{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T12:13:43Z","timestamp":1790252023594,"version":"4.1.0"},"reference-count":43,"publisher":"Rockefeller University Press","issue":"3","license":[{"start":{"date-parts":[[2023,1,12]],"date-time":"2023-01-12T00:00:00Z","timestamp":1673481600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01 HL141564"],"award-info":[{"award-number":["R01 HL141564"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100007899","name":"University of Arizona","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100007899","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["rupress.org"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2023,3,6]]},"abstract":"<jats:p>Cardiac muscle contraction is regulated by Ca2+-induced structural changes of the thin filaments to permit myosin cross-bridge cycling driven by ATP hydrolysis in the sarcomere. In congestive heart failure, contraction is weakened, and thus targeting the contractile proteins of the sarcomere is a promising approach to therapy. However, development of novel therapeutic interventions has been challenging due to a lack of precise discovery tools. We have developed a fluorescence lifetime-based assay using an existing site-directed probe, N,N\u2032-dimethyl-N-(iodoacetyl)-N\u2032-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD) attached to human cardiac troponin C (cTnC) mutant cTnCT53C, exchanged into porcine cardiac myofibrils. We hypothesized that IANBD-cTnCT53C fluorescence lifetime measurements provide insight into the activation state of the thin filament. The sensitivity and precision of detecting structural changes in cTnC due to physiological and therapeutic modulators of thick and thin filament functions were determined. The effects of Ca2+ binding to cTnC and myosin binding to the thin filament were readily detected by this assay in mock high-throughput screen tests using a fluorescence lifetime plate reader. We then evaluated known effectors of altered cTnC-Ca2+ binding, W7 and pimobendan, and myosin-binding drugs, mavacamten and omecamtiv mecarbil, used to treat cardiac diseases. Screening assays were determined to be of high quality as indicated by the Z\u2032 factor. We conclude that cTnC lifetime-based probes allow for precise evaluation of the thin filament activation in functioning myofibrils that can be used in future high-throughput screens of small-molecule modulators of function of the thin and thick filaments.<\/jats:p>","DOI":"10.1085\/jgp.202113054","type":"journal-article","created":{"date-parts":[[2022,12,21]],"date-time":"2022-12-21T02:20:33Z","timestamp":1671589233000},"update-policy":"https:\/\/doi.org\/10.1085\/jgp.crossmarkpolicy","source":"Crossref","is-referenced-by-count":3,"title":["Fluorescence lifetime-based assay reports structural changes in cardiac muscle mediated by effectors of contractile regulation"],"prefix":"10.1085","volume":"155","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1514-4747","authenticated-orcid":true,"given":"Alexey V.","family":"Dvornikov","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03m2x1q45","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Cellular and Molecular Medicine, University of Arizona 1 , Tucson, AZ, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas A.","family":"Bunch","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03m2x1q45","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Cellular and Molecular Medicine, University of Arizona 1 , Tucson, AZ, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Victoria C.","family":"Lepak","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03m2x1q45","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Cellular and Molecular Medicine, University of Arizona 1 , Tucson, AZ, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5721-4320","authenticated-orcid":true,"given":"Brett A.","family":"Colson","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03m2x1q45","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Cellular and Molecular Medicine, University of Arizona 1 , Tucson, AZ, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"291","published-online":{"date-parts":[[2023,1,12]]},"reference":[{"key":"2023011215045899500_bib1","doi-asserted-by":"publisher","first-page":"14634","DOI":"10.1074\/jbc.RA118.005098","article-title":"FRET-based analysis of the cardiac troponin T linker region reveals the structural basis of the hypertrophic cardiomyopathy-causing \u0394160E mutation","volume":"294","author":"Abdullah","year":"2019","journal-title":"J. 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