K. H. Myburgh

In Vitro myoblast motility models: investigating migration dynamics for the study of skeletal muscle repair

Skeletal muscle repair requires the migration of myoblasts (activated satellite cells) both to the injury site and then within the wound to facilitate cellular alignment in preparation for differentiation, fusion and eventual healing. Along this journey, the cells encounter a range of soluble and extracellular matrix factors which regulate their movement and ultimately determine how successful the repair process will be. Sub-optimal migration can lead to a number of scenarios, including reduced myoblast numbers entering the wound, poor alignment and insufficient differentiation to correctly repair the damage. It is therefore critical that all aspects of myoblast migration are understood, particularly in response to the changing growth and matrix factor profile prevalent following skeletal muscle injury. Since 1962, when Boyden first introduced his chemotactic chamber, numerous in vitro migration assays have been developed to mimic the wound more closely. These have increased in complexity to account for the complex micro-environment found in vivo during muscle repair and include a range of modified cell exclusion, chemotactic and three-dimensional assays. This review describes and discusses these advances and highlights the importance they have in expanding our understanding of myoblast migration dynamics.

Satellite cell count, VO2max, and p38 MAPK in inactive to moderately active young men

Satellite cells (SCs) are responsible for muscle repair following strenuous exercise or injury. SC responses to intervention have been studied, but most studies do not discuss or take into account the substantial variability in SC number among young individuals. We hypothesized that an active lifestyle reflected in higher VO2max may be associated with greater SC number. As training alters basal p38‐mitogen‐activated protein kinase (MAPK) activity, which is associated with SC proliferation, SC count may also correlate with this stress signaling kinase. Muscle biopsies from vastus lateralis of eight male participants were analyzed for fiber type, myogenin, and p38/phospho‐p38 MAPK using SDS‐PAGE and Western blotting. Immunofluorescence was used to detect Pax7+ SCs. Two weeks following the biopsy, subjects underwent an incremental treadmill test to determine VO2max. A strong positive correlation (P = 0.0087) was found between the number of Pax7+ nuclei and VO2max. Pax7+ cell number correlated negatively with phospho‐p38/p38 MAPK (P = 0.0006), but had no correlation with fiber type or myogenin. SC number is proportional to VO2max, and hence it can be postulated that higher levels of physical activity activate SC proliferation but not fusion, underlining the relevance of exercise in stimulating SC pool size even without injury.

ROCK‐2 Is Associated With Focal Adhesion Maturation During Myoblast Migration

Satellite cell migration is critical for skeletal muscle growth and regeneration. Controlled cell migration is dependent on the formation of mature focal adhesions between the cell and the underlying extracellular matrix (ECM). These cell–ECM interactions trigger the activation of signalling events such as the Rho/ROCK pathway. We have previously identified a specific role for ROCK‐2 during myoblast migration. In this study we report that ROCK inhibition with Y‐27632 increases C2C12 myoblast velocity, but at the expense of directional migration. In response to Y‐27632 an increased number of smaller focal adhesions were distributed across adhesion sites that in turn were clearly larger than sites in untreated cells, suggesting a reduction in focal adhesion maturation. We also confirm ROCK‐2 localisation to the focal adhesion sites in migrating myoblasts and demonstrate a change in the distribution of these ROCK‐2 containing adhesions in response to Y‐27632. Taken together, our observations provide further proof that ROCK‐2 regulates directional myoblast migration through focal adhesion formation and maturation. J. Cell. Biochem. 115: 1299–1307, 2014.

Neutrophil and monocyte responses to downhill running: Intracellular contents of MPO, IL‐6, IL‐10, pstat3, and SOCS3

High‐intensity exercise results in immune activation. This study determined whether (a) there is concordance between serum MPO and neutrophil and/or monocyte intracellular MPO content; (b) peripheral blood mononuclear cells respond to inflammatory interleukins (ILs) by increasing intracellular signaling. Healthy male (n = 12) volunteers participated in high‐intensity running (12 × 5 min, 10% decline, 15 km/h). Blood sample (pre, post, 4 h) analyses included serum concentrations of IL‐1β, IL‐1ra, IL‐4, IL‐6, IL‐8, IL‐10, matrix metalloprotease‐9 (MMP‐9) and creatine kinase (CK). Intracellular IL‐6, IL‐10, MPO and STAT3/SOCS3 signaling were assessed in mononuclear cells. CK (1573 ± 756 u/L), MMP‐9 (101 ± 27 ng/mL), neutrophil (9.89 ± 0.76 × 109 cells/L) and monocyte counts (1 ± 0.08 × 109 cells/L) increased at 4 h. At 4 h serum (7.1 ± 1.3 ng/mL) and monocyte MPO (1.7‐fold) increased, whereas neutrophil MPO decreased (0.8‐fold). Intracellular monocyte IL‐10 and IL‐6 decreased by 15% and 20–30%, respectively, coinciding with elevations in serum IL‐10 of 14.5 ± 4.7 pg/mL and IL‐6 of 5.4 ± 2.9 pg/mL, suggesting immune cell cytokine release in response to exercise. Intracellular PBMC p‐STAT3 to total STAT3 ratio increased from pre to 4 h. Circulating monocytes are responsive to increased serum IL‐6 suggesting a negative feedback loop via STAT3 signaling.

Variable inflammation and intramuscular STAT3 phosphorylation and myeloperoxidase levels after downhill running

Individual responses in creatine kinase (CK) release after eccentric exercise are divergent. This study aimed to identify whether this could be related to selected humoral or intramuscular inflammatory factors. Twenty‐three subjects were divided into non‐exercising (n = 5) and downhill run (DHR; n = 18) groups (12 × 5 min, 10% decline at 15 km/h). Blood samples were analyzed for white blood cell differential count, CK, myoglobin, tumor necrosis factor‐α, granulocyte colony‐stimulating factor, interleukin (IL)‐1β, IL‐6, and IL‐10. Muscle biopsies were analyzed for signal transducer and activator of transcription‐3 (STAT3), IκBα, and myeloperoxidase (MPO). DHR participants clustered as early (DHR1) recovery, biphasic response (DHR2), or classic delayed exaggerated CK response (DHR3), with a delayed CK peak (4784 ± 1496 U/L) on day 4. For DHR1 and DHR2, CK peaked on day 1 (DHR1: 1198 ± 837 U/L) or on day 1 and day 4 (DHR2: 1583 ± 448 U/L; 1878 ± 427 U/L), respectively. Immediately post‐DHR, IL‐6 increased in DHR2 and DHR3 whereas IL‐10 increased in all DHR groups. STAT3 signaling increased for DHR1 and DHR2 at 4 h, but MPO at day 2 only in DHR2. Objective cluster analysis uncovered a group of subjects with a characteristic biphasic CK release after DHR. The second elevation was related to their early cytokine response. The results provide evidence that early responses following eccentric exercise are indicative of later variation.

Greater Performance Impairment of Black Runners than White Runners when Running in Hypoxia

This study aimed to compare the response of performance-matched black and white runners during maximal and sub-maximal running in normoxic and hypoxic conditions. 14 well-trained runners (8 black, 6 white) performed 2 incremental maximal exercise tests and 2 fatigue resistance tests at 21% O2 (normoxia) or 14% O2 (hypoxia). Respiratory parameters, heart rate (HR), lactate concentration ([La(-)]) as well as arterial saturation (SpO2) were measured. Enzyme activities and myosin heavy chain content (MHC) were also measured. White runners reached a significantly greater peak treadmill speed and a higher HRmax than black runners in hypoxia (p<0.05). Additionally, White runners achieved a greater time to fatigue than black runners (p<0.05), with black runners displaying a greater decline in performance in hypoxia compared to normoxia (20.3% vs. 13.4%, black vs. white, respectively). However, black runners presented lower [La(-)] and higher SpO2 than white runners in hypoxia (p<0.05). Black runners had a higher proportion of MHC IIa and higher lactate dehydrogenase activity (p<0.05). The greater performance impairment observed in black runners in hypoxia suggests a greater performance sensitivity to this condition, despite the maintenance of physiological variables such as SpO2 and [La (-) ] within a smaller range than white runners.