Ca2+ leakage out of the sarcoplasmic reticulum is increased in type I skeletal muscle fibres in aged humans

Key points: The amount of Ca stored in the sarcoplasmic reticulum (SR) of muscle fibres is decreased in aged individuals, and an important question is whether this results from increased Ca leakage out through the Ca release channels (ryanodine receptors; RyRs). The present study examined the effects of blocking the RyRs with Mg, or applying a strong reducing treatment, on net Ca accumulation by the SR in skinned muscle fibres from Old (∼70 years) and Young (∼24 years) adults. Raising cytoplasmic [Mg] and reducing treatment increased net SR Ca accumulation in type I fibres of Old subjects relative to that in Young. The densities of RyRs and dihydropyridine receptors were not significantly changed in the muscle of Old subjects. These findings indicate that oxidative modification of the RyRs causes increased Ca leakage from the SR in muscle fibres in Old subjects, which probably deleteriously affects normal muscle function both directly and indirectly. The present study examined whether the lower Ca storage levels in the sarcoplasmic reticulum (SR) in vastus lateralis muscle fibres in Old (70 ± 4 years) relative to Young (24 ± 4 years) human subjects is the result of increased leakage of Ca out of the SR through the Ca release channels/ryanodine receptors (RyRs) and due to oxidative modification of the RyRs. SR Ca accumulation in mechanically skinned muscle fibres was examined in the presence of 1, 3 or 10 mm cytoplasmic Mg because raising [Mg] strongly inhibits Ca efflux through the RyRs. In type I fibres of Old subjects, SR Ca accumulation in the presence of 1 mm Mg approached saturation at shorter loading times than in Young subjects, consistent with Caleakage limiting net uptake, and raising [Mg] to 10 mm in such fibres increased maximal SR Ca accumulation. No significant differences were seen in type II fibres. Treatment with dithiothreitol (10 mm for 5 min), a strong reducing agent, also increased maximal SR Ca accumulation at 1 mm Mg in type I fibres of Old subjects but not in other fibres. The densities of dihydropyridine receptors and RyRs were not significantly different in muscles of Old relative to Young subjects. These findings indicate that Ca leakage from the SR is increased in type I fibres in Old subjects by reversible oxidative modification of the RyRs; this increased SR Ca leak is expected to have both direct and indirect deleterious effects on Ca movements and muscle function

The concentration of free Ca 2+ in the sarcoplasmic reticulum of frog cut twitch skeletal muscle fibers estimated with tetramethylmurexide

The contractile response in skeletal muscle is controlled by the time course and magnitude of Ca2+ release from the sarcoplasmic reticulum (SR) into the myoplasm. In order to gain an understanding of how SR Ca2+ release is regulated, it is often useful to estimate the permeability of the SR to Ca2+ as this provides a measure of the extent of activation of SR Ca2+ release channels. The permeability of the SR is proportional to the rate of Ca2+ release normalized by the driving force for release which is primarily determined by the concentration of free calcium in the SR (denoted [Ca2+]SR). In past studies (e.g. [1–4]), the driving force for Ca2+ release was taken to be the concentration of total calcium in the SR (denoted [CaT]SR) rather than [Ca2+]SR owing to the lack of information about [Ca2+]SR. The primary aims of the experiments in this article were to establish the resting value of [Ca2+]SR (denoted [Ca2+]SR,R) with the absorbance indicator tetramethylmurexide (TMX) and, perhaps more importantly, the ratio of [Ca2+]SR,R and TMX’s apparent dissociation constant. This latter information is needed to establish the time course of [Ca2+]SR with TMX during SR Ca2+ release and, thereby, the time course of the true release permeability

Effects of β-hydroxy-β-methylbutyrate on aerobic-performance components and body composition in college students

The aim of this study was to determine the effects of oral ß-hydroxy-ß-methylbutyrate (HMB) supplementation (3 g/d) on selected components of aerobic performance and body composition of active college students. Subjects were randomly assigned to either an HMB (n = 8) or a placebo (PLA) group (n = 8) for a 5-wk supplementation period during which they underwent interval training 3 times a week on a treadmill. Aerobic-performance components were measured using a respiratory-gas analyzer. Body composition was determined using dual-energy X-ray absorptiometry. After the intervention, there were significant differences (P < 0.05) between the 2 groups in gains in maximal oxygen consumption (+8.4% for PLA and +15.5% for HMB) and in respiratory-compensation point (+8.6% for PLA and +13.4% for HMB). Regarding body composition, there were no significant differences. The authors concluded that HMB supplementation positively affects selected components of aerobic performance in active college students

Effects of S-glutathionylation on the passive force-length relationship in skeletal muscle fibres of rats and humans

This study investigated the effect of S-glutathionylation on passive force in skeletal muscle fibres, to determine whether activity-related redox reactions could modulate the passive force properties of muscle. Mechanically-skinned fibres were freshly obtained from human and rat muscle, setting sarcomere length (SL) by laser diffraction. Larger stretches were required to produce passive force in human fibres compared to rat fibres, but there were no fibre-type differences in either species. When fibres were exposed to glutathione disulfide (GSSG; 20 mM, 15 min) whilst stretched (at a SL where passive force reached similar to 20% of maximal Ca2+-activated force, denoted as SL20 % max), passive force was subsequently decreased at all SLs in both type I and type II fibres of rat and human (e.g., passive force at SL20 % max decreased by 12 to 25%). This decrease was fully reversed by subsequent reducing treatment with dithiothreitol (DTT; 10 mM for 10 min). If freshly skinned fibres were initially treated with DTT, there was an increase in passive force in type II fibres (by 10 +/- 3% and 9 +/- 2% in rat and human fibres, respectively), but not in type I fibres. These results indicate that (i) S-glutathionylation, presumably in titin, causes a decrease in passive force in skeletal muscle fibres, but the reduction is relatively smaller than that reported in cardiac muscle, (ii) in rested muscle in vivo, there appears to be some level of reversible oxidative modification, probably involving S-glutathionylation of titin, in type II fibres, but not in type I fibres

Endogenous and maximal sarcoplasmic reticulum calcium content and calsequestrin expression in type I and type II human skeletal muscle fibres

The relationship between sarcoplasmic reticulum (SR) Ca2+ content and calsequestrin (CSQ) isoforms was investigated in human skeletal muscle. A fibre-lysing assay was used to quantify the endogenous Ca2+ content and maximal Ca2+ 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 Ca2+ content was slightly lower in type I compared to type II fibres (0.76 ± 0.03 and 0.85 ± 0.02 mmol Ca2+ per litre of fibre, respectively), with virtually all of this Ca2+ evidently being in the SR, as it could be rapidly released with a caffeine-low [Mg2+] solution (only 0.08 ± 0.01 and <0.07 mmol l−1, respectively, remaining). The maximal Ca2+ content that could be reached with SR Ca2+ 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 Ca2+ release and subsequent Ca2+ reloading similarly indicated that (i) maximal SR Ca2+ content was lower in type I fibres than in type II fibres (P < 0.05), and (ii) the endogenous Ca2+ 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 Ca2+ content characteristics in human type II fibres being primarily determined by the CSQ1 abundance, and in type I fibres by the combined amounts of both CSQ1 and CSQ2

Sarcoplasmic reticulum Ca2+ uptake and leak properties, and SERCA isoform expression, in type I and type II fibres of human skeletal muscle

The Ca2+ uptake properties of the sarcoplasmic reticulum (SR) were compared between type I and type II fibres of vastus lateralis muscle of young healthy adults. Individual mechanically skinned muscle fibres were exposed to solutions with the free [Ca2+] heavily buffered in the pCa range (–log10[Ca2+]) 7.3–6.0 for set times and the amount of net SR Ca2+ accumulation determined from the force response elicited upon emptying the SR of all Ca2+. Western blotting was used to determine fibre type and the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) isoform present in every fibre examined. Type I fibres contained only SERCA2 and displayed half-maximal Ca2+ uptake rate at ∼pCa 6.8, whereas type II fibres contained only SERCA1 and displayed half-maximal Ca2+ uptake rate at ∼pCa 6.6. Maximal Ca2+ uptake rate was ∼0.18 and ∼0.21 mmol Ca2+ (l fibre)–1 s–1 in type I and type II fibres, respectively, in good accord with previously measured SR ATPase activity. Increasing free [Mg2+] from 1 to 3 mm had no significant effect on the net Ca2+ uptake rate at pCa 6.0, indicating that there was little or no calcium-induced calcium release occurring through the Ca2+ release channels during uptake in either fibre type. Ca2+ leakage from the SR at pCa 8.5, which is thought to occur at least in part through the SERCA, was ∼2-fold lower in type II fibres than in type I fibres, and was little affected by the presence of ADP, in marked contrast to the larger SR Ca2+ leak observed in rat muscle fibres under the same conditions. The higher affinity of Ca2+ uptake in the type I human fibres can account for the higher relative level of SR Ca2+ loading observed in type I compared to type II fibres, and the SR Ca2+ leakage characteristics of the human fibres suggest that the SERCAs are regulated differently from those in rat and contribute comparatively less to resting metabolic rate

Acute effects of taurine on sarcoplasmic reticulum Ca2+ accumulation and contractility in human type I and type II skeletal muscle fibers

Taurine occurs in high concentrations in muscle and is implicated in numerous physiological processes, yet its effects on many aspects of contractility remain unclear. Using mechanically skinned segments of human vastus lateralis muscle fibers, we characterized the effects of taurine on sarcoplasmic reticulum (SR) Ca2+ accumulation and contractile apparatus properties in type I and type II fibers. Prolonged myoplasmic exposure (&gt;10 min) to taurine substantially increased the rate of accumulation of Ca2+ by the SR in both fiber types, with no change in the maximum amount accumulated; no such effect was found with carnosine. SR Ca2+ accumulation was similar with 10 or 20 mM taurine, but was significantly slower at 5 mM taurine. Cytoplasmic taurine (20 mM) had no detectable effects on the responsiveness of the Ca2+ release channels in either fiber type. Taurine caused a small increase in Ca2+ sensitivity of the contractile apparatus in type I fibers, but type II fibers were unaffected; maximum Ca2+-activated force was unchanged in both cases. The effects of taurine on SR Ca2+ accumulation 1) only became apparent after prolonged cytoplasmic exposure, and 2) persisted for some minutes after complete removal of taurine from the cytoplasm, consistent with the hypothesis that the effects were due to an action of taurine from inside the SR. In summary, taurine potentiates the rate of SR Ca2+ uptake in both type I and type II human fibers, possibly via an action from within the SR lumen, with the degree of potentiation being significantly reduced at low physiological taurine levels. </jats:p