Dual Therapy Deflazacort/Doxycyclyne Is Better Than Deflazacort Monotherapy to Alleviate Cardiomyopathy in Dystrophin-Deficient mdx Mice

Abstract

Cardiomyopathy related to the absence of dystrophin is an important feature in Duchenne muscular dystrophy (DMD) and in the mdx mouse. Doxycycline (DOX) could be a potential therapy for mdx skeletal muscles dystrophy. We investigated whether the corticoid deflazacort (DFZ) plus DOX could improve cardiac mdx dystrophy better than DFZ alone, later (17 months) in dystrophy. Mdx mice (8 months old) received DFZ/DOX or DFZ for 9 months. The combined therapy was greater than DFZ in reducing fibrosis (60% decrease with DFZ/DOX and 40% with DFZ alone) in the right ventricle and transforming growth factor β levels (6.8 ± 3.2 in untreated mdx mice, 2.8 ± 1.4 in combined therapy, and 4.6 ± 1.7 in DFZ; P < .05). Combined therapy more effectively ameliorated cardiac dysfunction (electrocardiogram [ECG]) than DFZ. Improvements were seen in the cardiomyopathy index (0.8 ± 0.1 in combined therapy and 1.0 ± 0.2 in DFZ), heart rate (418 ± 46 bpm in combined therapy and 457 ± 29 bpm in DFZ), QRS interval (11.3 ± 2 in combined therapy and 13.6 ± 1 in DFZ), and Q wave amplitude (−40.7 ± 21 in combined therapy and −90.9 ± 36 in DFZ). Both therapies decreased markers of inflammation (tumor necrosis factor α, nuclear factor κB, and metalloproteinase 9). DFZ/DOX improved mdx cardiomyopathy at this stage of the disease, supporting further clinical investigations.

Keywords

calsequestrin cardiac muscle ECG TGF-β 4-HNE

Introduction

Duchenne muscular dystrophy (DMD) is an incurable myopathy that primarily affects boys, with approximately 20 000 new cases worldwide annually.¹ Loss of dystrophin, the protein that binds the cytoskeleton of skeletal and cardiac muscle fibers to the extracellular matrix,² is the main cause of DMD. Deficiency of dystrophin leads to cardiomyocyte damage and inflammation, resulting in progressive myocardial fibrosis and dilated cardiomyopathy.^3,4 Because life expectancy can be prolonged through mechanical respiratory support, addressing dystrophic cardiomyopathy is important,⁵ given that almost all patients will show cardiac damage by the end of their life. Cardiomyopathy is often the main cause of death in patients with DMD.^6

–9 Although no cure is currently available for DMD, glucocorticoid (GC) therapy with deflazacort (DFZ), a derivative of prednisolone, is used to slow the progression of muscle debility.^10,11 Nevertheless, the beneficial effects of GC therapy on cardiac muscle are controversial,^8,12
–14 and new pharmacological strategies with ready-to-use compounds are of marked importance in DMD cardiomyopathy.

We previously showed that a combined treatment of DFZ and the tetracycline analog doxycycline (DOX) at the early onset of the disease ameliorated myonecrosis in the mdx mouse model for DMD.¹⁵ Considering that DMD therapy is long lasting and that cardiac alterations are seen later in dystrophy progression, in the present study, we examined whether long-term treatment with DFZ and DOX can alleviate dystrophic cardiomyopathy in aged (17 months of age) mdx mice. We evaluated functional (electrocardiogram), morphological (areas of inflammation, fibrosis, and myonecrosis), and molecular (metalloproteinase 9 [MMP-9], tumor necrosis factor α [TNF-α ], nuclear transcription factor κB [NF-κB], transforming growth factor β [TGF-β], calcium-binding protein calsequestrin, and the oxidative stress marker 4-HNE) aspects of dystrophy, at a later stage (17 months) of the disease, when mdx pathology more closely resembles the human disease.

Materials and Methods

Animals and Treatment

Male (n = 8) and female (n = 7) dystrophic mdx (C57BL/10-Dmd^mdx/PasUnib) and nondystrophic (C57BL/10ScCr/PasUnib) 17-month-old mice from our institutional animal care facility received food and water ad libitum. All experiments were performed in accordance with the Brazilian College for Animal Experimentation guidelines (protocol # 2662-1). The experimental groups were as follows: (1) C57BL/10 control (n = 13), (2) untreated mdx (n = 15), (3) Deflazacort (DFZ) treated (Libbis, Brazil; 1.2 mg/kg body weight/day in drinking water; n = 15), and (4) DFZ and doxycycline (DOX) combined treatment (DFZ at 1.2 mg/kg body weight/day plus DOX; Sandoz, Brazil; at 6.0 mg/mL in drinking water; n = 15). Each experimental group contained about half males and half females. No differences were observed regarding gender.¹⁶ The dosages and duration (9 months) of the treatment were based on previous studies.^16
–18 The ages of 8 and 17 months allowed the analysis of the effects of the drug therapy on inflammatory and fibrosis pathways and on functional changes in the dystrophic heart.

Electrocardiography

Electrocardiography (ECG) was performed as reported before.¹⁹ In short, heart function was evaluated (n = 9 for each group) at 17 months of age. Mice were anesthetized with 50.0 mg/kg ketamine hydrochloride (Francotar; Virbac, São Paulo, Brazil) and 5.0 mg/kg xylazine hydrochloride (2% Virbaxyl; Virbac). Three needle electrodes were inserted subcutaneously at the ventral junction between the chest and the right and left forelimbs and at the junction between the lower abdomen and left hind limb. All measurements were made by a blinded observer. The ECG parameters were analyzed based on lead I tracings (acquisition system PL3504/P PowerLab 4/35; AD Instruments, AU). Averaged ECG parameters were obtained from continuous 5 minutes recording used to determine heart rate; PR, QRS, and QT intervals; and the amplitudes of the waves. The cardiomyopathy index was evaluated given that previous studies have shown a significant increase in this index in the old mdx.²⁰ Clinical studies have also used this index to verify subclinical cardiac alterations in patients with DMD.^21,22 The cardiomyopathy index was evaluated by dividing the QT interval by the PQ segment (QT/PQ).^19,20

Histopathological and Morphometric Analysis: Areas of Inflammation, Fibrosis, and Myonecrosis

For histopathological and morphometric analysis, C57BL/10 (n = 3), untreated mdx (n = 5), DFZ treated (n = 5), and DFZ/DOX treated (n = 5) mdx mice were used. After 9 months of treatment, mice were anesthetized with a mixture of ketamine hydrochloride (130 mg/kg; Francotar, Virbac) and xylazine hydrochloride (6.8 mg/kg; 2% Virbaxyl, Virbac). The hearts were dissected out, snap frozen in isopentane, cooled in liquid nitrogen, and stored at −80°C. Cryostat transverse cross-sections (8-µm-thick) were cut from the midportion of the heart at the level of the papillary muscles. Inflammatory areas were identified by hematoxylin–eosin staining, and fibrosis was identified using Masson trichrome stain.¹⁶ The total cross-sectional area and the fibrosis and inflammatory areas from the entire section were measured in the heart muscle using a microscope (Nikon Eclipse, Nikon Instruments Inc, Melville, New York) with 10× magnification attached to a personal computer. The means of the inflammatory area and of the fibrosis area were expressed as the percentage of the mean total cross-sectional area.¹⁶ Some sections were labeled with anti-mouse immunoglobulin G (IgG; whole molecule) fluorescein isothiocyanate conjugate antibody developed in goats (F-0257; Sigma; 1:100) to verify myonecrosis as reported earlier.²³ The density of positively labeled patches of fibers was quantified with a hand counter, and the results are expressed as patches per mm.^2,24 All measurements were made by a blinded observer.

Western Blot Analysis

The MMP-9, TNF-α, NF-kB, TGF-β (profibrotic factor), calcium-binding protein calsequestrin (CSQ2), and 4-HNE protein adducts were quantified as described elsewhere.¹⁵ In short, mice (n = 10 for each group) were anesthetized, their muscles were frozen, and lysed in an assay lysis buffer. Protein homogenate (60 μg) was run on 8% to 15% sodium dodecyl sulfate-polyacrylamide gels and transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, California). Membranes were incubated with primary antibodies followed by proper peroxidase-conjugated secondary antibodies and developed using the SuperSignal West Pico Chemiluminescent Substrate kit (Pierce Biotechnology, Rockford, Illinois). Blots were stripped and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as described before.¹⁵ Immunoblotting bands were captured (G: Box iChemi Camera; Syngene, Cambridge, United Kingdom) and quantified using analysis software (GeneTools, version 4.01; Syngene). The primary antibodies used were MMP-9 (goat polyclonal anti-MMP-9; R&D Systems, Minnesota,), TNF-α (rabbit anti-mouse polyclonal anti-TNF-α; Merck Millipore, Massachusetts), NF-kB (goat polyclonal anti-NF-kB p65: sc-372; Santa Cruz Biotechnology, California), TGF-β (mouse monoclonal anti-TGF-β; Sigma-Aldrich, Missouri), CSQ2 (rabbit polyclonal anti-CSQ2; Thermo Scientific, Massachusetts), 4-HNE (sc-130083; Santa Cruz Biotechnology), and GAPDH (rabbit polyclonal; Santa Cruz Biotechnology). The corresponding secondary antibody used was peroxidase-labeled, affinity-purified mouse, rabbit, or goat IgG antibody (Peroxidase-labeled affinity purified IgG; KPL, Maryland).

DFZ/DOX Toxicity

To examine whether treatment with DFZ/DOX induced kidney damage, the total protein content of urine samples (n = 6) was analyzed using a commercially available kit (Urisys 1100 Combur¹⁰ Test UX; Roche, GE, USA). Liver samples from C57BL/10, untreated mdx, DFZ-, and DOX/DFZ-treated mice (n = 5) for each group were processed for histopathology and hematoxylin and eosin (H&E) staining.

Statistical Analysis

All analyses were performed using BioEstat 5.0 (PA, Brazil). Data were expressed as the mean ± standard deviation. Normality was assessed for each measurement using the Kolmogorov-Smirnov normality test. All measurements were normal. Analysis of variance (ANOVA) was used to compare means among groups (C57BL/10, untreated-mdx, DFZ, and DFZ/DOX). Statistical comparisons between the 4 groups for each condition were determined by 1-way, except the comparison between the right and left ventricle, which was determined by 2-way ANOVA, followed by Bonferroni post hoc for multiple comparisons. In all analyses, P values were considered statistically significant only if they were less than .05.

Results

Functional Analysis

While both therapies ameliorated the ECG parameters, the combined therapy was more efficient than DFZ alone in changing ECG parameter values. Polyphasic R-waves (Figure 1) were present in approximately 76% of the untreated mdx mice, in 22% of the DFZ, and in 11% of the DFZ/DOX treated mdx mice (Figure 1). Greater changes in the amplitudes of the PQRS complex were detected in the untreated mdx than in normal mice (Figure 2); DFZ/DOX improved the amplitudes of the Q and R waves (Figure 2). The S/R ratio was 9% in untreated mdx mice, representing an 82% decrease in C57BL/10 mice (P < .05, S/R ratio: 50%; Figure 3). The PR interval lengths were significantly shorter in the untreated mdx than in the C57BL/10 mice at 17 months of age (Figure 3). The PR interval length was 37.0 ± 4.4 in controls, 36.6 ± 4.3 in DFZ/DOX, and 30.5 ± 5.0 in DFZ (P < 0.05). The QRS interval in the combined therapy was similar to that observed in the control mice (Figure 3). Combined therapy decreased the cardiomyopathy index (0.81 ± 0.13 with combined vs. 1.04 ± 0.25 with DFZ), which was significantly increased in untreated mdx mice (1.58 ± 0.34) compared to normal mice (0.58 ± 0.09; Figure 3).

Figure 1.

Characteristic lead I electrocardiogram (ECG) tracings from 17-month-old C57BL/10, untreated mdx, DFZ, and DFZ/DOX mice. Note the changes in PR interval, suggesting alterations in conduction through the atrioventricular node. Approximately 76% of the untreated mdx mice had polyphasic R waves.

Figure 2.

Quantitative evaluation of P-, Q-, R-, S-, and T-wave amplitudes from lead I electrocardiogram (ECG) tracings in C57BL/10, untreated mdx, DFZ, and DFZ/DOX therapy mdx mice. The S/R wave ratio is expressed as the amplitude of the S wave as a percentage of the respective R wave. *Significantly different from C57BL/10. §Significantly different from untreated mdx. #Significantly different from DFZ (P < .05, analysis of variance [ANOVA]).

Figure 3.

Quantitative evaluation of heart rate (HR), PR interval, QRS duration, QT interval (QT), QT interval corrected by the heart rate (QTc), and the cardiomyopathy index (C. Index calculated as the value of the QT interval divided by the PQ segment) from lead I tracings. *Significantly different from C57BL/10. §Significantly different from untreated mdx. #Significantly different from DFZ. (P <.05, analysis of variance [ANOVA]).

Histopathology

Histopathological analysis showed areas of inflammation and areas of fibrosis in the hearts of DFZ-treated mdx mice (Figure 4). In 17-month-old mice, fibrosis levels seemed higher than inflammation levels; this observation was supported by the morphometric quantification, which showed lower levels of inflammation (5% inflammation in mdx heart total area) than fibrosis (approximately 14% fibrosis in mdx heart total area; P < .05). The right ventricle presented more fibrosis and inflammation than the left ventricle (Figure 4). Combined therapy reduced inflammation at rates comparable to DFZ alone (82% reduction with DFZ/DOX compared to 66% reduction with DFZ alone; Figure 4). Fibrosis was more efficiently reduced by the combined therapy (DFZ/DOX; 58% reduction in total fibrosis area) than by monotherapy (DFZ; 34% reduction in total fibrosis area).

Figure 4.

Cardioprotection by dual therapy with deflazacort (DFZ) and doxycycline (DOX) in dystrophin-deficient hearts after 9 months of treatment. Representative transversal histological sections of heart from C57BL/10, untreated-mdx, DFZ, and DFZ/DOX mdx mice (A). Fibrosis (Masson trichrome; green) is observed among the cardiomyocytes in whole heart sections (A) and in histological sections (B). Inflammation (shown by asterisk) is observed in the hematoxylin and eosin (H&E)-stained section (B). Immunofluorescence showed patches of cardiomyocytes positively labeled with immunoglobulin G (IgG; shown by asterisk), indicative of muscle damage (B). Quantitative analysis (bar graphs) revealed that heart histopathological features improved significantly in mdx mice treated with DFZ/DOX (C). Quantitative evaluation of the heart histopathology from C57BL/10, untreated-mdx, DFZ, and DFZ/DOX. LV indicates left ventricle; RV, right ventricle. *Significantly different from C57BL/10. §Significantly different from untreated mdx. #Significantly different from DFZ. **Significantly different from LV (P < .05, analysis of variance [ANOVA]). Data are presented as the mean ± standard deviation (SD). Scale bar (shown only in DFZ/DOX IgG): 40 μm.

Molecular Analysis

The levels of TNF-α, NF-kB, and MMP-9 and the levels of the pro-fibrotic marker (TGF-β) were significantly decreased by both therapies (Figure 5). The combined therapy was better than DFZ alone in reducing TGF-β. Calsequestrin was downregulated in the untreated mdx mice to a greater degree than in the control mice (Figure 5), and DFZ/DOX returned CSQ2 to almost normal levels (Figure 5), leading to a better result than DFZ therapy alone. The combination DFZ/DOX was also more efficient in reducing the oxidative stress marker levels (4-HNE; Figure 5) than DFZ alone.

Figure 5.

Western blot analysis of tumor necrosis factor α(TNF-α), metalloproteinase 9 (MMP-9), nuclear transcription factor kappa β (NF)-κβ, transforming growth factor β (TGF-β), calsequestrin 2 (CSQ2), and 4-hydroxynonenal-protein adducts (4HNE). The molecular weight (kDa) for each protein is indicated. The blots of the proteins (top row) and of glyceraldehyde-3-phosphate dehydrogenase (loading control, bottom row; 37 kDa) are shown. The graphs indicate the level of each protein in the heart. *Significantly different from C57BL/10. §Significantly different from untreated mdx. #Significantly different from deflazacort (DFZ; P < .05, analysis of variance [ANOVA]).

DFZ/DOX Toxicity

Total protein content in urine was similar in C57BL/10, untreated mdx, and DFZ/DOX-treated mdx mice (0.3 g/L). Most urine samples did not show ketones. The combined therapy did not affect liver morphology as shown by the H&E-stained transversal sections (Figure 6).

Figure 6.

Histological analysis of the liver in mdx mice after 9 months of combined treatment with deflazacort (DFZ) at 1.2 mg/kg body weight/day plus doxycycline (DOX)at 6.0 mg/mL. Hepatocyte from C57BL/10 (A), untreated-mdx (B), DFZ (C), and DFZ/DOX (D). hematoxylin and eosin (H&E) scale bar: 100 µm.

Discussion

In the mdx mice treated with DFZ/DOX, the ECG was characterized by a return of heart rate to levels comparable to normal and shorter PR, QT, and QRS intervals than those achieved through DFZ monotherapy. The cardiomyopathy index (QT/PQ) is an indicator of cardiac damage ^21,22 and was shown to be significantly reduced by the combined therapy. Such functional observations were concomitant with a significant decrease in fibrosis, as shown by the morphometric analysis of fibrosis area, which was accompanied by lower levels of the fibrosis biomarker (TGF-β). Both therapies similarly reduced inflammation, as indicated by the morphometric analysis (area of inflammation) and the levels of the inflammatory markers (MMP-9, NF-κB, and TNF-α). These findings are of interest because progressive cardiomyopathy is directly related to persistent inflammation in mdx mice and in human disease; it is therefore important to develop therapeutic strategies for DMD that target inflammation.^25,26

We also observed that 4-HNE protein adducts (markers of oxidative stress)²⁷ were more effectively reduced by DFZ/DOX therapy than DFZ alone. In addition to inflammation, oxidative stress contributes to cardiomyocyte lesion in mdx mice,²⁸ and 4-HNE has been used as an indicator of cardiomyopathy.²⁹ The effectiveness of DFZ/DOX therapy in reducing inflammation (similar effect with DFZ/DOX and DFZ) and fibrosis (better effect with DFZ/DOX than DFZ) was more pronounced in the right than the left ventricle, which may be related to the fact that each ventricle is distinctly affected, as recently proposed by 1 study³⁰ and confirmed in another.²⁴ The finding that inflammation and fibrosis are better resolved in the right than the left ventricle has also been demonstrated by other drugs¹⁶ and demonstrated here with DFZ monotherapy. Overall, the present results suggest that DFZ/DOX combination is more effective than DFZ monotherapy, the gold standard treatment of DMD, in slowing the progression of cardiomyopathy in the mdx.

Previous studies have shown that long-term GC therapy aggravates myocardial fibrosis in the mdx mouse model³¹ and in other models of cardiomyopathy.^32,33 In agreement with our own previous observations,³⁴ 17-month-old mdx mice that received a daily dose of 1.2 mg/kg of DFZ in water for 9 months did not experience any worsening of cardiomyopathy. Differences regarding age, time and delivery of therapy, and drug/dose used (prednisone or DFZ) may explain the disagreement in results. We cannot exclude the possibility that delivering higher doses of DFZ than those used in this study over a greater period of time could also lead to the worsening of mdx mouse cardiac muscle if administered under the same conditions used in this study. Notably, boys with DMD who receive corticoid therapy usually show improvement in cardiac function.^8,13

A striking finding is the observation that DFZ/DOX returned the levels of CSQ2 to almost normal, a result comparable to that observed in nondystrophic hearts (C57BL/10 mice). Although inflammation is important in the progression of cardiomyopathy,²⁸ damage in dystrophin-deficient cardiomyocytes is primarily due to a dysfunction in Ca²⁺ homeostasis.^26,35 Calsequestrin, the major sarcoplasmic reticulum (SR) Ca²⁺ reservoir protein in cardiac fibers, was reduced in dystrophic hearts, suggesting poor calcium handling in the SR of the dystrophic cardiac fiber. Loss of CSQ2 has been associated with fibrosis in the atrial pacemaker complex, leading to ECG conduction abnormalities as demonstrated in knockout mice.³⁶ Elevations in the levels of CSQ2 ameliorated mdx cardiac phenotypes.³⁵ The present finding of increased CSQ2 suggests that the combined therapy can also affect calcium homeostasis and thereby protect against cardiomyocyte damage. This protective effect against myonecrosis was illustrated by the decreased patches of IgG-positive fibers, an indicator of sarcolemma injury and myonecrosis.³⁷ Hence, the present results suggest that in addition to its superior anti-inflammatory effect, DFZ/DOX therapy also treats cardiomyocyte injury better than DFZ monotherapy by regulating calcium-related proteins. This finding is similar to the results previously reported in research on dystrophic skeletal muscle.¹⁵

The translational potential of the present investigation lies in the fact that both DFZ and DOX have been recently used as a therapy to chronic human diseases, such as obstructive pulmonary disease, being well tolerated, and to have no side effects.^38
–40 In addition, we simulated DMD’s natural course and clinical end points in mdx mice by beginning therapy at the age of 8 months and treating the animals for 9 months. Long-term administration of DFZ/DOX was well tolerated, with no deaths and no hepatic or renal lesions. DFZ is widely used in Europe in DMD therapy, and DOX has been approved by the Food and Drug Administration as a tetracycline-class antimicrobial drug indicated for several human infections, such as rickettsial, ophthalmic, respiratory, and specific bacterial infections.

In the majority of patients with DMD, ECG abnormalities, such as tachycardia, short PR interval, prominent Q waves, and flat/biphasic ST segments are usually observed in childhood.⁴¹ Diagnosis is made between 3 and 5 years of age, with death by the late 20s.⁴² In mdx mice, myocardial fibrosis begins to increase from 6 months of age,^43,44 and the typical life span of an mdx mouse is approximately 22 months.⁴⁵ Preclinical studies focusing on myocardial fibrosis and inflammation ideally begin between 6 and 9 months of age in mdx mice.⁴⁴ To simulate DMDs natural course and clinical end points in mdx mice, we began therapy at the age of 8 months and continued treatment for 9 months. In average, mdx mice lives about 22 months, thus the age of 17 months studied here corresponds to approximately 85% of mdx life.

Limitations

Limitations are inherently associated with the experimental model. Although mdx mice have been widely used in preclinical studies, caution must be exercised when transposing findings to humans, even when dystrophic parameters are drastically improved.³¹ Some pharmacological and nonpharmacological strategies have been shown to benefit mdx mice but not human patients with DMD.⁴⁶ The doses of the dual therapy (DFZ/DOX) administered to mdx mice are significantly higher than the doses used to treat human diseases,^8,46 possibly due to differences in the pharmacokinetics between rodents and humans regarding oral administration.⁴⁷

Conclusion

This study suggests that DFZ/DOX dual therapy was superior to DFZ monotherapy for alleviating progressive cardiac damage in mdx mice. Long-term administration of DFZ/DOX was well tolerated, with no deaths and no hepatic or renal lesions. If the cardiac benefits of DFZ/DOX therapy are confirmed in DMD, this dual therapy represents a promising transition to the clinical arena.

Footnotes

Author Contributions

J. Alves Pereira contributed to conception, design, acquisition, analysis, and interpretation and drafted the manuscript. A. Fogagnolo Mauricio contributed to acquisition, analysis, and interpretation. M. J. Marques contributed to conception, design, analysis, and interpretation and drafted the manuscript. H. Santo Neto contributed to conception, design, analysis, and interpretation and drafted the manuscript. All authors critically revised the manuscript, gave final approval, and agree to be accountable for all aspects of work ensuring integrity and accuracy.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grants 04/15526-9, 08/58491-1, 11/51697-6, 2014/04782-6). HSN and MJM are recipients of fellowships from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grants 302831/2013-4, 303320/2013-3, 474708/06-3). JAP was the recipient of CNPq fellowships (grants 142935/2011-5). AFM was the recipient of FAPESP fellowships (grants 2012/13577-1).

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