Structural Basis for Gating and Activation of RyR1.

The type-1 ryanodine receptor (RyR1) is an intracellular calcium (Ca(2+)) release channel required for skeletal muscle contraction. Here, we present cryo-EM reconstructions of RyR1 in multiple functional states revealing the structural basis of channel gating and ligand-dependent activation. Binding sites for the channel activators Ca(2+), ATP, and caffeine were identified at interdomain interfaces of the C-terminal domain. Either ATP or Ca(2+) alone induces conformational changes in the cytoplasmic assembly ("priming"), without pore dilation. In contrast, in the presence of all three activating ligands, high-resolution reconstructions of open and closed states of RyR1 were obtained from the same sample, enabling analyses of conformational changes associated with gating. Gating involves global conformational changes in the cytosolic assembly accompanied by local changes in the transmembrane domain, which include bending of the S6 transmembrane segment and consequent pore dilation, displacement, and deformation of the S4-S5 linker and conformational changes in the pseudo-voltage-sensor domain.

Role of leaky neuronal ryanodine receptors in stress-induced cognitive dysfunction.

The type 2 ryanodine receptor/calcium release channel (RyR2), required for excitation-contraction coupling in the heart, is abundant in the brain. Chronic stress induces catecholamine biosynthesis and release, stimulating ß-adrenergic receptors and activating cAMP signaling pathways in neurons. In a murine chronic restraint stress model, neuronal RyR2 were phosphorylated by protein kinase A (PKA), oxidized, and nitrosylated, resulting in depletion of the stabilizing subunit calstabin2 (FKBP12.6) from the channel complex and intracellular calcium leak. Stress-induced cognitive dysfunction, including deficits in learning and memory, and reduced long-term potentiation (LTP) at the hippocampal CA3-CA1 connection were rescued by oral administration of S107, a compound developed in our laboratory that stabilizes RyR2-calstabin2 interaction, or by genetic ablation of the RyR2 PKA phosphorylation site at serine 2808. Thus, neuronal RyR2 remodeling contributes to stress-induced cognitive dysfunction. Leaky RyR2 could be a therapeutic target for treatment of stress-induced cognitive dysfunction.

Structural insights into the regulation of RyR1 by S100A1.

S100A1, a small homodimeric EF-hand Ca2+-binding protein (~21 kDa), plays an important regulatory role in Ca2+ signaling pathways involved in various biological functions including Ca2+ cycling and contractile performance in skeletal and cardiac myocytes. One key target of the S100A1 interactome is the ryanodine receptor (RyR), a huge homotetrameric Ca2+ release channel (~2.3 MDa) of the sarcoplasmic reticulum. Here, we report cryoelectron microscopy structures of S100A1 bound to RyR1, the skeletal muscle isoform, in absence and presence of Ca2+. Ca2+-free apo-S100A1 binds beneath the bridging solenoid (BSol) and forms contacts with the junctional solenoid and the shell-core linker of RyR1. Upon Ca2+-binding, S100A1 undergoes a conformational change resulting in the exposure of the hydrophobic pocket known to serve as a major interaction site of S100A1. Through interactions of the hydrophobic pocket with RyR1, Ca2+-bound S100A1 intrudes deeper into the RyR1 structure beneath BSol than the apo-form and induces sideways motions of the C-terminal BSol region toward the adjacent RyR1 protomer resulting in tighter interprotomer contacts. Interestingly, the second hydrophobic pocket of the S100A1-dimer is largely exposed at the hydrophilic surface making it prone to interactions with the local environment, suggesting that S100A1 could be involved in forming larger heterocomplexes of RyRs with other protein partners. Since S100A1 interactions stabilizing BSol are implicated in the regulation of RyR-mediated Ca2+ release, the characterization of the S100A1 binding site conserved between RyR isoforms may provide the structural basis for the development of therapeutic strategies regarding treatments of RyR-related disorders.

Ryanodine receptor remodeling in cardiomyopathy and muscular dystrophy caused by lamin A/C gene mutation.

Mutations in the lamin A/C gene (LMNA), which encodes A-type lamins, cause several diseases called laminopathies, the most common of which is dilated cardiomyopathy with muscular dystrophy. The role of Ca2+ regulation in these diseases remain poorly understood. We now show biochemical remodeling of the ryanodine receptor (RyR)/intracellular Ca2+ release channel in heart samples from human subjects with LMNA mutations, including protein kinase A-catalyzed phosphorylation, oxidation and depletion of the stabilizing subunit calstabin. In the LmnaH222P/H222P murine model of Emery-Dreifuss muscular dystrophy caused by LMNA mutation, we demonstrate an age-dependent biochemical remodeling of RyR2 in the heart and RyR1 in skeletal muscle. This RyR remodeling is associated with heart and skeletal muscle dysfunction. Defective heart and muscle function are ameliorated by treatment with a novel Rycal small molecule drug (S107) that fixes 'leaky' RyRs. SMAD3 phosphorylation is increased in hearts and diaphragms of LmnaH222P/H222P mice, which enhances NADPH oxidase binding to RyR channels, contributing to their oxidation. There is also increased generalized protein oxidation, increased calcium/calmodulin-dependent protein kinase II-catalyzed phosphorylation of RyRs and increased protein kinase A activity in these tissues. Our data show that RyR remodeling plays a role in cardiomyopathy and skeletal muscle dysfunction caused by LMNA mutation and identify these Ca2+ channels as a potential therapeutic target.

Defective cerebellar ryanodine receptor type 1 and endoplasmic reticulum calcium 'leak' in tremor pathophysiology.

Essential Tremor (ET) is a prevalent neurological disease characterized by an 8-10 Hz action tremor. Molecular mechanisms of ET remain poorly understood. Clinical data suggest the importance of the cerebellum in disease pathophysiology, and pathological studies indicate Purkinje Cells (PCs) incur damage. Our recent cerebellar cortex and PC-specific transcriptome studies identified alterations in calcium (Ca2+) signaling pathways that included ryanodine receptor type 1 (RyR1) in ET. RyR1 is an intracellular Ca2+ release channel located on the Endoplasmic Reticulum (ER), and in cerebellum is predominantly expressed in PCs. Under stress conditions, RyR1 undergoes several post-translational modifications (protein kinase A [PKA] phosphorylation, oxidation, nitrosylation), coupled with depletion of the channel-stabilizing binding partner calstabin1, which collectively characterize a "leaky channel" biochemical signature. In this study, we found markedly increased PKA phosphorylation at the RyR1-S2844 site, increased RyR1 oxidation and nitrosylation, and calstabin1 depletion from the RyR1 complex in postmortem ET cerebellum. Decreased calstabin1-RyR1-binding affinity correlated with loss of PCs and climbing fiber-PC synapses in ET. This 'leaky' RyR1 signature was not seen in control or Parkinson's disease cerebellum. Microsomes from postmortem cerebellum demonstrated excessive ER Ca2+ leak in ET vs. controls, attenuated by channel stabilization. We further studied the role of RyR1 in tremor using a mouse model harboring a RyR1 point mutation that mimics constitutive site-specific PKA phosphorylation (RyR1-S2844D). RyR1-S2844D homozygous mice develop a 10 Hz action tremor and robust abnormal oscillatory activity in cerebellar physiological recordings. Intra-cerebellar microinfusion of RyR1 agonist or antagonist, respectively, increased or decreased tremor amplitude in RyR1-S2844D mice, supporting a direct role of cerebellar RyR1 leakiness for tremor generation. Treating RyR1-S2844D mice with a novel RyR1 channel-stabilizing compound, Rycal, effectively dampened cerebellar oscillatory activity, suppressed tremor, and normalized cerebellar RyR1-calstabin1 binding. These data collectively support that stress-associated ER Ca2+ leak via RyR1 may contribute to tremor pathophysiology.

PTEN arginine methylation by PRMT6 suppresses PI3K-AKT signaling and modulates pre-mRNA splicing.

Arginine methylation is a ubiquitous posttranslational modification that regulates critical cellular processes including signal transduction and pre-mRNA splicing. Here, we report that the tumor-suppressor PTEN is methylated by protein arginine methyltransferase 6 (PRMT6). Mass-spectrometry analysis reveals that PTEN is dimethylated at arginine 159 (R159). We found that PTEN is mutated at R159 in cancers, and the PTEN mutant R159K loses its capability to inhibit the PI3K-AKT cascade. Furthermore, PRMT6 is physically associated with PTEN, promotes asymmetrical dimethylation of PTEN, and regulates the PI3K-AKT cascade through PTEN R159 methylation. In addition, using transcriptome analyses, we found that PTEN R159 methylation is involved in modulation of pre-mRNA alternative splicing. Our results demonstrate that PTEN is functionally regulated by arginine methylation. We propose that PTEN arginine methylation modulates pre-mRNA alternative splicing and influences diverse physiologic processes.

Alzheimer's-like signaling in brains of COVID-19 patients.

INTRODUCTION: The mechanisms that lead to cognitive impairment associated with COVID-19 are not well understood. METHODS: Brain lysates from control and COVID-19 patients were analyzed for oxidative stress and inflammatory signaling pathway markers, and measurements of Alzheimer's disease (AD)-linked signaling biochemistry. Post-translational modifications of the ryanodine receptor/calcium (Ca2+ ) release channels (RyR) on the endoplasmic reticuli (ER), known to be linked to AD, were also measured by co-immunoprecipitation/immunoblotting of the brain lysates. RESULTS: We provide evidence linking SARS-CoV-2 infection to activation of TGF-ß signaling and oxidative overload. The neuropathological pathways causing tau hyperphosphorylation typically associated with AD were also shown to be activated in COVID-19 patients. RyR2 in COVID-19 brains demonstrated a "leaky" phenotype, which can promote cognitive and behavioral defects. DISCUSSION: COVID-19 neuropathology includes AD-like features and leaky RyR2 channels could be a therapeutic target for amelioration of some cognitive defects associated with SARS-CoV-2 infection and long COVID.

Ca2+ Release Channels Join the 'Resolution Revolution'.

Ryanodine receptors (RyRs) are calcium release channels expressed in the sarcoendoplasmic reticula of many cell types including cardiac and skeletal muscle cells. In recent years Ca2+ leak through RyRs has been implicated as a major contributor to the development of diseases including heart failure, muscle myopathies, Alzheimer's disease, and diabetes, making it an important therapeutic target. Recent mammalian RyR1 cryoelectron microscopy (cryo-EM) structures of multiple functional states have clarified longstanding questions including the architecture of the transmembrane (TM) pore and cytoplasmic domains, the location and architecture of the channel gate, ligand-binding sites, and the gating mechanism. As we advance toward complete models of RyRs this new information enables the determination of domain-domain interfaces and the location and structural effects of disease-causing RyR mutations.

Structure of a mammalian ryanodine receptor.

Ryanodine receptors (RyRs) mediate the rapid release of calcium (Ca(2+)) from intracellular stores into the cytosol, which is essential for numerous cellular functions including excitation-contraction coupling in muscle. Lack of sufficient structural detail has impeded understanding of RyR gating and regulation. Here we report the closed-state structure of the 2.3-megadalton complex of the rabbit skeletal muscle type 1 RyR (RyR1), solved by single-particle electron cryomicroscopy at an overall resolution of 4.8 Å. We fitted a polyalanine-level model to all 3,757 ordered residues in each protomer, defining the transmembrane pore in unprecedented detail and placing all cytosolic domains as tertiary folds. The cytosolic assembly is built on an extended α-solenoid scaffold connecting key regulatory domains to the pore. The RyR1 pore architecture places it in the six-transmembrane ion channel superfamily. A unique domain inserted between the second and third transmembrane helices interacts intimately with paired EF-hands originating from the α-solenoid scaffold, suggesting a mechanism for channel gating by Ca(2+).

Single-channel recordings of RyR1 at microsecond resolution in CMOS-suspended membranes.

Single-channel recordings are widely used to explore functional properties of ion channels. Typically, such recordings are performed at bandwidths of less than 10 kHz because of signal-to-noise considerations, limiting the temporal resolution available for studying fast gating dynamics to greater than 100 µs. Here we present experimental methods that directly integrate suspended lipid bilayers with high-bandwidth, low-noise transimpedance amplifiers based on complementary metal-oxide-semiconductor (CMOS) integrated circuits (IC) technology to achieve bandwidths in excess of 500 kHz and microsecond temporal resolution. We use this CMOS-integrated bilayer system to study the type 1 ryanodine receptor (RyR1), a Ca2+-activated intracellular Ca2+-release channel located on the sarcoplasmic reticulum. We are able to distinguish multiple closed states not evident with lower bandwidth recordings, suggesting the presence of an additional Ca2+ binding site, distinct from the site responsible for activation. An extended beta distribution analysis of our high-bandwidth data can be used to infer closed state flicker events as fast as 35 ns. These events are in the range of single-file ion translocations.

Late Ventilator-Induced Diaphragmatic Dysfunction After Extubation.

OBJECTIVES: Mechanical ventilation is associated with primary diaphragmatic dysfunction, also termed ventilator-induced diaphragmatic dysfunction. Studies evaluating diaphragmatic function recovery after extubation are lacking. We evaluated early and late recoveries from ventilator-induced diaphragmatic dysfunction in a mouse model. DESIGN: Experimental randomized study. SETTING: Research laboratory. SUBJECTS: C57/BL6 mice. INTERVENTIONS: Six groups of C57/BL6 mice. Mice were ventilated for 6 hours and then euthanatized immediately (n = 18), or 1 (n = 18) or 10 days after extubation with (n = 5) and without S107 (n = 16) treatment. Mice euthanatized immediately after 6 hours of anesthesia (n = 15) or after 6 hours of anesthesia and 10 days of recovery (n = 5) served as controls. MEASUREMENTS AND MAIN RESULTS: For each group, diaphragm force production, posttranslational modification of ryanodine receptor, oxidative stress, proteolysis, and cross-sectional areas were evaluated. After 6 hours of mechanical ventilation, diaphragm force production was decreased by 25-30%, restored to the control levels 1 day after extubation, and secondarily decreased by 20% 10 days after extubation compared with controls. Ryanodine receptor was protein kinase A-hyperphosphorylated, S-nitrosylated, oxidized, and depleted of its stabilizing subunit calstabin-1 6 hours after the onset of the mechanical ventilation, 1 and 10 days after extubation. Post extubation treatment with S107, a Rycal drug that stabilizes the ryanodine complex, did reverse the loss of diaphragmatic force associated with mechanical ventilation. Total protein oxidation was restored to the control levels 1 day after extubation. Markers of proteolysis including calpain 1 and calpain 2 remained activated 10 days after extubation without significant changes in cross-sectional areas. CONCLUSIONS: We report that mechanical ventilation is associated with a late diaphragmatic dysfunction related to a structural alteration of the ryanodine complex that is reversed with the S107 treatment.

Intracellular calcium leak as a therapeutic target for RYR1-related myopathies.

RYR1 encodes the type 1 ryanodine receptor, an intracellular calcium release channel (RyR1) on the skeletal muscle sarcoplasmic reticulum (SR). Pathogenic RYR1 variations can destabilize RyR1 leading to calcium leak causing oxidative overload and myopathy. However, the effect of RyR1 leak has not been established in individuals with RYR1-related myopathies (RYR1-RM), a broad spectrum of rare neuromuscular disorders. We sought to determine whether RYR1-RM affected individuals exhibit pathologic, leaky RyR1 and whether variant location in the channel structure can predict pathogenicity. Skeletal muscle biopsies were obtained from 17 individuals with RYR1-RM. Mutant RyR1 from these individuals exhibited pathologic SR calcium leak and increased activity of calcium-activated proteases. The increased calcium leak and protease activity were normalized by ex-vivo treatment with S107, a RyR stabilizing Rycal molecule. Using the cryo-EM structure of RyR1 and a new dataset of > 2200 suspected RYR1-RM affected individuals we developed a method for assigning pathogenicity probabilities to RYR1 variants based on 3D co-localization of known pathogenic variants. This study provides the rationale for a clinical trial testing Rycals in RYR1-RM affected individuals and introduces a predictive tool for investigating the pathogenicity of RYR1 variants of uncertain significance.

Ryanodine Receptor Calcium Leak in Circulating B-Lymphocytes as a Biomarker in Heart Failure.

BACKGROUND: Advances in congestive heart failure (CHF) management depend on biomarkers for monitoring disease progression and therapeutic response. During systole, intracellular Ca2+ is released from the sarcoplasmic reticulum into the cytoplasm through type-2 ryanodine receptor/Ca2+ release channels. In CHF, chronically elevated circulating catecholamine levels cause pathological remodeling of type-2 ryanodine receptor/Ca2+ release channels resulting in diastolic sarcoplasmic reticulum Ca2+ leak and decreased myocardial contractility. Similarly, skeletal muscle contraction requires sarcoplasmic reticulum Ca2+ release through type-1 ryanodine receptors (RyR1), and chronically elevated catecholamine levels in CHF cause RyR1-mediated sarcoplasmic reticulum Ca2+ leak, contributing to myopathy and weakness. Circulating B-lymphocytes express RyR1 and catecholamine-responsive signaling cascades, making them a potential surrogate for defects in intracellular Ca2+ handling because of leaky RyR channels in CHF. METHODS: Whole blood was collected from patients with CHF, CHF following left-ventricular assist device implant, and controls. Blood was also collected from mice with ischemic CHF, ischemic CHF+S107 (a drug that specifically reduces RyR channel Ca2+ leak), and wild-type controls. Channel macromolecular complex was assessed by immunostaining RyR1 immunoprecipitated from lymphocyte-enriched preparations. RyR1 Ca2+ leak was assessed using flow cytometry to measure Ca2+ fluorescence in B-lymphocytes in the absence and presence of RyR1 agonists that empty RyR1 Ca2+ stores within the endoplasmic reticulum. RESULTS: Circulating B-lymphocytes from humans and mice with CHF exhibited remodeled RyR1 and decreased endoplasmic reticulum Ca2+ stores, consistent with chronic intracellular Ca2+ leak. This Ca2+ leak correlated with circulating catecholamine levels. The intracellular Ca2+ leak was significantly reduced in mice treated with the Rycal S107. Patients with CHF treated with left-ventricular assist devices exhibited a heterogeneous response. CONCLUSIONS: In CHF, B-lymphocytes exhibit remodeled leaky RyR1 channels and decreased endoplasmic reticulum Ca2+ stores consistent with chronic intracellular Ca2+ leak. RyR1-mediated Ca2+ leak in B-lymphocytes assessed using flow cytometry provides a surrogate measure of intracellular Ca2+ handling and systemic sympathetic burden, presenting a novel biomarker for monitoring response to pharmacological and mechanical CHF therapy.

Ryanodine Receptor Structure and Function in Health and Disease.

Ryanodine receptors (RyRs) are ubiquitous intracellular calcium (Ca2+) release channels required for the function of many organs including heart and skeletal muscle, synaptic transmission in the brain, pancreatic beta cell function, and vascular tone. In disease, defective function of RyRs due either to stress (hyperadrenergic and/or oxidative overload) or genetic mutations can render the channels leaky to Ca2+ and promote defective disease-causing signals as observed in heat failure, muscular dystrophy, diabetes mellitus, and neurodegerative disease. RyRs are massive structures comprising the largest known ion channel-bearing macromolecular complex and exceeding 3 million Daltons in molecular weight. RyRs mediate the rapid release of Ca2+ from the endoplasmic/sarcoplasmic reticulum (ER/SR) to stimulate cellular functions through Ca2+-dependent processes. Recent advances in single-particle cryogenic electron microscopy (cryo-EM) have enabled the determination of atomic-level structures for RyR for the first time. These structures have illuminated the mechanisms by which these critical ion channels function and interact with regulatory ligands. In the present chapter we discuss the structure, functional elements, gating and activation mechanisms of RyRs in normal and disease states.

Leaky ryanodine receptors contribute to diaphragmatic weakness during mechanical ventilation.

Ventilator-induced diaphragmatic dysfunction (VIDD) refers to the diaphragm muscle weakness that occurs following prolonged controlled mechanical ventilation (MV). The presence of VIDD impedes recovery from respiratory failure. However, the pathophysiological mechanisms accounting for VIDD are still not fully understood. Here, we show in human subjects and a mouse model of VIDD that MV is associated with rapid remodeling of the sarcoplasmic reticulum (SR) Ca(2+) release channel/ryanodine receptor (RyR1) in the diaphragm. The RyR1 macromolecular complex was oxidized, S-nitrosylated, Ser-2844 phosphorylated, and depleted of the stabilizing subunit calstabin1, following MV. These posttranslational modifications of RyR1 were mediated by both oxidative stress mediated by MV and stimulation of adrenergic signaling resulting from the anesthesia. We demonstrate in the murine model that such abnormal resting SR Ca(2+) leak resulted in reduced contractile function and muscle fiber atrophy for longer duration of MV. Treatment with ß-adrenergic antagonists or with S107, a small molecule drug that stabilizes the RyR1-calstabin1 interaction, prevented VIDD. Diaphragmatic dysfunction is common in MV patients and is a major cause of failure to wean patients from ventilator support. This study provides the first evidence to our knowledge of RyR1 alterations as a proximal mechanism underlying VIDD (i.e., loss of function, muscle atrophy) and identifies RyR1 as a potential target for therapeutic intervention.

Maintenance of normal blood pressure is dependent on IP3R1-mediated regulation of eNOS.

Endothelial cells (ECs) are critical mediators of blood pressure (BP) regulation, primarily via the generation and release of vasorelaxants, including nitric oxide (NO). NO is produced in ECs by endothelial NO synthase (eNOS), which is activated by both calcium (Ca(2+))-dependent and independent pathways. Here, we report that intracellular Ca(2+) release from the endoplasmic reticulum (ER) via inositol 1,4,5-trisphosphate receptor (IP3R) is required for Ca(2+)-dependent eNOS activation. EC-specific type 1 1,4,5-trisphosphate receptor knockout (IP3R1(-/-)) mice are hypertensive and display blunted vasodilation in response to acetylcholine (ACh). Moreover, eNOS activity is reduced in both isolated IP3R1-deficient murine ECs and human ECs following IP3R1 knockdown. IP3R1 is upstream of calcineurin, a Ca(2+)/calmodulin-activated serine/threonine protein phosphatase. We show here that the calcineurin/nuclear factor of activated T cells (NFAT) pathway is less active and eNOS levels are decreased in IP3R1-deficient ECs. Furthermore, the calcineurin inhibitor cyclosporin A, whose use has been associated with the development of hypertension, reduces eNOS activity and vasodilation following ACh stimulation. Our results demonstrate that IP3R1 plays a crucial role in the EC-mediated vasorelaxation and the maintenance of normal BP.

Amyloid ß production is regulated by ß2-adrenergic signaling-mediated post-translational modifications of the ryanodine receptor.

Alteration of ryanodine receptor (RyR)-mediated calcium (Ca2+) signaling has been reported in Alzheimer disease (AD) models. However, the molecular mechanisms underlying altered RyR-mediated intracellular Ca2+ release in AD remain to be fully elucidated. We report here that RyR2 undergoes post-translational modifications (phosphorylation, oxidation, and nitrosylation) in SH-SY5Y neuroblastoma cells expressing the ß-amyloid precursor protein (ßAPP) harboring the familial double Swedish mutations (APPswe). RyR2 macromolecular complex remodeling, characterized by depletion of the regulatory protein calstabin2, resulted in increased cytosolic Ca2+ levels and mitochondrial oxidative stress. We also report a functional interplay between amyloid ß (Aß), ß-adrenergic signaling, and altered Ca2+ signaling via leaky RyR2 channels. Thus, post-translational modifications of RyR occur downstream of Aß through a ß2-adrenergic signaling cascade that activates PKA. RyR2 remodeling in turn enhances ßAPP processing. Importantly, pharmacological stabilization of the binding of calstabin2 to RyR2 channels, which prevents Ca2+ leakage, or blocking the ß2-adrenergic signaling cascade reduced ßAPP processing and the production of Aß in APPswe-expressing SH-SY5Y cells. We conclude that targeting RyR-mediated Ca2+ leakage may be a therapeutic approach to treat AD.

Mitochondrial calcium overload is a key determinant in heart failure.

Calcium (Ca2+) released from the sarcoplasmic reticulum (SR) is crucial for excitation-contraction (E-C) coupling. Mitochondria, the major source of energy, in the form of ATP, required for cardiac contractility, are closely interconnected with the SR, and Ca2+ is essential for optimal function of these organelles. However, Ca2+ accumulation can impair mitochondrial function, leading to reduced ATP production and increased release of reactive oxygen species (ROS). Oxidative stress contributes to heart failure (HF), but whether mitochondrial Ca2+ plays a mechanistic role in HF remains unresolved. Here, we show for the first time, to our knowledge, that diastolic SR Ca2+ leak causes mitochondrial Ca2+ overload and dysfunction in a murine model of postmyocardial infarction HF. There are two forms of Ca2+ release channels on cardiac SR: type 2 ryanodine receptors (RyR2s) and type 2 inositol 1,4,5-trisphosphate receptors (IP3R2s). Using murine models harboring RyR2 mutations that either cause or inhibit SR Ca2+ leak, we found that leaky RyR2 channels result in mitochondrial Ca2+ overload, dysmorphology, and malfunction. In contrast, cardiac-specific deletion of IP3R2 had no major effect on mitochondrial fitness in HF. Moreover, genetic enhancement of mitochondrial antioxidant activity improved mitochondrial function and reduced posttranslational modifications of RyR2 macromolecular complex. Our data demonstrate that leaky RyR2, but not IP3R2, channels cause mitochondrial Ca2+ overload and dysfunction in HF.

Intracellular calcium release channels: an update.

Ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3 Rs) are calcium (Ca2+ ) release channels on the endo/sarcoplasmic reticulum (ER/SR). Here we summarize the latest advances in the field, describing the recently discovered mechanistic roles of intracellular Ca2+ release channels in the regulation of mitochondrial fitness and endothelial function, providing novel therapeutic options for the treatment of heart failure, hypertension, and diabetes mellitus.

Post-translational remodeling of ryanodine receptor induces calcium leak leading to Alzheimer's disease-like pathologies and cognitive deficits.

The mechanisms underlying ryanodine receptor (RyR) dysfunction associated with Alzheimer disease (AD) are still not well understood. Here, we show that neuronal RyR2 channels undergo post-translational remodeling (PKA phosphorylation, oxidation, and nitrosylation) in brains of AD patients, and in two murine models of AD (3 × Tg-AD, APP +/- /PS1 +/-). RyR2 is depleted of calstabin2 (KFBP12.6) in the channel complex, resulting in endoplasmic reticular (ER) calcium (Ca2+) leak. RyR-mediated ER Ca2+ leak activates Ca2+-dependent signaling pathways, contributing to AD pathogenesis. Pharmacological (using a novel RyR stabilizing drug Rycal) or genetic rescue of the RyR2-mediated intracellular Ca2+ leak improved synaptic plasticity, normalized behavioral and cognitive functions and reduced Aß load. Genetically altered mice with congenitally leaky RyR2 exhibited premature and severe defects in synaptic plasticity, behavior and cognitive function. These data provide a mechanism underlying leaky RyR2 channels, which could be considered as potential AD therapeutic targets.