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In this study, we compared the effects of nNOS inhibition on contractile function, Ca2+ signaling, and myofilament-associated regulation in RVCMs and left ventricular cardiomyocytes (LVCMs). Pharmacological inhibition of nNOS with S-methyl-L-thiocitrulline (SMTC) enhanced sarcomere shortening and impaired relaxation in both cell types, with markedly greater effects observed in RVCMs. In contrast, SMTC-induced changes in Ca2+ transients and L-type Ca2+ current were more pronounced in LVCMs than in RVCMs, indicating a dissociation between Ca2+ signaling and mechanical responses in the SMTC-treated RVCMs. Analysis of dynamic Ca2+-sarcomere length coupling indicated that nNOS inhibition in RVCMs preferentially altered the relationship between intracellular Ca2+ signaling and sarcomere response, particularly during relaxation, rather than simply reflecting changes in Ca2+ magnitude. Consistent with this interpretation, inhibition of nNOS reduced cardiac troponin I (cTnI) Ser23\/24 phosphorylation and was associated with altered endogenous Ca2+ buffering properties in RVCMs. Moreover, myofilament-enriched fractions from RVCMs exhibited higher levels of Ser1417-phosphorylated nNOS\u03b2. Collectively, these data indicate that myofilament-associated nNOS\u03b2 predominantly regulates RV contractility through cTnI phosphorylation-mediated Ca2+ desensitization.<\/jats:p>","DOI":"10.1085\/jgp.202613982","type":"journal-article","created":{"date-parts":[[2026,9,5]],"date-time":"2026-09-05T06:20:41Z","timestamp":1788589241000},"update-policy":"https:\/\/doi.org\/10.1085\/jgp.crossmarkpolicy","source":"Crossref","is-referenced-by-count":0,"title":["Chamber-specific regulation of myofilament Ca2+ sensitivity by nNOS\u03b2 via troponin I phosphorylation: Predominance in the right ventricle"],"prefix":"10.1085","volume":"158","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4635-319X","authenticated-orcid":true,"given":"Jae Won","family":"Kwon","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04h9pn542","id-type":"ROR","asserted-by":"publisher"}],"name":"Seoul National University College of Medicine 1 Department of Physiology, , Seoul,","place":["Republic of 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