Phospholamban is concentrated in the nuclear envelope of cardiomyocytes and involved in perinuclear/nuclear calcium handling

Abstract Aims Phospholamban (PLB) regulates the cardiac Ca 2 + -ATPase (SERCA2a) in sarcoplasmic reticulum (SR). However, the localization of PLB at subcellular sites outside the SR and possible contributions to Ca 2 + cycling remain unknown. We examined the intracellular distribution of PLB and tested whether a pool of PLB exists in the nuclear envelope (NE) that might regulate perinuclear/nuclear Ca 2 + (nCa 2 + ) handling in cardiomyocytes (CMs). Methods and results Using confocal immunofluorescence microscopy and immunoblot analyses of CMs and CM nuclei, we discovered that PLB was highly concentrated in NE. Moreover, the ratio of PLB levels to SERCA levels was greater in NE than in SR. The increased levels of PLB in NE were a consistent finding using a range of antibodies, tissue samples, and species. To address a possible role in affecting Ca 2 + handling, we used Fluo-4 based confocal Ca 2 + imaging, with scan-lines across cytosol and nuclei, and evaluated the effects of PLB on cytosolic and nCa 2 + uptake and release in mouse CMs. In intact CMs, isoproterenol increased amplitude and decreased the decay time of Ca 2 + transients not only in cytosol but also in nuclear regions. In saponin-permeabilized mouse CMs ([Ca 2 + ] i  = 400 nM), we measured spontaneous Ca 2 + waves after specific reversal of PLB activity by addition of the Fab fragment of an anti-PLB monoclonal antibody (100 μg/ml). This highly selective immunological reagent enhanced Ca 2 + uptake (faster decay times) and Ca 2 + release (greater intensity) in both cytosol and across the nuclear regions. Conclusions Besides SR, PLB is concentrated in NE of CMs, and may be involved in modulation of nCa 2 + dynamics.