Bryon R. McKay

Co-expression of IGF-1 family members with myogenic regulatory factors following acute damaging muscle-lengthening contractions in humans

Muscle regeneration following injury is dependent on the ability of muscle satellite cells to activate, proliferate and fuse with damaged fibres. This process is controlled by the myogenic regulatory factors (MRF). Little is known about the temporal relation of the MRF with the expression of known myogenic growth factors (i.e. IGF‐1) in humans following muscle damage. Eight subjects (20.6 ± 2.1 years; 81.4 ± 9.8 kg) performed 300 lengthening contractions (180 deg s−1) of their knee extensors in one leg on a dynamometer. Blood and muscle samples were collected before and at 4 (T4), 24 (T24), 72 (T72) and 120 h (T120) post‐exercise. Mechano growth factor (MGF), IGF‐1Ea and IGF‐1Eb mRNA were quantified. Serum IGF‐1 did not change over the post‐exercise time course. IGF‐1Ea and IGF‐1Eb mRNA increased ∼4‐ to 6‐fold by T72 (P < 0.01) and MGF mRNA expression peaked at T24 (P= 0.005). MyoD mRNA expression increased ∼2‐fold at T4 (P < 0.05). Myf5 expression peaked at T24 (P < 0.05), while MRF4 and myogenin mRNA expression peaked at T72 (P < 0.05). Myf5 expression strongly correlated with the increase in MGF mRNA (r2= 0.83; P= 0.03), while MRF4 was correlated with both IGF‐1Ea and ‐Eb (r2= 0.90; r2= 0.81, respectively; P < 0.05). Immunofluorescence analysis showed IGF‐1 protein expression localized to satellite cells at T24, and to satellite cells and the myofibre at T72 and T120; IGF‐1 was not detected at T0 or T4. These results suggest that the temporal response of MGF is probably related to the activation/proliferation phase of the myogenic programme as marked by an increase in both Myf5 and MyoD, while IGF‐1Ea and ‐Eb may be temporally related to differentiation as marked by an increase in MRF4 and myogenin expression following acute muscle damage.

Association of interleukin-6 signalling with the muscle stem cell response following muscle-lengthening contractions in humans.

Background The regulation of muscle stem cells in humans in response to muscle injury remains largely undefined. Recently, interleukin-6 (IL-6) has been implicated in muscle stem cell (satellite cell)-mediated muscle hypertrophy in animals; however, the role of IL-6 in the satellite cell (SC) response following muscle-lengthening contractions in humans has not been studied. Methodology/Principal Findings Eight subjects (age 22±1 y; 79±8 kg) performed 300 maximal unilateral lengthening contractions (3.14 rad.s−1) of the knee extensors. Blood and muscle samples were collected before and at 4, 24, 72, and 120 hours post intervention. IL-6, IL-6 receptor (IL-6Rα), cyclin D1, suppressor of cytokine signling-3 (SOCS3) mRNA were measured using quantitative RT-PCR and serum IL-6 protein was measured using an ELISA kit. JAK2 and STAT3 phosphorylated and total protein was measured using western blotting techniques. Immunohistochemical analysis of muscle cross-sections was performed for the quantification of SCs (Pax7+ cells) as well as the expression of phosphorylated STAT3, IL-6, IL-6Rα, and PCNA across all time-points. The SC response, as defined by an amplification of Pax7+ cells, was rapid, increasing by 24 h and peaking 72 h following the intervention. Muscle IL-6 mRNA increased following the intervention, which correlated strongly (R2 = 0.89, p<0.002) with an increase in serum IL-6 concentration. SC IL-6Rα protein was expressed on the fiber, but was also localized to the SC, and IL-6+ SC increased rapidly following muscle-lengthening contractions and returned to basal levels by 72 h post-intervention, demonstrating an acute temporal expression of IL-6 with SC. Phosphorylated STAT3 was evident in SCs 4 h after lengthening contraction, and the downstream genes, cyclin D1 and SOCS3 were significantly elevated 24 hours after the intervention. Conclusions/Significance The increased expression of STAT3 responsive genes and expression of IL-6 within SCs demonstrate that IL-6/STAT3 signaling occurred in SCs, correlating with an increase in SC proliferation, evidenced by increased Pax7+/PCNA+ cell number in the early stages of the time-course. Collectively, these data illustrate that IL-6 is an important signaling molecule associated with the SC response to acute muscle-lengthening contractions in humans.

Myostatin is associated with age-related human muscle stem cell dysfunction

Human aging is accompanied by a progressive loss of muscle mass (sarcopenia). We tested the hypothesis that older males (OMs, 70 ±4 yr, n=9) would have a blunted myogenic response to a physiological stimulus compared to younger controls (21 ±3 yr, n=9). Subjects completed an acute bout of intense unilateral muscle loading. Young healthy males matched for body mass and activity level served as the control group. Muscle biopsies and blood were obtained before and at 3, 24, and 48 h after muscle loading. The muscle stem cell response was analyzed using flow cytometry, immunofluorescent microscopy, and standard protein and mRNA analysis. OMs had 35% fewer basal stem cells and a type II fiber‐specific impairment in stem cell content and proliferation. Myogenic determination factor staining and cell cycle analysis illustrated a severely blunted progression through the myogenic program. Myostatin protein and mRNA were 2‐fold higher in OMs. Stem cell‐specific myostatin levels were not different at baseline; however, there were 67% more myostatin‐positive type II‐associated stem cells in OMs at 24 h. These data illustrate an age‐related impairment of stem cell function in a fiber type‐specific manner. The greater colocalization of myostatin with stem cells provides a mechanism for the impaired myogenic capacity of aged muscle.—McKay, B. R., Ogborn, D. I., Bellamy, L. M., Tarnopolsky, M. A., Parise, G. Myostatin is associated with age‐related human muscle stem cell dysfunction. FASEB J. 26, 2509‐2521 (2012). www.fasebj.org

IL-6 induced STAT3 signalling is associated with the proliferation of human muscle satellite cells following acute muscle damage.

Background Although the satellite cell (SC) is a key regulator of muscle growth during development and muscle adaptation following exercise, the regulation of human muscle SC function remains largely unexplored. STAT3 signalling mediated via interleukin-6 (IL-6) has recently come to the forefront as a potential regulator of SC proliferation. The early response of the SC population in human muscle to muscle-lengthening contractions (MLC) as mediated by STAT3 has not been studied. Methodology/Principal Findings Twelve male subjects (21±2 y; 83±12 kg) performed 300 maximal MLC of the quadriceps femoris at 180°•s−1 over a 55° range of motion with muscle samples (vastus lateralis) and blood samples (antecubital vein) taken prior to exercise (PRE), 1 hour (T1), 3 hours (T3) and 24 hours (T24) post-exercise. Cytoplasmic and nuclear fractions of muscle biopsies were purified and analyzed for total and phosphorylated STAT3 (p-STAT3) by western blot. p-STAT3 was detected in cytoplasmic fractions across the time course peaking at T24 (p<0.01 vs. PRE). Nuclear total and p-STAT3 were not detected at appreciable levels. However, immunohistochemical analysis revealed a progressive increase in the proportion of SCs expressing p-STAT3 with ∼60% of all SCs positive for p-STAT3 at T24 (p<0.001 vs. PRE). Additionally, cMyc, a STAT3 downstream gene, was significantly up-regulated in SCs at T24 versus PRE (p<0.05). Whole muscle mRNA analysis revealed induction of the STAT3 target genes IL-6, SOCS3, cMyc (peaking at T3, p<0.05), IL-6Rα and GP130 (peaking at T24, p<0.05). In addition, Myf5 mRNA was up-regulated at T24 (p<0.05) with no appreciable change in MRF4 mRNA. Conclusions/Significant Findings We demonstrate that IL-6 induction of STAT3 signaling occurred exclusively in the nuclei of SCs in response to MLC. An increase in the number of cMyc+ SCs indicated that human SCs were induced to proliferate under the control of STAT3 signaling.

Hepatocyte growth factor (HGF) and the satellite cell response following muscle lengthening contractions in humans

The time‐courses of satellite cell (SC) activation and protein expression of hepatocyte growth factor (HGF), HGF activator (HGFA), HGFA inhibitor‐1 (HAI‐1), and HGFA inhibitor‐2 (HAI‐2) in human skeletal muscle, as well as serum HGF following a single bout of muscle lengthening contractions, were determined. Eight recreationally active participants were recruited for the study. Subjects performed 300 lengthening contractions involving the quadriceps femoris muscles of a single leg at a fixed velocity of 180°/s. Percutaneous muscle biopsies were taken before (PRE) and at 4 h (T4), 24 h (T24), 72 h (T72), and 120 h (T120) following the exercise. The protocol resulted in an increase in the number of SCs [neural cell adhesion molecule (NCAM)‐labeled cells] expressed relative to total myonuclei, at T24, compared with both PRE and T4 (P < 0.05), and peaked at T72 (∽80% increase vs. PRE, P < 0.05). HGF protein increased significantly in serum from baseline (PRE) to T4 (P < 0.05). Active HGF protein was detected in skeletal muscle at rest [14.4 ± 1.3 average integrated density value (IDV)/actin average IDV] and tended to increase at early time‐points (P = 0.12). HGFA protein increased significantly from PRE to T24 (P < 0.05). HAI‐1 protein increased significantly from PRE to T24 (P < 0.05). HAI‐2 (32 kDa) increased significantly from baseline (PRE) by T24 (P < 0.05), and also by T72 and T120 (P < 0.05). We conclude that a single bout of lengthening muscle contractions is sufficient to activate SCs, which may involve both a local and systemic HGF response to contraction‐induced injury. Muscle Nerve, 2008

The Acute Satellite Cell Response and Skeletal Muscle Hypertrophy following Resistance Training

The extent of skeletal muscle hypertrophy in response to resistance training is highly variable in humans. The main objective of this study was to explain the nature of this variability. More specifically, we focused on the myogenic stem cell population, the satellite cell (SC) as a potential mediator of hypertrophy. Twenty-three males (aged 18–35 yrs) participated in 16 wk of progressive, whole body resistance training, resulting in changes of 7.9±1.6% (range of −1.9–24.7%) and 21.0±4.0% (range of −7.0 to 51.7%) in quadriceps volume and myofibre cross-sectional area (CSA), respectively. The SC response to a single bout of resistance exercise (80% 1RM), analyzed via immunofluorescent staining resulted in an expansion of type II fibre associated SC 72 h following exercise (pre: 11.3±0.9; 72 h: 14.8±1.4 SC/type II fibre; p<0.05). Training resulted in an expansion of the SC pool associated with type I (pre: 10.7±1.1; post: 12.1±1.2 SC/type I fibre; p<0.05) and type II fibres (pre: 11.3±0.9; post: 13.0±1.2 SC/type II fibre; p<0.05). Analysis of individual SC responses revealed a correlation between the relative change in type I associated SC 24 to 72 hours following an acute bout of resistance exercise and the percentage increase in quadriceps lean tissue mass assessed by MRI (r2 = 0.566, p = 0.012) and the relative change in type II associated SC following 16 weeks of resistance training and the percentage increase in quadriceps lean tissue mass assessed by MRI (r2 = 0.493, p = 0.027). Our results suggest that the SC response to resistance exercise is related to the extent of muscular hypertrophy induced by training.

Satellite cell number and cell cycle kinetics in response to acute myotrauma in humans: immunohistochemistry versus flow cytometry

In humans, muscle satellite cell (SC) enumeration is an important measurement used to determine the myogenic response to various stimuli. To date, the standard practice for enumeration is immunohistochemistry (IHC) using antibodies against common SC markers (Pax7, NCAM). Flow cytometry (FC) analysis may provide a more rapid and quantitative determination of changes in the SC pool with potential for additional analysis not easily achievable with standard IHC. In this study, FC analysis revealed that the number of Pax7+ cells per milligram isolated from ∼50 mg of fresh tissue increased 36% 24 h after exercise‐induced muscle injury (300 unilateral maximal eccentric contractions). IHC analysis of Pax7 and neural cell adhesion molecule (NCAM) appeared to sufficiently and similarly represent the expansion of SCs after injury (28–36% increase). IHC and FC data illustrated that Pax7 was the most widely expressed SC marker in muscle cross‐sections and represented the majority of positive cells, while NCAM was expressed to a lesser degree. Moreover, FC and IHC demonstrated a similar percentage change 24 h after injury (36% increase, Pax7; 28% increase, NCAM). FC analysis of isolated SCs revealed that the number of Pax7+ cells per milligram in G2/M phase of the cell cycle increased 202% 24 h after injury. Number of cells per milligram in G0/G1 and cells in S‐phase increased 32% and 59% respectively. Here we illustrate the use of FC as a method for enumerating SC number on a per milligram tissue basis, providing a more easily understandable relation to muscle mass (vs. percentage of myonuclei or per myofibre). Although IHC is a powerful tool for SC analysis, FC is a fast, reliable and effective method for SC quantification as well as a more informative method for cell cycle kinetics of the SC population in humans.

Satellite cells in human skeletal muscle plasticity

Skeletal muscle satellite cells are considered to play a crucial role in muscle fiber maintenance, repair and remodeling. Our knowledge of the role of satellite cells in muscle fiber adaptation has traditionally relied on in vitro cell and in vivo animal models. Over the past decade, a genuine effort has been made to translate these results to humans under physiological conditions. Findings from in vivo human studies suggest that satellite cells play a key role in skeletal muscle fiber repair/remodeling in response to exercise. Mounting evidence indicates that aging has a profound impact on the regulation of satellite cells in human skeletal muscle. Yet, the precise role of satellite cells in the development of muscle fiber atrophy with age remains unresolved. This review seeks to integrate recent results from in vivo human studies on satellite cell function in muscle fiber repair/remodeling in the wider context of satellite cell biology whose literature is largely based on animal and cell models.

Eccentric exercise increases satellite cell content in type II muscle fibers.

INTRODUCTION Satellite cells (SCs) are of key importance in skeletal muscle tissue growth, repair, and regeneration. A single bout of high-force eccentric exercise has been demonstrated to increase mixed muscle SC content after 1-7 d of postexercise recovery. However, little is known about fiber type-specific changes in SC content and their activation status within 24 h of postexercise recovery. METHODS Nine recreationally active young men (23 ± 1 yr) performed 300 eccentric actions of the knee extensors on an isokinetic dynamometer. Skeletal muscle biopsies from the vastus lateralis were collected preexercise and 24 h postexercise. Muscle fiber type-specific SC content and the number of activated SCs were determined by immunohistochemical analyses. RESULTS There was no difference between Type I and Type II muscle fiber SC content before exercise. SC content significantly increased 24 h postexercise in Type II muscle fibers (from 0.085 ± 0.012 to 0.133 ± 0.016 SCs per fiber, respectively; P < 0.05), whereas there was no change in Type I fibers. In accordance, activation status increased from preexercise to 24 h postexercise as demonstrated by the increase in the number of DLK1+ SCs in Type II muscle fibers (from 0.027 ± 0.008 to 0.070 ± 0.017 SCs per muscle fiber P < 0.05). Although no significant changes were observed in the number of Ki-67+ SCs, we did observe an increase in the number of proliferating cell nuclear antigen-positive SCs after 24 h of postexercise recovery. CONCLUSION A single bout of high-force eccentric exercise increases muscle fiber SC content and activation status in Type II but not Type I muscle fibers.

Evidence for the contribution of muscle stem cells to nonhypertrophic skeletal muscle remodeling in humans

The purpose of this study was to explore the possible role of muscle stem cells, also referred to as satellite cells (SCs), in adaptation and remodeling following a nonhypertrophic stimulus in humans. Muscle biopsies were obtained from the vastus lateralis of previously untrained women (n=15; age: 27±8 yr, BMI: 29±6 kg/m2) before and after 6 wk of aerobic interval training. The fiber type‐specific SC response to training was analyzed using immunofluorescent microscopy of muscle cross sections. Following training, the number of SCs associated with fibers expressing myosin heavy‐chain type I and II isoforms (hybrid fibers) increased (pre: 0.062±0.035 SC/hybrid fiber; post: 0.38±0.063 SC/hybrid fiber; P<0.01). In addition, there was a greater number of MyoD+/Pax7– SCs, indicative of differentiating SCs, associated with hybrid fibers (0.18±0.096 MyoD+/Pax7– SC/hybrid fiber) compared to type I (0.015±0.00615 MyoD+/Pax7– SC/type I fiber) or II (0.012±0.00454 MyoD+/Pax7– SC/type II fiber) fibers (P<0.05). There was also a training‐induced increase in the number of hybrid fibers containing centrally located nuclei (15.1%) compared to either type I (3.4%) or II fibers (3.6%) (P<0.01). These data are consistent with the hypothesis that SCs contribute to the remodeling of muscle fibers even in the absence of hypertrophy.—Joanisse, S., Gillen, J. B., Bellamy, L. M., McKay, B. R., Tarnopolsky, M. A., Gibala, M. J., Parise, G. Evidence for the contribution of muscle stem cells to nonhypertrophic skeletal muscle remodeling in humans. FASEB J. 27, 4596–4605 (2013). www.fasebj.org