Phospholamban regulates nuclear Ca 2+ stores and inositol 1,4,5-trisphosphate mediated nuclear Ca 2+ cycling in cardiomyocytes

摘要

AimsPhospholamban (PLB) is the key regulator of the cardiac Ca2+pump (SERCA2a)-mediated sarcoplasmic reticulum Ca2+stores. We recently reported that PLB is highly concentrated in the nuclear envelope (NE) from where it can modulate perinuclear Ca2+handling of the cardiomyocytes (CMs). Since inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) mediates nuclear Ca2+release, we examined whether the nuclear pool of PLB regulates IP3-induced nuclear Ca2+handling. Methods and resultsFluo-4 based confocal Ca2+imaging was performed to measure Ca2+dynamics across both nucleus and cytosol in saponin-permeabilized CMs isolated from wild-type (WT) or PLB-knockout (PLB-KO) mice. At diastolic intracellular Ca2+([Ca2+]i?=?100?nM), the Fab fragment of the monoclonal PLB antibody (anti-PLB Fab) facilitated the formation and increased the length of spontaneous Ca2+waves (SCWs) originating from the nuclear region in CMs from WT but not from PLB-KO mice. We next examined nuclear Ca2+activities at basal condition and after sequential addition of IP3, anti-PLB Fab, and the IP3R inhibitor 2-aminoethoxydiphenyl borate (2-APB) at a series of [Ca2+]i. In WT mice, at 10?nM [Ca2+]iwhere ryanodine receptor (RyR2) based spontaneous Ca2+sparks rarely occurred, IP3increased fluorescence amplitude (F/F0) of overall nuclear region to 1.19?±?0.02. Subsequent addition of anti-PLB Fab significantly decreased F/F0to 1.09?±?0.02. At 50?nM [Ca2+]i, anti-PLB Fab not only decreased the overall nuclear F/F0previously elevated by IP3, but also increased the amplitude and duration of spark-like nuclear Ca2+release events. These nuclear Ca2+releases were blocked by 2-APB. At 100?nM [Ca2+]i, IP3induced short SCWs originating from nucleus. Anti-PLB Fab transformed those short waves into long SCWs with propagation from the nucleus into the cytosol. In contrast, neither nuclear nor cytosolic Ca2+dynamics was affected by anti-PLB Fab in CMs from PLB-KO mice in all these conditions. Furthermore, in WT CMs pretreated with RyR2 blocker tetracaine, IP3and anti-PLB Fab still increased the magnitude of nuclear Ca2+release but failed to regenerate SCWs. Finally, anti-PLB Fab increased low Ca2+affinity mag-fluo 4 fluorescence intensity in the lumen of NE of nuclei isolated from WT but not in PLB-KO mice. ConclusionPLB regulates nuclear Ca2+handling. By increasing Ca2+uptake into lumen of the NE and perhaps other perinuclear membranes, the acute reversal of PLB inhibition decreases global Ca2+concentration at rest in the nucleoplasm, and increases Ca2+release into the nucleus, through mechanisms involving IP3R and RyR2 in the vicinity.