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.

Efficient High-Throughput Screening by Endoplasmic Reticulum Ca2+ Measurement to Identify Inhibitors of Ryanodine Receptor Ca2+-Release Channels.

Genetic mutations in ryanodine receptors (RyRs), Ca2+-release channels in the sarcoplasmic reticulum essential for muscle contractions, cause various skeletal muscle and cardiac diseases. Because the main underlying mechanism of the pathogenesis is overactive Ca2+ release by gain-of-function of the RyR channel, inhibition of RyRs is expected to be a promising treatment of these diseases. Here, to identify inhibitors specific to skeletal muscle type 1 RyR (RyR1), we developed a novel high-throughput screening (HTS) platform using time-lapse fluorescence measurement of Ca2+ concentrations in the endoplasmic reticulum (ER) ([Ca2+]ER). Because expression of RyR1 carrying disease-associated mutation reduces [Ca2+]ER in HEK293 cells through Ca2+ leakage from RyR1 channels, specific drugs that inhibit RyR1 will increase [Ca2+]ER by preventing such Ca2+ leakage. RyR1 carrying the R2163C mutation and R-CEPIA1er, a genetically encoded ER Ca2+ indicator, were stably expressed in HEK293 cells, and time-lapse fluorescence was measured using a fluorometer. False positives were effectively excluded by using cells expressing wild-type (WT) RyR1. By screening 1535 compounds in a library of well characterized drugs, we successfully identified four compounds that significantly increased [Ca2+]ER They include dantrolene, a known RyR1 inhibitor, and three structurally different compounds: oxolinic acid, 9-aminoacridine, and alexidine. All the hit compounds, except for oxolinic acid, inhibited [3H]ryanodine binding of WT and mutant RyR1. Interestingly, they showed different dose dependencies and isoform specificities. The highly quantitative nature and good correlation with the channel activity validated this HTS platform by [Ca2+]ER measurement to explore drugs for RyR-related diseases.

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.

Nitric oxide-induced calcium release via ryanodine receptors regulates neuronal function.

Mobilization of intracellular Ca(2+) stores regulates a multitude of cellular functions, but the role of intracellular Ca(2+) release via the ryanodine receptor (RyR) in the brain remains incompletely understood. We found that nitric oxide (NO) directly activates RyRs, which induce Ca(2+) release from intracellular stores of central neurons, and thereby promote prolonged Ca(2+) signalling in the brain. Reversible S-nitrosylation of type 1 RyR (RyR1) triggers this Ca(2+) release. NO-induced Ca(2+) release (NICR) is evoked by type 1 NO synthase-dependent NO production during neural firing, and is essential for cerebellar synaptic plasticity. NO production has also been implicated in pathological conditions including ischaemic brain injury, and our results suggest that NICR is involved in NO-induced neuronal cell death. These findings suggest that NICR via RyR1 plays a regulatory role in the physiological and pathophysiological functions of the brain.

Dual role of the S5 segment in type 1 ryanodine receptor channel gating.

The type 1 ryanodine receptor (RyR1) is a Ca2+ release channel in the sarcoplasmic reticulum that is essential for skeletal muscle contraction. RyR1 forms a channel with six transmembrane segments, in which S5 is the fifth segment and is thought to contribute to pore formation. However, its role in channel gating remains unclear. Here, we performed a functional analysis of several disease-associated mutations in S5 and interpreted the results with respect to the published RyR1 structures to identify potential interactions associated with the mutant phenotypes. We demonstrate that S5 plays a dual role in channel gating: the cytoplasmic side interacts with S6 to reduce the channel activity, whereas the luminal side forms a rigid structural base necessary for S6 displacement in channel opening. These results deepen our understanding of the molecular mechanisms of RyR1 channel gating and provide insight into the divergent disease phenotypes caused by mutations in S5.

Overexpression of ryanodine receptor type 1 enhances mitochondrial fragmentation and Ca2+-induced ATP production in cardiac H9c2 myoblasts.

Ca(+) influx to mitochondria is an important trigger for both mitochondrial dynamics and ATP generation in various cell types, including cardiac cells. Mitochondrial Ca(2+) influx is mainly mediated by the mitochondrial Ca(2+) uniporter (MCU). Growing evidence also indicates that mitochondrial Ca(2+) influx mechanisms are regulated not solely by MCU but also by multiple channels/transporters. We have previously reported that skeletal muscle-type ryanodine receptor (RyR) type 1 (RyR1), which expressed at the mitochondrial inner membrane, serves as an additional Ca(2+) uptake pathway in cardiomyocytes. However, it is still unclear which mitochondrial Ca(2+) influx mechanism is the dominant regulator of mitochondrial morphology/dynamics and energetics in cardiomyocytes. To investigate the role of mitochondrial RyR1 in the regulation of mitochondrial morphology/function in cardiac cells, RyR1 was transiently or stably overexpressed in cardiac H9c2 myoblasts. We found that overexpressed RyR1 was partially localized in mitochondria as observed using both immunoblots of mitochondrial fractionation and confocal microscopy, whereas RyR2, the main RyR isoform in the cardiac sarcoplasmic reticulum, did not show any expression at mitochondria. Interestingly, overexpression of RyR1 but not MCU or RyR2 resulted in mitochondrial fragmentation. These fragmented mitochondria showed bigger and sustained mitochondrial Ca(2+) transients compared with basal tubular mitochondria. In addition, RyR1-overexpressing cells had a higher mitochondrial ATP concentration under basal conditions and showed more ATP production in response to cytosolic Ca(2+) elevation compared with nontransfected cells as observed by a matrix-targeted ATP biosensor. These results indicate that RyR1 possesses a mitochondrial targeting/retention signal and modulates mitochondrial morphology and Ca(2+)-induced ATP production in cardiac H9c2 myoblasts.

Prediction and structural equation model of sertraline treatment response in Japanese patients with major depressive disorder.

OBJECTIVE: The aim of this study was to extract the factors possibly associated with sertraline treatment response and elucidate their interactions and extent of influence. METHODS: Demographic state, stress state, personality, and eight genetic polymorphisms at baseline and clinical symptoms at baseline and 8 weeks were analyzed and examined by logistic regression and a structural equation model in sertraline treatment study of 96 Japanese patients with major depressive disorder. RESULTS: Non-responders were associated with higher scores of harm avoidance in Temperament and Character Inventory, higher scores (≥24) of 17-item Hamilton Rating Scale for Depression at baseline, recurrence, and 12/12 genotype of the serotonin transporter variable number of tandem repeat polymorphism in intron 2 (5HTTSTin2). When we calculated the response index using four factors extracted, the mean response index value of non-responders was significantly higher than that of responders. The symptoms at baseline, personality, recurrence, and polymorphism of 5HTTSTin2 showed significantly direct and positive influences on the symptoms at 8 weeks in our final structural equation model with a good model fit. CONCLUSION: Considering the combination of four factors extracted may be useful for predicting a worse response to sertraline treatment and selecting different treatment other than sertraline.

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.

MAOA, MTHFR, and TNF-ß genes polymorphisms and personality traits in the pathogenesis of migraine.

Migraine is a multifactorial disease with various factors, such as genetic polymorphisms and personality traits, but the contribution of those factors is not clear. To clarify the pathogenesis of migraine, the contributions of genetic polymorphisms and personality traits were simultaneously investigated using multivariate analysis. Ninety-one migraine patients and 119 non-headache healthy volunteers were enrolled. The 12 gene polymorphisms analysis and NEO-FFI personality test were performed. At first, the univariate analysis was performed to extract the contributing factors to pathogenesis of migraine. We then extracted the factors that independently contributed to the pathogenesis of migraine using multivariate stepwise logistic regression analysis. Using the multivariate analysis, three gene polymorphisms including monoamine oxidase A (MAOA) T941G, methylenetetrahydrofolate reductase (MTHFR) C677T, and tumor necrosis factor beta (TNF-ß) G252Α, and the neuroticism and conscientiousness scores in NEO-FFI were selected as significant factors that independently contributed to the pathogenesis of migraine. Their odds ratios were 1.099 (per point of neuroticism score), 1.080 (per point of conscientiousness score), 2.272 (T and T/T or T/G vs G and G/G genotype of MAOA), 1.939 (C/T or T/T vs C/C genotype of MTHFR), and 2.748 (G/A or A/A vs G/G genotype of TNF-ß), respectively. We suggested that multiple factors, such as gene polymorphisms and personality traits, contribute to the pathogenesis of migraine. The contribution of polymorphisms, such as MAOA T941G, MTHFR C677T, and TNF-ß G252A, were more important than personality traits in the pathogenesis of migraine, a multifactorial disorder.

Serum cystatin C and CRP are early predictive biomarkers for emergence of hypoxia in COVID-19.

BACKGROUND: In Japan, during the coronavirus disease 2019 (COVID-19) pandemic, patients with non-hypoxia are recommended to recuperate at home or in pre-hospital facilities. However, it was observed that unexpected hypoxia may occur and become severe subsequently in patients whose symptoms were initially expected to improve naturally. The aim of this study is to validate biomarkers that can predict at an early stage the emergence of hypoxia in COVID-19 patients without hypoxia. METHODS: We retrospectively enrolled 193 patients with COVID-19, excluding patients with hypoxia and severe disease from the onset. Participants were classified into two groups according to the emergence of hypoxia during the clinical course, and the laboratory data were compared to identify biomarkers that could predict early the emergence of hypoxia. RESULTS: The areas under the curve for serum cystatin C (CysC) and C-reactive protein (CRP) levels for the emergence of hypoxia during the clinical course were higher than those for other biomarkers (CysC, 0.84 and CRP, 0.83). Multivariate analysis showed that high serum CysC and CRP levels were associated with the emergence of hypoxia during the clinical course. CONCLUSIONS: Elevated serum CysC and CRP levels were associated with the emergence of hypoxia during the clinical course in COVID-19 patients without hypoxia. These findings may help determine the need for hospitalization in initially non-hypoxic COVID-19 patients.