The dynamic role of cardiac myosin binding protein-C during ischemia.

Cardiac myosin binding protein C (cMyBP-C) is a myofibrillar protein important for normal myocardial contractility and stability. In mutated form it can cause cardiomyopathy and heart failure. cMyBP-C appears to have separate regions for different functions. Three phosphorylation sites near the N terminus modulate contractility by their effect on both the kinetics of contraction and the binding site of the N-terminus. The C terminal region binds to myosin rods and stabilizes thick filament structure. The aim of the study reported here was to test whether cMyBPC is important in producing the structural and functional changes that result from ischemia/reperfusion. In this study the sequential changes in cMyBP-C, contractility, and thick filament structure following dephosphorylation of cMyBP-C associated with ischemia and reperfusion have been studied in biopsied specimens from chronically instrumented dogs. One and two dimensional electrophoresis, electron microscopy and immunocytochemistry with multiple antibodies generated against different domains in cMyBP-C have been used to follow structural changes in cMyBP-C. Ischemia produced dephosphorylation of cMyBP-C. Subsequent reperfusion released the dephosphorylated cMyBP-C from myofibrils and activated proteolysis of the cytoplasmic cMyBP-C. This in turn leads to increased vulnerability of cMyBP-C to proteolysis and increased degradation of thick filaments. The state of cMyBP-C appears to be closely related to phosphorylation and dephosphorylation of serine 282. In the absence of the stabilizing action of cMyBP-C either as a consequence of genetic mutation or dephosphorylation, premature degradation of thick filaments occurs and is accompanied by persistent contractile dysfunction.

Myosin-binding protein C phosphorylation, myofibril structure, and contractile function during low-flow ischemia.

BACKGROUND: Contractile dysfunction develops in the chronically instrumented canine myocardium after bouts of low-flow ischemia and persists after reperfusion. The objective of this study is to identify whether changes in the phosphorylation state of myosin-binding protein C (MyBP-C) are a potential cause of dysfunction. METHODS AND RESULTS: During low-flow ischemia, MyBP-C is dephosphorylated, and the number of actomyosin cross-bridges in the central core of the sarcomere decreases as thick filaments dissemble from the periphery of the myofibril. During reperfusion, MyBP-C remains dephosphorylated, and its degradation is accelerated. CONCLUSIONS: Dephosphorylation of MyBP-C may initiate changes in myofibril thick filament structure that decrease the interaction of myosin heads with actin thin filaments. Limiting the formation of actomyosin cross-bridges may contribute to the contractile dysfunction that is apparent after low-flow ischemia. Breakdown of MyBP-C during reperfusion may prolong myocardial stunning.

Phosphorylation of contractile proteins in response to alpha- and beta-adrenergic stimulation in neonatal cardiomyocytes.

alpha- and beta-Adrenergic receptor agonists induce an inotropic response in the adult heart by promoting the phosphorylation of several regulatory proteins, including myosin-binding protein C (MyBP-C), cardiac troponin I (cTnI), and phospholamban (PLB). However, the adrenergic-induced phosphorylation of these proteins has not been characterized in the developing heart. Accordingly, we evaluated MyBP-C, cTnI, and PLB phosphorylation in cultured neonatal rat cardiomyocytes (NRCMs) after alpha- and beta-receptor activation with phenylephrine and isoproterenol. alpha-Receptor stimulation increased, whereas beta-receptor activation reduced MyBP-C phosphorylation. Isoelectric-focusing experiments indicated that the amount of monophosphorylated MyBP-C was sensitive to alpha-adrenergic activation, but diphosphorylated and triphosphorylated MyBP-C levels were largely unaffected. The phosphorylation of cTnI and PLB was consistent with the mechanism observed in adult hearts: alpha- and beta-Receptor stimulation phosphorylated both proteins. For cTnI, the greatest difference associated with beta-receptor activation was observed in the diphosphorylated state, whereas alpha-receptor activation was associated with a marked increase in the tetraphosphorylated protein and absence of the unphosphorylated state. Despite these apparent changes in cTnI and PLB phosphorylation, beta-receptor activation failed to alter calcium transients in NRCMs. Collectively, these findings suggest that, unlike cTnI and PLB, MyBP-C and inotropy are not coupled to beta-adrenergic stimulation in NRCMs. Therefore, cTnI and PLB probably play a more central role in modulating contractile function in NRCMs in response to catecholamines than does MyBP-C, and MyBP-C may have a structural role in stabilizing thick filament assembly rather than influencing cross-bridge formation in developing hearts.

HSC73-tubulin complex formation during low-flow ischemia in the canine myocardium.

Canine myocardium was exposed to bouts of low-flow ischemia to identify the interactions that develop between the microtubule-based cytoskeleton and the heat shock protein 70 (HSP70) family of heat shock proteins in viable cardiomyocytes. "Moderate" or "severe" low-flow ischemia was produced in chronically instrumented dogs by reducing circumflex coronary flow by 50% for 2 h or by 75% for 5 h followed by reperfusion for 2 and 24 h, respectively. Electron and immunofluorescence microscopy demonstrated either partial or nearly complete depolymerization of the intermyofibrillar microtubules in areas of myofibril disruption and partial dissolution of the perinuclear microtubule girdle. In contrast, centrosomal tubulin arrays appeared to remain intact following low-flow ischemia. In cardiomyocytes displaying myofibril disruption, constitutively expressed HSP73 (HSC73) colocalized with intact but not disrupted microtubules and with perinuclear and centrosomal tubulin following moderate ischemia. Microtubule depolymerization and high molecular weight tubulin-HSC73 complexes were present in more severely ischemic tissue. These results suggest that HSC73 directly interacts with tubulin and may protect selected elements of the microtubule network and limit myofibril disruption during reversible low-flow ischemia.