Structural and functional interactions between the EF hand domain and S2-S3 loop in the type-1 ryanodine receptor ion channel.

Previous cryo-electron micrographs suggested that the skeletal muscle Ca2+ release channel, ryanodine receptor (RyR)1, is regulated by intricate interactions between the EF hand Ca2+ binding domain and the cytosolic loop (S2-S3 loop). However, the precise molecular details of these interactions and functional consequences of the interactions remain elusive. Here, we used molecular dynamics simulations to explore the specific amino acid pairs involved in hydrogen bond interactions within the EF hand-S2-S3 loop interface. Our simulations unveiled two key interactions: (1) K4101 (EF hand) with D4730 (S2-S3 loop) and (2) E4075, Q4078, and D4079 (EF hand) with R4736 (S2-S3 loop). To probe the functional significance of these interactions, we constructed mutant RyR1 complementary DNAs and expressed them in HEK293 cells for [3H]ryanodine binding assays. Our results demonstrated that mutations in the EF hand, specifically K4101E and K4101M, resulted in reduced affinities for Ca2+/Mg2+-dependent inhibitions. Interestingly, the K4101E mutation increased the affinity for Ca2+-dependent activation. Conversely, mutations in the S2-S3 loop, D4730K and D4730N, did not significantly change the affinities for Ca2+/Mg2+-dependent inhibitions. Our previous finding that skeletal disease-associated RyR1 mutations, R4736Q and R4736W, impaired Ca2+-dependent inhibition, is consistent with the current results. In silico mutagenesis analysis aligned with our functional data, indicating altered hydrogen bonding patterns upon mutations. Taken together, our findings emphasize the critical role of the EF hand-S2-S3 loop interaction in Ca2+/Mg2+-dependent inhibition of RyR1 and provide insights into potential therapeutic strategies targeting this domain interaction for the treatment of skeletal myopathies.

Vitamin D and calcium co-therapy mitigates pre-established cadmium nephropathy by regulating renal calcium homeostatic molecules and improving anti-oxidative and anti-inflammatory activities in rat.

BACKGROUND: Cadmium (Cd) is a major environmental pollutant and chronic toxicity could induce nephropathy by increasing renal oxidative stress and inflammation. Although vitamin D (VD) and calcium (Ca) prophylactic treatments attenuated Cd-induced cell injury, none of the prior studies measure their renoprotective effects against pre-established Cd-nephropathy. AIMS: To measure the alleviating effects of VD and/or Ca single and dual therapies against pre-established nephrotoxicity induced by chronic Cd toxicity prior to treatment initiation. METHODS: Forty male adult rats were allocated into: negative controls (NC), positive controls (PC), Ca, VD and VC groups. The study lasted for eight weeks and all animals, except the NC, received CdCl2 in drinking water (44 mg/L) throughout the study. Ca (100 mg/kg) and/or VD (350 IU/kg) were given (five times/week) during the last four weeks to the designated groups. Subsequently, the expression of transforming growth factor-ß (TGF-ß1), inducible nitric oxide synthase (iNOS), neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), VD synthesising (Cyp27b1) and catabolizing (Cyp24a1) enzymes with VD receptor (VDR) and binding protein (VDBP) was measured in renal tissues. Similarly, renal expression of Ca voltage-dependent channels (CaV1.1/CaV3.1), store-operated channels (RyR1/ITPR1), and binding proteins (CAM/CAMKIIA/S100A1/S100B) were measured. Serum markers of renal function alongside several markers of oxidative stress (MDA/H2O2/GSH/GPx/CAT) and inflammation (IL-6/TNF-α/IL-10) together with renal cell apoptosis and expression of caspase-3 were also measured. RESULTS: The PC group exhibited hypovitaminosis D, hypocalcaemia, hypercalciuria, proteinuria, reduced creatinine clearance, and increased renal apoptosis/necrosis with higher caspase-3 expression. Markers of renal tissue damage (TGF-ß1/iNOS/NGAL/KIM-1), oxidative stress (MDA/H2O2), and inflammation (TNF-α/IL-1ß/IL-6) increased, whilst the antioxidants (GSH/GPx/CAT) and IL-10 decreased, in the PC group. The PC renal tissues also showed abnormal expression of Cyp27b1, Cyp24a1, VDR, and VDBP, alongside Ca-membranous (CaV1.1/CaV3.1) and store-operated channels (RyR1/ITPR1) and cytosolic Ca-binding proteins (CAM/CAMKIIA/S100A1/S100B). Although VD was superior to Ca monotherapy, their combination revealed the best mitigation effects by attenuating serum and renal tissue Cd concentrations, inflammation and oxidative stress, alongside modulating the expression of VD/Ca-molecules. CONCLUSIONS: This study is the first to show improved alleviations against Cd-nephropathy by co-supplementing VD and Ca, possibly by better regulation of Ca-dependent anti-oxidative and anti-inflammatory actions.

Synergistic FRET assays for drug discovery targeting RyR2 channels.

A key therapeutic target for heart failure and arrhythmia is the deleterious leak through sarcoplasmic reticulum (SR) ryanodine receptor 2 (RyR2) calcium release channels. We have previously developed methods to detect the pathologically leaky state of RyR2 in adult cardiomyocytes by monitoring RyR2 binding to either calmodulin (CaM) or a biosensor peptide (DPc10). Here, we test whether these complementary binding measurements are effective as high-throughput screening (HTS) assays to discover small molecules that target leaky RyR2. Using FRET, we developed and validated HTS procedures under conditions that mimic a pathological state, to screen the library of 1280 pharmaceutically active compounds (LOPAC) for modulators of RyR2 in cardiac SR membrane preparations. Complementary FRET assays with acceptor-labeled CaM and DPc10 were used for Hit prioritization based on the opposing binding properties of CaM vs. DPc10. This approach narrowed the Hit list to one compound, Ro 90-7501, which altered FRET to suggest increased RyR2-CaM binding and decreased DPc10 binding. Follow-up studies revealed that Ro 90-7501 does not detrimentally affect myocyte Ca2+ transients. Moreover, Ro 90-7501 partially inhibits overall Ca2+ leak, as assessed by Ca2+ sparks in permeabilized rat cardiomyocytes. Together, these results demonstrate (1) the effectiveness of our HTS approach where two complementary assays synergize for Hit ranking and (2) a drug discovery process that combines high-throughput, high-precision in vitro structural assays with in situ myocyte assays of the pathologic RyR2 leak. These provide a drug discovery platform compatible with large-scale HTS campaigns, to identify agents that inhibit RyR2 for therapeutic development.

Crosstalk between ERO1α and ryanodine receptor in arsenite-dependent mitochondrial ROS formation.

Arsenite, a well-established human carcinogen and toxic compound, promotes the formation of mitochondrial superoxide (mitoO2-) via a Ca2+-dependent mechanism, in which an initial stimulation of the inositol 1, 4, 5-trisphosphate receptor (IP3R) is followed by the activation of the ryanodine receptor (RyR), critical for providing Ca2+ to the mitochondria. We now report that, under the same conditions, arsenite triggers endoplasmic reticulum (ER) stress and a threefold increase in ER oxidoreductin 1α (ERO1 α) levels in proliferating U937 cells. EN460, an inhibitor of ERO1 α, recapitulated all the effects associated with RyR inhibition or downregulation, including prevention of RyR-induced Ca2+ accumulation in mitochondria and the resulting O2-. formation. Quantitatively similar results were obtained in inhibitor studies performed in terminally differentiated wild type C2C12 cells. Moreover, ERO1 α knockout C2C12 myotubes responded to arsenite as their wild type counterpart supplemented with EN460. As a final note, arsenite enhanced the expression of ERO1 α via a mechanism mediated by Ca2+ release from both the IP3R and RyR. We therefore conclude that arsenite activates a positive feedback amplification cycle between Ca2+ levels and ERO1 α in the ER, by which IP3R-dependent Ca2+ induces ERO1 α and ERO1 α promotes Ca2+ release via RyR, thereby amplifying the initial Ca2+ load and causing the mitochondrial accumulation of the cation, critical for mitoO2- formation.

Pachymic Acid Attenuated Doxorubicin-Induced Heart Failure by Suppressing miR-24 and Preserving Cardiac Junctophilin-2 in Rats.

Defects in cardiac contractility and heart failure (HF) are common following doxorubicin (DOX) administration. Different miRs play a role in HF, and their targeting was suggested as a promising therapy. We aimed to target miR-24, a suppressor upstream of junctophilin-2 (JP-2), which is required to affix the sarcoplasmic reticulum to T-tubules, and hence the release of Ca2+ in excitation-contraction coupling using pachymic acid (PA) and/or losartan (LN). HF was induced with DOX (3.5 mg/kg, i.p., six doses, twice weekly) in 24 rats. PA and LN (10 mg/kg, daily) were administered orally for four weeks starting the next day of the last DOX dose. Echocardiography, left ventricle (LV) biochemical and histological assessment and electron microscopy were conducted. DOX increased serum BNP, HW/TL, HW/BW, mitochondrial number/size and LV expression of miR-24 but decreased EF, cardiomyocyte fiber diameter, LV content of JP-2 and ryanodine receptors-2 (RyR2). Treatment with either PA or LN reversed these changes. Combined PA + LN attained better results than monotherapies. In conclusion, HF progression following DOX administration can be prevented or even delayed by targeting miR-24 and its downstream JP-2. Our results, therefore, suggest the possibility of using PA alone or as an adjuvant therapy with LN to attain better management of HF patients, especially those who developed tolerance toward LN.

Water-soluble alkaloids extracted from Aconiti Radix lateralis praeparata protect against chronic heart failure in rats via a calcium signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Many studies have shown the beneficial effects of aconite water-soluble alkaloid extract (AWA) in experimental models of heart disease, which have been ascribed to the presence of aconine, hypaconine, talatisamine, fuziline, neoline, and songorine. This study evaluated the effects of a chemically characterized AWA by chemical content, evaluated its effects in suprarenal abdominal aortic coarctation surgery (AAC)-induced chronic heart failure (CHF) in rats, and revealed the underlying mechanisms of action by proteomics. METHODS: Rats were distributed into different groups: sham, model, and AWA-treated groups (10, 20, and 40 mg/kg/day). Sham rats received surgery without AAC, whereas model rats an AWA-treated groups underwent AAC surgery. after 8 weeks, the treatment group was fed AWA for 4 weeks, and body weight was assessed weekly. At the end of the treatment, heart function was tested by echocardiography. AAC-induced chronic heart failure, including myocardial fibrosis, cardiomyocyte hypertrophy, and apoptosis, was evaluated in heart tissue and plasma by RT-qPCR, ELISA, hematoxylin and eosin (H&E) staining, Masson's trichrome staining, TUNEL staining, and immunofluorescence staining of α-SMA, Col Ⅰ, and Col Ⅲ. Then, a proteomics approach was used to explore the underlying mechanisms of action of AWA in chronic heart failure. RESULTS: AWA administration reduced body weight gain, myocardial fibrosis, cardiomyocyte hypertrophy, and apoptosis, and rats showed improvement in cardiac function compared to model group. The extract significantly ameliorated the AAC-induced altered expression of heart failure markers such as ANP, NT-proBNP, and ß-MHC, as well as fibrosis, hypertrophy markers MMP-2 and MMP-9, and other heart failure-related factors including plasma levels of TNF-α and IL-6. Furthermore, the extract reduced the protein expression of α-SMA, Col Ⅰ, and Col Ⅲ in the left ventricular (LV), thus inhibiting the LV remodeling associated with CHF. In addition, proteomics characterization of differentially expressed proteins showed that AWA administration inhibited left ventricular remodeling in CHF rats via a calcium signaling pathway, and reversed the expression of RyR2 and SERCA2a. CONCLUSIONS: AWA extract exerts beneficial effects in an AAC-induced CHF model in rats, which was associated with an improvement in LV function, hypertrophy, fibrosis, and apoptotic status. These effects may be related to the regulation of calcium signaling by the altered expression of RyR2 and SERCA2a.

Design, synthesis, and biological activity of novel semicarbazones as potent Ryanodine receptor1 inhibitors of Alzheimer's disease.

Ryanodine receptors (RyRs) are important ligand-gated Ca2+ channels; their excessive activation leads to Ca2+ leakage in the sarcoplasmic reticulum that may cause neurological diseases. In this study, three series of novel potent RyR1 inhibitors based on dantrolene and bearing semicarbazone and imidazolyl moieties were designed and synthesized, and their biological activity was evaluated. Using a single-cell calcium imaging method, the calcium overload inhibitory activities of 26 target compounds were tested in the R614C cell line, using dantrolene as a positive control. The preliminary investigation showed that compound 12a suppressed Ca2+ release as evidenced by store overload-induced Ca2+release (SOICR) (31.5 ± 0.1%, 77.2 ± 0.1%, 93.7 ± 0.2%) at 0.1 µM, 3 µM and 10 µM, respectively. Docking simulation results showed that compound 12a could bind at the active site of the RyR1 protein. The Morris water-maze test showed that compound 12a significantly improved the cognitive behavior of AD-model mice. Further studies on the structural optimization of this series of derivatives are currently underway in our laboratory.

Discovery of a polysubstituted phenyl containing novel N-phenylpyrazole scaffold as potent ryanodine receptor activator.

To develop the novel ryanodine receptors (RyRs) insecticides, encouraged by our previous research work, a series of novel N-phenylpyrazole derivatives containing a polysubstituted phenyl ring scaffold were designed and synthesized. The bioassays results indicated that some title compounds exhibited excellent insecticidal activity. For oriental armyworm (Mythimna separata), compounds 7f, 7g, 7i and 7o at 0.5 mg L-1 displayed 100% larvicidal activity, and even at 0.1 mg L-1, 7o was 30% larvicidal activity, comparable to chlorantraniliprole (30%) and better than cyantraniliprole (10%). Compounds 7f and 7o had the median lethal concentrations (LC50) of 8.83 × 10-2 and 7.12 × 10-2 mg L-1, respectively, close to chlorantraniliprole (6.79 × 10-2 mg L-1). Additionally, for diamondback moth (Plutella xylostella), the larvicidal activity of compounds 7f and 7i were 90% and 70% at 0.01 mg L-1, respectively, better than chlorantraniliprole (50%) and cyantraniliprole (40%). More impressively, the LC50 value of 7f was 4.2 × 10-3 mg L-1, slightly lower than that of chlorantraniliprole (5.0 × 10-3 mg L-1). The molecular docking between compound 7f and RyRs of diamondback moth validated our molecular designation. Furthermore, the calcium imaging experiment explored the influence of compound 7o on the calcium homeostasis in the central neurons of the third larvae of oriental armyworm. The results of this study indicated that 7o is a potent novel lead targeting at RyRs.

Nitro-fatty acids suppress ischemic ventricular arrhythmias by preserving calcium homeostasis.

Nitro-fatty acids are electrophilic anti-inflammatory mediators which are generated during myocardial ischemic injury. Whether these species exert anti-arrhythmic effects in the acute phase of myocardial ischemia has not been investigated so far. Herein, we demonstrate that pretreatment of mice with 9- and 10-nitro-octadec-9-enoic acid (nitro-oleic acid, NO2-OA) significantly reduced the susceptibility to develop acute ventricular tachycardia (VT). Accordingly, epicardial mapping revealed a markedly enhanced homogeneity in ventricular conduction. NO2-OA treatment of isolated cardiomyocytes lowered the number of spontaneous contractions upon adrenergic isoproterenol stimulation and nearly abolished ryanodine receptor type 2 (RyR2)-dependent sarcoplasmic Ca2+ leak. NO2-OA also significantly reduced RyR2-phosphorylation by inhibition of increased CaMKII activity. Thus, NO2-OA might be a novel pharmacological option for the prevention of VT development.

Mixed methods analysis of Health-Related Quality of Life in ambulant individuals affected with RYR1-related myopathies pre-post-N-acetylcysteine therapy.

PURPOSE: To characterize Health-Related Quality of Life (HRQoL) in ambulant individuals with RYR1-RM and to determine if a qualitative PRO tool (subjective self-assessment) complements PROMIS and Neuro-QoL scales to detect changes in HRQoL in ambulant individuals with RYR1-RM post N-acetylcysteine (NAC) treatment. METHODS: The study used a mixed methods research (MMR) design applying methodological triangulation. Qualitative data were collected via semi-structured interviews using open-ended questions. Quantitative data were gathered through PROMIS and Neuro-QoL instruments. Additionally, qualitative data were transformed into quantitative data for subjective self-assessment and frequency analyses. RESULTS: Qualitative results identified five domains and 33 subdomains as areas of interest. The most valuable were the importance of social impacts, the development of several coping strategies, both physical and psychological, and the identification of fatigue and weakness as key symptoms. Data transformation then categorized more than 3100 citations on frequency analyses, globally and by domain, visit, and participant. Regarding quantitative results, there was no clear evidence that any of the three PRO tools captured positive changes as a result of NAC treatment. CONCLUSION: Qualitative results showed a comprehensive characterization of HRQoL in this population based on a symptom/patient-centered approach. These findings will inform future studies. Furthermore, given the similar findings across our multiple methods and endpoints, the introduction of MMR may be a valuable, complementary approach to clinical trials. MMR may be especially useful to incorporate in order to address and follow the FDA's guidance and prioritization on the inclusion of affected individuals' perspectives in clinical trials.

Improved Calcium Homeostasis and Force by Selenium Treatment and Training in Aged Mouse Skeletal Muscle.

During aging reduction in muscle mass (sarcopenia) and decrease in physical activity lead to partial loss of muscle force and increased fatigability. Deficiency in the essential trace element selenium might augment these symptoms as it can cause muscle pain, fatigue, and proximal weakness. Average voluntary daily running, maximal twitch and tetanic force, and calcium release from the sarcoplasmic reticulum (SR) decreased while reactive oxygen species (ROS) production associated with tetanic contractions increased in aged - 22-month-old - as compared to young - 4-month-old - mice. These changes were accompanied by a decline in the ryanodine receptor type 1 (RyR1) and Selenoprotein N content and the increased amount of a degraded RyR1. Both lifelong training and selenium supplementation, but not the presence of an increased muscle mass at young age, were able to compensate for the reduction in muscle force and SR calcium release with age. Selenium supplementation was also able to significantly enhance the Selenoprotein N levels in aged mice. Our results describe, for the first time, the beneficial effects of selenium supplementation on calcium release from the SR and muscle force in old age while point out that increased muscle mass does not improve physical performance with aging.

Stachydrine hydrochloride alleviates pressure overload-induced heart failure and calcium mishandling on mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine Leonurus japonicus Houtt. has a long history in the treatment of cardiovascular diseases. Stachydrine hydrochloride, the main bioactive ingredient extracted from Leonurus japonicus Houtt., has been shown to have cardioprotective effects. However, the underlying mechanisms of stachydrine hydrochloride haven't been comprehensively studied so far. AIM OF THE STUDY: The aim of this study was to investigate the protective role of stachydrine hydrochloride in heart failure and elucidate its possible mechanisms of action. MATERIALS AND METHODS: In vivo, transverse aorta constriction was carried out in C57BL/6J mice, and thereafter, 7.2 mg/kg telmisartan (a selective AT1R antagonist as positive control) and 12 mg/kg stachydrine hydrochloride was administered daily intragastrically for 4 weeks. Cardiac function was evaluated by assessing morphological changes as well as echocardiographic and haemodynamic parameters. In vitro, neonatal rat cardiomyocytes or adult mice cardiomyocytes were treated with stachydrine hydrochloride and challenged with phenylephrine (α-AR agonist). Ventricular myocytes were isolated from the hearts of C57BL/6J mice by Langendorff crossflow perfusion system. Intracellular calcium was measured by an ion imaging system. The length and movement of sarcomere were traced to evaluate the systolic and diastolic function of single myocardial cells. RESULTS: Stachydrine hydrochloride improved the cardiac function and calcium transient amplitudes, and inhibited the SR leakage and the amount of sparks in cardiac myocytes isolated from TAC mice. We also demonstrated that stachydrine hydrochloride could ameliorated phenylephrine-induced enhance in sarcomere contraction, calcium transients and calcium sparks. Moreover, our data shown that stachydrine hydrochloride blocked the hyper-phosphorylation of CaMKII, RyR2, PLN, and prevented the disassociation of FKBP12.6 from RyR2. CONCLUSION: Our results suggest that stachydrine hydrochloride exerts beneficial therapeutic effects against heart failure. These cardioprotective effects may be associated with the regulation of calcium handling by stachydrine hydrochloride through inhibiting the hyper-phosphorylation of CaMKII.

Neferine induces autophagy-dependent cell death in apoptosis-resistant cancers via ryanodine receptor and Ca2+-dependent mechanism.

Resistance of cancer cells to chemotherapy is a significant clinical concern and mechanisms regulating cell death in cancer therapy, including apoptosis, autophagy or necrosis, have been extensively investigated over the last decade. Accordingly, the identification of medicinal compounds against chemoresistant cancer cells via new mechanism of action is highly desired. Autophagy is important in inducing cell death or survival in cancer therapy. Recently, novel autophagy activators isolated from natural products were shown to induce autophagic cell death in apoptosis-resistant cancer cells in a calcium-dependent manner. Therefore, enhancement of autophagy may serve as additional therapeutic strategy against these resistant cancers. By computational docking analysis, biochemical assays, and advanced live-cell imaging, we identified that neferine, a natural alkaloid from Nelumbo nucifera, induces autophagy by activating the ryanodine receptor and calcium release. With well-known apoptotic agents, such as staurosporine, taxol, doxorubicin, cisplatin and etoposide, utilized as controls, neferine was shown to induce autophagic cell death in a panel of cancer cells, including apoptosis-defective and -resistant cancer cells or isogenic cancer cells, via calcium mobilization through the activation of ryanodine receptor and Ulk-1-PERK and AMPK-mTOR signaling cascades. Taken together, this study provides insights into the cytotoxic mechanism of neferine-induced autophagy through ryanodine receptor activation in resistant cancers.

Dantrolene Requires Mg2+ and ATP To Inhibit the Ryanodine Receptor.

Dantrolene is a ryanodine receptor (RyR) inhibitor, which is used to relax muscles in malignant hyperthermia syndrome. Although dantrolene binds to the RyR protein, its mechanism of action is unknown, mainly because of the controversial results showing that dantrolene inhibited Ca2+ release from intact fibers and sarcoplasmic reticulum (SR) vesicles, but failed to inhibit single RyR channel currents in bilayers. Accordingly, it was concluded that an important factor for dantrolene's action was lost during the purification procedure of RyR. Recently, Mg2+ was demonstrated to be the essential factor for dantrolene to inhibit Ca2+ release in skinned muscle fibers. The aim of the present study was to confirm these results in Ca2+ release and bilayer experiments, using SR vesicles and solubilized channels, respectively. Our Ca2+ release experiments demonstrated that the effect of dantrolene and Mg2+ was cooperative and that ATP enhanced the inhibiting effect of dantrolene. Namely, 10 µM dantrolene reduced RyR channel open probability by ∼50% in the presence of 3 mM free Mg2+ and 1 mM ATP, whereas channel activity further decreased to ∼20% of control when [ATP] was increased to 2 mM. Our data provide important complementary information that supports the direct, Mg2+-dependent mechanism of dantrolene's action and suggests that dantrolene also requires ATP to inhibit RyR.

Ca2+-induced sarcoplasmic reticulum Ca2+ release in myotubularin-deficient muscle fibers.

Skeletal muscle deficiency in the 3-phosphoinositide (PtdInsP) phosphatase myotubularin (MTM1) causes myotubular myopathy which is associated with severe depression of voltage-activated sarcoplasmic reticulum Ca2+ release through ryanodine receptors. In the present study we aimed at further understanding how Ca2+ release is altered in MTM1-deficient muscle fibers, at rest and during activation. While in wild-type muscle fibers, SR Ca2+ release exhibits fast stereotyped kinetics of activation and decay throughout the voltage range of activation, Ca2+ release in MTM1-deficient muscle fibers exhibits slow and unconventional kinetics at intermediate voltages, suggestive of partial loss of the normal control of ryanodine receptor Ca2+ channel activity. In addition, the diseased muscle fibers at rest exhibit spontaneous elementary Ca2+ release events at a frequency 30 times greater than that of control fibers. Eighty percent of the events have spatiotemporal properties of archetypal Ca2+ sparks while the rest take either the form of lower amplitude, longer duration Ca2+ release events or of a combination thereof. The events occur at preferred locations in the fibers, indicating spatially uneven distribution of the parameters determining spontaneous ryanodine receptor 1 opening. Spatially large Ca2+ release sources were obviously involved in some of these events, suggesting that opening of ryanodine receptors in one cluster can activate opening of ryanodine receptors in a neighboring one. Overall results demonstrate that opening of Ca2+-activated ryanodine receptors is promoted both at rest and during excitation-contraction coupling in MTM1-deficient muscle fibers. Because access to this activation mode is denied to ryanodine receptors in healthy skeletal muscle, this may play an important role in the associated disease situation.

Lead exposure-induced cognitive impairment through RyR-modulating intracellular calcium signaling in aged rats.

Lead is widely distributed in the environment and has become a global public health issue. It is well known that lead exposure induces not only neurodevelopmental toxicity but also neurodegenerative diseases, with learning and memory impairment in the later stage. However, the molecular mechanisms remain elusive. The present study investigated the effects of early life and lifetime lead exposure on cognition and identified the molecular mechanisms involved in aged rats. The results herein demonstrated that the lead concentration in peripheral blood and brain tissues in aged rats was significantly increased in a lead dose-dependent manner. High-dose lead exposure caused cognitive functional impairment in aged rats, concomitant with a longer escape latency and a lower frequency of crossing the platform via Morris water maze testing compared to those in the control and low-dose lead exposure groups. Importantly, neuron functional defects were still observed even in early life lead exposure during the prenatal and weaning periods in aged rats. The neurotoxicity induced by lead exposure was morphologically evidenced by a recessed nuclear membrane, a swollen endoplasmic reticulum, and mitochondria in the neurons. Mechanistically, the exposure of aged rats to lead resulted in increasing free calcium concentration, reactive oxygen species, and apoptosis in the hippocampal neurons. Lead exposure increased RyR3 expression and decreased the levels of p-CaMKIIα/CaMKIIα and p-CREB/CREB in the hippocampus of aged rats. These findings indicated that early life lead exposure-induced cognition disorder was irreversible in aged rats. Lead-induced neurotoxicity might be related to the upregulation of RyR3 expression and high levels of intracellular free calcium with increasing lead concentration in injured neurons.

The harmful effects of acute PM2.5 exposure to the heart and a novel preventive and therapeutic function of CEOs.

Epidemiological researches have demonstrated the relationship between PM2.5 exposure and increased morbidity and mortality of cardiovascular injury. However, no effective therapeutic method was established. The purpose of this study is to investigate the effect of acute PM2.5 exposure on the mice heart tissue and explore the therapeutic effects of compound essential oils (CEOs) in this model. In this study, after mice were exposed to PM2.5 intratracheally, some obvious histopathological changes as well as some great alterations of proinflammatory cytokines were observed in the heart tissue. The imbalance of oxidative stress, the altered Ca2+ channel related proteins and the increased intracellular free Ca2+ were all involved in the heart impairment and would also be investigated in this model. The CEOs alleviated the heart impairment via its antioxidant effect rather than its anti-inflammatory function because our results revealed that oxidative stress related indicators were restored after CEOs administration. At the same time, increased concentration of intracellular free Ca2+ and ROS induced by PM2.5 were reduced after NAC (N-Acetyl-L-cysteine) administration. These data suggested that the acute PM2.5 exposure would damage heart tissue by inducing the inflammatory response, oxidative stress and intracellular free Ca2+ overload. PM2.5-induced oxidative stress probably increase intracellular free Ca2+ via RYR2 and SERCA2a. CEOs have the potential to be a novel effective and convenient therapeutic method to prevent and treat the acute heart impairment induced by PM2.5 via its antioxidant function.

Iron-deficiency anemia reduces cardiac contraction by downregulating RyR2 channels and suppressing SERCA pump activity.

Iron deficiency is present in ~50% of heart failure (HF) patients. Large multicenter trials have shown that treatment of iron deficiency with i.v. iron benefits HF patients, but the underlying mechanisms are not known. To investigate the actions of iron deficiency on the heart, mice were fed an iron-depleted diet, and some received i.v. ferric carboxymaltose (FCM), an iron supplementation used clinically. Iron-deficient animals became anemic and had reduced ventricular ejection fraction measured by magnetic resonance imaging. Ca2+ signaling, a pathway linked to the contractile deficit in failing hearts, was also significantly affected. Ventricular myocytes isolated from iron-deficient animals produced smaller Ca2+ transients from an elevated diastolic baseline but had unchanged sarcoplasmic reticulum (SR) Ca2+ load, trigger L-type Ca2+ current, or cytoplasmic Ca2+ buffering. Reduced fractional release from the SR was due to downregulated RyR2 channels, detected at protein and message levels. The constancy of diastolic SR Ca2+ load is explained by reduced RyR2 permeability in combination with right-shifted SERCA activity due to dephosphorylation of its regulator phospholamban. Supplementing iron levels with FCM restored normal Ca2+ signaling and ejection fraction. Thus, 2 Ca2+-handling proteins previously implicated in HF become functionally impaired in iron-deficiency anemia, but their activity is rescued by i.v. iron supplementation.

Oxidative stress creates a unique, CaMKII-mediated substrate for atrial fibrillation in heart failure.

The precise mechanisms by which oxidative stress (OS) causes atrial fibrillation (AF) are not known. Since AF frequently originates in the posterior left atrium (PLA), we hypothesized that OS, via calmodulin-dependent protein kinase II (CaMKII) signaling, creates a fertile substrate in the PLA for triggered activity and reentry. In a canine heart failure (HF) model, OS generation and oxidized-CaMKII-induced (Ox-CaMKII-induced) RyR2 and Nav1.5 signaling were increased preferentially in the PLA (compared with left atrial appendage). Triggered Ca2+ waves (TCWs) in HF PLA myocytes were particularly sensitive to acute ROS inhibition. Computational modeling confirmed a direct relationship between OS/CaMKII signaling and TCW generation. CaMKII phosphorylated Nav1.5 (CaMKII-p-Nav1.5 [S571]) was located preferentially at the intercalated disc (ID), being nearly absent at the lateral membrane. Furthermore, a decrease in ankyrin-G (AnkG) in HF led to patchy dropout of CaMKII-p-Nav1.5 at the ID, causing its distribution to become spatially heterogeneous; this corresponded to preferential slowing and inhomogeneity of conduction noted in the HF PLA. Computational modeling illustrated how conduction slowing (e.g., due to increase in CaMKII-p-Nav1.5) interacts with fibrosis to cause reentry in the PLA. We conclude that OS via CaMKII leads to substrate for triggered activity and reentry in HF PLA by mechanisms independent of but complementary to fibrosis.

Promiscuous attraction of ligands within the ATP binding site of RyR2 promotes diverse gating behaviour.

ATP is an essential constitutive regulator of cardiac ryanodine receptors (RyR2), enabling small changes in cytosolic Ca2+ to trigger large changes in channel activity. With recent landmark determinations of the full structures of RyR1 (skeletal isoform) and RyR2 using cryo-EM, and identification of the RyR1 ATP binding site, we have taken the opportunity to model the binding of fragments of ATP into RyR2 in order to investigate how the structure of the ATP site dictates the functional responses of ligands attracted there. RyR2 channel gating was assessed under voltage-clamp conditions and by [3H]ryanodine binding studies. We show that even the triphosphate (PPPi) moiety alone was capable of activating RyR2 but produced two distinct effects (activation or irreversible inactivation) that we suggest correspond to two preferred binding locations within the ATP site. Combinations of complementary fragments of ATP (Pi + ADP or PPi + AMP) could not reproduce the effects of ATP, however, the presence of adenosine prevented the inactivating PPPi effects, allowing activation similar to that of ATP. RyR2 appears to accommodate diverse types of molecules, including PPPi, deep within the ATP binding site. The most effective ligands, however, have at least three phosphate groups that are guided into place by a nucleoside.