Length Dependence of Cardiac Myocyte Force Generation is Mediated by CTnI Phosphorylation

Abstract

According to the Frank-Starling relationship, greater ventricular volume increases ventricular output. The Frank-Starling relationship is based partly on the length-tension relationship in cardiac myocytes. Recently, we identified two populations of length-tension relationships in mammalian cardiac myocytes, which was dependent on PKA-induced myofibrillar phosphorylation. We also observed a relationship between cardiac troponin I (cTnI) phosphorylation and steepness of ventricular function curves in rat working hearts. Thus, we tested the hypothesis that phosphorylation of cTnI is sufficient and/or necessary to control the length dependence of force generation. Our approach took advantage of the fact that troponin can be readily exchanged in permeabilized striated muscle cell preparations. We first used permeabilized 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 nearly all the endogenous slow 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 and a final sarcomere length-tension relationship measured. Contrary to our prediction, the length-tension relationship remained shallow after PKA phosphorylation of exogeneous cTnI. We next tested whether cTnI phosphorylation is necessary to steepen length-tension relationships in a cardiac myofibrillar background. For these experiments, we exchanged unphosphorylated cTn into a PKA treated myocyte that displayed a steep length-tension relationship. In this case cTn exchange shifted the length-tension relationship from a steep to a shallow relation. These results indicate that cTnI phosphorylation is not sufficient to alter length dependence of force generation in slow-twitch skeletal muscle fibers but appears necessary to convert the length-tension relationship from steep to shallow in cardiac myocytes.