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

Aims: Phospholamban (PLB) is the key regulator of the cardiac Ca pump (SERCA2a)-mediated sarcoplasmic reticulum Ca stores. We recently reported that PLB is highly concentrated in the nuclear envelope (NE) from where it can modulate perinuclear Ca handling of the cardiomyocytes (CMs). Since inositol 1,4,5-trisphosphate (IP) receptor (IPR) mediates nuclear Ca release, we examined whether the nuclear pool of PLB regulates IP-induced nuclear Ca handling.

Methods And Results: Fluo-4 based confocal Ca imaging was performed to measure Ca dynamics across both nucleus and cytosol in saponin-permeabilized CMs isolated from wild-type (WT) or PLB-knockout (PLB-KO) mice. At diastolic intracellular Ca ([Ca] = 100 nM), the Fab fragment of the monoclonal PLB antibody (anti-PLB Fab) facilitated the formation and increased the length of spontaneous Ca waves (SCWs) originating from the nuclear region in CMs from WT but not from PLB-KO mice. We next examined nuclear Ca activities at basal condition and after sequential addition of IP, anti-PLB Fab, and the IPR inhibitor 2-aminoethoxydiphenyl borate (2-APB) at a series of [Ca]. In WT mice, at 10 nM [Ca] where ryanodine receptor (RyR2) based spontaneous Ca sparks rarely occurred, IP increased fluorescence amplitude (F/F) of overall nuclear region to 1.19 ± 0.02. Subsequent addition of anti-PLB Fab significantly decreased F/F to 1.09 ± 0.02. At 50 nM [Ca], anti-PLB Fab not only decreased the overall nuclear F/F previously elevated by IP, but also increased the amplitude and duration of spark-like nuclear Ca release events. These nuclear Ca releases were blocked by 2-APB. At 100 nM [Ca], IP induced 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 Ca 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, IP and anti-PLB Fab still increased the magnitude of nuclear Ca release but failed to regenerate SCWs. Finally, anti-PLB Fab increased low Ca affinity mag-fluo 4 fluorescence intensity in the lumen of NE of nuclei isolated from WT but not in PLB-KO mice.

Conclusion: PLB regulates nuclear Ca handling. By increasing Ca uptake into lumen of the NE and perhaps other perinuclear membranes, the acute reversal of PLB inhibition decreases global Ca concentration at rest in the nucleoplasm, and increases Ca release into the nucleus, through mechanisms involving IPR and RyR2 in the vicinity.