The Role of Calcium Affinity and C-I Interaction in Length-Dependent Activation

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The Frank-Starling response is an intrinsic property of cardiac muscle that helps to modulate cardiac output on a beat-to-beat basis. At the cellular level an increase in pressure during ventricle loading stretches the myocytes in the ventricle increasing sarcomere length (SL) and results in increased Ca2+ sensitivity and force of contraction. This mechanism is known as length-dependent activation (LDA), and multiple components of the sarcomere are believed to be involved in modulating this response. This includes changes in myofilament lattice spacing and/or myosin crossbridge orientation, affecting the probability of myosin binding to actin at longer SLs. Previous work from our lab, using the L48Q-cTnC variant, demonstrated that the inherent properties of cardiac troponin C (cTnC) maybe important in determining LDA. LDA is measured as a difference between pCa50 in force-pCa relationship at SL=2.3 and 2.0 in demembranated rat trabecula after a passive exchange of troponin complexes containing cTnC variants. Incorporation of L48Q-cTnC variant (with higher Ca2+binding affinity and increased strength of cTnC-cTnI interaction, denoted KCa and KC-I, respectively) into rat cardiac trabeculae diminished LDA, ΔpCa50= ∼0.03 compared to ∼0.1 for WT-cTn. Alternatively, L57Q-cTnC (which decreases KCa and KC-I) increases LDA, ΔpCa50= ∼0.2. The aim of the current study is to isolate changes in Ca2+ binding affinity of cTnC vs. C-I interaction on LDA using a series of cTnC variants that affect KCa and/or KC-I, as determined using fluorescently labeled cTnC. Our initial findings suggest that salmonid cTnC (ScTnC) with higher KCa, increases LDA (ΔpCa50= ∼0.2), in contrast to effects of L48Q-cTnC. In ongoing experiments we are examining the strength of ScTnC-cTnI interaction and investigating the changes in LDA caused by L48A-cTnC, which does not alter KCa but decreases KC-I.