DMD carrier model with mosaic dystrophin expression in the heart reveals complex vulnerability to myocardial injury

Abstract

Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease that causes progressive muscle wasting and cardiomyopathy. This X-linked disease results from mutations of the DMD allele on the X-chromosome resulting in the loss of expression of the protein dystrophin. Dystrophin loss causes cellular dysfunction that drives the loss of healthy skeletal muscle and cardiomyocytes. As gene therapy strategies strive toward dystrophin restoration through micro-dystrophin delivery or exon skipping, preclinical models have shown that incomplete restoration in the heart results in heterogeneous dystrophin expression throughout the myocardium. This outcome prompts the question of how much dystrophin restoration is sufficient to rescue the heart from DMD-related pathology. Female DMD carrier hearts can shed light on this question, due to their mosaic cardiac dystrophin expression resulting from random X-inactivation. In this work, a dystrophinopathy carrier mouse model was derived by breeding male or female dystrophin-null mdx mice with a wild type mate. We report that these carrier hearts are significantly susceptible to injury induced by one or multiple high doses of isoproterenol, despite expressing ~57% dystrophin. Importantly, only carrier mice with dystrophic mothers showed mortality after isoproterenol. These findings indicate that dystrophin restoration in approximately half of the heart still allows for marked vulnerability to injury. Additionally, the discovery of divergent stress-induced mortality based on parental origin in mice with equivalent dystrophin expression underscores the need for better understanding of the epigenetic, developmental, and even environmental factors that may modulate vulnerability in the dystrophic heart.

Figure 1

Dystrophinopathy carrier hearts display mosaic dystrophin expression throughout the myocardium. (A) Heterozygous DMD carrier mice were generated from two different breeding strategies. Paternal carrier (Carrier^P) mice were bred from mdx sires and wild type (C57Bl/10) dams, and maternal carrier (Carrier^M) mice were derived from mdx dams and wild type sires. (B) Representative whole heart and magnified images of dystrophin distribution in C10, carrier and mdx hearts, showing the mosaicism of carrier dystrophin expression. Magnified image scale = 0.5 × 0.5 mm. (C) Heart weights were not different between C10, carrier and mdx mice at baseline when normalized to tibial length to control for body size (n = 6–14 per group; P = 0.21).

Figure 2

Dystrophin is expressed in just over half of the heart on average in both groups of dystrophinopathy carriers. (A) Histological quantification of dystrophin-positive signal in DMD carrier hearts normalized to dystrophin signal in wild type (C10) hearts. Both groups of carriers express dystrophin in ~57% of the heart area (n = 7–13 per group). (B) Representative images of biological variability in dystrophin expression in carrier hearts, ranging from 40.0% to 78.4%. (C) Immunoblot quantification of dystrophin content in ventricular tissue, showing ~55–56% average expression of dystrophin in carrier hearts normalized to wild type hearts (n = 3–12 per group). (D) Representative immunoblot bands used for dystrophin quantification.

Figure 3

Dystrophinopathy carrier cardiac susceptibility is extensive and partially dependent on parental origin of DMD alleles. (A) Three injections of low-dose Iso (0.35 mg/kg each) induced slight injury in C10, carrier and mdx hearts 30 h after the first Iso injection, with 2-fold higher injury area in mdx hearts (n = 6–9 per group; # P = 0.04 vs. mdx). (B) Representative images of hearts 30 h after starting a course of 3 low-dose Iso injections, showing slight IgG-positive (red) cardiomyocyte injury. (C) A single injection of high-dose Iso (10 mg/kg) caused widespread lesions in carrier and mdx hearts and mortality in 21% of Carrier^M mice (n = 17–24 per group; § P = 0.027). Among survivors, both groups of carriers displayed significantly higher injury than C10 hearts, and Carrier^P hearts displayed significantly lower injury than mdx (n = 10–14 per group; ^*P = 0.04 vs. C10; ^***P < 0.001 vs. C10; # P = 0.04 vs. mdx; ## P = 0.001 vs. mdx). (D) Representative images of acute injury distribution in wild type, carrier and mdx hearts after a single high-dose Iso injection. (E) Most of the injured cardiomyocytes (red) lack dystrophin (green), including in wild type hearts; white arrows indicate injured myocytes with significant dystrophin signal. Magnified image scale = 0.3 × 0.3 mm.

Figure 4

DMD carrier hearts are highly susceptible to damage with repeated bouts of injurious stressor. (A) The repeated Iso challenge is comprised of 14 injections of high-dose (10 mg/kg) Iso over the span of 5 days. (B) All of the C10 and Carrier^P mice survived the repeated Iso challenge, but 31% of Carrier^M and 38% of mdx mice died during the challenge (^*P = 0.01; ^**P = 0.005). (C) Among survivors, repeated high-dose Iso caused similarly extensive replacement fibrosis in the hearts of carrier and mdx mice, which was significantly higher than C10 injury (n = 6–17 per group; ^**P = 0.01; ^***P < 0.001). (D) Representative images of fibrosis in wild type (C10), carrier and mdx hearts after repeated injury with high-dose Iso. Fibrosis is red, and intact myocardium is green.