Abstract 16191: Phospholamban Regulates Perinuclear/nuclear Calcium Dynamics in Cardiomyocytes
Introduction: Phospholamban (PLB) regulates cardiac sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a), thus modulating SR Ca2+ dynamics. Recent studies demonstrated that SERCA is involved in Ca2+ uptake into the lumen of nuclear envelope (NE) of cardiomyocytes (CMs). However, the regulatory role of PLB on Ca2+ uptake into NE remains unknown.
Hypothesis: We hypothesize that PLB is also responsible for modulating nuclear Ca2+ dynamics.
Methods: Confocal immunofluorescence microscopy was used to determine subcellular expression of PLB. By using fluo-4 based confocal line-scan Ca2+ imaging, we measured spontaneous Ca2+ 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 Ca2+ uptake and release. SCWs through the nuclear regions had typically relative low fluorescent amplitude (F/F0) and slow decay time (t1/2) compared to that in the cytoplasmic region. At the free intracellular Ca2+ concentration ([Ca2+]i) of 400 nM, Fab (100 μg/mL) significantly enhanced F/F0 and decreased t1/2 of SCWs in both cytoplasmic and nuclear regions. After addition of Fab, F/F0of SCWs through the nuclear regions increased from 0.91±0.16 to 1.27±0.19 (n=9, p<.05) while t1/2 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/F0=1.43±0.11 vs. 1.04±0.14 in control, p<.05; t1/2=107.82±10.9 ms vs. 139.21±20.1 ms in control, n=6, p<.05). At high [Ca2+]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 Ca2+ uptake and release. PLB is critically involved in nuclear Ca2+ signaling modulation.
- spontaneous Ca2+ waves
- Fab fragment of anti-PLB monoclonal antibody
- nuclear Ca2+ signaling
Author Disclosures: A.Z. Wu: None. Y. Chan: None. S. Lin: None. P. Chen: None. Z. Chen: None.
- © 2015 by American Heart Association, Inc.