Hanne Gissel

The Role of Ca2+ in Muscle Cell Damage

Abstract: Skeletal muscle is the largest single organ of the body. Skeletal muscle damage may lead to loss of muscle function, and widespread muscle damage may have serious systemic implications due to leakage of intracellular constituents to the circulation. Ca2+ acts as a second messenger in all muscle and may activate a whole range of processes ranging from activation of contraction to degradation of the muscle cell. It is therefore of vital importance for the muscle cell to control [Ca2+] in the cytoplasm ([Ca2+]c). If the permeability of the sarcolemma for Ca2+ is increased, the muscle cell may suffer Ca2+ overload, defined as an inability to control [Ca2+]c. This could lead to the activation of calpains, resulting in proteolysis of cellular constituents, activation of phospholipase A2 (PLA2), affecting membrane integrity, an increased production of reactive oxygen species (ROS), causing lipid peroxidation, and possibly mitochondrial Ca2+ overload, all of which may further worsen the damage in a self‐reinforcing process. An increased influx of Ca2+ leading to Ca2+ overload in muscle may occur in a range of situations such as exercise, mechanical and electrical trauma, prolonged ischemia, Duchenne muscular dystrophy, and cachexia. Counteractions include membrane stabilizing agents, Ca2+ channel blockers, calpain inhibitors, PLA2 inhibitors, and ROS scavengers.

Erythropoietin Over-Expression Protects against Diet-Induced Obesity in Mice through Increased Fat Oxidation in Muscles

Erythropoietin can be over-expressed in skeletal muscles by gene electrotransfer, resulting in 100-fold increase in serum EPO and significant increases in haemoglobin levels. Earlier studies have suggested that EPO improves several metabolic parameters when administered to chronically ill kidney patients. Thus we applied the EPO over-expression model to investigate the metabolic effect of EPO in vivo. At 12 weeks, EPO expression resulted in a 23% weight reduction (P<0.01) in EPO transfected obese mice; thus the mice weighed 21.9±0.8 g (control, normal diet,) 21.9±1.4 g (EPO, normal diet), 35.3±3.3 g (control, high-fat diet) and 28.8±2.6 g (EPO, high-fat diet). Correspondingly, DXA scanning revealed that this was due to a 28% reduction in adipose tissue mass. The decrease in adipose tissue mass was accompanied by a complete normalisation of fasting insulin levels and glucose tolerance in the high-fat fed mice. EPO expression also induced a 14% increase in muscle volume and a 25% increase in vascularisation of the EPO transfected muscle. Muscle force and stamina were not affected by EPO expression. PCR array analysis revealed that genes involved in lipid metabolism, thermogenesis and inflammation were increased in muscles in response to EPO expression, while genes involved in glucose metabolism were down-regulated. In addition, muscular fat oxidation was increased 1.8-fold in both the EPO transfected and contralateral muscles. In conclusion, we have shown that EPO when expressed in supra-physiological levels has substantial metabolic effects including protection against diet-induced obesity and normalisation of glucose sensitivity associated with a shift to increased fat metabolism in the muscles.

Ca2+ accumulation and cell damage in skeletal muscle during low frequency stimulation

Abstract Electrical stimulation has been shown to produce a marked increase in Ca2+ influx and Ca2+ content in rat skeletal muscle. Long-term low-frequency stimulation (1 Hz, 240 min) increased 45Ca uptake by 30% and 154% in soleus and extensor digitorum longus muscles, respectively. Studies using Ca2+-fluorescent dyes have shown that intracellular concentrations of free Ca2+ are increased up to threefold during long-term low-frequency stimulation, suggesting that muscle cells have difficulties in handling the Ca2+ taken up during stimulation. Furthermore, long-term low-frequency stimulation induces leakage of the intracellular enzyme lactate dehydrogenase from the muscles. This leakage may reflect degradation of membrane proteins by the Ca2+-activated neutral protease calpain. This, in turn, leads to further influx of Ca2+ and further acceleration of protein breakdown. Membrane leakages are likely to result in sensations of pain in the damaged muscle. It is suggested that Ca2+ plays a central role in the development of muscle fibre injury during prolonged muscle activity of workers using a computer mouse.

Sensitive and precise regulation of haemoglobin after gene transfer of erythropoietin to muscle tissue using electroporation

Electroporation-based gene transfer (electro gene transfer (EGT)) is gaining increasing momentum, in particular for muscle tissue, where long-term high-level expression is obtainable. Induction of expression using the Tet-On system was previously established; however, attempts to reach a predefined target dose – a prescription, have not been reported. We set three target haemoglobin levels (10, 12 and 14 mmol/l, base level was 8.2 mmol/l) and aimed at them by transferring the erythropoietin (EPO) gene to mouse tibialis cranialis (TC) muscle, and varying (1) DNA amount, (2) muscle mass transfected and (3) induction with the Tet-On system. Results showed that (a) using GFP, luciferase and EPO low DNA amounts were needed. In fact, 0.5 μg of DNA to one TC muscle led to significant Hgb elevation – this amount extrapolates to 1.4 mg of DNA in humans, (b) three prescribers hit the targets with average Hgb of 10.5, 12.0 and 13.7 mmol/l, (c) different approaches could be used, (d) undershooting could be corrected by retransferring, and (e) overshooting could be alleviated by reducing dose of inducer (doxycycline (dox)). In conclusion, this study shows that using EGT to muscle, a preset level of protein expression can be reached. This is of great interest for future clinical use.

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.

Effects of Electrical Stimulation and Insulin on Na+–K+-ATPase ([3H]Ouabain Binding) in Rat Skeletal Muscle

Exercise has been reported to increase the Na+–K+‐ATPase (Na+–K+ pump) α2 isoform in the plasma membrane 1.2‐ to 1.9‐fold, purportedly reflecting Na+–K+ pump translocation from an undefined intracellular pool. We examined whether Na+–K+ pump stimulation, elicited by muscle contraction or insulin, increases the plasma membrane Na+–K+ pump content ([3H]ouabain binding) in muscles from young rats. Stimulation of isolated soleus muscle for 10 s at 120 Hz caused a rapid rise in intracellular Na+ content, followed by an 18‐fold increase in the Na+ re‐extrusion rate (80 % of theoretical maximum). Muscles frozen immediately or 120 s after 10–120 s stimulation showed 10–22 % decrease in [3H]ouabain binding expressed per gram wet weight, but with no significant change expressed per gram dry weight. In soleus muscles from adult rats, [3H]ouabain binding was unaltered after 10 s stimulation at 120 Hz. Extensor digitorum longus (EDL) muscles stimulated for 10–60 s at 120 Hz showed no significant change in [3H]ouabain binding. Insulin (100 mU ml−1) decreased intracellular Na+ content by 27 % and increased 86Rb uptake by 23 % soleus muscles, but [3H]ouabain binding was unchanged. After stimulation for 30 s at 60 Hz soleus muscle showed a 30% decrease in intracellular Na+ content, demonstrating increased Na+–K+ pump activity, but [3H]ouabain binding measured 5 to 120 min after stimulation was unchanged. Stimulation of soleus or EDL muscles for 120–240 min at 1 Hz (continuously) or 10 Hz (intermittently) produced no change in [3H]ouabain binding per gram dry weight. In conclusion, the stimulating effects of electrical stimulation or insulin on active Na+, K+‐transport in rat skeletal muscle could not be even partially accounted for by an acute increase in the content of functional Na+–K+ pumps in the plasma membrane.

Excitation-induced Ca2+uptake in rat skeletal muscle

In isolated rat extensor digitorum longus (EDL) muscle mounted for isometric contractions, chronic low-frequency electrical stimulation was found to lead to an increased uptake of45Ca (154% above control after 240 min) and a progressive accumulation of Ca2+ (85% above control after 240 min). In soleus, however, this treatment led to a small, but significant, increase in 45Ca uptake (30% above control after 180 min) but no significant accumulation of Ca2+. In muscles mounted for isotonic contractions without any external load, electrical stimulation gave rise to a larger45Ca uptake and accumulation of Ca2+ in both EDL and soleus. These uptakes of Ca2+ coincided with an accumulation of Na+. During isometric or isotonic contractions, stimulation at 40 Hz increased the initial (60 s) rate of 45Ca uptake in soleus muscle 15- and 30-fold, respectively. The stimulation-induced increase in 45Ca uptake was only reduced by 17% by the Ca2+-channel blockers nifedipine and verapamil but was blocked by tetrodotoxin. The initial rate of stimulation-induced 22Na and45Ca uptake was correlated ( r = 0.80; P < 0.003). Stimulation of Na+ channels with veratridine increased 45Ca uptake by 93 and 139% in soleus and EDL, respectively ( P < 0.001), effects that were abolished by tetrodotoxin. The results indicate that in skeletal muscle, excitation induces a considerable influx of Ca2+, mediated by Na+ channels.

Gene expression profiles in skeletal muscle after gene electrotransfer

BackgroundGene transfer by electroporation (DNA electrotransfer) to muscle results in high level long term transgenic expression, showing great promise for treatment of e.g. protein deficiency syndromes. However little is known about the effects of DNA electrotransfer on muscle fibres. We have therefore investigated transcriptional changes through gene expression profile analyses, morphological changes by histological analysis, and physiological changes by force generation measurements. DNA electrotransfer was obtained using a combination of a short high voltage pulse (HV, 1000 V/cm, 100 μs) followed by a long low voltage pulse (LV, 100 V/cm, 400 ms); a pulse combination optimised for efficient and safe gene transfer. Muscles were transfected with green fluorescent protein (GFP) and excised at 4 hours, 48 hours or 3 weeks after treatment.ResultsDifferentially expressed genes were investigated by microarray analysis, and descriptive statistics were performed to evaluate the effects of 1) electroporation, 2) DNA injection, and 3) time after treatment. The biological significance of the results was assessed by gene annotation and supervised cluster analysis.Generally, electroporation caused down-regulation of structural proteins e.g. sarcospan and catalytic enzymes. Injection of DNA induced down-regulation of intracellular transport proteins e.g. sentrin. The effects on muscle fibres were transient as the expression profiles 3 weeks after treatment were closely related with the control muscles. Most interestingly, no changes in the expression of proteins involved in inflammatory responses or muscle regeneration was detected, indicating limited muscle damage and regeneration. Histological analysis revealed structural changes with loss of cell integrity and striation pattern in some fibres after DNA+HV+LV treatment, while HV+LV pulses alone showed preservation of cell integrity. No difference in the force generation capacity was observed in the muscles 2 weeks after DNA electrotransfer.ConclusionThe small and transient changes found in the gene expression profiles are of great importance, as this demonstrates that DNA electrotransfer is safe with minor effects on the muscle host cells. These findings are essential for introducing the DNA electrotransfer to muscle for clinical use. Indeed the HV+LV pulse combination used has been optimised to ensure highly efficient and safe DNA electrotransfer.

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.

Calcium electroporation in three cell lines: a comparison of bleomycin and calcium, calcium compounds, and pulsing conditions.

BACKGROUND Electroporation with calcium (calcium electroporation) can induce ATP depletion-associated cellular death. In the clinical setting, the cytotoxic drug bleomycin is currently used with electroporation (electrochemotherapy) for palliative treatment of tumors. Calcium electroporation offers several advantages over standard treatment options: calcium is inexpensive and may readily be applied without special precautions, as is the case with cytostatic drugs. Therefore, details on the use of calcium electroporation are essential for carrying out clinical trials comparing calcium electroporation and electrochemotherapy. METHODS The effects of calcium electroporation and bleomycin electroporation (alone or in combination) were compared in three different cell lines (DC-3F, transformed Chinese hamster lung fibroblast; K-562, human leukemia; and murine Lewis Lung Carcinoma). Furthermore, the effects of electrical pulsing parameters and calcium compound on treatment efficacy were determined. RESULTS Electroporation with either calcium or bleomycin significantly reduced cell survival (p<0.0001), without evidence of a synergistic effect. Cellular death following calcium or bleomycin treatment occurred at similar applied voltages, suggesting that similar parameters should be applied. At equimolar concentrations, calcium chloride and calcium glubionate resulted in comparable decreases in cell viability. CONCLUSIONS Calcium electroporation and bleomycin electroporation significantly reduce cell survival at similar applied voltage parameters. The effect of calcium electroporation is independent of calcium compound. GENERAL SIGNIFICANCE This study strongly supports the use of calcium electroporation as a potential cancer therapy and the results may aid in future clinical trials.