Abstract
The precise mechanism of cardiac troponin I (cTnI) proteolysis in myocardial stunning is not fully understood. Accordingly, we determined the effect of cTnI C terminus truncation on chemo-mechanical transduction in isolated skinned rat trabeculae. Recombinant troponin complex (cTn), containing either mouse cTnI-(1-193) or human cTnI-(1-192) was exchanged into skinned cardiac trabeculae; Western blot analysis confirmed that 60-70% of the endogenous cTn was replaced by recombinant Tn. Incorporation of truncated cTnI induced significant reductions ( approximately 50%) in maximum force and cooperative activation as well as increases ( approximately 50%) in myofilament Ca(2+) sensitivity and tension cost. Similar results were obtained with either mouse or human truncated cTn. Presence of truncated cTnI increased maximum actin-activated S1 ATPase activity as well as its Ca(2+) sensitivity in vitro. Partial exchange (50%) for truncated cTnI resulted in similar reductions in maximum force and cooperativity; tension cost was increased in proportion to truncated cTnI content. In vitro, to determine the molecular mechanism responsible for the enhanced myofilament Ca(2+) sensitivity, we measured Ca(2+) binding to cTn as reported using a fluorescent probe. Incorporation of truncated cTnI did not affect Ca(2+) binding affinity to cTn alone. However, when cTn was incorporated into thin filaments, cTnI truncation induced a significant increase in Ca(2+) binding affinity to cTn. We conclude that cTnI truncation induces depressed myofilament function. Decreased cardiac function after ischemia/reperfusion injury may directly result, in part, from proteolytic degradation of cTnI, resulting in alterations in cross-bridge cycling kinetics.
Highlights
Of normal coronary flow in the absence of irreversible damage
Measurement of Ca2ϩ binding to Tn demonstrated a decrease in the affinity induced by the presence of truncated cTn I (cTnI) but only when Tn was reconstituted into the thin filament
The absence of 17 amino acids at the C terminus induced a higher mobility of cTnI-(1–193) as compared with cTnIWT, as is apparent both in the Coomassie-stained gel shown in A and in the cTnI Western blot shown in B
Summary
Of normal coronary flow in the absence of irreversible damage. It has become increasingly evident that myocardial stunning may contribute significantly to the morbidity associated with coronary artery disease (1). We found increases in cross-bridge cycle kinetics and myofilament Ca2ϩ sensitivity and reductions in maximum force development and cooperativity of activation.
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