Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disorder resulting from a mutation in the dystrophin gene. Loss of dystrophin function leads to progressive muscle wasting and cardiomyopathy. In 2018, advanced cardiomyopathy is the primary mode of death, despite the application of standard of care CHF therapies. Our group has recently undertaken a cardiac MRI study demonstrating that adult DMD patients have small hearts with very low LV mass as compared to patients with non-ischemic cardiomyopathy or healthy patients. The central hypothesis is DMD-associated cardiomyopathy develops secondary to dysregulation of various cardiac signaling pathways that modulate cardiac growth, namely cardiomyocyte proliferation. Utilizing the mdx mouse, a model of DMD, decreased heart/body weight ratios were noted from birth (P1: Hrt/BW 5.6 ± 0.16 vs. 4.8 ± 0.18 NTg vs mdx p<0.005 n=32). NTg and m dx hearts have similar cardiomyocyte cell size (WGA staining: 0.08 ± 0.001 vs 0.08 ± 0.002 NTg vs mdx p<0.05 n=3), suggesting there are fewer cells in mdx hearts. FACS analysis indicated mdx hearts have fewer cardiomyocytes than NTg hearts (35% reduction; 51.7e 4 ± 4.9e 4 vs. 33.8e 4 ± 3.5e 4 NTg vs mdx p<0.05 n=6). Transcriptome profiling demonstrated that molecular pathways governing cardiac proliferation were significantly reduced in mdx hearts (Ki67: 4.8 ± 0.3 vs 2.0 ± 0.1 NTg vs mdx p<0.005 n=3), with a corresponding increase in cardiac atrophy gene expression at later time points (Foxo3 1.4 ± 0.2 vs 2.8 ± 0.3 NTg vs mdx p<0.005 n=3). Furthermore, immunohistochemical analyses showed significantly reduced proliferation in mdx hearts (Ki67: 13.7 ± 1.2 vs. 6.0 ± 1.0 NTg vs mdx p<0.005, n=3; pH3: 118 ± 5.3 vs 59 ± 4.5 p<0.005 n=3). Finally, RNA-Seq data revealed a disruption of the YAP signaling pathway, an important mediator of cardiomyocyte proliferation, in mdx P4 hearts. Cell cycle targets of YAP were reduced in P4 mdx hearts (20/27 genes, n=3) suggesting a disruption of the Hippo-Yap pathways in mdx hearts. Collectively, the current study provides a unique insight into the mechanism leading to the development DMD cardiomyopathy and directs investigation into potential therapeutic targets for the amelioration of DMD-associated cardiomyopathy.

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Rationale: Oxidative stress is a major determinant of DNA damage within cardiomyocytes both at baseline and following cardiac injury. Cytoglobin (Cygb), a stress responsive hemoprotein, serves as a redox modulator and imparts cytoprotection in a variety of cell types. In addition, Cygb is involved in regulation of myocyte proliferation and differentiation due to its ability to scavenge free radicals and maintain redox equilibrium within the myocytes. Objective: The goal of this study was to investigate the role of Cygb in cardiomyocytes and identify the molecular pathways underlying its cytoprotective function. Method and Results: Utilizing transgenic mice with cardiac specific overexpression of Cygb (TgCygb), we demonstrated that Cygb overexpression promoted cardiomyocyte proliferation as compared to the control hearts (NTg) (phospho-Histone 3 IHC: 17 ± 0.7 vs 7 ± 0.5 positive cells/mm 2 TgCygb vs NTg; p<0.05, n=3) . The enhanced cellular proliferation in TgCygb hearts was associated with larger hearts at baseline. However, with exposure to acute cardiac stress (subcutaneous Angiotensin II infusion), TgCygb hearts showed significantly reduced cell death, oxidative stress and an overall lower induction of the DNA damage response pathway as compared to the NTg hearts [TUNEL: 7 ± 0.8 vs 10 ± 1.2 TUNEL positive nuclei/mm 2 , p<0.05, n=3; phospho-ATM IHC: 11 ± 2 vs 22 ± 1.5 phospho-ATM positive cells/mm 2 , p<0.05, n=5, dihydroethidium (DHE) staining: 78 ± 0.6% vs 98 ± 0.7% DHE positive nuclei/mm 2 , p<0.05, n=5]. Using single cell electrophoresis (Comet assay), we demonstrated that Cygb overexpressing cardiomyocytes underwent significantly lower DNA damage due to single and double stranded DNA breaks following exposure to oxidative stress as compared to controls cardiomyocytes (36 ± 2.1 vs 48 ± 3.1; p<0.05, n=6). Finally, chromatin immunoprecipitation, quantitative PCR and immunoblotting indicated that Cygb activates the expression of apurinic/apyrimidinic endonuclease 1 ( Ape1 ) and thus promotes DNA base excision repair following acute cardiac injury. Conclusions: Our data demonstrate that Cygb promotes cellular proliferation and protects cardiomyocytes against oxidative stress-mediated DNA damage by directly activating Ape1 expression.

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