Abstract 16191: Phospholamban Regulates Perinuclear/nuclear Calcium Dynamics in Cardiomyocytes

Abstract

Introduction: Phospholamban (PLB) regulates cardiac sarcoplasmic reticulum (SR) Ca 2+ -ATPase (SERCA2a), thus modulating SR Ca 2+ dynamics. Recent studies demonstrated that SERCA is involved in Ca 2+ uptake into the lumen of nuclear envelope (NE) of cardiomyocytes (CMs). However, the regulatory role of PLB on Ca 2+ uptake into NE remains unknown. Hypothesis: We hypothesize that PLB is also responsible for modulating nuclear Ca 2+ dynamics. Methods: Confocal immunofluorescence microscopy was used to determine subcellular expression of PLB. By using fluo-4 based confocal line-scan Ca 2+ imaging, we measured spontaneous Ca 2+ waves (SCWs) across both cytoplasmic and nuclear regions in isolated permeabilized mouse CMs. Results: Several anti-PLB antibodies strongly stained PLB at both SR and the perinuclear membranes in CMs. A PLB peptide (residues 1-31) eliminated all these anti-PLB antibody stains. To identify the functional role of PLB expressed in the perinuclear membranes, we took advantage of our recently established method that a Fab fragment of anti-PLB monoclonal antibody (Fab) reversed PLB inhibition specifically and increased SR Ca 2+ uptake and release. SCWs through the nuclear regions had typically relative low fluorescent amplitude (F/F 0 ) and slow decay time (t 1/2 ) compared to that in the cytoplasmic region. At the free intracellular Ca 2+ concentration ([Ca 2+ ] i ) of 400 nM, Fab (100 μg/mL) significantly enhanced F/F 0 and decreased t 1/2 of SCWs in both cytoplasmic and nuclear regions. After addition of Fab, F/F 0 of SCWs through the nuclear regions increased from 0.91±0.16 to 1.27±0.19 (n=9, p<.05) while t 1/2 decreased from 137.6±18.5 ms to 105.0±11.3 ms, (p<.05). Similar effects were also observed after phosphorylation of PLB by addition of 20 μM cAMP (F/F 0 =1.43±0.11 vs. 1.04±0.14 in control, p<.05; t 1/2 =107.82±10.9 ms vs. 139.21±20.1 ms in control, n=6, p<.05). At high [Ca 2+ ] i of 1000 nM where PLB does not inhibit SERCA2a, addition of cAMP or Fab had no significant effect on SCWs in both cytoplasmic and nuclear regions. Conclusions: We demonstrated that PLB is expressed in and around NE. Acute removal of PLB inhibition increased perinuclear/nuclear Ca 2+ uptake and release. PLB is critically involved in nuclear Ca 2+ signaling modulation.