The ryanodine receptor as a sensor for cellular environments in muscles

The ryanodine receptor (RyR) is a Ca2+ release channel in the sarcoplasmic reticulum of skeletal and cardiac muscles and plays a key role in excitation-contraction coupling. The activity of the RyR is regulated by many intracellular factors such as divalent cations (Ca2+ and Mg2+), nucleotides, associated proteins, and reactive oxygen species. Since these intracellular factors change depending on the condition of the muscle, e.g., exercise, fatigue, or disease states, the RyR channel activity will be altered accordingly. In this review, we describe how the RyR channel is regulated under various conditions and discuss the possibility that the RyR acts as a sensor for change in the cellular environment of muscles.


Introduction
Skeletal and cardiac muscles are essential organs for the maintenance of life, which embodies all activities, such as walking, eating, and creating. Skeletal muscles are required to perform flexible and complex movements, generate and maintain force, and respond rapidly to different conditions, while the heart muscle sustains and regulates the heartbeat according to physical activity. In addition, during human physical activities, muscles are subjected to high-loads and fatigue and various external stimuli, such as injury, stress, and excitement. These external stimuli cause intricate changes in the intracellular environment of muscle cells. If a control unit in muscle can sense the intracellular environment, the muscle can immediately respond to various stimuli without waiting for commands from the brain.
The contractile activity of skeletal and cardiac muscles is induced by a series of events, including action potential changes, Ca 2+ release from the sarcoplasmic reticulum (SR), and contractile force generation. The ryanodine receptor (RyR) is a Ca 2+ release channel in the SR. In skeletal muscle, the type 1 RyR (RyR1) interacts with the dihydropyridine receptor (DHPR or Cav1.1), an L-type Ca 2+ channel in the transverse tubule (T-tubule). As a result, channel opening is triggered by a conformational change in the DHPR after depolarization of the T-tubule (referred to as depolarization-induced Ca 2+ release, DICR) [1][2] [3]. In cardiac muscle, the type 2 RyR (RyR2) is opened by the binding of Ca 2+ that is introduced from the extracellular space through the DHPR (Cav1.2) (referred to as Ca 2+ -induced Ca 2+ release, CICR). However, in cardiac muscle, a direct interaction between the RyR2 and DHPR is lacking. The opening of RyR channels releases massive Ca 2+ from the SR. Thus, the intracellular Ca 2+ concentration rapidly increases from 50 nmol/L during the resting state to 10-20 μmol/L as maximum peak in twitch contraction [4][5] [6]. A structural change in tropomyosin on the thin muscle filament is caused by Ca 2+ binding to troponin C, and this change facilitates the interaction between myosin and actin, resulting in muscle contraction [7] [8]. Thus, the after exercise is 8.5 mmol/L, 0.15 mmol/L and 2.7 μmol/L, respectively [25]. RyR is predicted to have two ATP-binding sites in one protomer [26], but structural studies have revealed that only one ATP is identified at the interface between Central, S6 and CTD which is near the Ca 2+ binding site [10]. Because other adenine nucleotides can also bind to the site, displacement of ATP by ADP might occur when ADP concentration is increased. In consequence, an increase in ADP concentration causes a reduction of Ca 2+ release.
The change in concentrations of the factors described above is mainly involved in high-load exercises. We will discuss these changes further in the exercise and fatigue section of this review.

SR luminal Ca 2+ and calsequestrin
Single-channel recording experiments showed that the activity of RyRs increased when the concentration of luminal Ca 2+ increased above 1 mmol/L in the SR, whereas trypsin treatment of the luminal side of the SR membrane decreased RyR activity after Ca 2+ concentrations increased [27]. Therefore, it was proposed that RyRs have at least two types of luminal Ca 2+ sensor sites in the SR lumen: one for activation and one for inhibition. A recent study of RyR2 showed that the E4872A mutation abolished luminal Ca 2+  In an imaging study of the flexor digitorum brevis (FDB) muscle, the concentration of luminal Ca 2+ was 0.4 mmol/L at rest and fell to 0.08 mmol/L after tetanic stimulation at 50 Hz. This result indicated that approximately 88% of the luminal Ca 2+ was released during tetanic contractions [39].
The change in concentration of these factors is mainly involved in high-load exercises. We will discuss them in the exercise and fatigue section of this review.

pH
Similar to many proteins in the cell, pH regulates the activity of RyRs [40]. The maximum activity of RyR1 is exhibited at pH 8.0, and sensitivity to Ca 2+ decreases with decreasing pH [33]. The cytosolic pH is usually 7.0 in resting muscle, and exercise induces an acidic shift of pH to 6.7 [25] [41]. The change in this factor is mainly involved in high-load exercises. We will discuss this in the exercise and fatigue section of this review.

Dihydropyridine receptor (DHPR)
In skeletal muscle, RyR1 is under the control of the DHPR. Therefore, abnormalities in the DHPR are directly related to RyR1 function [42][43] [44]. Amino acid mutations in the DHPR cause diseases such as malignant hyperthermia and myopathy [45]. In addition, drugs that cause depolarization of cell membranes, such as depolarizing muscle relaxants, have been implicated in myopathy. Malignant hyperthermia can be induced not only by inhalational anesthetics but also by depolarizing muscle relaxants, e.g., suxamethonium [46]. This is caused by Ca 2+ release from RyR1 after activation of the DHPR by depolarization. Furthermore, changes in blood potassium ion concentrations may also alter the cellular membrane potential and affect the excitation-contraction coupling and Ca 2+ release from RyR1.

FKBP12
FKBP12 is a protein that binds to the immunosuppressive agent FK506, which is used in patients with organ transplants or autoimmune diseases. FKBP12  . The changes in these factors are mainly involved in exercise, oxidative stress and ischemia/reperfusion injury. We will discuss them in the exercise and fatigue, skeletal muscle crisis and modulation of heart function in health and disease section of this review. . S100A1, a 21 kDa Ca 2+ -binding protein, can also bind to the calmodulin binding site of the RyR, and it has been reported that the peak of Ca 2+ transients is reduced in skeletal muscle lacking this protein [62]. The change in concentration of this factor is mainly involved in tachycardia. We will discuss this change in the modulation of heart function in health and disease section of this review.

Phosphorylation
Phosphorylation is an important modification in intracellular signal transduction. It has been demonstrated that the RyR is phosphorylated [63]. Although the mechanism is not fully understood, phosphorylation may alter the activation of the RyR that is mediated by ATP [64]. In RyR2, there are currently three serine residues that are targets for phosphorylation (S2030, S2808, and S2814). These amino acids are thought to be phosphorylated by protein kinase A (PKA), protein kinase C (PKC) or Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) [65]. The change in this factor is mainly involved in exercise and excitement, ischemia/reperfusion injury, catecholaminergic polymorphic ventricular tachycardia (CPVT) and diabetes. We will discuss these factors in the modulation of heart function in health and disease and diet, diabetes section of this review.

S-nitrosylation and oxidation
There are thiols in the RyR that undergo oxidative modifications [66], and the channel activity increases when these thiol groups undergo S-nitrosylation or S-glutathionylation. In experiments using various nitric oxide (NO) donors, some donors enhanced activity, while others showed an inhibitory tendency [67]. These results suggest that there may be multiple target cysteine residues that undergo oxidative modification [68]. The RyR1 Cys3635 residue has been identified as an S-nitrosylation target [69]. It has been reported that thiol modifications promote not only the activity of the RyR itself but also the dissociation of FKBP12 and Ca 2+ -bound calmodulin [70], thereby leading to an increase in channel activity by relieving FKBP12-mediated suppression (Fig. 1A).
It is also known that RyRs can be oxidized by reactive oxygen species (ROS), especially hydroxyl radicals (─OH), which dissociate calmodulin from RyR2 and release the receptor from calmodulin-induced inhibition [71] [72]. Hydroxyl radicals are produced from H2O2 by the Fenton reaction: therefore, the concentration of ─OH is closely related to that of H2O2. H2O2 is generated in mitochondria and permeates the cell membrane easily. The production of H2O2 in normal tissues has been reported to be 100-400 pmol/min/mg [73] [74][75] [76]. H2O2-induced RyR activation occurs at concentrations below 1 mM, and higher concentrations cause inhibition. The inhibitory effect is abrogated by thiol modification [77], and it has been reported that S-nitrosylation protects the RyR from oxidation by ROS [78]. Oxidative modifications occur not only on the RyR but also on FKBP12.6, which causes dissociation from RyR2 and relieves the receptor from FKBP12.6-mediated suppression [79] [80]. Furthermore, treatment of RyR2 with dithiothreitol altered the activity of RyR2 in response to luminal Ca 2+ concentration. Therefore, it is possible that cytoplasmic redox status affects the luminal Ca 2+ sensor as well [81]. The changes in these factors are mainly involved in high load-bearing work, muscle injury, heat stress, and individuals who consume a high-fat diet. We will discuss these changes in the exercise and fatigue, skeletal muscle crisis, modulation of heart function in health and disease and diet, diabetes section of this review.

NADH/NAD
Nicotinamide adenine dinucleotide (NADH) is a cofactor in redox reactions, and the ratio of NADH to NAD changes in cells during energy metabolism. NADH and NAD have been shown to increase RyR1 activity [82]. The NADH was reported as 0.1-0.5 mmol/L in resting skeletal muscle [83] [84], and the ratio of NADH/NAD was 0.002-0.003 [85] [86]. The EC50 of NADH and NAD for activation of RyR1 was shown to be 0.23 mmol/L and 0.31 mmol/L, respectively [82]. For RyR2, activity is decreased by NADH, and NAD counteracts the effect of NADH and restores the the RyR2 channel activity [87]. Because the effects of NADH and NAD are opposite to each other, the ratio of NADH/NAD is a key factor in heart function. The ratio of NADH/NAD was reported to be 0.04-0.05 in heart muscle [88][89] [90]. The change in this factor is mainly involved in myocardial metabolism. We will discuss this change in the modulation of heart function in health section of this review.

Acyl-CoA
Acyl-CoA [91] and acly-carnitine [92], which are metabolic intermediates of lipid metabolism, have been shown to directly increase RyR activity. In particular, palmitoyl-CoA is thought to reduce RyR inhibition mediated by Mg 2+ [93]. Furthermore, acyl-CoA and acyl-CoA-binding protein may interact with FKBPs to regulate RyR activity [94]. Changes in these factors are mainly involved in individuals who consume a high-fat diet. We will discuss these factors further in the diet, diabetes section of this review.

Lipid
Several lipids are known to affect the activity of RyRs, but the majority of these are not biological substances and are not expected to directly interact with RyRs. Several sphingolipids have been shown to inhibit RyR1 activity in physiological situations [95]. However, it is not clear whether they actually interact in vivo.

Exercise and fatigue
Exercise and high load-bearing work cause various changes in skeletal muscle. In particular, during eccentric contraction, the expression of the NADPH oxidase (Nox)4, which produces superoxide from oxygen is increased and substantial ROS are generated [96] [97]. ROS scavengers abolish the exercise-induced increase in Ca 2+ release in skeletal muscle [98]. Although these studies on ROS generation were not always directly related to RyRs, ROS production was shown to correlate with increased Ca 2+ release and muscle tension. It has also been reported that the binding of FKBP12 to RyRs in muscle is reduced by eccentric contraction [99], and as noted above, dissociation reduces FKBP12-mediated suppression of RyR activity (Fig. 1A). In most cases, exercise increases RyR activity, which in turn increases muscle tension (Fig. 1B).
When high-frequency stimulation is sustained, muscle tension gradually declines and the muscle falls into a state of fatigue. Under these conditions, a large amount of ATP is consumed by myosin and sarcoendoplasmic reticulum calcium ATPase (SERCA) in the muscle. Studies measuring ATP concentrations in creatine-kinase inhibited muscle fibers have shown that the ATP concentration in resting muscle was estimated to at 6-8 mmol/L, but decreased to approximately 3 mmol/L during contraction and 1.7 mmol/L when consumption was high [100]. Experiments using skinned fiber have shown that contractility decreased when the ATP concentration fell below 2 mmol/L [101]. The concentration of Mg 2+ , which was released from MgATP also increased with consumption of ATP [102]. However, normal exercise does not substantially reduce the ATP concentration in muscles [103][25] [104], but rather a large amount of ADP is generated as a by-product [104]. ADP does not maintain RyR activity as efficiently as ATP [24]. Therefore, the abundant increase in ADP may suppress channel activity induced by ATP. When high load-bearing exercise increases the ADP/ATP ratio, the impact of ADP may be enhanced.
The SR luminal Ca 2+ concentration decreased from 1mmol/L to 0.08 mmol/L after exercise and high load-bearing work in one study [39]. The RyR senses a reduction in luminal Ca 2+ concentration and channel activity decreases. In addition, when luminal Ca 2+ is depleted, calsequestrin binds to the RyR and suppresses its activity. Moreover, acidification of the muscle cytosol by exercise [25] causes suppression of RyR activity. Thus, high load-bearing work results in muscle fatigue that suppresses RyR activity (Fig.  1C).

Skeletal muscle crises
Skeletal muscles are close to the body surface and are exposed to external environments. Therefore, strong pressure or blows can lead to muscle contusions. It is known that muscle injury increases the production of H2O2 [105] and various other ROS [106] [107]. Although ROS, including H2O2 are known to increase RyR activity, an increase in RyR activity is not generated in damaged muscle fibers during exercise because damaged fibers are unable to contract [106] [107]. However, it has been shown that ROS can spread from the site of injury and affect other muscle fibers and tissues. In particular, H2O2 has a large impact because it can easily permeate the cell membrane. Excessive contraction of the surrounding fibers because of increased RyR activity may increase damage to the contusion site. It has been reported that cooling of the contusion site reduces ROS production [107]. Thus, preventing the increase in RyR activity after muscle injury may be an important aspect of early treatment. Other external stresses include heat stress, which is known to cause a transient increase in oxidation [108], increased mitochondrial ROS production [109], and increased expression of endothelial nitric oxide synthase (NOS) [110]. As in the case of muscle contusion, heat stress also leads to an increase in ROS production. In this case, Ca 2+ release from RyRs is increased and ATP-dependent Ca 2+ uptake by SERCA is enhanced, which may lead to fever, and subsequent heat stroke. In fact, it has been reported that mice with a malignant hyperthermia mutation in RyR1 displayed increased RyR activity and rapid fever after the induction of heat stress, with unfortunate feedback that enhanced mitochondrial ROS production, and resulted in sudden death [111].
Changes in redox state are commonly observed with stress. For example, restraint induced disuse is well known to cause atrophy, which increases H2O2 [112] and neuronal NOS [113] [114]. Changes in redox state occur during hypoxia at high altitude. Under these conditions, an increase in oxidized proteins [115] and a decrease in FKBP12 accompanied by an increase in ROS and NO [116] has been reported. At high altitudes, muscle injury may be induced by intense contractions because the activity of RyR1 is already increased (Fig. 1D).
Aging must also be discussed as one of the crises coming to the muscle. Although many studies have been performed on aging and muscle atrophy, there are not many reports on factors that regulate RyR activity. However, it was demonstrated that calsequestrin increases with age [117]. Additionally, the coupling of the DHPR and RyR1 is also decreased with aging [118].

Modulation of heart function in health and disease
The heartbeat is formed by regular Ca 2+ release from the RyR2 channel, which is controlled by an action potential. The rate of the heartbeat is usually 60-100 beats per minute in humans, but it is known to be higher during exercise and psychological stress. When the sympathetic nervous system is stimulated by exercise or excitement, β-adrenergic signaling activates PKA in the myocardium and increases amplitude of cardiac action-potential-induced Ca 2+ release from RyR2 by direct and indirect mechanisms. The factors that enhance RyR2 activity are elevation of SR luminal Ca 2+ , which results from an increase in Ca 2+ influx via L-type Ca 2+ channels and enhanced SERCA activity, and increased phosphorylation of RyR2 (S2030 and S2808) [119] [120]. NO production is also increased under exercise and stress conditions. These factors all potentiate RyR2 activity and result in strong contractions [121] [122].
Heart failure is a highly prevalent progressive cardiac disorder with high morbidity and mortality. It arises from various causes, including coronary artery disease leading to cardiac arrest, high blood pressure, atrial fibrillation, valvular heart disease, and cardiomyopathy. When the ejection force is reduced during heart failure, the heart is unable to sufficiently maintain blood flow to meet the oxygen demands of tissues. The sympathetic nervous system is activated in the early stage of heart failure, which accelerates phosphorylation of RyR2 by PKA. NO production is increased during heart failure [123]. It was reported that inducible NOS expression increased in failing cardiomyocytes [124]. In addition to the increase in NOS, an increase in Nox expression has also been reported during heart failure [125] [126]. Nox is an enzyme involved in the generation of ROS [127] [128], and ROS promotes RyR oxidation and contributes to increased channel activity. As noted above, phosphorylation, S-nitrosylation, and oxidation may contribute to the activation of RyR2. Moreover, it has been reported that FKBP12.6 dissociates from RyR2 as heart failure progresses [129] [130], which likely contributes to increased RyR2 activity (Fig. 1E). The immediate activation of RyR2 by early compensatory mechanisms that involve these factors may restore cardiac function and physical safety.
The chronic activation of RyR2 has deleterious effects on cardiac function. The chronic and inordinate activation of RyR2 causes diastolic Ca 2+ leakage from the SR and/or generation of Ca 2+ waves. The former leads to contractile dysfunction and the latter causes arrhythmias. Elevated intracellular calcium levels activate CaMKII, which is also involved in the phosphorylation of RyRs. CaMKII was found to be increased in failing cardiomyocytes [131] and may be involved in the subsequent development of disease-related ventricular remodeling [132] or arrhythmia [133]. Therefore, excessive activation of RyR2 may lead to worsening of heart failure rather than improvement of cardiac function. Interestingly, introduction of calmodulin that had high binding affinity for RyR2 into animal models prevented progression to arrhythmia [134] and reduced the symptoms of CPVT [135]. These results indicate that it may be desirable to return to a moderate level of RyR2 activity in the heart after the emergency period has ended. Similar to heart failure, impaired cardiac function caused by ischemia-reperfusion injury also promotes ROS and NO production, phosphorylation, and dissociation of FKBP12.6 from RyR2 [136]. Ischemia-reperfusion is defined as the restoration of coronary blood flow after an ischemic episode. This injury to the myocardium causes the reduction of ejection force, and the increased demand for oxygen in tissues is similar to that observed for intense exercise and heart failure. It has been reported that phosphorylation of S2814 by CaMKII is enhanced after ischemia-reperfusion [137]. Increased oxidation of RyR2 is a result of the increase in ROS generation in cardiomyocytes [138][139] (Fig. 1D and E). The influence of ROS reaches the cardiomyocytes that surround the injured myocytes. Furthermore, ROS produced from tissues other than heart tissue are also involved in RyR2 modulation. Strong oxidative stress responses occur after burns and include activation of RyR2 and leakage of calcium from the SR in cardiomyocytes, which leads to heart failure [140].
Heart failure has been shown to have a significant effect on skeletal muscle. Biopsies of skeletal muscle from patients with heart failure have shown that RyR1 binding to FKBP12 is reduced [141]. It has been reported that phosphorylation of the S2843 residue of RyR1 by PKA dissociates FKBP12 from the receptor, and phosphorylation and dissociation of FKBP12 were also observed in an animal model of heart failure [142].

Diet, Diabetes
RyRs may also be sensitive to the effects of diet. It has been shown that a high-fat diet enhances β-oxidation, which in turn enhances ROS generation in the mitochondria [143] and S-nitrosylation of RyR1, which increases RyR1 activity. Prolonged S-nitrosylation, however, leads to a state of sustained high Ca 2+ concentration, which activates CaMKII and enhances RyR1 phosphorylation [144]. Since increased cytosolic levels of acyl-CoA and acyl-carnitine increase RyR1 activity [91][92] [93], a high-fat diet also acts to increase RyR1 activity (Fig. 1F). Increased RyR1 activity in skeletal muscle leads to increased mobility; therefore, exercise after a high-fat meal is advantageous. In particular, swimming increased the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in skeletal muscle [145], which may suppress excessive diet-induced activation of RyR1. A high-fat diet also affects the myocardium. Accumulation of palmitoyl-carnitine, a long-chain fatty ester of acyl-carnitine, has been shown to increase mitochondrial ROS production, RyR2 oxidation and S-nitrosylation and FKBP12.6 dissociation, which in turn may lead to increased Ca 2+ spark frequency and development of tachycardia [146] (Fig. 1E).
Long-chain unsaturated fatty acids, such as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and oleic acid, in contrast, have been shown to decrease the activity of RyR2 [147] [148]. Although the mechanism is not fully understood, DHA has been shown to enhance the stability of RyR2-FKBP12.6 binding through its oxidized neuroprostane by-product (NeuroP) [148]. Furthermore, DHA has been found to reduce RyR2 mRNA expression [149].
It has been noted that Ca 2+ release is abnormal in diabetes mellitus [150] [151]. Decreased glucose utilization and increased β-oxidation increase ROS, as does the consumption of a high-fat diet. The production of H2O2 is increased 1.5 times in hyperinsulinemic premenopausal women [152]. When ROS scavengers and CaMKII inhibitors were added to the myocardium from diabetic models, the peak Ca 2+ transients were normalized and spontaneous Ca 2+ release was suppressed [151] [153]. It is also known that phosphorylation occurs to a high degree in diabetic cardiac RyRs [154] [150]. In particular, it has been shown that S2809 is phosphorylated [155].

Conclusion
The RyR may not be a simple ion channel, it may act as a sensor and receptor that actively regulates muscular mechanisms. When skeletal muscle is subjected to high-load exercise, the channel activity becomes reduced by alteration of nucleotide composition. After a critical situation occurs because of external stress, injury, or excitement of skeletal or cardiac muscle, changes in redox state and dissociation of inhibitory factors increase channel activity. Since RyRs are submaximally active under normal muscle conditions, they can sense changes in the intracellular environment caused by external influences and perform biphasic modulation of Ca 2+ release. The balance of RyR channel activity and biphasic modulation associated with the intracellular environment may be the key to maintaining muscle function, and an imbalance in RyR activity may lead to skeletal muscle disease and/or cardiomyopathy.