Ryanodine receptors (RyRs) are located in the sarcoplasmic/endoplasmic reticulum membrane and are responsible for the release of Ca 2þ from intracellular stores during excitation-contraction coupling in both cardiac and skeletal muscle. RyRs are the largest known ion channels (.2MDa) and exist as three mammalian isoforms (RyR 1 -3), all of which are homotetrameric proteins that interact with and are regulated by phosphorylation, redox modifications, and a variety of small proteins and ions. Most RyR channel modulators interact with the large cytoplasmic domain whereas the carboxy-terminal portion of the protein forms the ion-conducting pore. Mutations in RyR2 are associated with human disorders such as catecholaminergic polymorphic ventricular tachycardia whereas mutations in RyR1 underlie diseases such as central core disease and malignant hyperthermia. This chapter examines the current concepts of the structure, function and regulation of RyRs and assesses the current state of understanding of their roles in associated disorders.

High-intensity interval training (HIIT) is a time-efficient way of improving physical performance in healthy subjects and in patients with common chronic diseases, but less so in elite endurance athletes. The mechanisms underlying the effectiveness of HIIT are uncertain. Here, recreationally active human subjects performed highly demanding HIIT consisting of 30-s bouts of all-out cycling with 4-min rest in between bouts (≤3 min total exercise time). Skeletal muscle biopsies taken 24 h after the HIIT exercise showed an extensive fragmentation of the sarcoplasmic reticulum (SR) Ca 2+ release channel, the ryanodine receptor type 1 (RyR1). The HIIT exercise also caused a prolonged force depression and triggered major changes in the expression of genes related to endurance exercise. Subsequent experiments on elite endurance athletes performing the same HIIT exercise showed no RyR1 fragmentation or prolonged changes in the expression of endurance-related genes. Finally, mechanistic experiments performed on isolated mouse muscles exposed to HIIT-mimicking stimulation showed reactive oxygen/nitrogen species (ROS)-dependent RyR1 fragmentation, calpain activation, increased SR Ca 2+ leak at rest, and depressed force production due to impaired SR Ca 2+ release upon stimulation. In conclusion, HIIT exercise induces a ROSdependent RyR1 fragmentation in muscles of recreationally active subjects, and the resulting changes in muscle fiber Ca 2+ -handling trigger muscular adaptations. However, the same HIIT exercise does not cause RyR1 fragmentation in muscles of elite endurance athletes, which may explain why HIIT is less effective in this group.ryanodine receptor 1 | high-intensity exercise | skeletal muscle | Ca 2+ | reactive oxygen species

Obesity and insulin resistance are associated with enhanced fatty acid utilization, which may play a central role in diabetic cardiomyopathy. We now assess the effect of the saturated fatty acid palmitate (1.2 mmol/l) on Ca 2؉ handling, cell shortening, and mitochondrial production of reactive oxygen species (ROS) in freshly isolated ventricular cardiomyocytes from normal (wild-type) and obese, insulin-resistant ob/ob mice. Cardiomyocytes were electrically stimulated at 1 Hz, and the signal of fluorescent indicators was measured with confocal microscopy. Palmitate decreased the amplitude of cytosolic Ca 2؉ transients (measured with fluo-3), the sarcoplasmic reticulum Ca 2؉ load, and cell shortening by ϳ20% in wild-type cardiomyocytes; these decreases were prevented by the general antioxidant N-acetylcysteine. In contrast, palmitate accelerated Ca 2؉ transients and increased cell shortening in ob/ob cardiomyocytes. Application of palmitate rapidly dissipated the mitochondrial membrane potential (measured with tetra-methyl rhodamine-ethyl ester) and increased the mitochondrial ROS production (measured with MitoSOX Red) in wild-type but not in ob/ob cardiomyocytes. In conclusion, increased saturated fatty acid levels impair cellular Ca 2؉ handling and contraction in a ROSdependent manner in normal cardiomyocytes. Conversely, high fatty acid levels may be vital to sustain cardiac Ca 2؉ handling and contraction in obesity and insulin-resistant conditions. Diabetes 56: 1136 -1142, 2007 C ardiac muscle cells generate ATP at a high rate to support the continuous contractile function of the beating heart. Cardiac cells use various substrates to generate ATP, and the extent of substrates utilized depends on the substrate availability, the energy demand, and the physiological or pathological condition (1). In humans as well as in different animal models, obesity, insulin resistance, and type 2 diabetes are associated with an altered cardiac metabolism characterized by an enhanced reliance on fatty acids and a decreased glucose utilization. These changes play a central role in the development of diabetic cardiomyopathy (2). For instance, application of the saturated fatty acid palmitate had markedly different effects on power output and oxygen consumption in hearts of control mice and ob/ob mice, which are obese, insulin resistant, and have increased serum free fatty acid concentrations (3). Moreover, ob/ob hearts displayed a decreased mitochondrial oxidative capacity and an increased fatty acid-induced mitochondrial uncoupling (4).Cellular Ca 2ϩ handling is altered in type 2 diabetes (5), and diabetic cardiomyopathy is characterized by defective sarcoplasmic reticulum (SR) function, which results in smaller and slower cytoplasmic Ca 2ϩ transients (6,7). Mitochondria play a central role in the development of diabetes complications, and the mitochondrial dysfunction is characterized by decreased mitochondrial Ca 2ϩ loading capacity and increased production of reactive oxygen species (ROS) (8 -10). Increased ROS production...

Key pointsr Increased free radical production may contribute to decreased muscle force production during fatiguing exercise, and might delay recovery from fatigue. r We exposed mouse fast-twitch single fibres to antioxidants targeting specific cellular sites to determine whether these compounds delay fatigue development and/or improve the recovery from fatigue.r Antioxidants had no effect on the fatigue-induced decrease in contractile force. r During recovery from fatigue, a mitochondria-targeted antioxidant, SS-31, restored the fatigue-induced decrease in sarcoplasmic reticulum Ca 2+ release, but did not improve force recovery.r We conclude that antioxidants cannot counteract the force decline during or after induction of muscle fatigue, although they may affect the underlying mechanisms.Abstract The contractile performance of skeletal muscle declines during intense activities, i.e. fatigue develops. Fatigued muscle can enter a state of prolonged low-frequency force depression (PLFFD). PLFFD can be due to decreased tetanic free cytosolic [Ca 2+ ] ([Ca 2+ ] i ) and/or decreased myofibrillar Ca 2+ sensitivity. Increases in reactive oxygen and nitrogen species (ROS/RNS) may contribute to fatigue-induced force reductions. We studied whether pharmacological ROS/RNS inhibition delays fatigue and/or counteracts the development of PLFFD. Mechanically isolated mouse fast-twitch fibres were fatigued by sixty 150 ms, 70 Hz tetani given every 1 s. Experiments were performed in standard Tyrode solution (control) or in the presence of: NADPH oxidase (NOX) 2 inhibitor (gp91ds-tat); NOX4 inhibitor (GKT137831); mitochondria-targeted antioxidant (SS-31); nitric oxide synthase (NOS) inhibitor (L-NAME); the general antioxidant N-acetylcysteine (NAC); a cocktail of SS-31, L-NAME and NAC. Spatially and temporally averaged [Ca 2+ ] i and peak force were reduced by ß20% and ß70% at the end of fatiguing stimulation, respectively, with no marked differences between groups. PLFFD was similar in all groups, with 30 Hz force being decreased by ß60% at 30 min of recovery. PLFFD was mostly due to decreased tetanic [Ca 2+ ] i in control fibres and in the presence of NOX2 or NOX4 inhibitors. Conversely, in fibres exposed to

Mice with a knock-in mutation (Y524S) in the type I ryanodine receptor (RyR1) die when exposed to short periods of temperature elevation (≥ 37 °C). We demonstrate that treatment with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) prevents heat-induced sudden death in Y524S mice. The AICAR protection is independent of AMPK activation and results from a newly identified action on the mutant RyR1 to reduce Ca2+ leak, preventing Ca2+ dependent increases in both reactive oxygen and reactive nitrogen species that act to further increase resting Ca2+ concentrations. If unchecked, the temperature driven increases in resting Ca2+ and ROS/RNS create an amplifying cycle that ultimately triggers sustained muscle contractions, rhabdomyolysis and death. Although antioxidants are effective in reducing this cycle in vitro, only AICAR prevents the heat induced death in vivo. Our findings suggest that AICAR is likely to be effective in prophylactic treatment of humans with enhanced susceptibility to exercise/heat-induced sudden death associated with RyR1 mutations.

The production of reactive oxygen/nitrogen species (ROS/RNS) is generally considered to increase during physical exercise. Nevertheless, direct measurements of ROS/RNS often show modest increases in ROS/RNS in muscle fibres even during intensive fatiguing stimulation, and the major source(s) of ROS/RNS during exercise is still being debated. In rested muscle fibres, mild and acute exposure to exogenous ROS/RNS generally increases myofibrillar submaximal force, whereas stronger or prolonged exposure has the opposite effect. Endogenous production of ROS/RNS seems to preferentially decrease submaximal force and positive effects of antioxidants are mainly observed during fatigue induced by submaximal contractions. Fatigued muscle fibres frequently enter a prolonged state of reduced submaximal force, which is caused by a ROS/RNS-dependent decrease in sarcoplasmic reticulum Ca(2+) release and/or myofibrillar Ca(2+) sensitivity. Increased ROS/RNS production during exercise can also be beneficial and recent human and animal studies show that antioxidant supplementation can hamper the beneficial effects of endurance training. In conclusion, increased ROS/RNS production have both beneficial and detrimental effects on skeletal muscle function and the outcome depends on a combination of factors: the type of ROS/RNS; the magnitude, duration and location of ROS/RNS production; and the defence systems, including both endogenous and exogenous antioxidants.

ObjectiveSkeletal muscle weakness is a prominent clinical feature in patients with rheumatoid arthritis (RA), but the underlying mechanism(s) is unknown. Here we investigate the mechanisms behind arthritis-induced skeletal muscle weakness with special focus on the role of nitrosative stress on intracellular Ca2+ handling and specific force production.MethodsNitric oxide synthase (NOS) expression, degree of nitrosative stress and composition of the major intracellular Ca2+ release channel (ryanodine receptor 1, RyR1) complex were measured in muscle. Changes in cytosolic free Ca2+ concentration ([Ca2+]i) and force production were assessed in single-muscle fibres and isolated myofibrils using atomic force cantilevers.ResultsThe total neuronal NOS (nNOS) levels were increased in muscles both from collagen-induced arthritis (CIA) mice and patients with RA. The nNOS associated with RyR1 was increased and accompanied by increased [Ca2+]i during contractions of muscles from CIA mice. A marker of peroxynitrite-derived nitrosative stress (3-nitrotyrosine, 3-NT) was increased on the RyR1 complex and on actin of muscles from CIA mice. Despite increased [Ca2+]i, individual CIA muscle fibres were weaker than in healthy controls, that is, force per cross-sectional area was decreased. Furthermore, force and kinetics were impaired in CIA myofibrils, hence actin and myosin showed decreased ability to interact, which could be a result of increased 3-NT content on actin.ConclusionsArthritis-induced muscle weakness is linked to nitrosative modifications of the RyR1 protein complex and actin, which are driven by increased nNOS associated with RyR1 and progressively increasing Ca2+ activation.

Manipulation of muscle temperature is believed to improve post-exercise recovery, with cooling being especially popular among athletes. However, it is unclear whether such temperature manipulations actually have positive effects. Accordingly, we studied the effect of muscle temperature on the acute recovery of force and fatigue resistance after endurance exercise. One hour of moderate-intensity arm cycling exercise in humans was followed by 2 h recovery in which the upper arms were either heated to 38°C, not treated (33°C), or cooled to ∼15°C. Fatigue resistance after the recovery period was assessed by performing 3 × 5 min sessions of all-out arm cycling at physiological temperature for all conditions (i.e. not heated or cooled). Power output during the all-out exercise was better maintained when muscles were heated during recovery, whereas cooling had the opposite effect. Mechanisms underlying the temperature-dependent effect on recovery were tested in mouse intact single muscle fibres, which were exposed to ∼12 min of glycogen-depleting fatiguing stimulation (350 ms tetani given at 10 s interval until force decreased to 30% of the starting force). Fibres were subsequently exposed to the same fatiguing stimulation protocol after 1-2 h of recovery at 16-36°C. Recovery of submaximal force (30 Hz), the tetanic myoplasmic free [Ca ] (measured with the fluorescent indicator indo-1), and fatigue resistance were all impaired by cooling (16-26°C) and improved by heating (36°C). In addition, glycogen resynthesis was faster at 36°C than 26°C in whole flexor digitorum brevis muscles. We conclude that recovery from exhaustive endurance exercise is accelerated by raising and slowed by lowering muscle temperature.