Molecular Function of the C-terminal Domain of Cardiac Troponin I

Abstract

Ca2+ regulation of cardiac muscle contraction is dependent upon regulation by tropomyosin (Tm) and troponin (Tn); the extreme C-terminus of the inhibitory subunit of Troponin (cTnI) binds to actin at low [Ca2+] and is presumed to hold Tm in a closed position preventing actomyosin interaction. cTnI's C terminus (“mobile domain” (MD)) is the site of several human mutations that lead to familial hypertrophic cardiomyopathy (FHC), therefore it is of interest to clarify the specific function and importance of this domain in cardiac muscle contraction. We have demonstrated that even in the absence of Tm, Tn is able to enhance thin filament sliding speed and heavy meromyosin ATPase activity. To explore the possibility that the MD plays a role in enhancement of myosin activity in cardiac muscle, we have utilized an all-cardiac protein (porcine cardiac actin and myosin, recombinant human cardiac Tm-Tn) In Vitro Motility assay to detect alterations in Ca2+ regulation of cardiac actomyosin interaction in the presence of two specific human recombinant cTn MD structural mutants. “K164Δ” is truncated after cTnI K164 and “LINK 2c2” has an inserted 8-amino acid linker before cTnI K164. At pCa5, K164Δ showed no significant difference from WT in filament sliding speed at most Tn-Tm concentrations tested, while sliding speed with LINK 2c2 was significantly slower than WT. Conversely, at pCa9, K164Δ was unable to stop actomyosin interaction, with sliding speeds significantly faster than WT; LINK 2c2 regulated the same as WT at pCa9 for most concentrations tested. Our in vitro cardiac muscle experimental data suggest that (1) the MD of TnI is a key player in Ca2+ regulation of cardiac muscle contraction, and (2) the C-terminal Mobile Domain of cTnI is not responsible for observed functional enhancement of myosin at saturating [Ca2+].