Novel Calmodulin Variant p.E46K Associated With Severe Catecholaminergic Polymorphic Ventricular Tachycardia Produces Robust Arrhythmogenicity in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

BACKGROUND: CaM (calmodulin) is a ubiquitously expressed, multifunctional Ca2+ sensor protein that regulates numerous proteins. Recently, CaM missense variants have been identified in patients with malignant inherited arrhythmias, such as long QT syndrome and catecholaminergic polymorphic ventricular tachycardia (CPVT). However, the exact mechanism of CaM-related CPVT in human cardiomyocytes remains unclear. In this study, we sought to investigate the arrhythmogenic mechanism of CPVT caused by a novel variant using human induced pluripotent stem cell (iPSC) models and biochemical assays. METHODS: We generated iPSCs from a patient with CPVT bearing CALM2 p.E46K. As comparisons, we used 2 control lines including an isogenic line, and another iPSC line from a patient with long QT syndrome bearing CALM2 p.N98S (also reported in CPVT). Electrophysiological properties were investigated using iPSC-cardiomyocytes. We further examined the RyR2 (ryanodine receptor 2) and Ca2+ affinities of CaM using recombinant proteins. RESULTS: We identified a novel de novo heterozygous variant, CALM2 p.E46K, in 2 unrelated patients with CPVT accompanied by neurodevelopmental disorders. The E46K-cardiomyocytes exhibited more frequent abnormal electrical excitations and Ca2+ waves than the other lines in association with increased Ca2+ leakage from the sarcoplasmic reticulum via RyR2. Furthermore, the [3H]ryanodine binding assay revealed that E46K-CaM facilitated RyR2 function especially by activating at low [Ca2+] levels. The real-time CaM-RyR2 binding analysis demonstrated that E46K-CaM had a 10-fold increased RyR2 binding affinity compared with wild-type CaM which may account for the dominant effect of the mutant CaM. Additionally, the E46K-CaM did not affect CaM-Ca2+ binding or L-type calcium channel function. Finally, antiarrhythmic agents, nadolol and flecainide, suppressed abnormal Ca2+ waves in E46K-cardiomyocytes. CONCLUSIONS: We, for the first time, established a CaM-related CPVT iPSC-CM model which recapitulated severe arrhythmogenic features resulting from E46K-CaM dominantly binding and facilitating RyR2. In addition, the findings in iPSC-based drug testing will contribute to precision medicine.

Drug development for the treatment of RyR1-related skeletal muscle diseases.

Type 1 ryanodine receptor (RyR1) is an intracellular Ca2+ release channel on the sarcoplasmic reticulum of skeletal muscle, and it plays a central role in excitation-contraction (E-C) coupling. Mutations in RyR1 are implicated in various muscle diseases including malignant hyperthermia, central core disease, and myopathies. Currently, no specific treatment exists for most of these diseases. Recently, high-throughput screening (HTS) assays have been developed for identifying potential candidates for treating RyR-related muscle diseases. Currently, two different methods, namely a FRET-based assay and an endoplasmic reticulum Ca2+-based assay, are available. These assays identified several compounds as novel RyR1 inhibitors. In addition, the development of a reconstituted platform permitted HTS assays for E-C coupling modulators. In this review, we will focus on recent progress in HTS assays and discuss future perspectives of these promising approaches.

A reconstituted depolarization-induced Ca2+ release platform for validation of skeletal muscle disease mutations and drug discovery.

In skeletal muscle excitation-contraction (E-C) coupling, depolarization of the plasma membrane triggers Ca2+ release from the sarcoplasmic reticulum (SR), referred to as depolarization-induced Ca2+ release (DICR). DICR occurs through the type 1 ryanodine receptor (RyR1), which physically interacts with the dihydropyridine receptor Cav1.1 subunit in specific machinery formed with additional essential components including ß1a, Stac3 adaptor protein, and junctophilins. Exome sequencing has accelerated the discovery of many novel mutations in genes encoding DICR machinery in various skeletal muscle diseases. However, functional validation is time-consuming because it must be performed in a skeletal muscle environment. In this study, we established a platform of the reconstituted DICR in HEK293 cells. The essential components were effectively transduced into HEK293 cells expressing RyR1 using baculovirus vectors, and Ca2+ release was quantitatively measured with R-CEPIA1er, a fluorescent ER Ca2+ indicator, without contaminant of extracellular Ca2+ influx. In these cells, [K+]-dependent Ca2+ release was triggered by chemical depolarization with the aid of inward rectifying potassium channel, indicating a successful reconstitution of DICR. Using the platform, we evaluated several Cav1.1 mutations that are implicated in malignant hyperthermia and myopathy. We also tested several RyR1 inhibitors; whereas dantrolene and Cpd1 inhibited DICR, procaine had no effect. Furthermore, twitch potentiators such as perchlorate and thiocyanate shifted the voltage dependence of DICR to more negative potentials without affecting Ca2+-induced Ca2+ release. These results well reproduced the findings with the muscle fibers and the cultured myotubes. Since the procedure is simple and reproducible, the reconstituted DICR platform will be highly useful for the validation of mutations and drug discovery for skeletal muscle diseases.

Mice with R2509C-RYR1 mutation exhibit dysfunctional Ca2+ dynamics in primary skeletal myocytes.

Type 1 ryanodine receptor (RYR1) is a Ca2+ release channel in the sarcoplasmic reticulum (SR) of the skeletal muscle and plays a critical role in excitation-contraction coupling. Mutations in RYR1 cause severe muscle diseases, such as malignant hyperthermia, a disorder of Ca2+-induced Ca2+ release (CICR) through RYR1 from the SR. We recently reported that volatile anesthetics induce malignant hyperthermia (MH)-like episodes through enhanced CICR in heterozygous R2509C-RYR1 mice. However, the characterization of Ca2+ dynamics has yet to be investigated in skeletal muscle cells from homozygous mice because these animals die in utero. In the present study, we generated primary cultured skeletal myocytes from R2509C-RYR1 mice. No differences in cellular morphology were detected between wild type (WT) and mutant myocytes. Spontaneous Ca2+ transients and cellular contractions occurred in WT and heterozygous myocytes, but not in homozygous myocytes. Electron microscopic observation revealed that the sarcomere length was shortened to ∼1.7 µm in homozygous myocytes, as compared to ∼2.2 and ∼2.3 µm in WT and heterozygous myocytes, respectively. Consistently, the resting intracellular Ca2+ concentration was higher in homozygous myocytes than in WT or heterozygous myocytes, which may be coupled with a reduced Ca2+ concentration in the SR. Finally, using infrared laser-based microheating, we found that heterozygous myocytes showed larger heat-induced Ca2+ transients than WT myocytes. Our findings suggest that the R2509C mutation in RYR1 causes dysfunctional Ca2+ dynamics in a mutant-gene dose-dependent manner in the skeletal muscles, in turn provoking MH-like episodes and embryonic lethality in heterozygous and homozygous mice, respectively.

Development of a water-soluble ryanodine receptor 1 inhibitor.

Ryanodine receptor 1 (RyR1) is a Ca2+-release channel expressed on the sarcoplasmic reticulum (SR) membrane. RyR1 mediates release of Ca2+ from the SR to the cytoplasm to induce muscle contraction, and mutations associated with overactivation of RyR1 cause lethal muscle diseases. Dantrolene sodium salt (dantrolene Na) is the only approved RyR inhibitor to treat malignant hyperthermia patients with RyR1 mutations, but is poorly water-soluble. Our group recently developed a bioassay system and used it to identify quinoline derivatives such as 1 as potent RyR1 inhibitors. In the present study, we focused on modification of these inhibitors with the aim of increasing their water-solubility. First, we tried reducing the hydrophobicity by shortening the N-octyl chain at the quinolone ring of 1; the N-heptyl compound retained RyR1-inhibitory activity, but the N-hexyl compound showed decreased activity. Next, we introduced a more hydrophilic azaquinolone ring in place of quinolone; in this case, only the N-octyl compound retained activity. The sodium salt of N-octyl azaquinolone 7 showed similar inhibitory activity to dantrolene Na with approximately 1,000-fold greater solubility in saline.

Heat-hypersensitive mutants of ryanodine receptor type 1 revealed by microscopic heating.

Thermoregulation is an important aspect of human homeostasis, and high temperatures pose serious stresses for the body. Malignant hyperthermia (MH) is a life-threatening disorder in which body temperature can rise to a lethal level. Here we employ an optically controlled local heat-pulse method to manipulate the temperature in cells with a precision of less than 1 °C and find that the mutants of ryanodine receptor type 1 (RyR1), a key Ca2+ release channel underlying MH, are heat hypersensitive compared with the wild type (WT). We show that the local heat pulses induce an intracellular Ca2+ burst in human embryonic kidney 293 cells overexpressing WT RyR1 and some RyR1 mutants related to MH. Fluorescence Ca2+ imaging using the endoplasmic reticulum-targeted fluorescent probes demonstrates that the Ca2+ burst originates from heat-induced Ca2+ release (HICR) through RyR1-mutant channels because of the channels' heat hypersensitivity. Furthermore, the variation in the heat hypersensitivity of four RyR1 mutants highlights the complexity of MH. HICR likewise occurs in skeletal muscles of MH model mice. We propose that HICR contributes an additional positive feedback to accelerate thermogenesis in patients with MH.

Involvement of kallikrein-PAR2-proinflammatory pathway in severe trastuzumab-induced cardiotoxicity.

Trastuzumab-induced cardiotoxicity interferes with continued treatment in approximately 10% of patients with ErbB2-positive breast cancer, but its mechanism has not been fully elucidated. In this study, we recruited trastuzumab-treated patients with ≥30% reduction in left ventricular ejection fraction (SP) and noncardiotoxic patients (NP). From each of these patients, we established three cases of induced pluripotent stem cell-derived cardiomyocytes (pt-iPSC-CMs). Reduced contraction and relaxation velocities following trastuzumab treatment were more evident in SP pt-iPSC-CMs than NP pt-iPSC-CMs, indicating the cardiotoxicity phenotype could be replicated. Differences in ATP production, reactive oxygen species, and autophagy activity were observed between the two groups. Analysis of transcripts revealed enhanced kallikrein5 expression and pro-inflammatory signaling pathways, such as interleukin-1ß, in SP pt-iPSC-CMs after trastuzumab treatment. The kallilkrein5-protease-activated receptor 2 (PAR2)-MAPK signaling pathway was more activated in SP pt-iPSC-CMs, and treatment with a PAR2-antagonist suppressed interleukin-1ß expression. Our data indicate enhanced pro-inflammatory responses through kallikrein5-PAR2 signaling and vulnerability to external stresses appear to be the cause of trastuzumab-induced cardiotoxicity in SP.

[Therapeutic effects of novel type1 ryanodine receptor inhibitor on skeletal muscle diseases].

Type 1 ryanodine receptor (RyR1) plays a key role in Ca2+ release from the sarcoplasmic reticulum (SR) during excitation-contraction coupling of skeletal muscle. Mutations in RyR1 hyperactivate the channel to cause malignant hyperthermia (MH). MH is a serious complication characterized by skeletal muscle rigidity and elevated body temperature in response to commonly used inhalational anesthetics. Thus far, more than 300 mutations in RyR1 gene have been reported in patients with MH. Some heat stroke triggered by exercise or environmental heat stress is also related to MH mutations in the RyR1 gene. The only drug approved for ameliorating the symptoms of MH is dantrolene, which has been first developed in 1960s as a muscle relaxant. However, dantrolene has several disadvantages for clinical use: poor water solubility which makes rapid preparation difficult in emergency situations and long plasma half-life, which causes long-lasting side effects such as muscle weakness. Here we show that a novel RyR1-selective inhibitor, 6,7-(methylenedioxy)-1-octyl-4-quinolone-3-carboxylic acid (Compound 1, Cpd1), effectively rescues MH and heat stroke in new mouse model relevant to MH. Cpd1 has great advantages of higher water solubility and shorter plasma half-life compared to dantrolene. Our data suggest that Cpd1 has the potential to be a promising new candidate for effective treatment of patients carrying RyR1 mutations.

A novel RyR1-selective inhibitor prevents and rescues sudden death in mouse models of malignant hyperthermia and heat stroke.

Mutations in the type 1 ryanodine receptor (RyR1), a Ca2+ release channel in skeletal muscle, hyperactivate the channel to cause malignant hyperthermia (MH) and are implicated in severe heat stroke. Dantrolene, the only approved drug for MH, has the disadvantages of having very poor water solubility and long plasma half-life. We show here that an oxolinic acid-derivative RyR1-selective inhibitor, 6,7-(methylenedioxy)-1-octyl-4-quinolone-3-carboxylic acid (Compound 1, Cpd1), effectively prevents and treats MH and heat stroke in several mouse models relevant to MH. Cpd1 reduces resting intracellular Ca2+, inhibits halothane- and isoflurane-induced Ca2+ release, suppresses caffeine-induced contracture in skeletal muscle, reduces sarcolemmal cation influx, and prevents or reverses the fulminant MH crisis induced by isoflurane anesthesia and rescues animals from heat stroke caused by environmental heat stress. Notably, Cpd1 has great advantages of better water solubility and rapid clearance in vivo over dantrolene. Cpd1 has the potential to be a promising candidate for effective treatment of patients carrying RyR1 mutations.

Insights into channel modulation mechanism of RYR1 mutants using Ca2+ imaging and molecular dynamics.

Type 1 ryanodine receptor (RYR1) is a Ca2+ release channel in the sarcoplasmic reticulum in skeletal muscle and plays an important role in excitation-contraction coupling. Mutations in the RYR1 gene cause severe muscle diseases such as malignant hyperthermia (MH), which is a disorder of CICR via RYR1. Thus far, >300 mutations in RYR1 have been reported in patients with MH. However, owing to a lack of comprehensive analysis of the structure-function relationship of mutant RYR1, the mechanism remains largely unknown. Here, we combined functional studies and molecular dynamics (MD) simulations of RYR1 bearing disease-associated mutations at the N-terminal region. When expressed in HEK293 cells, the mutant RYR1 caused abnormalities in Ca2+ homeostasis. MD simulations of WT and mutant RYR1s were performed using crystal structure of the N-terminal domain (NTD) monomer, consisting of A, B, and C domains. We found that the mutations located around the interdomain region differentially affected hydrogen bonds/salt bridges. Particularly, mutations at R402, which increase the open probability of the channel, cause clockwise rotation of BC domains with respect to the A domain by alteration of the interdomain interactions. Similar results were also obtained with artificial mutations that mimic alteration of the interactions. Our results reveal the importance of interdomain interactions within the NTD in the regulation of the RYR1 channel and provide insights into the mechanism of MH caused by the mutations at the NTD.

Structural development of a type-1 ryanodine receptor (RyR1) Ca2+-release channel inhibitor guided by endoplasmic reticulum Ca2+ assay.

Type-1 ryanodine receptor (RyR1) is a calcium-release channel localized on sarcoplasmic reticulum (SR) of the skeletal muscle, and mediates muscle contraction by releasing Ca2+ from the SR. Genetic mutations of RyR1 are associated with skeletal muscle diseases such as malignant hyperthermia and central core diseases, in which over-activation of RyR1 causes leakage of Ca2+ from the SR. We recently developed an efficient high-throughput screening system based on the measurement of Ca2+ in endoplasmic reticulum, and used it to identify oxolinic acid (1) as a novel RyR1 channel inhibitor. Here, we designed and synthesized a series of quinolone derivatives based on 1 as a lead compound. Derivatives bearing a long alkyl chain at the nitrogen atom of the quinolone ring and having a suitable substituent at the 7-position of quinolone exhibited potent RyR1 channel-inhibitory activity. Among the synthesized compounds, 14h showed more potent activity than dantrolene, a known RyR1 inhibitor, and exhibited high RyR1 selectivity over RyR2 and RyR3. These compounds may be promising leads for clinically applicable RyR1 channel inhibitors.

A novel strategy for treatment of cancer cachexia targeting xanthine oxidase in the brain.

Cancer cachexia is a systemic wasting syndrome characterized by anorexia and loss of body weight. The xanthine oxidase (XO) inhibitor febuxostat is one of the promising candidates for cancer cachexia treatment. However, cachexic symptoms were not alleviated by oral administration of febuxostat in our cancer cachexia model. Metabolomic analysis with brains of our cachexic model showed that purine metabolism was activated and XO activity was increased, and thus suggested that febuxostat would not reach the brain. Accordingly, targeting XO in the brain, which controls appetite, may be an effective strategy for treatment of cancer cachexia.

Therapeutic potential of ghrelin and des-acyl ghrelin against chemotherapy-induced cardiotoxicity.

Cancer was considered an incurable disease for many years; however, with the development of anticancer drugs and state-of-the art technologies, it has become curable. Cardiovascular diseases in patients with cancer or induced by cancer chemotherapy have recently become a great concern. Certain anticancer drugs and molecular targeted therapies cause cardiotoxicity, which limit the widespread implementation of cancer treatment and decrease the quality of life in cancer patients significantly. The anthracycline doxorubicin (DOX) causes cardiotoxicity. The cellular mechanism underlying DOX-induced cardiotoxicity include free-radical damage to cardiac myocytes, leading to mitochondrial injury and subsequent death of myocytes. Recently, circulating orexigenic hormones, ghrelin and des-acyl ghrelin, have been reported to inhibit DOX-induced cardiotoxicity. However, little is known about the molecular mechanisms underlying their preventive effects. In the present study, we show the possible mechanisms underlying the effects of ghrelin and des-acyl ghrelin against DOX-induced cardiotoxicity through in vitro and in vivo researches.

Genotype-Phenotype Correlations of Malignant Hyperthermia and Central Core Disease Mutations in the Central Region of the RYR1 Channel.

Type 1 ryanodine receptor (RYR1) is a Ca2+ release channel in the sarcoplasmic reticulum of skeletal muscle and is mutated in some muscle diseases, including malignant hyperthermia (MH) and central core disease (CCD). Over 200 mutations associated with these diseases have been identified, and most mutations accelerate Ca2+ -induced Ca2+ release (CICR), resulting in abnormal Ca2+ homeostasis in skeletal muscle. However, it remains largely unknown how specific mutations cause different phenotypes. In this study, we investigated the CICR activity of 14 mutations at 10 different positions in the central region of RYR1 (10 MH and four MH/CCD mutations) using a heterologous expression system in HEK293 cells. In live-cell Ca2+ imaging, the mutant channels exhibited an enhanced sensitivity to caffeine, a reduced endoplasmic reticulum Ca2+ content, and an increased resting cytoplasmic Ca2+ level. The three parameters for CICR (Ca2+ sensitivity for activation, Ca2+ sensitivity for inactivation, and attainable maximum activity, i.e., gain) were obtained by [3 H]ryanodine binding and fitting analysis. The mutant channels showed increased gain and Ca2+ sensitivity for activation in a site-specific manner. Genotype-phenotype correlations were explained well by the near-atomic structure of RYR1. Our data suggest that divergent CICR activity may cause various disease phenotypes by specific mutations.

Divergent Activity Profiles of Type 1 Ryanodine Receptor Channels Carrying Malignant Hyperthermia and Central Core Disease Mutations in the Amino-Terminal Region.

The type 1 ryanodine receptor (RyR1) is a Ca2+ release channel in the sarcoplasmic reticulum of skeletal muscle and is mutated in several diseases, including malignant hyperthermia (MH) and central core disease (CCD). Most MH and CCD mutations cause accelerated Ca2+ release, resulting in abnormal Ca2+ homeostasis in skeletal muscle. However, how specific mutations affect the channel to produce different phenotypes is not well understood. In this study, we have investigated 11 mutations at 7 different positions in the amino (N)-terminal region of RyR1 (9 MH and 2 MH/CCD mutations) using a heterologous expression system in HEK293 cells. In live-cell Ca2+ imaging at room temperature (~25 °C), cells expressing mutant channels exhibited alterations in Ca2+ homeostasis, i.e., an enhanced sensitivity to caffeine, a depletion of Ca2+ in the ER and an increase in resting cytoplasmic Ca2+. RyR1 channel activity was quantitatively evaluated by [3H]ryanodine binding and three parameters (sensitivity to activating Ca2+, sensitivity to inactivating Ca2+ and attainable maximum activity, i.e., gain) were obtained by fitting analysis. The mutations increased the gain and the sensitivity to activating Ca2+ in a site-specific manner. The gain was consistently higher in both MH and MH/CCD mutations. Sensitivity to activating Ca2+ was markedly enhanced in MH/CCD mutations. The channel activity estimated from the three parameters provides a reasonable explanation to the pathological phenotype assessed by Ca2+ homeostasis. These properties were also observed at higher temperatures (~37 °C). Our data suggest that divergent activity profiles may cause varied disease phenotypes by specific mutations. This approach should be useful for diagnosis and treatment of diseases with mutations in RyR1.

Role of amino-terminal half of the S4-S5 linker in type 1 ryanodine receptor (RyR1) channel gating.

The type 1 ryanodine receptor (RyR1) is a Ca(2+) release channel found in the sarcoplasmic reticulum of skeletal muscle and plays a pivotal role in excitation-contraction coupling. The RyR1 channel is activated by a conformational change of the dihydropyridine receptor upon depolarization of the transverse tubule, or by Ca(2+) itself, i.e. Ca(2+)-induced Ca(2+) release (CICR). The molecular events transmitting such signals to the ion gate of the channel are unknown. The S4-S5 linker, a cytosolic loop connecting the S4 and S5 transmembrane segments in six-transmembrane type channels, forms an α-helical structure and mediates signal transmission in a wide variety of channels. To address the role of the S4-S5 linker in RyR1 channel gating, we performed alanine substitution scan of N-terminal half of the putative S4-S5 linker (Thr(4825)-Ser(4829)) that exhibits high helix probability. The mutant RyR1 was expressed in HEK cells, and CICR activity was investigated by caffeine-induced Ca(2+) release, single-channel current recordings, and [(3)H]ryanodine binding. Four mutants (T4825A, I4826A, S4828A, and S4829A) had reduced CICR activity without changing Ca(2+) sensitivity, whereas the L4827A mutant formed a constitutive active channel. T4825I, a disease-associated mutation for malignant hyperthermia, exhibited enhanced CICR activity. An α-helical wheel representation of the N-terminal S4-S5 linker provides a rational explanation to the observed activities of the mutants. These results suggest that N-terminal half of the S4-S5 linker may form an α-helical structure and play an important role in RyR1 channel gating.

Behavior of Ca(2+) waves in multicellular preparations from guinea pig ventricle.

Ca(+) waves have been implicated in Ca(2+) overload-induced cardiac arrhythmias. To deepen understanding of the behavior of Ca(2+) waves in a multicellular system, consecutive two-dimensional Ca(2+) images were obtained with a confocal microscope from surface cells of guinea pig ventricular papillary muscles loaded with fluo 3 or rhod 2. In intact muscles, no Ca(2+) waves were detected under the resting condition, whereas they were frequently observed during the rest immediately after high-frequency stimulations where cytoplasmic Ca(2+) concentration and Ca(2+) stored in the sarcoplasmic reticulum (SR) were gradually decreasing. The intervals of Ca(2+) waves increased as they occurred later, their amplitudes and velocities remaining unchanged. A SERCA inhibitor reversibly prolonged the wave intervals. In Na(+)-free/Ca(2+)-free medium where neither Ca(2+) influx nor Na(+)/Ca(2+) exchange took place, recurrent Ca(2+) waves emerged at constant intervals in each cell. These results are consistent with the conclusion that the loading level of the SR is critical for induction of Ca(2+) waves. Each cell independently exhibited its own regular rhythm of Ca(2+) wave with a distinct interval. These waves propagated in either direction along the longitudinal axis within a muscle cell, but seldom beyond the cell boundary. In contrast, in partially damaged muscles that showed spontaneous Ca(2+) waves at rest in normal Krebs solution, their propagation often was unidirectional, decreasing in frequency. In these cases, however, Ca(2+) waves rarely moved beyond the cellular boundary. The gradient of the cytoplasmic Ca(2+) concentration was suggested to be the cause of the one-way propagation.

Ryanodine receptor isoforms of non-Mammalian skeletal muscle.

Whereas mammalian skeletal muscles express primarily a single isoform of ryanodine receptor (RyR) as the Ca2+ releasing channel, many non-mammalian vertebrate skeletal muscles express two isoforms in almost similar amount, alpha- and beta-RyR which are homologues of mammalian isoforms RyR1 and 3, respectively. alpha-RyR is believed to be directly involved in excitation-contraction coupling in skeletal muscles and is variable in its properties among animals and fibers, while beta-RyR shows similar properties and is variable in its content. alpha- and beta-RyR purified from frog skeletal muscle, a favorite material for physiological and morphological experiments, are very similar in Ca2+ dependent [3H]ryanodine binding. On the SR membrane, however, alpha-RyR is selectively suppressed in the ligand binding, indicating that the Ca2+-induced Ca2+ release (CICR) activity in skeletal muscle is conducted primarily by beta-RyR. We also stressed here that Ca2+ binding to the activating site is a necessary but not a sufficient condition for CICR. The maximum activity attainable under a specified condition is also a critical parameter to be determined. Taking these findings into consideration, we conclude that CICR is too slow to explain the physiological Ca2+ release on depolarization.