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Muscle fiber force production is determined by the excitation frequency of motor nerves, which induce transient increases in cytoplasmic free Ca2+ concentration ([Ca2+]i) and the force-generating capacity of the actomyosin cross-bridges. Previous studies suggest that, in addition to altered cross-bridge properties, force changes during dynamic (concentric or eccentric) contraction might be affected by Ca2+-dependent components. Here we investigated this by measuring [Ca2+]i and force in mouse muscle fibers undergoing isometric, concentric, and eccentric contractions. Intact single muscle fibers were dissected from the flexor digitorum brevis muscle of mice. Fibers were electrically activated isometrically at 30–100 Hz and after reaching the isometric force plateau, they were actively shortened or stretched. We calculated the ratio (relative changes) in force and [Ca2+]i attained in submaximal (30 Hz) and near-maximal (100 Hz) contractions under isometric or dynamic conditions. Tetanic [Ca2+]i was similar during isometric, concentric and eccentric phases of contraction at given stimulation frequencies while the forces were clearly different depending on the contraction types. The 30/100 Hz force ratio was significantly lower in the concentric (44.1 ± 20.3%) than in the isometric (50.3 ± 20.4%) condition (p = 0.005), whereas this ratio did not differ between eccentric and isometric conditions (p = 0.186). We conclude that the larger force decrease by decreasing the stimulation frequency during concentric than during isometric contraction is caused by decreased myofibrillar Ca2+ sensitivity, not by the decreased [Ca2+]i. [ABSTRACT FROM AUTHOR]

It has long been debated whether changes in [Ca.sup.2+] are involved in insulin-stimulated glucose uptake in skeletal muscle. We have now investigated the effect of insulin on the global free myoplasmic [Ca.sup.2+] concentration and the near-membrane free [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.mem]) in intact, single skeletal muscle fibers from mice by using fluorescent [Ca.sup.2+] indicators. Insulin has no effect on the global free myoplasmic [Ca.sup.2+] concentration. However, insulin increases [[[Ca.sup.2+]].sub.mem] by [approximately equal to] 70% and the half-maximal increase in [[[Ca.sup.2+]].sub.mem] occurs at an insulin concentration of 110 microunits per ml. The increase in [[[Ca.sup.2+]].sub.mem] by insulin persists when sarcoplasmic reticulum [Ca.sup.2+] release is inhibited but is lost by perfusing the fiber with a low [Ca.sup.2+] medium or by addition of L-type [Ca.sup.2+] channel inhibitors. Thus, insulin appears to stimulate [Ca.sup.2+] entry into muscle cells via L-type [Ca.sup.2+] channels. Wortmannin, which inhibits insulin-mediated activation of glucose transport in isolated skeletal muscle, also inhibits the insulin-mediated increase in [[[Ca.sup.2+]].sub.mem]. These data demonstrate a new facet of insulin signaling and indicate that insulin-mediated increases in [[[Ca.sup.2+]].sub.mem] in skeletal muscle may underlie important actions of the hormone.

Key points: Changes in intramuscular Ca2+ handling contribute to development of fatigue and disease‐related loss of muscle mass and function. To date, no data on human intact living muscle fibres have been described.We manually dissected intact single fibres from human intercostal muscle and simultaneously measured force and myoplasmic free [Ca2+] at physiological temperature.Based on their fatigue resistance, two distinct groups of fibres were distinguished: fatigue sensitive and fatigue resistant. Force depression in fatigue and during recovery was due to impaired sarcoplasmic reticulum Ca2+ release in both groups of fibres.Acidification did not affect force production in unfatigued fibres and did not affect fatigue development in fatigue‐resistant fibres.The current study provides novel insight into the mechanisms of fatigue in human intercostal muscle. Changes in intracellular Ca2+ handling of individual skeletal muscle fibres cause a force depression following physical activity and are also implicated in disease‐related loss of function. The relation of intracellular Ca2+ handling with muscle force production and fatigue tolerance is best studied in intact living single fibres that allow continuous measurements of force and myoplasmic free [Ca2+] during repeated contractions. To this end, manual dissections of human intercostal muscle biopsies were performed to isolate intact single fibres. Based on the ability to maintain tetanic force at >40% of the initial value during 500 fatiguing contractions, fibres were classified as either fatigue sensitive or fatigue resistant. Following fatigue all fibres demonstrated a marked reduction in sarcoplasmic reticulum Ca2+ release, while myofibrillar Ca2+ sensitivity was either unaltered or increased. In unfatigued fibres, acidosis caused a reduction in myofibrillar Ca2+ sensitivity that was offset by increased tetanic myoplasmic free [Ca2+] so that force remained unaffected. Acidification did not affect the fatigue tolerance of fatigue‐resistant fibres, whereas uncertainties remain whether or not fatigue‐sensitive fibres were affected. Following fatigue, a prolonged force depression at preferentially low‐frequency stimulation was evident in fatigue‐sensitive fibres and this was caused exclusively by an impaired sarcoplasmic reticulum Ca2+ release. We conclude that impaired sarcoplasmic reticulum Ca2+ release is the predominant mechanism of force depression both in the development of, and recovery from, fatigue in human intercostal muscle. Key points: Changes in intramuscular Ca2+ handling contribute to development of fatigue and disease‐related loss of muscle mass and function. To date, no data on human intact living muscle fibres have been described.We manually dissected intact single fibres from human intercostal muscle and simultaneously measured force and myoplasmic free [Ca2+] at physiological temperature.Based on their fatigue resistance, two distinct groups of fibres were distinguished: fatigue sensitive and fatigue resistant. Force depression in fatigue and during recovery was due to impaired sarcoplasmic reticulum Ca2+ release in both groups of fibres.Acidification did not affect force production in unfatigued fibres and did not affect fatigue development in fatigue‐resistant fibres.The current study provides novel insight into the mechanisms of fatigue in human intercostal muscle. [ABSTRACT FROM AUTHOR]