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Background: PKA (protein kinase A)–mediated phosphorylation of cardiac RyR2 (ryanodine receptor 2) has been extensively studied for decades, but the physiological significance of PKA phosphorylation of RyR2 remains poorly understood. Recent determination of high-resolution 3-dimensional structure of RyR2 in complex with CaM (calmodulin) reveals that the major PKA phosphorylation site in RyR2, serine-2030 (S2030), is located within a structural pathway of CaM-dependent inactivation of RyR2. This novel structural insight points to a possible role of PKA phosphorylation of RyR2 in CaM-dependent inactivation of RyR2, which underlies the termination of Ca 2+ release and induction of cardiac Ca 2+ alternans. Methods: We performed single-cell endoplasmic reticulum Ca 2+ imaging to assess the impact of S2030 mutations on Ca 2+ release termination in human embryonic kidney 293 cells. Here we determined the role of the PKA site RyR2-S2030 in a physiological setting, we generated a novel mouse model harboring the S2030L mutation and carried out confocal Ca 2+ imaging. Results: We found that mutations, S2030D, S2030G, S2030L, S2030V, and S2030W reduced the endoplasmic reticulum luminal Ca 2+ level at which Ca 2+ release terminates (the termination threshold), whereas S2030P and S2030R increased the termination threshold. S2030A and S2030T had no significant impact on release termination. Furthermore, CaM–wild-type increased, whereas Ca 2+ binding deficient CaM mutant (CaM-M [a loss-of-function CaM mutation with all 4 EF-hand motifs mutated]), PKA, and Ca 2+ /CaMKII (CaM-dependent protein kinase II) reduced the termination threshold. The S2030L mutation abolished the actions of CaM–wild-type, CaM-M, and PKA, but not CaMKII, in Ca 2+ release termination. Moreover, we showed that isoproterenol and CaM-M suppressed pacing-induced Ca 2+ alternans and accelerated Ca 2+ transient recovery in intact working hearts, whereas CaM–wild-type exerted an opposite effect. The impact of isoproterenol was partially and fully reversed by the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide and the CaMKII inhibitor N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide individually and together, respectively. S2030L abolished the impact of CaM–wild-type, CaM-M, and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide–sensitive component, but not the N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide–sensitive component, of isoproterenol.

PKA (protein kinase A)-mediated phosphorylation of cardiac RyR2 (ryanodine receptor 2) has been extensively studied for decades, but the physiological significance of PKA phosphorylation of RyR2 remains poorly understood. Recent determination of high-resolution 3-dimensional structure of RyR2 in complex with CaM (calmodulin) reveals that the major PKA phosphorylation site in RyR2, serine-2030 (S2030), is located within a structural pathway of CaM-dependent inactivation of RyR2. This novel structural insight points to a possible role of PKA phosphorylation of RyR2 in CaM-dependent inactivation of RyR2, which underlies the termination of CaWe performed single-cell endoplasmic reticulum CaThese data demonstrate, for the first time, that the PKA phosphorylation site RyR-S2030 is an important determinant of PKA-regulated, CaM-dependent Ca

The suppression mechanism of regulatory T cells is an intensely investigated topic. As our focus has shifted towards a model centered on indirect inhibition of dendritic cells, a universally applicable effector mechanism controlled by FoxP3 expression has not been found. Here, we report that FoxP3 blocks the transcription of ER Ca2+-release channel ryanodine receptor 2. Reduced RyR2 shuts down basal Ca2+oscillation in Tregs, which reduces m-Calpain activities that is needed for T cells to disengage from DCs, suggesting a persistent blockage of DC antigen presentation. RyR2 deficiency renders the CD4+T cell pool to become immune suppressive, and behave in the same manner as FoxP3+Tregs in viral infection, asthma, hypersensitivity, colitis and tumor development. In the absence of FoxP3, RyR2-deficient CD4+T cells rescue the systemic autoimmunity associated with Scurfy mice. Therefore, FoxP3-mediated Ca2+signaling inhibition may be a central effector mechanism of Treg immune suppression.One Sentence SummaryCalcium channel RyR2 dictates Treg adhesion-based suppression

Inositol 1,4,5-trisphosphate receptor 1 (ITPR1) is an intracellular Ca2+ release channel critical for numerous cellular processes. Despite its ubiquitous physiological significance, ITPR1 mutations have thus far been linked to primarily movement disorders. Surprisingly, most disease-associated ITPR1 mutations generate a loss of function. This leaves our understanding of ITPR1-associated pathology oddly one-sided, as little is known about the pathological consequences of ITPR1 gain of function (GOF). To this end, we generated an ITPR1 gating domain mutation (D2594K) that substantially enhanced the inositol trisphosphate (IP3)-sensitivity of ITPR1, and a mouse model expressing this ITPR1-D2594K+/− GOF mutation. We found that heterozygous ITPR1-D2594K+/− mutant mice exhibited male infertility, azoospermia, and acrosome loss. Furthermore, we functionally characterized a human ITPR1 variant V494I identified in the UK Biobank database as potentially associated with disorders of the testis. We found that the ITPR1-V494I variant significantly enhanced IP3-induced Ca2+ release in HEK293 cells. Thus, ITPR1 hyperactivity may increase the risk of testicular dysfunction.

Aims In 2003, an Australian woman was convicted by a jury of smothering and killing her four children over a 10-year period. Each child died suddenly and unexpectedly during a sleep period, at ages ranging from 19 days to 18 months. In 2019 we were asked to investigate if a genetic cause could explain the children’s deaths as part of an inquiry into the mother’s convictions. Methods and results Whole genomes or exomes of the mother and her four children were sequenced. Functional analysis of a novel CALM2 variant was performed by measuring Ca2+-binding affinity, interaction with calcium channels and channel function. We found two children had a novel calmodulin variant (CALM2 G114R) that was inherited maternally. Three genes (CALM1-3) encode identical calmodulin proteins. A variant in the corresponding residue of CALM3 (G114W) was recently reported in a child who died suddenly at age 4 and a sibling who suffered a cardiac arrest at age 5. We show that CALM2 G114R impairs calmodulin's ability to bind calcium and regulate two pivotal calcium channels (CaV1.2 and RyR2) involved in cardiac excitation contraction coupling. The deleterious effects of G114R are similar to those produced by G114W and N98S, which are considered arrhythmogenic and cause sudden cardiac death in children. Conclusion A novel functional calmodulin variant (G114R) predicted to cause idiopathic ventricular fibrillation, catecholaminergic polymorphic ventricular tachycardia, or mild long QT syndrome was present in two children. A fatal arrhythmic event may have been triggered by their intercurrent infections. Thus, calmodulinopathy emerges as a reasonable explanation for a natural cause of their deaths., Graphical Abstract

In-depth microbial community characterization, a community-level metabolic function analysis, and biogeochemical assessments of residues were performed to understand the principles governing microbial community assembly in wheat straw during decomposition with different N fertilization rates in soil. We identified a suite of decomposition-associated bacterial and fungal groups in straw that contribute to C and N cycling. The decomposition-associated microbial community in straw is likely mainly derived from the original straw, and the bacterial and fungal communities showed different patterns along with the decomposition. Overall, the microbial community composition and function were not substantially affected by the N fertilization rate, but N fertilization significantly increased the straw microbial assembly speed and had significant effects on the abundances of certain taxa and C- and N-related genes, leading to different decomposition rates of straw under different N fertilization rates. Furthermore, the straw quality, especially dissolved organic C (DOC) and lignin, accounted for most observed effects on microbial community development and decomposition. The results provide new insight into the roles of the microbial community in straw during crop residue decomposition for nutrient cycling in farmland ecosystems.

Ryanodine receptor 2 (RyR2) is abundantly expressed in the heart and brain. Mutations in RyR2 are associated with both cardiac arrhythmias and intellectual disability. While the mechanisms of RyR2-linked arrhythmias are well characterized, little is known about the mechanism underlying RyR2-associated intellectual disability. Here, we employed a mouse model expressing a green fluorescent protein (GFP)-tagged RyR2 and a specific GFP probe to determine the subcellular localization of RyR2 in hippocampus. GFP-RyR2 was predominantly detected in the soma and dendrites, but not the dendritic spines of CA1 pyramidal neurons or dentate gyrus granular neurons. GFP-RyR2 was also detected within the mossy fibers in the stratum lucidum of CA3, but not in the presynaptic terminals of CA1 neurons. An arrhythmogenic RyR2-R4496C+/− mutation downregulated the A-type K+ current and increased membrane excitability, but had little effect on the afterhyperpolarization current or presynaptic facilitation of CA1 neurons. The RyR2-R4496C+/− mutation also impaired hippocampal long-term potentiation, learning, and memory. These data reveal the precise subcellular distribution of hippocampal RyR2 and its important role in neuronal excitability, learning, and memory.

Background: A novel familial arrhythmia syndrome, cardiac ryanodine receptor (RyR2) calcium release deficiency syndrome (CRDS), has recently been described. We evaluated a large and well characterized family to assess provocation testing, risk factor stratification and response to therapy in CRDS. Methods: We present a family with multiple unheralded sudden cardiac deaths and aborted cardiac arrests, primarily in children and young adults, with no clear phenotype on standard clinical testing. Results: Genetic analysis, including whole genome sequencing, firmly established that a missense mutation in RYR2 , Ala4142Thr, was the underlying cause of disease in the family. Functional study of the variant in a cell model showed RyR2 loss-of-function, indicating that the family was affected by CRDS. EPS (Electrophysiological Study) was undertaken in 9 subjects known to carry the mutation, including a survivor of aborted sudden cardiac death, and the effects of flecainide alone and in combination with metoprolol were tested. There was a clear gradation in inducibility of nonsustained and sustained ventricular arrhythmia between subjects at EPS, with the survivor of aborted sudden cardiac death being the most inducible subject. Administration of flecainide substantially reduced arrhythmia inducibility in this subject and abolished arrhythmia in all others. Finally, the effects of additional metoprolol were tested; it increased inducibility in 4/9 subjects. Conclusions: The Ala4142Thr mutation of RYR2 causes the novel heritable arrhythmia syndrome CRDS, which is characterized by familial sudden death in the absence of prior symptoms or a recognizable phenotype on ambulatory monitoring or exercise stress testing. We increase the experience of a specific EPS protocol in human subjects and show that it is helpful in establishing the clinical status of gene carriers, with potential utility for risk stratification. Our data provide evidence that flecainide is protective in human subjects with CRDS, consistent with the effect previously shown in a mouse model.

Meta-analysis provides a quantitative method for combining results from independent studies with the same treatment. However, existing estimation methods are sensitive to the presence of outliers in the datasets. In this paper we study the robust estimation for the parameters in meta-regression, including the between-study variance and regression parameters. Huber’s rho function and Tukey’s biweight function are adopted to derive the formulae of robust maximum likelihood ( ML ) estimators. The corresponding algorithms are developed. The asymptotic confidence interval and second-order-corrected confidence interval are investigated. Extensive simulation studies are conducted to assess the performance of the proposed methodology, and our results show that the robust estimators are promising and outperform the conventional ML and restricted maximum likelihood estimators when outliers exist in the dataset. The proposed methods are applied in three case studies and the results further support the eligibility of our methods in practical situations.

Mutations in cardiac ryanodine receptor (RyR2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). Most CPVT RyR2 mutations characterized are gain-of-function (GOF), indicating enhanced RyR2 function as a major cause of CPVT. Loss-of-function (LOF) RyR2 mutations have also been identified and are linked to a distinct entity of cardiac arrhythmia termed RyR2 Ca2+ release deficiency syndrome (CRDS). Exercise stress testing (EST) is routinely used to diagnose CPVT, but it is ineffective for CRDS. There is currently no effective diagnostic tool for CRDS in humans. An alternative strategy to assess the risk for CRDS is to directly determine the functional impact of the associated RyR2 mutations. To this end, we have functionally screened 18 RyR2 mutations that are associated with idiopathic ventricular fibrillation (IVF) or sudden death. We found two additional RyR2 LOF mutations E4146K and G4935R. The E4146K mutation markedly suppressed caffeine activation of RyR2 and abolished store overload induced Ca2+ release (SOICR) in human embryonic kidney 293 (HEK293) cells. E4146K also severely reduced cytosolic Ca2+ activation and abolished luminal Ca2+ activation of single RyR2 channels. The G4935R mutation completely abolished caffeine activation of and [3H]ryanodine binding to RyR2. Co-expression studies showed that the G4935R mutation exerted dominant negative impact on the RyR2 wildtype (WT) channel. Interestingly, the RyR2-G4935R mutant carrier had a negative EST, and the E4146K carrier had a family history of sudden death during sleep, which are different from phenotypes of typical CPVT. Thus, our data further support the link between RyR2 LOF and a new entity of cardiac arrhythmias distinct from CPVT.

Ryanodine receptors (RyRs) are ion channels that mediate the release of Ca2+ from the sarcoplasmic reticulum/endoplasmic reticulum, mutations of which are implicated in a number of human diseases. The adjacent C-terminal domains (CTDs) of cardiac RyR (RyR2) interact with each other to form a ring-like tetrameric structure with the intersubunit interface undergoing dynamic changes during channel gating. This mobile CTD intersubunit interface harbors many disease-associated mutations. However, the mechanisms of action of these mutations and the role of CTD in channel function are not well understood. Here, we assessed the impact of CTD disease-associated mutations P4902S, P4902L, E4950K, and G4955E on Ca2+- and caffeine-mediated activation of RyR2. The G4955E mutation dramatically increased both the Ca2+-independent basal activity and Ca2+-dependent activation of [3H]ryanodine binding to RyR2. The P4902S and E4950K mutations also increased Ca2+ activation but had no effect on the basal activity of RyR2. All four disease mutations increased caffeine-mediated activation of RyR2 and reduced the threshold for activation and termination of spontaneous Ca2+ release. G4955D dramatically increased the basal activity of RyR2, whereas G4955K mutation markedly suppressed channel activity. Similarly, substitution of P4902 with a negatively charged residue (P4902D), but not a positively charged residue (P4902K), also dramatically increased the basal activity of RyR2. These data suggest that electrostatic interactions are involved in stabilizing the CTD intersubunit interface and that the G4955E disease mutation disrupts this interface, and thus the stability of the closed state. Our studies shed new insights into the mechanisms of action of RyR2 CTD disease mutations.

Background The cardiac ryanodine receptor type 2 (RyR2) is a large homotetramer, located in the sarcoplasmic reticulum (SR), which releases Ca 2+ from the SR during systole. The molecular mechanism underlying Ca 2+ sensing and gating of the RyR2 channel in health and disease is only partially elucidated. Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT1) is the most prevalent syndrome caused by RyR2 mutations. Methods and Results This study involves investigation of a family with 4 cases of ventricular fibrillation and sudden death and physiological tests in HEK 293 cells and normal mode analysis (NMA) computation. We found 4 clinically affected members who were homozygous for a novel RyR2 mutation, G3118R, whereas their heterozygous relatives are asymptomatic. G3118R is located in the periphery of the protein, far from the mutation hotspot regions. HEK293 cells harboring G3118R mutation inhibited Ca 2+ release in response to increasing doses of caffeine, but decreased the termination threshold for store‐overload‐induced Ca 2+ release, thus increasing the fractional Ca 2+ release in response to increasing extracellular Ca 2+ . NMA showed that G3118 affects RyR2 tetramer in a dose‐dependent manner, whereas in the model of homozygous mutant RyR2, the highest entropic values are assigned to the pore and the central regions of the protein. Conclusions RyR2 G3118R is related to ventricular fibrillation and sudden death in recessive mode of inheritance and has an effect of gain of function on the protein. Despite a peripheral location, it has an allosteric effect on the stability of central and pore regions in a dose‐effect manner.

Cardiac ryanodine receptor (RyR2) gain-of-function mutations cause catecholaminergic polymorphic ventricular tachycardia, a condition characterized by prominent ventricular ectopy in response to catecholamine stress, which can be reproduced on exercise stress testing (EST). However, reports of sudden cardiac death (SCD) have emerged in EST-negative individuals who have loss-of-function (LOF) RyR2 mutations. The clinical relevance of RyR2 LOF mutations including their pathogenic mechanism, diagnosis, and treatment are all unknowns. Here, we performed clinical and genetic evaluations of individuals who suffered from SCD and harbored an LOF RyR2 mutation. We carried out electrophysiological studies using a programed electrical stimulation protocol consisting of a long-burst, long-pause, and short-coupled (LBLPS) ventricular extra-stimulus. Linkage analysis of RyR2 LOF mutations in six families revealed a combined logarithm of the odds ratio for linkage score of 11.479 for a condition associated with SCD with negative EST. A RyR2 LOF mouse model exhibited no catecholamine-provoked ventricular arrhythmias as in humans but did have substantial cardiac electrophysiological remodeling and an increased propensity for early afterdepolarizations. The LBLPS pacing protocol reliably induced ventricular arrhythmias in mice and humans having RyR2 LOF mutations, whose phenotype is otherwise concealed before SCD. Furthermore, treatment with quinidine and flecainide abolished LBLPS-induced ventricular arrhythmias in model mice. Thus, RyR2 LOF mutations underlie a previously unknown disease entity characterized by SCD with normal EST that we have termed RyR2 Ca2+ release deficiency syndrome (CRDS). Our study provides insights into the mechanism of CRDS, reports a specific CRDS diagnostic test, and identifies potentially efficacious anti-CRDS therapies., This work was supported by research grants from the Canadian Institutes of Health Research (PJT-155940) to S.R.W.C. and (PJT 166105 and MOP 142486) to R.A.R.; the National Natural Science Foundation of China (81903611) to B.S.; the Spanish Ministry of Science Innovation and Universities SAF2017-88019-C3-R to L.H.-M. and R.B. and Marato-2015-20-30 to L.H.-M.; the Royal Netherlands Academy of Sciences (CVON 2012-10 PREDICT) to A.A.M.W.; the E-Rare Joint Transnational Call for Proposals 2015 “Improving Diagnosis and Treatment of Catecholaminergic Polymorphic Ventricular Tachycardia: Integrating Clinical and Basic Science” to S.R.W.C., S.S., and A.A.M.W.; the European Research Council Grant “EU-rhythmy” ERC-ADG-2014 (ID: 669387) to S.G.P.; the Novo Nordisk Foundation, Denmark (NNF18OC0031258) to H.K.J.; and the NIH (R01HL057832) to M.F. B.S. and J.Y. are recipients of the Alberta Innovates-Health Solutions (AIHS) Fellowship Award. J.W. is a recipient of the Libin Cardiovascular Institute of Alberta and Cumming School of Medicine Postdoctoral Fellowship Award. X.Z. and W.G. are recipients of the AIHS Studentship Award. S.R.W.C. holds the Heart and Stroke Foundation Chair in Cardiovascular Research.

Rationale: Ca2+ alternans plays an essential role in cardiac alternans that can lead to ventricular fibrillation, but the mechanism underlying Ca2+ alternans remains undefined. Increasing evidence suggests that Ca2+ alternans results from alternations in the inactivation of cardiac RyR2 (ryanodine receptor 2). However, what inactivates RyR2 and how RyR2 inactivation leads to Ca2+ alternans are unknown. Objective: To determine the role of CaM (calmodulin) on Ca2+ alternans in intact working mouse hearts. Methods and results: We used an in vivo local gene delivery approach to alter CaM function by directly injecting adenoviruses expressing CaM-wild type, a loss-of-function CaM mutation, CaM (1-4), and a gain-of-function mutation, CaM-M37Q, into the anterior wall of the left ventricle of RyR2 wild type or mutant mouse hearts. We monitored Ca2+ transients in ventricular myocytes near the adenovirus-injection sites in Langendorff-perfused intact working hearts using confocal Ca2+ imaging. We found that CaM-wild type and CaM-M37Q promoted Ca2+ alternans and prolonged Ca2+ transient recovery in intact RyR2 wild type and mutant hearts, whereas CaM (1-4) exerted opposite effects. Altered CaM function also affected the recovery from inactivation of the L-type Ca2+ current but had no significant impact on sarcoplasmic reticulum Ca2+ content. Furthermore, we developed a novel numerical myocyte model of Ca2+ alternans that incorporates Ca2+-CaM-dependent regulation of RyR2 and the L-type Ca2+ channel. Remarkably, the new model recapitulates the impact on Ca2+ alternans of altered CaM and RyR2 functions under 9 different experimental conditions. Our simulations reveal that diastolic cytosolic Ca2+ elevation as a result of rapid pacing triggers Ca2+-CaM dependent inactivation of RyR2. The resultant RyR2 inactivation diminishes sarcoplasmic reticulum Ca2+ release, which, in turn, reduces diastolic cytosolic Ca2+, leading to alternations in diastolic cytosolic Ca2+, RyR2 inactivation, and sarcoplasmic reticulum Ca2+ release (ie, Ca2+ alternans). Conclusions: Our results demonstrate that inactivation of RyR2 by Ca2+-CaM is a major determinant of Ca2+ alternans, making Ca2+-CaM dependent regulation of RyR2 an important therapeutic target for cardiac alternans., This work was supported by research grants from the Heart and Stroke Foundation of Canada (G-19-0026444), the Heart and Stroke Foundation Chair in Cardiovascular Research (END611955), the Canadian Institutes of Health Research to S.R.W. Chen (PJT-155940), the Spanish Ministry of Science Innovation and Universities SAF2017-88019-C3-1R, 2R, and 3R (to L. Hove-Madsen, R. Benitez, and B. Echebarria), Marato-TV3 20152030 (to L. Hove-Madsen) and 20151110 (to B. Echebarria), and Generalitat de Catalunya SGR2017-1769 (to L. Hove-Madsen).

Increasing environmental stress strongly affects soil microbial communities, but the responses of the microbial assembly and the functional potential of the dominant microbial community in the presence of environmental stress in drylands are still poorly understood. Here, we undertook a broad appraisal of the abundance, diversity, similarity, community assembly, network properties and functions of soil microbiomes in 82 dryland grasslands along environmental gradients. We found that the bacterial and fungal diversity and community similarity showed different sensitivities to environmental stress (decreased mean annual precipitation (MAP) and soil nutrient levels and increased soil pH), and MAP was the most important factor influencing microbial community patterns. In addition, the dominant subcommunity of both bacteria and fungi was more sensitive to environmental stress than the nondominant subcommunity. Although increasing environmental stress decreased microbial phylogenetic clustering, it had no effects on the stochastic and deterministic assembly process balance. Moreover, we identified 101 bacterial and 34 fungal environmental stress-discriminatory taxa that were sensitive to environmental stress, and these bacterial markers showed a high correlation with the abundance of carbon (C) and nitrogen (N) cycling-related genes, whereas the taxa classified as connectors in the network were mainly correlated with C degradation genes. Our study shows that the different responses of bacteria and fungi to environmental stress bring challenges to predicting microbial function, but a relatively small number of taxa play an important role in driving C and N cycling-related functional genes, indicating that identifying an organism's phenotypic characteristics or traits of key taxa may improve our knowledge of the microbial response to ongoing global changes.

CaTo determine the role of CaM (calmodulin) on CaWe used an in vivo local gene delivery approach to alter CaM function by directly injecting adenoviruses expressing CaM-wild type, a loss-of-function CaM mutation, CaM (1-4), and a gain-of-function mutation, CaM-M37Q, into the anterior wall of the left ventricle of RyR2 wild type or mutant mouse hearts. We monitored CaOur results demonstrate that inactivation of RyR2 by Ca

Structural analyses identified the central domain of ryanodine receptor (RyR) as a transducer converting conformational changes in the cytoplasmic platform to the RyR gate. The central domain is also a regulatory hub encompassing the Ca(2+)-, ATP-, and caffeine-binding sites. However, the role of the central domain in RyR activation and regulation has yet to be defined. Here, we mutated five residues that form the Ca(2+) activation site and 10 residues with negatively charged or oxygen-containing side chains near the Ca(2+) activation site. We also generated eight disease-associated mutations within the central domain of RyR2. We determined the effect of these mutations on Ca(2+), ATP, and caffeine activation and Mg(2+) inhibition of RyR2. Mutating the Ca(2+) activation site markedly reduced the sensitivity of RyR2 to Ca(2+) and caffeine activation. Unexpectedly, Ca(2+) activation site mutation E3848A substantially enhanced the Ca(2+)-independent basal activity of RyR2, suggesting that E3848A may also affect the stability of the closed state of RyR2. Mutations in the Ca(2+) activation site also abolished the effect of ATP/caffeine on the Ca(2+)-independent basal activity, suggesting that the Ca(2+) activation site is also a critical determinant of ATP/caffeine action. Mutating residues with negatively charged or oxygen-containing side chains near the Ca(2+) activation site significantly altered Ca(2+) and caffeine activation and reduced Mg(2+) inhibition. Furthermore, disease-associated RyR2 mutations within the central domain significantly enhanced Ca(2+) and caffeine activation and reduced Mg(2+) inhibition. Our data demonstrate that the central domain plays an important role in channel activation, channel regulation, and closed state stability.

The succession of microbial community structure and function is a central ecological topic; however, the mechanisms that underlie community assembly and promote temporal succession remain unclear. We studied microbial community-associated functional dynamics in a well-established secondary successional chronosequence that spans approximately 160 years of ecosystem development on the Loess Plateau of China, by sequencing both 16S and ITS rRNA genes and soil metagenomes, resulting in a total of 132.5 Gb of data. Notably, both bacterial and fungal communities shifted with succession, but the microbial community changed little from the pioneer forest stage (approximately 110 years) to the latter successional forest stage. Fungi showed higher variability with succession than bacteria, and the shift of both the bacterial and fungal communities was related more to the soil characteristics than to the litter characteristics. Shifts in soil microbial functions were associated with microbial phylogenetic changes, but microbial gene function also showed changes in the absence of phylogenetic changes at the late successional stages. The reduction in microbial C cycle genes was related to a decrease in litter decomposition ability, thus resulting in a steady state of nutrient cycle in the ecosystem. In addition, high microbial respiration in nutrient-rich soil does not necessarily indicate high microbial decomposition functions; the latter also depend on the abundance of related genes, on enzyme activity and on the physicochemical properties of the litter. Our study provides a metagenome profile of a successional chronosequence and provides insight into the mechanisms underlying the soil microbe-driven functional changes in nutrient cycles during succession.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a condition that is characterized by an abnormal heart rhythm in response to physical or emotional stress. The majority CPVT patients carry mutations in the RYR2 gene that encodes the calcium release channel/ryanodine receptor (RyR2) in cardiomyocytes. The pathogenic mechanisms that account for the clinical phenotypes of CPVT are still elusive. We have identified a de novo mutation, A165D, from a CPVT patient. We found that CPVT phenotypes are recapitulated in A165D knock-in mice. The mutant RyR2 channels enhanced sarcoplasmic reticulum Ca2+ release, triggered delayed afterdepolarization in cardiomyocytes. Structural analysis revealed that the A165D mutation is located in a loop that is involved in inter-subunit interactions in the RyR2 tetrameric structure, it disrupted conformational stability of the RyR2, which favored a closed-to-open state transition, resulting in a leaky channel. The loop also harbors several other CPVT mutations, which suggests a common pathogenic molecular mechanism of CPVT-causing mutations. Our data illustrated disease-relevant functional defects and provide a deeper mechanistic understanding of a life-threatening cardiac arrhythmia.

Neuronal hyperactivity is an early, common manifestation of Alzheimer's disease (AD), and is believed to drive AD progression. Neuronal hyperactivity in the form of baseline activity (or spontaneous Ca2+ transients) has consistently been demonstrated in mouse models of AD using two-photon in vivo Ca2+ imaging of cortical or hippocampal neurons in anesthetized animals. Notably, these AD-related spontaneous Ca2+ transients were hardly detected in acute hippocampal slices, probably due to neuronal damage during brain slicing. To better preserve neuronal activity, we employed the N-methyl-D-glucamine (NMDG) protective brain slicing protocol. We performed confocal in vitro Ca2+ imaging of hippocampal CA1 neurons in optimized hippocampal slices. Consistent with previous in vivo studies, our in vitro studies using optimized brain slices also showed that limiting the open duration of the ryanodine receptor 2 (RyR2) by the RyR2 mutation E4872Q or by the R-carvedilol enantiomer prevented and rescued neuronal hyperactivity of hippocampal CA1 neurons from 5xFAD mice. Thus, genetically and pharmacologically limiting RyR2 open time prevented and rescued AD-related neuronal hyperactivity in vitro in optimized brain slices in the absence of anesthetics' influence. Our data also suggest that the NMDG protective brain slicing preparation offers an alternative means to study neuronal hyperactivity of various cell types in different brain regions, especially in regions that are not readily accessible to two-photon in vivo Ca2+ imaging.

The litter decomposition, nutrient patterns, as well as nutrient release and soil nutrient contents were determined in response to nitrogen (N) and phosphorus (P) addition and drought treatments following long-term vegetation recovery. The litter decomposition rate decreased with vegetation recovery, due to changes in litter quality, soil nutrient availability, and soil enzyme activity. Nitrogen addition promoted litter decomposition in the early recovery stages but inhibited decomposition in the later stages, indicating a shift in the nutrient limitations to litter decomposition with succession. Neither N nor P addition had any effect on the release of litter carbon (C), whereas N addition inhibited litter N release. In addition, drought decreased litter decomposition and nutrient release during the vegetation recovery process. Our findings suggest that litter quality, soil nutrient availability, and moisture at different vegetation recovery stages should be considered when modeling the C cycle and nutrient dynamics in these ecosystems.

Mutations in the skeletal muscle ryanodine receptor (RyR1) cause malignant hyperthermia (MH) and central core disease (CCD), whereas mutations in the cardiac ryanodine receptor (RyR2) lead to catecholaminergic polymorphic ventricular tachycardia (CPVT). Most disease-associated RyR1 and RyR2 mutations are located in the N-terminal, central, and C-terminal regions of the corresponding ryanodine receptor (RyR) isoform. An increasing body of evidence demonstrates that CPVT-associated RyR2 mutations enhance the propensity for spontaneous Ca2+ release during store Ca2+ overload, a process known as store overload-induced Ca2+ release (SOICR). Considering the similar locations of disease-associated RyR1 and RyR2 mutations in the RyR structure, we hypothesize that like CPVT-associated RyR2 mutations, MH/CCD-associated RyR1 mutations also enhance SOICR. To test this hypothesis, we determined the impact on SOICR of 12 MH/CCD-associated RyR1 mutations E2347-del, R2163H, G2434R, R2435L, R2435H, and R2454H located in the central region, and Y4796C, T4826I, L4838V, A4940T, G4943V, and P4973L located in the C-terminal region of the channel. We found that all these RyR1 mutations reduced the threshold for SOICR. Dantrolene, an acute treatment for MH, suppressed SOICR in HEK293 cells expressing the RyR1 mutants R164C, Y523S, R2136H, R2435H, and Y4796C. Interestingly, carvedilol, a commonly used β-blocker that suppresses RyR2-mediated SOICR, also inhibits SOICR in these RyR1 mutant HEK293 cells. Therefore, these results indicate that a reduced SOICR threshold is a common defect of MH/CCD-associated RyR1 mutations, and that carvedilol, like dantrolene, can suppress RyR1-mediated SOICR. Clinical studies of the effectiveness of carvedilol as a long-term treatment for MH/CCD or other RyR1-associated disorders may be warranted.

Summary: Neuronal hyperactivity is an early primary dysfunction in Alzheimer’s disease (AD) in humans and animal models, but effective neuronal hyperactivity-directed anti-AD therapeutic agents are lacking. Here we define a previously unknown mode of ryanodine receptor 2 (RyR2) control of neuronal hyperactivity and AD progression. We show that a single RyR2 point mutation, E4872Q, which reduces RyR2 open time, prevents hyperexcitability, hyperactivity, memory impairment, neuronal cell death, and dendritic spine loss in a severe early-onset AD mouse model (5xFAD). The RyR2-E4872Q mutation upregulates hippocampal CA1-pyramidal cell A-type K+ current, a well-known neuronal excitability control that is downregulated in AD. Pharmacologically limiting RyR2 open time with the R-carvedilol enantiomer (but not racemic carvedilol) prevents and rescues neuronal hyperactivity, memory impairment, and neuron loss even in late stages of AD. These AD-related deficits are prevented even with continued β-amyloid accumulation. Thus, limiting RyR2 open time may be a hyperactivity-directed, non-β-amyloid-targeted anti-AD strategy.

BACKGROUND Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an uncommon inherited arrhythmia disorder characterized by adrenergically evoked ventricular arrhythmias. Mutations in the cardiac calcium release channel/ryanodine receptor gene (RYR2) are identified in the majority of patients with CPVT. RyR2 is also the major RyR isoform expressed in the brain.OBJECTIVE The purpose of this study was to estimate the prevalence of intellectual disability (ID) and other neurodevelopmental disorders (NDDs) in RYR2-associated CPVT (CPVT1) and to study the characteristics of these patients.METHODS We reviewed the medical records of all CPVT1 patients from 12 international centers and analyzed the characteristics of all CPVT1 patients with concomitant NDDs. We functionally characterized the mutations to assess their response to caffeine activation. We did not correct for potential confounders.RESULTS Among 421 CPVT1 patients, we identified 34 patients with ID (8%; 95% confidence interval 6%-11%). Median age at diagnosis was 9.3 years (interquartile range 7.0-14.5). Parents for 24 of 34 patients were available for genetic testing, and 13 of 24 (54%) had a de novo mutation. Severity of ID ranged from mild to severe and was accompanied by other NDDs in 9 patients (26%). Functionally, the ID-associated mutations showed a markedly enhanced response of RyR2 to activation by caffeine. Seventeen patients (50%) also had supraventricular arrhythmias. During median follow-up of 8.4 years (interquartile range 1.8-12.4), 15 patients (45%) experienced an arrhythmic event despite adequate therapy.CONCLUSION Our study indicates that ID is more prevalent among CPVT1 patients (8%) than in the general population (1%-3%). This subgroup of CPVT1 patients reveals a malignant cardiac phenotype with marked supraventricular and ventricular arrhythmias.

The high-conductance intracellular calcium (Ca

Improving rice yield potential is crucial for global food security. Taoyuan, China, is famous worldwide as a special ecosite for ultrahigh rice yield. Climatological factors affecting this phenomenon have been identified, but the potential molecular processes and environmental mechanisms promoting ultrahigh yield remain mysteries. This study identifies soil biotic factors affecting ultrahigh yield, considering soil microbial community structure and metagenomic functions during four key rice growth stages, together with results from nitrogen enrichment experiments and rice root transcriptome analysis. Our results show that Taoyuan has more diverse bacterial taxa, less diverse fungal taxa, and a 10-fold-stronger connection among microbial taxa as well as a significantly higher proportion of nutrient transport functions than a regular site. Notably, our metagenomic analysis shows that Taoyuan contains more taxa with nitrogen metabolism functions and a higher abundance of genes involved in the nitrification process (e.g., hydroxylamine oxidoreductase and nitric oxide dioxygenase), promoting effective transformation of ammonium (NH4+) to nitrate (NO3−) in rice fields and stimulating high expression of nitrate transporters in rice roots, leading to ultrahigh yields. Our results indicate that soil microbiota contribute to ultrahigh rice yield in Taoyuan, and indicate that nitrogen metabolism functions could be one of the mechanism for the ultrahigh yield of rice.

The Ca2+ -sensing protein calmodulin (CaM) inhibits cardiac ryanodine receptor (RyR2)-mediated Ca2+ release. CaM mutations associated with arrhythmias and sudden cardiac death have been shown to diminish CaM-dependent inhibition of RyR2, but the underlying mechanisms are not well understood. Nearly all arrhythmogenic CaM mutations identified are located in the C-domain of CaM and exert marked effects on Ca2+ binding to CaM and on the CaM C-domain interaction with the CaM-binding domain 2 (CaMBD2) in RyR2. Interestingly, the arrhythmogenic N-domain mutation CaM-N54I has little or no effect on Ca2+ binding to CaM or the CaM C-domain-RyR2 CaMBD2 interaction, unlike all CaM C-domain mutations. This suggests that CaM-N54I may diminish CaM-dependent RyR2 inhibition by affecting CaM N-domain interactions with RyR2 CaMBDs other than CaMBD2. To explore this possibility, we assessed the effects of deleting each of the four known CaMBDs in RyR2 (CaMBD1a, -1b, -2, or -3) on the CaM-dependent inhibition of RyR2-mediated Ca2+ release in HEK293 cells. We found that removing CaMBD1a, CaMBD1b, or CaMBD3 did not alter the effects of CaM-N54I or CaM-WT on RyR2 inhibition. On the other hand, deleting RyR2-CaMBD2 abolished the effects of both CaM-N54I and CaM-WT. Our results support that CaM-N54I causes aberrant RyR2 regulation via an uncharacterized CaMBD or less likely CaMBD2, and that RyR2 CaMBD2 is required for the actions of both N- and C-domain CaM mutations. Moreover, our results show that CaMBD1a is central to RyR2 regulation, but CaMBD1a, CaMBD1b, and CaMBD3 are not required for CaM-dependent inhibition of RyR2 in HEK293 cells.

Sarcoplasmic reticulum (SR) Ca2+ cycling is governed by the cardiac ryanodine receptor (RyR2) and SR Ca2+-ATPase (SERCA2a). Abnormal SR Ca2+ cycling is thought to be the primary cause of Ca2+ alternans that can elicit ventricular arrhythmias and sudden cardiac arrest. Although alterations in either RyR2 or SERCA2a function are expected to affect SR Ca2+ cycling, whether and to what extent altered RyR2 or SERCA2a function affects Ca2+ alternans is unclear. Here, we employed a gain-of-function RyR2 variant (R4496C) and the phospholamban-knockout (PLB-KO) mouse model to assess the effect of genetically enhanced RyR2 or SERCA2a function on Ca2+ alternans. Confocal Ca2+ imaging revealed that RyR2-R4496C shortened SR Ca2+ release refractoriness and markedly suppressed rapid pacing–induced Ca2+ alternans. Interestingly, despite enhancing RyR2 function, intact RyR2-R4496C hearts exhibited no detectable spontaneous SR Ca2+ release events during pacing. Unlike for RyR2, enhancing SERCA2a function by ablating PLB exerted a relatively minor effect on Ca2+ alternans in intact hearts expressing RyR2 WT or a loss-of-function RyR2 variant, E4872Q, that promotes Ca2+ alternans. Furthermore, partial SERCA2a inhibition with 3 μM 2,5-di-tert-butylhydroquinone (tBHQ) also had little impact on Ca2+ alternans, whereas strong SERCA2a inhibition with 10 μM tBHQ markedly reduced the amplitude of Ca2+ transients and suppressed Ca2+ alternans in intact hearts. Our results demonstrate that enhanced RyR2 function suppresses Ca2+ alternans in the absence of spontaneous Ca2+ release and that RyR2, but not SERCA2a, is a key determinant of Ca2+ alternans in intact working hearts, making RyR2 an important therapeutic target for cardiac alternans., This work was supported by research grants from the Canadian Institutes of Health Research; the Heart and Stroke Foundation of Alberta, Northwest Territories, and Nunavut; the Canada Foundation for Innovation (CFI); the Heart and Stroke Foundation Chair in Cardiovascular Research; the Alberta Innovates-Health Solutions (to S. R. W. C.); Spanish Ministry of Economy and Competitiveness (MINECO) Grants DPI2013-44584-R (to R. B.) and SAF2014-58286-C2-1R (to L. H. M.), and Generalitat de Catalunya Grant SGR2014-1465 (to L. H. M. and R. B.).

Membrane proteins known as ryanodine receptors (RyRs) display large conductance of ∼1 nS and nearly ideal charge selectivity. Both properties are inversely correlated in other large-conductance but nonselective biological nanopores (i.e., α-hemolysin) used as industrial biosensors. Although recent cryo-electron microscopy structures of RyR2 show similarities to K+- and Na+-selective channels, it remains unclear whether similar ion conduction mechanisms occur in RyR2. Here, we combine microseconds of all-atom molecular dynamics (MD) simulations with mutagenesis and electrophysiology experiments to investigate large K+ conductance and charge selectivity (cation vs anion) in an open-state structure of RyR2. Our results show that a water-mediated knock-on mechanism enhances the cation permeation. The polar Q4863 ring may function as a confinement zone amplifying charge selectivity, while the cytoplasmic vestibule can contribute to the efficiency of the cation attraction. We also provide direct evidence that t...

Sarcoplasmic reticulum (SR) Ca(2+) cycling is governed by the cardiac ryanodine receptor (RyR2) and SR Ca(2+)-ATPase (SERCA2a). Abnormal SR Ca(2+) cycling is thought to be the primary cause of Ca(2+) alternans that can elicit ventricular arrhythmias and sudden cardiac arrest. Although alterations in either RyR2 or SERCA2a function are expected to affect SR Ca(2+) cycling, whether and to what extent altered RyR2 or SERCA2a function affects Ca(2+) alternans is unclear. Here, we employed a gain-of-function RyR2 variant (R4496C) and the phospholamban-knockout (PLB-KO) mouse model to assess the effect of genetically enhanced RyR2 or SERCA2a function on Ca(2+) alternans. Confocal Ca(2+) imaging revealed that RyR2-R4496C shortened SR Ca(2+) release refractoriness and markedly suppressed rapid pacing–induced Ca(2+) alternans. Interestingly, despite enhancing RyR2 function, intact RyR2-R4496C hearts exhibited no detectable spontaneous SR Ca(2+) release events during pacing. Unlike for RyR2, enhancing SERCA2a function by ablating PLB exerted a relatively minor effect on Ca(2+) alternans in intact hearts expressing RyR2 WT or a loss-of-function RyR2 variant, E4872Q, that promotes Ca(2+) alternans. Furthermore, partial SERCA2a inhibition with 3 μm 2,5-di-tert-butylhydroquinone (tBHQ) also had little impact on Ca(2+) alternans, whereas strong SERCA2a inhibition with 10 μm tBHQ markedly reduced the amplitude of Ca(2+) transients and suppressed Ca(2+) alternans in intact hearts. Our results demonstrate that enhanced RyR2 function suppresses Ca(2+) alternans in the absence of spontaneous Ca(2+) release and that RyR2, but not SERCA2a, is a key determinant of Ca(2+) alternans in intact working hearts, making RyR2 an important therapeutic target for cardiac alternans.

We explored the clinico-genetic basis of spinocerebellar ataxia 29 (SCA29) by determining the frequency, phenotype, and functional impact of ITPR1 missense variants associated with early-onset ataxia (EOA). Three hundred thirty one patients from a European EOA target cohort (n = 120), US-American EOA validation cohort (n = 72), and early-onset epileptic encephalopathy (EOEE) control cohort (n = 139) were screened for de novo ITPR1 variants. The target cohort was also screened for inherited ITPR1 variants. The variants' functional impact was determined by IP3-induced Ca2+ release in HEK293 cells. 3/120 patients (2.5%) from the target cohort and 4/72 patients (5.5%) from the validation cohort, but none from the EOEE control cohort, carried de novo ITPR1 variants. However, most ITPR1 variants (7/10 = 70%) in the target cohort were inherited from a healthy parent, with 3/6 patients carrying disease-causing variants in other genes. This suggests limited or no phenotypic impact of many ITPR1 missense variants, even if ultra-rare and well-conserved. While common bioinformatics tools did not discriminate de novo from other ITPR1 variants, functional characterization demonstrated reduced IP3-induced Ca2+ release for all de novo variants, including the recurrent c.805C>T (p.(R269W)) variant. In sum, these findings show that de novo ITPR1 missense variants are a recurrent cause of EOA (SCA29) across independent cohorts, acting via loss of IP3 channel function. Inherited ITPR1 variants are also enriched in EOA, but often without strong impact, albeit rare and well-conserved. Functional studies allow identifying ITPR1 variants with large impact, likely disease-causing. Such functional confirmation is warranted for inherited ITPR1 variants before making a SCA29 diagnosis.

The cardiac Ca(2+) release channel (ryanodine receptor, RyR2) plays an essential role in excitation-contraction coupling in cardiac muscle cells. Effective and stable excitation-contraction coupling critically depends not only on the expression of RyR2, but also on its distribution. Despite its importance, little is known about the distribution and organization of RyR2 in living cells. To study the distribution of RyR2 in living cardiomyocytes, we generated a knock-in mouse model expressing a GFP-tagged RyR2 (GFP-RyR2). Confocal imaging of live ventricular myocytes isolated from the GFP-RyR2 mouse heart revealed clusters of GFP-RyR2 organized in rows with a striated pattern. Similar organization of GFP-RyR2 clusters was observed in fixed ventricular myocytes. Immunofluorescence staining with the anti-α-actinin antibody (a z-line marker) showed that nearly all GFP-RyR2 clusters were localized in the z-line zone. There were small regions with dislocated GFP-RyR2 clusters. Interestingly, these same regions also displayed dislocated z-lines. Staining with di-8-ANEPPS revealed that nearly all GFP-RyR2 clusters were co-localized with transverse but not longitudinal tubules, whereas staining with MitoTracker Red showed that GFP-RyR2 clusters were not co-localized with mitochondria in live ventricular myocytes. We also found GFP-RyR2 clusters interspersed between z-lines only at the periphery of live ventricular myocytes. Simultaneous detection of GFP-RyR2 clusters and Ca(2+) sparks showed that Ca(2+) sparks originated exclusively from RyR2 clusters. Ca(2+) sparks from RyR2 clusters induced no detectable changes in mitochondrial Ca(2+) level. These results reveal, for the first time, the distribution of RyR2 clusters and its functional correlation in living ventricular myocytes.

The cardiac Ca2+ release channel [ryanodine receptor type 2 (RyR2)] is modulated by thiol reactive agents, but the molecular basis of RyR2 modulation by thiol reagents is poorly understood. Cys3635 in the skeletal muscle RyR1 is one of the most hyper-reactive thiols and is important for the redox and calmodulin (CaM) regulation of the RyR1 channel. However, little is known about the role of the corresponding cysteine residue in RyR2 (Cys3602) in the function and regulation of the RyR2 channel. In the present study, we assessed the impact of mutating Cys3602 (C3602A) on store overload-induced Ca2+ release (SOICR) and the regulation of RyR2 by thiol reagents and CaM. We found that the C3602A mutation suppressed SOICR by raising the activation threshold and delayed the termination of Ca2+ release by reducing the termination threshold. As a result, C3602A markedly increased the fractional Ca2+ release. Furthermore, the C3602A mutation diminished the inhibitory effect of N-ethylmaleimide on Ca2+ release, but it had no effect on the stimulatory action of 4,4′-dithiodipyridine (DTDP) on Ca2+ release. In addition, Cys3602 mutations (C3602A or C3602R) did not abolish the effect of CaM on Ca2+-release termination. Therefore, RyR2–Cys3602 is a major site mediating the action of thiol alkylating agent N-ethylmaleimide, but not the action of the oxidant DTDP. Our data also indicate that residue Cys3602 plays an important role in the activation and termination of Ca2+ release, but it is not essential for CaM regulation of RyR2.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is one of the most lethal inherited cardiac arrhythmias mostly linked to cardiac ryanodine receptor (RyR2) mutations with high disease penetrance. Interestingly, a novel RyR2 mutation G357S discovered in a large family of more than 1400 individuals has reduced penetrance. The molecular basis for the incomplete disease penetrance in this family is unknown. To gain insights into the variable disease expression in this family, we determined the impact of the G357S mutation on RyR2 function and expression. We assessed spontaneous Ca2+ release in HEK293 cells expressing RyR2 wildtype and the G357S mutant during store Ca2+ overload, also known as store overload induced Ca2+ release (SOICR). We found that the G357S mutation reduced the percentage of RyR2-expressing cells that showed SOICR. However, in cells that displayed SOICR, G357S reduced the thresholds for the activation and termination of SOICR. Furthermore, G357S decreased the thermal stability of the N-terminal domain of RyR2, and markedly reduced the protein expression of the full-length RyR2. On the other hand, the G357S mutation did not alter the Ca2+ activation of [3H]ryanodine binding or the Ca2+ induced release of Ca2+ from the intracellular stores in HEK293 cells. These data indicate that the CPVT-associated G357S mutation enhances the arrhythmogenic SOICR and reduces RyR2 protein expression, which may be attributable to the incomplete penetrance of CPVT in this family.

Mutations in the skeletal muscle ryanodine receptor (RyR1) cause malignant hyperthermia (MH) and central core disease (CCD), whereas mutations in the cardiac ryanodine receptor (RyR2) lead to catecholaminergic polymorphic ventricular tachycardia (CPVT). Most disease-associated RyR1 and RyR2 mutations are located in the N-terminal, central, and C-terminal regions of the corresponding ryanodine receptor (RyR) isoform. An increasing body of evidence demonstrates that CPVT-associated RyR2 mutations enhance the propensity for spontaneous Ca2+ release during store Ca2+ overload, a process known as store overload-induced Ca2+ release (SOICR). Considering the similar locations of disease-associated RyR1 and RyR2 mutations in the RyR structure, we hypothesize that like CPVT-associated RyR2 mutations, MH/CCD-associated RyR1 mutations also enhance SOICR. To test this hypothesis, we determined the impact on SOICR of 12 MH/CCD-associated RyR1 mutations E2347-del, R2163H, G2434R, R2435L, R2435H, and R2454H located in the central region, and Y4796C, T4826I, L4838V, A4940T, G4943V, and P4973L located in the C-terminal region of the channel. We found that all these RyR1 mutations reduced the threshold for SOICR. Dantrolene, an acute treatment for MH, suppressed SOICR in HEK293 cells expressing the RyR1 mutants R164C, Y523S, R2136H, R2435H, and Y4796C. Interestingly, carvedilol, a commonly used β-blocker that suppresses RyR2-mediated SOICR, also inhibits SOICR in these RyR1 mutant HEK293 cells. Therefore, these results indicate that a reduced SOICR threshold is a common defect of MH/CCD-associated RyR1 mutations, and that carvedilol, like dantrolene, can suppress RyR1-mediated SOICR. Clinical studies of the effectiveness of carvedilol as a long-term treatment for MH/CCD or other RyR1-associated disorders may be warranted.

A number of point mutations in the intracellular Ca2+-sensing protein calmodulin (CaM) are arrhythmogenic, yet their underlying mechanisms are not clear. These mutations generally decrease Ca2+ binding to CaM and impair inhibition of CaM-regulated Ca2+ channels like the cardiac Ca2+-release channel (ryanodine receptor, RyR2), and it appears that attenuated CaM Ca2+-binding correlates with impaired CaM-dependent RyR2 inhibition. Here, we investigated the RyR2 inhibitory action of the CaM p.Phe142Leu mutation (F142L; numbered including the start methionine), which markedly reduces CaM Ca2+-binding. Surprisingly, CaM-F142L had little to no aberrant effect on RyR2-mediated store-overload induced Ca2+-release in HEK293 cells compared to CaM-WT. Furthermore, CaM-F142L enhanced CaM-dependent RyR2 inhibition at the single channel level, compared to CaM-WT. This is in stark contrast to the actions of arrhythmogenic CaM mutations N54I, D96V, N98S and D130G, which all diminish CaM-dependent RyR2 inhibition. Thermodynamic analysis shows that apoCaM-F142L converts an endothermal interaction between CaM and the CaM-binding domain (CaMBD) of RyR2 into an exothermal one. Moreover, NMR spectra reveals that the CaM-F142L-CaMBD interaction is structurally different from that of CaM-WT at low Ca2+. These data indicate a distinct interaction between CaM-F142L and RyR2s CaMBD, which may explain the stronger CaM-dependent RyR2 inhibition by CaM-F142L, despite its reduced Ca2+-binding. Collectively, these results add to our understanding of CaM-dependent regulation of RyR2 as well as the mechanistic effects of arrhythmogenic CaM mutations. The unique properties of the CaM-F142L mutation may provide novel clues on how to suppress excessive RyR2 Ca2+-release by manipulating the CaM-RyR2 interaction.

RyR2 (cardiac ryanodine receptor)-mediated Ca2+ release in cardiomyocytes terminates when the sarcoplasmic reticulum Ca2+ content depletes to a threshold level, known as the termination threshold. Despite its importance, little is known about the mechanism that regulates the termination threshold. CaM (calmodulin), by inhibiting RyR2, has been implicated in Ca2+-release termination, but whether CaM modulates the termination threshold is unknown. To this end, we monitored the endoplasmic reticulum Ca2+ dynamics in RyR2-expressing HEK (human embryonic kidney)-293 cells transfected with WT (wild-type) CaM or mutants. We found that WT CaM or CaM mutations which abolish Ca2+ binding to the N-lobe (N-terminal lobe) of CaM increased the termination threshold (i.e. facilitated termination), but had no effect on the activation threshold at which spontaneous Ca2+ release occurs. On the other hand, CaM mutations that diminish Ca2+ binding to both the N-lobe and C-lobe (C-terminal lobe), or the C-lobe only, decreased the termination threshold (i.e. delayed termination) with a similar activation threshold. Furthermore, deletion of residues 3583–3603 or point mutations (W3587A/L3591D/F3603A, W3587A, or L3591D) in the CaM-binding domain of RyR2 that are known to abolish or retain CaM binding all reduced the termination threshold without having a significant impact on the activation threshold. Interestingly, the RyR2-F3603A mutation affected both the activation and termination threshold. Collectively, these data indicate that CaM facilitates the termination of Ca2+ release by increasing the termination threshold, and that this action of CaM depends on Ca2+ binding to the C-lobe, but not to the N-lobe, of CaM. The results of the present study also suggest that the CaM-binding domain of RyR2 is an important determinant of Ca2+-release termination and activation.

Summary Calmodulin (CaM), a 16 kDa ubiquitous calcium-sensing protein, is known to bind tightly to the calcium release channel/ryanodine receptor (RyR), and modulate RyR function. CaM binding studies using RyR fragments or synthetic peptides have revealed the presence of multiple, potential CaM-binding regions in the primary sequence of RyR. In the present study, we inserted GFP into two of these proposed CaM-binding sequences and mapped them onto the three-dimensional structure of intact cardiac RyR2 by cryo-electron microscopy. Interestingly, we found that the two potential CaM-binding regions encompassing, Arg3595 and Lys4269, respectively, are in close proximity and are adjacent to the previously mapped CaM-binding sites. To monitor the conformational dynamics of these CaM-binding regions, we generated a fluorescence resonance energy transfer (FRET) pair, a dual CFP- and YFP-labeled RyR2 (RyR2R3595-CFP/K4269-YFP) with CFP inserted after Arg3595 and YFP inserted after Lys4269. We transfected HEK293 cells with the RyR2R3595-CFP/K4269-YFP cDNA, and examined their FRET signal in live cells. We detected significant FRET signals in transfected cells that are sensitive to the channel activator caffeine, suggesting that caffeine is able to induce conformational changes in these CaM-binding regions. Importantly, no significant FRET signals were detected in cells co-transfected with cDNAs encoding the single CFP (RyR2R3595-CFP) and single YFP (RyR2K4269-YFP) insertions, indicating that the FRET signal stemmed from the interaction between R3595–CFP and K4269–YFP that are in the same RyR subunit. These observations suggest that multiple regions in the RyR2 sequence may contribute to an intra-subunit CaM-binding pocket that undergoes conformational changes during channel gating.

CPVT (catecholaminergic polymorphic ventricular tachycardia) is an inherited life-threatening arrhythmogenic disorder. CPVT is caused by DADs (delayed after-depolarizations) that are induced by spontaneous Ca 2+ release during SR (sarcoplasmic reticulum) Ca 2+ overload, a process also known as SOICR (store-overload-induced Ca 2+ release). A number of mutations in the cardiac ryanodine receptor RyR2 are linked to CPVT. Many of these CPVT-associated RyR2 mutations enhance the propensity for SOICR and DADs by sensitizing RyR2 to luminal or luminal/cytosolic Ca 2+ activation. Recently, a novel CPVT RyR2 mutation, G230C, was found to increase the cytosolic, but not the luminal, Ca 2+ sensitivity of single RyR2 channels in lipid bilayers. This observation led to the suggestion of a SOICR-independent disease mechanism for the G230C mutation. However, the cellular impact of this mutation on SOICR is yet to be determined. To this end, we generated stable inducible HEK (human embryonic kidney)-293 cell lines expressing the RyR2 WT (wild-type) and the G230C mutant. Using single-cell Ca 2+ imaging, we found that the G230C mutation markedly enhanced the propensity for SOICR and reduced the SOICR threshold. Furthermore, the G230C mutation increased the sensitivity of single RyR2 channels to both luminal and cytosolic Ca 2+ activation and the Ca 2+ -dependent activation of [ 3 H]ryanodine binding. In addition, the G230C mutation decreased the thermal stability of the N-terminal region (amino acids 1–547) of RyR2. These data suggest that the G230C mutation enhances the propensity for SOICR by sensitizing the channel to luminal and cytosolic Ca 2+ activation, and that G230C has an intrinsic structural impact on the N-terminal domains of RyR2.

Global conformational changes in the three-dimensional structure of the Ca(2+) release channel/ryanodine receptor (RyR) occur upon ligand activation. A number of ligands are able to activate the RyR channel, but whether these structurally diverse ligands induce the same or different conformational changes in the channel is largely unknown. Here we constructed a fluorescence resonance energy transfer (FRET)-based probe by inserting a CFP after residue Ser-2367 and a YFP after residue Tyr-2801 in the cardiac RyR (RyR2) to yield a CFP- and YFP-dual labeled RyR2 (RyR2(Ser-2367-CFP/Tyr-2801-YFP)). Both of these insertion sites have previously been mapped to the "clamp" region in the four corners of the square-shaped cytoplasmic assembly of the three-dimensional structure of RyR2. Using this novel FRET probe, we monitored the extent of conformational changes in the clamp region of RyR2(Ser-2367-CFP/Tyr-2801-YFP) induced by various ligands. We also monitored the extent of Ca(2+) release induced by the same ligands in HEK293 cells expressing RyR2(Ser-2367-CFP/Tyr-2801-YFP). We detected conformational changes in the clamp region for the ligands caffeine, aminophylline, theophylline, ATP, and ryanodine but not for Ca(2+) or 4-chloro-m-cresol, although they all induced Ca(2+) release. Interestingly, caffeine is able to induce further conformational changes in the clamp region of the ryanodine-modified channel, suggesting that ryanodine does not lock RyR in a fixed conformation. Our data demonstrate that conformational changes in the clamp region of RyR are ligand-dependent and suggest the existence of multiple ligand dependent RyR activation mechanisms associated with distinct conformational changes.

The ryanodine receptor (RyR) channel pore is formed by four S6 inner helices, with its intracellular gate located at the S6 helix bundle crossing region. The cytoplasmic region of the extended S6 helix is held by the U motif of the central domain and is thought to control the opening and closing of the S6 helix bundle. However, the functional significance of the S6 cytoplasmic region in channel gating is unknown. Here we assessed the role of the S6 cytoplasmic region in the function of cardiac RyR (RyR2) via structure-guided site-directed mutagenesis. We mutated each residue in the S6 cytoplasmic region of the mouse RyR2 (4876QQEQVKEDM4884) and characterized their functional impact. We found that mutations Q4876A, V4880A, K4881A, and M4884A, located mainly on one side of the S6 helix that faces the U motif, enhanced basal channel activity and the sensitivity to Ca2+ or caffeine activation, whereas mutations Q4877A, E4878A, Q4879A, and D4883A, located largely on the opposite side of S6, suppressed channel activity. Furthermore, V4880A, a cardiac arrhythmia-associated mutation, markedly enhanced the frequency of spontaneous openings and the sensitivity to cytosolic and luminal Ca2+ activation of single RyR2 channels. V4880A also increased the propensity and reduced the threshold for arrhythmogenic spontaneous Ca2+ release in HEK293 cells. Collectively, our data suggest that interactions between the cytoplasmic region of S6 and the U motif of RyR2 are important for stabilizing the closed state of the channel. Mutations in the S6/U motif domain interface likely destabilize the closed state of RyR2, resulting in enhanced basal channel activity and sensitivity to activation and increased propensity for spontaneous Ca2+ release and cardiac arrhythmias.

Recent three-dimensional structural studies reveal that the central domain of ryanodine receptor (RyR) serves as a transducer that converts long-range conformational changes into the gating of the channel pore. Interestingly, the central domain encompasses one of the mutation hotspots (corresponding to amino acid residues 3778–4201) that contains a number of cardiac RyR (RyR2) mutations associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) and atrial fibrillation (AF). However, the functional consequences of these central domain RyR2 mutations are not well understood. To gain insights into the impact of the mutation and the role of the central domain in channel function, we generated and characterized eight disease-associated RyR2 mutations in the central domain. We found that all eight central domain RyR2 mutations enhanced the Ca2+-dependent activation of [3H]ryanodine binding, increased cytosolic Ca2+-induced fractional Ca2+ release, and reduced the activation and termination thresholds for spontaneous Ca2+ release in HEK293 cells. We also showed that racemic carvedilol and the non-beta-blocking carvedilol enantiomer, (R)-carvedilol, suppressed spontaneous Ca2+ oscillations in HEK293 cells expressing the central domain RyR2 mutations associated with CPVT and AF. These data indicate that the central domain is an important determinant of cytosolic Ca2+ activation of RyR2. These results also suggest that altered cytosolic Ca2+ activation of RyR2 represents a common defect of RyR2 mutations associated with CPVT and AF, which could potentially be suppressed by carvedilol or (R)-carvedilol.

Background Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an ion channelopathy usually caused by gain-of-function mutations ryanodine receptor type-2 (RyR2). Left ventricular non-compaction (LVNC) is an often genetic cardiomyopathy. A rare LVNC-CPVT overlap syndrome may be caused by exon 3 deletion in RyR2. We sought to characterize the phenotypic spectrum and molecular basis of a novel RyR2 mutation identified in a family with both conditions. Methods Several members of an affected family underwent clinical and genetic assessments. A homology model of the RyR2 pore-region was generated to predict the location and potential impact of their RyR2 mutation. Ca 2+ -release assays were performed to characterize the functional impact of the RyR2 mutant expressed in HEK293 cells. Results A multigenerational family presented with a history of sudden death and a phenotype of atypical CPVT and LVNC. Genetic testing revealed a RYR2 mutation (I4855M) in two affected individuals. A homology model of the RyR2 pore-region showed that the I4855M mutant reside is located in the highly conserved ‘inner vestibule', a water-filled cavity. I4855M may interfere with Ca 2+ permeation and affect interactions between RyR2 pore subunits, and is thus predicted in silico to be damaging. Expression and functional studies in HEK293 cells revealed that I4855M inhibited caffeine-induced Ca 2+ release and exerted a dominant-negative impact on wild type RyR2. Conclusions This study identifies a potentially lethal overlapping syndrome of LVNC and atypical CPVT related to a novel RYR2 variant. Structural and functional studies suggest that this is a loss-of-function mutation, which exerts a dominant-negative effect on wild type RyR2.

Beat-to-beat alternations in the amplitude of the cytosolic Ca2+ transient (Ca2+ alternans) are thought to be the primary cause of cardiac alternans that can lead to cardiac arrhythmias and sudden death. Despite its important role in arrhythmogenesis, the mechanism underlying Ca2+ alternans remains poorly understood. Here, we investigated the role of cardiac ryanodine receptor (RyR2), the major Ca2+ release channel responsible for cytosolic Ca2+ transients, in cardiac alternans. Using a unique mouse model harboring a suppression-of-function (SOF) RyR2 mutation (E4872Q), we assessed the effect of genetically suppressing RyR2 function on Ca2+ and action potential duration (APD) alternans in intact hearts, and electrocardiogram (ECG) alternans in vivo. We found that RyR2-SOF hearts displayed prolonged sarcoplasmic reticulum Ca2+ release refractoriness and enhanced propensity for Ca2+ alternans. RyR2-SOF hearts/mice also exhibited increased propensity for APD and ECG alternans. Caffeine, which enhances RyR2 activity and the propensity for catecholaminergic polymorphic ventricular tachycardia (CPVT), suppressed Ca2+ alternans in RyR2-SOF hearts, whereas carvedilol, a β-blocker that suppresses RyR2 activity and CPVT, promoted Ca2+ alternans in these hearts. Thus, RyR2 function is an important determinant of Ca2+, APD, and ECG alternans. Our data also indicate that the activity of RyR2 influences the propensity for cardiac alternans and CPVT in an opposite manner. Therefore, overly suppressing or enhancing RyR2 function is pro-arrhythmic.