Effect of levosimendan on the contractility of muscle fibers from nemaline myopathy patients with mutations in the nebulin gene.

BACKGROUND: Nemaline myopathy (NM), the most common non-dystrophic congenital myopathy, is characterized by generalized skeletal muscle weakness, often from birth. To date, no therapy exists that enhances the contractile strength of muscles of NM patients. Mutations in NEB, encoding the giant protein nebulin, are the most common cause of NM. The pathophysiology of muscle weakness in NM patients with NEB mutations (NEB-NM) includes a lower calcium-sensitivity of force generation. We propose that the lower calcium-sensitivity of force generation in NEB-NM offers a therapeutic target. Levosimendan is a calcium sensitizer that is approved for use in humans and has been developed to target cardiac muscle fibers. It exerts its effect through binding to slow skeletal/cardiac troponin C. As slow skeletal/cardiac troponin C is also the dominant troponin C isoform in slow-twitch skeletal muscle fibers, we hypothesized that levosimendan improves slow-twitch muscle fiber strength at submaximal levels of activation in patients with NEB-NM. METHODS: To test whether levosimendan affects force production, permeabilized slow-twitch muscle fibers isolated from biopsies of NEB-NM patients and controls were exposed to levosimendan and the force response was measured. RESULTS: No effect of levosimendan on muscle fiber force in NEB-NM and control skeletal muscle fibers was found, both at a submaximal calcium level using incremental levosimendan concentrations, and at incremental calcium concentrations in the presence of levosimendan. In contrast, levosimendan did significantly increase the calcium-sensitivity of force in human single cardiomyocytes. Protein analysis confirmed that the slow skeletal/cardiac troponin C isoform was present in the skeletal muscle fibers tested. CONCLUSIONS: These findings indicate that levosimendan does not improve the contractility in human skeletal muscle fibers, and do not provide rationale for using levosimendan as a therapeutic to restore muscle weakness in NEB-NM patients. We stress the importance of searching for compounds that improve the calcium-sensitivity of force generation of slow-twitch muscle fibers. Such compounds provide an appealing approach to restore muscle force in patients with NEB-NM, and also in patients with other neuromuscular disorders.

PKCα-specific phosphorylation of the troponin complex in human myocardium: a functional and proteomics analysis.

AIMS: Protein kinase Cα (PKCα) is one of the predominant PKC isoforms that phosphorylate cardiac troponin. PKCα is implicated in heart failure and serves as a potential therapeutic target, however, the exact consequences for contractile function in human myocardium are unclear. This study aimed to investigate the effects of PKCα phosphorylation of cardiac troponin (cTn) on myofilament function in human failing cardiomyocytes and to resolve the potential targets involved. METHODS AND RESULTS: Endogenous cTn from permeabilized cardiomyocytes from patients with end-stage idiopathic dilated cardiomyopathy was exchanged (∼69%) with PKCα-treated recombinant human cTn (cTn (DD+PKCα)). This complex has Ser23/24 on cTnI mutated into aspartic acids (D) to rule out in vitro cross-phosphorylation of the PKA sites by PKCα. Isometric force was measured at various [Ca(2+)] after exchange. The maximal force (Fmax) in the cTn (DD+PKCα) group (17.1±1.9 kN/m(2)) was significantly reduced compared to the cTn (DD) group (26.1±1.9 kN/m(2)). Exchange of endogenous cTn with cTn (DD+PKCα) increased Ca(2+)-sensitivity of force (pCa50 = 5.59±0.02) compared to cTn (DD) (pCa50 = 5.51±0.02). In contrast, subsequent PKCα treatment of the cells exchanged with cTn (DD+PKCα) reduced pCa50 to 5.45±0.02. Two PKCα-phosphorylated residues were identified with mass spectrometry: Ser198 on cTnI and Ser179 on cTnT, although phosphorylation of Ser198 is very low. Using mass spectrometry based-multiple reaction monitoring, the extent of phosphorylation of the cTnI sites was quantified before and after treatment with PKCα and showed the highest phosphorylation increase on Thr143. CONCLUSION: PKCα-mediated phosphorylation of the cTn complex decreases Fmax and increases myofilament Ca(2+)-sensitivity, while subsequent treatment with PKCα in situ decreased myofilament Ca(2+)-sensitivity. The known PKC sites as well as two sites which have not been previously linked to PKCα are phosphorylated in human cTn complex treated with PKCα with a high degree of specificity for Thr143.

Gene expression profile in the diaphragm following contractile inactivity during thoracic surgery.

Introduction. Recent work revealed the development of marked muscle fiber weakness in the diaphragm, but not in the non-respiratory latissimus dorsi, during thoracic surgery. To disentangle the molecular processes that underlie the development of diaphragm muscle fiber weakness during thoracic surgery, we studied changes in the gene expression profile. Methods. Serial biopsies from the diaphragm and the latissimus dorsi muscle were obtained from four patients during thoracotomy for resection of a tumor in the right lung. Biopsies were taken as soon as the diaphragm had been exposed (t0) and again after two hours (t2). Gobal differences in gene expression in diaphragm biopsies were assessed by microarray analysis. Results. 346 differentially expressed gene transcripts were found in the diaphragm at t2 vs. t0. Pathway analysis revealed that genes associated with inflammation (83 genes; p<0.0001) and cell death (118 genes, p<0.0001) pathways were significantly overexpressed at t2. Of the 346 differentially expressed genes in the diaphragm at t2, 258 were also differential in the latissimus dorsi muscle, with the direction of change being identical for all differentially expressed genes. In addition, latissimus dorsi showed exclusive upregula-ton of negative regulators of cell death. Conclusions. Two hours of thoracic surgery result in rapid and profound changes in expression of inflammatory response and apoptotic genes in the diaphragm. The apoptotic response was stronger in the diaphragm than in the latissiums dorsi. These findings suggest that the development of selective diaphragm muscle fiber weakness in these patients might be related to an exaggerated apoptotic response.