Length Dependence of Force Generation is Controlled by Phosphorylation of cTnI at Serines 23/24

Abstract

The steepness of the Frank-Starling relationship is modulated by numerous physiological factors including beta-adrenergic stimulation, which steepens the relationship. This arises in part from increased myofibrillar length dependence of force and power by PKA, a downstream signaling molecule of the beta-adrenergic system. Since PKA has multiple myofibrillar substrates including titin, myosin binding protein-C (MyBP-C), and cardiac troponin I (cTnI), we sought to define if phosphorylation of one of these molecules was sufficient to control length-tension relationships. We focused on cTnI since (i) we previously observed a relationship between cTnI phosphorylation and the steepness of ventricular function curves in rat working hearts, (ii) 2D-DIGE indicated a distribution of cTnI phosphorylation states consistent with our previous observation of two populations of length-tension relationships (one shallow the other steep) in rat skinned cardiac myocytes, and (iii) troponin can be more readily exchanged in permeabilized striated muscle cell preparations. To test if PKA-mediated phosphorylation of cTnI is sufficient to mediate length dependence of force we used skinned rat slow-twitch skeletal muscle fibers, which exhibit shallow length-tension relationships that are unaltered by PKA. A sarcomere length-tension relationship was measured during submaximal Ca2+ activation, then the fiber was incubated in a troponin (Tn) exchange solution, which replaced ∼80% of the endogenous slow-skeletal Tn with cTn. After cTn incorporation the length-tension relationship remained shallow as predicted since the purified cTnI lacks phosphate incorporation. Next, the fiber was incubated in PKA, which caused the length-tension relationship to shift from shallow to steep. In addition, exchange with cTn having cTnI serines 23/24 modified to aspartic acids to mimic phosphorylation steepened the length-tension relationship. These results indicate that phosphorylation of cTnI serines 23/24 is sufficient to control length dependence of force generation in striated muscle.