Improvement of cardiac fibrosis in dystrophic mice by rAAV9-mediated microdystrophin transduction

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Duchenne muscular dystrophy (DMD) is the most common form of the progressive muscular dystrophies characterized by defects of the dystrophin gene. Although primarily characterized by degeneration of the limb muscles, cardiomyopathy is a major cause of death. Therefore, the development of curative modalities such as gene therapy is imperative. We evaluated the cardiomyopathic features of mdx mice to observe improvements in response to intravenous administration of recombinant adeno-associated virus (AAV) type 9 encoding microdystrophin. The myocardium was extensively transduced with microdystrophin to significantly prevent the development of fibrosis, and expression persisted for the duration of the study. Intraventricular conduction patterns, such as the QRS complex duration and S/R ratio in electrocardiography, were also corrected, indicating that the transduced microdystrophin has a protective effect on the dystrophin-deficient myocardium. Furthermore, BNP and ANP levels were reduced to normal, suggesting the absence of cardiac dysfunction. In aged mice, prevention of ectopic beats as well as echocardiographic amelioration was also demonstrated with improved exercise performance. These findings indicate that AAV-mediated cardiac transduction with microdystrophin might be a promising therapeutic strategy for the treatment of dystrophin-deficient cardiomyopathy.


Duchenne muscular dystrophy (DMD) is a devastating X-linked inherited disease and the most frequent form of progressive muscular dystrophy, affecting roughly 1 in 3500 male births.1 DMD is caused by a variety of mutations in the dystrophin gene.2 Often overshadowed by the manifestation of limb muscle weakness, cardiomyopathy afflicts almost all patients with DMD,3 and is a major cause of death from this disease.4 Cardiomyopathy is also prevalent in Becker muscular dystrophy (BMD), which is a milder form of muscular dystrophy also caused by defects in the dystrophin gene. Although the progression of BMD is slower than that of DMD, the symptoms of cardiac failure may be more severe in BMD than in DMD due to the greater functional demands placed on the heart by persistent patient mobility.5 At the onset of refractory congestive heart failure, these patients become candidates for heart transplantation6 and alternative treatment options are limited to corticosteroid or symptomatic therapy.

Recombinant adeno-associated viruses (rAAV) are promising gene transfer vectors with no known pathogenicity.7 However, dystrophin is the largest human gene identified to date and its cDNA measures 14 kb,8 well beyond the packaging capacity of rAAV.9 Therefore, microdystrophin, in which most of the rod domains of dystrophin are deleted, was developed to fit into rAAV.10, 11 We and others have reported successful improvement of the dystrophic phenotype in skeletal muscles of DMD mice models following introduction of this transgene.12, 13 To achieve transduction of an entire extremity, a limb perfusion by retrograde intravenous administration of the rAAV in larger animal models is being developed.14 For cardiac transduction, we utilized rAAV serotype 9, which can be administered systemically with excellent cardiac tropism.15

In this study, we used mdx mice which are considered to be a representative animal model of DMD.16 They lack full-length dystrophin in their muscles, due to a nonsense mutation in exon 23 of the dystrophin gene.17, 18 The mdx mice show initial evidence of cardiac dysfunction at 8–12 weeks,19 and develop moderate myocardial necrosis and fibrosis by 6–8 months of age,20 which progresses continuously thereafter. Correction of basic electrocardiographic (ECG) profiles was suggested after microdystrophin transduction in neonatal mdx mice,21 although limited hemodynamic improvement was observed following microdystrophin transduction.22 Here, we explored rAAV9-mediated myocardial microdystrophin transduction for the prevention of cardiac pathology as well as dysfunction. Long-term extensive cardiac transduction with microdystrophin demonstrated successful improvement of cardiac fibrosis with amelioration of DMD cardiomyopathy.


Extensive long-term expression of microdystrophin by rAAV9-mediated transduction

Transduction of the mdx mice with the rAAV9 containing CMV promoter and microdystrophin were evaluated, following administration of the vector at 3.0 × 1012 viral genomes/mouse via the tail vein at 4 weeks of age. The microdystrophin contains the N-terminal, actin-binding domain, four rod repeats (R1, 2, 3, 24) and three hinges (H1, 2, 4), the cysteine-rich domain, and the truncated C-terminal domain.12 Intravenous systemic delivery of the rAAV9-microdystrophin effectively transduced the cardiac tissue of mdx mice, and the expression of microdystrophin persisted until they were killed at 24 (Figures 1A and Bc–e) or 74 weeks (Figure 1Bf) after transduction. A large percentage of cardiomyocytes were transduced in both ventricles, with no difference in the number of transduced cardiomyocytes between endomyocardial and epimyocardial tissues. Immunofluorescence microscopy revealed that expressed microdystrophin localized at the sarcolemma of cardiomyocytes (Figure 1Bc–f).

Figure 1

Expression of dystrophin/microdystrophin in cardiac muscles. (A) Transverse section of mdx mouse heart at mid-ventricular level 24 weeks after transduction of microdystrophin, stained with anti-dystrophin antibody NCL-DysB. Scale bar, 500 μm. (B) Expression of dystrophin in C57BL10 hearts at the sarcolemma (a), while it is absent in mdx hearts (b). Magnified views of sections from the center of the left ventricle at 28 weeks (c–e) show microdystrophin expression in the areas indicated in (A) (scale bar, 100 μm). At 74 weeks after transduction, mdx mice still retain extensive expression of microdystrophin (f). (C) In Western blot analysis of hearts at 28 weeks old, dystrophin is detected in the C57BL10 mice (B10), while microdystrophin is detected in the transduced mdx mice (mdx-tx). (D) Dystrophin/microdystrophin mRNA expression levels in the heart or quadriceps of 28-week-old C57BL10, untransduced mdx, and transduced mdx mice were measured by quantitative RT–PCR (n=3 per group). mRNA levels were normalized to 18S rRNA. *P<0.01; **P<0.05.

We also examined the expression of dystrophin in C57BL10 mice and microdystrophin in the transduced mdx mice by western blot analysis (Figure 1C), and the expression levels of dystrophin/microdystrophin mRNA in the heart and quadriceps muscle by quantitative RT–PCR (n=3 per group, Figure 1D). The amount of microdystrophin expression in the transduced cardiac tissue of the mdx mice came near to the level of dystrophin expression in C57BL10 mice. In contrast, expression level of dystrophin/microdystrophin mRNA in the untransduced mdx mice was negligible. In quadriceps, the copy number of microdystrophin mRNA in transduced mdx mice was significantly lower than that of dystrophin in C57BL10 mice (n=3 per group, Figure 1D) and the expression of microdystrophin in the skeletal muscle was marginal at 24 weeks after transduction (Supplementary Figure 1), which is below the estimated therapeutic level.

Transduced mdx mice do not develop sinus tachycardia or QRS abnormalities

Mice were injected with 3.0 × 1012 viral genomes/mouse via the tail vein at 4 weeks of age for periodical evaluation (n=3–4 per group at each time point). The ECG data obtained from C57BL10, untransduced mdx mice, and transduced mdx mice are summarized in Table 1. Untransduced mdx mice demonstrated higher heart rates and shorter PR intervals than C57BL10 mice (Figures 2a, b, d and e) at 8 weeks of age. These abnormalities persisted until the age of 28 weeks (P<0.01 compared with C57BL10 mice). When mdx mice were transduced with microdystrophin, their heart rates slowed and the PR intervals lengthened in comparison to those of the untransduced mdx mice at 4 weeks after transduction (Figures 2c–e).

Table 1 Baseline ECG parameters in C57BL10, untransduced mdx and transduced mdx mice
Figure 2

ECGs of C57BL10, untransduced mdx and transduced mdx mice. (ac) Representative ECG tracings from mice at the age of 8 weeks. Compared with the ECG tracing of C57BL10 mice (a), mdx mice (b) show a shorter RR interval, a shorter PR interval, and a smaller S/R ratio. These ECG abnormalities are prevented in the transduced mdx mice (c). Vertical scale bar, 200 μV; horizontal scale bar, 20 ms. (dg) Graphical representation of ECG profiles with age (n=3–4 per group at each time point); (d) heart rate, (e) PR interval, (f) QRS duration, and (g) S/R ratio. The horizontal axis represents age. C57BL10 mice (B10) are represented by open circles with solid lines, untransduced mdx mice (mdx) by filled squares with solid lines, and mdx mice transduced with microdystrophin (mdx-tx) by open squares with broken lines. Data are shown as mean±s.e.m; *P<0.01 between C57BL10 mice and untransduced mdx mice, and also between transduced mdx mice and untransduced mdx mice; **P<0.01 between C57BL10 mice and untransduced mdx mice, as well as P<0.05 between transduced mdx mice and untransduced mdx mice.

Untransduced mdx mice also showed longer QRS durations (Figure 2f) compared with those of C57BL10 mice at 12 weeks of age (P<0.01), and the S/R ratios were significantly lower in untransduced mdx mice than in C57BL10 mice at 8 weeks of age (P<0.01; Figures 2a, b and g). The S/R ratio of the untransduced mdx mice continued to decrease with age up to 28 weeks. These QRS complex abnormalities significantly improved in transduced mdx mice at 28 weeks of age, and the improvements persisted for the duration of the study (Figures 2c, f and g). The improved values measured in the transduced mdx mice did not differ significantly from those of C57BL10 mice. The differences in ECG parameters between C57BL10 and untransduced mdx mice were diminished in mice at 76 weeks old.

Parasympathetic influence determines the difference in heart rate between normal and dystrophic mice

Heart rate and PR interval changed dynamically in accordance with body temperature in normal and mdx mice (Figure 3a). These data suggest that sinus tachycardia with a short PR interval in mdx mice is more likely to be a regulatory response of the autonomic nervous system than a consequence of structural abnormalities. Pharmacological blockade with the atropine treatment induced a paradoxical heart rate drop in untransduced mdx mice, as has been reported previously,23 while transduced mdx mice exhibited a normal tachycardic response comparable to that of C57BL10 mice (n=3–4 per group at each time point, Figure 3b). In contrast, the response to propranolol was not significantly different between the groups (Figure 3c). In addition, in order to estimate the amount of sympathetic tone in the awake state, we measured the urinary excretion of catecholamine and found that the levels of epinephrine and norepinephrine were in the same range in all three groups (n=7 per group, Supplementary Figure 2). We also tested the mRNA levels of BNP and ANP in the ventricular myocardium at 24 weeks after transduction (n=3 per group). Transcripts of both peptides were significantly increased in untransduced mdx mice (P<0.01), but were decreased in the transduced mdx mice to levels approaching that of C57BL10 mice (Figure 4).

Figure 3

Dynamics of heart rate and PR intervals. (a) Heart rate and PR interval changes according to body temperature. Changes in heart rate (upper graph) and PR interval (lower graph) were monitored serially with increasing body temperature in C57BL10 and untransduced mdx mice (mdx) at the age of 12 weeks, Scale bar, 1 min. (b) Heart rate changes in response to atropine. The horizontal axis represents age. Data are shown as mean±s.e.m. *P<0.01 between C57BL10 mice (B10) and untransduced mdx mice, and also between transduced mdx mice (mdx-tx) and untransduced mdx mice (n=3–4 per group at each time point). (c) Heart rate changes of the mice in response to propranolol (n=3–4 per group at each time point).

Figure 4

BNP and ANP expression in cardiac tissues. mRNA levels of BNP (a) and ANP (b) in C57BL10 (B10), untransduced mdx (mdx) and transduced mdx (mdx-tx) mice, relative to the expression of 18S rRNA. mdx Mice 24 weeks after transduction and control mice at the same age were evaluated. Data are shown as the mean±s.e.m., n=3 per group.

Transduction with microdystrophin prevents cardiac fibrosis in mdx mice

Widespread cardiac fibrosis is indicative of advanced cardiomyopathy in DMD patients. In this study, the therapeutic effect of microdystrophin was associated with reduced cardiac fibrosis (Figures 5a–f). When compared with mdx mice at 24 weeks after transduction, age-matched untransduced mdx mice showed marked infiltration of fibrosis in the cardiac tissue. Although the distribution of fibrosis varied among the mice, fibrotic changes in untransduced mdx mice were most prominently observed in the epimyocardial tissues of the left ventricle. Right ventricles were also infiltrated with fibrous tissue to varying degrees. In contrast, microdystrophin transduction of mdx mice successfully improved the pathologic changes in the heart muscle. The amount of fibrous tissue measured by Sirius red staining was statistically equivalent to that of C57BL10 control mice (n=3 per group, Figure 5g). Furthermore, TGF-β upregulation was prevented in the hearts of mdx mice at 24 weeks after microdystrophin transduction, while mRNA levels of TGF-β were significantly increased in the untransduced mdx mice (n=3 per group, Figure 5h). Long-term analysis of the cardiac fibrosis demonstrated that the untransduced mdx mice at 18 months after transduction showed extensive fibrosis in the epimyocardial area, while fibrosis was not evident in the transduced mdx mice (Supplementary Figure 4).

Figure 5

Fibrosis in the epimyocardium. Transverse sections of mid-ventricular level at 24 weeks after transduction. Red areas in the unpolarized views (ac) and bright areas in the polarized views (df) represent fibrosis. Untransduced mdx mice (b, e) show extensive fibrosis in the epimyocardial area, while fibrosis is not evident in transduced mdx mice (c, f). (g) Quantification of the areas of these cardiac fibrosis, n=3 per group. (h) mRNA levels of TGF-β in each group, n=3 per group.

ECG findings in aged mice

Various ECG abnormalities appeared in untransduced mdx mice at 76 weeks of age. Ventricular premature contractions (VPCs) were frequently recorded in aged untransduced mdx mice (Figures 6a and c), while VPCs were observed only at frequencies of <1 VPC every 10 min in C57BL10 and transduced mdx mice at the same or younger ages. In addition, QRS complexes with a pre-excitation pattern were recorded on ECGs in half of the aged untransduced mdx mice (Figures 6b and c).

Figure 6

Various ECG abnormalities in aged mdx mice. (a) Various ECG abnormalities were evident in untransduced mdx mice at 76 weeks of age. Representative ECG tracings show the presence of VPCs (*). Horizontal scale bar, 20 ms; vertical scale bar, 100 μV. (b) Pre-excitation patterns in the QRS complex. Arrows, δ wave; horizontal scale bar, 5 ms; vertical scale bar, 100 μV. (c) Summary of the ECG abnormalities in aged mice.

Dilated cardiomyopathy is prevented in transduced mdx mice

Left ventricular dimensions and Doppler tissue velocities were measured in 76 weeks old mice. In untransduced mdx mice, dilated cardiomyopathy was evident and fractional shortening was markedly reduced. In contrast, ventricular function and heart muscle structure were preserved in the transduced mdx mice (n=4–5 per group, Figure 7a). Systolic and diastolic Doppler tissue velocities were also significantly reduced in the untransduced mdx mice (n=4–5 per group, Figure 7b) compared with C57BL10 and transduced mdx mice.

Figure 7

Echocardiograms of aged mice. (a) Fractional shortening (FS, %) of the left ventricle at 76 weeks of age, n=4–5 per group. (b) Doppler tissue velocities at 76 weeks of age, n=4–5 per group. DTVS, systolic Doppler tissue velocity; DTVD, diastolic Doppler tissue velocity. B10, C57BL10 mice; mdx, untransduced mdx mice; mdx-tx, mdx mice transduced with microdystrophin.

Transduction with microdystrophin improves voluntary wheel running performance

To evaluate if microdystrophin transduction improves the exercise performance of mdx mice, voluntary wheel running performances of the untransduced and transduced mdx mice were analyzed for 13 days (Figure 8). Microdystrophin transduction of the mdx mice resulted in a significant and consistent improvement of daily running distances (Figure 8a). Cumulative running distance of the microdystrophin-transduced mdx mice (Figure 8b, n=4 per group, mean 28.67 km, 2.17 km as daily running) during the 13 days was longer than that of the untransduced mdx mice (n=4 per group, P<0.01, unpaired t-test, mean 9.94 km, 0.76 km as daily running). The differences in cumulative running distances also reflected daily running distances between untransduced and transduced mdx mice. Microdystrophin transduction did not significantly improve running speed (data not shown, untransduced mice, 12.14±8.60 m per minute; microdystrophin transduced mice, 23.01±2.88 m per minute).

Figure 8

Voluntary wheel running. A computerized wheel system was utilized to estimate daily running distance (a) and cumulative running distance (b). Mice were housed in cages equipped with 12 cm diameter stainless steel running wheels and allowed for free access to the wheel 24 h everyday. Each wheel rotation resulted in a micro-switch closure that was recorded and stored on a computer-based data acquisition system. The running distance was recorded every 2 min. *P<0.05.

Improved survival by microdystrophin transduction

The cumulative survival rate of the microdystrophin-transduced mdx mice at the age of 88 weeks was 81.8% (n=11, data not shown), which was higher than that of the untransduced mdx mice (36.3%, n=68, P<0.01, Kaplan–Meier analysis).


In this study, we demonstrated effective prevention of cardiac fibrosis by transduction of mdx mice with microdystrophin. Furthermore, improvement of various ECG abnormalities as well as echocardiographic parameters was also demonstrated in the aged mdx mice. The transduction efficiency achieved with rAAV9 was nearly complete, with persistent expression for 74 weeks after transduction. This performance is likely due to both the strong affinity of the rAAV9 for cardiac tissue and the therapeutic effect of the expressed microdystrophin in preventing the degeneration of the cardiomyocytes. However, in quadriceps, the copy number of microdystrophin mRNA in transduced mdx mice was significantly lower than that of dystrophin in C57BL10 mice and the expression of microdystrophin in the skeletal muscle was <10% at 24 weeks after transduction which is below the estimated therapeutic level. Therefore, at the transduction condition used in this study, the heart is supposed to be the only organ benefited by transduced microdystrophin. Others performed time course assay of the rAAV9-mediated transgene expression in cardiac and skeletal muscles of neonatal mice.24 After the intravenous administration of the rAAV9, the amount of transgene expression in cardiac tissue was consistently as well as significantly higher than that in skeletal muscle and continued to steadily increase throughout the duration of the experiment. Intravenous administration of the rAAV9 into adult mice also demonstrated a marker gene expression profile similar to that found in newborns. The vector genome bio-distribution profile observed in the nonhuman primate demonstrated a dramatic preference for cardiac tissue over skeletal muscle.24 Although theories to explain the phenomenon have currently been investigated, the preferential expression might be magnified by an increased kinetics of double-stranded DNA synthesis, annealing, uncoating and/or trafficking.

The mdx mice exhibited increased heart rates and shortened PR intervals compared with the background strain beginning at 8 weeks of age, which coincides with the earliest instances of cardiac dysfunction reported in mdx mice.25 During this period, hemodynamic changes are not evident unless the mice are stressed,26, 27 and an increased heart rate is suggestive of compensation for compromised cardiac output. We showed that at 28 weeks of age, mdx mice develop heart failure that is associated with increased levels of BNP and ANP (Figure 4) as well as fibrotic infiltration (Figure 5). The autonomic nervous system has a major role in the effort to offset the effects of heart failure, primarily by influencing heart rate through the activation of the sympathetic nervous system or the inactivation of the parasympathetic nervous system.28 However, increased sympathetic activity does not appear to be the primary cause of increased heart rate in this study. Furthermore, we observed a paradoxical heart rate drop in mdx mice in response to sympathetic blockade, as has been reported elsewhere.23 In a dog model of induced heart failure, the onset of heart failure attenuates parasympathetic control of the heart and upregulates muscarinic receptor expression in the heart.29 We believe this explains both the increase in resting heart rate in the mdx mice and the paradoxical response to atropine.30

The shortened PR interval is likely the result of a compensatory autonomic influence rather than an indication of a pre-excitation syndrome such as Lown–Ganong–Levine syndrome.31 In this study, the shortened PR interval was clearly accompanied by sinus tachycardia and changes in the dynamic range of heart rate in response to temperature (Figure 3a). The predominant pathologic feature in the mdx mouse heart is dilated cardiomyopathy, rather than cardiac hypertrophy such as in Pompe32 or Fabry disease.33 A pre-excitation pattern in QRS complexes, which is a sign of structural AV node involvement, appeared only in older mice (Figures 6b and c).

With regard to intraventricular conduction patterns, prolongation of the QRS complex duration and reduced S/R ratio were prominent. In our series of mdx mice, these abnormalities became evident later than abnormalities in cardiac rhythm, but did not develop in C57BL10 or transduced mdx mice. Because prolongation of the QRS complex duration occurs as a manifestation of heart failure due to left ventricular dyssynchrony,34 its prevention in the transduced mdx mice can be assumed to be a therapeutic effect of microdystrophin expression against the development of dilated cardiomyopathy. Reduction of the S/R ratio is another indication of intraventricular conduction abnormalities in mdx mice.35 It initially appeared at 8 weeks of age and progressively worsened (Figure 2g).35 It is not likely to be the result of cardiac axis deviation, as it can be detected to a similar degree in any of the limb leads (Supplementary Figure 3). Therefore, changes in the S/R ratio might be due to epimyocardial distribution of cardiac fibrosis, which is evident in untransduced mdx mice in this study as well as in autopsy cases of DMD patients.36 Expression of microdystrophin in mdx mice successfully repressed myocardial damage and inhibited the development of cardiac fibrosis (Figure 5) throughout the myocardium.

ECG parameters in the aged mice group were somewhat misleading, possibly confounded by decompensation of heart failure (Figure 2). In aged mice, ECG findings such as VPCs and pre-excitation patterns of the QRS complex were more indicative of the therapeutic effects of microdystrophin (Figure 6). In addition, we detected echocardiographic abnormalities in aged mdx mice, as has been reported previously,26 and demonstrated prevention of cardiac dysfunction in aged mice by microdystrophin transduction (Figure 7).

Voluntary exercise performance was monitored on the untransduced and microdystrophin-transduced mdx mice. Compared with the untransduced mdx mice, daily running distances were significantly longer in the microdystrophin-transduced mdx mice. Voluntary running is an endurance exercise that reflects cardiac function. rAAV9-mediated long-term expression of microdystrophin might be cardioprotective and ameliorate running performance in the dystrophin-deficient mdx mouse with improved survival.

We demonstrated that ECG findings reveal early signs of dystrophin-deficient cardiomyopathy5 and are helpful for monitoring the progression of cardiomyopathy in the course of gene transduction experiments. In addition, this is the first long-term study describing the therapeutic effects of microdystrophin transduction against dystrophin-deficient cardiomyopathy. The results presented here are promising in that microdystrophin was able to prevent both functional and pathologic deterioration until the mice reached advanced age. Considering the fact that cardiac fibrosis was prevented up to the period of advanced age, our results present the possibility that microdystrophin might be effective in the treatment of both DMD and BMD-related cardiomyopathy.

Materials and methods

Construction of proviral plasmid and recombinant AAV vector production

The AAV vector plasmid, pAAV hΔCS2, harboring a human microdystrophin gene hΔCS2 expression cassette flanked by ITRs was described before.12, 37 The proviral vector plasmid pAAV hΔCS2, an AAV9 helper plasmid pAAV2/9 (a gift from Dr James M Wilson),38 and an adenoviral helper plasmid pAdeno39 were transfected into HEK293 cells at 60% confluence in a 10-tray Cell Factory (Nunc, Thermo Fisher, Roskilde Site, Denmark), and incubated for 48 h at 37 °C and 5% CO2 with use of the active gassing technique.40 The rAAV particles were purified by the dual ion-exchange procedures with the high-performance membrane adsorbers.41 The viral titers were determined by quantitative PCR using the MyiQ single-color detection system (Bio-Rad, Hercules, CA, USA) with primer sets designed to specifically target the N-terminus of hΔCS2 (forward 5′-IndexTermCCAAAAGAAAAAGGATCCACAA-3′, reverse 5′-IndexTermTTCCAAATCAAACCAAGAGTCA-3′).

Animal transduction

All experimental procedures were approved by the Experimental Animal Care and Use Committee at the National Institute of Neuroscience (NIN, Tokyo, Japan). mdx Mice and background strain C57BL10 mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and maintained under the standard protocol for animal care at the NIN. We performed systemic microdystrophin transduction in 4 weeks old mdx mice, so that the time of gene transduction correlated with the stage of DMD/BMD progression in patients at the time of initial diagnosis, at which point skeletal muscle pathology has already developed, but cardiomyopathy is not yet evident. Mice were injected with 3.0 × 1012 viral genomes/body via the tail vein at 4 weeks old. An equal volume of PBS was injected into mice in the control groups. The mice were killed at 24 or 74 weeks after the injection. Samples were taken from the heart, diaphragm and quadriceps and frozen in liquid nitrogen-cooled isopentane. Six to eight mice from each group were used for each time point.


All ECGs were performed under general inhalation anesthesia with 2% isoflurane using a precision vaporizer (Shinano, Tokyo, Japan) at 4, 8, 24 and 72 weeks after transduction. Mice were placed in a closed box to minimize light and noise, and abdominal skin temperature was controlled at 35±0.1 °C using an electric heating pad under the mouse in the prone position. Electrodes were placed subcutaneously on the distal limbs and readings from lead II were recorded. The PowerLab data acquisition system with Chart-Pro 6 software (AD Instruments, Bella Vista, NSW, Australia) was used for recording and analysis. ECG tracings were accepted as valid only when the values remained stabilized for 2 min. Pharmacological autonomic blockade was performed immediately after the baseline recording. Propranolol (1 mg kg–1 bodyweight) or atropine (0.5 mg kg–1 bodyweight) was injected intraperitoneally to induce sympathetic or parasympathetic blockade, respectively. Mice subjected to autonomic blockade were excluded from ECG recordings for the following 48 h. The S/R ratio represents the ratio of the amplitude of S waves to R waves on lead II.35

Immunofluorescence staining of dystrophin/microdystrophin

Slide mounted sections measuring 8 μm thick from the heart, diaphragm and quadriceps were stained according to the following procedure. First, the blocking solution from the Mouse-on-Mouse kit (Vector Laboratories, Burlingame, CA, USA) was applied for 1 h at room temperature. The slides were then washed with PBS and incubated with the mouse anti-dystrophin monoclonal antibody NCL-DysB (Novocastra, Newcastle, UK) at a 1:100 dilution for 1 h at room temperature, and then with Alexa Fluor 568 goat anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) diluted to 1:1000. Fluorescence imaging was performed with a BZ-9000 microscope (Keyence, Osaka, Japan) using a tiling method.

Quantitative RT–PCR of dystrophin/microdystrophin, BNP, ANP and TGF-β

Total RNA was extracted from frozen mouse heart and quadriceps muscle tissue using TRIzol reagent (Invitrogen), and converted to cDNA with the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). Expression levels were determined by quantitative PCR using the MyiQ single-color detection system (Bio-Rad), and normalized to the expression of 18S rRNA. Primers for each gene were designed based on the mouse genome in the NCBI Reference Sequence database42 as follows: dystrophin/microdystrophin, forward 5′-IndexTermCCAAACAAAGTGCCCTACTATATCAA-3′, reverse 5′-IndexTermGCTCAAGAGATCCAAGCAAAGG-3′; BNP, forward 5′-IndexTermGGTCCAGCAGAGACCTCAAAAT-3′, reverse 5′-IndexTermAGACCCAGGCAGAGTCAGAAAC-3′; ANP, forward 5′-IndexTermGATTGGAGCCCAGAGTGGACTA-3′, reverse 5′-IndexTermTCGTGATAGATGAAGGCAGGAA-3′; TGF-β, forward 5′-IndexTermGGAGAGCCCTGGATACCAACTA-3′, reverse 5′-IndexTermCTGTGTGTCCAGGCTCCAAATA-3′; 18S rRNA, forward 5′-IndexTermACCGCAGCTAGGAATAATGGAA-3′, reverse 5′-IndexTermCCTCCGACTTTCGTTCTTGATT-3′.

Western blot analysis

Samples from hearts were taken from the mid-ventricle and suspended in 100 μl of sodium dodecyl sulfate–polyacrylamide gel electrophoresis lysis buffer (10% sodium dodecyl sulfate, 70 mM Tris–HCl, 5% β-mercaptoethanol, 10 mM ethylenediaminetetraacetic acid) at 90 °C for 10 min. In all, 10 μg of protein from each sample was separated on an sodium dodecyl sulfate–polyacrylamide gel. The separated proteins were then transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA) using semi-dry transfer for 1 h. The membrane was then incubated with the following primary antibodies: NCL-DysB for dystrophin/microdystrophin, and sarcomeric α-actinin antibody EA-53 (Abcam, Cambridge, UK) for internal control. The membrane was further washed and incubated with secondary antibody: anti-mouse IgG conjugated with HRP (Amersham Biosciences, Fairfield, CT, USA). The signals were detected using enhanced chemiluminescence (Amersham Biosciences).

Estimation of cardiac fibrosis

Sirius red staining solution was prepared by dissolving 0.5 g of Sirius red F3B (Sigma Aldrich, St Louis, MO, USA) in a saturated aqueous solution of picric acid (Sigma Aldrich). Sections taken from frozen tissues samples were stained in the Sirius red solution for 2 min and washed twice with 0.5% acetic acid solution. Images obtained by unpolarized light microscopy were used for morphologic examination, and polarized light microscopy (Eclipse E6000, Nikon, Tokyo, Japan) was used for quantification of fibrosis. For each mouse, three mid-ventricular transverse sections were analyzed with Paint.NET image processing software (dotPDN LLC, Kirkland, WA, USA). Bright areas were measured as areas of fibrosis, and these values were then divided by the total cross-sectional area to determine the degree of cardiac fibrosis.


Under 2% isoflurane general inhalation anesthesia, mice were placed in the left decubitus position over an electric heating pad. A 10-MHz echocardiographic probe (EUB-6500, Hitachi, Tokyo, Japan) was gently applied to the left hemithorax, with care taken not to induce bradycardia by chest compression. Left ventricular dimensions were measured at the papillary muscle level on the parasternal long-axis angle. Doppler tissue velocities of the left ventricular posterior wall were evaluated from the parasternal short-axis angle.43

Voluntary wheel running

Running activity was analyzed with a computerized wheel system. Mice were housed in cages equipped with 12 cm diameter stainless steel running wheels (WW-3302, O’ Hara & Co., Ltd., Tokyo, Japan) and allowed for free access to the wheel 24 h per day. Each wheel rotation resulted in a micro-switch closure that was recorded and stored on a computer-based data acquisition system. The running distance and average running speed were recorded every 2 min.

Statistical analysis

Statistical significance was determined on the basis of an unpaired two-tailed Student's t-test or Kaplan–Meier analysis using Statview software (Statview; SAS Institute Inc., Cary, NC, USA). A P-value of <0.05 was considered significant.


  1. 1

    Emery AE . Population frequencies of inherited neuromuscular diseases--a world survey. Neuromuscul Disord 1991; 1: 19–29.

  2. 2

    Aartsma-Rus A, Van Deutekom JCT, Fokkema IF, Van Ommen G-JB, Den Dunnen JT . Entries in the Leiden Duchenne muscular dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule. Muscle Nerve 2006; 34: 135–144.

  3. 3

    Bushby K, Muntoni F, Bourke JP . 107th ENMC international workshop: the management of cardiac involvement in muscular dystrophy and myotonic dystrophy. 7th–9th June 2002, Naarden, the Netherlands. Neuromuscul Disord 2003; 13: 166–172.

  4. 4

    Moxley RT . Clinical overview of Duchenne muscular dystrophy. In: Chamberlain J, Rando T (eds). Duchenne Muscular Dystrophy: Advances in Therapeutics. Taylor & Francis Group: New York, 2006, pp 1–20.

  5. 5

    Nigro G, Comi LI, Politano L, Nigro G . Cardiomyopathies associated with muscular dystrophies. In: Engel A, Franzini-Armstrong C (eds). Myology: Basic and Clinical. McGraw-Hill: New York, 2004, pp 1239–1256.

  6. 6

    Connuck DM, Sleeper LA, Colan SD, Cox GF, Towbin JA, Lowe AM et al. Characteristics and outcomes of cardiomyopathy in children with Duchenne or Becker muscular dystrophy: a comparative study from the Pediatric Cardiomyopathy Registry. Am Heart J 2008; 155: 998–1005.

  7. 7

    Gray SJ, Samulski RJ . Optimizing gene delivery vectors for the treatment of heart disease. Expert Opin Biol Ther 2008; 8: 911–922.

  8. 8

    Hoffman EP, Brown RH, Kunkel LM . Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 1987; 51: 919–928.

  9. 9

    Dong JY, Fan PD, Frizzell RA . Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Hum Gene Therapy 1996; 7: 2101–2112.

  10. 10

    Yuasa K, Miyagoe Y, Yamamoto K, Nabeshima Y, Dickson G, Takeda S . Effective restoration of dystrophin-associated proteins in vivo by adenovirus-mediated transfer of truncated dystrophin cDNAs. FEBS Lett 1998; 425: 329–336.

  11. 11

    Sakamoto M, Yuasa K, Yoshimura M, Yokota T, Ikemoto T, Suzuki M et al. Micro-dystrophin cDNA ameliorates dystrophic phenotypes when introduced into mdx mice as a transgene. Biochem Biophys Res Commun 2002; 293: 1265–1272.

  12. 12

    Yoshimura M, Sakamoto M, Ikemoto M, Mochizuki Y, Yuasa K, Miyagoe-Suzuki Y et al. AAV vector-mediated microdystrophin expression in a relatively small percentage of mdx myofibers improved the mdx phenotype. Mol Ther 2004; 10: 821–828.

  13. 13

    Gregorevic P, Allen JM, Minami E, Blankinship MJ, Haraguchi M, Meuse L et al. rAAV6-microdystrophin preserves muscle function and extends lifespan in severely dystrophic mice. Nat Med 2006; 12: 787–789.

  14. 14

    Ohshima S, Shin JH, Yuasa K, Nishiyama A, Kira J, Okada T et al. Transduction efficiency and immune response associated with the administration of AAV8 vector into dog skeletal muscle. Mol Ther 2009; 17: 73–80.

  15. 15

    Inagaki K, Fuess S, Storm TA, Gibson GA, McTiernan CF, Kay MA et al. Robust systemic transduction with AAV9 vectors in mice: efficient global cardiac gene transfer superior to that of AAV8. Mol Ther 2006; 14: 45–53.

  16. 16

    Bulfield G, Siller WG, Wight PA, Moore KJ . X chromosome-linked muscular dystrophy (mdx) in the mouse. Proc Natl Acad Sci USA 1984; 81: 1189–1192.

  17. 17

    Sicinski P, Geng Y, Ryder-Cook AS, Barnard EA, Darlison MG, Barnard PJ . The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science 1989; 244: 1578–1580.

  18. 18

    Im WB, Phelps SF, Copen EH, Adams EG, Slightom JL, Chamberlain JS . Differential expression of dystrophin isoforms in strains of mdx mice with different mutations. Hum Mol Genet 1996; 5: 1149–1153.

  19. 19

    Sapp JL, Bobet J, Howlett SE . Contractile properties of myocardium are altered in dystrophin-deficient mdx mice. J Neurol Sci 1996; 142: 17–24.

  20. 20

    Bridges LR . The association of cardiac muscle necrosis and inflammation with the degenerative and persistent myopathy of MDX mice. J Neurol Sci 1986; 72: 147–157.

  21. 21

    Bostick B, Yue Y, Lai Y, Long C, Li D, Duan D . Adeno-associated virus serotype-9 microdystrophin gene therapy ameliorates electrocardiographic abnormalities in mdx mice. Hum Gene Therapy 2008; 19: 851–856.

  22. 22

    Townsend D, Blankinship MJ, Allen JM, Gregorevic P, Chamberlain JS, Metzger JM . Systemic administration of micro-dystrophin restores cardiac geometry and prevents dobutamine-induced cardiac pump failure. Mol Ther 2007; 15: 1086–1092.

  23. 23

    Chu V, Otero JM, Lopez O, Sullivan MF, Morgan JP, Amende I et al. Electrocardiographic findings in mdx mice: a cardiac phenotype of Duchenne muscular dystrophy. Muscle Nerve 2002; 26: 513–519.

  24. 24

    Pacak CA, Mah CS, Thattaliyath BD, Conlon TJ, Lewis MA, Cloutier DE et al. Recombinant adeno-associated virus serotype 9 leads to preferential cardiac transduction in vivo. Circ Res 2006; 99: e3–e9.

  25. 25

    Janssen PML, Hiranandani N, Mays TA, Rafael-Fortney JA . Utrophin deficiency worsens cardiac contractile dysfunction present in dystrophin-deficient mdx mice. Am J Physiol Heart Circ Physiol 2005; 289: H2373–H2378.

  26. 26

    Quinlan JG, Hahn HS, Wong BL, Lorenz JN, Wenisch AS, Levin LS . Evolution of the mdx mouse cardiomyopathy: physiological and morphological findings. Neuromuscul Disord 2004; 14: 491–496.

  27. 27

    Yue Y, Skimming JW, Liu M, Strawn T, Duan D . Full-length dystrophin expression in half of the heart cells ameliorates beta-isoproterenol-induced cardiomyopathy in mdx mice. Hum Mol Genet 2004; 13: 1669–1675.

  28. 28

    Mann DL . Heart failure and cor pulmonale. In: Fauci AS, Braunwald E, Kasper DL et al. (eds) Harrison's Principles of Internal Medicine. 17th edn. chapter 227. McGraw Hill: New York, 2008, pp 1443–1457.

  29. 29

    Dunlap ME, Bibevski S, Rosenberry TL, Ernsberger P . Mechanisms of altered vagal control in heart failure: influence of muscarinic receptors and acetylcholinesterase activity. Am J Physiol Heart Circ Physiol 2003; 285: H1632–H1640.

  30. 30

    Das G, Talmers FN, Weissler AM . New observations on the effects of atropine on the sinoatrial and atrioventricular nodes in man. Am J Cardiol 1975; 36: 281–285.

  31. 31

    Benditt DG, Pritchett LC, Smith WM, Wallace AG, Gallagher JJ . Characteristics of atrioventricular conduction and the spectrum of arrhythmias in Lown-Ganong-Levine syndrome. Circulation 1978; 57: 454–465.

  32. 32

    Ansong AK, Li JS, Nozik-Grayck E, Ing R, Kravitz RM, Idriss SF et al. Electrocardiographic response to enzyme replacement therapy for Pompe disease. Genet Med 2006; 8: 297–301.

  33. 33

    Linhart A, Lubanda JC, Palecek T, Bultas J, Karetová D, Ledvinová J et al. Cardiac manifestations in Fabry disease. J Inherit Metab Dis 2001; 24 (Suppl 2): 75–83.

  34. 34

    Kashani A, Barold SS . Significance of QRS complex duration in patients with heart failure. J Am Coll Cardiol 2005; 46: 2183–2192.

  35. 35

    Bia BL, Cassidy PJ, Young ME, Rafael JA, Leighton B, Davies KE et al. Decreased myocardial nNOS, increased iNOS and abnormal ECGs in mouse models of Duchenne muscular dystrophy. J Mol Cell Cardiol 1999; 31: 1857–1862.

  36. 36

    Frankel KA, Rosser RJ . The pathology of the heart in progressive muscular dystrophy: epimyocardial fibrosis. Hum Pathol 1976; 7: 375–386.

  37. 37

    Yuasa K, Sakamoto M, Miyagoe-Suzuki Y, Tanouchi A, Yamamoto H, Li J et al. Adeno-associated virus vector-mediated gene transfer into dystrophin-deficient skeletal muscles evokes enhanced immune response against the transgene product. Gene Therapy 2002; 9: 1576–1588.

  38. 38

    Lin J, Zhi Y, Mays L, Wilson JM . Vaccines based on novel adeno-associated virus vectors elicit aberrant CD8+ T-cell responses in mice. J Virol 2007; 81: 11840–11849.

  39. 39

    Matsushita T, Elliger S, Elliger C, Podsakoff G, Villarreal L, Kurtzman GJ et al. Adeno-associated virus vectors can be efficiently produced without helper virus. Gene Therapy 1998; 5: 938–945.

  40. 40

    Okada T, Nomoto T, Yoshioka T, Nonaka-Sarukawa M, Ito T, Ogura T et al. Large-scale production of recombinant viruses by use of a large culture vessel with active gassing. Hum Gene Ther 2005; 16: 1212–1218.

  41. 41

    Okada T, Nonaka-Sarukawa M, Uchibori R, Kinoshita K, Hayashita-Kinoh H, Nitahara-Kasahara Y et al. Scalable purification of adeno-associated virus serotype 1 (AAV1) and AAV8 vectors, using dual ion-exchange adsorptive membranes. Hum Gene Ther 2009; 20: 1013–1021.

  42. 42

    Pruitt KD, Tatusova T, Klimke W, Maglott DR . NCBI reference sequences: current status, policy and new initiatives. Nucleic Acids Res 2009; 37: D32–D36.

  43. 43

    Fayssoil A . Tissue Doppler characterization of cardiac phenotype in mouse. Eur J Radiol 2009; 72: 82–84.

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We thank Dr James M Wilson for providing a helper plasmid pAAV2/9. This work was supported by a Grant for Research on Nervous and Mental Disorders, by Health Science Research Grants for Research on the Human Genome and Gene Therapy, by Research on Brain Science from the Ministry of Health, Labor, and Welfare, and by Grants in Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology.

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Correspondence to T Okada or S Takeda.

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Shin, J., Nitahara-Kasahara, Y., Hayashita-Kinoh, H. et al. Improvement of cardiac fibrosis in dystrophic mice by rAAV9-mediated microdystrophin transduction. Gene Ther 18, 910–919 (2011) doi:10.1038/gt.2011.36

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  • Duchenne muscular dystrophy
  • cardiomyopathy
  • AAV vector
  • microdystrophin
  • electrocardiography
  • cardiac fibrosis

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