Ian M. MacLean

Skeletal muscle fibre type transformation following spinal cord injury.

Following spinal cord injury (SCI), upper motor neuron paralysed muscles lose the normal type I (slow) and II (fast) fibre mosaic pattern and become predominantly composed of type II (fast glycolytic) fibres. The majority of the research demonstrating this fibre type shift was based on pH sensitive myofibrillar ATPase staining techniques on muscle from longstanding paraplegics and quadriplegics. The purpose of this study was to describe muscle fibre type changes over a wide time spectrum post SCI using immunofluorescent techniques which may be more sensitive to change. A total of 19 vastus lateralis muscle biopsy specimens were obtained from 12 SCI subjects representing time points of 0.5 – 219 months post SCI. Fast and slow myosin heavy chain isoform distribution was determined on single muscle fibres for each of the biopsy specimens. Early post SCI (<1 month) myosin heavy chain (MCH) isoform composition remained relatively stable. A transitional period was seen between 1 and 20 months post SCI wherein there was a progressive drop in the proportion of slow MHC isoform fibres and a rise in the proportion that co-expressed both the fast and slow MHC isoform. By approximately 70 months post SCI a new steady state had been reached characterized by almost exclusively fast MHC isoform expression. This research has demonstrated that post SCI muscle type II transformation occurs in stages and commences earlier than previously appreciated. Interventions aimed at preventing or minimizing the transformation would need to be instituted within weeks post SCI.

AMPK activation increases uncoupling protein‐3 expression and mitochondrial enzyme activities in rat muscle without fibre type transitions

The present study examined the effect of chronic activation of 5′‐AMP‐activated protein kinase (AMPK) on the metabolic profile, including uncoupling protein‐3 (UCP‐3) and myosin heavy chain (MHC)‐based fibre phenotype of rodent fast‐twitch tibialis anterior muscle. Sprague‐Dawley rats were given daily injections of 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside (AICAR), a known activator of AMPK, or vehicle (control) for 28 days. After AICAR treatment, UCP‐3 expression at the mRNA level was elevated 1.6 ± 0.1‐fold (P < 0.006) and corresponded to a 3.3 ± 0.2‐fold increase in UCP‐3 protein content (P < 0.0001). In addition, the activities of the mitochondrial reference enzymes citrate synthase (EC 4.1.3.7) and 3‐hydroxyacyl‐CoA‐dehydrogenase (EC 1.1.1.35), which are known to increase in proportion to mitochondrial volume density, were elevated 1.6‐fold (P < 0.006), while the activity of lactate dehydrogenase (EC 1.1.1.27) was reduced to 80 % of control (P < 0.02). No differences were detected after AICAR treatment in the activities of the glycolytic reference enzymes glyceraldehydephosphate dehydrogenase (EC 1.2.1.12) or phosphofructokinase (EC 2.7.1.11), nor were MHC‐based fibre‐type transitions observed, using immunohistochemical or electrophoretic analytical methods. These changes could not be attributed to variations in inter‐organ signalling by metabolic substrates or insulin. We conclude that an AMPK‐dependent pathway of signal transduction does mimic some of the metabolic changes associated with chronic exercise training, but does not affect expression of the MHC‐based structural phenotype. Thus, the metabolic and MHC‐based fibre types do not appear to be regulated in a co‐ordinated way, but may be independently modified by different signalling pathways.

Functional electrical stimulation exercise increases GLUT-1 and GLUT-4 in paralyzed skeletal muscle

The study purpose was to determine the effect of functional electrical stimulation (FES)-leg cycle ergometer training (30 minutes on 3 d/wk for 8 weeks) on the GLUT-1 and GLUT-4 content of paralyzed skeletal muscle. Biopsy samples of vastus lateralis muscle were obtained pre- and post-training from five individuals with motor-complete spinal cord injury ([SCI] four men and one woman aged 31 to 50 years, 3 to 25 years postinjury involving C5-T8). Western blot analysis indicated that GLUT-1 increased by 52% and GLUT-4 increased by 72% with training (P < .05). This coincided with an increase in the muscle oxidative capacity as indicated by a 56% increase in citrate synthase (CS) activity (P < .05) and an improvement in the insulin sensitivity index as determined from oral glucose tolerance tests (P < .05). It is concluded that FES endurance training is effective to increase glucose transporter protein levels in paralyzed skeletal muscle of individuals with SCI.

Testosterone, cortisol and catecholamine responses to exercise stress and autonomic dysreflexia in elite quadriplegic athletes

Episodes of short high intensity exercise are associated with an increase in circulating total testosterone (T) in men. Mechanisms may include hemoconcentration, decreased metabolic clearance and/or increased synthesis. Beta-blockade abolishes the T response suggesting a direct beta-adrenergic effect on the testes. Some spinal cord injured (SCI) athletes deliberately induce autonomic dysreflexia (boosting) to enhance performance. Associated with this practice are elevated catecholamine (CA) levels and exaggerated responses to serum catecholamine levels. Since basal T levels are reported to be normal in the SCI male, the T response to acute high intensity exercise might be expected to be exaggerated by boosting and associated elevated CA levels. The acute exercise T response has not been examined in SCI men to date. To determine whether the increased CA values associated with boosting enhanced the exercise-induced T elevation we measured circulating levels of T, Cortisol (C), norepinephrine (NE) and epinephrine (E) before and after maximal exertion and a simulated 7.5 km race with and without boosting in eight elite quadriplegic athletes. Maximal incremental exercise and a simulated 7.5 km race resulted in a rise in T similar to able bodied men under normal exercise conditions. Under boosted conditions the rise in T was eliminated while NE levels were significantly elevated above unboosted levels. The data may suggest an inhibitory role for CA on T production or release under conditions of extreme stress. Other possible mechanisms include C induced suppression, impaired gonadotropin stimulation of the Leydig cell and CA mediated alterations in gonadal blood supply.

Effects of 10-ppm hydrogen sulfide inhalation in exercising men and women. Cardiovascular, metabolic, and biochemical responses.

This study examined the acute effects of 10-ppm hydrogen sulfide (H2S) inhalation, a concentration equal to its occupational exposure limit, on the cardiovascular, metabolic, and biochemical responses in healthy volunteers. Fifteen men and 13 women completed two 30-minute exercise sessions at 50% of their maximal oxygen uptake, during which they inhaled medical air or 10 ppm H2S in a blind manner. Arterial and finger-prick blood samples were obtained before and during the final minute of exercise. Muscle biopsies were withdrawn from the right vastus lateralis immediately after exercise. Cardiorespiratory measurements were monitored using an automated metabolic cart interfaced with an electrocardiogram and blood pressure apparatus. A significant decrease in oxygen uptake (VO2), with a concomitant increase in blood lactate, was observed in men and women as a result of H2S exposure. No significant changes were observed in arterial blood parameters and the cardiovascular responses under these conditions. Muscle lactate, as well as the activities of lactate dehydrogenase, citrate synthase, and cytochrome oxidase, were not significantly altered by H2S exposure. However, there was a tendency for muscle lactate to increase and citrate synthase activity to decrease in both genders in the presence of H2S. It appeared that 10-ppm H2S inhalation reduced VO2 during exercise, most likely by inhibiting the aerobic capacity of the exercising muscle. These findings question the scientific validity of the current occupational exposure limit for H2S.

Effects of 10-ppm hydrogen sulfide inhalation on pulmonary function in healthy men and women.

This study examined the acute effects of oral inhalation of 10-ppm hydrogen sulfide (H2S) inhalation (a concentration equal to its occupational exposure limit) on the pulmonary function in healthy men and women. Nine men and ten women consented to inhale medical air or 10 ppm H2S for 15 minutes each during cycle exercise at 50% of their maximal aerobic power. Routine pulmonary function tests were administered at rest and immediately after the two exposure conditions. The results indicated no significant changes in any of the variables derived from the flow volume loop, maximum ventilation volume, and diffusion capacity of the lung for carbon monoxide in both genders. None of the subjects experienced any signs and symptoms as a result of H2S exposure. It was concluded that oral inhalation of 10 ppm H2S at an elevated metabolic and ventilation rate does not significantly alter pulmonary function in healthy men and women.

Effects of 5 ppm Hydrogen Sulfide Inhalation on Biochemical Properties of Skeletal Muscle in Exercising Men and Women

This study compared the acute effects of 5 ppm hydrogen sulfide (H2S) inhalation (50 % of its occupational exposure limit) on the biochemical properties of skeletal muscle in exercising men and women. Twenty-five healthy volunteers, 13 men and 12 women, completed two 30-minute submaximal tests at 50% of their predetermined maximal aerobic power (VO2max) while breathing 0 ppm (control) or 5 ppm H2S from a specially designed flow system in a single-blind manner. Immediately after exercise, biopsies were obtained from the vastus lateralis muscle under local anaesthesia. They were subsequently analyzed for concentrations of the following markers of anaerobic and aerobic metabolism: lactate (La), lactate dehydrogenase (LDH), citrate synthase (CS), and cytochrome oxidase (CytOx). Repeated measures analysis of variance indicated that in men, the CS concentration decreased significantly (p = 0.006) as a result of H2S exposure. There was also a tendency for their La and LDH concentrations to increase and CytOx concentration to decrease in the presence of H2S, but these changes were not significant (p > 0.05). In women no significant changes were observed in any of these biochemical properties. These results suggest that (1) exposure to H2S at 50% of its OEL might inhibit aerobic metabolism during exercise in healthy men, thereby increasing their dependency on anaerobic metabolism; and (2) there could be a significant gender difference in the acute response to sub-OEL exposures of H2S.

Effect of satellite cell ablation on low‐frequency‐stimulated fast‐to‐slow fibre‐type transitions in rat skeletal muscle

The purpose of this study was to determine whether satellite cell ablation within rat fast‐twitch muscles exposed to chronic low‐frequency stimulation (CLFS) would limit fast‐to‐slow fibre‐type transitions. Twenty‐nine male Wistar rats were randomly assigned to one of three groups. Satellite cells of the left tibialis anterior were ablated by weekly exposure to a 25 Gy dose of γ‐irradiation during 21 days of CLFS (IRR‐Stim), whilst a second group received only 21 days of CLFS (Stim). A third group received weekly doses of γ‐irradiation (IRR). Non‐irradiated right legs served as internal controls. Continuous infusion of 5‐bromo‐2′‐deoxyuridine (BrdU) revealed that CLFS induced an 8.0‐fold increase in satellite cell proliferation over control (mean ±s.e.m.: 23.9 ± 1.7 versus 3.0 ± 0.5 mm−2, P < 0.0001) that was abolished by γ‐irradiation. M‐cadherin and myogenin staining were also elevated 7.7‐ and 3.8‐fold (P < 0.0001), respectively, in Stim compared with control, indicating increases in quiescent and terminally differentiating satellite cells; these increases were abolished by γ‐irradiation. Myonuclear content was elevated 3.3‐fold (P < 0.0001) in Stim, but remained unchanged in IRR‐Stim. Immunohistochemical analyses revealed attenuation of fast‐to‐slow fibre‐type transitions in IRR‐Stim compared with Stim. Comparable changes were observed at the protein level by SDS‐PAGE. It is concluded that although considerable adaptive potential exists within myonuclei, satellite cells play a role in facilitating fast‐to‐slow fibre‐type transitions.

Nitric oxide synthase inhibition prevents activity-induced calcineurin–NFATc1 signalling and fast-to-slow skeletal muscle fibre type conversions

•  Exercise is known to trigger skeletal muscle structural and functional adaptations. •  Control of these adaptive alterations is a complex process involving multiple signalling pathways and levels of regulation. •  The well‐characterized calcineurin–nuclear factor of activated T‐cells (NFATc1) signalling pathway is involved in the regulation of activity‐dependent alterations in skeletal muscle myosin heavy chain expression. Myosin heavy chain is a contractile protein that largely dictates a muscles speed of contraction. •  We show that a signalling molecule called nitric oxide may be regulating alterations in myosin heavy chain expression via activity‐modulated calcineurin–NFATc1 signalling. •  These findings increase our understanding of how skeletal muscle adaptive alterations are regulated.

Adaptive responses to creatine loading and exercise in fast-twitch rat skeletal muscle

We investigated the effects of chronic creatine loading and voluntary running (Run) on muscle fiber types, proteins that regulate intracellular Ca2+, and the metabolic profile in rat plantaris muscle to ascertain the bases for our previous observations that creatine loading results in a higher proportion of myosin heavy chain (MHC) IIb, without corresponding changes in contractile properties. Forty Sprague-Dawley rats were assigned to one of four groups: creatine-fed sedentary, creatine-fed run-trained, control-fed sedentary, and control-fed run-trained animals. Proportion and cross-sectional area increased 10% and 15% in type IIb fibers and the proportion of type IIa fibers decreased 11% in the creatine-fed run-trained compared with the control-fed run-trained group (P < 0.03). No differences were observed in fast Ca2+-ATPase isoform SERCA1 content (P > 0.49). Creatine feeding alone induced a 41% increase (P < 0.03) in slow Ca2+-ATPase (SERCA2) content, which was further elevated by 33% with running (P < 0.02). Run training alone reduced parvalbumin content by 50% (P < 0.05). By comparison, parvalbumin content was dramatically decreased by 75% (P < 0.01) by creatine feeding alone but was not further reduced by run training. These adaptive changes indicate that elevating the capacity for high-energy phosphate shuttling, through creatine loading, alleviates the need for intracellular Ca2+ buffering by parvalbumin and increases the efficiency of Ca2+ uptake by SERCAs. Citrate synthase and 3-hydroxyacyl-CoA dehydrogenase activities were elevated by run training (P < 0.003) but not by run training + creatine feeding. This indicates that creatine loading during run training supports a faster muscle phenotype that is adequately supported by the existing glycolytic potential, without changes in the capacity for terminal substrate oxidation.