MicroRNAs in skeletal muscle biology and exercise adaptation

MicroRNAs (miRNAs) have emerged as important players in the regulation of gene expression, being involved in most biological processes examined to date. The proposal that miRNAs are primarily involved in the stress response of the cell makes miRNAs ideally suited to mediate the response of skeletal muscle to changes in contractile activity. Although the field is still in its infancy, the studies presented in this review highlight the promise that miRNAs will have an important role in mediating the response and adaptation of skeletal muscle to various modes of exercise. The roles of miRNAs in satellite cell biology, muscle regeneration, and various myopathies are also discussed.     

GNE is involved in the early development of skeletal and cardiac muscle

UDP-N-acetylglucosamine 2 epimerase/N-acetylmannosamime kinase (GNE) is a bifunctional enzyme which catalyzes the two key sequential steps in the biosynthetic pathway of sialic acid, the most abundant terminal monosaccharide on glycoconjugates of eukaryotic cells. GNE knock out (GNE KO) mice are embryonically lethal at day E8.5. Although the role of GNE in the sialic pathway has been well established as well as the importance of sialylation in many diverse biological pathways, less is known about the involvement of GNE in muscle development. To address this issue we have studied the role of GNE during in vitro embryogenesis by comparing the developmental profile in culture of embryonic stem cells (ES) from wild type and from GNE KO E3.5 mice embryos, during 45 days. Neuronal cells appeared rarely in GNE KO ES cultures and did not reach an advanced differentiated stage. Although primary cardiac cells appeared at the same time in both normal and GNE KO ES cultures, GNE KO cardiac cells degraded very soon and their beating capacity decayed rapidly. Furthermore very rare skeletal muscle committed cells were detected in the GNE KO ES cultures at any stage of differentiation, as assessed by analysis of the expression of either Pax7, MyoD and MyHC markers. Beyond the supporting evidence that GNE plays an important role in neuronal cell and brain development, these results show that GNE is strongly involved in cardiac tissue and skeletal muscle early survival and organization. These findings could open new avenues in the understanding of muscle function mechanisms in health and in disease.     

Ganglioside-binding proteins in skeletal and cardiac muscle

Several ganglioside-binding proteins have been identified inguinea pig skeletal and cardiac muscle. In the cytosolic fractionsof both tissues, a 130-kD protein was found to have the highestpropensity to bind lucifer yellow CH-labelled G_M1. This bindingcould be abolished by prior incubation of the protein with G_M2.Polysialogangliosides including G_D1a, G_D1b, G_T1b, and G_Q1b wereless effective. The 130-kD protein migrated as a doublet withapparent isoelectric points (pI) of 6.3 and 6.5, respectively,in isoelectric focusing gel, but as a single species with anapparent M_r of 43000 in SDS-polyacrylamide gel. Both the ganglioside-bindingand the immunological properties of the 43-kD subunit proteinwere different from those of rabbit skeletal muscle actin. Cardiacmuscle extract also contained a 77-kD minor ganglioside-bindingprotein that was absent in skeletal muscle. This protein hadan apparent pI of 5.4 and migrated as a 39-kD species in SDSgels. By contrast, only the particulate fraction of skeletalmuscle was found to contain a 180-kD major ganglioside-bindingprotein. Binding of fluorescent G_M1 to this protein was blockedby pre-incubation of the protein with G_M1 or G_M2. The 180-kDprotein migrated as a 98-kD species in SDS gels. However, itspropensity to bind lucifer yellow CH-G_M1 was at least 10 timesgreater than that of rabbit skeletal muscle phosphorylase b(M_r = 97400). The apparent pI (6.5) of the 180-kD protein alsowas slightly higher than that of rabbit phosphorylase. Tissuedistribution studies revealed that both the 130-kD and the 180-kDmajor ganglioside-binding proteins were muscle specific. Itis, therefore, possible that these two proteins may play someunique roles in ganglioside-related functions in muscle tissues. gangliosides ganglioside-binding proteins muscle     

Localization of calcium in skeletal and cardiac muscle

Summary The requirement of calcium (Ca²⁺) in the excitation-contraction coupling of both skeletal and cardiac muscle is well established. However, the exact location of the intracellular storage sites of Ca²⁺ is not firmly established. We report here on the ultrastructural distribution of Ca²⁺ in white and red skeletal muscle and in cardiac muscle of the rat using combined phosphate-pyroantimonate (PPA) and oxalate-pyroantimonate (OPA) procedures. The methods are based on (a) stabilization and/or trapping of Ca²⁺ during the primary fixation step in glutaraldehyde by potassium phosphate or oxalate; (b) subsequent wash-out of all non-trapped cations such as Na⁺ and Mg²⁺ in potassium phosphate or oxalate; (c) conversion of the complexed or trapped Ca²⁺ into an electron-dense calcium pyroantimonate salt in 100 m-thick tissue sections; and (d) wash-out of the excess potassium pyroantimonate at alkaline pH.With the OPA procedure, mitochondria of all muscle types showed little precipitate. The junctional sarcoplasmic reticulum was stongly reactive in relaxed white skeletal muscle, negative in contracted white fibres and negative in red skeletal and cardiac muscle, independent of the state of relaxation-contraction. Other organelles were essentially free of deposits.With the PPA method, the precipitate was almost exclusively confined to the sarcolemma and its T-tubular invaginations in cardiac and slow skeletal muscle, and was absent in fast skeletal muscle. Apart from occasional deposits in mitochondria, all other organelles were free of precipitate. The sarcolemma-associated deposits were clearly confined to the inner leaflet of the lipid bilayer. The amount of precipitate varied within the contraction cycle, relaxed cells possessing the highest density.Exposure of the tissue to La³⁺ resulted in the complete absence of sarcolemma-bound precipitate suggesting that the Ca²⁺ is exchangeable. Furthermore, these cytological data suggest a basic difference in Ca²⁺ storage between white skeletal muscle on the one hand, and red skeletal and cardiac muscle on the other.     

Postnatal development of palmitate oxidation and mitochondrial enzyme activities in rat cardiac and skeletal muscle

1. The palmitate oxidation rate was measured in intact diaphragm and m. flexor digitorum brevis and in whole homogenates of heart, diaphragm and m. quadriceps of developing rats between late foetal life and maturity. Activities of the mitochondrial enzymes cytochrome c oxidase and citrate synthase were also determined. 2. Immediately after birth the palmitate oxidation rate increases markedly in both intact diaphragm and m. flexor digitorum brevis and falls gradually after day 1 to adult values which are about 35% of those at birth. 3. The oxidation capacities of diaphragm and m. quadriceps, but especially of heart, increase steadily during development, starting before birth and reaching adult values at 15-20 days postnatally. The activities of the mitochondrial enzymes show a similar developmental pattern. 4. In heart the increase of oxidative capacity is the result of an increase of both mitochondrial content and mitochondrial activity. The mitochondrial contents of diaphragm and m. quadriceps, on the other hand, decrease with age and the increase of their oxidative capacities is due to a large rise of the mitochondrial activity.     

Developmental changes of cardiac and slow skeletal muscle troponin T expression in chicken cardiac and skeletal muscles

Numerous troponin T (TnT) isoforms are produced by alternative splicing from three genes characteristic of cardiac, fast skeletal, and slow skeletal muscles. Apart from the developmental transition of fast skeletal muscle TnT isoforms, switching of TnT expression during muscle development is poorly understood. In this study, we investigated precisely and comprehensively developmental changes in chicken cardiac and slow skeletal muscle TnT isoforms by two-dimensional gel electrophoresis and immunoblotting with specific antisera. Four major isoforms composed of two each of higher and lower molecular weights were found in cardiac TnT (cTnT). Expression of cTnT changed from high- to low-molecular-weight isoforms during cardiac muscle development. On the other hand, such a transition was not found and only high-molecular-weight isoforms were expressed in the early stages of chicken skeletal muscle development. Two major and three minor isoforms of slow skeletal muscle TnT (sTnT), three of which were newly found in this study, were expressed in chicken skeletal muscles. The major sTnT isoforms were commonly detected throughout development in slow and mixed skeletal muscles, and at developmental stages until hatching-out in fast skeletal muscles. The expression of minor sTnT isoforms varied from muscle to muscle and during development.     

Smooth muscle myosin light chain kinase expression in cardiac and skeletal muscle

The purpose of this study was to characterize myosin light chain kinase (MLCK) expression in cardiac and skeletal muscle. The only classic MLCK detected in cardiac tissue, purified cardiac myocytes, and in a cardiac myocyte cell line (AT1) was identical to the 130-kDa smooth muscle MLCK (smMLCK). A complex pattern of MLCK expression was observed during differentiation of skeletal muscle in which the 220-kDa-long or "nonmuscle" form of MLCK is expressed in undifferentiated myoblasts. Subsequently, during myoblast differentiation, expression of the 220-kDa MLCK declines and expression of this form is replaced by the 130-kDa smMLCK and a skeletal muscle-specific isoform, skMLCK in adult skeletal muscle. These results demonstrate that the skMLCK is the only tissue-specific MLCK, being expressed in adult skeletal muscle but not in cardiac, smooth, or nonmuscle tissues. In contrast, the 130-kDa smMLCK is ubiquitous in all adult tissues, including skeletal and cardiac muscle, demonstrating that, although the 130-kDa smMLCK is expressed at highest levels in smooth muscle tissues, it is not a smooth muscle-specific protein.     

Chloride activity and its control in skeletal and cardiac muscle

Ion-selective microelectrodes have been used to compare the mechanisms controlling intracellular Cl- activity in skeletal and cardiac muscle. In frog Sartorius skeletal muscle fibres, Cl- levels are low (about 3 mM) and are determined mainly passively. The effect of any Cl- transport system will be quickly short-circuited through the high membrane Cl- conductance. In contrast, the sheep-heart Purkinje fibre, like other cardiac tissues, contains higher than passive levels of intracellular Cl- (20-30 mM). Many Cl- movements occur, not through Cl- channels (the permeability for Cl- is low), but by a Cl- -HCO3- countertransport system. High internal Cl- levels are achieved by an exchange of extracellular Cl- for intracellular HCO3-, which acidifies the fibre by 0.3 pH. Anion exchange in heart differs from that proposed for other excitable cells in that it is not specialized to compensate for an intracellular acidosis. Instead, it can prevent the fibres from becoming too alkaline by promoting a bicarbonate efflux and a chloride influx whenever internal bicarbonate levels rise. Possible reasons for this are briefly discussed.     

Heparan sulfates in skeletal muscle development and physiology

Recent years have seen an emerging interest in the composition of the skeletal muscle extracellular matrix (ECM) and in the developmental and physiological roles of its constituents. Many cell surface-associated and ECM-embedded molecules occur in highly organized spatiotemporal patterns, suggesting important roles in the development and functioning of skeletal muscle. Glycans are historically underrepresented in the study of skeletal muscle ECM, even though studies from up to 30 years ago have demonstrated specific carbohydrates and glycoproteins to be concentrated in neuromuscular junctions (NMJs). Changes in glycan profile and distribution during myogenesis and synaptogenesis hint at an active involvement of glycoconjugates in muscle development. A modest amount of literature involves glycoconjugates in muscle ion housekeeping, but a recent surge of evidence indicates that glycosylation defects are causal for many congenital (neuro)muscular disorders, rendering glycosylation essential for skeletal muscle integrity. In this review, we focus on a single class of ECM-resident glycans and their emerging roles in muscle development, physiology, and pathology: heparan sulfate proteoglycans (HSPGs), notably their heparan sulfate (HS) moiety.     

Sarcotubular development in dystrophic skeletal muscle cells

Summary Dilations of the sarcotubular system and misaligned myofilaments have been reported as early indicators of muscular dystrophy in skeletal muscle. Since the developing tubular component is believed instrumental in initial myofilament alignment during myogenesis, tubular development is evaluated using normal and dystrophic chick embryo skeletal muscle and cultures of normal and dystrophic embryonic pectoral muscle incubated in the presence of horse spleen ferritin. Comparisons of the findings show that periodic tubules are absent from dystrophic somitic muscle and that invaginating tubules from the sarcolemma are found in fewer, randomly located areas of dystrophic pectoral muscle cells. The results indicate that the tubular component is not involved in the bizarre vesiculations seen in mature dystrophic muscle, however, the malalignment of dystrophic myofilaments is probably the result of the poorer development of the T system in this muscle.