Inflammatory cells in rat skeletal muscle are elevated after electrically stimulated contractions.

We determined the effect of muscle contractions resulting from high-frequency electrical stimulation (HFES) on inflammatory cells in rat tibialis anterior (TA), plantaris (Pln), and soleus (Sol) muscles at 6, 24, and 72 h post-HFES. A minimum of four and a maximum of seven rats were analyzed at each time point. HFES, applied to the sciatic nerve, caused the Sol and Pln to contract concentrically and the TA to contract eccentrically. Neutrophils were higher (P < 0.05) at 6 and 24 h after HFES in the Sol, Pln, and TA muscles relative to control muscles. ED1(+) macrophages in the Pln were elevated at 6 and 24 h after HFES and were also elevated in the Sol and TA after HFES relative to controls. ED2(+) macrophages in the Sol and TA were elevated at 24 and 72 h after HFES, respectively, and were also elevated in the Pln after HFES relative to controls. In contrast to the TA muscles, the Pln and Sol muscles showed no gross histological abnormalities. Collectively, these data indicate that both eccentric and concentric contractions can increase inflammatory cells in muscle, regardless of whether overt histological signs of injury are apparent.     

The expression of myogenic regulatory factors and muscle growth factors in the masticatory muscles of dystrophin-deficient (MDX) mice

The activities of myogenic regulatory factors (MRF) and muscle growth factors increase in muscle that is undergoing regeneration, and may correspond to some specific changes. Little is known about the role of MRFs in masticatory muscles in mdx mice (the model of Duchenne muscular dystrophy) and particularly about their mRNA expression during the process of muscle regeneration. Using Taqman RT-PCR, we examined the mRNA expression of the MRFs myogenin and MyoD1 (myogenic differentiation 1), and of the muscle growth factors myostatin, IGF1 (insulin-like growth factor) and MGF (mechanogrowth factor) in the masseter, temporal and tongue masticatory muscles of mdx mice (n = 6 to 10 per group). The myogenin mRNA expression in the mdx masseter and temporal muscle was found to have increased (P < 0.05), whereas the myostatin mRNA expressions in the mdx masseter (P < 0.005) and tongue (P < 0.05) were found to have diminished compared to those for the controls. The IGF and MGF mRNA amounts in the mdx mice remained unchanged. Inside the mdx animal group, gender-related differences in the mRNA expressions were also found. A higher mRNA expression of myogenin and MyoD1 in the mdx massterer and temporal muscles was found in females in comparison to males, and the level of myostatin was higher in the masseter and tongue muscle (P < 0.001 for all comparisons). Similar gender-related differences were also found within the control groups. This study reveals the intermuscular differences in the mRNA expression pattern of myogenin and myostatin in mdx mice. The existence of these differences implies that dystrophinopathy affects the skeletal muscles differentially. The finding of gender-related differences in the mRNA expression of the examined factors may indicate the importance of hormonal influences on muscle regeneration.     

Skeletal myogenic progenitors originating from embryonic dorsal aorta coexpress endothelial and myogenic markers and contribute to postnatal muscle growth and regeneration

Skeletal muscle in vertebrates is derived from somites, epithelial structures of the paraxial mesoderm, yet many unrelated reports describe the occasional appearance of myogenic cells from tissues of nonsomite origin, suggesting either transdifferentiation or the persistence of a multipotent progenitor. Here, we show that clonable skeletal myogenic cells are present in the embryonic dorsal aorta of mouse embryos. This finding is based on a detailed clonal analysis of different tissue anlagen at various developmental stages. In vitro, these myogenic cells show the same morphology as satellite cells derived from adult skeletal muscle, and express a number of myogenic and endothelial markers. Surprisingly, the latter are also expressed by adult satellite cells. Furthermore, it is possible to clone myogenic cells from limbs of mutant c-Met-/- embryos, which lack appendicular muscles, but have a normal vascular system. Upon transplantation, aorta-derived myogenic cells participate in postnatal muscle growth and regeneration, and fuse with resident satellite cells.The potential of the vascular system to generate skeletal muscle cells may explain observations of nonsomite skeletal myogenesis and raises the possibility that a subset of satellite cells may derive from the vascular system.     

Selection of multipotent cells and enhanced muscle reconstruction by myogenic macrophage-secreted factors

Skeletal muscle regeneration relies on satellite cells, a population of myogenic precursors. Inflammation also plays a determinant role in the process, as upon injury, macrophages are attracted by the damaged myofibers and the activated satellite cells and act as key elements of dynamic muscle supportive stroma. Yet, it is not known how macrophages interact with the more profound stem cells of the satellite cell niche. Here we show that in the presence of a murine macrophage conditioned medium (mMCM) a subpopulation of multipotent cells could be selected and expanded from adult rat muscle. These cells were small, round, poorly adhesive, slow-growing and showed mesenchymal differentiation plasticity. At the same time, mMCM showed clear myogenic capabilities, as experiments with satellite cells mechanically isolated from suspensions of single myofibers showed that the macrophagic factors inhibited their tendency to shift towards adipogenesis. In vivo, intramuscular administrations of concentrated mMCM in a rat model of extensive surgical ablation dramatically improved muscle regeneration. Altogether, these findings suggest that macrophagic factors could be of great help in developing therapeutic protocols with myogenic stem cells.     

Ultra rapid calcium events in electrically stimulated frog nerve terminals.

Fast calcium events occurring in cytoplasmic organelles after a single electrical stimulus were investigated by electron spectroscopic imaging (an electron microscope technique that reveals total calcium with high sensitivity and spatial resolution) in quick frozen presynaptic terminals of the frog neuromuscular junction. In resting preparations synaptic vesicles showed a prominent calcium signal whereas mitochondria were mostly negative and only some of the cisternae of the endoplasmic reticulum were clearly positive. In preparations quick frozen 10 ms after the application to the nerve of a single, supramaximal electric stimulus, no obvious change was observed in synaptic vesicles, while calcium levels rose to high values in the endoplasmic reticulum cisternae and in the matrix of mitochondria. Voltage-induced influx of Ca(2+) within synaptic terminals appears therefore to induce an extremely rapid uptake into selected organelles. The possible physiological role of this response is discussed.     

Gene expression profiles during differentiation and transdifferentiation of bovine myogenic satellite cells

The molecular mechanisms underlying myogenic satellite cells (MSCs) differentiation into myotube-formed cells (MFCs) and transdifferentiation into adipocyte-like cells (ALCs) are unclear. As a step towards understanding the molecular mechanisms underlying MSC differentiation and transdifferentiation, we attempted to identify the genes differentially expressed during differentiation and transdifferentiation using gene microarray analysis (GMA). Thirty oligonucleotide arrays were used with two technical replicates and nine and six biological replicates for MFCs vs. MSCs and ALCs vs. MSCs, respectively, to contrast expression profile differences. GMA identified 1,224 differentially expressed genes by at least 2-fold during differentiation and transdifferentiation of MSCs. To select the highly expressed genes for future functional study, genes with a 4-fold expression difference were selected for validation by real time RT-PCR and approximately 96.9% of the genes were validated. The up-regulation of marker genes for myogenesis (MYL2, MYH3) and adipogenesis (PPAR??, and FABP4) was observed during the differentiation and transdifferentiation of MSCs into MFCs and ALCs, respectively. KOG analysis revealed that the most of the genes up-regulated during differentiation and transdifferentiation of MSCs were related to signal transduction. Again the exact location of 109 differentially expressed genes by 4-fold were analyzed by chromosome mapping. Among those, co-localization of 29 genes up-regulated during transdifferentiation with QTL for marbling score and intramuscular fat percentage supports the involvement of these genes in cellular transdifferentiation. Interestingly, some genes with unknown function were also identified during the process. Functional studies on these genes may unfold the molecular mechanisms controlling MSC differentiation and transdifferentiation.     

Increased expression of the nicotinic acetylcholine receptor in stimulated muscle

Chronic low-frequency stimulation has been used as a model for investigating responses of skeletal muscle fibres to enhanced neuromuscular activity under conditions of maximum activation. Fast-to-slow isoform shifting of markers of the sarcoplasmic reticulum and the contractile apparatus demonstrated successful fibre transitions prior to studying the effect of chronic electro-stimulation on the expression of the nicotinic acetylcholine receptor. Comparative immunoblotting revealed that the alpha- and delta-subunits of the receptor were increased in 10-78 day stimulated specimens, while an associated component of the surface utrophin-glycoprotein complex, beta-dystroglycan, was not drastically changed in stimulated fast skeletal muscle. Previous studies have shown that electro-stimulation induces degeneration of fast glycolytic fibres, trans-differentiation leading to fast-to-slow fibre transitions and activation of muscle precursor cells. In analogy, our results indicate a molecular modification of the central functional unit of the post-synaptic muscle surface within existing neuromuscular junctions and/or during remodelling of nerve-muscle contacts.     

Bacterial growth in ground beef prepared from electrically stimulated and nonstimulated muscles.

Ground beef samples prepared from electrically stimulated and nonstimulated biceps femoris and infraspinatus muscles were inoculated with Lactobacillus sp., Pseudomonas sp., Acinetobacter sp., or a mixture of Lactobacillus spp., Pseudomonas spp., Acinetobacter spp., Moraxella sp., Microbacterium thermosphactum, and Erwinia herbicola. There were no significant differences in growth of various bacteria in ground beef made from electrically stimulated and nonstimulated muscles.     

Analysis of fiber type transformation and histology in chronic electrically stimulated canine rectus abdominis muscle island-flap stomal sphincters

Dynamic skeletal muscle flaps are designed to perform a specific functional task through contraction and relaxation of their muscle fibers. The most commonly used dynamic skeletal flaps today are for cardiomyoplasty and anal or urinary myoplasty. Low-frequency chronic stimulation of these flaps enables them to use their intrinsic energy stores in a more efficient manner through aerobic metabolic pathways for increased endurance and improved work capacity. The purpose of this study was to (1) determine whether fiber type transformation from fatigue-prone (type II) muscle fibers to fatigue-resistant (type I) muscle fibers could be demonstrated in the authors' chronic canine stomal sphincter model where the rectus abdominis muscle was used to create a functional stomal sphincter, (2) assess whether there is any correlation between the degree of muscle fiber type transformation and the continence times, and (3) examine the long-term effects of the training regimens on the skeletal muscle fibers through histologic and volumetric analysis. Eight dynamic island-flap sphincters were created from a part of the rectus abdominis muscle in mongrel dogs by preserving the deep inferior epigastric vascular pedicle and the most caudal investing intercostal nerve. The muscular sphincters were wrapped around a blind loop of distal ileum and trained with pacing electrodes. Two different training protocols were used. In group A (n = 4), a preexisting anal dynamic graciloplasty training protocol was used. A revised protocol was used in group B (n = 4). Muscle biopsy specimens were obtained before and after training from the rectus abdominis muscle sphincter. Fiber type transformation was assessed using a monoclonal antibody directed against the fatigue-prone type II fibers. Pretraining and posttraining skeletal muscle specimens were examined histologically. A significant fiber type conversion was achieved in both group A and group B animals, with each group achieving greater than 50 percent conversion from fatigue-prone (type II) muscle fibers to fatigue-resistant (type I) muscle fibers. The continence time was different for both groups. Biopsy specimens 1 cm from the electrodes revealed that fiber type transformation was uniform throughout this region of the sphincters. Skeletal muscle fibers within both groups demonstrated a reduction in their fiber diameter and volume. Fiber type transformation is possible in this unique canine island-flap rectus abdominis sphincter model. The relative design of the flap with preservation of the skeletal muscle resting length and neuronal and vascular supply are important characteristics when designing a functional dynamic flap for stomal continence.     

Time course of myogenic and metabolic gene expression in response to acute exercise in human skeletal muscle.

The aim of this study was to examine the time course activation of select myogenic (MRF4, Myf5, MyoD, myogenin) and metabolic (CD36, CPT1, HKII, and PDK4) genes after an acute bout of resistance (RE) or run (Run) exercise. Six RE subjects [25 +/- 4 yr (mean +/- SD), 74 +/- 14 kg, 1.71 +/- 0.11 m] and six Run subjects (25 +/- 4 yr, 72 +/- 5 kg, 1.81 +/- 0.07 m, 63 +/- 8 ml.kg(-1).min(-1)) were studied. Eight muscle biopsies were taken from the vastus lateralis (RE) and gastrocnemius (Run) before, immediately after, and 1, 2, 4, 8, 12 and 24 h after exercise. RE increased mRNA of MRF4 (3.7- to 4.5-fold 2-4 h post), MyoD (5.8-fold 8 h post), myogenin (2.6- and 3.5-fold 8-12 h post), HKII (3.6- to 10.5-fold 2-12 h post), and PDK4 (14- to 26-fold 2-8 h post). There were no differences in Myf5, CD36, and CPT1 mRNA levels 0-24 h post-RE. Run increased mRNA of MyoD (5.0- to 8.0-fold), HKII (12- to 16-fold), and PDK4 (32- to 52-fold) at 8-12 h postexercise. There were no differences in MRF4, Myf5, myogenin, CD36 and CPT1 mRNA levels 0-24 h post-Run. These data indicate a myogenic and metabolic gene induction with RE and Run exercise. The timing of the gene induction is variable and generally peaks 4-8 h postexercise with all gene expression not significantly different from the preexercise levels by 24 h postexercise. These data provide basic information for the timing of human muscle biopsy samples for gene-expression studies involving exercise.     

Changes in tropomyosin subunit pattern in chronic electrically stimulated rabbit fast muscles.

The tropomyosin subunit ratio of rabbit fast muscle (α:β = 80:20) changes to that characteristic of skeletal slow muscles (α:β = 55:45) on continuous (10 Hz) stimulation for 3 weeks. The altered myosin light chain pattern and histochemical ATPase stain also show clear changes of fast → slow transformation. However, the rate of changes in the light chain patterns of myosin are slower than those of tropomyosin subunits. These results do not support the previous finding (Amphlett et al., Nature $\text{257}$, 602, 1975) that the tropomyosin subunit pattern remains unaltered during transformation of skeletal muscles and the conclusion that the genetic expression of tropomyosin is regulated under separate control from other myofibrillar proteins. Rather, our results suggest that the polymorphic patterns of all myofibrillar proteins in skeletal muscles undergo changes in a temporal manner during skeletal muscle transformation.     

Expression of myogenic factors in denervated chicken breast muscle: isolation of the chicken Myf5 gene.

In this study, we have isolated and characterized the chicken Myf5 gene, and cDNA clones encoding chicken MyoD1 and myogenin. The chicken Myf5 and MRF4 genes are tandemly located on a single genomic DNA fragment, and the chicken Myf5 gene is organized into at least three exons. Using genomic and cDNA probes, we further analyzed the mRNA levels of four myogenic factors during chicken breast muscle development. This analysis revealed that myogenin expression is restricted to in ovo stages in breast muscle, and is not detectable in neonatal and adult stages. On the other hand, Myf5 expression is detectable until day 7 post-hatching, and is not found in adult muscle, whereas high levels of MyoD1 and MRF4 are detectable at all stages. To further understand the roles of innervation on muscle maturation, we analyzed the expression of the four myogenic factors in denervated adult breast muscle. We found that MyoD1, myogenin, and MRF4 are induced at high levels in denervated muscle, whereas no change occurs in the level of Myf5. These studies suggest that innervation controls the relative abundance and type of myogenic factors that are expressed in adult muscle, and that when nerve control is removed, the muscle reverts to a neonatal phenotype, with the enhanced expression of three myogenic factors (MyoD1, myogenin, and MRF4).     

Metabolic and myogenic factors in local regulation of the microcirculation.

Patterns of flow were recorded from individual capillaries of mesentery and muscle during autoregulation and reactive hyperemia. In cat mesentery at normal arterial pressure capillary blood flow was often periodic in nature. When arterial pressure was reduced periodicity was abolished and in certain cases mean flow increased. Elevation of venous pressure at this time caused restoration of flow periodicity and simultaneously a large fall in mean flow. Vasomotion and autoregulation in mesentery appear to be dependent on intravascular pressure per se. In cat sartorius muscle substantial increase in flow was seen in most capillaries during reactive hyperemia. In certain capillaries the pattern resembled the gross flow pattern while others showed a brief hyperemia and then a period of flow arrest that is presumably due to a strong precapillary vasoconstriction. The latter response is suggestive of a myogenic control while the former may be due to accumulation of metabolites. In frog pectoralis muscle reactive hyperemia was very prolonged in comparison to cat sartorius muscle. The general pattern of flow was consistent with the notion of a strong metabolic control mechanism. The three tissues studied provide examples of strong myogenic, strong metabolic, and combined metabolic and myogenic control of the microcirculation.