The heart’s response to varying demands of the body is regulated by signaling pathways that activate protein kinases which phosphorylate sarcomeric proteins. Although phosphorylation of cardiac myosin binding protein-C (cMyBP-C) has been recognized as a key regulator of myocardial contractility, little is known about its mechanism of action. Here, we used protein kinase A (PKA) and Cε (PKCε), as well as ribosomal S6 kinase II (RSK2), which have different specificities for cMyBP-C’s multiple phosphorylation sites, to show that individual sites are not independent, and that phosphorylation of cMyBP-C is controlled by positive and negative regulatory coupling between those sites. PKA phosphorylation of cMyBP-C’s N terminus on 3 conserved serine residues is hierarchical and antagonizes phosphorylation by PKCε, and vice versa. In contrast, RSK2 phosphorylation of cMyBP-C accelerates PKA phosphorylation. We used cMyBP-C’s regulatory N-terminal domains in defined phosphorylation states for protein–protein interaction studies with isolated cardiac native thin filaments and the S2 domain of cardiac myosin to show that site-specific phosphorylation of this region of cMyBP-C controls its interaction with both the actin-containing thin and myosin-containing thick filaments. We also used fluorescence probes on the myosin-associated regulatory light chain in the thick filaments and on troponin C in the thin filaments to monitor structural changes in the myofilaments of intact heart muscle cells associated with activation of myocardial contraction by the N-terminal region of cMyBP-C in its different phosphorylation states. Our results suggest that cMyBP-C acts as a sarcomeric integrator of multiple signaling pathways that determines downstream physiological function.

The Frank-Starling relation is a fundamental auto-regulatory property of the heart that ensures the volume of blood ejected in each heartbeat is matched to the extent of venous filling. At the cellular level, heart muscle cells generate higher force when stretched, but despite intense efforts the underlying molecular mechanism remains unknown. We applied a fluorescence-based method, which reports structural changes separately in the thick and thin filaments of rat cardiac muscle, to elucidate that mechanism. The distinct structural changes of troponin C in the thin filaments and myosin regulatory light chain in the thick filaments allowed us to identify two aspects of the Frank-Starling relation. Our results show that the enhanced force observed when heart muscle cells are maximally activated by calcium is due to a change in thick filament structure, but the increase in calcium sensitivity at lower calcium levels is due to a change in thin filament structure.DOI: http://dx.doi.org/10.7554/eLife.24081.001

Time-resolved changes in the conformation of troponin in the thin filaments of skeletal muscle were followed during activation in situ by photolysis of caged calcium using bifunctional fluorescent probes in the regulatory and the coiled-coil (IT arm) domains of troponin. Three sequential steps in the activation mechanism were identified. The fastest step (1,100 s −1 ) matches the rate of Ca 2+ binding to the regulatory domain but also dominates the motion of the IT arm. The second step (120 s −1 ) coincides with the azimuthal motion of tropomyosin around the thin filament. The third step (15 s −1 ) was shown by three independent approaches to track myosin head binding to the thin filament, but is absent in the regulatory head. The results lead to a four-state structural kinetic model that describes the molecular mechanism of muscle activation in the thin filament-myosin head complex under physiological conditions. muscle regulation | excitation-contraction coupling | muscle signaling C ontraction of skeletal and cardiac muscle is initiated by a transient increase in the concentration of intracellular Ca 2+ ions, which bind to troponin in the thin filaments of the muscle sarcomere. This leads to azimuthal movement of tropomyosin around the thin filament, which uncovers the myosin binding sites on actin and allows the head domain of myosin from the thick filaments to bind to actin and generate force (1, 2). In vitro studies using isolated protein components showed that myosin head binding can produce a further motion of tropomyosin, at least in low [ATP] or rigor-like conditions (2-4), but the functional significance of this effect in physiological conditions and intact sarcomeres is not clear.To elucidate the molecular structural basis of muscle regulation and the role of myosin binding in situ, we introduced bifunctional fluorescent probes into the calcium-binding subunit of troponin, troponin C (TnC) (Fig. 1, yellow), in demembranated fibers from skeletal muscle (5-7). One probe cross-linked a pair of cysteines introduced into the C helix of TnC (Fig. 1, green), close to the regulatory Ca 2+ binding sites (Fig. 1, black spheres) in its N-terminal lobe, and reports the rotation and opening of this lobe on binding Ca 2+ (5). The N-lobe opening is associated with binding of the switch peptide of troponin I (TnI) (Fig. 1, blue) to a hydrophobic pocket on its surface, and this is a key step in the signaling pathway of calcium regulation (8, 9).A second probe was attached to the E helix of TnC (Fig. 1, magenta) in its C-terminal lobe, which contains a pair of divalent cation binding sites (Fig. 1, gray spheres) that can bind Mg 2+ as well as Ca 2+ . The C lobe of TnC is clasped between two long helices of TnI, one of which forms a coiled coil with part of the tropomyosin-binding component of troponin, troponin T (TnT) (Fig. 1, orange). The C lobe of TnC and these long TnI and TnT helices form a well-defined structural domain called the "IT arm" (9, 10). Although the C-lobe E helix of TnC is continuous with the N-lob...