Poster abstracts

Poster number 15 submitted by Yotam Blech-Hermoni

Effects of CELF1 knockdown on myofibril structure and function in cultured primary embryonic cardiomyocytes

Yotam Blech-Hermoni (Program in Cell Biology, Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine), Michael W. Jenkins (Department of Pediatrics, Case Western Reserve University School of Medicine), Oliver Wessely (Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic), Andrea N. Ladd (Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic)

Abstract:
CUG-BP and Elav-like family member 1 (CELF1) is an RNA-binding protein expressed in a variety of tissues, including the embryonic and adult heart, where it is restricted to the cardiomyocytes. CELF1 has been shown to be involved in the regulation of alternative pre-mRNA splicing in the nucleus, as well as transcript degradation and translation control in the cytoplasm. While it is predominantly cytoplasmic in other embryonic tissues, its expression in skeletal and cardiac muscle cells is predominantly nuclear, and this pattern is conserved from frog to mouse. Primary embryonic cardiomyocytes are spontaneously contractile in culture, and this system yields robust knockdown of CELF1 at the transcript and protein levels following siRNA transfection. In a transgenic mouse model in which nuclear CELF activity is repressed in the myocardium as a result of the expression of a dominant-negative CELF protein, profound dilated cardiomyopathy and contractile dysfunction follow. In order to investigate the role of CELF1 in cardiomyocyte contraction, we transfected cultured cells with siRNAs targeting CELF1 and evaluated myofibril structure and function. We observed profound disorganization of myofibrillar structure following siRNA-mediated knockdown of CELF1. By visualizing sarcomeric markers (ACTN2, TTN, and TNNT2) by immunofluorescence, we found a shift in sarcomere appearance from rod-like striations along thick, long fibrils to globular puncta arranged along thin, often web-like, matrices. The consequences of CELF1 abrogation in vivo are currently being investigated. Effects of morpholino-mediated CELF1 knockdown on myofibrillar organization, heart morphology, and cardiac functional parameters are being evaluated in the developing frog embryo by immunofluorescence and optical coherence tomography (OCT).

Keywords: RNA binding protein, myofibrillar structure, cardiac function