Reducing Thin Filament Ca 2+ Affinity with a cTnC Variant (L57Q) Reduces Force but Enhances Cross-Bridge Dependence of Cooperative Activation in Demembranated Rat Trabeculae

Abstract

Activation of cardiac contraction is initiated by binding of Ca2+ to troponin C (cTnC) and regulated by cooperative strong cross-bridge binding. We previously showed that passive exchange with cTn containing a cTnC variant, L48Q, increased Ca2+ sensitivity of force development and eliminated sarcomere length (SL) dependence of Ca2+ sensitivity of force in rat cardiac trabeculae. This was shown to be due to decreased reliance on strong cross-bridge binding for full thin-filament activation, possibly due to stronger cTnC-cTnI interaction. We also showed that PKA phosphorylation decreased Ca2+ sensitivity of force and eliminated SL dependence of force-pCa relations, by unknown specific mechanisms. Here we test the hypothesis that incorporation of a cTnC variant with decreased Ca2+ binding affinity, L57Q cTnC, will result in increased reliance on cross-bridge binding for full activation, thus increasing SL-dependence of Ca2+ sensitivity. As expected, results indicate trabeculae passively exchanged with L57Q cTnC-cTn displayed decreased Ca2+ sensitivity and rate of force production compared to WT cTnC at a given [Ca2+]. Interestingly, preliminary results indicate L57Q cTnC-cTn mildly increased the SL-dependence of Ca2+ sensitivity of force and also significantly decreased maximal force. Both of these effects were not observed in PKA-treated trabeculae, which had a comparable reduction in Ca2+ sensitivity of force. These findings suggest that reducing cTnC Ca2+ affinity per se can reduce Ca2+ sensitivity of contractile activation to the point of limited overall force production, which may enhance cross-bridge dependence of cooperative thin filament activation. Current experiments aim to increase force in L57Q cTnC (to near WT cTnC) by increasing cross-bridge attachment using 2’-deoxy-ATP, which has previously been shown to increase force and Ca2+ sensitivity while maintaining SL dependence. NIH-HL65497(MR), AHA-2310117(FSK).