We conclude that DCM-causing mutations in thin filament proteins abolish the relationship between myofilament Ca(2+) sensitivity and troponin I phosphorylation by PKA. We propose that this blunts the response to β-adrenergic stimulation and could be the cause of DCM in the long term.

Background: Cardiac myosin regulatory light chain (RLC) phosphorylation alters cardiac muscle function.Results: Phosphorylation affects mechanical parameters of cardiac muscle contraction during shortening.Conclusion: Phosphorylation impacts mechanical function of cardiac muscle and is altered during cardiac disease.Significance: Understanding RLC regulation by phosphorylation in cardiac muscle contraction is crucial for understanding changes in disease.

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Hypertrophic cardiomyopathy (HCM) is a genetic form of left ventricular hypertrophy, primarily caused by mutations in sarcomere proteins. The cardiac remodeling that occurs as the disease develops can mask the pathogenic impact of the mutation. Here, to discriminate between mutation-induced and disease-related changes in myofilament function, we investigate the pathogenic mechanisms underlying HCM in a patient carrying a homozygous mutation (K280N) in the cardiac troponin T gene (TNNT2), which results in 100% mutant cardiac troponin T. We examine sarcomere mechanics and energetics in K280N-isolated myofibrils and demembranated muscle strips, before and after replacement of the endogenous troponin. We also compare these data to those of control preparations from donor hearts, aortic stenosis patients (LVHao), and HCM patients negative for sarcomeric protein mutations (HCMsmn). The rate constant of tension generation following maximal Ca2+ activation (kACT) and the rate constant of isometric relaxation (slow kREL) are markedly faster in K280N myofibrils than in all control groups. Simultaneous measurements of maximal isometric ATPase activity and Ca2+-activated tension in demembranated muscle strips also demonstrate that the energy cost of tension generation is higher in the K280N than in all controls. Replacement of mutant protein by exchange with wild-type troponin in the K280N preparations reduces kACT, slow kREL, and tension cost close to control values. In donor myofibrils and HCMsmn demembranated strips, replacement of endogenous troponin with troponin containing the K280N mutant increases kACT, slow kREL, and tension cost. The K280N TNNT2 mutation directly alters the apparent cross-bridge kinetics and impairs sarcomere energetics. This result supports the hypothesis that inefficient ATP utilization by myofilaments plays a central role in the pathogenesis of the disease.

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 Ca 2þ 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.