Calcineurin regulates ryanodine receptor/Ca2+-release channels in rat heart

Abstract

The present study was undertaken to examine the physical and physiological interaction of protein phosphatase 2B, calcineurin, with the ryanodine receptor (RyR) in rat cardiac tissue and neonatal cardiomyocytes. The presence of calcineurin, the RyR and FK506-binding protein (FKBP)12.6 in rat cardiac sarcoplasmic reticulum (SR) was identified by Western blot analysis. The possible interactions between calcineurin, the RyR and FKBP12.6 were further studied by co-immunoprecipitation using CHAPS-solubilized cardiac-membrane fractions (CSMFs) or SR preparations. Physical interactions between the RyR and calcineurin were found in the CSMF in the presence of added 100µM Ca2+; however, the interactions were interrupted in the presence of 20mM EGTA, 1µM rapamycin or 1µM FK506, suggesting that the interaction is Ca2+-dependent, and is mediated by FKBP12.6. The Ca2+-dependent interaction between FKBP12.6 and the RyR was also found by co-immunoprecipitation. Effects of calcineurin inhibitors were tested on neonatal-rat-heart cardiomyocytes. Treatment of neonatal cardiomyocytes with 20µM deltamethrin, 10µM cyclosporin A (CsA), or 10µM FK506 led to Ca2+ oscillations in originally quiescent cardiomyocytes. Preincubation of cardiomyocytes with 20µM rapamycin which dissociates FKBP12.6 from the RyR, evoked Ca2+ oscillations, probably due to the leakiness of the RyR. However, Ca2+ oscillations by rapamycin were not further affected by 10µM CsA or 10µM deltamethrin, suggesting that only RyR-associated calcineurin could regulate the channel activities. In spontaneously Ca2+-oscillating cardiomyocytes, CsA or FK506 treatments increased the frequency of oscillations. In 10µM ryanodine-treated cardiomyocytes, CsA failed to induce Ca2+ oscillations. These data show evidence that calcineurin associated with the RyR could modulate Ca2+ release in rat heart.