The effect of insulin on skeletal muscle contractions and its relation to the effect produced by β-adrenoceptor stimulation

The soleus, a slow-contracting, and the extensor digitorum longus (EDL), a fast-contracting muscle, from the guinea-pig were prepared for measurement of isometric contractions in vitro. Insulin, 2.5–55 mu/ml, caused a dose-dependent depression of twitches and subtetanic concentrations of the soleus muscle similar to and additive with that produced by the β₂-adrenoceptor agonist, terbutaline. The effect of terbutaline but not that of insulin was blocked by propranolol. Insulin had no apparent effect on the contractions of the EDL, whereas terbutaline increased the force of contraction. When depressed by KC1, however, insulin partially restored the twitch tension in both muscles. The possible role of effects on the Na⁺-K⁺ transport is discussed.     

Classification of β-adrenoceptors in the microcirculation of skeletal muscle

Nervous and humoral β-adrenergic, postjunctional effects on microvascular resistance, on precapillary sphincter tone, and on transcapillary fluid exchange in cat skeletal muscle (Lundvall & Järhult 1974, 1976 a, Lundvall & Hillman 1978 a, b) were evaluated with regard to the β₁-or β₂,-specificity of the adrenoceptors. Marked β₂-dilator responses but no significant β₁-effects were observed. The conclusion was therefore reached that neurogenic as well as humoral β-adrenergic control of the microcirculation in skeletal muscle is exerted via activation of β₂-adrenoceptors.     

Slow recovery of force in single skeletal muscle fibres

After a bout of intense exercise, especially in untrained persons, recovery of muscle force is often slow. Force depression is much more marked at low frequencies of stimulation than at high frequencies (recovery is also seen in single muscle fibres from frog and mouse after fatigue induced by repeated, brief contractions. Evidence from our own and other laboratories indicates that the impairment is unlikely to result from metabolic changes and points to a defect in excitation–contraction coupling. We demonstrate that the likely site of failure is in the coupling between t-tubule depolarization and release of Ca²⁺ from the SR. The causative agent appears to be a localized increase in cytoplasmic Ca²⁺ which initiates some disruptive process, which can, however, be fully reversed, albeit slowly. Our experimental evidence does not support the involvement of Ca²⁺-activated proteases. Attempts to clarify the possible role of Ca²⁺-activated lipases (phospholipase A₂) and Ca²⁺/calmodulin have been hampered by side-effects of available inhibitors. Efforts to clarify how Ca²⁺ exerts its effects are continuing.     

β-adrenoceptor agonists do not cause tremor in the conscious rat

The ability of some β-adrenoceptor agonists to induce tremor via skeletal muscle β₂-adrenoceptors in conscious unrestrained rats has been investigated. Tremor was assessed by visual observation and by an objective method based on accelerometry. Infusion of isoprenaline or terbutaline did not cause tremor, neither did β-stimulation potentiate an established mild tremor produced by central muscarinic receptor stimulation. Since β-agonists readily produce tremor in man via skeletal muscle β₂-adrenoceptor stimulation, our findings indicate that these receptors have a different function in the rat.     

Prejunctional β2-adrenoreceptor blockade reduces nerve stimulation evoked release of endogenous noradrenaline in skeletal muscle in situ

Prejunctional β-adrenoceptor-mediated modulation of endogenous noradrenaline (NA) overflow elicited by sympathetic nerve stimulation was studied in blood-perfused canine gracilis muscle in situ. An attempt was made to subclassify these β-adrenoceptors by comparing the effects of β₁-selective (metoprolol) and non-selective (propranolol) β-adrenoceptor blockade. Animals were pre-treated with desipramine and phenoxybenzamine in order to counteract possible influences of neuronal uptake and stimulation-evoked changes in vascular resistance on the diffusion of NA into the blood stream. Metoprolol did not decrease stimulation-evoked NA overflow, as compared with control experiments (?10 and ?8 %, respectively). However, propranolol reduced stimulation-evoked NA overflow by 30% in metoprolol pre-treated animals (P < 0.05 vs. control experiments). Both antagonists elevated basal perfusion pressure, suggesting that vascular post-junctional β₁-as well as β₂-adrenoceptors are present. Propranolol increased stimulation-evoked vasoconstriction in metoprolol pre-treated animals, indicating that neuronally released NA may activate postjunctional β₂-adrenoceptors under these experimental conditions. In conclusion, our findings suggest that NA release can be enhanced by activation of prejunctional β₂-adrenoceptors in vivo.     

Glutathione metabolism in skeletal muscle derived cells of the L6 line

Skeletal muscle derived L6 myoblasts possess a considerably high resting total glutathione (TGSH) pool. Exposure to l -buthionine-[S,R]-sulphoximine resulted in a 90% depletion of the intracellular TGSH pool. All the key enzymes of glutathione metabolism, especially glutathione S-transferase, were observed to be considerably active in the undifferentiated cells. Se-dependent glutathione peroxidase activity appeared to account for most of the total GSH peroxidase activity of the cells. A significant contribution of γ-glutamyl transpeptidase-independent (5 mm acivicin insensitive) mechanism to the extracellular GSH uptake capacity of the muscle cells was evident. Efflux of oxidized glutathione from the cells exposed to t-butyl hydroperoxide was rapid and appeared to be energy linked.     

Effects of noradrenaline and flow on lactate uptake in the perfused rat hindlimb

Skeletal muscle can release or take up lactate depending on the lactate concentration gradient across the cell membrane. In the perfused rat hindlimb without arterial lactate, both noradrenaline (NA) infusion and increased flow promote lactate release and oxygen consumption (VO ₂). However, it is unclear whether NA or increased flow rate have similar effects on lactate uptake. The present study compares these effects in the rat hindlimb perfused at a basal flow rate of 0.33 mL min^?1 g^?1 and 25 °C in the presence of added arterial lactate. When 10 mmol L^?1 L-(+)-lactate was added to the arterial perfusate, lactate was taken up (16 ± 1.0 μmol g^?1 h^?1, n = 13) by the hindlimb with a 35% higher VO ₂ than that without added lactate. Doubling perfusion flow rate enhanced lactate uptake and VO ₂ by 120% and 40%, respectively. Glucose uptake was also increased (by 253%) with increased flow. Infusion of NA increased perfusion pressure, VO ₂ and glucose uptake similarly to those induced by increased flow rate. However, lactate uptake was inhibited by NA. This inhibition was not altered by the β-adrenergic antagonist propranolol. Vasopressin also showed similar effects to NA to decrease lactate uptake associated with increased VO ₂ and vasoconstriction. These data indicate that in the presence of a high arterial lactate concentration, NA has opposite effects from increased flow rate on skeletal muscle lactate uptake although both have similar effects on lactate release in the absence of arterial lactate. Inhibition of lactate uptake may relate to the vasoconstrictive action of NA.     

Events of the excitation–contraction–relaxation (E–C–R) cycle in fast- and slow-twitch mammalian muscle fibres relevant to muscle fatigue

The excitation–contraction–relaxation cycle (E–C–R) in the mammalian twitch muscle comprises the following major events: (1) initiation and propagation of an action potential along the sarcolemma and transverse (T)-tubular system; (2) detection of the T-system depolarization signal and signal transmission from the T-tubule to the sarcoplasmic reticulum (SR) membrane; (3) Ca²⁺ release from the SR; (4) transient rise of myoplasmic [Ca²⁺]; (5) transient activation of the Ca²⁺-regulatory system and of the contractile apparatus; (6) Ca²⁺ reuptake by the SR Ca²⁺ pump and Ca²⁺ binding to myoplasmic sites. There are many steps in the E–C–R cycle which can be seen as potential sites for muscle fatigue and this review explores how structural and functional differences between the fast- and slow-twitch fibres with respect to the E–C–R cycle events can explain to a great extent differences in their fatiguability profiles.     

Mutations and polymorphisms of the skeletal muscle α‐actin gene (ACTA1)

The ACTA1 gene encodes skeletal muscle α‐actin, which is the predominant actin isoform in the sarcomeric thin filaments of adult skeletal muscle, and essential, along with myosin, for muscle contraction. ACTA1 disease‐causing mutations were first described in 1999, when a total of 15 mutations were known. In this article we describe 177 different disease‐causing ACTA1 mutations, including 85 that have not been described before. ACTA1 mutations result in five overlapping congenital myopathies: nemaline myopathy; intranuclear rod myopathy; actin filament aggregate myopathy; congenital fiber type disproportion; and myopathy with core‐like areas. Mixtures of these histopathological phenotypes may be seen in a single biopsy from one patient. Irrespective of the histopathology, the disease is frequently clinically severe, with many patients dying within the first year of life. Most mutations are dominant and most patients have de novo mutations not present in the peripheral blood DNA of either parent. Only 10% of mutations are recessive and they are genetic or functional null mutations. To aid molecular diagnosis and establishing genotype–phenotype correlations, we have developed a locus‐specific database for ACTA1 variations ( http://waimr.uwa.edu.au ). Hum Mutat 30:1–11, 2009. © 2009 Wiley‐Liss, Inc.     

Effects of chronic nicotine exposure on contractile enzyme-histochemical and biochemical properties of fast- and slow-twitch skeletal muscles in the rat

Nicotine-exposed and control rats were compared with respect to contractile, enzyme-histochemical and biochemical properties of fast- and slow-twitch skeletal muscles in order to elucidate the mechanisms underlying previously observed effects of tobacco smoking on skeletal muscle. The nicotine was administered in drinking water, since this approach has been shown to result in a plasma nicotine pattern similar to that seen in tobacco smokers. In a pilot study, fibre-type proportions and mitochondrial enzyme activities tended to change in the slow-twitch soleus muscle after 9 weeks of nicotine exposure in a way similar to that previously reported in tobacco smokers. In the present study, the duration of nicotine exposure was more prolonged (18 weeks) and the number of studied animals was increased. In this series neither contractile, enzyme-histochemical nor biochemical properties were affected by the nicotine exposure. It is thus concluded that prolonged nicotine exposure has no significant effect on the skeletal muscle characteristics studied, and that other aetiological agent(s) for the observed differences in such characteristics between smokers and non-smokers should be searched for.     

Modulation of oxotremorine-induced tremor by central β-adrenoceptors

The muscarinic agonist oxotremorine was used to induce tremor in rats pretreated with methylatropine. An objective assessment of tremor intensity was accomplished by means of an accelerometer-based recording system. The non-selective, lipophilic β-adrenoceptor antagonist propranolol dose-dependently suppressed tremor intensity, whereas the r -isomer of propranolol was without effect, verifying β-adrenoceptor involvement. Since the hydrophilic, non-selective β-antagonist nadolol was ineffective, the effect appears to be located inside the blood-brain barrier. The β₂-selective antagonist ICI 118, 551 dose-dependently reduced tremor intensity, whereas selective blockade of β₁-adrenoceptors with metoprolol had no effect, indicating the participation of a β₂-adrenoceptor. On the other hand, the lipophilic β₂-agonist clenbuterol dose-dependently enhanced tremor induced by oxotremorine. Determination of circulating plasma catecholamine concentrations revealed that the effect of β-antagonists on tremor was not secondary to an effect on the oxotremorine-induced rise in catecholamine levels. Thus, the results suggest that β₂-adrenocpetors located inside the blood-brain barrier are able to modulate oxotremorine-induced tremor in rats.     

VIP inhibition of vascular smooth muscle: complementary to β2-adrenoceptor mediated relaxation in the isolated rat portal vein

Exogenous VIP caused a concentration dependent inhibition of the spontaneous mechanical activity in the isolated rat mesenteric-portal vein preparation via a mechanism which was completely independent of the propranolol-blocked β-adrenoceptor, of high K⁺ in the medium and of exogenous bovine pancreatic polypeptide, neurotensin and opioids. The potency of VIP ((pD₂=7.52±0.18, n=6) was about 30 times higher than that of isoprenaline in the atropine and phentolamine-blocked preparation. The isoprenaline inhibition was mediated via a β₂-type of adrenoceptor with low apparent affinity for noradrenaline (intrinsic activity (a) = 0.27±0.01, n=8). Opposite effects of exogenous VIP and noradrenaline were on the other hand observed in the atropinized and β-blocked preparation. These results suggest that in the rat portal vein neuronal VIP and circulating adrenaline may be complementary in their antagonism of the α-adrenoceptor mediated increase in contractility.     

Exogenous reactive oxygen and nitric oxide alter intracellular oxidant status of skeletal muscle fibres

To test whether exogenous oxidants alter intracellular oxidant levels in skeletal muscle fibres, we exposed rat diaphragm to donors of nitric oxide (NO_x), reactive oxygen species (ROS) or hyperoxia, and monitored intracellular oxidant levels using a fluorescent probe. Fibre bundles were dissected from the diaphragm and loaded with 2’,7’-dichlorodihydrofluorescein (DCFH); emissions were monitored using a fluorescence microscope. DCFH-loaded muscles were exposed to either a NO_x donor (1 m M S-nitroso-N-acetyl penicillamine, SNAP; 1 m M sodium nitroprusside, SNP; 400 μM 1-hydroxy-2-oxo-3-(N-3-methyl-aminopropyl)-3-methyl-1-triazen, NOC-7), an ROS donor (100 μM hydrogen peroxide, H₂O₂; 100 μM tert-butyl hydroperoxide; 1 m M hypoxanthine plus 0.01 U mL^–1 xanthine oxidase, HXXO) or a range of PO ₂s (25, 60 or 95% O₂ oxygenating Krebs–Ringer solution) for 40 min; time-matched control bundles remained in Krebs–Ringer solution. Control muscles oxidized DCFH at a rate of 0.32 ± 0.1 greyscale units min^–1. SNAP (766%), SNP (1244%), NOC-7 (851%), H₂O₂ (543%), and HXXO (541%) increased DCFH oxidation from control levels. The increase in emissions caused by NOC-7 and SNP were blunted by the NO_x scavenger haemoglobin (1 μM ). DCFH oxidation by HXXO was unaffected by 1000 U mL^–1 superoxide dismutase but was significantly decreased by 1000 U mL^–1 catalase and 1 m M salicylate. PO ₂ had no effect on intracellular oxidant levels. Therefore, extracellular NO_x and ROS can alter intracellular oxidant status in skeletal muscle fibres. These observations suggest that intrafibre oxidant levels could be the result of both intracellular and extracellular oxidant production.     

Apoptosis and changes in contractile protein pattern in the skeletal muscle in heart failure

Chronic heart failure is characterized as a clinical disorder by exercise intolerance. There are two factors that are independently responsible for the reduced exercise capacity: (a) a shift from myosin heavy chain 1 (MHC1) to MHC2a and MHC2b and (b) muscle atrophy. We have demonstrated, both in experimental models of heart failure and in man, that the more severe the heart failure, the greater the magnitude of skeletal muscle apoptosis. In the monocrotaline treated rat, that develops a severe right‐sided heart failure, the increased number of apoptotic nuclei was paralleled by increasing levels of circulating TNFα. In agreement with some recent observations showing that sphingolipids can mediate programmed cell death, we found that in animals with heart failure and high number of apoptotic nuclei, circulating levels of sphingosine were significantly increased. In a study conducted in patients with heart failure we found a correlation between exercise capacity limitation and skeletal myocytes apoptosis. There was also a correlation between degree of muscle atrophy and magnitude of apoptosis. The shift in MHCs, although with a different mechanism, is also responsible for the reduced exercise capacity in these patients. In fact there is a strong correlation between indices of severity of CHF and MHC composition. Muscle fatigue, appears earlier in patients that have a greater skeletal muscle expression of ‘fast’ MHCs. We have also demonstrated that MHCs shift and apoptosis can be prevented by using angiotensin II converting enzyme inhibitors and angiotensin II receptor blockers.     

Dietary effects on glycogen and lipoprotein lipase activity in skeletal muscle in man

The influence of short-term adaptation to a fat and protein enriched diet (F+P) and a carbohydrate enriched diet (CHO) on skeletal muscle lipoprotein lipase (LPL) activity and muscle glycogen levels was evaluated in 7 males. Muscle biopsies were taken from the m. vastus lateralis after an uncontrolled, mixed diet (M), after a 3 day F+P diet preceded by intense exercise, and after a 3 day CHO diet. After the F+P diet glycogen concentration was 55% that of the M diet while LPL activity increased by 21% (n. s.). After the CHO diet glycogen levels increased by 82% and LPL activity decreased by 55% compared to the M diet (p<0.01). The changes in LPL after the CHO diet were related to the changes in glycogen concentration (r = 0.98, p<0.01). LPL activity in the control situation was directly related to percent slow twitch (ST) muscle fibre type (r = 0.95, p<0.01). The results suggest that the uptake of fat from the circulation may be actively regulated by the muscle as a function of intramuscular substrate availability and that this regulation may be related to muscle fibre type composition.     

Structure, distribution and innervation of muscle spindles in avian fast and slow skeletal muscle

Muscle spindles in 2 synergistic avian skeletal muscles, the anterior (ALD) and posterior (PLD) latissimus dorsi, were studied by light and electron microscopy to determine whether morphological or quantitative differences existed between these sensory receptors. Differences were found in the density, distribution and location of muscle spindles in the 2 muscles. They also differed with respect to the morphology of their capsules and intracapsular components. The slow ALD possessed muscle spindles which were evenly distributed throughout the muscle, whereas in the fast PLD they were mainly concentrated around the single nerve entry point into the muscle. The muscle spindle index (number of spindles per gram wet muscle weight) in the ALD was more than double that of its fast-twitch PLD counterpart (130.5±2.0 vs 55.4±2.0 respectively, n=6). The number of intrafusal fibres per spindle ranged from 1 to 8 in the ALD and 2 to 9 in the PLD, and their diameters varied from 5.0 to 16.0 μm and 4.5 to 18.5 μm, respectively. Large diameter intrafusal fibres were more frequently encountered in spindles of the PLD. Unique to the ALD was the presence of monofibre muscle spindles (12.7% of total spindles observed in ALD) which contained a solitary intrafusal fibre. In muscle spindles of both the ALD and PLD, sensory nerve endings terminated in a spiral fashion on the intrafusal fibres in their equatorial regions. Motor innervation was restricted to either juxtaequatorial or polar regions of the intrafusal fibres. Outer capsule components were extensive in polar and juxtaequatorial regions of ALD spindles, whereas inner capsule cells of PLD spindles were more numerous in juxtaequatorial and equatorial regions. Overall, muscle spindles of the PLD exhibited greater complexity with respect to the number of intrafusal fibres per spindle, range of intrafusal fibre diameters and development of their inner capsules. It is postulated that the differences in muscle spindle density and structure observed in this study reflect the function of the muscles in which they reside.