PMID- 32353354 OWN - NLM STAT- MEDLINE DCOM- 20210712 LR - 20210712 IS - 1095-8584 (Electronic) IS - 0022-2828 (Linking) VI - 143 DP - 2020 Jun TI - Association with SERCA2a directs phospholamban trafficking to sarcoplasmic reticulum from a nuclear envelope pool. PG - 107-119 LID - S0022-2828(20)30116-4 [pii] LID - 10.1016/j.yjmcc.2020.04.025 [doi] AB - AIMS: Phospholamban (PLB) stoichiometrically regulates the cardiac Ca(2+) pump (SERCA2a) in the sarcoplasmic reticulum (SR); but in the nuclear envelope (NE) of cardiomyocytes (CMs), the PLB to SERCA2a molar ratio is higher, which highlights our poor understanding of how SR proteins distribute to their functional subcompartments. By tracking newly made PLB and SERCA2a in CMs, we will elucidate underlying cellular pathways responsible for their unique intracellular distributions. METHODS AND RESULTS: Highly specific monoclonal antibodies were used to compare the subcellular distributions of SERCA2a, PLB, and junctin (JCN) in dog heart tissue. The data supported a view that both non-junctional and junctional SR proteins are all prominently enriched in transverse stretches of SR tubules, along the edges of sarcomeres (SR z-tubules). To understand the genesis of these steady state distributions, we analyzed confocal immunofluorescence images of adult rat CMs after acute expression (12-48 h) of the dog ortholog of PLB (dPLB) or dSERCA2a. Newly made dog proteins in rat CMs were detected using dog-specific monoclonal antibodies. By 12-24 h, dSERCA2a had accumulated within the NE in a punctate pattern, presumably reflecting initial sites of biosynthesis. Over the next 24-48 h, higher levels of dSERCA2a immunofluorescence accumulated in transverse/radial SR tubules, aligned along sarcolemmal transverse (T)-tubules, and extending from NE puncta. The patterns of SR tubules carrying dSERCA2a overlapped with those for newly made JCN, suggesting a common Nuclear Envelope to SR along T-tubules or NEST pathway for SR proteins. In contrast to the SERCA2a distribution pattern, dPLB accumulated uniformly in the NE, without visible puncta. With co-expression of dSERCA2a, however, PLB no longer uniformly filled the NE, but instead moved together with SERCA2a to form bright NE puncta, from which the two proteins then trafficked anterogradely. CONCLUSION: Expression of dog SR protein orthologs (dSERCA2a, dPLB, and dJCN) for as little as 48 h reproduces their characteristic steady state distributions. Detailed analyses of the time courses of protein accumulation suggest a possible mechanism by which PLB distributes to both the NE and SR, unlike SERCA2a. SERCA2a moves in SR z-tubules directly from rough ER, along pathways that are in common with those used by junctional SR proteins. A different trafficking route for PLB away the rough ER/NE led to its accumulation in the NE, a process that may account for its enrichment in NE in situ. Association of SERCA2a with PLB from this NE pool enhanced PLB trafficking along the NEST pathway, contributing to steady state stoichiometry and physiologically regulated SERCA2a. CI - Copyright (c) 2020 The Authors. Published by Elsevier Ltd.. All rights reserved. FAU - He, Wenbo AU - He W AD - Krannert Institute of Cardiology, Indiana University, Indianapolis, IN, USA; Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, China. FAU - Huang, Dayang AU - Huang D AD - Krannert Institute of Cardiology, Indiana University, Indianapolis, IN, USA; Department of Cardiology, The First Affiliated Hospital of Harbin Medical University, Harbin, China. FAU - Guo, Shuai AU - Guo S AD - Krannert Institute of Cardiology, Indiana University, Indianapolis, IN, USA; Department of Cardiology, The First Affiliated Hospital of Harbin Medical University, Harbin, China. FAU - Wang, Danning AU - Wang D AD - Krannert Institute of Cardiology, Indiana University, Indianapolis, IN, USA. FAU - Guo, Jin AU - Guo J AD - Krannert Institute of Cardiology, Indiana University, Indianapolis, IN, USA. FAU - Cala, Steven E AU - Cala SE AD - Krannert Institute of Cardiology, Indiana University, Indianapolis, IN, USA; Department of Physiology, Wayne State University, Detroit, MI, USA. FAU - Chen, Zhenhui AU - Chen Z AD - Krannert Institute of Cardiology, Indiana University, Indianapolis, IN, USA. Electronic address: zhechen@iu.edu. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20200427 PL - England TA - J Mol Cell Cardiol JT - Journal of molecular and cellular cardiology JID - 0262322 RN - 0 (Biomarkers) RN - 0 (Calcium-Binding Proteins) RN - 0 (phospholamban) RN - EC 3.6.3.8 (Sarcoplasmic Reticulum Calcium-Transporting ATPases) RN - SH1WY3R615 (Nocodazole) SB - IM MH - Amino Acid Sequence MH - Animals MH - Biomarkers MH - Calcium-Binding Proteins/*metabolism MH - Female MH - Fluorescent Antibody Technique MH - Humans MH - Myocytes, Cardiac/*metabolism MH - Nocodazole/pharmacology MH - Nuclear Envelope/*metabolism MH - Protein Transport/drug effects MH - Rats MH - Sarcoplasmic Reticulum/*metabolism MH - Sarcoplasmic Reticulum Calcium-Transporting ATPases/*metabolism MH - Signal Transduction/drug effects OTO - NOTNLM OT - Cardiomyocyte OT - Phospholamban OT - SERCA2a OT - Sarcoplasmic reticulum OT - Trafficking COIS- Declaration of Competing Interest None declared. EDAT- 2020/05/01 06:00 MHDA- 2021/07/13 06:00 CRDT- 2020/05/01 06:00 PHST- 2020/01/28 00:00 [received] PHST- 2020/04/08 00:00 [revised] PHST- 2020/04/23 00:00 [accepted] PHST- 2020/05/01 06:00 [pubmed] PHST- 2021/07/13 06:00 [medline] PHST- 2020/05/01 06:00 [entrez] AID - S0022-2828(20)30116-4 [pii] AID - 10.1016/j.yjmcc.2020.04.025 [doi] PST - ppublish SO - J Mol Cell Cardiol. 2020 Jun;143:107-119. doi: 10.1016/j.yjmcc.2020.04.025. Epub 2020 Apr 27.