Isolation and quantitative immunocytochemical characterization of primary myogenic cells and fibroblasts from human skeletal 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.

Molecular motor MYO1C, acetyltransferase KAT6B and osteogenetic transcription factor RUNX2 expression in human masseter muscle contributes to development of malocclusion.

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.

Human skeletal muscle fibroblasts, but not myogenic cells, readily undergo adipogenic differentiation.

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.

Epigenetic influence of KAT6B and HDAC4 in the development of skeletal malocclusion.

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.

Human masseter muscle fiber type properties, skeletal malocclusions, and muscle growth factor expression.

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.

Effects of endurance and strength-directed electrical stimulation training on the performance and histological properties of paralyzed human muscle: a pilot study.

Electrical stimulation (ES) improves muscle properties after spinal cord injury (SCI), but cycling power output (PO) remains low. We investigated the effect of endurance and strength ES training on these parameters. Assessments of quadriceps strength and fatigue resistance, cycling PO, and muscle biopsies were made in four well-trained SCI subjects (three cyclists and one rower) before and after additional weight training in the cyclists and once in the rower. Weight training improved muscle strength, but cycling PO was low in all subjects. There was no effect of training type on biopsy data. Biopsies showed non-specific signs of pathology, predominance of type IIa fibers, and uniform metabolic activity. Oxidative activity was low, as were capillary:fiber ratios in the cyclists. Cycling PO is limited by factors other than muscle strength. Future ES training studies should attempt to improve muscle oxidative capacity to optimize the potential benefits of ES exercise.

Specialized cranial muscles: how different are they from limb and abdominal muscles?

Mammalian skeletal muscle fibers can be classified into functional types by the heavy chain (MyHC) and light chain (MyLC) isoforms of myosin (the primary motor protein) that they contain. Most human skeletal muscle contains fiber types and myosin isoforms I, IIA and IIX. Some highly specialized muscle fibers in human extraocular and jaw-closing muscles express either novel myosins or unusual combinations of isoforms of unknown functional significance. Extrinsic laryngeal muscles may express the extraocular MyHC isoform for rapid contraction and a tonic MyHC isoform for slow tonic contractions. In jaw-closing muscles, fiber phenotypes and myosin expression have been characterized as highly unusual. The jaw-closing muscles of most carnivores and primates have tissue-specific expression of the type IIM or 'type II masticatory' MyHC. Human jaw-closing muscles, however, do not contain IIM myosin. Rather, they express myosins typical of developing or cardiac muscle in addition to type I, IIA and IIX myosins, and many of their fibers are hybrids, expressing two or more isoforms. Fiber morphology is also unusual in that the type II fibers are mostly of smaller diameter than type I. By combining physiological and biochemical techniques it is possible to determine the maximum velocity of unloaded shortening (V(o)) of an individual skeletal muscle fiber and subsequently determine the type and amount of myosin isoform. When analyzed, some laryngeal fibers shorten at much faster rates than type II fibers from limb and abdominal muscle. Yet some type I fibers in masseter show an opposite trend towards speeds 10-fold slower than type I fibers of limb muscle. These unusual shortening velocities are most probably regulated by MyHC isoforms in laryngeal fibers and by MyLC isoforms in masseter. For the jaw-closing muscles, this finding represents the first case in human muscle of physiological regulation of kinetics by light chains. Together, these results demonstrate that, compared to other skeletal muscles, cranial muscles have a wider repertoire of contractile protein expression and function. Molecular techniques for reverse transcription of mRNA and amplification by polymerase chain reaction have been applied to typing of single fibers isolated from limb muscles, successfully identifying pure type I, IIA and IIX and hybrid type I/IIA and IIA/IIX fibers. This demonstrates the potential for future studies of the regulation of gene expression in jaw-closing and laryngeal muscles, which have such a variety of complex fiber types fitting them for their roles in vivo.