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

The present study was undertaken to examine the physical and physiological interaction of protein phosphatase 2B, calcineurin, with the ryanodine rece ptor (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 p ossible 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 Ry R and calcineurin were found in the CSMF in the presence of added 100 muM C a2+; however, the interactions we:re interrupted in the presence of 20 mM E GTA, 1 muM rapamycin or 1 muM FK506, suggesting that the interaction is Ca2 +-dependent, and is mediated by FKBP12.6, The Ca2+-dependent interaction be tween FKBP12.6 and the RyR was also found by co-immunoprecipitation. Effect s of calcineurin inhibitors were tested on neonatal-rat-heart cardiomyocyte s, Treatment of neonatal cardiomyocytes with 20 muM deltamethrin, 10 muM cy closporin A (CsA), or 10 muM FK506 led to Ca2+ oscillations in originally q uiescent cardiomyocytes. Preincubation of cardiomyocytes with 20 muM rapamy cin which dissociates FKBP12.6 from the RyR, evoked Ca2+ oscillations, prob ably due to the leakiness of the RyR. However, Ca2+ oscillations by rapamyc in were not further affected by 10 muM CsA or 10 muM deltamethrin, suggesti ng that only RyR-associated calcineurin could regulate the channel activiti es. In spontaneously Ca2+-oscillating cardiomyocytes, CsA or FK506 treatmen ts increased the frequency of oscillations. In 10 muM ryanodine-treated car diomyocytes, CsA failed to induce Ca2+ oscillations. These data show eviden ce that calcineurin associated with the RyR could modulate Ca2+ release in rat heart.