title: Acute reversal of phospholamban inhibition facilitates the rhythmic whole-cell propagating calcium waves in isolated ventricular myocytes.
abstract: Phospholamban (PLB) inhibits the activity of cardiac sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a). Phosphorylation of PLB during sympathetic activation reverses SERCA2a inhibition, increasing SR Ca(2+) uptake. However, sympathetic activation also modulates multiple other intracellular targets in ventricular myocytes (VMs), making it impossible to determine the specific effects of the reversal of PLB inhibition on the spontaneous SR Ca(2+) release. Therefore, it remains unclear how PLB regulates rhythmic activity in VMs. Here, we used the Fab fragment of 2D12, a monoclonal anti-PLB antibody, to test how acute reversal of PLB inhibition affects the spontaneous SR Ca(2+) release in normal VMs. Ca(2+) sparks and spontaneous Ca(2+) waves (SCWs) were recorded in the line-scan mode of confocal microscopy using the Ca(2+) fluorescent dye Fluo-4 in isolated permeabilized mouse VMs. Fab, which reverses PLB inhibition, significantly increased the frequency, amplitude, and spatial/temporal spread of Ca(2+) sparks in VMs exposed to 50 nM free [Ca(2+)]. At physiological diastolic free [Ca(2+)] (100-200 nM), Fab facilitated the formation of whole-cell propagating SCWs. At higher free [Ca(2+)], Fab increased the frequency and velocity, but decreased the decay time of the SCWs. cAMP had little additional effect on the frequency or morphology of Ca(2+) sparks or SCWs after Fab addition. These findings were complemented by computer simulations. In conclusion, acute reversal of PLB inhibition alone significantly increased the spontaneous SR Ca(2+) release, leading to the facilitation and organization of whole-cell propagating SCWs in normal VMs. PLB thus plays a key role in subcellular Ca(2+) dynamics and rhythmic activity of VMs.
authors.affiliation: Krannert Institute of Cardiology and Division of Cardiology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA; Division of Cardiology, Department of Internal Medicine, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Linkou, Taoyuan, Taiwan.
authors.forename: Yi-Hsin
authors.initials: YH
authors.lastname: Chan
authors.affiliation: Krannert Institute of Cardiology and Division of Cardiology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA; Division of Cardiology, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung University College of Medicine, Kaohsiung, Taiwan.
authors.forename: Wei-Chung
authors.initials: WC
authors.lastname: Tsai
authors.affiliation: Division of Cardiology, Department of Medicine, University of California, Los Angeles, CA, USA.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Song
authors.affiliation: Division of Cardiology, Department of Medicine, University of California, Los Angeles, CA, USA.
authors.forename: Christopher Y
authors.initials: CY
authors.lastname: Ko
authors.affiliation: Division of Cardiology, Department of Medicine, University of California, Los Angeles, CA, USA.
authors.forename: Zhilin
authors.initials: Z
authors.lastname: Qu
authors.affiliation: Division of Cardiology, Department of Medicine, University of California, Los Angeles, CA, USA.
authors.forename: James N
authors.initials: JN
authors.lastname: Weiss
authors.affiliation: Krannert Institute of Cardiology and Division of Cardiology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA; Institute of Biomedical Engineering, National Chiao-Tung University, Hsin-Chu, Taiwan.
authors.forename: Shien-Fong
authors.initials: SF
authors.lastname: Lin
authors.affiliation: Krannert Institute of Cardiology and Division of Cardiology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA.
authors.forename: Peng-Sheng
authors.initials: PS
authors.lastname: Chen
authors.affiliation: Krannert Institute of Cardiology and Division of Cardiology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA.
authors.forename: Larry R
authors.initials: LR
authors.lastname: Jones
authors.affiliation: Krannert Institute of Cardiology and Division of Cardiology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA. Electronic address: zhechen@iu.edu.
authors.forename: Zhenhui
authors.initials: Z
authors.lastname: Chen
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D002135:Calcium-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002478:Cells, Cultured; D003198:Computer Simulation; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D007140:Immunoglobulin Fab Fragments / Q000494:pharmacology; D051379:Mice; D008954:Models, Biological; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism; D000099211:Phospholamban
chemical_list: D000911:Antibodies, Monoclonal; D002118:Calcium; D002135:Calcium-Binding Proteins; D000242:Cyclic AMP; D007140:Immunoglobulin Fab Fragments; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban
keywords: Calcium; Phospholamban; Sarcoplasmic reticulum; Spontaneous calcium waves