A rapid and early loss of skeletal muscle mass underlies the physical disability common amongst survivors of critical illness. However, skeletal muscle function depends not only on its quantity but ...its quality, which may be adversely affected. We set out to characterise the changes in macroscopic muscle echogenicity and fascial characteristics that occur early in critical illness, and to relate these to microscopic histologically defined myofibre necrosis and fascial pathology.
Prospective two center observational study.
Thirty subjects comprising a subgroup of patients recruited to the Musculoskeletal Ultrasound in Critical Illness: Longitudinal Evaluation (MUSCLE) study.
Comparisons were made between sequential Vastus Lateralis histological specimens and ultrasound assessment of Rectus Femoris echogenicity. Change in muscle echogenicity was greater in patients who developed muscle necrosis (n = 15) than in those who did not (8.2% 95% CI, -5.3 to 21.7 vs -15.0% 95% CI, -28.9 to -1.09; p = 0.016). The area under receiver operator curve for ultrasound echogenicity's prediction of myofiber necrosis was 0.74 (95% CI, 0.565 to 0.919; p = 0.024) increasing to 0.85 (95% CI, 0.703 to -0.995; p = 0.003) with the removal of those with potential iatrogenic muscle damage. Fasciitis was observed in 18 of 30 biopsies (60%).
Myofiber necrosis and fascial inflammation can be detected noninvasively using ultrasound in the critically ill. Fasciitis precedes and frequently accompanies muscle necrosis. These findings may have functional implications for survivors of critical illness.
We characterised the adherent cell types isolated from human skeletal muscle by enzymatic digestion, and demonstrated that even at 72 hours after isolation these cultures consisted predominantly of ...myogenic cells (CD56(+), desmin(+)) and fibroblasts (TE-7(+), collagen VI(+), PDGFRα(+), vimentin(+), fibronectin(+)). To evaluate the behaviour of the cell types obtained, we optimised a double immuno-magnetic cell-sorting method for the separation of myogenic cells from fibroblasts. This procedure gave purities of >96% for myogenic (CD56(+), desmin(+)) cells. The CD56(-) fraction obtained from the first sort was highly enriched in TE-7(+) fibroblasts. Using quantitative analysis of immunofluorescent staining for lipid content, lineage markers and transcription factors, we tested if the purified cell populations could differentiate into adipocytes in response to treatment with either fatty acids or adipocyte-inducing medium. Both treatments caused the fibroblasts to differentiate into adipocytes, as shown by loss of intracellular TE-7, upregulation of the adipogenic transcription factors PPARγ and C/EBPα, and adoption of a lipid-laden adipocyte morphology. By contrast, myogenic cells did not undergo adipogenesis and showed differential regulation of PPARγ and C/EBPα in response to these adipogenic treatments. Our results show that human skeletal muscle fibroblasts are at least bipotent progenitors that can remain as extracellular-matrix-producing cells or differentiate into adipocytes.
Summary
In this study, results are reported from the analyses of vastus lateralis muscle biopsy samples obtained from a subset (n = 90) of 125 previously phenotyped, highly active male and female ...cyclists aged 55–79 years in regard to age. We then subsequently attempted to uncover associations between the findings in muscle and in vivo physiological functions. Muscle fibre type and composition (ATPase histochemistry), size (morphometry), capillary density (immunohistochemistry) and mitochondrial protein content (Western blot) in relation to age were determined in the biopsy specimens. Aside from an age‐related change in capillary density in males (r = −.299; p = .02), no other parameter measured in the muscle samples showed an association with age. However, in males type I fibres and capillarity (p < .05) were significantly associated with training volume, maximal oxygen uptake, oxygen uptake kinetics and ventilatory threshold. In females, the only association observed was between capillarity and training volume (p < .05). In males, both type II fibre proportion and area (p < .05) were associated with peak power during sprint cycling and with maximal rate of torque development during a maximal voluntary isometric contraction. Mitochondrial protein content was not associated with any cardiorespiratory parameter in either males or females (p > .05). We conclude in this highly active cohort, selected to mitigate most of the effects of inactivity, that there is little evidence of age‐related changes in the properties of VL muscle across the age range studied. By contrast, some of these muscle characteristics were correlated with in vivo physiological indices.
Introduction Genetic influences on the development of malocclusion include heritable effects on both masticatory muscles and jaw skeletal morphology. Beyond genetic variations, however, the ...characteristics of muscle and bone are also influenced by epigenetic mechanisms that produce differences in gene expression. We studied 2 enzymes known to change gene expressions through histone modifications, chromatin-modifying histone acetyltransferase KAT6B and deacetylase HDAC4 , to determine their associations with musculoskeletal variations in jaw deformation malocclusions. Methods Samples of masseter muscle were obtained from subjects undergoing orthognathic surgery from 6 malocclusion classes based on skeletal sagittal and vertical dysplasia. The muscles were characterized for fiber type properties by immunohistochemistry, and their total RNA was isolated for gene expression studies by microarray analysis and quantitative real-time polymerase chain reaction. Results Gene expressions for fast isoforms of myosins and contractile regulatory proteins and for KAT6B and HDAC4 were severalfold greater in masseter muscles from a patient with a deepbite compared with one with an open bite, and genes related to exercise and activity did not differ substantially. In the total population, expressions of HDAC4 ( P = 0.03) and KAT6B ( P = 0.004) were significantly greater in subjects with sagittal Class III than in Class II malocclusion, whereas HDAC4 tended to correlate negatively with slow myosin type I and positively with fast myosin gene, especially type IIX. Conclusions These data support other published reports of epigenetic regulation in the determination of skeletal muscle fiber phenotypes and bone growth. Further investigations are needed to elucidate how this regulatory model might apply to musculoskeletal development and malocclusion.
Nuclear receptor signaling plays an important role in energy metabolism. In this study we demonstrate that the nuclear receptor corepressor RIP140 is a key regulator of metabolism in skeletal muscle. ...RIP140 is expressed in a fiber type-specific manner, and manipulation of its levels in null, heterozygous, and transgenic mice demonstrate that low levels promote while increased expression suppresses the formation of oxidative fibers. Expression profiling reveals global changes in the expression of genes implicated in both myofiber phenotype and metabolic functions. Genes involved in fatty-acid oxidation, oxidative phosphorylation, and mitochondrial biogenesis are upregulated in the absence of RIP140. Analysis of cultured myofibers demonstrates that the changes in expression are intrinsic to muscle cells and that nuclear receptor-regulated genes are direct targets for repression by RIP140. Therefore RIP140 is an important signaling factor in the regulation of skeletal muscle function and physiology.
Purpose We identified masseter muscle fiber type property differences in subjects with dentofacial deformities. Patients and Methods Samples of masseter muscle were collected from 139 young adults ...during mandibular osteotomy procedures to assess mean fiber areas and percent tissue occupancies for the 4 fiber types that comprise the muscle. Subjects were classified into 1 of 6 malocclusion groups based on the presence of a skeletal Class II or III sagittal dimension malocclusion and either a skeletal open, deep, or normal bite vertical dimension malocclusion. In a subpopulation, relative quantities of the muscle growth factors IGF-I and GDF-8 gene expression were quantified by real-time polymerase chain reaction. Results Fiber properties were not different in the sagittal malocclusion groups, but were very different in the vertical malocclusion groups ( P ≤ .0004). There were significant mean fiber area differences for type II ( P ≤ .0004) and type neonatal—atrial ( P = .001) fiber types and for fiber percent occupancy differences for both type I-II hybrid fibers and type II fibers ( P ≤ .0004). Growth factor expression differed by gender for IGF-I ( P = .02) and GDF-8 ( P < .01). The ratio of IGF-I:GDF-8 expression associates with type I and II mean fiber areas. Conclusion Fiber type properties are very closely associated with variations in vertical growth of the face, with statistical significance for overall comparisons at P ≤ .0004. An increase in masseter muscle type II fiber mean fiber areas and percent tissue occupancies is inversely related to increases in vertical facial dimension.
Abstract Objective Type I myosins are molecular motors necessary for glucose transport in the cytoplasm and initiation of transcription in the nucleus. Two of these, MYO1H and MYO1C , are paralogs ...which may be important in the development of malocclusion. The objective of this study was to investigate their gene expression in the masseter muscle of malocclusion subjects. Two functionally related proteins known to contribute to malocclusion were also investigated: KAT6B (a chromatin remodelling epigenetic enzyme which is activated by MYO1C ) and RUNX2 (a transcription factor regulating osteogenesis which is activated by KAT6B ). Design Masseter muscle samples and malocclusion classifications were obtained from orthognathic surgery subjects. Muscle was sectioned and immunostained to determine fibre type properties. RNA was isolated from the remaining sample to determine expression levels for the four genes by TaqMan® RT-PCR. Fibre type properties, gene expression quantities and malocclusion classification were compared. Results There were very significant associations ( P < 0.0000001) between MYO1C and KAT6B expressions. There were also significant associations ( P < 0.005) between RUNX2 expression and masseter muscle type II fibre properties. Very few significant associations were identified between MYO1C and masseter muscle fibre type properties. Conclusions The relationship between MYO1C and KAT6B suggests that the two are interacting in chromatin remodelling for gene expression. This is the nuclear myosin1 (NM1) function of MYO1C. A surprising finding is the relationship between RUNX2 and type II masseter muscle fibres, since RUNX2 expression in mature muscle was previously unknown. Further investigations are necessary to elucidate the role of RUNX2 in adult masseter muscle.
The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic ...digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56(+) and later as CD56(+)/desmin(+) cells and (ii) muscle-derived fibroblasts, identified as CD56(-) and TE-7(+). Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (>95% myogenic cells) and good yield (~2.8 x 10(6) ± 8.87 x 10(5) cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56(+) cells bound to microbeads are retained by the field whereas CD56(-) cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.
We recently reported insulin resistance in adult offspring of obese C57BL/6J mice. We have now evaluated whether parameters of skeletal muscle structure and function may play a role in insulin ...resistance in this model of developmental programming. Obesity was induced in female mice by feeding a highly palatable sugar and fat-rich diet for 6 wk prior to pregnancy, and during pregnancy and lactation. Offspring of obese dams were weaned onto standard laboratory chow. At 3 mo of age, skeletal muscle insulin signaling protein expression, mitochondrial electron transport chain activity (ETC), muscle fiber type, fiber density, and fiber cross-sectional area were compared with that of offspring of control dams weaned onto the chow diet. Female offspring of obese dams demonstrated decreased skeletal muscle expression of p110beta, the catalytic subunit of PI3K (P < 0.01), as well as reduced Akt phosphorylation at Serine residue 473 compared with control offspring. Male offspring of obese dams demonstrated increased skeletal muscle Akt2 and PKCzeta expression (P < 0.01; P < 0.001, respectively). A decrease in mitochondrial-linked complex II-III was observed in male offspring of obese dams (P < 0.01), which was unrelated to CoQ deficiency. This was not observed in females. There were no differences in muscle fiber density between offspring of obese dams and control offspring in either sex. Sex-related alterations in key insulin-signaling proteins and in mitochondrial ETC may contribute to a state of insulin resistance in offspring of obese mice.
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