Abstract 3278: Myoscape (Muscle Specific Calcium-Channel Associated Protein) is a Novel Striated Muscle Enriched Gene Regulating Calcium Cycling and Contractile Function
Calcium induced calcium release and EC Coupling is a fundamental process how muscle cells generate contractile force. Although many proteins modulating calcium cycling and force transmission are well characterised, the precise mechanism and the fine-tuning of these processes are still unclear. In an effort to identify novel heart and muscle specific genes using a bioinformatic approach we found a 100kd protein we named Myoscape, which is highly conserved between the species of human, mouse, rat and zebrafish. The expression of Myoscape is strongly enriched in heart and skeletal muscle as determined by quantative real-time PCR, northern- and western-blot analyses. Myoscape protein is equally detectable in cytosolic as well as membrane fractions. Immunolocalization of Myoscape using a rabbit anti-Myoscape-Antibody showed a distinct expression at sarcomeric Z-bands colocalizing with Alpha-actinin both in cardiac and skeletal muscle tissue. In a yeast two-hybrid screen we identified the C-terminus of the L-Type Ca-Channel as an interaction partner for Myoscape. Adenoviral knockdown of Myoscape in adult rat ventricular cardiomyocytes in vitro using a specific GFP-tagged synthetic micro-RNA led to a significant decrease in calcium transients assessed by fura-2 with smaller calcium amplitudes (120 cells, N=4, 0.138 vs. 0.108, p<0.001), lower diastolic calcium contents (120 cells, N=4, 0.342. vs. 0.293, p<0.01), and a prolonged time to peak (60 cells, N=3, 0.12ms±0.03 vs. 0.099ms±0.02, p<0.002). Consistently, adenoviral knockdown significantly reduced fractional shortening of adult rat ventricular cardiomyocytes (60 cells, N=3, 21%±1% vs. 14%±1%, p<0.0001). In vivo antisense-morpholino based knockdown of the Myoscape orthologue in zebrafish (N=60) led to impaired heart function, pericardial edema and hypertrophy of heart chambers 72 hours post fertilisation. In summary, we identified a novel heart and muscle enriched protein termed Myoscape which localizes to the Z-Band/T-tubule interface and modulates cardiac calcium cycling and force generation both in vitro and in vivo. Together these findings indicate an important role for Myoscape in heart function.