Myonuclear Content Research Articles

Mesenchymal stem cells augment the adaptive response to eccentric exercise.

The α7β1 integrin is a transmembrane protein expressed in the skeletal muscle that can link the actin cytoskeleton to the surrounding basal lamina. We have previously demonstrated that transgenic mice overexpressing the α7B integrin in the skeletal muscle (MCK:α7B; α7Tg) mount an enhanced satellite cell and growth response to single or multiple bouts of eccentric exercise. In addition, interstitial stem cells characterized as mesenchymal stem cells (MSCs) accumulate in α7Tg muscle (mMSCs) in the sedentary state and after exercise. The results from these studies prompted us to determine the extent to which mMSC underlie the beneficial adaptive responses observed in α7Tg skeletal muscle after exercise. mMSCs (Sca-1CD45) were isolated from α7Tg mice, dye-labeled, and intramuscularly injected into adult wild type recipient mice. After injection of mMSCs or saline, mice remained sedentary (SED) or were subjected to eccentric exercise training (TR) (downhill running) on a treadmill (three times per week) for 2 or 4 wk. Gastrocnemius-soleus complexes were collected 24 h after the last bout of exercise. mMSCs did not directly fuse with existing fibers; however, mMSCs injection enhanced Pax7 satellite cell number and myonuclear content compared with all other groups at 2 wk after exercise. Mean CSA, percentage of larger caliber fibers (>3000 μm), and grip strength were increased in mMSCs/TR compared with saline/SED and mMSCs/SED at 4 wk. mMSC transplantation did not enhance repair or growth in the absence of exercise. The results from this study demonstrate that mMSCs contribute to beneficial changes in satellite cell expansion and growth in α7Tg muscle after eccentric exercise. Thus, MSCs that naturally accumulate in the muscle after eccentric contractions may enhance the adaptive response to exercise.

Fibre type‐specific hypertrophy mechanisms in human skeletal muscle: potential role of myonuclear addition

Fibre type‐specific hypertrophy mechanisms in human skeletal muscle: potential role of myonuclear addition

Fibre type‐specific satellite cell response to aerobic training in sedentary adults

In the present study, we sought to determine the effect of a traditional, 12 week aerobic training protocol on skeletal muscle fibre type distribution and satellite cell content in sedentary subjects. Muscle biopsies were obtained from the vastus lateralis [n = 23 subjects (six male and 17 female); body mass index 30.7 ± 1.2 kg m(-2)] before and after 12 weeks of aerobic training performed on a cycle ergometer. Immunohistochemical analyses were used to quantify myosin heavy chain (MyHC) isoform expression, cross-sectional area and satellite cell and myonuclear content. Following training, a decrease in MyHC hybrid type IIa/IIx fibre frequency occurred, with a concomitant increase in pure MyHC type IIa fibres. Pretraining fibre type correlated with body mass index, and the change in fibre type following training was associated with improvements in maximal oxygen consumption. Twelve weeks of aerobic training also induced increases in mean cross-sectional area in both MyHC type I and type IIa fibres. Satellite cell content was also increased following training, specifically in MyHC type I fibres, with no change in the number of satellite cells associated with MyHC type II fibres. With the increased satellite cell content following training, an increase in myonuclear number per fibre also occurred in MyHC type I fibres. Hypertrophy of MyHC type II fibres occurred without detectable myonuclear addition, suggesting that the mechanisms underlying growth in fast and slow fibres differ. These data provide intriguing evidence for a fibre type-specific role of satellite cells in muscle adaptation following aerobic training.

Automated fiber-type-specific cross-sectional area assessment and myonuclei counting in skeletal muscle

Skeletal muscle is an exceptionally adaptive tissue that compromises 40% of mammalian body mass. Skeletal muscle functions in locomotion, but also plays important roles in thermogenesis and metabolic homeostasis. Thus characterizing the structural and functional properties of skeletal muscle is important in many facets of biomedical research, ranging from myopathies to rehabilitation sciences to exercise interventions aimed at improving quality of life in the face of chronic disease and aging. In this paper, we focus on automated quantification of three important morphological features of muscle: 1) muscle fiber-type composition; 2) muscle fiber-type-specific cross-sectional area, and 3) myonuclear content and location. We experimentally prove that the proposed automated image analysis approaches for fiber-type-specific assessments and automated myonuclei counting are fast, accurate, and reliable.

Arachidonic acid supplementation enhances in vitro skeletal muscle cell growth via a COX-2-dependent pathway

Arachidonic acid (AA) is the metabolic precursor to a diverse range of downstream bioactive lipid mediators. A positive or negative influence of individual eicosanoid species [e.g., prostaglandins (PGs), leukotrienes, and hydroxyeicosatetraenoic acids] has been implicated in skeletal muscle cell growth and development. The collective role of AA-derived metabolites in physiological states of skeletal muscle growth/atrophy remains unclear. The present study aimed to determine the direct effect of free AA supplementation and subsequent eicosanoid biosynthesis on skeletal myocyte growth in vitro. C2C12 (mouse) skeletal myocytes induced to differentiate with supplemental AA exhibited dose-dependent increases in the size, myonuclear content, and protein accretion of developing myotubes, independent of changes in cell density or the rate/extent of myogenic differentiation. Nonselective (indomethacin) or cyclooxygenase 2 (COX-2)-selective (NS-398) nonsteroidal anti-inflammatory drugs blunted basal myogenesis, an effect that was amplified in the presence of supplemental free AA substrate. The stimulatory effects of AA persisted in preexisting myotubes via a COX-2-dependent (NS-389-sensitive) pathway, specifically implying dependency on downstream PG biosynthesis. AA-stimulated growth was associated with markedly increased secretion of PGF(2α) and PGE(2); however, incubation of myocytes with PG-rich conditioned medium failed to mimic the effects of direct AA supplementation. In vitro AA supplementation stimulates PG release and skeletal muscle cell hypertrophy via a COX-2-dependent pathway.

A single bout of exercise activates skeletal muscle satellite cells during subsequent overnight recovery

Skeletal muscle satellite cell (SC) content has been reported to increase following a single bout of exercise. Data on muscle fibre type-specific SC content and/or SC activation status are presently lacking. The objective of the study was to determine the impact of a single bout of exercise on muscle fibre type-specific SC content and activation status following subsequent overnight recovery. Eight healthy men (age, 20 ± 1 years) performed a single bout of combined endurance- and resistance-type exercise. Muscle biopsies were collected before and immediately after exercise, and following 9 h of postexercise, overnight recovery. Muscle fibre type-specific SC and myonuclear content and SC activation status were determined by immunohistochemical analyses. Satellite cell activation status was assessed by immunohistochemical staining for both Delta-like homologue 1 (DLK1) and Ki-67. Muscle fibre size and fibre area per nucleus were greater in type II compared with type I muscle fibres (P < 0.05). At baseline, no differences were observed in the percentage of SCs staining positive for DLK1 and/or Ki67 between fibre types. No significant changes were observed in SC content following 9 h of postexercise, overnight recovery; however, the percentage of DLK1-positive SCs increased significantly during overnight recovery, from 22 ± 5 to 41 ± 5% and from 24 ± 6 to 51 ± 9% in the type I and II muscle fibres, respectively. No changes were observed in the percentage of Ki-67-positive SCs. A single bout of exercise activates both type I and II skeletal muscle fibre SCs within a single night of postexercise recovery, preceding the subsequent increase in SC content.

Voluntary resistance wheel exercise during post‐natal growth in rats enhances skeletal muscle satellite cell and myonuclear content at adulthood

To determine whether voluntary free wheel (FW) or resistance wheel (RW) exercise or reduced muscle activity would influence maturational increases in muscle mass and the number of satellite cells (SCs) and myonuclei (MN) accrued by adulthood. Hind limb muscles of male rats housed with, or without, FWs from 4 to 5, 7 or 10 weeks of age, and rats housed with RWs from 4 to 10 week of age, were evaluated. To assess the effect of reduced muscle activity, gastrocnemius muscles of 4-week-old rats were injected with botulinum toxin (Btx) and collected at 7 weeks of age. Muscle fibre size and the frequency of Pax7-positive SCs and MN were determined in 7- and 10-week-old muscles via immunohistochemical methods. Free wheel exercise enhanced muscle growth and the frequency of SCs in the medial gastrocnemius (MG) (threefold) and vastus lateralis (VL) (twofold) of rats at 10 week of age. Resistance wheel exercise increased the number of SCs and MN (22-30%), with more muscle fibre nuclei being associated with larger fibre size, in the soleus, MG and VL muscles. Btx impaired the normal increases in muscle fibre size and the accrual of MN but not SCs. A greater volume of exercise during maturational growth was important for enhancing SC numbers, whereas their conversion to MN required higher-intensity exercise. The enhanced muscle fibre nuclear populations may influence the capacity of the muscle to adapt to exercise, injury or disuse in later adulthood.

Continuous endurance‐type exercise training does not modulate satellite cell content in obese type 2 diabetes patients

Endurance-type exercise training represents a cornerstone in type 2 diabetes treatment. However, the effects of prolonged continuous, endurance-type exercise on muscle fiber characteristics remain equivocal. Fifteen obese male type 2 diabetes patients (61 ± 6 years) participated in a 6-month continuous, endurance-type exercise program. Muscle biopsies were collected before, and after 2 and 6 months of intervention. Muscle fiber type-specific composition, size, and satellite cell (SC) and myonuclear content were determined by immunohistochemistry. Although continuous endurance-type exercise training lowered total body weight and reduced fat mass, no changes were observed in leg lean mass. At baseline, SC content was significantly lower in type II compared with type I muscle fibers. No change in SC content was observed after exercise training. Continuous endurance-type exercise training lowers fat mass, but it does not increase leg lean mass and/or modulate muscle fiber characteristics in type 2 diabetes patients.

Characteristics of Muscle Fiber Type Are Predictive of Skeletal Muscle Mass and Strength in Elderly Men

To investigate the relationship between skeletal muscle fiber type-specific characteristics, circulating hormone concentrations, and skeletal muscle mass and strength in older men. Cross-sectional analyses. University research center. Forty-one community dwelling elderly men (≥ 65). Leg strength (1-repetition maximum, 1RM) and whole-body and limb muscle mass were determined, and muscle fiber type composition, cross-sectional area (CSA), myonuclear content, and satellite cell (SC) content were assessed in skeletal muscle biopsy samples. In addition, blood samples were collected to determine serum testosterone, sex hormone-binding globulin, insulinlike growth factor (IGF)-1, and IGF binding protein-3 concentrations. Muscle mass correlated with muscle strength (0.41 ≤ correlation coefficient (r) ≤ 0.72; P < .01). Muscle fiber CSA, myonuclear content, and SC content were significantly lower in type II than in type I muscle fibers. Myonuclear and SC content were positively correlated with muscle fiber CSA. Furthermore, greater muscle fiber CSA (type I and II) was associated with greater thigh muscle area and muscle strength (0.30 ≤ r ≤ 0.45; P < .05). Testosterone concentration was positively correlated with muscle mass and muscle fiber CSA. Regression analysis showed that SC content, myonuclear content, and testosterone concentration are predictive of muscle fiber CSA. Furthermore, muscle mass and type II muscle fiber CSA are predictive of muscle strength. Skeletal muscle mass and strength in elderly men are positively correlated with muscle fiber type-specific CSA, myonuclear content, and SC content. These findings support the assumption that a decline in SC content plays an important role in age-related decline in muscle mass and strength.

Point:Counterpoint: Satellite cell addition is/is not obligatory for skeletal muscle hypertrophy

Myofiber size is dynamically regulated, increasing and decreasing depending on muscle use. Hypertrophy is defined by increases in myofiber cross-sectional area and mass as well as myofibrillar protein content. Myofibers contain many hundreds of nuclei, each of which has a nuclear domain. A nuclear

Caspase-3 Regulation of Diaphragm Myonuclear Domain during Mechanical Ventilation–induced Atrophy

Unloading the diaphragm via mechanical ventilation (MV) results in rapid diaphragmatic fiber atrophy. It is unknown whether the myonuclear domain (cytoplasmic myofiber volume/myonucleus) of diaphragm myofibers is altered during MV. We tested the hypothesis that MV-induced diaphragmatic atrophy is associated with a loss of myonuclei via a caspase-3-mediated, apoptotic-like mechanism resulting in a constant myonuclear domain. To test this postulate, Sprague-Dawley rats were randomly assigned to a control group or to experimental groups exposed to 6 or 12 h of MV with or without administration of a caspase-3 inhibitor. After 12 h of MV, type I and type IIa diaphragm myofiber areas were decreased by 17 and 23%, respectively, and caspase-3 inhibition attenuated this decrease. Diaphragmatic myonuclear content decreased after 12 h of MV and resulted in the maintenance of a constant myonuclear domain in all fiber types. Both 6 and 12 h of MV resulted in caspase-3-dependent increases in apoptotic markers in the diaphragm (e.g., number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling positive nuclei and DNA fragmentation). Caspase-3-dependent increases in apoptotic markers occurred after 6 h of MV, before the onset of myofiber atrophy. Collectively, these data support the hypothesis that the myonuclear domain of diaphragm myofibers is maintained during prolonged MV and that caspase-3-mediated myonuclear apoptosis contributes to this process.

Caspase-3 Regulation of Diaphragmatic Myonuclear Apoptosis during prolonged Mechanical Ventilation

PURPOSE: Unloading the diaphragm via mechanical ventilation (MV) results in a rapid decrease in myonuclear content and diaphragmatic myofiber atrophy. We tested the hypothesis that caspase-3 activation is a critical component of the MV-induced myonuclear loss. METHODS: To test this postulate, Sprague-Dawley rats were randomly assigned to one of five experimental groups: control, 6-hour MV, 12-hour MV, 6-hour MV with a highly specific caspase-3 inhibitor (DEVD-CHO), and 12-hour MV with DEVD-CHO. RESULTS: Decreases in myonuclear content and increases in the number of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) positive nuclei were observed in diaphragm muscle fibers following 6-and 12-hours of MV. DEVD-CHO administration attenuated the decrease in both myonuclear content and the increase in TUNEL positive myonuclei at both 6-and 12-hours of MV. In addition, decreases in both Type I and Type IIa diaphragm myofiber cross sectional areas with 12-hours of MV were attenuated by DEVD-CHO administration. CONCLUSIONS: Collectively, these data support the hypothesis that caspase-3 activation is a critical component of myonuclear loss occurring during rapid MV induced myofiber atrophy. This work was supported by National Institutes of Health (R01 HL072789).

Differential effects of muscle fibre length and insulin on muscle-specific mRNA content in isolated mature muscle fibres during long-term culture

The aims of this study were (1) to determine the relationship between muscle fibre cross-sectional area and cytoplasmic density of myonuclei in high- and low-oxidative Xenopus muscle fibres and (2) to test whether insulin and long-term high fibre length caused an increase in the number of myonuclei and in the expression of alpha-skeletal actin and of myogenic regulatory factors (myogenin and MyoD) in these muscle fibres. In high- and low-oxidative muscle fibres from freshly frozen iliofibularis muscles, the number of myonuclei per millimetre fibre length was proportional to muscle fibre cross-sectional area. The in vivo myonuclear density thus seemed to be strictly regulated, suggesting that the induction of hypertrophy required the activation of satellite cells. The effects of muscle fibre length and insulin on myonuclear density and myonuclear mRNA content were investigated on high-oxidative single muscle fibres cultured for 4-5 days. Muscle fibres were kept at a low length (~15% below passive slack length) in culture medium with a high insulin concentration (~6 nmol/l: "high insulin medium") or without insulin, and at a high length (~5% above passive slack length) in high insulin medium. High fibre length and high insulin medium did not change the myonuclear density of isolated muscle fibres during culture. High insulin increased the myonuclear alpha-skeletal actin mRNA content, whereas fibre length had no effect on alpha-skeletal actin mRNA content. After culture at high fibre length in high insulin medium, the myonuclear myogenin mRNA content was 2.5-fold higher than that of fibres cultured at low length in high insulin medium or in medium without insulin. Myonuclear MyoD mRNA content was not affected by fibre length or insulin. These in vitro experiments indicate that high muscle fibre length and insulin enhance muscle gene expression but that other critical factors are required to induce adaptation of muscle fibre size and performance.

Effect of satellite cell ablation on low‐frequency‐stimulated fast‐to‐slow fibre‐type transitions in rat skeletal muscle

The purpose of this study was to determine whether satellite cell ablation within rat fast-twitch muscles exposed to chronic low-frequency stimulation (CLFS) would limit fast-to-slow fibre-type transitions. Twenty-nine male Wistar rats were randomly assigned to one of three groups. Satellite cells of the left tibialis anterior were ablated by weekly exposure to a 25 Gy dose of gamma-irradiation during 21 days of CLFS (IRR-Stim), whilst a second group received only 21 days of CLFS (Stim). A third group received weekly doses of gamma-irradiation (IRR). Non-irradiated right legs served as internal controls. Continuous infusion of 5-bromo-2'-deoxyuridine (BrdU) revealed that CLFS induced an 8.0-fold increase in satellite cell proliferation over control (mean +/-s.e.m.: 23.9 +/- 1.7 versus 3.0 +/- 0.5 mm(-2), P < 0.0001) that was abolished by gamma-irradiation. M-cadherin and myogenin staining were also elevated 7.7- and 3.8-fold (P < 0.0001), respectively, in Stim compared with control, indicating increases in quiescent and terminally differentiating satellite cells; these increases were abolished by gamma-irradiation. Myonuclear content was elevated 3.3-fold (P < 0.0001) in Stim, but remained unchanged in IRR-Stim. Immunohistochemical analyses revealed attenuation of fast-to-slow fibre-type transitions in IRR-Stim compared with Stim. Comparable changes were observed at the protein level by SDS-PAGE. It is concluded that although considerable adaptive potential exists within myonuclei, satellite cells play a role in facilitating fast-to-slow fibre-type transitions.

Maintenance of myonuclear domain during mechanical ventilation induced diaphragmatic atrophy

Unloading the diaphragm via mechanical ventilation (MV) results in rapid diaphragmatic fiber atrophy. We tested the hypothesis that MV-induced diaphragmatic atrophy is associated with a loss of myonuclei via an apoptotic-like mechanism resulting in a constant myonuclear domain. To test this postulate, Sprague-Dawley rats were randomly assigned to one of three experimental groups: control, 6-, or 12-hour MV. Following 12-hours of MV, Type I and Type IIa diaphragm myofiber cross sectional areas were decreased by 17% and 23%, respectively. Myonuclear content was also decreased after 12-hours of MV and resulted in the maintenance of a constant myonuclear domain in all fiber types. Both 6- and 12-hour MV resulted in significant increases in the number of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) positive nuclei in diaphragm fibers. Collectively, these data support the concept that the myonuclear domain of diaphragm myofibers is maintained during MV-induced atrophy and that myonuclear apoptosis contributes to this maintenance. This work was supported by National Institutes of Health (R01 HL62361).

Fiber-type transitions and satellite cell activation in low-frequency-stimulated muscles of young and aging rats.

We examined satellite cell content and the activity of satellite cell progeny in tibialis anterior muscles of young (15 weeks) and aging (101 weeks) Brown Norway (BN) rats, after they were exposed for 50 days to a standardized and highly reproducible regime of chronic low-frequency electrical stimulation. Chronic low-frequency electrical stimulation was successful in inducing fast-to-slow fiber-type transformation, characterized by a 2.3-fold increase in the proportion of IIA fibers and fourfold and sevenfold decreases in the proportion of IID/X and IIB fibers in both young and aging BN rats. These changes were accompanied by a twofold increase in the satellite cell content in both the young and aging groups; satellite cell content reached a level that was significantly higher in the young group (p <.04). The total muscle precursor cell content (i.e., satellite cells plus progeny), however, did not differ between groups, because there was a greater number of satellite cell progeny passing through the proliferative and differentiative compartments of the aging group. The resulting 1.5-fold increase in myonuclear content was similar in the young and aging groups. We conclude that satellite cells and satellite cell progeny of aging BN rats possess an unaltered capacity to contribute to the adaptive response.