Leeann M. Bellamy

Acute Post-Exercise Myofibrillar Protein Synthesis Is Not Correlated with Resistance Training-Induced Muscle Hypertrophy in Young Men

Muscle hypertrophy following resistance training (RT) involves activation of myofibrillar protein synthesis (MPS) to expand the myofibrillar protein pool. The degree of hypertrophy following RT is, however, highly variable and thus we sought to determine the relationship between the acute activation of MPS and RT-induced hypertrophy. We measured MPS and signalling protein activation after the first session of resistance exercise (RE) in untrained men (n = 23) and then examined the relation between MPS with magnetic resonance image determined hypertrophy. To measure MPS, young men (24±1 yr; body mass index  = 26.4±0.9 kg•m2) underwent a primed constant infusion of L-[ring-13C6] phenylalanine to measure MPS at rest, and acutely following their first bout of RE prior to 16 wk of RT. Rates of MPS were increased 235±38% (P<0.001) above rest 60–180 min post-exercise and 184±28% (P = 0.037) 180–360 min post exercise. Quadriceps volume increased 7.9±1.6% (−1.9–24.7%) (P<0.001) after training. There was no correlation between changes in quadriceps muscle volume and acute rates of MPS measured over 1–3 h (r = 0.02), 3–6 h (r = 0.16) or the aggregate 1–6 h post-exercise period (r = 0.10). Hypertrophy after chronic RT was correlated (r = 0.42, P = 0.05) with phosphorylation of 4E-BP1Thr37/46 at 1 hour post RE. We conclude that acute measures of MPS following an initial exposure to RE in novices are not correlated with muscle hypertrophy following chronic RT.

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

Muscular and Systemic Correlates of Resistance Training-Induced Muscle Hypertrophy

Purpose To determine relationships between post-exercise changes in systemic [testosterone, growth hormone (GH), insulin like grow factor 1 (IGF-1) and interleukin 6 (IL-6)], or intramuscular [skeletal muscle androgen receptor (AR) protein content and p70S6K phosphorylation status] factors in a moderately-sized cohort of young men exhibiting divergent resistance training-mediated muscle hypertrophy. Methods Twenty three adult males completed 4 sessions•wk-1 of resistance training for 16 wk. Muscle biopsies were obtained before and after the training period and acutely 1 and 5 h after the first training session. Serum hormones and cytokines were measured immediately, 15, 30 and 60 minutes following the first and last training sessions of the study. Results Mean fiber area increased by 20% (range: -7 to 80%; P<0.001). Protein content of the AR was unchanged with training (fold change = 1.17 ± 0.61; P=0.19); however, there was a significant correlation between the changes in AR content and fiber area (r=0.60, P=0.023). Phosphorylation of p70S6K was elevated 5 hours following exercise, which was correlated with gains in mean fiber area (r=0.54, P=0.007). There was no relationship between the magnitude of the pre- or post-training exercise-induced changes in free testosterone, GH, or IGF-1 concentration and muscle fiber hypertrophy; however, the magnitude of the post exercise IL-6 response was correlated with muscle hypertrophy (r=0.48, P=0.019). Conclusion Post-exercise increases in circulating hormones are not related to hypertrophy following training. Exercise-induced changes in IL-6 correlated with hypertrophy, but the mechanism for the role of IL-6 in hypertrophy is not known. Acute increases, in p70S6K phosphorylation and changes in muscle AR protein content correlated with muscle hypertrophy implicating intramuscular rather than systemic processes in mediating hypertrophy.

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.

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

Regulation of Muscle Satellite Cell Activation and Chemotaxis by Angiotensin II

The role of angiotensin II (Ang II) in skeletal muscle is poorly understood. We report that pharmacological inhibition of Ang II signaling or ablation of the AT1a receptor significantly impaired skeletal muscle growth following myotrauma, in vivo, likely due to impaired satellite cell activation and chemotaxis. In vitro experiments demonstrated that Ang II treatment activated quiescent myoblasts as evidenced by the upregulation of myogenic regulatory factors, increased number of β-gal+, Myf5-LacZ myoblasts and the acquisition of cellular motility. Furthermore, exogenous treatment with Ang II significantly increased the chemotactic capacity of C2C12 and primary cells while AT1a−/− myoblasts demonstrated a severe impairment in basal migration and were not responsive to Ang II treatment. Additionally, Ang II interacted with myoblasts in a paracrine-mediated fashion as 4 h of cyclic mechanical stimulation resulted in Ang II-induced migration of cocultured myoblasts. Ang II-induced chemotaxis appeared to be regulated by multiple mechanisms including reorganization of the actin cytoskeleton and augmentation of MMP2 activity. Collectively, these results highlight a novel role for Ang II and ACE inhibitors in the regulation of skeletal muscle growth and satellite cell function.

Skeletal muscle-endothelial cell cross talk through angiotensin II

The role of angiotensin II (ANG II) in postnatal vasculogenesis and angiogenesis during skeletal muscle (SKM) regeneration is unknown. We examined the capacity of ANG II to stimulate capillary formation and growth during cardiotoxin-induced muscle regeneration in ACE inhibitor-treated ANG II type 1a receptor knockout (AT1a(-/-)) and C57Bl/6 control mice. Analysis of tibialis anterior (TA) cross-sections revealed 17% and 23% reductions in capillarization in AT1a(-/-) and captopril treated mice, respectively, when compared with controls, 21 days postinjury. Conversely, no differences in capillarization were detected at early time points (7 and 10 days). These results identify ANG II as a regulator of angiogenesis but not vasculogenesis in vivo. In vitro angiogenesis assays of human umbilical vein endothelial cells (HUVECs) further confirmed ANG II as proangiogeneic as 71% and 124% increases in tube length and branch point number were observed following ANG II treatment. Importantly, treatment of HUVECs with conditioned media from differentiated muscle cells resulted in an 84% and 203% increase in tube length and branch point number compared with controls, which was abolished following pretreatment of the cells with an angiotensin-converting enzyme inhibitor. The pro-angiogenic effect of ANG II can be attributed to an enhanced endothelial cell migration because both transwell and under agarose migration assays revealed a 37% and 101% increase in cell motility, respectively. Collectively, these data highlight ANG II as a proangiogenic regulator during SKM regeneration in vivo and more importantly demonstrates that ANG II released from SKM can signal endothelial cells and regulate angiogenesis through the induction of endothelial cell migration.

IGF-1 colocalizes with muscle satellite cells following acute exercise in humans

Insulin-like growth factor-1 (IGF-1) regulates stem cell proliferation and differentiation in vitro. The aim of this study was to quantify the change in satellite cell (SC) specific IGF-1 colocalization following exercise. We observed a significant increase (p < 0.05) in the percentage of SC with IGF-1 colocalization from baseline to 72 h after a bout of resistance exercise. This strongly supports a role for IGF-1 in human SC function following exercise.

Captopril treatment induces hyperplasia but inhibits myonuclear accretion following severe myotrauma in murine skeletal muscle

The role of ANG II in skeletal muscle and satellite cell regulation is largely unknown. Cardiotoxin (CTX) was used to investigate whether muscle injury activates a local ANG II signaling system. Following injury, immunohistochelmistry (IHC) analysis revealed a robust increase in the intensity of angiotensinogen and angiotensin type 1 (AT(1)) receptor expression. As regeneration proceeded, however, AT(1) and angiotensinogen were downregulated. Nuclear accretion and fiber formation were also assessed during muscle regeneration in mice treated with captopril (an angiotensin-converting enzyme inhibitor). When ANG II formation was blocked through the use of captopril, we observed a significantly reduced accretion of nuclei into myofibers (-25%), while tibialis anterior total fiber number was significantly increased +37%. This phenotype appeared to be due to alterations in satellite cell differentiation kinetics; captopril treatment led to sustained mRNA expression of markers associated with quiescence and proliferation (Myf5, Pax7) and simultaneously delayed or inhibited the expression of myogenin. IHC staining supported these findings, revealing that captopril treatment resulted in a strong trend (P = 0.06) for a decrease in the proportion of myogenin-positive myoblasts. Furthermore, these observations were associated with a delay in muscle fiber maturation; captopril treatment resulted in sustained expression of embryonic myosin heavy chain. Collectively, these findings demonstrate that localized skeletal muscle angiotensin signaling is important to muscle fiber formation, myonuclear accretion, and satellite cell function.