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

Abstract

Aims: Phospholamban (PLB) regulates the cardiac Ca²⁺-ATPase (SERCA2a) in sarcoplasmic reticulum (SR). However, the localization of PLB at subcellular sites outside the SR and possible contributions to Ca²⁺ 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 Ca²⁺ (nCa²⁺) 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 Ca²⁺ handling, we used Fluo-4 based confocal Ca²⁺ imaging, with scan-lines across cytosol and nuclei, and evaluated the effects of PLB on cytosolic and nCa²⁺ uptake and release in mouse CMs. In intact CMs, isoproterenol increased amplitude and decreased the decay time of Ca²⁺ transients not only in cytosol but also in nuclear regions. In saponin-permeabilized mouse CMs ([Ca²⁺]_i=400nM), we measured spontaneous Ca²⁺ waves after specific reversal of PLB activity by addition of the Fab fragment of an anti-PLB monoclonal antibody (100μg/ml). This highly selective immunological reagent enhanced Ca²⁺ uptake (faster decay times) and Ca²⁺ 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 nCa²⁺ dynamics.

Keywords: Calcium signaling; Cardiomyocyte; Nuclear membranes; Perinuclear Ca(2+) dynamics; Phospholamban; Sarcoplasmic reticulum Ca(2+) ATPase.