Myoblast proliferation and myotube formation are critical early events in skeletal muscle regeneration.The attending inflammation and cytokine signaling are involved in regulation of myoblast proliferation and differentiation. The aim of this study was to find out whether secretion of muscle derived cytokines resulting from the exposure to inflammatory factors depends on the differentiation stage of regenerating muscle cells. Cultured human myoblasts and myotubes were exposed to 24-hour pre-treatment with tumor necrosis factor (TNF)- alfa or lipopolysaccharide (LPS). Secretion of interleukin 6 (IL-6), a major muscle derived cytokine, and interleukin 1 (IL-1), an important regulator of inflammatory response, were measured 24 hours after termination of TNF- alfa or LPS pre-treatment. Myoblasts pretreated with TNF- alfa or LPS displayed robustly increased IL-6 secretion during the 24h post-treatment period, while IL- 1 secretion remained unaltered. IL-6 secretion was also increased in myotubes, but the response was less pronounced in comparison to myoblasts. In contrast to myoblasts, IL-1 secretion was markedly stimulated in LPS-pretreated myotubes. We demonstrate that pre- exposure to inflammatory factors results in prolonged upregulation of muscle-derived IL-6 and/or IL-1 in cultured skeletal muscle cells. Our findings also indicate that cytokine response to inflammatory factors in regenerating skeletal muscle depends on the differentiation stage of myogenic cells.
COBISS.SI-ID: 36675589
Proteins in living organisms have names that are usually derived from their function in the biochemical system which was investigaed first. Typical examples are acetylcholinesterase and agrin; however, both have been demonstrated to have also various other functions not related to the cholinergic system. Our investigations have been focused on the alternative roles of acetylcholinesterase and agrin in the processes of muscle development and regeneration. Previously, we discovered a role for agrin in the development of excitability of skeletal muscle. In this study, we found that agrin increases IL-6 secretion from myoblasts which are the earliest precursors of muscle regeneration. This effect seems to be general as it was observed in all cell models investigated: human and mouse primary muscle cultures and mouse C2C12 cell line. After fusion of myoblasts into myotubes, this effect of agrin is no longer evident, however in the in vitro innervated human muscle it reappears at the innervation stage . These effects of agrin are another demonstration of its non-synaptic and non-cholinergic roles which are apparently developmental-stage specific. Our data additionally support the view that acetylcholinesterase and agrin participate in various processes during development of skeletal muscle.
COBISS.SI-ID: 31114201
Acetylcholinesterase (AChE) and agrin play unique functional roles in the neuromuscular junction (NMJ). AChE is a cholinergic and agrin a synaptogenetic component. In spite of their different functions, they share several common features: their targeting is determined by alternative splicing; unlike most other NMJ components they are expressed in both, muscle and motor neuron and both reside on the synaptic basal lamina of the NMJ. Also, both were reported to play various nonjunctional roles. However, while the origin of basal lamina bound agrin is undoubtedly neural, the neural origin of AChE, which is anchored to the basal lamina with collagenic tail ColQ, is elusive. Hypothesizing that motor neuron proteins targeted to the NMJ basal lamina share common temporal pattern of expression, which is coordinated with the formation of basal lamina, we compared expression of agrin isoforms with the expression of AChE-T and ColQ in the developing rat spinal cord at the stages before and after the formation of NMJ basal lamina. Cellular origin of AChE-T and agrin was determined by in situ hybridization and their quantitative levels by RT PCR. We found parallel increase in expression of the synaptogenetic (agrin 8) isoform of agrin and ColQ after the formation of basal lamina supporting the view that ColQ bound AChE and agrin 8 isoform are destined to the basal lamina. Catalytic AChE-T subunit and agrin isoforms 19 and 0 followed different expression patterns. In accordance with the reports of other authors, our investigations also revealed various alternative functions for AChE and agrin. We have already demonstrated participation of AChE in myoblast apoptosis; in this study we presented the evidence that agrin promotes maturation of heavy myosin chains and the excitation-contraction coupling. These results show that common features of AChE and agrin extend to their capacity to play multiple roles in muscle development.
COBISS.SI-ID: 30270681
Three fast myosin heavy chain (MyHC) isoforms, i.e. MyHC-2a, -2x and -2b, are expressed in skeletal muscles of smaller mammals. In contrast, only MyHC-2a and -2x have been revealed in humans so far. The expression of MyHC isoforms is known to be wider in the functionally more specialized laryngeal muscles. Though mRNA transcripts of the MyHC-2b gene were found to be expressed in certain human skeletal and laryngeal muscles, the corresponding isoform has not been demonstrated in these muscles. To our knowledge, we are the first to demonstrate not only the expression of MyHC-2b transcripts using an in situ hybridization technique but also the corresponding protein, i.e. the MyHC-2b isoform, in some human laryngeal muscles by immunohistochemistry but not by polyacrylamide gel electrophoresis. Using a set of antibodies specific to MyHC isoforms, we demonstrated that MyHC-2b was always co-expressed with the major MyHC isoforms, not only with the fast ones (MyHC-2a and -2x) but with the slow isoform (MyHC-1) as well.
COBISS.SI-ID: 31191001
The effect of ageing on the capillary network in skeletal muscles has produced conflicting results in both,human and animals studies. Some of the inconsistencies are due to non-comparable and biased methods thatwere applied on thin transversal sections, especially in muscles with complicated morphological structures,such as in human masseter muscle. We present a new immunohistochemical method for staining capillariesand muscle fibres in 100 m thick sections as well as novel approach to 3D visualization of capillaries andmuscle fibres. Applying confocal microscopy and virtual 3D stereological grids, or tracing capillaries invirtual reality, length of capillaries within a muscle volume or length of capillaries adjacent to muscle fibre perfibre length, fibre surface or fibre volume were evaluated in masseter muscle of young and old subjects byan unbiased approach. Our findings show that anatomic capillarity is well maintained in masseter muscle in oldsubjects; however, vascular remodelling occurs with age, which could be a response to changed musclefunction and age-related muscle fibre type transformations.
COBISS.SI-ID: 30985945