In this thesis we investigated a novel Z-disc protein, cytoskeletal heart-enriched actin-associated protein (CHAP). Two isoforms of CHAP exist, encoded by one gene. The longer isoform CHAPa is... Show moreIn this thesis we investigated a novel Z-disc protein, cytoskeletal heart-enriched actin-associated protein (CHAP). Two isoforms of CHAP exist, encoded by one gene. The longer isoform CHAPa is predominately expressed in adult tissues, whereas CHAPb is expressed during cardiac and skeletal development. First, we investigated CHAP expression in adult mouse tissues in more detail and show that both CHAP isoforms are highest expressed in slow skeletal muscle fibers and vascular smooth muscle cells. In addition, we sequenced and investigated the expression of the chick CHAP isoform and show that CHAP expression in chick is similar to the expression observed in mouse embryo__s. To investigate the role of CHAP in vivo, we generated CHAP knockout embryonic stem cells, which can be used to generate CHAP knockout mice and we investigated the effect of morpolino mediated knockdown of CHAP in chick embryo__s. In addition, we generated heart specific CHAP transgenic (Tg) mice. Whereas in CHAPa Tg mice no phenotype was observed, CHAPb Tg mice developed cardiomyopathy with diastolic dysfunction. In addition, in the hearts of CHAPb Tg mice stress fibers were observed and the actin signaling pathway was activated. To study the effects of CHAP overexpression in vitro, we generated CHAP adenoviruses. We showed that although CHAPb also induced stress fibers in vitro, the actin signaling pathway was not activated, suggesting that this was not a direct effect in Tg mice. In addition, we showed that CHAPa is important for Z-disc integrity in vitro. Finally, we also showed that CHAP is expressed in the small intestine and kidney, and this expression is correlated with F-actin expression. These data show that CHAP is important for muscle development, Z-disc integrity and stability and actin signaling. Show less
Duchenne muscular dystrophy (DMD) is the most prevalent neuromuscular disorder, caused by mutations in the DMD gene that prevent synthesis of dystrophin. Fibers that lack dystrophin are sensitive... Show moreDuchenne muscular dystrophy (DMD) is the most prevalent neuromuscular disorder, caused by mutations in the DMD gene that prevent synthesis of dystrophin. Fibers that lack dystrophin are sensitive to exercise-induced damage, resulting in progressive muscle wasting, loss of ambulation and premature death. There is no cure, but several therapeutic approaches are clinically tested. At best, these clinical interventions result in the expression of low dystrophin levels. Fortunately, expression of wild type levels is not needed, as both humans and mice expressing ~50% of dystrophin do not show pathology. Detailed studies on which dystrophin levels are needed to prevent pathology and improve muscle function have been performed in this thesis. After the set-up of good outcome measures and serum biomarkers to monitor disease progression, two new innovative mouse models expressing low levels of dystrophin based on skewed X-inactivation were generated. In the mdx-Xist__hs model we observed that <15% dystrophin already improved muscle performance, while histopathology was largely with >15% dystrophin. To protect muscles from exercise-induced damage >22% dystrophin was needed. Dystrophin levels between 3-21% prevent the development of dilated cardiomyopathy in 10 months old mice. Mice lacking both dystrophin and its homologue utrophin, mimic the human phenotype and die before the age of 12 weeks. In these mice, <10% dystrophin improved life expectancy and muscle function while >10% dystrophin was needed to improve histopathology. These findings are encouraging for ongoing and future clinical trails. Show less
