An inherited life-threatening arrhythmia model established by screening randomly mutagenized mice.

Inherited arrhythmia syndromes (IASs) can cause life-threatening arrhythmias and are responsible for a significant proportion of sudden cardiac deaths (SCDs). Despite progress in the development of devices to prevent SCDs, the precise molecular mechanisms that induce detrimental arrhythmias remain to be fully investigated, and more effective therapies are desirable. In the present study, we screened a large-scale randomly mutagenized mouse library by electrocardiography to establish a disease model of IASs and consequently found one pedigree that exhibited spontaneous ventricular arrhythmias (VAs) followed by SCD within 1 y after birth. Genetic analysis successfully revealed a missense mutation (p.I4093V) of the ryanodine receptor 2 gene to be a cause of the arrhythmia. We found an age-related increase in arrhythmia frequency accompanied by cardiomegaly and decreased ventricular contractility in the Ryr2I4093V/+ mice. Ca2+ signaling analysis and a ryanodine binding assay indicated that the mutant ryanodine receptor 2 had a gain-of-function phenotype and enhanced Ca2+ sensitivity. Using this model, we detected the significant suppression of VA following flecainide or dantrolene treatment. Collectively, we established an inherited life-threatening arrhythmia mouse model from an electrocardiogram-based screen of randomly mutagenized mice. The present IAS model may prove feasible for use in investigating the mechanisms of SCD and assessing therapies.

Molecular mechanism of the severe MH/CCD mutation Y522S in skeletal ryanodine receptor (RyR1) by cryo-EM.

Ryanodine receptors (RyRs) are main regulators of intracellular Ca2+ release and muscle contraction. The Y522S mutation of RyR1 causes central core disease, a weakening myopathy, and malignant hyperthermia, a sudden and potentially fatal response to anesthetics or heat. Y522 is in the core of the N-terminal subdomain C of RyR1 and the mechanism of how this mutation orchestrates malfunction is unpredictable for this 2-MDa ion channel, which has four identical subunits composed of 15 distinct cytoplasmic domains each. We expressed and purified the RyR1 rabbit homolog, Y523S, from HEK293 cells and reconstituted it in nanodiscs under closed and open states. The high-resolution cryogenic electron microscopic (cryo-EM) three-dimensional (3D) structures show that the phenyl ring of Tyr functions in a manner analogous to a "spacer" within an α-helical bundle. Mutation to the much smaller Ser alters the hydrophobic network within the bundle, triggering rearrangement of its α-helices with repercussions in the orientation of most cytoplasmic domains. Examining the mutation-induced readjustments exposed a series of connected α-helices acting as an ∼100 Å-long lever: One end protrudes toward the dihydropyridine receptor, its molecular activator (akin to an antenna), while the other end reaches the Ca2+ activation site. The Y523S mutation elicits channel preactivation in the absence of any activator and full opening at 1.5 µM free Ca2+, increasing by ∼20-fold the potency of Ca2+ to activate the channel compared with RyR1 wild type (WT). This study identified a preactivated pathological state of RyR1 and a long-range lever that may work as a molecular switch to open the channel.

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.

Discovery and Structure-Activity Relationship of a Ryanodine Receptor 2 Inhibitor.

Ryanodine receptor 2 (RyR2) is a large Ca2+-release channel in the sarcoplasmic reticulum (SR) of cardiac muscle cells. It serves to release Ca2+ from the SR into the cytosol to initiate muscle contraction. RyR2 overactivation is associated with arrhythmogenic cardiac disease, but few specific inhibitors have been reported so far. Here, we identified an RyR2-selective inhibitor 1 from the chemical compound library and synthesized it from glycolic acid. Synthesis of various derivatives to investigate the structure-activity relationship of each substructure afforded another two RyR2-selective inhibitors 6 and 7, among which 6 was the most potent. Notably, compound 6 also inhibited Ca2+ release in cells expressing the RyR2 mutants R2474S, R4497C and K4750Q, which are associated with cardiac arrhythmias such as catecholaminergic polymorphic ventricular tachycardia (CPVT). This inhibitor is expected to be a useful tool for research on the structure and dynamics of RyR2, as well as a lead compound for the development of drug candidates to treat RyR2-related cardiac disease.

Screening for Novel Type 2 Ryanodine Receptor Inhibitors by Endoplasmic Reticulum Ca2+ Monitoring.

Type 2 ryanodine receptor (RyR2) is a Ca2+ release channel on the endoplasmic (ER)/sarcoplasmic reticulum that plays a central role in the excitation-contraction coupling in the heart. Hyperactivity of RyR2 has been linked to ventricular arrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia and heart failure, where spontaneous Ca2+ release via hyperactivated RyR2 depolarizes diastolic membrane potential to induce triggered activity. In such cases, drugs that suppress RyR2 activity are expected to prevent the arrhythmias, but there is no clinically available RyR2 inhibitors at present. In this study, we searched for RyR2 inhibitors from a well-characterized compound library using a recently developed ER Ca2+-based assay, where the inhibition of RyR2 activity was detected by the increase in ER Ca2+ signals from R-CEPIA1er, a genetically encoded ER Ca2+ indicator, in RyR2-expressing HEK293 cells. By screening 1535 compounds in the library, we identified three compounds (chloroxylenol, methyl orsellinate, and riluzole) that greatly increased the ER Ca2+ signal. All of the three compounds suppressed spontaneous Ca2+ oscillations in RyR2-expressing HEK293 cells and correspondingly reduced the Ca2+-dependent [3H]ryanodine binding activity. In cardiomyocytes from RyR2-mutant mice, the three compounds effectively suppressed abnormal Ca2+ waves without substantial effects on the action-potential-induced Ca2+ transients. These results confirm that ER Ca2+-based screening is useful for identifying modulators of ER Ca2+ release channels and suggest that RyR2 inhibitors have potential to be developed as a new category of antiarrhythmic drugs. SIGNIFICANCE STATEMENT: We successfully identified three compounds having RyR2 inhibitory action from a well-characterized compound library using an endoplasmic reticulum Ca2+-based assay, and demonstrated that these compounds suppressed arrhythmogenic Ca2+ wave generation without substantially affecting physiological action-potential induced Ca2+ transients in cardiomyocytes. This study will facilitate the development of RyR2-specific inhibitors as a potential new class of drugs for life-threatening arrhythmias induced by hyperactivation of RyR2.

Differential effects of the formin inhibitor SMIFH2 on contractility and Ca2+ handling in frog and mouse cardiomyocytes.

Genetic mutations in actin regulators have been emerging as a cause of cardiomyopathy, although the functional link between actin dynamics and cardiac contraction remains largely unknown. To obtain insight into this issue, we examined the effects of pharmacological inhibition of formins, a major class of actin-assembling proteins. The formin inhibitor SMIFH2 significantly enhanced the cardiac contractility of isolated frog hearts, thereby augmenting cardiac performance. SMIFH2 treatment had no significant effects on the Ca2+ sensitivity of frog muscle fibers. Instead, it unexpectedly increased Ca2+ concentrations of isolated frog cardiomyocytes, suggesting that the inotropic effect is due to enhanced Ca2+ transients. In contrast to frog hearts, the contractility of mouse cardiomyocytes was attenuated by SMIFH2 treatment with decreasing Ca2+ transients. Thus, SMIFH2 has opposing effects on the Ca2+ transient and contractility between frog and mouse cardiomyocytes. We further found that SMIFH2 suppressed Ca2+ -release via type 2 ryanodine receptor (RyR2); this inhibitory effect may explain the species differences, since RyR2 is critical for Ca2+ transients in mouse myocardium but absent in frog myocardium. Although the mechanisms underlying the enhancement of Ca2+ transients in frog cardiomyocytes remain unclear, SMIFH2 differentially affects the cardiac contraction of amphibian and mammalian by differentially modulating their Ca2+ handling.

Structural basis for diamide modulation of ryanodine receptor.

The diamide insecticide class is one of the top-selling insecticides globally. They are used to control a wide range of pests by targeting their ryanodine receptors (RyRs). Here, we report the highest-resolution cryo-electron microscopy (cryo-EM) structure of RyR1 in the open state, in complex with the anthranilic diamide chlorantraniliprole (CHL). The 3.2-Å local resolution map facilitates unambiguous assignment of the CHL binding site. The molecule induces a conformational change by affecting the S4-S5 linker, triggering channel opening. The binding site is further corroborated by mutagenesis data, which reveal how diamide insecticides are selective to the Lepidoptera group of insects over honeybee or mammalian RyRs. Our data reveal that several pests have developed resistance via two mechanisms, steric hindrance and loss of contact. Our results provide a foundation for the development of highly selective pesticides aimed at overcoming resistance and therapeutic molecules to treat human myopathies.

TRIC-A Channel Maintains Store Calcium Handling by Interacting With Type 2 Ryanodine Receptor in Cardiac Muscle.

RATIONALE: Trimeric intracellular cation (TRIC)-A and B are distributed to endoplasmic reticulum/sarcoplasmic reticulum intracellular Ca2+ stores. The crystal structure of TRIC has been determined, confirming the homotrimeric structure of a potassium channel. While the pore architectures of TRIC-A and TRIC-B are conserved, the carboxyl-terminal tail (CTT) domains of TRIC-A and TRIC-B are different from each other. Aside from its recognized role as a counterion channel that participates in excitation-contraction coupling of striated muscles, the physiological function of TRIC-A in heart physiology and disease has remained largely unexplored. OBJECTIVE: In cardiomyocytes, spontaneous Ca2+ waves, triggered by store overload-induced Ca2+ release mediated by the RyR2 (type 2 ryanodine receptor), develop extrasystolic contractions often associated with arrhythmic events. Here, we test the hypothesis that TRIC-A is a physiological component of RyR2-mediated Ca2+ release machinery that directly modulates store overload-induced Ca2+ release activity via CTT. METHODS AND RESULTS: We show that cardiomyocytes derived from the TRIC-A-/- (TRIC-A knockout) mice display dysregulated Ca2+ movement across sarcoplasmic reticulum. Biochemical studies demonstrate a direct interaction between CTT-A and RyR2. Modeling and docking studies reveal potential sites on RyR2 that show differential interactions with CTT-A and CTT-B. In HEK293 (human embryonic kidney) cells with stable expression of RyR2, transient expression of TRIC-A, but not TRIC-B, leads to apparent suppression of spontaneous Ca2+ oscillations. Ca2+ measurements using the cytosolic indicator Fura-2 and the endoplasmic reticulum luminal store indicator D1ER suggest that TRIC-A enhances Ca2+ leak across the endoplasmic reticulum by directly targeting RyR2 to modulate store overload-induced Ca2+ release. Moreover, synthetic CTT-A peptide facilitates RyR2 activity in lipid bilayer reconstitution system, enhances Ca2+ sparks in permeabilized TRIC-A-/- cardiomyocytes, and induces intracellular Ca2+ release after microinjection into isolated cardiomyocytes, whereas such effects were not observed with the CTT-B peptide. In response to isoproterenol stimulation, the TRIC-A-/- mice display irregular ECG and develop more fibrosis than the WT (wild type) littermates. CONCLUSIONS: In addition to the ion-conducting function, TRIC-A functions as an accessory protein of RyR2 to modulate sarcoplasmic reticulum Ca2+ handling in cardiac muscle.

Loss-of-function mutations in cardiac ryanodine receptor channel cause various types of arrhythmias including long QT syndrome.

AIMS: Gain-of-function mutations in RYR2, encoding the cardiac ryanodine receptor channel (RyR2), cause catecholaminergic polymorphic ventricular tachycardia (CPVT). Whereas, genotype-phenotype correlations of loss-of-function mutations remains unknown, due to a small number of analysed mutations. In this study, we aimed to investigate their genotype-phenotype correlations in patients with loss-of-function RYR2 mutations. METHODS AND RESULTS: We performed targeted gene sequencing for 710 probands younger than 16-year-old with inherited primary arrhythmia syndromes (IPAS). RYR2 mutations were identified in 63 probands, and 3 probands displayed clinical features different from CPVT. A proband with p.E4146D developed ventricular fibrillation (VF) and QT prolongation whereas that with p.S4168P showed QT prolongation and bradycardia. Another proband with p.S4938F showed short-coupled variant of torsade de pointes (scTdP). To evaluate the functional alterations in these three mutant RyR2s and p.K4594Q previously reported in a long QT syndrome (LQTS), we measured Ca2+ signals in HEK293 cells and HL-1 cardiomyocytes as well as Ca2+-dependent [3H]ryanodine binding. All mutant RyR2s demonstrated a reduced Ca2+ release, an increased endoplasmic reticulum Ca2+, and a reduced [3H]ryanodine binding, indicating loss-of-functions. In HL-1 cells, the exogenous expression of S4168P and K4594Q reduced amplitude of Ca2+ transients without inducing Ca2+ waves, whereas that of E4146D and S4938F evoked frequent localized Ca2+ waves. CONCLUSION: Loss-of-function RYR2 mutations may be implicated in various types of arrhythmias including LQTS, VF, and scTdP, depending on alteration of the channel activity. Search of RYR2 mutations in IPAS patients clinically different from CPVT will be a useful strategy to effectively discover loss-of-function RYR2 mutations.

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.

Regulatory mechanisms of ryanodine receptor/Ca2+ release channel revealed by recent advancements in structural studies.

Ryanodine receptors (RyRs) are huge homotetrameric Ca2+ release channels localized to the sarcoplasmic reticulum. RyRs are responsible for the release of Ca2+ from the SR during excitation-contraction coupling in striated muscle cells. Recent revolutionary advancements in cryo-electron microscopy have provided a number of near-atomic structures of RyRs, which have enabled us to better understand the architecture of RyRs. Thus, we are now in a new era understanding the gating, regulatory and disease-causing mechanisms of RyRs. Here we review recent advances in the elucidation of the structures of RyRs, especially RyR1 in skeletal muscle, and their mechanisms of regulation by small molecules, associated proteins and disease-causing mutations.

Sudden death after inappropriate shocks of implantable cardioverter defibrillator in a catecholaminergic polymorphic ventricular tachycardia case with a novel RyR2 mutation.

BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic syndrome and a cause of exercise-related sudden death. CPVT has been reported to be caused by gain of function underlying a mutation of cardiac ryanodine receptor (RyR2). METHODS: In a family with a CPVT patient, genomic DNA was extracted from peripheral blood lymphocytes, and the RyR2 gene underwent target gene sequence using MiSeq. The activity of wild-type (WT) and mutant RyR2 channel were evaluated by monitoring Ca2+ signals in HEK293 cells expressing WT and mutant RyR2. We investigated a role of a RyR2 mutation in the recent tertiary structure of RyR2. RESULTS: Though a 17-year-old man diagnosed as CPVT had implantable cardioverter defibrillator (ICD) and was going to undergo catheter ablation for the control of paroxysmal atrial fibrillation, he suddenly died at the age of twenty-one because of ventricular fibrillation which was spontaneously developed after maximum inappropriate ICD shocks against rapid atrial fibrillation. The genetic test revealed a de novo RyR2 mutation, Gln4936Lys in mosaicism which was located at the α-helix interface between U-motif and C-terminal domain. In the functional analysis, Ca2+ release from endoplasmic reticulum via the mutant RyR2 significantly increased than that from WT. CONCLUSION: A RyR2 mutation, Gln4936Lys, to be documented in a CPVT patient with exercise-induced ventricular tachycardias causes an excessive Ca2+ release from the sarcoplasmic reticulum which corresponded to clinical phenotypes of CPVT. The reduction of inappropriate shocks of ICD is essential to prevent unexpected sudden death in patients with CPVT.

The Ryanodine Receptor as a Sensor for Intracellular 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 the changes in the level of 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 changes in the intracellular environments in muscles.

Co-Phenotype of Left Ventricular Non-Compaction Cardiomyopathy and Atypical Catecholaminergic Polymorphic Ventricular Tachycardia in Association With R169Q, a Ryanodine Receptor Type 2 Missense Mutation.

BACKGROUND: Left ventricular non-compaction (LVNC) is a cardiomyopathy characterized by prominent trabeculae and intertrabecular recesses. We present the cases of 3 girls with the sameryanodine receptor type 2(RYR2) mutation who had phenotypes of both catecholaminergic polymorphic ventricular tachycardia (CPVT) and LVNC .Methods and Results:Clinical characteristics and genetic background of the 3 patients were analyzed retrospectively. Age at onset was 5, 6, and 7 years, respectively. Clinical presentation included syncope during exercise in all 3 patients and cardiac arrest in 2 patients. LVNC diagnosis was confirmed on echocardiography according to previously defined criteria. Exercise stress testing provoked ventricular arrhythmia in two of the patients. Beta-blockers (n=3) and flecainide (n=2) were given, and an implantable cardioverter defibrillator was used in 1 patient. Genotyping identified the sameRYR2-R169Q missense mutation and no other CPVT- or LVNC-related gene mutations. Functional analysis of the mutation using HEK293 cells with single-cell Ca2+imaging and [3H]ryanodine binding analysis, indicated a gain of function: a reduced threshold for overload-induced Ca2+release from the sarcoplasmic reticulum and increased fractional Ca2+release. CONCLUSIONS: The rare association of LVNC and CPVT phenotypes withRYR2mutations is less likely to be coincidental. Screening for life-threatening arrhythmias using exercise or pharmacologic stress tests is recommended in LVNC patients to prevent sudden cardiac death in those with preserved LV function.

Effects of intravesicular loading of a Ca2+ chelator and depolymerization of actin fibers on neurotransmitter release in frog motor nerve terminals.

Ca2+ -induced Ca2+ release (CICR) via type-3 ryanodine receptor enhances neurotransmitter release in frog motor nerve terminals. To test a possible role of synaptic vesicle in CICR, we examined the effects of loading of EGTA, a Ca2+ chelator, into synaptic vesicles and depolymerization of actin fibers. Intravesicular EGTA loading via endocytosis inhibited the ryanodine sensitive enhancement of transmitter release induced by tetanic stimulation and the associated rises in intracellular-free Ca2+ ([Ca2+ ]i : Ca2+ transients). Latrunculin A, a depolymerizer of actin fibers, enhanced both spontaneous and stimulation-induced transmitter release, but inhibited the enhancement of transmitter release elicited by successive tetanic stimulation. The results suggest a possibility that the activation of CICR from mobilized synaptic vesicles caused the enhancement of neurotransmitter release.

Conversion of Ca2+ oscillation into propagative electrical signals by Ca2+-activated ion channels and connexin as a reconstituted Ca2+ clock model for the pacemaker activity.

Conversion of intracellular Ca2+ signals to electrical activity results in multiple and differing physiological impacts depending on cell types. In some organs such as gastrointestinal and urinary systems, spontaneous Ca2+ oscillation in pacermaker cells can function essentially as a Ca2+ clock mechanism, which has been originally found in pacemaking in sinoatrial node cell of the heart. The conversion of discrete Ca2+ clock events to spontaneous electrical activity is an essential step for the initiation and propagation of pacemaker activity through the multicellular organs resulting in synchronized physiological functions. Here, a model of intracellular signal transduction from a Ca2+ oscillation to initiation of electrical slow waves and their propagation were reconstituted in HEK293 cells. This was accomplished based on ryanodine receptor (RyR) type 3, Ca2+-activated ion channels, i.e. small conductance Ca2+-activated K+ channel (SK2) or Ca2+-activated Cl- channel (TMEM16A), and connexin43 being heterologously co-expressed. The propagation of electrical waves was abolished or substantially reduced by treatment with selective blockers of the expressed channels and 18ß-glycyrrhetinic acid, a gap junction inhibitor, respectively. Thus, we demonstrated that the conversion of Ca2+ oscillation to electrical signals with cell to cell propagation can be reconstituted as a model of Ca2+ clock pacemaker activity by combinational expression of critical elements in heterologous expression system.

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.

Changes in serum interleukin-6 levels as possible predictor of efficacy of tocilizumab treatment in rheumatoid arthritis.

OBJECTIVES: We aimed to evaluate the association between the change in serum IL-6 during the clinical course of tocilizumab (TCZ) therapy and rheumatoid arthritis (RA) disease activity or occurrence of adverse events. METHODS: General laboratory data including serum IL-6 levels and physical findings were obtained every 4 weeks, and, in addition, at the time when any adverse events occurred. RESULTS: The proportion achieving Clinical Disease Activity Index (CDAI) remission at 52 weeks was significantly lower in 20 patients with serum IL-6 ≥ 30 pg/ml at 12 weeks than 24 patients with serum IL-6 < 30 pg/ml. In 17 patients with serum IL-6 ≥ 30 pg/ml at 24 weeks, the proportion achieving CDAI remission was also significantly lower than 27 patients with serum IL-6 < 30 pg/ml then. In these 17 patients, Disease Activity Score (DAS) 28-ESR and CDAI at 52 weeks were significantly higher than those with serum IL-6 < 30 pg/ml. Age- and sex-adjusted logistic regression analysis showed logIL-6 at 12 weeks to be a predictive factor for DAS28-ESR remission at 52 weeks. CONCLUSION: Serum IL-6 levels from 12 to 24 weeks after TCZ initiation better reflect the efficacy of TCZ at 52 weeks.

[Rapid exacerbation of thrombotic microangiopathy accompanied by electrocardiogram abnormality leading to death].

A 73-year-old female was hospitalized with thrombotic microangiopathy (TMA) diagnosis because of consciousness disturbance, anemia, thrombocytopenia, renal dysfunction, and electrocardiogram abnormality. The patient died on day 12 of the symptom onset. The immunohistochemical analysis of microclot found in the autopsy of coronary artery confirmed TMA. It was suggested that the relationship to collagen disease by antinuclear antibody positive and the necessity of initiating circulation management and plasma exchange immediately before approximately 1×104 of platelets for the prognosis. The findings suggested considering TMA at the time of an unidentified shock, particularly acute adrenal insufficiency.

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.