Excessive Accumulation of Ca2 + in Mitochondria of Y522S-RYR1 Knock-in Mice: A Link Between Leak From the Sarcoplasmic Reticulum and Altered Redox State.

Mice (Y522S or YS), carrying a mutation of the sarcoplasmic reticulum (SR) Ca2+ release channel of skeletal muscle fibers (ryanodine receptor type-1, RyR1) which causes Ca2+ leak, are a widely accepted and intensively studied model for human malignant hyperthermia (MH) susceptibility. Since the involvement of reactive oxygen species (ROS) and of mitochondria in MH crisis has been previously debated, here we sought to determine Ca2+ uptake in mitochondria and its possible link with ROS production in single fibers isolated from flexor digitorum brevis (FDB) of YS mice. We found that Ca2+ concentration in the mitochondrial matrix, as detected with the ratiometric FRET-based 4mtD3cpv probe, was higher in YS than in wild-type (WT) fibers at rest and after Ca2+ release from SR during repetitive electrical stimulation or caffeine administration. Also mitochondrial ROS production associated with contractile activity (detected with Mitosox probe) was much higher in YS fibers than in WT. Importantly, the inhibition of mitochondrial Ca2+ uptake achieved by silencing MCU reduced ROS accumulation in the matrix and Ca2+ release from SR. Finally, inhibition of mitochondrial ROS accumulation using Mitotempo reduced SR Ca2+ release in YS fibers exposed to caffeine. The present results support the view that mitochondria take up larger amounts of Ca2+ in YS than in WT fibers and that mitochondrial ROS production substantially contributes to the increased caffeine-sensitivity and to the enhanced Ca2+ release from SR in YS fibers.

Age-dependent variations in the expression of myosin isoforms and myogenic factors during the involution of the proximal sesamoidean ligament of sheep.

In ungulates the stability of the fetlock joint is dependent on several muscles, which are exposed to high stress and strain. Among those muscles, the proximal sesamoidean ligament or PSL (also known as the suspensory ligament or Ruini's elasto-tendinous organ) is organized at birth in layers of muscle fibres alternated with abundant tendinous tissue that, during the postnatal development, becomes the predominant tissue. In this study we analysed the PSL of the sheep at the age of 1, 30 and 180 days and determined the expression of several genes which either (a) are markers of muscle fibre growth and maturation, or (b) play a role as signal molecules. We observed an accelerated maturation, as indicated by the transition of MyHC isoform expression towards the slow isoforms and a reduced regenerative potential indicated by the low Pax7 expression and the altered Wnt signalling. We also found a specific myogenic expression pattern of MyoD, Myf5 and Myogenin in the developing PSL and high mRNA levels of specific fibrogenic factors, as TGF-ß1, that, undoubtedly, stimulate the growth of connective tissue. Our observations confirmed, at molecular level, the peculiarity of the fast involution observed in PSL a muscle that undergoes a very specific active differentiation process during early development, which implies myofibres involution and their replacement with connective tissue.

Loss of maximal explosive power of lower limbs after 2 weeks of disuse and incomplete recovery after retraining in older adults.

KEY POINTS: Disuse in older adults can critically decrease lower limb muscle power, leading to compromised mobility and overall quality of life. We studied how muscle power and its determinants (muscle mass, single muscle fibre properties and motor control) adapted to 2 weeks of disuse and subsequent 2 weeks of physical training in young and older people. Disuse decreased lower limb muscle power in both groups; however, different adaptations in single muscle fibre properties and co-contraction of leg muscles were observed between young and older individuals. Six physical training sessions performed after disuse promoted the recovery of muscle mass and power. However, they were not sufficient to restore muscle power to pre-disuse values in older individuals, suggesting that further countermeasures are required to counteract the disuse-induced loss of muscle power in older adults. ABSTRACT: Disuse-induced loss of muscle power can be detrimental in older individuals, seriously impairing functional capacity. In this study, we examined the changes in maximal explosive power (MEP) of lower limbs induced by a 14-day disuse (bed-rest, BR) and a subsequent 14-day retraining, to assess whether the impact of disuse was greater in older than in young men, and to analyse the causes of such adaptations. Sixteen older adults (Old: 55-65 years) and seven Young (18-30 years) individuals participated in this study. In a subgroup of eight Old subjects, countermeasures based on cognitive training and protein supplementation were applied. MEP was measured with an explosive ergometer, muscle mass was determined by magnetic resonance, motor control was studied by EMG, and single muscle fibres were analysed in vastus lateralis biopsy samples. MEP was ∼33% lower in Old than in Young individuals, and remained significantly lower (-19%) when normalized by muscle volume. BR significantly affected MEP in Old (-15%) but not in Young. Retraining tended to increase MEP; however, this intervention was not sufficient to restore pre-BR values in Old. Ankle co-contraction increased after BR in Old only, and remained elevated after retraining (+30%). Significant atrophy occurred in slow fibres in Old, and in fast fibres in Young. After retraining, the recovery of muscle fibre thickness was partial. The proposed countermeasures were not sufficient to affect muscle mass and power. The greater impact of disuse and smaller retraining-induced recovery observed in Old highlight the importance of designing suitable rehabilitation protocols for older individuals.

The disorders of the calcium release unit of skeletal muscles: what have we learned from mouse models?

Calcium storage, release, and reuptake are essential for normal physiological function of muscle. Several human skeletal muscle disorders can arise from dysfunction in the control and coordination of these three critical processes. The release from the Sarcoplasmic Reticulum stores (SR) is handled by a multiprotein complex called Calcium Release Unit and composed of DiHydroPyridine Receptor or DHPR, Ryanodine Receptor or RYR, Calsequestrin or CASQ, junctin, Triadin, Junctophilin and Mitsugumin 29. Malignant hyperthermia (MH), Central Core Disease (CCD), Exertional/environmental Heat Stroke (EHS) and Multiminicore disease (MmD) are inherited disorders of calcium homeostasis in skeletal muscles directly related to mutations of genes coding for proteins of the CRU, primarily ryanodine receptor (RYR1). To understand the pathophysiology of MH and CCD, four murine lines carrying point mutations of human RYR1 have been developed: Y524S, R163C, I4898T and T4826I. Mice carrying those mutations show a phenotype with the traits of MH and/or CCD. Interestingly, also ablation of skeletal muscle calsequestrin (CASQ1) leads to a phenotype with MH-like lethal episodes in response to halothane and heat stress and development of central cores. In this review, we aim to describe the murine lines with RYR mutations or CASQ ablation, which show a phenotype similar to human MH or CCD, to underline their specific phenotypes and their differences and to discuss their contribution to the understanding of the pathophysiology of the disorders and the development of therapeutic strategies.

Effects of local vibrations on skeletal muscle trophism in elderly people: mechanical, cellular, and molecular events.

Several studies have examined the effects of vibrations on muscle mass and performance in young healthy people. We studied the effects of vibrations on muscles of elderly male and female volunteers (65-85 years of age) diagnosed with sarcopenia. We applied mechanical vibrations locally (local vibrational training) to the thigh muscles at 300 Hz for a period of 12 weeks, starting with a session of 15 min stimulation once a week and increasing to three sessions of 15 min per week. Treated muscles displayed enhanced maximal isometric strength and increased content of fast MyHC-2X myosin. Single muscle fiber analysis did not show any change in cross-sectional area or in specific tension. Analysis of transcriptional profiles by microarray revealed changes in gene expression after 12 weeks of local vibrational training. In particular, pathways related with energy metabolism, sarcomeric protein balance and oxidative stress response were affected. We conclude that vibration treatment is effective in counteracting the loss of muscular strength associated with sarcopenia and the mode of action of vibration is based on cellular and molecular changes which do not include increase in fiber or muscle size.