Anne Fredsted

Effects of Running Distance and Training on Ca2+ Content and Damage in Human Muscle

PURPOSE Muscle damage and soreness are well-known adverse effects of running, especially when covering distances in excess of habitual running activity. Loss of Ca homeostasis is hypothesized to initiate the development of exercise-induced muscle damage. We tested the hypothesis that the Ca content of vastus lateralis muscle increases after a 10- or 20-km run and studied the relations between Ca accumulation and running distance, endurance training, and fiber type distribution. METHODS Twenty-four healthy young men and women were divided into two groups who ran either 10 or 20 km. Muscle biopsies and blood samples were collected before, immediately after, and in the days after the run. RESULTS : The Ca content in muscle biopsies increased from 0.70 +/- 0.02 to 0.93 +/- 0.04 micromol x g wet weight after the 20-km run (P < 0.001) and was still significantly elevated at 4 and 48 h after the run. In the 10-km runners, however, no significant increase in Ca was found (0.81 +/- 0.03 vs 0.91 +/- 0.06 micromol x g wet weight, P = 0.08). Plasma levels of lactate dehydrogenase and creatine kinase increased after both running distances, the increase being greatest after the 20-km run. Eight of the 10-km runners completed an endurance-training program and subsequently repeated the 10-km run. The response to a new 10-km run with regard to muscle Ca content and parameters of muscle damage was essentially unchanged by training. CONCLUSIONS The degree of muscle damage depends on running distance, and a significant Ca accumulation in muscle is seen after 20 km. Ten weeks of endurance training does not influence Ca homeostasis and muscle damage after 10-km running.

Chronic hypoxia increases rat diaphragm muscle endurance and sodium–potassium ATPase pump content

The effects of chronic hypoxia (CH) on respiratory muscle are poorly understood. The aim of the present study was to examine the effects of CH on respiratory muscle structure and function, and to determine whether nitric oxide is implicated in respiratory muscle adaptation to CH. Male Wistar rats were exposed to CH for 1–6 weeks. Sternohyoid and diaphragm muscle contractile properties, muscle fibre type and size, the density of fibres expressing sarco/endoplasmic reticulum calcium-ATPase (SERCA) 2 and sodium–potassium ATPase (Na+,K+-ATPase) pump content were determined. Muscle succinate dehydrogenase (SDH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) dehydrogenase activities were also assessed. Acute and chronic blockade of nitric oxide synthase (NOS) was employed to determine whether or not NO is critically involved in functional remodelling in CH muscles. CH improved diaphragm, but not sternohyoid, fatigue tolerance in a time-dependent fashion. This adaptation was not attributable to increased SDH or NADPH dehydrogenase activities. The areal density of muscle fibres and relative area of fibres expressing SERCA2 were unchanged. Na+,K+-ATPase pump content was significantly increased in CH diaphragm. Chronic NOS inhibition decreased diaphragm Na+,K+-ATPase pump content and prevented CH-induced increase in muscle endurance. This study provides novel insight into the mechanisms involved in CH-induced muscle plasticity. The results may be of relevance to respiratory disorders characterised by CH, such as chronic obstructive pulmonary disease.

Reactive oxygen species mediated diaphragm fatigue in a rat model of chronic intermittent hypoxia.

What is the central question of this study? The effects of chronic intermittent hypoxia (CIH) on respiratory muscles are relatively underexplored. It is speculated that muscle dysfunction and other key morbidities associated with sleep apnoea are the result of CIH‐induced oxidative stress. We sought to investigate the putative role of CIH‐induced reactive oxygen species in the development of respiratory muscle dysfunction. What is the main finding and its importance? The CIH‐induced diaphragm muscle fatigue is time and intensity dependent and is associated with a modest oxidative stress. Supplementation with N‐acetyl cysteine prevents CIH‐induced diaphragm muscle dysfunction, suggesting that antioxidant supplementation may have therapeutic value in respiratory muscle disorders characterized by CIH, such as obstructive sleep apnoea.

Excitation-induced Ca2+ influx and muscle damage in the rat: loss of membrane integrity and impaired force recovery

Prolonged or unaccustomed exercise leads to loss of contractility and muscle cell damage. The possible role of an increased uptake of Ca2+ in this was explored by examining how graded fatiguing stimulation, leading to a graded uptake of Ca2+, results in progressive loss of force, impairment of force recovery, and loss of cellular integrity. The latter is indicated by increased [14C]sucrose space and lactic acid dehydrogenase (LDH) release. Isolated rat extensor digitorum longus (EDL) muscles were allowed to contract isometrically using a fatiguing protocol with intermittent stimulation at 40 Hz. Force declined rapidly, reaching 11% of the initial level after 10 min and stayed low for up to 60 min. During the initial phase (2 min) of stimulation 45Ca uptake showed a 10‐fold increase, followed by a 4‐ to 5‐fold increase during the remaining period of stimulation. As the duration of stimulation increased, the muscles subsequently regained gradually less of their initial force. Following 30 or 60 min of stimulation, resting 45Ca uptake, [14C]sucrose space, and LDH release were increased 4‐ to 7‐fold, 1.4‐ to 1.7‐fold and 3‐ to 9‐fold, respectively (P < 0.001). The contents of Ca2+ and Na+ were also increased (P < 0.01), a further indication of loss of cellular integrity. When fatigued at low [Ca2+]o (0.65 mm), force recovery was on average twofold higher than that of muscles fatigued at high [Ca2+]o (2.54 mm). Muscles showing the best force recovery also had a 41% lower total cellular Ca2+ content (P < 0.01). In conclusion, fatiguing stimulation leads to a progressive functional impairment and loss of plasma membrane integrity which seem to be related to an excitation‐induced uptake of Ca2+. Mechanical strain on the muscle fibres does not seem a likely mechanism since very little force was developed beyond 10 min of stimulation.

Anoxia induces Ca2+ influx and loss of cell membrane integrity in rat extensor digitorum longus muscle

Anoxia can lead to skeletal muscle damage. In this study we have investigated whether an increased influx of Ca2+, which is known to cause damage during electrical stimulation, is a causative factor in anoxia‐induced muscle damage. Isolated extensor digitorum longus (EDL) muscles from 4‐week‐old Wistar rats were mounted at resting length and were either resting or stimulated (30 min, 40 Hz, 10 s on, 30 s off) in the presence of standard oxygenation (95% O2, 5% CO2), anoxia (95% N2, 5% CO2) or varying degrees of reduced oxygenation. At varying extracellular Ca2+ concentrations ([Ca2+]o), 45Ca influx and total cellular Ca2+ content were measured and the release of lactic acid dehydrogenase (LDH) was determined as an indicator of cell membrane leakage. In resting muscles, incubated at 1.3 mm Ca2+, 15–75 min of exposure to anoxia increased 45Ca influx by 46–129% (P < 0.001) and Ca2+ content by 20–50% (P < 0.001). Mg2+ (11.2 mm) reduced the anoxia‐induced increase in 45Ca influx by 43% (P < 0.001). In muscles incubated at 20 and 5% O2, 45Ca influx was also stimulated (P < 0.001). Increasing [Ca2+]o to 5 mm induced a progressive increase in both 45Ca uptake and LDH release in resting anoxic muscles. When electrical stimulation was applied during anoxia, Ca2+ content and LDH release increased markedly and showed a significant correlation (r2= 0.55, P < 0.001) . In conclusion, anoxia or incubation at 20 or 5% O2 leads to an increased influx of 45Ca. This is associated with a loss of cell membrane integrity, possibly initiated by Ca2+. The loss of cell membrane integrity further increases Ca2+ influx, which may elicit a self‐amplifying process of cell membrane leakage.

Effects of step exercise on muscle damage and muscle Ca2+ content in men and women.

Fredsted, A, Clausen, T, Overgaard, K. Effects of step exercise on muscle damage and muscle Ca2+ content in men and women. J Strength Cond Res 22: 1136-1146, 2008-Eccentric exercise often produces severe muscle damage, whereas concentric exercise of a similar load elicits a minor degree of muscle damage. The cellular events initiating muscle damage are thought to include an increase in cytosolic Ca2+. It was hypothesized that eccentric muscle activity in humans would lead to a larger degree of cell damage and increased intracellular Ca2+ accumulation in skeletal muscle than concentric activity would. Furthermore, possible differences between men and women in muscle damage were investigated following step exercise. Thirty-three healthy subjects (18 men and 15 women) participated in a 30-minute step exercise protocol involving concentric contractions with 1 leg and eccentric contractions with the other leg. Muscle Ca2+ content, maximal voluntary contraction (MVC), and muscle enzymes in the plasma were measured. In a subgroup of the subjects, T2 relaxation time was measured by magnetic resonance imaging. No significant changes were found in muscle Ca2+ content in vastus lateralis biopsy specimens in women or in men. Following step exercise, MVC decreased in both legs of both genders. The women had a significantly larger strength decrease in the eccentric leg than the men had on postexercise day 2 (p < 0.01). Plasma creatine kinase increased following step exercise, with a sevenfold higher response in women than in men on day 3 (p < 0.001). The women, but not the men, had an increase in T2 relaxation time in the eccentrically working adductor magnus muscle, peaking on day 3 (75%) (p < 0.001). In conclusion, step exercise does not lead to Ca2+ accumulation in the vastus lateralis but does induce muscle damage preferentially in the eccentrically working muscles, considerably more in women than in men. This indicates that gender-specific step training programs may be warranted to avoid excessive muscle damage.

Diaphragm Muscle Remodeling in a Rat Model of Chronic Intermittent Hypoxia

Respiratory muscle remodeling occurs in human sleep apnea—a common respiratory disorder characterized by chronic intermittent hypoxia (CIH) due to recurrent apnea during sleep. We sought to determine if CIH causes remodeling in rat sternohyoid (upper airway dilator) and diaphragm muscles. Adult male Wistar rats were exposed to CIH (n=8), consisting of 90 sec of hypoxia (5% at the nadir; SaO2 ~80%)/90 sec of normoxia, 8 hr per day, for 7 consecutive days. Sham animals (n=8) were exposed to alternating air/air cycles in parallel. The effect of CIH on myosin heavy-chain (MHC) isoform (1, 2a, 2x, 2b) distribution, sarcoplasmic reticulum calcium ATPase (SERCA) isoform distribution, succinate dehydrogenase activity, glycerol phosphate dehydrogenase activity, and Na+/K+ ATPase pump content was determined. Sternohyoid muscle structure was unaffected by CIH treatment. CIH did not alter oxidative/glycolytic capacity or the Na+/K+-ATPase pump content of the diaphragm. CIH significantly increased the areal density of MHC 2b fibers in the rat diaphragm, and this was associated with a shift in SERCA proteins from SERCA2 to SERCA1. We conclude that CIH causes a slow-to-fast fiber transition in the rat diaphragm after just 7 days of treatment. Respiratory muscle functional remodeling may drive aberrant functional plasticity such as decreased muscle endurance, which is a feature of human sleep apnea.

Effects of β2-agonists on force during and following anoxia in rat extensor digitorum longus muscle

Electrical stimulation of isolated muscles may lead to membrane depolarization, gain of Na(+), loss of K(+) and fatigue. These effects can be counteracted with β(2)-agonists possibly via activation of the Na(+)-K(+) pumps. Anoxia induces loss of force; however, it is not known whether β(2)-agonists affect force and ion homeostasis in anoxic muscles. In the present study isolated rat extensor digitorum longus (EDL) muscles exposed to anoxia showed a considerable loss of force, which was markedly reduced by the β(2)-agonists salbutamol (10(-6) M) and terbutaline (10(-6) M). Intermittent stimulation (15-30 min) clearly increased loss of force during anoxia and reduced force recovery during reoxygenation. The β(2)-agonists salbutamol (10(-7)-10(-5) M) and salmeterol (10(-6) M) improved force development during anoxia (25%) and force recovery during reoxygenation (55-262%). The effects of salbutamol on force recovery were prevented by blocking the Na(+)-K(+) pumps with ouabain or by blocking glycolysis with 2-deoxyglucose. Dibutyryl cAMP (1 mM) or theophylline (1 mM) also improved force recovery remarkably. In anoxic muscles, salbutamol decreased intracellular Na(+) and increased (86)Rb uptake and K(+) content, indicating stimulation of the Na(+)-K(+) pumps. In fatigued muscles salbutamol induced recovery of excitability. Thus β(2)-agonists reduce the anoxia-induced loss of force, leading to partial force recovery. These data strongly suggest that this effect is mediated by cAMP stimulation of the Na(+)-K(+) pumps and that it is not related to recovery of energy status (PCr, ATP, lactate).