Potassium chloride released from contracting skeletal muscle may stimulate development of its hypertrophy

The effects of potassium chloride on the expression of IGF-1 splice forms and myoblast proliferation were investigated. KCl at the concentrations of 7–12 mM stimulated the synthesis of IGF-1 and mechano growth factor (MGF) in murine myoblasts as well as in myotubes both at the mRNA and protein levels. Pan-calcium channel blocker CdCl₂ completely abolished stimulation of growth factor expression, whereas blocker of HCN and Na_v1.4 channels ZD7288 drastically reduced it. In addition, potassium chloride stimulated myoblast proliferation, while IGF-1 autocrine signaling inhibition partially suppressed these mitogenic effects.     

Retinoic Acid and Its Geometrical Isomers Block Both Growth and Fusion of L6 Myoblasts by Modulating the Expression of Protein Kinase A

All-trans retinoic acid (RA) and its geometrical isomers, such as 9-cis RA, 13-cis RA, and 9,13-di-cis RA, strongly inhibited both growth and fusion of L6 myoblasts. However, illumination of white light diminished their inhibitory activity on membrane fusion with little effect on cell growth. During myogenic differentiation, the intracellular level of cAMP decreased whereas the total activity of protein kinase A as well as the protein level of its regulatory subunit I (RI) and catalytic subunit (C) increased. RAs raised the intracellular level of cAMP by over 3-fold, but decreased the total activity of protein kinase A. Like RAs, dibutyryl-cAMP inhibited myoblast fusion and reduced the expression of both RI and C subunits. These results suggest that RAs negatively modulate the differentiation of L6 myoblasts by increasing the intracellular level of cAMP, which may in turn down-regulate the expression of protein kinase A and hence its activity.     

Aging induced loss of stemness with concomitant gain of myogenic properties of a pure population of CD34+/CD45− muscle derived stem cells

Aging is accompanied by the functional decline of cells, tissues, and organs, as well as, a striking increase in susceptibility to a wide range of diseases. Within a tissue, both differentiated cells and adult stem cells are susceptible to intrinsic and extrinsic changes while aging. Muscle derived stem cells (MDSCs) are tissue specific stem cells which have been studied well for their multipotential nature. Although there are reports relating to diminished function and regenerative capacity of aged MDSCs as compared to their young counterparts, not much has been reported relating to the concomitant gain in unipotent nature of aged MDSCs. In this study, we report an inverse correlation between aging and expression of adult/mesenchymal stem cell markers and a direct correlation between aging and myogenecity in MDSCs. Aged MDSCs were able to generate a greater number of dystrophin positive myofibres, as compared to, the young MDSCs when transplanted in muscle of dystrophic mice. Our data, therefore, suggests that aging stress adds to the decline in stem cell characteristics with a concomitant increase in unipotency, in terms of, myogenecity of MDSCs. This study, hence, also opens the possibilities of using unipotent aged MDSCs as potential candidates for transplantation in patients with muscular dystrophies.     

Differential response of fetal and neonatal myoblasts to topographical guidance cues in vitro

 Fusion of mononucleated myoblasts into parallel arrays of mutinucleated myotubes is an essential step in skeletal myogenesis. The formation of such a highly ordered structure requires myoblasts to come together, orient and align in the correct location prior to fusion. We report here that fetal and neonatal myoblasts can use topographical features as strong guidance cues in vitro. Myoblasts were cultured on multiple grooved substrata of varying dimensions, and the axial orientations of individual cells were recorded. Both fetal and neonatal myoblasts aligned parallel with the direction of deep grooves (2.3–6.0 μm), which is correlated well with the location of myoblasts in similar sized grooves during secondary myogenesis. Fetal myoblasts also responded to shallower grooves (0.04–0.14 μm) by aligning parallel or perpendicular to the direction of the grooves, indicating the ability of these cells to respond to fine elements normally encountered within the developing muscle architecture. In contrast, neonatal myoblasts failed to respond to shallow grooves, adding to the suggestion that fetal and neonatal myoblasts may represent separate populations of myoblasts. Overall, the results demonstrate that myoblasts respond to large and small features of the physical topography in vitro and indicate that structural elements in the microenvironment of the muscle may play a critical role in myoblast spatial organization during myogenesis. Received: 29 May 1998 / Accepted: 17 February 1999     

Biochemical lesions of respiratory enzymes and configurational changes of mitochondria in vivo

Summary Chick embryo heart fragments in primary hanging-drop culture were treated with sodium fluoroacetate to induce inhibition of aconitate hydratase, a mitochondrial enzyme of the tricarboxylic acid cycle. The mitochondria were analyzed in the living myoblasts by phase-contrast time-lapse cinemicrography. The results were recorded in a 16 mm film. After 20–30 minutes contact of the cells with the inhibitor some mitochondria became thickened and swollen. The swelling was polymorphous, asynchronous and reversible; the same mitochondrion could swell and shrink many times. Some mitochondria seemed not to respond to fluoroacetate and remained rod-like. Mitochondria appeared the only cell components to be morphologically affected by fluoroacetate and the changes were specifically caused by the inhibitor. The type of mitochondrial swelling differed from the large-amplitude respiration-dependent swelling of the isolated mitochondria in vitro and from the configurational changes of isolated mitochondria associated with the respiratory states. The evidence pointed to a specific connection between the biochemical lesion caused by fluoroacetate and the configurational changes of the mitochondria. The mitochondrial swelling was to a large extent reversed by washing the cultures with Tyrode physiological saline solution and the reversal was further accentuated by incubation of the cultures in fresh nutrient medium.This work was supported by grants of the Consiglio Nazionale delle Richerce of Italy to both Institutes.     

Biochemical and morphological differences between fibroblasts and myoblasts from embryonic chicken skeletal muscle

Summary Non-myogenic cells were isolated from the breast muscle of 10-day-old chicken embryos employing Percoll density centrifugation. In culture, these cells exhibited the spread out, stellate morphology of fibroblast-like cells. They also exhibited receptor-mediated binding of plateletderived growth factor (PDGF). Such binding was not detected in cultures of predominantly myogenic cells isolated by the Percoll density centrifugation from the same muscle. Percoll-isolated myogenic and fibrogenic cell populations were also analyzed by two-dimensional polyacrylamide gel electrophoresis immediately after removal from the muscle. This analysis revealed at least six polypeptides specific to the fibroblasts but not detected in the myogenic cell population. In addition, at least eight polypeptides found in the myogenic population were barely detectable, or lacking altogether from the fibroblast-like cells. Ultrastructural analysis of the freshly isolated cells demonstrated that the fibroblasts were larger than the myoblasts and that their cytoplasm contained many vesicles. We conclude that the fibrogenic and myogenic cells isolated by Percoll from embryonic muscle express cell type-specific characteristics. Moreover, based on the PDGF binding studies, the fibrogenic cells can be categorized as true fibroblasts.     

C2C12 myoblasts release micro-vesicles containing mtDNA and proteins involved in signal transduction

Micro-vesicles can be released by different cell types and operate as ‘safe containers’ mediating inter-cellular communication. In this work we investigated whether cultured myoblasts could release exosomes. The reported data demonstrate, for the first time, that C2C12 myoblasts release micro-vesicles as shown by the presence of two exosome markers (Tsg101 and Alix proteins). Using real-time PCR analysis it was shown that these micro-vesicles, like other cell types, carry mtDNA. Proteomic characterization of the released micro-vesicle contents showed the presence of many proteins involved in signal transduction. The bioinformatics assessment of the Disorder Index and Aggregation Index of these proteins suggested that C2C12 micro-vesicles mainly deliver the machinery for signal transduction to target cells rather than key proteins involved in hub functions in molecular networks. The presence of IGFBP-5 in the purified micro-vesicles represents an exception, since this binding protein can play a key role in the modulation of the IGF-1 signalling pathway.In conclusion, the present findings demonstrate that skeletal muscle cells release micro-vesicles, which probably have an important role in the communication processes within skeletal muscles and between skeletal muscles and other organs. In particular, the present findings suggest possible new diagnostic approaches to skeletal muscle diseases.