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Biomedical subjects

Håkan Westerblad

Publications and source records attributed to Håkan Westerblad.

30 records · Page 2Linked to original sources
PubMed2003

Creatine kinase injection restores contractile function in creatine-kinase-deficient mouse skeletal muscle fibres.

Viable genetically engineered animals generally exhibit adaptations to the altered genotype, which may mask the role of the protein of interest. We now describe a novel method by which the direct effects of the altered genotype can be distinguished from secondary adaptive changes in isolated adult skeletal muscle cells. We studied contractile function and intracellular Ca2+ handling in single skeletal muscle fibres that are completely deficient of creatine kinase (CK; CK-/-) before and after microinjection of purified CK (injected together with the fluorescent Ca2+ indicator indo-1). The mean total CK activity after CK injection was estimated to be approximately 4 mM s-1, which is approximately 5 % of the activity in wild-type muscle fibres. After CK injection, CK-/- fibres approached the wild-type phenotype in several aspects: (a) the free myoplasmic [Ca2+] ([Ca2+]i) increased and force showed little change during a period of high-intensity stimulation (duty cycle, i.e. tetanic duration divided by tetanic interval = 0.67); (b) [Ca2+]i did not decline during a brief (350 ms) tetanus; (c) during low-intensity fatiguing stimulation (duty cycle = 0.14), tetanic [Ca2+]i increased over the first 10 tetani, and thereafter it decreased; (d) tetanic [Ca2+]i and force did not display a transient reduction in the second tetanus of low-intensity fatiguing stimulation. Conversely, tetanic force in the unfatigued state was lower in CK-/- than in wild-type fibres, and this difference persisted after CK injection. Injection of inactivated CK had no obvious effect on any of the measured parameters. In conclusion, microinjection of CK into CK-/- fibres markedly restores many, but not all, aspects of the wild-type phenotype.

Animals↗
PubMed2003

CBA/J mice infected with Trypanosoma cruzi: an experimental model for inflammatory myopathies.

Idiopathic inflammatory myopathies are inflammatory disorders of unknown origin, characterized by muscle weakness. The aim of our study was to establish and characterize an animal model for chronic inflammatory myopathy which would permit investigations of the role of T-cells in chronic myositis as well as of the mechanisms for muscle weakness in chronic inflammatory muscle disorders. CBA/J mice were infected with the protozoan parasite Trypanosoma cruzi. Immunohistochemistry was used to characterize the distribution and composition of inflammatory infiltrates, and demonstrated a chronic focal inflammation comprised mainly of T-cells and macrophages in infected mice. The inflammatory infiltrates were predominantly found in the endomysium and, to a lesser extent, in perivascular areas. CD8(+) T-cells were found to have invaded nonnecrotic muscle fibers. Degenerating muscle fibers were also found, as well as an increased number of central muscle nuclei. The murine model described in this article may be useful in studying certain aspects of idiopathic inflammatory myopathies such as the role of T-cells in chronic muscle inflammation and chronic myocytotoxicity.

Animals↗
PubMed2002

Ryanodine receptors of pancreatic beta-cells mediate a distinct context-dependent signal for insulin secretion.

The ryanodine (RY) receptors in beta-cells amplify signals by Ca2+-induced Ca2+ release (CICR). The role of CICR in insulin secretion remains unclear in spite of the fact that caffeine is known to stimulate secretion. This effect of caffeine is attributed solely to the inhibition of cAMP-phosphodiesterases (cAMP-PDEs). We demonstrate that stimulation of insulin secretion by caffeine is due to a sensitization of the RY receptors. The dose-response relationship of caffeine-induced inhibition of cAMP-PDEs was not correlated with the stimulation of insulin secretion. Sensitization of the RY receptors stimulated insulin secretion in a context-dependent manner, that is, only in the presence of a high concentration of glucose. This effect of caffeine depended on an increase in [Ca2+]i. Confocal images of beta-cells demonstrated an increase in [Ca2+]i induced by caffeine but not by forskolin. 9-Methyl-7-bromoeudistomin D (MBED), which sensitizes RY receptors, did not inhibit cAMP-PDEs, but it stimulated secretion in a glucose-dependent manner. The stimulation of secretion by caffeine and MBED involved both the first and the second phases of secretion. We conclude that the RY receptors of beta-cells mediate a distinct glucose-dependent signal for insulin secretion and may be a target for developing drugs that will stimulate insulin secretion only in a glucose-dependent manner.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2002

Increased mitochondrial mass in mitochondrial myopathy mice.

We have generated an animal model for mitochondrial myopathy by disrupting the gene for mitochondrial transcription factor A (Tfam) in skeletal muscle of the mouse. The knockout animals developed a myopathy with ragged-red muscle fibers, accumulation of abnormally appearing mitochondria, and progressively deteriorating respiratory chain function in skeletal muscle. Enzyme histochemistry, electron micrographs, and citrate synthase activity revealed a substantial increase in mitochondrial mass in skeletal muscle of the myopathy mice. Biochemical assays demonstrated that the increased mitochondrial mass partly compensated for the reduced function of the respiratory chain by maintaining overall ATP production in skeletal muscle. The increased mitochondrial mass thus was induced by the respiratory chain deficiency and may be beneficial by improving the energy homeostasis in the affected tissue. Surprisingly, in vitro experiments to assess muscle function demonstrated that fatigue development did not occur more rapidly in myopathy mice, suggesting that overall ATP production is sufficient. However, there were lower absolute muscle forces in the myopathy mice, especially at low stimulation frequencies. This reduction in muscle force is likely caused by deficient formation of force-generating actin-myosin cross bridges and/or disregulation of Ca(2+) homeostasis. Thus, both biochemical measurements of ATP-production rate and in vitro physiological studies suggest that reduced mitochondrial ATP production might not be as critical for the pathophysiology of mitochondrial myopathy as thought previously.

Animals↗
PubMed2002

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.

Actin Cytoskeleton↗
PubMed2002

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.

Animals↗
PubMed2002

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.

Animals↗
PubMed2002

Recent advances in the understanding of skeletal muscle fatigue.

Prolonged or repeated contractions of skeletal muscles lead to impaired muscle function, fatigue develops. Fatigue may be caused by factors within the muscle cells (peripheral fatigue) and diminished activation from the central nervous system (central fatigue). The relative importance of peripheral central fatigue depends on the type of physical activity. Central fatigue may be more prominent in elderly subjects. Increased concentration of inorganic phosphate seems to be of major importance for acute peripheral fatigue. There is frequently a long-lasting depression of force production after fatiguing muscle activity, especially at low stimulation frequencies. This low-frequency fatigue seems to be due to "structural" changes in proteins involved in intracellular Ca handling. Contractions in which the muscle is stretched (eccentric contractions) cause muscle weakness and damage. The initial defect induced by eccentric contractions is overstretched sarcomeres, but these appear to cause localized membrane tears that subsequently contribute to muscle weakness and damage.

Aging↗
PubMed2002

Effects of glucose on contractile function, [Ca2+]i, and glycogen in isolated mouse skeletal muscle.

Extensor digitorum longus muscles were stimulated to contract to fatigue and allowed to recover for 2 h in the absence or presence of 5.5 or 11 mM extracellular glucose. This was followed by a second fatigue run, which ended when the absolute force was the same as at the end of the first run. During the first fatigue run, the fluorescence ratio for indo 1 increased [reflecting an increase in myoplasmic free Ca2+ concentration ([Ca2+]i)] during the initial tetani, peaking at approximately 115% of the first tetanic value, followed by a continuous decrease to approximately 90% at fatigue. During the first fatigue run, myofibrillar Ca2+ sensitivity was significantly decreased. During the second run, the number of tetani was 57 +/- 6% of initial force in muscles that recovered in the absence of glucose and 110 +/- 6 and 119 +/- 2% of initial force in muscles that recovered in 5.5 and 11 mM glucose, respectively. Fluorescence ratios during the first, peak, and last tetani did not differ significantly between the first and second fatigue runs during any of the three conditions. Glycogen decreased by almost 50% during the first fatigue run and did not change further after recovery in the absence of glucose. After recovery in the presence of 5.5 and 11 mM glucose, glycogen increased 32 and 42% above the nonstimulated control value (P < 0.01). These data demonstrate that extracellular glucose delays the decrease of tetanic force and [Ca2+]i during fatiguing stimulation and that glycogen supercompensation following contraction can occur in the absence of insulin.

Animals↗
PubMed2002

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.

Animals↗
PubMed2001

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.

Adenosine Triphosphate↗