Poster Session 2 - C4
1,2Xavier Lee, 1,2Allen C.T. Teng, 1,2Anthony Gramolini
1 Dept. of Physiology, University of Toronto; 2 Translational Biology and Engineering Program, Ted Rogers Centre for Heart Research
Heart failure is the leading cause of death worldwide. Despite its prevalence however, the cellular mechanisms underlying heart failure remain unknown. A commonality in failing hearts is a loss of control over Ca2+ cycling, thus implicating Ca2+ regulatory proteins in disease pathogenesis. Additionally, protein trafficking and degradation is commonly impaired in failing cardiomyocytes making the elucidation of such mechanisms, especially those involving Ca2+ handling proteins, crucial for understanding heart failure. Phospholamban, an inhibitor of sarco(endo)plasmic reticulum Ca2+ ATPase-2, is a key regulator of Ca2+ cycling and contractility in cardiac muscle. Three mutations in phospholamban have been identified: a missense mutant (R9C), a deletion mutant (R∆14), and a nonsense mutant (L39Stop), all of which unequivocally result in dilated cardiomyopathy in both humans and mice. Since disrupting the clearance of misfolded or mutated proteins is known to have damaging effects on cardiomyocytes, we investigated the degradation mechanisms of phospholamban and its mutants. While our lab has identified the degradation mechanism behind wild-type phospholamban, whether mutation perturbs this process remains unclear. The L39Stop mutant has been shown to be absent at the protein level whilst still being present at the transcript level in cardiac muscle of human patients, and the R∆14 mutant has been shown to form perinuclear and cytosolic aggregates; these data suggest that mutant phospholamban contributes to pathology through aberrant degradation. I hypothesize that L39Stop and R∆14 will be subject to greater degradation in cardiomyocytes. We will use immunofluorescence on cultured neonatal mouse cardiomyocytes to determine the degree of colocalization between the phospholamban mutants and degradation markers: ubiquitin and p62, as well as immunoprecipitations and western blotting to assess the molecular interactions between the phospholamban mutants and said degradation markers. Our current immunoprecipitation results indicate that all three phospholamban mutants are tagged with ubiquitin, a requisite modification that leads to autophagy of phospholamban. We are furthering this study with the addition of autophagy and proteasome inhibitors to our immunoprecipitation experiments along with the development of our immunofluorescence data in cultured neonatal mouse cardiomyocytes.