Disuse and passive stretch cause rapid alterations in expression of developmental and adult contractile protein genes in skeletal muscle

Contractile proteins exist as a number of isoforms that show a developmental and tissue-specific pattern of expression. Using gene-specific cDNA probes, the expression of the sarcomeric myosin heavy chain (MHC) multi-gene family and of cardiac (foetal) alpha-actin was analysed in three different rat hindlimb muscles immobilised for 5 days in either the shortened or lengthened positions. For each of the MHC genes normally expressed in adult muscle (slow, IIA and IIB), the effect of disuse alone (immobilisation in the shortened position) upon expression was markedly different to that of passive stretch (immobilisation in the lengthened position) in each of the three muscles. However, the same adult sarcomeric myosin heavy chain gene can be affected in a different, or even opposite, manner by either disuse or passive stretch depending on the muscle in which it is being expressed. The fast IIB MHC gene, for example, exhibits a rapid induction in the slow postural soleus muscle, in response to disuse but no such induction occurs in the faster plantaris and gastrocnemius muscles. Furthermore, the induction of this gene in the soleus was prevented by passive stretch. The MHC gene, normally only expressed in embryonic skeletal muscle, showed a similar response in all three muscles and was reinduced in adult muscle in response to passive stretch but not by disuse alone. In contrast, the isoform of alpha-actin which is normally only present in significant quantities in embryonic skeletal muscle and which is reduced postnatally, is not reinduced by passive stretch but is reduced still further by immobilisation in the shortened position.     

The effects of starvation upon protein turnover in red and white myotomal muscle of rainbow trout, Salmo gairdneri Richardson

Protein synthesis rates of both red and white muscle were measured using a constant infusion technique in fed and starved rainbow trout over a period of 2 months. In both tissues, rates of protein synthesis fell during starvation although the fall was more rapid in white than in red muscle. The reduced rates of protein synthesis were correlated to a reduced level of RNA in the tissues and a lower rate of translation of the RNA present. Estimated rates for protein degradation in white muscle showed a marked initial increase but with more prolonged starvation the degradation rate become only slightly greater than that in the fed fish.     

Muscle origin of porcine satellite cells affects in vitro differentiation potential

Post‐natal muscle regeneration relies on the activation of tissue stem cells known as satellite cells, to repair damage following exercise trauma and disease. Satellite cells from individual muscles are known to be heterogeneous with regard to proliferation, fusion and transplantation abilities, although the muscle origin has rarely been considered pertinent to their differentiation capabilities. In this study we compared the potential of two functionally distinct skeletal muscle satellite cell populations from porcine diaphragm and hind‐limb semi‐membranosus muscles. These two muscles were chosen primarily for differences in metabolic and contractile properties: the diaphragm is more continuously active and has a greater oxidative capacity. Cells were induced to differentiate towards myogenic and adipogenic lineages, and here we have shown that cells from diaphragm exhibit a significantly greater degree of myogenesis compared with those from semi‐membranosus, while the converse was true for adipogenesis. Unexpectedly, both conditions generated small numbers of cells with neuronal characteristics for both muscle types, although more so in cells derived from the diaphragm. With increased interest in muscle adiposity with age and disease, these findings suggest that muscle origin of satellite cells does affect lineage fate, however whether differences in developmental origin or metabolic activity of the parent tissue govern this, remains to be determined. Copyright © 2010 John Wiley & Sons, Ltd.     

Knockdown of embryonic myosin heavy chain reveals an essential role in the morphology and function of the developing heart

The expression and function of embryonic myosin heavy chain (eMYH) has not been investigated within the early developing heart. This is despite the knowledge that other structural proteins, such as alpha and beta myosin heavy chains and cardiac alpha actin, play crucial roles in atrial septal development and cardiac function. Most cases of atrial septal defects and cardiomyopathy are not associated with a known causative gene, suggesting that further analysis into candidate genes is required. Expression studies localised eMYH in the developing chick heart. eMYH knockdown was achieved using morpholinos in a temporal manner and functional studies were carried out using electrical and calcium signalling methodologies. Knockdown in the early embryo led to abnormal atrial septal development and heart enlargement. Intriguingly, action potentials of the eMYH knockdown hearts were abnormal in comparison with the alpha and beta myosin heavy chain knockdowns and controls. Although myofibrillogenesis appeared normal, in knockdown hearts the tissue integrity was affected owing to apparent focal points of myocyte loss and an increase in cell death. An expression profile of human skeletal myosin heavy chain genes suggests that human myosin heavy chain 3 is the functional homologue of the chick eMYH gene. These data provide compelling evidence that eMYH plays a crucial role in important processes in the early developing heart and, hence, is a candidate causative gene for atrial septal defects and cardiomyopathy.     

Inward relocation of exogenous phosphatidylserine triggered by IGF-1 in non-apoptotic C2C12 cells is concentration dependent

The plasma membrane is composed of two leaflets that are asymmetric with regard to their phospholipid composition with phosphatidylserine (PS) predominantly located within the inner leaflet whereas other phospholipids such as phosphatidylcholine (PC) are preferentially located in the outer leaflet. An intimate relationship between cellular physiology and the composition of the plasma membrane has been demonstrated, with for example apoptosis requiring PS exposure for macrophage recognition. In skeletal muscle development, differentiation also requires PS exposure in myoblasts to create cell-cell contact areas allowing the formation of multinucleate myotubes. Although it is clearly established that membrane composition/asymmetry plays an important role in cellular physiology, the role of cytokines in regulating this asymmetry is still unclear. When incubated with myoblasts, insulin-like growth factor I (IGF-1) has been shown to promote proliferation versus differentiation in a concentration dependent manner and therefore, may be a potential candidate regulating cell membrane asymmetry. We show, in non-apoptotic C2C12 cells, that relocation of an exogenous PS analogue, from the outer into the inner leaflet, is accelerated by IGF-1 in a concentration-dependent manner and that maintenance of membrane asymmetry triggered by IGF-1 is however independent of the PI3K inhibitor wortmannin.     

Stretch-induced activation of ERK in myocytes is p38 and calcineurin-dependent

Activation of specific mitogen-activated protein kinases (MAPKs) has been suggested to be involved in phenotype modulation of cells subjected to mechanical strain, which may be common to different mechano-sensitive cell types. We have submitted C2C12 myocytes to a static stretch and examined its effect upon the activation of ERK. Stretch induced a rapid but transient activation of ERK. This activation was however prevented when cells were pre-treated with inhibitors of p38 and calcineurin. The dependence of strain-induced ERK activation upon p38 suggests a cross-talk between these two pathways when mediating a response to a mechanical stimulus in this cell type. This suggests that cross relationships between these MAP kinases may be of crucial importance for myocyte phenotype modulation and differentiation in response to a mechanical stimulus.     

Angiopoietin and Tie signaling pathways in vascular development.

The angiopoietin ligands and Tie receptors belong to a novel class of ligand/receptor families, which play critical roles in blood vessel formation. They are considered to control numerous signaling pathways that are involved in diverse cellular processes, such as cell migration, proliferation and survival, and reorganization of the actin cytoskeleton. In this review, we summarize the important biochemical and biological properties of this interesting ligand/receptor family. Particular emphasis will be made on potential downstream targets and consequences of the endothelial cell behavior, due to regulation by the angiopoietin/Tie pathway.     

Endotoxin transiently inhibits protein synthesis through Akt and MAPK mediating pathways in C2C12 myotubes

In this study, the effect of lipopolysaccharide (LPS) on protein synthesis (PS) and intracellular signaling factors that regulate it have been investigated in C2C12 murine-derived myotubes. In particular, the role of Akt/mammalian target of rapamycin (mTOR) and the mitogen-activated protein kinases (MAPKs) [p38 and extracelluar regulated protein kinase (ERK1/2)] have been examined. The direct effect of LPS on PS was measured at 3 and 18 h. LPS significantly decreased PS at 3 h but not at the 18-h time point. This effect was preceded by decreased Akt phosphorylation at 5 and 30 min after LPS administration. The mTOR phosphorylation exhibited a long time dose-dependent increase at all the time points. Similarly, the activity-related phosphorylation of p38 and ERK1/2 significantly increased in a time- and dose-dependent manner at all the time points. Polymyxin B abolished the LPS-induced decrease in PS rate. The phosphatidylinositol 3-kinase inhibitor LY-0294002 in combination with LPS significantly decreased the rate of PS by 81% and alone by 66%, respectively, for the 3- and 18-h time points, whereas p38 and ERK inhibitors in combination with LPS significantly decreased the rate PS rate at the 18-h time point by 41% and 59%, respectively, compared with control cells. In conclusion, LPS alone transiently decreased the rate of PS by 50% at 3 h; this effect is most likely mediated via the Toll-like receptor 4 (TLR4)-Akt/mTOR pathway, and both p38 and ERK when inhibited in the presence of LPS at 3 h have a similar effect in preventing the LPS-induced reduction in PS.