scale bar, 50 μm.(C) Number of LC3 positive dots per 0.1mm2 in each LV section.*: p<0.05.https://doi.org/10.1371/journal.pone.0297083.g002 (a) and (b)).Interestingly, simultaneous administration of chloroquine with isoproterenol significantly inhibited LV fibrosis in 16-week mdx mice (Fig 4A (c)).The calculated fibrosis area was also significantly larger in the LV of mdx mice.The fibrotic area of mdx mice was significantly reduced after the administration of chloroquine (Fig 4B).No mice treated with chloroquine exhibited side effects during the study.Furthermore, we evaluated the expression of caspase 3 in the apoptotic pathway to