Full-length dystrophin expression in half of the heart cells ameliorates beta-isoproterenol-induced cardiomyopathy in mdx mice

Abstract

Gene therapy holds great promise for curing Duchenne muscular dystrophy (DMD), the most common fatal inherited childhood muscle disease. Success of DMD gene therapy depends upon functional improvement in both skeletal and cardiac muscle. Numerous gene transfer studies have been performed to correct skeletal muscle pathology, yet little is known about cardiomyopathy gene therapy. Since complete transduction of the entire heart is an impractical goal, it becomes critical to determine the minimal level of correction needed for successful DMD cardiomyopathy gene therapy. To address this question, we generated heterozygous mice that persistently expressed the full-length dystrophin gene in 50% of the cardiomyocytes of mdx mice, a model for DMD. We questioned whether dystrophin expression in half of the heart cells was sufficient to prevent stress-induced cardiomyopathy. Heart function of mdx mouse is normal in the absence of external stress. To determine the therapeutic effect, we challenged 3-month-old mice with beta-isoproterenol. Cardiomyocyte sarcolemma integrity was significantly impaired in mdx but not in heterozygous and C57Bl/10 mice. Importantly, in vivo closed-chest hemodynamic assays revealed normal left ventricular function in beta-isoproterenol-stimulated heterozygous mice. Since the expression profile in the heterozygous mice mimicked viral transduction, we conclude that gene therapy correction in 50% of the heart cells may be sufficient to treat cardiomyopathy in mdx mice. This finding may also apply to the gene therapy of other inherited cardiomyopathies.

Figure 1

Breeding scheme and dystrophin expression profile. (A) mdx mice were crossed with BL10 mice to generate paternal and maternal female heterozygous mice. In maternal heterozygous mice, mutated dystrophin gene is inherited from the mdx mother. In paternal, the mutated gene is from the mdx father. (B) Morphometric quantification of dystrophin-positive cardiomyocytes in the hearts. There is no significant difference between maternal and paternal mice. However, values in BL10 mice are significantly higher than those in heterozygous mice. N = 3 for BL10, N = 7 for maternal heterozygous, N = 6 for paternal heterozygous. (C) Representative western blot of full-length dystrophin expression (427 kDa, arrow) in the hearts of different mouse strains. Hetero, heart lysate from heterozygous mouse. (D) Top panels are representative immunofluorescence staining of dystrophin expression in anterior tibialis muscle (TA, top left) and the heart (top right) of heterozygous mice. Higher power photomicrographs of two representative areas in the heart are displayed in bottom panels. Box 1 depicts a region of high dystrophin expression. Box 2 is a low dystrophin expression area. Scale bar in the top left panel (TA) is 300 μm. Scale bar in the bottom right panel (Box 2) is 100 μm. This scale bar also applies to the photomicrograph in the bottom left panel (Box 1).

Figure 2

Quantitative evaluation of sarcolemma integrity in the hearts. (A) Representative photomicrographs of the entire heart sections from BL10, mdx and heterozygous mice. Scale bar, 1 mm. EBD uptake is visualized under Texas Red channel. Bar graph shows the percentage of injured area (EBD-positive region) in the hearts. The results are significantly different among all groups and between any paired strains. N = 17 for BL10, N = 11 for mdx, N = 10 for heterozygous. Hetero, heterozygous mice. (B) Representative higher magnification photomicrographs of the heart sections. Dystrophin expression was detected by indirect immunofluorescence staining (FITC channel). EBD uptake in the same field is shown in Texas Red channel photomicrographs. EBD uptake can also be visualized under FITC channel as cells that were filled up with fluorescence signal. However, this is different from continuous, peripheral membrane staining of dystrophin. Arrow, an EBD positive-region in a heterozygous mouse heart section. Scale bar, 300 μm.

Figure 3

In vivo hemodynamic evaluation of the heart function. (A) Representative tracing following β-isoproterenol challenge in BL10, mdx and heterozygous mice. dP/dt, rate of pressure change in left ventricle; LVP, left ventricular pressure. (B) Hemodynamic profile in different mouse strains in the absence (open bar) or presence (filled bar) of β-isoproterenol challenge. Developed pressure was calculated from the difference between minimal diastolic pressure and systolic pressure. For β-isoproterenol treated group, N = 10 for BL10, N = 9 for mdx and N = 11 for heterozygous. For non-treated group, N = 3 for BL10, N = 10 for mdx and N = 3 for heterozygous. There is no statistical difference among BL10, mdx and heterozygous mice in the absence of β-isoproterenol stimulation. *, β-isoproterenol treated mdx was significantly different from similarly treated BL10 or heterozygous. #, β-isoproterenol treated mdx was significantly different from the non-treated mdx.

Figure 3

In vivo hemodynamic evaluation of the heart function. (A) Representative tracing following β-isoproterenol challenge in BL10, mdx and heterozygous mice. dP/dt, rate of pressure change in left ventricle; LVP, left ventricular pressure. (B) Hemodynamic profile in different mouse strains in the absence (open bar) or presence (filled bar) of β-isoproterenol challenge. Developed pressure was calculated from the difference between minimal diastolic pressure and systolic pressure. For β-isoproterenol treated group, N = 10 for BL10, N = 9 for mdx and N = 11 for heterozygous. For non-treated group, N = 3 for BL10, N = 10 for mdx and N = 3 for heterozygous. There is no statistical difference among BL10, mdx and heterozygous mice in the absence of β-isoproterenol stimulation. *, β-isoproterenol treated mdx was significantly different from similarly treated BL10 or heterozygous. #, β-isoproterenol treated mdx was significantly different from the non-treated mdx.