Muscular force production after concentric contraction.

The steady-state force following active shortening does not reach the maximum isometric force associated with the final length. Isolated extensor digitorum longus and soleus muscles from mice (NMRI strain) were used to investigate the force produced by a muscle, and some parameters hypothetically influencing this shortening-induced force depression. The muscles were pre-stimulated at fixed length, shortened and then held isometrically to give maximum post-shortening forces, before de-stimulation. The shortening magnitude was 0.18, 0.36 or 0.72mm (about 2-7% of optimal length), time of shortening was chosen as 0.03, 0.06 and 0.12s, and final length as +0.72, 0 and -0.72mm, related to optimal length. The mechanical work during active shortening was evaluated by integrating the product of force and shortening velocity over the shortening period. The results show a positive correlation between the force depression and the mechanical work, whereas the force depression was not correlated to the velocity of shortening. Depression of the passive force component was also observed following all stimulations. Experiments show that the fully stimulated redevelopment of isometric force following concentric contraction follows a time function similar to the creation of force when isometric muscle is initially stimulated. The conclusion is that the isometric force development after active shortening can be well described by an asymptotic force which is decided by the produced work, and the initial isometric time constant.

The role of Ca2+ influx for insulin-mediated glucose uptake in skeletal muscle.

The involvement of Ca(2+) in insulin-mediated glucose uptake is uncertain. We measured Ca(2+) influx (as Mn(2+) quenching or Ba(2+) influx) and 2-deoxyglucose (2-DG) uptake in single muscle fibers isolated from limbs of adult mice; 2-DG uptake was also measured in isolated whole muscles. Exposure to insulin increased the Ca(2+) influx in single muscle cells. Ca(2+) influx in the presence of insulin was decreased by 2-aminoethoxydiphenyl borate (2-APB) and increased by the membrane-permeable diacylglycerol analog 1-oleyl-2-acetyl-sn-glycerol (OAG), agents frequently used to block and activate, respectively, nonselective cation channels. Maneuvers that decreased Ca(2+) influx in the presence of insulin also decreased 2-DG uptake, whereas increased Ca(2+) influx was associated with increased insulin-mediated glucose uptake in isolated single cells and whole muscles from both normal and insulin-resistant obese ob/ob mice. 2-APB and OAG affected neither basal nor hypoxia- or contraction-mediated 2-DG uptake. 2-APB did not inhibit the insulin-mediated activation of protein kinase B or extracellular signal-related kinase 1/2 in whole muscles. In conclusion, alterations in Ca(2+) influx specifically modulate insulin-mediated glucose uptake in both normal and insulin-resistant skeletal muscle. Moreover, the present results indicate that Ca(2+) acts late in the insulin signaling pathway, for instance, in the GLUT4 translocation to the plasma membrane.

Impeded interaction between Schwann cells and axons in the absence of laminin alpha4.

The Schwann cell basal lamina (BL) is required for normal myelination. Loss or mutations of BL constituents, such as laminin-2 (alpha2beta1gamma1), lead to severe neuropathic diseases affecting peripheral nerves. The function of the second known laminin present in Schwann cell BL, laminin-8 (alpha4beta1gamma1), is so far unknown. Here we show that absence of the laminin alpha4 chain, which distinguishes laminin-8 from laminin-2, leads to a disturbance in radial sorting, impaired myelination, and signs of ataxia and proprioceptive disturbances, whereas the axonal regenerative capacity is not influenced. In vitro studies show poor axon growth of spinal motoneurons on laminin-8, whereas it is extensive on laminin-2. Schwann cells, however, extend longer processes on laminin-8 than on laminin-2, and, in contrast to the interaction with laminin-2, solely use the integrin receptor alpha6beta1 in their interaction with laminin-8. Thus, laminin-2 and laminin-8 have different critical functions in peripheral nerves, mediated by different integrin receptors.

Mitochondrial and myoplasmic [Ca2+] in single fibres from mouse limb muscles during repeated tetanic contractions.

Previous studies on single fast-twitch fibres from mouse toe muscles have shown marked fatigue-induced changes in the free myoplasmic [Ca2+] ([Ca2+]i), while mitochondrial [Ca2+] remained unchanged. We have now investigated whether muscle fibres from the legs of mice respond in a similar way. Intact, single fibres were dissected from the soleus and extensor digitorum longus (EDL) muscles of adult mice. To measure [Ca2+]i, indo-1 was injected into the isolated fibres. Mitochondrial [Ca2+] was measured using Rhod-2 and confocal laser microscopy. Fatigue was induced by up to 1000 tetanic contractions (70 Hz) given at 2 s intervals. In soleus fibres, there was no significant decrease in tetanic [Ca2+]i at the end of the fatiguing stimulation, whereas tetanic force was significantly reduced by about 30 %. In 10 out of 14 soleus fibres loaded with Rhod-2 and subjected to fatigue, mitochondrial [Ca2+] increased to a maximum after about 50 tetani; this increase was fully reversed within 20 min after the end of stimulation. The force-frequency curve of the non-responding soleus fibres was shifted to higher frequencies compared to that of the responding fibres. In addition, eight out of nine Rhod-2-loaded EDL fibres showed similar changes in mitochondrial [Ca2+] during and after a period of fatiguing stimulation. The stimulation-induced increase in mitochondrial [Ca2+] was reduced when mitochondria were depolarised by application of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, whereas it was increased by application of an inhibitor of the mitochondrial Na+/Ca2+ exchange (CGP-37157). In conclusion, isolated slow-twitch muscle fibres show only modest changes in tetanic force and [Ca2+]i during repeated contractions. The increase in mitochondrial Ca2+ does not appear to be essential for activation of mitochondrial ATP production, nor does it cause muscle damage.

Isometric force and endurance in skeletal muscle of mice devoid of all known thyroid hormone receptors.

The importance of thyroid hormone receptors for isometric force, endurance and content of specific muscle enzymes was studied in isolated slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles in mice deficient in all known subtypes of thyroid hormone receptors (i.e. TR alpha1, beta1, beta2 and beta3). The weights of soleus and EDL muscles were lower in TR-deficient (TRalpha1-/-beta-/-) mice than in wild-type controls. The force per cross-sectional area was not significantly different between TRalpha1-/-beta-/- and wild-type muscles. Soleus muscles of TRalpha1-/-beta-/- mice showed increased contraction and relaxation times and the force-frequency relationship was shifted to the left. Soleus muscles of TRalpha1-/-beta-/- mice were more fatigue resistant than wild-type controls. Protein analysis of TRalpha1-/-beta-/- soleus muscles showed a marked increase in expression of the slow isoform of the sarcoplasmic reticulum Ca2+ pump (SERCa2), whilst expression of the fast type (SERCa1) was decreased. There was also a major decrease in the alpha2-subunit of the Na+-K+ pump in TRalpha1-/-beta-/- soleus muscles. EDL muscles from TRalpha1-/-beta-/- and wild-type mice showed no significant difference in contraction and relaxation times, fatigue resistance and protein expression. In conclusion, the present data show changes in contractile characteristics of skeletal muscles of TRalpha1-/-beta-/- mice similar to those seen in hypothyroidism. We have previously shown that muscles of mice deficient in TRalpha1 or TRbeta display modest changes in muscle function. Thus, in skeletal muscle there seems to be functional overlap between TRalpha1 and TRbeta, so that the lack of one of the receptors to some extent can be compensated for by the presence of the other.

Dynamic vacuolation in skeletal muscle fibres after fatigue.

Vacuoles develop after fatiguing stimulation in frog skeletal muscle fibres. Experiments on isolated Xenopus muscle fibres show that this vacuolation is a dynamic process that reaches its maximum about 20 min after the end of fatiguing stimulation and then recedes. Fatigue-induced vacuoles originate from the t-tubular system. Recent data indicate that vacuoles are formed because of lactate accumulation in the t-tubules resulting in increased osmotic pressure and subsequent water influx. There is no obligatory connection between the presence of vacuoles and force depression, which is another common feature during the recovery from fatigue. Nevertheless, extensive vacuolation may exaggerate this force depression.

Regulation of myoplasmic Ca(2+) in genetically obese (ob/ob) mouse single skeletal muscle fibres.

The present study examined whether calcium handling in skeletal muscle fibres from ob/ob mice was abnormal compared to normal mice. Simultaneous measurements of free myoplasmic calcium and force were made in mouse single intact muscle fibres at rest, during repetitive stimulation and for 30 min afterwards. Fibres were subjected to two bouts of intermittent tetanic contractions 1 h apart. The first bout consisted of 50 tetani only, while during the second bout stimulation was continued until force fell to 40% of control. During a bout of 50 repeated contractions, muscle fibres from ob/ob mice were unable to maintain basal calcium and tetanic calcium transients. During a second series of contractions, muscle fibres from ob/ob mice showed a marked improvement in calcium handling compared to the first series but still fatigued more rapidly than control fibres. It is concluded that calcium handling in skeletal muscle fibres from ob/ob mice is abnormal compared to fibres from normal mice and this contributes to premature fatigue.

Respiratory and limb muscle weakness induced by tumor necrosis factor-alpha: involvement of muscle myofilaments.

The respiratory and limb skeletal muscles become weakened in sepsis, congestive heart failure, and other inflammatory diseases. A potential mediator of muscle weakness is tumor necrosis factor (TNF)-alpha, a cytokine that can stimulate muscle wasting and also can induce contractile dysfunction without overt catabolism. This study addressed the latter process. Murine diaphragm and limb muscle (flexor digitorum brevis [FDB]) preparations were used to determine the relative sensitivities of these muscles to TNF-alpha. Intact muscle fibers were isolated from FDB and microinjected with indo-1 to measure changes in sarcoplasmic calcium regulation. We found that TNF-alpha depressed tetanic force of the diaphragm and FDB to comparable degrees across a range of stimulus frequencies. In isolated muscle fibers, TNF-alpha decreased tetanic force without altering tetanic calcium transients or resting calcium levels. We conclude that (1) TNF-alpha compromises contractile function of diaphragm and limb muscle similarly, and (2) TNF-alpha decreases force by blunting the response of muscle myofilaments to calcium activation.

Intracellular ATP measured with luciferin/luciferase in isolated single mouse skeletal muscle fibres.

The firefly luciferin/luciferase reaction was utilized to monitor intracellular ATP concentration ([ATP](i)). Single fibres of mouse skeletal muscle were dissected and injected with luciferase. Luciferin was added to the perfusate and light emission from the fibres was monitored as an indication of [ATP](i). Inhibition of oxidative phosphorylation with cyanide and anaerobic glycolysis with iodoacetate caused light emission to fall to zero within 10 min and the fibres developed a rigor contraction. Inhibition of creatine kinase with 2,4-dinitro-1-fluorobenzene produced a small transient fall in light emission in association with each tetanus. Muscle fibres were fatigued by repeated tetani and 5/12 fibres showed a fall in light emission in the late phase of fatigue. If fibres were allowed to recover from fatigue in the absence of glucose and then restimulated in the absence of glucose they fatigued much more rapidly. However, such fibres showed no obvious change in light emission. We conclude that the luciferin/luciferase system can be used to monitor [ATP](i) in functioning single skeletal muscle cells. A depletion of global [ATP](i) is not observed in all fatiguing fibres and cannot be the sole cause of the final phase of fatigue.

Muscle fatigue: lactic acid or inorganic phosphate the major cause?

Intracellular acidosis due mainly to lactic acid accumulation has been regarded as the most important cause of skeletal muscle fatigue. Recent studies on mammalian muscle, however, show little direct effect of acidosis on muscle function at physiological temperatures. Instead, inorganic phosphate, which increases during fatigue due to breakdown of creatine phosphate, appears to be a major cause of muscle fatigue.