Key pointsr Muscle weakness in old age is due in large part to an overall loss of skeletal muscle tissue, but it remains uncertain how much also stems from alterations in the properties of the individual muscle fibres.r This study examined the contractile properties and amount of stored intracellular calcium in single muscle fibres of Old (70 ± 4 years) and Young (22 ± 3 years) adults.r The maximum level of force production (per unit cross-sectional area) in fast twitch fibres in Old subjects was lower than in Young subjects, and the fibres were also less sensitive to activation by calcium.r The amount of calcium stored inside muscle fibres and available to trigger contraction was also lower in both fast-and slow-twitch muscle fibres in the Old subjects.r These findings indicate that muscle weakness in old age stems in part from an impaired capacity for force production in the individual muscle fibres.Abstract This study examined the contractile properties and sarcoplasmic reticulum (SR) Ca 2+ content in mechanically skinned vastus lateralis muscle fibres of Old (70 ± 4 years) and Young (22 ± 3 years) humans to investigate whether changes in muscle fibre properties contribute to muscle weakness in old age. In type II fibres of Old subjects, specific force was reduced by ß17% and Ca 2+ sensitivity was also reduced (pCa 50 decreased ß0.05 pCa units) relative to that in Young. S-Glutathionylation of fast troponin I (TnI f ) markedly increased Ca 2+ sensitivity in type II fibres, but the increase was significantly smaller in Old versus Young (+0.136 and +0.164 pCa unit increases, respectively). Endogenous and maximal SR Ca 2+ content were significantly smaller in both type I and type II fibres in Old subjects. In fibres of Young, the SR could be nearly fully depleted of Ca 2+ by a combined caffeine and low Mg 2+ stimulus, whereas in fibres of Old the amount of non-releasable Ca 2+ was significantly increased (by > 12% of endogenous Ca

Key points• Ca 2+ release from the sarcoplasmic reticulum (SR) controls contraction in vertebrate skeletal muscle. Calsequestrin (CSQ) is thought to be the principal Ca 2+ binding protein in the SR but little is known about SR Ca 2+ content and loading characteristics, or CSQ isoform distribution, in human skeletal muscle fibres.• Type I (slow-twitch) and type II (fast-twitch) skeletal muscle fibres in young healthy adults show highly-stereotyped patterns of isoform expression of CSQ and SR Ca 2+ pumps, in tight correspondence with isoform expression of the contractile proteins, which probably facilitates optimal contractile function in the individual fibre types.• Endogenous Ca 2+ content of the SR is slightly larger in type II fibres than in type I fibres, but its maximal capacity is substantially greater, probably due to the larger amount of the CSQ1 isoform present. SR Ca 2+ content and capacity in type I fibres is probably determined by their content of both CSQ1 and CSQ2.Abstract The relationship between sarcoplasmic reticulum (SR) Ca 2+ content and calsequestrin (CSQ) isoforms was investigated in human skeletal muscle. A fibre-lysing assay was used to quantify the endogenous Ca 2+ content and maximal Ca 2+ capacity of the SR in skinned segments of type I and type II fibres from vastus lateralis muscles of young healthy adults. Western blotting of individual fibres showed the great majority contained either all fast or all slow isoforms of myosin heavy chain (MHC), troponins C and I, tropomyosin and SERCA, and that the strontium sensitivity of the force response was closely indicative of the troponin C isoform present. The endogenous SR Ca 2+ content was slightly lower in type I compared to type II fibres (0.76 ± 0.03 and 0.85 ± 0.02 mmol Ca 2+ per litre of fibre, respectively), with virtually all of this Ca 2+ evidently being in the SR, as it could be rapidly released with a caffeine-low [Mg 2+ ] solution (only 0.08 ± 0.01 and <0.07 mmol l −1 , respectively, remaining). The maximal Ca 2+ content that could be reached with SR Ca 2+ loading was 1.45 ± 0.04 and 1.79 ± 0.03 mmol l −1 in type I and type II fibres, respectively (P < 0.05). In non-lysed skinned fibres, where the SR remained functional, repeated cycles of caffeine-induced Ca 2+ release and subsequent Ca 2+ reloading similarly indicated that (i) maximal SR Ca 2+ content was lower in type I fibres than in type II fibres (P < 0.05), and (ii) the endogenous Ca 2+ content represented a greater percentage of maximal content in type I fibres compared to type II fibres (∼59% and 41%, respectively, P < 0.05). Type II fibres were found on average to contain ∼3-fold more CSQ1 and ∼5-fold less CSQ2 than type I fibres (P < 0.001). The findings are consistent with the SR Ca 2+ content characteristics in human type II

There is considerable interest in potential ergogenic and therapeutic effects of increasing skeletal muscle carnosine content, although its effects on excitation-contraction (EC) coupling in human muscle have not been defined. Consequently, we sought to characterize what effects carnosine, at levels attained by supplementation, has on human muscle fiber function, using a preparation with all key EC coupling proteins in their in situ positions. Fiber segments, obtained from vastus lateralis muscle of human subjects by needle biopsy, were mechanically skinned, and their Ca(2+) release and contractile apparatus properties were characterized. Ca(2+) sensitivity of the contractile apparatus was significantly increased by 8 and 16 mM carnosine (increase in pCa(50) of 0.073 ± 0.007 and 0.116 ± 0.006 pCa units, respectively, in six type I fibers, and 0.063 ± 0.018 and 0.103 ± 0.013 pCa units, respectively, in five type II fibers). Caffeine-induced force responses were potentiated by 8 mM carnosine in both type I and II fibers, with the potentiation in type II fibers being entirely explicable by the increase in Ca(2+) sensitivity of the contractile apparatus caused by carnosine. However, the potentiation of caffeine-induced responses caused by carnosine in type I fibers was beyond that expected from the associated increase in Ca(2+) sensitivity of the contractile apparatus and suggestive of increased Ca(2+)-induced Ca(2+) release. Thus increasing muscle carnosine content likely confers benefits to muscle performance in both fiber types by increasing the Ca(2+) sensitivity of the contractile apparatus and possibly also by aiding Ca(2+) release in type I fibers, helping to lessen or slow the decline in muscle performance during fatiguing stimulation.

Key points• Reactive oxygen-based molecules generated within muscle fibres in both exercise and pathological conditions can greatly affect muscle function. These and consequent reactions can lead to either decreased or increased force response by the contractile proteins, but the mechanisms are unknown.• This study demonstrates that the increase in force response appears to be due to a specific chemical process, known as S-glutathionylation, of a particular cysteine residue present on the troponin I molecule in fast-twitch muscle fibres, which is involved in sensing and responding to changes in intracellular calcium levels.• S-Glutathionylation can occur when glutathione, the primary cellular anti-oxidant, reacts with oxidized cysteine residues.• S-Glutathionylation of troponin I not only helps protect the molecule from oxidative stress, but evidently also makes the contractile apparatus much more sensitive to calcium ions.• This process seemingly occurs in exercising humans and is likely to be an important mechanism helping delay onset of muscle fatigue.Abstract Oxidation can decrease or increase the Ca 2+ sensitivity of the contractile apparatus in rodent fast-twitch (type II) skeletal muscle fibres, but the reactions and molecular targets involved are unknown. This study examined whether increased Ca 2+ sensitivity is due to S-glutathionylation of particular cysteine residues. Skinned muscle fibres were directly activated in heavily buffered Ca 2+ solutions to assess contractile apparatus Ca 2+ sensitivity. Rat type II fibres were subjected to S-glutathionylation by successive treatments with 2,2 -dithiodipyridine (DTDP) and glutathione (GSH), and displayed a maximal increase in pCa 50 (−log 10 [Ca 2+ ] at half-maximal force) of ∼0.24 pCa units, with little or no effect on maximum force or Hill coefficient. Partial similar effect was produced by exposure to oxidized gluthathione (GSSG, 10 mM) for 10 min at pH 7.1, and near-maximal effect by GSSG treatment at pH 8.5. None of these treatments significantly altered Ca 2+ sensitivity in rat type I fibres. Western blotting showed that both the DTDP-GSH and GSSG-pH 8.5 treatments caused marked S-glutathionylation of the fast troponin I isoform (TnI f ) present in type II fibres, but not of troponin C (TnC) or myosin light chain 2. Both the increased Ca 2+ sensitivity and glutathionylation of TnI f were blocked by N-ethylmaleimide (NEM). S-Nitrosoglutathione (GSNO) also increased Ca 2+ sensitivity, but only in conditions where it caused S-glutathionylation of TnI f . In human type II fibres from

Muscle contraction depends on tightly regulated Ca 2+ release. Aberrant Ca 2+ leak through ryanodine receptor 1 (RyR1) on the sarcoplasmic reticulum (SR) membrane can lead to heatstroke and malignant hyperthermia (MH) susceptibility, as well as severe myopathy. However, the mechanism by which Ca 2+ leak drives these pathologies is unknown. Here, we investigate the effects of four mouse genotypes with increasingly severe RyR1 leak in skeletal muscle fibers. We find that RyR1 Ca 2+ leak initiates a cascade of events that cause precise redistribution of Ca 2+ among the SR, cytoplasm, and mitochondria through altering the Ca 2+ permeability of the transverse tubular system membrane. This redistribution of Ca 2+ allows mice with moderate RyR1 leak to maintain normal function; however, severe RyR1 leak with RYR1 mutations reduces the capacity to generate force. Our results reveal the mechanism underlying force preservation, increased ATP metabolism, and susceptibility to MH in individuals with gain-of-function RYR1 mutations.