{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,2]],"date-time":"2026-07-02T12:19:17Z","timestamp":1782994757597,"version":"3.54.5"},"posted":{"date-parts":[[2023,5,26]]},"group-title":"Physiology","reference-count":60,"publisher":"openRxiv","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2023,5,28]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  The sarco(endo)plasmic reticulum Ca\n                  <jats:sup>2+<\/jats:sup>\n                  ATPase (SERCA) is a membrane transporter that creates and maintains intracellular Ca\n                  <jats:sup>2+<\/jats:sup>\n                  stores. In the heart, SERCA is regulated by an inhibitory interaction with the monomeric form of the transmembrane micropeptide phospholamban (PLB). PLB also forms avid homo-pentamers, and dynamic exchange of PLB between pentamers and the regulatory complex with SERCA is an important determinant of cardiac responsiveness to exercise. Here, we investigated two naturally occurring pathogenic mutations of PLB, a cysteine substitution of arginine 9 (R9C) and an in-frame deletion of arginine 14 (R14del). Both mutations are associated with dilated cardiomyopathy. We previously showed that the R9C mutation causes disulfide crosslinking and hyperstabilization of pentamers. While the pathogenic mechanism of R14del is unclear, we hypothesized that this mutation may also alter PLB homo-oligomerization and disrupt the PLB-SERCA regulatory interaction. SDS-PAGE revealed a significantly increased pentamer:monomer ratio for R14del-PLB when compared to WT-PLB. In addition, we quantified homo-oligomerization and SERCA-binding in live cells using fluorescence resonance energy transfer (FRET) microscopy. R14del-PLB showed an increased affinity for homo-oligomerization and decreased binding affinity for SERCA compared to WT, suggesting that, like R9C, the R14del mutation stabilizes PLB in its pentameric form, decreasing its ability to regulate SERCA. Moreover, the R14del mutation reduces the rate of PLB unbinding from the pentamer after a transient Ca\n                  <jats:sup>2+<\/jats:sup>\n                  elevation, limiting the rate of re-binding to SERCA. A computational model predicted that hyperstabilization of PLB pentamers by R14del impairs the ability of cardiac Ca\n                  <jats:sup>2+<\/jats:sup>\n                  handling to respond to changing heart rates between rest and exercise. We postulate that impaired responsiveness to physiological stress contributes to arrhythmogenesis in human carriers of the R14del mutation.\n                <\/jats:p>","DOI":"10.1101\/2023.05.26.542463","type":"posted-content","created":{"date-parts":[[2023,5,27]],"date-time":"2023-05-27T00:25:12Z","timestamp":1685147112000},"source":"Crossref","is-referenced-by-count":7,"title":["Dilated cardiomyopathy variant R14del increases phospholamban pentamer stability, blunting dynamic regulation of cardiac calcium handling"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4789-0042","authenticated-orcid":false,"given":"Sean R.","family":"Cleary","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Allen C. T.","family":"Teng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Audrey Deyawe","family":"Kongmeneck","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xuan","family":"Fang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Taylor A.","family":"Phillips","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ellen E.","family":"Cho","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7286-3022","authenticated-orcid":false,"given":"Peter","family":"Kekenes-Huskey","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anthony O.","family":"Gramolini","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1193-9510","authenticated-orcid":false,"given":"Seth L.","family":"Robia","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2023053112000630000_2023.05.26.542463v2.1","doi-asserted-by":"publisher","DOI":"10.1161\/CIRCRESAHA.116.309396"},{"key":"2023053112000630000_2023.05.26.542463v2.2","doi-asserted-by":"publisher","DOI":"10.1093\/eurheartj\/ehm342"},{"key":"2023053112000630000_2023.05.26.542463v2.3","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa1110186"},{"key":"2023053112000630000_2023.05.26.542463v2.4","doi-asserted-by":"publisher","DOI":"10.1016\/j.ahj.2007.07.038"},{"key":"2023053112000630000_2023.05.26.542463v2.5","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0510519103"},{"key":"2023053112000630000_2023.05.26.542463v2.6","doi-asserted-by":"publisher","DOI":"10.1126\/science.1081578"},{"key":"2023053112000630000_2023.05.26.542463v2.7","doi-asserted-by":"publisher","DOI":"10.1093\/cvr\/cvv127"},{"key":"2023053112000630000_2023.05.26.542463v2.8","doi-asserted-by":"publisher","DOI":"10.1172\/JCI200317892"},{"key":"2023053112000630000_2023.05.26.542463v2.9","doi-asserted-by":"publisher","DOI":"10.1093\/eurjhf\/hfs119"},{"key":"2023053112000630000_2023.05.26.542463v2.10","doi-asserted-by":"publisher","DOI":"10.1038\/415198a"},{"key":"2023053112000630000_2023.05.26.542463v2.11","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1016\/j.yjmcc.2022.10.005","article-title":"Early calcium and cardiac contraction defects in a model of phospholamban R9C mutation in zebrafish","volume":"173","year":"2022","journal-title":"J Mol Cell Cardiol"},{"key":"2023053112000630000_2023.05.26.542463v2.12","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M114.630319"},{"key":"2023053112000630000_2023.05.26.542463v2.13","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1016535108"},{"key":"2023053112000630000_2023.05.26.542463v2.14","doi-asserted-by":"crossref","unstructured":"R. Aguayo-Ortiz , L. M. Espinoza-Fonseca , Linking Biochemical and Structural States of SERCA: Achievements, Challenges, and New Opportunities. Int J Mol Sci 21, p(2020).","DOI":"10.3390\/ijms21114146"},{"key":"2023053112000630000_2023.05.26.542463v2.15","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.RA120.012948"},{"key":"2023053112000630000_2023.05.26.542463v2.16","doi-asserted-by":"publisher","DOI":"10.1021\/bi961955q"},{"key":"2023053112000630000_2023.05.26.542463v2.17","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M804782200"},{"key":"2023053112000630000_2023.05.26.542463v2.18","doi-asserted-by":"publisher","DOI":"10.1161\/CIRCRESAHA.107.159947"},{"key":"2023053112000630000_2023.05.26.542463v2.19","doi-asserted-by":"crossref","first-page":"102060","DOI":"10.1016\/j.jbc.2022.102060","article-title":"Inhibitory and stimulatory micropeptides preferentially bind to different conformations of the cardiac calcium pump","volume":"298","year":"2022","journal-title":"J Biol Chem"},{"key":"2023053112000630000_2023.05.26.542463v2.20","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M112.382713"},{"key":"2023053112000630000_2023.05.26.542463v2.21","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M112.360859"},{"key":"2023053112000630000_2023.05.26.542463v2.22","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1013987108"},{"key":"2023053112000630000_2023.05.26.542463v2.23","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0106746"},{"key":"2023053112000630000_2023.05.26.542463v2.24","doi-asserted-by":"crossref","unstructured":"B. Badone et al., Characterization of the PLN p.Arg14del Mutation in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes. Int J Mol Sci 22, (2021).","DOI":"10.3390\/ijms222413500"},{"key":"2023053112000630000_2023.05.26.542463v2.25","doi-asserted-by":"crossref","unstructured":"C. Maniezzi et al., Early consequences of the phospholamban mutation PLN-R14del+\/-in a transgenic mouse model. bioRxiv, 2023.2004.2005.535536 (2023).","DOI":"10.1101\/2023.04.05.535536"},{"key":"2023053112000630000_2023.05.26.542463v2.26","doi-asserted-by":"crossref","unstructured":"J. Qin et al., Structures of PKA-phospholamban complexes reveal a mechanism of familial dilated cardiomyopathy. Elife 11, (2022).","DOI":"10.7554\/eLife.75346"},{"key":"2023053112000630000_2023.05.26.542463v2.27","doi-asserted-by":"crossref","first-page":"e13074","DOI":"10.15252\/emmm.202013074","article-title":"Impairment of the ER\/mitochondria compartment in human cardiomyocytes with PLN p.Arg14del mutation","volume":"13","year":"2021","journal-title":"EMBO Mol Med"},{"key":"2023053112000630000_2023.05.26.542463v2.28","doi-asserted-by":"crossref","first-page":"109448","DOI":"10.1016\/j.celrep.2021.109448","article-title":"Molecular noise filtering in the beta-adrenergic signaling network by phospholamban pentamers","volume":"36","year":"2021","journal-title":"Cell Rep"},{"key":"2023053112000630000_2023.05.26.542463v2.29","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1016\/j.bbamem.2014.09.007","article-title":"Effects of naturally occurring arginine 14 deletion on phospholamban conformational dynamics and membrane interactions","volume":"1848","year":"2015","journal-title":"Biochim Biophys Acta"},{"key":"2023053112000630000_2023.05.26.542463v2.30","doi-asserted-by":"crossref","unstructured":"E. Vafiadaki , K. Haghighi , D. A. Arvanitis , E. G. Kranias , D. Sanoudou , Aberrant PLN-R14del Protein Interactions Intensify SERCA2a Inhibition, Driving Impaired Ca(2+) Handling and Arrhythmogenesis. Int J Mol Sci 23, (2022).","DOI":"10.3390\/ijms23136947"},{"key":"2023053112000630000_2023.05.26.542463v2.31","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.RA118.002472"},{"key":"2023053112000630000_2023.05.26.542463v2.32","doi-asserted-by":"publisher","DOI":"10.1152\/ajpheart.00175.2016"},{"key":"2023053112000630000_2023.05.26.542463v2.33","first-page":"11468","article-title":"Phospholamban phosphorylation in intact ventricles. Phosphorylation of serine 16 and threonine 17 in response to beta-adrenergic stimulation","volume":"264","year":"1989","journal-title":"J Biol Chem"},{"key":"2023053112000630000_2023.05.26.542463v2.34","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2019.07.037"},{"key":"2023053112000630000_2023.05.26.542463v2.35","doi-asserted-by":"crossref","unstructured":"M. Kumar et al., Myofilament Alterations Associated with Human R14del-Phospholamban Cardiomyopathy. Int J Mol Sci 24, (2023).","DOI":"10.3390\/ijms24032675"},{"key":"2023053112000630000_2023.05.26.542463v2.36","doi-asserted-by":"crossref","unstructured":"K. Haghighi et al., Impaired Right Ventricular Calcium Cycling Is an Early Risk Factor in R14del-Phospholamban Arrhythmias. J Pers Med 11, (2021).","DOI":"10.3390\/jpm11060502"},{"key":"2023053112000630000_2023.05.26.542463v2.37","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-020-66656-9"},{"key":"2023053112000630000_2023.05.26.542463v2.38","doi-asserted-by":"publisher","DOI":"10.1016\/j.yjmcc.2011.11.012"},{"key":"2023053112000630000_2023.05.26.542463v2.39","doi-asserted-by":"publisher","DOI":"10.1161\/CIRCULATIONAHA.108.783506"},{"key":"2023053112000630000_2023.05.26.542463v2.40","unstructured":"H. P. Bowditch , \u00dcber die Eigenth\u00fcmlichkeiten der Reizbarkeit, welche die Muskelfasern des Herzens zeigen. Arbeiten aus der Physiologischen Anstalt zu Leipzig, (1871)."},{"key":"2023053112000630000_2023.05.26.542463v2.41","doi-asserted-by":"crossref","first-page":"H249","DOI":"10.1152\/ajpheart.2000.278.1.H249","article-title":"Phospholamban: a major determinant of the cardiac force-frequency relationship","volume":"278","year":"2000","journal-title":"Am J Physiol Heart Circ Physiol"},{"key":"2023053112000630000_2023.05.26.542463v2.42","first-page":"H779","article-title":"Phospholamban-to-SERCA2 ratio controls the force-frequency relationship","volume":"276","year":"1999","journal-title":"Am J Physiol"},{"key":"2023053112000630000_2023.05.26.542463v2.43","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-018-30638-9"},{"key":"2023053112000630000_2023.05.26.542463v2.44","doi-asserted-by":"publisher","DOI":"10.1161\/01.CIR.85.5.1743"},{"key":"2023053112000630000_2023.05.26.542463v2.45","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M111.266759"},{"key":"2023053112000630000_2023.05.26.542463v2.46","doi-asserted-by":"publisher","DOI":"10.1161\/CIRCGENETICS.114.001003"},{"key":"2023053112000630000_2023.05.26.542463v2.47","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMra1509267"},{"key":"2023053112000630000_2023.05.26.542463v2.48","doi-asserted-by":"publisher","DOI":"10.1161\/CIRCULATIONAHA.105.609719"},{"key":"2023053112000630000_2023.05.26.542463v2.49","doi-asserted-by":"crossref","first-page":"157","DOI":"10.1007\/s12471-017-0946-7","article-title":"Potential new mechanisms of pro-arrhythmia in arrhythmogenic cardiomyopathy: focus on calcium sensitive pathways","volume":"25","year":"2017","journal-title":"Neth Heart J"},{"key":"2023053112000630000_2023.05.26.542463v2.50","doi-asserted-by":"crossref","first-page":"1480","DOI":"10.1161\/CIRCULATIONAHA.121.057757","article-title":"Exercise Causes Arrhythmogenic Remodeling of Intracellular Calcium Dynamics in Plakophilin-2-Deficient Hearts","volume":"145","year":"2022","journal-title":"Circulation"},{"key":"2023053112000630000_2023.05.26.542463v2.51","doi-asserted-by":"publisher","DOI":"10.1529\/biophysj.103.022087"},{"key":"2023053112000630000_2023.05.26.542463v2.52","doi-asserted-by":"publisher","DOI":"10.1016\/0263-7855(96)00018-5"},{"key":"2023053112000630000_2023.05.26.542463v2.53","doi-asserted-by":"publisher","DOI":"10.1002\/jcc.20945"},{"key":"2023053112000630000_2023.05.26.542463v2.54","doi-asserted-by":"crossref","first-page":"044130","DOI":"10.1063\/5.0014475","article-title":"Scalable molecular dynamics on CPU and GPU architectures with NAMD","volume":"153","year":"2020","journal-title":"J Chem Phys"},{"key":"2023053112000630000_2023.05.26.542463v2.55","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.4067"},{"key":"2023053112000630000_2023.05.26.542463v2.56","doi-asserted-by":"publisher","DOI":"10.1016\/0021-9991(77)90098-5"},{"key":"2023053112000630000_2023.05.26.542463v2.57","doi-asserted-by":"crossref","unstructured":"W. N. Venables , B. D. Ripley , in Modern Applied Statistics with S-PLUS. (Springer New York, New York, NY, 1999), pp. 401\u2013431.","DOI":"10.1007\/978-1-4757-3121-7_13"},{"key":"2023053112000630000_2023.05.26.542463v2.58","doi-asserted-by":"publisher","DOI":"10.1002\/pro.3280"},{"key":"2023053112000630000_2023.05.26.542463v2.59","doi-asserted-by":"publisher","DOI":"10.1021\/ct400341p"},{"key":"2023053112000630000_2023.05.26.542463v2.60","doi-asserted-by":"publisher","DOI":"10.1016\/j.ceca.2017.11.006"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2023.05.26.542463","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T04:41:36Z","timestamp":1768452096000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2023.05.26.542463"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,5,26]]},"references-count":60,"URL":"https:\/\/doi.org\/10.1101\/2023.05.26.542463","relation":{},"subject":[],"published":{"date-parts":[[2023,5,26]]},"subtype":"preprint"}}