Zachary A. Graham

Focal adhesion kinase and its role in skeletal muscle

Skeletal muscle has a remarkable ability to respond to different physical stresses. Loading muscle through exercise, either anaerobic or aerobic, can lead to increases in muscle size and function while, conversely, the absence of muscle loading stimulates rapid decreases in size and function. A principal mediator of this load-induced change is focal adhesion kinase (FAK), a downstream non-receptor tyrosine kinase that translates the cytoskeletal stress and strain signals transmitted across the cytoplasmic membrane by integrins to activate multiple anti-apoptotic and cell growth pathways. Changes in FAK expression and phosphorylation have been found to correlate to specific developmental states in myoblast differentiation, muscle fiber formation and muscle size in response to loading and unloading. With the capability to regulate costamere formation, hypertrophy and glucose metabolism, FAK is a molecule with diverse functions that are important in regulating muscle cell health.

Instrument-assisted soft tissue mobilization: Effects on the properties of human plantar flexors

The effect of instrument-assisted soft tissue mobilization (ISTM) on passive properties and inflammation in human skeletal muscle has not been evaluated. Passive properties of muscle, inflammatory myokines and subjective reporting of functional ability were used to identify the effects of ISTM on the plantar flexors. 11 healthy men were measured for passive musculotendinous stiffness (MTS), passive range of motion (PROM), passive resistive torque (PASTQ) and maximum voluntary contraction peak torque (MVCPT) for plantar flexor muscles of the lower leg. Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were measured from muscle biopsies from the gastrocnemius, and subjective measurements of functional ability were taken using the perception of functional ability questionnaire (PFAQ). MTS, PROM, PRT and MVCPT were measured in the treatment leg (TL) and control leg (CL) before, immediately after, 24 h, 48 h and 72 h following IASTM. Biopsies for IL-6 and TNF-α and PFAQ responses were collected before as well as 24 h, 48 h and 72 h after IASTM. There were no significant differences in MTS, PROM, PASTQ, MVCPT, IL-6 and TNF-α between the TL or CL. A significant decrease in the perception of function and a significant increase in pain for the TL were found following IASTM.

A Soluble Activin Receptor IIB Fails to Prevent Muscle Atrophy in a Mouse Model of Spinal Cord Injury.

Myostatin (MST) is a potent regulator of muscle growth and size. Spinal cord injury (SCI) results in marked atrophy of muscle below the level of injury. Currently, there is no effective pharmaceutical treatment available to prevent sublesional muscle atrophy post-SCI. To determine whether inhibition of MST with a soluble activin IIB receptor (RAP-031) prevents sublesional SCI-induced muscle atrophy, mice were randomly assigned to the following groups: Sham-SCI; SCI+Vehicle group (SCI-VEH); and SCI+RAP-031 (SCI-RAP-031). SCI was induced by complete transection at thoracic level 10. Animals were euthanized at 56 days post-surgery. RAP-031 reduced, but did not prevent, body weight loss post-SCI. RAP-031 increased total lean tissue mass compared to SCI-VEH (14.8%). RAP-031 increased forelimb muscle mass post-SCI by 38% and 19% for biceps and triceps, respectively (p < 0.001). There were no differences in hindlimb muscle weights between the RAP-031 and SCI-VEH groups. In the gastrocnemius, messenger RNA (mRNA) expression was elevated for interleukin (IL)-6 (8-fold), IL-1β (3-fold), and tumor necrosis factor alpha (8-fold) in the SCI-VEH, compared to the Sham group. Muscle RING finger protein 1 mRNA was 2-fold greater in the RAP-031 group, compared to Sham-SCI. RAP-031 did not influence cytokine expression. Bone mineral density of the distal femur and proximal tibia were decreased post-SCI (-26% and -28%, respectively) and were not altered by RAP-031. In conclusion, MST inhibition increased supralesional muscle mass, but did not prevent sublesional muscle or bone loss, or the inflammation in paralyzed muscle.

Abundance in proteins expressed after functional electrical stimulation cycling or arm cycling ergometry training in persons with chronic spinal cord injury

Study design: Longitudinal design. Objectives: The study determined the effects of two forms of exercise training on the abundance of two proteins, (glucose transporter-4 [GLUT-4], adenosine monophosphate kinase [AMPK]) involved in glucose utilization and the transcriptional coactivator that regulates the genes involved in energy metabolism and mitochondrial biogenesis (peroxisome proliferator-activated receptor (PPAR) coactivator 1 alpha [PGC-1α]), in muscles in men with chronic motor-complete spinal cord injury (SCI). Settings: Clinical trial at a Medical Center. Methods: Nine men with chronic motor-complete SCI participated in functional electrical stimulation lower extremity cycling (FES-LEC; n = 4) or arm cycling ergometer (arm-cycling ergometer [ACE]; n = 5) 5 days/week for 16 weeks. Whole body composition was measured by dual energy X-ray absorptiometry. An intravenous glucose tolerance test was performed to measure glucose effectiveness (Sg) and insulin sensitivity (Si). Muscle biopsies of the right vastus lateralis (VL) and triceps muscles were collected one week prior to and post the exercise training intervention. Results: Neither training intervention altered body composition or carbohydrate metabolism. GLUT-4 increased by 3.8 fold in the VL after FES training and increased 0.6 fold in the triceps after ACE training. PGC-1α increased by 2.3 fold in the VL after FES training and 3.8 fold in the triceps after ACE training. AMPK increased by 3.4 fold in the VL after FES training and in the triceps after ACE training. Conclusion: FES-LEC and ACE training were associated with greater protein expressions in the trained muscles by effectively influencing the abundance of GLUT-4, AMPK and PGC-1α. Thus, FES-LEC training of paralyzed muscle can modulate protein expression similar to that of trained and innervated muscle.

The Signature of MicroRNA Dysregulation in Muscle Paralyzed by Spinal Cord Injury Includes Downregulation of MicroRNAs that Target Myostatin Signaling

Spinal cord injury (SCI) results in muscle atrophy, reduced force generation and an oxidative-to-glycolytic fiber type shift. The mechanisms responsible for these alterations remain incompletely understood. To gain new insights regarding mechanisms involved in deterioration of muscle after SCI, global expression profiles of miRs in paralyzed gastrocnemius muscle were compared between sham-operated (Sham) and spinal cord-transected (SCI) rats. Ingenuity Pathways Analysis of the altered miRs identified signaling via insulin, IGF-1, integrins and TGF-β as being significantly enriched for target genes. By qPCR, miRs 23a, 23b, 27b, 145, and 206, were downregulated in skeletal muscle 56 days after SCI. Using FISH, miR-145, a miR not previously implicated in the function of skeletal muscle, was found to be localized to skeletal muscle fibers. One predicted target of miR-145 was Cited2, a transcriptional regulator that modulates signaling through NF-κB, Smad3 and other transcription factors. The 3’ UTR of Cited2 mRNA contained a highly conserved miR-145 seed sequence. Luciferase reporter assays confirmed that miR-145 interacts with this seed sequence. However, Cited2 protein levels were similar between Sham and SCI groups, indicating a biochemical interaction that was not involved in the context of adaptations after SCI. Taken together, the findings indicate dysregulation of several highly expressed miRs in skeletal muscle after SCI and suggest that reduced expression of miR-23a, 145 and 206 may have roles in alteration in skeletal muscle mass and insulin responsiveness in muscle paralyzed by upper motor neuron injuries.

Muscle–bone interactions: movement in the field of mechano–humoral coupling of muscle and bone

Cyclical, mechanical loading of bone by skeletal muscle is widely recognized as a critical determinant of bone structure and mass. A growing body of evidence indicates that substances released from skeletal muscle into the bloodstream also regulate bone mass and metabolism. In this commentary, we discuss the status of research in the area of humoral regulation of bone mass by the skeletal muscle secretome, with an emphasis on the roles of myostatin, irisin, interleukin‐6, and exosomes. The interplay between muscle, bone, and other modulators of bone mass, including circadian rhythm and sympathetic tone, is also discussed.

Changes in α7β1 integrin signaling after eccentric exercise in heat-shocked rat soleus.

α7β1 integrin links the extracellular matrix to the focal adhesion (FA) in skeletal muscle and serves as a stabilizing and signal relayer. Heat shock (HS) induces expression of proteins that interact with the FA.

Short-wave diathermy pretreatment and inflammatory myokine response after high-intensity eccentric exercise

CONTEXT Various modalities have been used to pretreat skeletal muscle to attenuate inflammation. OBJECTIVE To determine the effects of short-wave diathermy (SWD) preheating treatment on inflammation and stress markers after eccentric exercise. DESIGN Controlled laboratory study. SETTING University laboratory setting. PATIENTS OR OTHER PARTICIPANTS Fifteen male (age = 22 ± 4.9 years, height = 179.75 ± 9.56 cm, mass = 82.22 ± 12.67 kg) college-aged students. INTERVENTION(S) Seven participants were selected randomly to receive 40 minutes of SWD heat treatment (HT), and 8 participants served as the control (CON) group and rested without SWD. Both groups completed 7 sets of 10 repetitions of a high-intensity eccentric exercise protocol (EEP) at 120% of the 1-repetition maximum (1-RM) leg extension. MAIN OUTCOME MEASURE(S) We biopsied muscles on days 1, 3 (24 hours post-EEP), and 4 (48 hours post-EEP) and collected blood samples on days 1, 2 (4 hours post-EEP), 3, and 4. We determined 1-RM on day 2 (24 hours post-SWD) and measured 1-RM on days 3 and 4. We analyzed the muscle samples for interleukin 6 (IL-6), tumor necrosis factor α, and heat shock protein 70 and the blood for serum creatine kinase. RESULTS We found a group × time interaction for intramuscular IL-6 levels after SWD (F2,26 = 7.13, P = .003). The IL-6 decreased in HT (F1,6 = 17.8, P = .006), whereas CON showed no change (P > .05). We found a group × time interaction for tumor necrosis factor α levels (F2,26 = 3.71, P = .04), which increased in CON (F2,14 = 7.16, P = .007), but saw no changes for HT (P > .05). No group × time interactions were noted for 1-RM, heat shock protein 70, or creatine kinase (P > .05). CONCLUSIONS The SWD preheating treatment provided a treatment effect for intramuscular inflammatory myokines induced through high-intensity eccentric exercise but did not affect other factors associated with intense exercise and inflammation.

Focal adhesion kinase signaling is decreased 56 days following spinal cord injury in rat gastrocnemius

Study design:Descriptive study.Objectives:The goal of this study was to determine the effects of spinal cord injury (SCI) on aspects of the focal adhesion kinase (FAK) signaling pathway 56 days post injury in rat gastrocnemius.Setting:This study was conducted in Bronx, NY, USA.Methods:Three-month-old male Wistar rats were exposed to either a sham surgery (n=10) or complete T4 spinal cord transection (n=10). Rats were killed 56 days following surgery and the muscle was collected. Following homogenization, proteins of the FAK pathway were analyzed by western immunoblotting or reverse transcription-qPCR. In addition, cellular markers for proteins that target the degradation of FAK were investigated.Results:SCI resulted in significantly lower levels of total and phosphorylated FAK, cSrc and p70S6k, and a trend for increased FRNK protein expression. SCI did not change levels of the α7 or β1 integrin subunits, total or phosphorylated ERK1/2, phosphorylated Akt and TSC2 or total p70S6k. SCI resulted in a greater expression of total Akt. mRNA expression of FAK and the α7 or β1 integrins remained unchanged between sham and SCI groups. Caspase-3/7 activity and Trim72 mRNA and protein expression remained unchanged following SCI.Conclusion:SCI results in diminished FAK signaling and is independent of ERK1/2 and Akt. SCI has no effect on mRNA levels for genes encoding components of the focal adhesion 56 days after injury.

Plasma adiponectin levels are correlated with body composition, metabolic profiles, and mitochondrial markers in individuals with chronic spinal cord injury

Study designCross-sectional design.ObjectivesThis study examined the relationships between circulating adiponectin levels, body composition, metabolic profile, and measures of skeletal muscle mitochondrial enzyme activity and biogenesis.SettingsClinical Research in a Medical Center.MethodsPlasma adiponectin was quantified in 19 individuals with chronic spinal cord injury (SCI). Body composition was evaluated by dual x-ray absorptiometry and magnetic resonance imaging. Metabolic profile was assessed by basal metabolic rate (BMR), oxygen uptake (VO2), and intravenous glucose tolerance testing. Mitochondrial enzyme activity of skeletal muscle was obtained by spectrophotometric assays and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and 5′ AMP-activated protein kinase (AMPK) protein expression was assessed by Western blots.ResultsAdiponectin was negatively related to both total and regional fat mass and positively related to lean mass and muscle mass. Furthermore, there were positive relationships between adiponectin and BMR (r = 0.52, P = 0.02) and VO2 (r = 0.73, P = 0.01). Furthermore, adiponectin was positively related to citrate synthase (r = 0.68, P = 0.002) and complex III activity (r = 0.57, P = 0.02). The relationships between adiponectin and body composition remained significant after accounting for age. The relationships between adiponectin, metabolic profile, and markers of mitochondria mass and activity were influenced by age.ConclusionsThe study demonstrated that adiponectin is closely related to body composition and metabolic profile in persons with SCI and further supports mechanistic studies suggesting that adiponectin may stimulate mitochondrial biogenesis.