Ratchakrit Srikuea

Effective fiber hypertrophy in satellite cell-depleted skeletal muscle.

An important unresolved question in skeletal muscle plasticity is whether satellite cells are necessary for muscle fiber hypertrophy. To address this issue, a novel mouse strain (Pax7-DTA) was created which enabled the conditional ablation of >90% of satellite cells in mature skeletal muscle following tamoxifen administration. To test the hypothesis that satellite cells are necessary for skeletal muscle hypertrophy, the plantaris muscle of adult Pax7-DTA mice was subjected to mechanical overload by surgical removal of the synergist muscle. Following two weeks of overload, satellite cell-depleted muscle showed the same increases in muscle mass (approximately twofold) and fiber cross-sectional area with hypertrophy as observed in the vehicle-treated group. The typical increase in myonuclei with hypertrophy was absent in satellite cell-depleted fibers, resulting in expansion of the myonuclear domain. Consistent with lack of nuclear addition to enlarged fibers, long-term BrdU labeling showed a significant reduction in the number of BrdU-positive myonuclei in satellite cell-depleted muscle compared with vehicle-treated muscle. Single fiber functional analyses showed no difference in specific force, Ca2+ sensitivity, rate of cross-bridge cycling and cooperativity between hypertrophied fibers from vehicle and tamoxifen-treated groups. Although a small component of the hypertrophic response, both fiber hyperplasia and regeneration were significantly blunted following satellite cell depletion, indicating a distinct requirement for satellite cells during these processes. These results provide convincing evidence that skeletal muscle fibers are capable of mounting a robust hypertrophic response to mechanical overload that is not dependent on satellite cells.

VDR and CYP27B1 are Expressed in C2C12 Cells and Regenerating Skeletal Muscle: Potential Role in Suppression of Myoblast Proliferation

1α,25(OH)(2)D(3), the active form of vitamin D(3), has been reported to regulate the cell biology of skeletal muscle. However, there has been some controversy about the expression of the vitamin D receptor (VDR) and thus the potential role of vitamin D(3) in skeletal muscle. In this study, we isolated and sequenced the full-length Vdr and Cyp27b1 transcripts in C2C12 myoblasts and myotubes. Western blots and immunocytochemistry confirmed protein expression in both myoblasts and myotubes clearly demonstrating that C2C12 cells express VDR and CYP27B1. To determine the vitamin D(3) action, we found that C2C12 myoblasts treated with either 1α,25(OH)(2)D(3) or 25(OH)D(3) inhibited cell proliferation and this was associated with increased Vdr expression. The observation that treatment of C2C12 myoblasts with the inactive form of vitamin D(3), [25(OH)D(3)], inhibited proliferation suggested that CYP27B1 was functionally active. We used small interfering RNA to knock down Cyp27b1 in myoblasts, and cells were treated with 25(OH)D(3). The growth-suppressive effects of 25(OH)D(3) were abolished, suggesting that CYP27B1 in myoblasts is necessary for the ability of 25(OH)D(3) to affect cell proliferation. Finally, we analyzed expression of VDR and CYP27B1 in regenerating skeletal muscle in vivo. We found that expression of VDR and CYP27B1 increased significantly at day 7 of regeneration, and these results confirm the expression of Vdr and Cyp27b1 in vivo and suggest a potential role for vitamin D(3) in skeletal muscle regeneration following injury.

Inducible Cre transgenic mouse strain for skeletal muscle-specific gene targeting

BackgroundThe use of the Cre/loxP system for gene targeting has been proven to be a powerful tool for understanding gene function. The purpose of this study was to create and characterize an inducible, skeletal muscle-specific Cre transgenic mouse strain.MethodsTo achieve skeletal muscle-specific expression, the human α-skeletal actin promoter was used to drive expression of a chimeric Cre recombinase containing two mutated estrogen receptor ligand-binding domains.ResultsWestern blot analysis, PCR and β-galactosidase staining confirmed that Cre-mediated recombination was restricted to limb and craniofacial skeletal muscles only after tamoxifen administration.ConclusionsA transgenic mouse was created that allows inducible, gene targeting of floxed genes in adult skeletal muscle of different developmental origins. This new mouse will be of great utility to the skeletal muscle community.

Intrinsic muscle clock is necessary for musculoskeletal health

The endogenous molecular clock in skeletal muscle is necessary for maintenance of phenotype and function. Loss of Bmal1 solely from adult skeletal muscle (iMSBmal1−/−) results in reductions in specific tension, increased oxidative fibre type and increased muscle fibrosis with no change in feeding or activity. Disruption of the molecular clock in adult skeletal muscle is sufficient to induce changes in skeletal muscle similar to those seen in the Bmal1 knockout mouse (Bmal1−/−), a model of advanced ageing. iMSBmal1−/− mice develop increased bone calcification and decreased joint collagen, which in combination with the functional changes in skeletal muscle results in altered gait. This study uncovers a fundamental role for the skeletal muscle clock in musculoskeletal homeostasis with potential implications for ageing.

Association of fibromyalgia with altered skeletal muscle characteristics which may contribute to postexertional fatigue in postmenopausal women

OBJECTIVE To identify muscle physiologic properties that may contribute to postexertional fatigue and malaise in women with fibromyalgia (FM). METHODS Healthy postmenopausal women with (n = 11) and without (n = 11) FM, ages 51-70 years, participated in this study. Physical characteristics and responses to self-reported questionnaires were evaluated. Strength loss and tissue oxygenation in response to a fatiguing exercise protocol were used to quantify fatigability and the local muscle hemodynamic profile. Muscle biopsies were performed to assess between-group differences in baseline muscle properties using histochemical, immunohistochemical, and electron microscopic analyses. RESULTS There was no significant difference between healthy controls and FM patients in muscle fatigue in response to exercise. However, self-reported fatigue and pain were correlated with prolonged loss of strength following 12 minutes of recovery in patients with FM. Although there was no difference in percent succinate dehydrogenase (SDH)-positive (type I) and SDH-negative (type II) fibers or in mean fiber cross-sectional area between groups, FM patients exhibited greater variability in fiber size and altered fiber size distribution. In healthy controls only, fatigue resistance was strongly correlated with the size of SDH-positive fibers and hemoglobin oxygenation. In contrast, FM patients with the highest percentage of SDH-positive fibers recovered strength most effectively, and this was correlated with capillary density. However, overall, capillary density was lower in the FM group. CONCLUSION Peripheral mechanisms, i.e., altered muscle fiber size distribution and decreased capillary density, may contribute to postexertional fatigue in FM. Understanding of these defects in fibromyalgic muscle may provide valuable insight with regard to treatment.

Noninvasive optical characterization of muscle blood flow, oxygenation, and metabolism in women with fibromyalgia.

IntroductionWomen with fibromyalgia (FM) have symptoms of increased muscular fatigue and reduced exercise tolerance, which may be associated with alterations in muscle microcirculation and oxygen metabolism. This study used near-infrared diffuse optical spectroscopies to noninvasively evaluate muscle blood flow, blood oxygenation and oxygen metabolism during leg fatiguing exercise and during arm arterial cuff occlusion in post-menopausal women with and without FM.MethodsFourteen women with FM and twenty-three well-matched healthy controls participated in this study. For the fatiguing exercise protocol, the subject was instructed to perform 6 sets of 12 isometric contractions of knee extensor muscles with intensity steadily increasing from 20 to 70% maximal voluntary isometric contraction (MVIC). For the cuff occlusion protocol, forearm arterial blood flow was occluded via a tourniquet on the upper arm for 3 minutes. Leg or arm muscle hemodynamics, including relative blood flow (rBF), oxy- and deoxy-hemoglobin concentration ([HbO2] and [Hb]), total hemoglobin concentration (THC) and blood oxygen saturation (StO2), were continuously monitored throughout protocols using a custom-built hybrid diffuse optical instrument that combined a commercial near-infrared oximeter for tissue oxygenation measurements and a custom-designed diffuse correlation spectroscopy (DCS) flowmeter for tissue blood flow measurements. Relative oxygen extraction fraction (rOEF) and oxygen consumption rate (rVO2) were calculated from the measured blood flow and oxygenation data. Post-manipulation (fatiguing exercise or cuff occlusion) recovery in muscle hemodynamics was characterized by the recovery half-time, a time interval from the end of manipulation to the time that tissue hemodynamics reached a half-maximal value.ResultsSubjects with FM had similar hemodynamic and metabolic response/recovery patterns as healthy controls during exercise and during arterial occlusion. However, tissue rOEF during exercise in subjects with FM was significantly lower than in healthy controls, and the half-times of oxygenation recovery (Δ[HbO2] and Δ[Hb]) were significantly longer following fatiguing exercise and cuff occlusion.ConclusionsOur results suggest an alteration of muscle oxygen utilization in the FM population. This study demonstrates the potential of using combined diffuse optical spectroscopies (i.e., NIRS/DCS) to comprehensively evaluate tissue oxygen and flow kinetics in skeletal muscle.

Satellite cell activity in muscle regeneration after contusion in rats

1. The role of satellite cells in muscle growth during development is well documented, but the involvement of these cells in muscle repair after contusion is less well known. In the present study, we investigated the time‐course of satellite cell activity (from 3 h to 7 days) after contusion of rat gastrocnemius muscle using specific molecular markers for immunofluorescence and real‐time polymerase chain reaction (PCR).

Leukaemia inhibitory factor is expressed in rat gastrocnemius muscle after contusion and increases proliferation of rat L6 myoblasts via c-Myc signalling.

1. Leukaemia inhibitory factor (LIF) has been shown to have an important role during muscle regeneration. The regenerative capacity of muscles after contusion injury in LIF‐knockout mice is impaired compared with that of wild‐type mice.

Erratum to: Inducible Cre transgenic mouse strain for skeletal muscle-specific gene targeting

Skeletal Muscle 2012, 2:8 http://dx.doi.org/10.1186/2044-5040-2-8. The electronic version of this article is the complete one and can be found online at: http://www.skeletalmusclejournal.com/content/2/1/8. Correction Upon publication of this work [1], it was brought to our attention that the reverse primer sequence for genotyping of HSA-MCM pups was incorrect. The correct reverse primer sequence is 5′-CGACCGGCAAACGGACAGAAGC −3′.