Abstract 16166: Divergent Effects of Calnexin on Serca2 Activity and Expression in Cardiomyocytes
Background: Calnexin (CNX) has been shown to inhibit SERCA2 activity in a phosphorylation dependent manner. As such we were interested in identifying the effect of calnexin loss on cardiomyocyte function.
Methods/Results: We assessed the effect of acute CNX deficiency at the cellular level by silencing CNX in mouse neonatal cardiomyocytes (mNCMs) using LentiVector mediated transduction of shRNAs. To characterize the effect of loss of CNX we quantified cell viability, evaluated cell morphology by immunofluorescent staining, and examined calcium transients. Acute CNX silencing in mNCMs led to a significant increase in both basal and H2O2 stimulated cell death. Immunofluorescent staining of these cells showed that they were rounded and significantly smaller compared to controls. Four days after CNX silencing in spontaneously beating mNCMs, calcium transients were larger in amplitude yet showed enhanced uptake kinetics. After 7 days of CNX silencing we found that while the increase in calcium amplitude persisted, the calcium uptake kinetics had returned to baseline levels. Gene expression analysis was carried out by qRT-PCR and showed that in CNX deficient mNCMs the sarcolemmal L-type calcium channel, DHPR, was significantly increased (>2-fold, p<0.001) while the SERCA2 expression was significantly decreased (>3-fold, p<0.001). Similarly, we found a significant loss of SERCA2 expression in adult hearts from CNX deficient mice. Furthermore, CNX was found to co-immunoprecipitate with SERCA2 supporting it's regulatory role on the calcium pump.
Conclusion: Here we show that CNX silencing in cardiomyocytes leads to prolonged calcium transients, likely due to increased DHPR expression and activity. Furthermore, partial relief of SERCA inhibition due to the loss of CNX leads to enhanced calcium uptake; however the increase in calcium uptake kinetics is abrogated after prolonged CNX silencing due to decreased SERCA2 expression.
- © 2011 by American Heart Association, Inc.