{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T16:59:06Z","timestamp":1785430746293,"version":"3.56.0"},"posted":{"date-parts":[[2025,10,1]]},"group-title":"Biochemistry","reference-count":27,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,10,1]],"date-time":"2025-10-01T00:00:00Z","timestamp":1759276800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"award":["HL141698"],"award-info":[{"award-number":["HL141698"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["HL165137"],"award-info":[{"award-number":["HL165137"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["HL181346"],"award-info":[{"award-number":["HL181346"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,10,1]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Approximately 40% of genetic hypertrophic cardiomyopathy cases involve mutations in\n                  <jats:italic>MYBPC3<\/jats:italic>\n                  , which encodes cardiac myosin binding protein-C (cMyBP-C), a key regulator of sarcomere contractility. The atrial-specific paralog, myosin binding protein-H like (MyBP-HL), has been associated with dilated cardiomyopathy in humans and mice. Both proteins bind to the same binding sites in the thick filament C-zone. In the atria, cMyBP-C and MyBP-HL are found at \u223c1:1 ratios, while ventricles only express cMyBP-C, which is found at twice the atrial level, indicating a stoichiometric relationship. In the atria, we hypothesize that missense variants in either gene may cause alterations in thick filament binding affinity and changes in the normal \u223c1:1 ratio. Notably, MyBP-HL deletion in atrial myofibrils accelerates relaxation kinetics, suggesting that altered stoichiometry impacts biophysical parameters.\n                <\/jats:p>\n                <jats:p>\n                  We hypothesized that deletion, overexpression, or missense variants in either gene would alter the abundance of the other protein in atrial sarcomeres, affecting sarcomere localization and function. To test this, we engineered two constructs: a mini-C construct comprising thick filament-binding domains of cMyBP-C and a MyBP-HL construct. Selected\n                  <jats:italic>MYBPC3<\/jats:italic>\n                  and\n                  <jats:italic>MYBPHL<\/jats:italic>\n                  missense variants were introduced and expressed in neonatal rat ventricular cardiomyocytes (NRVMs). Sarcomere localization was assessed by co-localization with endogenous cMyBP-C.\n                  <jats:italic>MYBPC3<\/jats:italic>\n                  variants were selected across a range of pathogenicity, while MYBPHL variants were based on evolutionary conservation of residues.\n                  <jats:italic>MYBPHL<\/jats:italic>\n                  variants Gly275Ser, Arg285His, and Ala342Thr induced significant sarcomere mislocalization, and\n                  <jats:italic>MYBPC3<\/jats:italic>\n                  variants Pro1181Ala and Asn1257Lys showed variable effects on sarcomere mislocalization.\n                <\/jats:p>\n                <jats:p>\n                  To assess stoichiometric effects, we developed a T2A\/P2A polycistronic construct to co-express mini-C, Td-Tomato, and MyBP-HL. Immunoblotting and mass spectrometry confirmed consistent and reproducible expression. We identified several\n                  <jats:italic>MYBPC3<\/jats:italic>\n                  and MYBPHL variants that reduced the affinity of their protein for myofilament incorporation. These results suggest that this 2A construct is a useful tool for measuring the effect of myosin binding protein missense variants on sarcomere affinity, with implications for assessing pathogenicity of these variants.\n                <\/jats:p>","DOI":"10.1101\/2025.09.29.679322","type":"posted-content","created":{"date-parts":[[2025,10,1]],"date-time":"2025-10-01T20:50:13Z","timestamp":1759351813000},"source":"Crossref","is-referenced-by-count":0,"title":["Missense variants in the myosin binding domains of\n                  <i>MYBPC3<\/i>\n                  and\n                  <i>MYBPHL<\/i>\n                  impair sarcomere incorporation"],"prefix":"10.64898","author":[{"given":"Kelly N.","family":"Araujo","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hannah E.","family":"Cizauskas","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yoldas","family":"Yildiz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Geena E.","family":"Fritzmann","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tuan","family":"Bui","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lucas M.","family":"Wittenkeller","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexandra","family":"Pe\u00f1a","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Toni R.","family":"Pak","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6166-640X","authenticated-orcid":false,"given":"David Y.","family":"Barefield","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"issue":"9","key":"2025100401001159000_2025.09.29.679322v1.1","first-page":"778","article-title":"Cardiomyopathy: an overview","volume":"79","year":"2009","journal-title":"Am Fam Physician"},{"key":"2025100401001159000_2025.09.29.679322v1.2","doi-asserted-by":"publisher","DOI":"10.1172\/JCI62862"},{"key":"2025100401001159000_2025.09.29.679322v1.3","doi-asserted-by":"publisher","DOI":"10.1007\/s00424-019-02259-2"},{"key":"2025100401001159000_2025.09.29.679322v1.4","doi-asserted-by":"publisher","DOI":"10.1016\/j.cmet.2015.01.013"},{"key":"2025100401001159000_2025.09.29.679322v1.5","doi-asserted-by":"publisher","DOI":"10.1161\/01.CIR.0000066323.15244.54"},{"issue":"1","key":"2025100401001159000_2025.09.29.679322v1.6","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1038\/s41598-019-56597-3","article-title":"Troponin destabilization impairs sarcomere-cytoskeleton interactions in iPSC-derived cardiomyocytes from dilated cardiomyopathy patients","volume":"10","year":"2020","journal-title":"Sci Rep"},{"key":"2025100401001159000_2025.09.29.679322v1.7","doi-asserted-by":"publisher","DOI":"10.1016\/j.yjmcc.2013.04.021"},{"key":"2025100401001159000_2025.09.29.679322v1.8","doi-asserted-by":"publisher","DOI":"10.1056\/NEJM200012073432304"},{"key":"2025100401001159000_2025.09.29.679322v1.9","doi-asserted-by":"publisher","DOI":"10.1161\/CIRCULATIONAHA.117.033200"},{"issue":"23","key":"2025100401001159000_2025.09.29.679322v1.10","doi-asserted-by":"crossref","first-page":"e021970","DOI":"10.1161\/JAHA.121.021970","article-title":"Atrial Fibrillation in Patients With Cardiomyopathy: Prevalence and Clinical Outcomes From Real-World Data","volume":"10","year":"2021","journal-title":"J Am Heart Assoc"},{"key":"2025100401001159000_2025.09.29.679322v1.11","doi-asserted-by":"crossref","first-page":"140","DOI":"10.1016\/j.ijcard.2020.08.062","article-title":"Atrial fibrillation in dilated cardiomyopathy: Outcome prediction from an observational registry","volume":"323","year":"2021","journal-title":"Int J Cardiol"},{"key":"2025100401001159000_2025.09.29.679322v1.12","doi-asserted-by":"publisher","DOI":"10.1136\/heart.86.5.516"},{"key":"2025100401001159000_2025.09.29.679322v1.13","doi-asserted-by":"publisher","DOI":"10.1016\/S0735-1097(01)01727-2"},{"key":"2025100401001159000_2025.09.29.679322v1.14","doi-asserted-by":"publisher","DOI":"10.1161\/hc4601.097997"},{"issue":"5","key":"2025100401001159000_2025.09.29.679322v1.15","first-page":"1200","article-title":"Predictors of Atrial Fibrillation Risk in Hypertrophic Cardiomyopathy","volume":"7","year":"2015","journal-title":"J Atr Fibrillation"},{"issue":"3","key":"2025100401001159000_2025.09.29.679322v1.16","doi-asserted-by":"crossref","first-page":"H568","DOI":"10.1152\/ajpheart.00252.2023","article-title":"Fine tuning contractility: atrial sarcomere function in health and disease","volume":"326","year":"2024","journal-title":"Am J Physiol Heart Circ Physiol"},{"issue":"51","key":"2025100401001159000_2025.09.29.679322v1.17","doi-asserted-by":"crossref","first-page":"e2314920120","DOI":"10.1073\/pnas.2314920120","article-title":"Myosin-binding protein H-like regulates myosin-binding protein distribution and function in atrial cardiomyocytes","volume":"120","year":"2023","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"9","key":"2025100401001159000_2025.09.29.679322v1.18","doi-asserted-by":"crossref","first-page":"1663","DOI":"10.1002\/ar.22966","article-title":"Cell biology of sarcomeric protein engineering: disease modeling and therapeutic potential","volume":"297","year":"2014","journal-title":"Anat Rec (Hoboken)"},{"issue":"5","key":"2025100401001159000_2025.09.29.679322v1.19","doi-asserted-by":"crossref","first-page":"396","DOI":"10.1161\/CIRCGEN.120.002929","article-title":"Spatial and Functional Distribution of MYBPC3 Pathogenic Variants and Clinical Outcomes in Patients With Hypertrophic Cardiomyopathy","volume":"13","year":"2020","journal-title":"Circ Genom Precis Med"},{"key":"2025100401001159000_2025.09.29.679322v1.20","doi-asserted-by":"publisher","DOI":"10.1007\/s11886-023-01876-9"},{"key":"2025100401001159000_2025.09.29.679322v1.21","doi-asserted-by":"publisher","DOI":"10.1016\/j.yjmcc.2014.11.018"},{"key":"2025100401001159000_2025.09.29.679322v1.22","doi-asserted-by":"crossref","unstructured":"Helms, A.S. , et al., Effects of MYBPC3 loss-of-function mutations preceding hypertrophic cardiomyopathy. JCI Insight, 2020. 5(2).","DOI":"10.1172\/jci.insight.133782"},{"key":"2025100401001159000_2025.09.29.679322v1.23","doi-asserted-by":"crossref","unstructured":"Barefield, D.Y. , Is haploinsufficiency a sufficient mechanism for MYBPC3 truncating mutations? J Gen Physiol, 2023. 155(4).","DOI":"10.1085\/jgp.202313351"},{"key":"2025100401001159000_2025.09.29.679322v1.24","doi-asserted-by":"crossref","unstructured":"De Lange, W.J. , et al., cMyBP-C ablation in human engineered cardiac tissue causes progressive Ca2+-handling abnormalities. J Gen Physiol, 2023. 155(4).","DOI":"10.1085\/jgp.202213204"},{"key":"2025100401001159000_2025.09.29.679322v1.25","doi-asserted-by":"publisher","DOI":"10.1089\/ars.2017.7236"},{"issue":"16","key":"2025100401001159000_2025.09.29.679322v1.26","doi-asserted-by":"crossref","first-page":"1238","DOI":"10.1161\/CIRCULATIONAHA.121.056265","article-title":"Muscle LIM Protein Force-Sensing Mediates Sarcomeric Biomechanical Signaling in Human Familial Hypertrophic Cardiomyopathy","volume":"145","year":"2022","journal-title":"Circulation"},{"key":"2025100401001159000_2025.09.29.679322v1.27","doi-asserted-by":"crossref","unstructured":"Clippinger, S.R. , et al., Mechanical dysfunction of the sarcomere induced by a pathogenic mutation in troponin T drives cellular adaptation. J Gen Physiol, 2021. 153(5).","DOI":"10.1085\/jgp.202012787"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.09.29.679322","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T09:42:15Z","timestamp":1768470135000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.09.29.679322"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,10,1]]},"references-count":27,"URL":"https:\/\/doi.org\/10.1101\/2025.09.29.679322","relation":{},"subject":[],"published":{"date-parts":[[2025,10,1]]},"subtype":"preprint"}}