Cardiomyopathy in the mouse model of Duchenne muscular dystrophy caused by disordered secretion of vascular endothelial growth factor

Abstract

Background: Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder that affects skeletal muscles and cardiac muscle tissue. In some cases, myocardial injury secondary to hypoxia can lead to dilative cardiomyopathy (DCM). A genetic defect in the dystrophin gene may increase the susceptibility of myocardium to hypoxia. Available data suggest that this may be caused by impaired secretion of NO, which is bound with secretion of VEGF-A.

Material/methods: Male mice C57BI/10ScSn mdx (animal model of DMD) and healthy mice C57BI/10ScSn were exposed to hypobaric hypoxia in low-pressure chambers. Their hearts were harvested immediately after and 1, 3, 7, and 21 days after exposure to hypoxia. Normobaric mice were used as controls. The expression of VEGF-A in myocardium and cardiac vessel walls was evaluated using immunohistochemistry, Western blotting, and in situ hybridization.

Results: VEGF-A expression in myocardium and vessel walls of healthy mice peaked 24 hours after exposure to hypoxia. The expression of VEGF-A in vessel walls was similar in dystrophic and healthy mice; however, VEGF-A expression in the myocardium of dystrophic mice was impaired, peaking around day 7. In the heart, the total level of VEGF depends on VEGF expression in myocardium, not in vessel endothelium, and our research demonstrates that the expression of VEGF is dystrophin-dependent.

Conclusions: Disordered secretion of VEGF-A in hypoxic myocardium caused the total level of this factor to be impaired in the heart. This factor, which in normal situations protect against hypoxia, promotes the gradual progression of cardiomyopathy.

Figure 1

Heart’s specimen from a control mouse. The measuring frame of 70×50 μm is marked. In the black frame – vessel. Scale bar = 50 μm.

Figure 2

(A) Western analysis of VEGF expression in the heart following hypoxia normal mice. Control group (1); immediately after (2); 1 day after (3); 3 days after (4); 7 days after (5), and 21 days after (6) hypoxia. (B) Western analysis of VEGF expression in the heart following hypobaric hypoxia in dystrophic mice. Control group (1); immediately after (2); 1 day after (3); 3 days after (4); 7 days after (5), and 21 days after (6) hypoxia. (C) Quantitative analysis of western blot signals in normal and mdx mice. Note the difference in the timing of maximum VEGF expression between normal and mdx mice. The control group signal is set to 100%. * Statistically significant compared to healthy mice. In each group was 10 mice.

Figure 3

In situ hybridization analysis of VEGF mRNA expression in cardiac vessel endothelial cells (A) and in cardiac myocytes (B) from normal (C57BI/10ScSn) and mdx (C57BI/10ScSn) mice (values are optical densities expressed as percentage of the signal for control mice). N – 5 samples from each group, 5 fields of myocardium in 6 preparations from each mouse were examined together 150 analysis. * Statistically significant compared to normal – healthy mice.

Figure 4

Immunohistochemical analysis of VEGF expression in cardiac vessel endothelial cells (A) and in cardiac myocytes (B) from normal (C57BI/10ScSn) and mdx (C57BI/10ScSn) mice (values are optical densities expressed as percentage of the signal for control mice).). N – 5 samples from each group, 5 fields of myocardium in 6 preparations from each mouse were examined together 150 analysis. * Statistically significant compared to normal – healthy mice.

Figure 5

(A) Heart’s specimen from normal mice obtained 1 day after exposure to hypoxia. Increased expression of VEGF in both myocardium and vessel walls. (B) Heart’s specimen from mdx mice obtained 1 day after exposure to hypoxia. Low expression of VEGF in myocardium and an enhanced signal in vessel walls. Scale bar =100 μm.

Figure 6

(A) Heart’s specimen from normal mice obtained 7 day after exposure to hypoxia. The expression of VEGF returns to initial levels. (B) Heart’s specimen from mdx mice obtained 7 day after exposure to hypoxia. The expression of VEGF in myocardium remains elevated but returns to normal in vessel walls. Scale bar =100 μm.