Circulating protein synthesis rates reveal skeletal muscle proteome dynamics.

Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders.

Activated Integrin-Linked Kinase Negatively Regulates Muscle Cell Enhancement Factor 2C in C2C12 Cells.

Our previous study reported that muscle cell enhancement factor 2C (MEF2C) was fully activated after inhibition of the phosphorylation activity of integrin-linked kinase (ILK) in the skeletal muscle cells of goats. It enhanced the binding of promoter or enhancer of transcription factor related to proliferation of muscle cells and then regulated the expression of these genes. In the present investigation, we explored whether ILK activation depended on PI3K to regulate the phosphorylation and transcriptional activity of MEF2C during C2C12 cell proliferation. We inhibited PI3K activity in C2C12 with LY294002 and then found that ILK phosphorylation levels and MEF2C phosphorylation were decreased and that MCK mRNA expression was suppressed significantly. After inhibiting ILK phosphorylation activity with Cpd22 and ILK-shRNA, we found MEF2C phosphorylation activity and MCK mRNA expression were increased extremely significantly. In the presence of Cpd22, PI3K activity inhibition increased MEF2C phosphorylation and MCK mRNA expression indistinctively. We conclude that ILK negatively and independently of PI3K regulated MEF2C phosphorylation activity and MCK mRNA expression in C2C12 cells. The results provide new ideas for the study of classical signaling pathway of PI3K-ILK-related proteins and transcription factors.

Hesperedin promotes MyoD-induced myogenic differentiation in vitro and in vivo.

BACKGROUND AND PURPOSE: The bioflavonoid, hesperedin, promotes osteoblast differentiation in human mesenchymal stem cells, indicating an anabolic effect of hesperedin on bone metabolism. Murine bone marrow mesenchymal stem cells undergo myogenic differentiation as well as osteogenic differentiation. We therefore explored whether hesperedin modulates muscle cell differentiation. EXPERIMENTAL APPROACH: Myoblast C2C12 cells were differentiated into muscle cells in the presence or absence of hesperedin. The effects of hesperedin on myogenic differentiation were determined by analysing specific muscle markers in vitro using reporter gene assays, immunoblotting, RT-PCR and DNA pull-down assays. In vivo, the effects of hesperedin were assessed using the freeze injury-induced muscle regeneration model in mice and daily injections of hesperedin for 6 days. KEY RESULTS: Hesperedin promoted myogenic differentiation, in a dose-dependent manner, by increasing myogenin gene expression. MyoD-induced myogenin gene transcription was enhanced by hesperedin, as this bioflavonoid augmented the nuclear localization and myogenin promoter-binding of MyoD. In addition, hesperedin increased myogenin and muscle creatine kinase gene expression during myogenic differentiation from C3H10T1/2 mesenchymal stem cells in a MyoD-dependent manner and accelerated in vivo muscle regeneration induced by muscle injury. CONCLUSIONS AND IMPLICATIONS: Our results demonstrate that hesperedin promoted myogenic differentiation in vitro and in vivo through activation of MyoD-mediated myogenin expression, suggesting a beneficial role in promoting muscle regeneration, following injury.

Expression of Gα(z) in C2C12 cells restrains myogenic differentiation.

The recent identification of Gα(z) expression in C2C12 myoblasts and its demonstrated interaction with the transcription factor Eya2 inferred an unanticipated role of Gα(z) in muscle development. In the present study, endogenous Gα(z) mRNA and protein expressions in C2C12 cells increased upon commencement of myogenesis and peaked at around 4-6days after induction but were undetectable in adult skeletal muscle. Surprisingly, stable expression of recombinant Gα(z) in C2C12 myoblasts strongly suppressed myotube formation upon serum deprivation, and the constitutively active mutant Gα(z)QL exerted more pronounced effects. Transcriptional activities of reporter genes responsive to early (MyoD, MEF2 and myogenin) and late (muscle creatine kinase and myosin heavy chain) myogenic markers were reduced by transiently expressed Gα(z)QL. Membrane attachment of Gα(z) was apparently required for the suppressive effects because a fatty acylation-deficient Gα(z) mutant could not inhibit myogenin expression. Introduction of siRNA against Gα(z) enhanced myogenin-driven luciferase activity and increased myosin heavy chain expression. Immunostaining of C2C12 cells over-expressing Gα(z) showed delayed nuclear expression of myogenin and severe myotube deformation. Gα(z) expression was accompanied by reduced levels of Rock2, RhoA and RhoGAP, enhanced expression of Rnd3, and a reduction of serum-responsive factor-driven reporter activity. These results support a novel role of Gα(z) in restraining myogenic differentiation through the disruption of Rho signaling.

Significance of CK-elevation in noncompaction with regard to cardiac and neuromuscular disease.

OBJECTIVES: This retrospective study aimed to find out how often CK is elevated in left-ventricular hypertrabeculation/noncompaction (LVHT), how often CK-elevation is due to neuromuscular disorders (NMDs), other cardiac disease, or causes other than cardiac or NMD, and how often CK-elevation is associated with troponin-T-positivity. METHODS: Electronic records from all LVHT-patients diagnosed between 1995-2005 were reviewed for CK, CK-MB, and troponin-T-values, frequency of CK-elevation, and causes of CK-elevation. RESULTS: Among 100 LVHT-patients, electronic records with laboratory data and final reports were available in 88. The highest CK-values ranged between 9-5547U/l. In 40 patients at least one CK-value was elevated (46%). Only 8 patients were troponin-T-positive. There was no significant difference of the clinical cardiologic, ECG, and echocardiographic parameters between patients with normal and elevated CK. Seventy of the 88 patients were seen by a neurologist. A NMD was diagnosed in 57 patients (81%). CK-elevation was attributed to NMD in 31 patients (79%), to causes other than cardiac or NMD in 6 patients (15%), and to cardiac causes in 4 patients (10%). CONCLUSIONS: CK-elevation occurs in half of the LVHT-patients. CK-elevation in LVHT is most frequently attributable to NMDs but hardly to cardiac disease. CK-elevation in LVHT suggests NMD and prompts neurological investigations.

[How should we manage patients with elevated preoperative creatine kinase? --A case of CK productive lung cancer].

A 70-year-old man with lung cancer was scheduled for partial resection of the right lung. Preoperative serum creatine kinase was elevated (1808 IU x l(-1), CK-MM 97%). Acute coronary syndrome was denied by the absence of significant stenosis of coronary artery and the normal segmental wall motion in echocardiography. The other examinations did not reveal the cause of CK elevation. He did not receive dantlorene preoperatively, but the surgical procedure was performed uneventfully without the use of the triggering agents, such as volatile inhalational anesthetic gases and suxamethonium. Thoracic wall was not invaded by the tumor. After the operation, CK went down quickly. It decreased further after the postoperative chemotherapy. We concluded that CK elevation might have been produced by adenocarcinoma itself, judging from the rapid decrease after surgery and the absence of thoracic wall invasion. Though CK elevation may indicate the malignant hyperthermia, we should not delay the surgery too long when there is possibility of CK elevation derived from cancer itself.