The role of SYNPO2L, a novel protein expressed in cardiac and skeletal muscle, in sarcomere formation and stabilization
06 / 2006 - 03 / 2011
Cardiomyopathies consist of a group of heart diseases in which the malfunctioning of the heart muscle itself is the primary cause and not the result of, for example, hypertensive and ischemic diseases. Mutations in sarcomeric and cytoskeletal proteins have been correlated with hypertrophic and dilated cardiomyopathy, respectively. Recently, we identified a novel cardiac gene, annotated as SYNPO2L, by whole genome transcriptome analysis on human embryonic stem cells, differentiating towards cardiomyocytes. SYNPO2L displays significant homology with cytoskeletal protein myopodin, which is involved in cytoskeletal organization and muscle differentiation. SYNPO2L is expressed in developing heart in skeletal muscle in zebrafish, mouse and human. Hypothesis We postulate that SYNPO2L plays a role in the formation and stabilization of sarcomeres in heart and skeletal muscle and that a cardiac-specific deletion of SYNPO2L in mice results in dilated cardiomyopathy. Aims/Methods/Expected results To test our hypothesis we will further characterize the expression and function of SYNPO2L in zebrafish, in particular in heart and skeletal muscle (aim 1). By whole mount ISH expression of SYNPO2L will be determined throughout zebrafish development, whereas injection of morpholino oligos in zebrafish eggs will be used to knock down specifically SYNPO2L expression, allowing us to study heart and skeletal muscle formation. Preliminary results demonstrate that heart function is impaired and skeletal muscle formation is disorganized. Next, we will study the role of SYNPO2L in sarcomere formation and stabilization in vitro (aim 2). For this we overexpress full length or truncated proteins of SYNPO2L in rat neonatal cardiomyocytes. This allows us to determine the subcellular localization and the functional domains of SYNPO2L. Knockdown of SYNPO2L will be achieved by RNA-interference in neonatal cardiomyocytes and cardiomyocytes derived from mouse or human embryonic stem cells, allowing us to study the role of SYNPO2L in sarcomere formation and stabilization. Analysis will be predominantly performed by immunofluorescence using sarcomeric antibodies. Possible interactions of SYNPO2L with sarcomeric proteins will be determined by immunoprecipitation. We expect an effect on cytoskeletal organization by affecting actin-bundling activity. Finally, we will characterize the in vivo role of SYNPO2L in heart and skeletal muscle development and function in mice (aim 3). Using conditional knockout technology we will generate mice with a heart-specific deletion of SYNPO2L. Heart and muscle defects will be studied during development and in adult mice. Morphology, immunohistochemical analysis, in situ hybridization and heart function will be studied ithese knockout mice. We expect that deletion of SYNPO2L will affect heart and muscle function during development. Using this approach we will be able to study the role of SYNPO2L in adult heart with or without inducing additional cardiac stress, such as myocardial infraction or aorta coarctation Objectives Objective 1. Characterizing the role of SYNPO2L in heart and skeletal muscle development in zebrafish Objective 2. Characterizing the in vitro role of SYNPO2L in sarcomere formation and stabilization Objective 3. Characterizing the in vivo role of SYNPO2L in heart and skeletal muscle development in mouse Relevance for cardiovascular diseases This research proposal focuses on the pathogenesis of cardiac disease, in particular dilated cardiomyopathy. In general, mutations or altered expression of cardiac sarcomeric (beta-myosin heavy chain or cardiac troponin T) or cytoskeletal (dystrophin) proteins lead to hypertrophic and dilated cardiomyopathy, respectively. Here we describe a novel cardiac cytoskeletal protein (SYNPO2L) and its role in heart disease. Preliminary data suggest a role in cardiomyocyte stabilization and heart disease. Functional studies will be performed in vitro as well as in vivo in different organisms, which enable us to study the role at a cellular level and in heart disease. Generation of a conditional heart-specific knockout for SYNPO2L in mice provides information whether SYNPO2L is necessary for normal cardiac development and whether SYNPO2L plays a role in the onset or progression of cardiac disease in adult mice. This will lead to a better understanding of the molecular mechanisms underlying cardiac diseases. Ultimately this may improve diagnosis and treatment of cardiac diseases.