Location of ryanodine receptor type 2 mutation predicts age of onset of sudden death in catecholaminergic polymorphic ventricular tachycardia - A systematic review and meta-analysis of case-based literature.

Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare inherited arrhythmia caused by mutations in the ryanodine receptor type 2 (RyR2). Diagnosis of CPVT often occurs after a major cardiac event, thus posing a severe threat to the patient's health. Methods: Publication databases, including PubMed, Scopus, and Embase, were searched for articles on patients with RyR2-CPVT mutations and their associated clinical presentation. Articles were reviewed by two independent reviewers and mutations were analyzed for demographic information, mutation distribution, and therapeutics. The human RyR2 cryo-EM structure was used to model CPVT mutations and predict the diagnosis and outcomes of CPVT patients. Findings: We present a database of 1008 CPVT patients from 227 papers. Data analyses revealed that patients most often experienced exercise-induced syncope in their early teenage years but the diagnosis of CPVT took a decade. Mutations located near key regulatory sites in the channel were associated with earlier onset of CPVT symptoms including sudden cardiac death. Interpretation: The present study provides a road map for predicting clinical outcomes based on the location of RyR2 mutations in CPVT patients. The study was partially limited by the inconsistency in the depth of information provided in each article, but nevertheless is an important contribution to the understanding of the clinical and molecular basis of CPVT and suggests the need for early diagnosis and creative approaches to disease management. Funding: The work was supported by grant NIH R01HL145473, P01 HL164319 R25HL156002, T32 HL120826.

Aberrant mitochondrial dynamics contributes to diaphragmatic weakness induced by mechanical ventilation.

In critical care patients, the ""temporary inactivity of the diaphragm caused by mechanical ventilation (MV) triggers a series of events leading to diaphragmatic dysfunction and atrophy, commonly known as ventilator-induced diaphragm dysfunction (VIDD). While mitochondrial dysfunction related to oxidative stress is recognized as a crucial factor in VIDD, the exact molecular mechanism remains poorly understood. In this study, we observe that 6 h of MV triggers aberrant mitochondrial dynamics, resulting in a reduction in mitochondrial size and interaction, associated with increased expression of dynamin-related protein 1 (DRP1). This effect can be prevented by P110, a molecule that inhibits the recruitment of DRP1 to the mitochondrial membrane. Furthermore, isolated mitochondria from the diaphragms of ventilated patients exhibited increased production of reactive oxygen species (ROS). These mitochondrial changes were associated with the rapid oxidation of type 1 ryanodine receptor (RyR1) and a decrease in the stabilizing subunit calstabin 1. Subsequently, we observed that the sarcoplasmic reticulum (SR) in the ventilated diaphragms showed increased calcium leakage and reduced contractile function. Importantly, the mitochondrial fission inhibitor P110 effectively prevented all of these alterations. Taken together, the results of our study illustrate that MV leads, in the diaphragm, to both mitochondrial fragmentation and dysfunction, linked to the up-/down-regulation of 320 proteins, as assessed through global comprehensive quantitative proteomics analysis, primarily associated with mitochondrial function. These outcomes underscore the significance of developing compounds aimed at modulating the balance between mitochondrial fission and fusion as potential interventions to mitigate VIDD in human patients.

Targeting ryanodine receptor type 2 to mitigate chemotherapy-induced neurocognitive impairments in mice.

Chemotherapy-induced cognitive dysfunction (chemobrain) is an important adverse sequela of chemotherapy. Chemobrain has been identified by the National Cancer Institute as a poorly understood problem for which current management or treatment strategies are limited or ineffective. Here, we show that chemotherapy treatment with doxorubicin (DOX) in a breast cancer mouse model induced protein kinase A (PKA) phosphorylation of the neuronal ryanodine receptor/calcium (Ca2+) channel type 2 (RyR2), RyR2 oxidation, RyR2 nitrosylation, RyR2 calstabin2 depletion, and subsequent RyR2 Ca2+ leakiness. Chemotherapy was furthermore associated with abnormalities in brain glucose metabolism and neurocognitive dysfunction in breast cancer mice. RyR2 leakiness and cognitive dysfunction could be ameliorated by treatment with a small molecule Rycal drug (S107). Chemobrain was also found in noncancer mice treated with DOX or methotrexate and 5-fluorouracil and could be prevented by treatment with S107. Genetic ablation of the RyR2 PKA phosphorylation site (RyR2-S2808A) also prevented the development of chemobrain. Chemotherapy increased brain concentrations of the tumor necrosis factor-α and transforming growth factor-ß signaling, suggesting that increased inflammatory signaling might contribute to oxidation-driven biochemical remodeling of RyR2. Proteomics and Gene Ontology analysis indicated that the signaling downstream of chemotherapy-induced leaky RyR2 was linked to the dysregulation of synaptic structure-associated proteins that are involved in neurotransmission. Together, our study points to neuronal Ca2+ dyshomeostasis via leaky RyR2 channels as a potential mechanism contributing to chemobrain, warranting further translational studies.

Personalized medicine in the dish to prevent calcium leak associated with short-coupled polymorphic ventricular tachycardia in patient-derived cardiomyocytes.

BACKGROUND: Polymorphic ventricular tachycardia (PMVT) is a rare genetic disease associated with structurally normal hearts which in 8% of cases can lead to sudden cardiac death, typically exercise-induced. We previously showed a link between the RyR2-H29D mutation and a clinical phenotype of short-coupled PMVT at rest using patient-specific hiPSC-derived cardiomyocytes (hiPSC-CMs). In the present study, we evaluated the effects of clinical and experimental anti-arrhythmic drugs on the intracellular Ca2+ handling, contractile and molecular properties in PMVT hiPSC-CMs in order to model a personalized medicine approach in vitro. METHODS: Previously, a blood sample from a patient carrying the RyR2-H29D mutation was collected and reprogrammed into several clones of RyR2-H29D hiPSCs, and in addition we generated an isogenic control by reverting the RyR2-H29D mutation using CRIPSR/Cas9 technology. Here, we tested 4 drugs with anti-arrhythmic properties: propranolol, verapamil, flecainide, and the Rycal S107. We performed fluorescence confocal microscopy, video-image-based analyses and biochemical analyses to investigate the impact of these drugs on the functional and molecular features of the PMVT RyR2-H29D hiPSC-CMs. RESULTS: The voltage-dependent Ca2+ channel inhibitor verapamil did not prevent the aberrant release of sarcoplasmic reticulum (SR) Ca2+ in the RyR2-H29D hiPSC-CMs, whereas it was prevented by S107, flecainide or propranolol. Cardiac tissue comprised of RyR2-H29D hiPSC-CMs exhibited aberrant contractile properties that were largely prevented by S107, flecainide and propranolol. These 3 drugs also recovered synchronous contraction in RyR2-H29D cardiac tissue, while verapamil did not. At the biochemical level, S107 was the only drug able to restore calstabin2 binding to RyR2 as observed in the isogenic control. CONCLUSIONS: By testing 4 drugs on patient-specific PMVT hiPSC-CMs, we concluded that S107 and flecainide are the most potent molecules in terms of preventing the abnormal SR Ca2+ release and contractile properties in RyR2-H29D hiPSC-CMs, whereas the effect of propranolol is partial, and verapamil appears ineffective. In contrast with the 3 other drugs, S107 was able to prevent a major post-translational modification of RyR2-H29D mutant channels, the loss of calstabin2 binding to RyR2. Using patient-specific hiPSC and CRISPR/Cas9 technologies, we showed that S107 is the most efficient in vitro candidate for treating the short-coupled PMVT at rest.

Heart failure-induced cognitive dysfunction is mediated by intracellular Ca2+ leak through ryanodine receptor type 2.

Cognitive dysfunction (CD) in heart failure (HF) adversely affects treatment compliance and quality of life. Although ryanodine receptor type 2 (RyR2) has been linked to cardiac muscle dysfunction, its role in CD in HF remains unclear. Here, we show in hippocampal neurons from individuals and mice with HF that the RyR2/intracellular Ca2+ release channels were subjected to post-translational modification (PTM) and were leaky. RyR2 PTM included protein kinase A phosphorylation, oxidation, nitrosylation and depletion of the stabilizing subunit calstabin2. RyR2 PTM was caused by hyper-adrenergic signaling and activation of the transforming growth factor-beta pathway. HF mice treated with a RyR2 stabilizer drug (S107), beta blocker (propranolol) or transforming growth factor-beta inhibitor (SD-208), or genetically engineered mice resistant to RyR2 Ca2+ leak (RyR2-p.Ser2808Ala), were protected against HF-induced CD. Taken together, we propose that HF is a systemic illness driven by intracellular Ca2+ leak that includes cardiogenic dementia.

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.

Zoledronic acid improves bone quality and muscle function in a high bone turnover state.

SUMMARY: Zoledronic acid (ZA) prevents muscle weakness in mice with bone metastases; however, its role in muscle weakness in non-tumor-associated metabolic bone diseases and as an effective treatment modality for the prevention of muscle weakness associated with bone disorders, is unknown. We demonstrate the role of ZA-treatment on bone and muscle using a mouse model of accelerated bone remodeling, which represents the clinical manifestation of non-tumor associated metabolic bone disease. ZA increased bone mass and strength and rescued osteocyte lacunocanalicular organization. Short-term ZA treatment increased muscle mass, whereas prolonged, preventive treatment improved muscle mass and function. In these mice, muscle fiber-type shifted from oxidative to glycolytic and ZA restored normal muscle fiber distribution. By blocking TGFß release from bone, ZA improved muscle function, promoted myoblast differentiation and stabilized Ryanodine Receptor-1 calcium channel. These data demonstrate the beneficial effects of ZA in maintaining bone health and preserving muscle mass and function in a model of metabolic bone disease. Context and significance: TGFß is a bone regulatory molecule which is stored in bone matrix, released during bone remodeling, and must be maintained at an optimal level for the good health of the bone. Excess TGFß causes several bone disorders and skeletal muscle weakness. Reducing excess TGFß release from bone using zoledronic acid in mice not only improved bone volume and strength but also increased muscle mass, and muscle function. Progressive muscle weakness coexists with bone disorders, decreasing quality of life and increasing morbidity and mortality. Currently, there is a critical need for treatments improving muscle mass and function in patients with debilitating weakness. Zoledronic acid's benefit extends beyond bone and could also be useful in treating muscle weakness associated with bone disorders.

Structural analyses of human ryanodine receptor type 2 channels reveal the mechanisms for sudden cardiac death and treatment.

Ryanodine receptor type 2 (RyR2) mutations have been linked to an inherited form of exercise-induced sudden cardiac death called catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT results from stress-induced sarcoplasmic reticular Ca2+ leak via the mutant RyR2 channels during diastole. We present atomic models of human wild-type (WT) RyR2 and the CPVT mutant RyR2-R2474S determined by cryo-electron microscopy with overall resolutions in the range of 2.6 to 3.6 Å, and reaching local resolutions of 2.25 Å, unprecedented for RyR2 channels. Under nonactivating conditions, the RyR2-R2474S channel is in a "primed" state between the closed and open states of WT RyR2, rendering it more sensitive to activation that results in stress-induced Ca2+ leak. The Rycal drug ARM210 binds to RyR2-R2474S, reverting the primed state toward the closed state. Together, these studies provide a mechanism for CPVT and for the therapeutic actions of ARM210.

A drug and ATP binding site in type 1 ryanodine receptor.

The ryanodine receptor (RyR)/calcium release channel on the sarcoplasmic reticulum (SR) is required for excitation-contraction coupling in skeletal and cardiac muscle. Inherited mutations and stress-induced post-translational modifications result in an SR Ca2+ leak that causes skeletal myopathies, heart failure, and exercise-induced sudden death. A class of therapeutics known as Rycals prevent the RyR-mediated leak, are effective in preventing disease progression and restoring function in animal models, and are in clinical trials for patients with muscle and heart disorders. Using cryogenic-electron microscopy, we present a model of RyR1 with a 2.45-Å resolution before local refinement, revealing a binding site in the RY1&2 domain (3.10 Å local resolution), where the Rycal ARM210 binds cooperatively with ATP and stabilizes the closed state of RyR1.

Role of oxidation of excitation-contraction coupling machinery in age-dependent loss of muscle function in Caenorhabditis elegans.

Age-dependent loss of body wall muscle function and impaired locomotion occur within 2 weeks in Caenorhabditis elegans (C. elegans); however, the underlying mechanism has not been fully elucidated. In humans, age-dependent loss of muscle function occurs at about 80 years of age and has been linked to dysfunction of ryanodine receptor (RyR)/intracellular calcium (Ca2+) release channels on the sarcoplasmic reticulum (SR). Mammalian skeletal muscle RyR1 channels undergo age-related remodeling due to oxidative overload, leading to loss of the stabilizing subunit calstabin1 (FKBP12) from the channel macromolecular complex. This destabilizes the closed state of the channel resulting in intracellular Ca2+ leak, reduced muscle function, and impaired exercise capacity. We now show that the C. elegans RyR homolog, UNC-68, exhibits a remarkable degree of evolutionary conservation with mammalian RyR channels and similar age-dependent dysfunction. Like RyR1 in mammals, UNC-68 encodes a protein that comprises a macromolecular complex which includes the calstabin1 homolog FKB-2 and is immunoreactive with antibodies raised against the RyR1 complex. Furthermore, as in aged mammals, UNC-68 is oxidized and depleted of FKB-2 in an age-dependent manner, resulting in 'leaky' channels, depleted SR Ca2+ stores, reduced body wall muscle Ca2+ transients, and age-dependent muscle weakness. FKB-2 (ok3007)-deficient worms exhibit reduced exercise capacity. Pharmacologically induced oxidization of UNC-68 and depletion of FKB-2 from the channel independently caused reduced body wall muscle Ca2+ transients. Preventing FKB-2 depletion from the UNC-68 macromolecular complex using the Rycal drug S107 improved muscle Ca2+ transients and function. Taken together, these data suggest that UNC-68 oxidation plays a role in age-dependent loss of muscle function. Remarkably, this age-dependent loss of muscle function induced by oxidative overload, which takes ~2 years in mice and ~80 years in humans, occurs in less than 2-3 weeks in C. elegans, suggesting that reduced antioxidant capacity may contribute to the differences in lifespan among species.

IP3 receptor orchestrates maladaptive vascular responses in heart failure.

Patients with heart failure (HF) have augmented vascular tone, which increases cardiac workload, impairing ventricular output and promoting further myocardial dysfunction. The molecular mechanisms underlying the maladaptive vascular responses observed in HF are not fully understood. Vascular smooth muscle cells (VSMCs) control vasoconstriction via a Ca2+-dependent process, in which the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) on the sarcoplasmic reticulum (SR) plays a major role. To dissect the mechanistic contribution of intracellular Ca2+ release to the increased vascular tone observed in HF, we analyzed the remodeling of IP3R1 in aortic tissues from patients with HF and from controls. VSMC IP3R1 channels from patients with HF and HF mice were hyperphosphorylated by both serine and tyrosine kinases. VSMCs isolated from IP3R1VSMC-/- mice exhibited blunted Ca2+ responses to angiotensin II (ATII) and norepinephrine compared with control VSMCs. IP3R1VSMC-/- mice displayed significantly reduced responses to ATII, both in vivo and ex vivo. HF IP3R1VSMC-/- mice developed significantly less afterload compared with HF IP3R1fl/fl mice and exhibited significantly attenuated progression toward decompensated HF and reduced interstitial fibrosis. Ca2+-dependent phosphorylation of the MLC by MLCK activated VSMC contraction. MLC phosphorylation was markedly increased in VSMCs from patients with HF and HF mice but reduced in VSMCs from HF IP3R1VSMC-/- mice and HF WT mice treated with ML-7. Taken together, our data indicate that VSMC IP3R1 is a major effector of increased vascular tone, which contributes to increased cardiac afterload and decompensation in HF.

Acute RyR1 Ca2+ leak enhances NADH-linked mitochondrial respiratory capacity.

Sustained ryanodine receptor (RyR) Ca2+ leak is associated with pathological conditions such as heart failure or skeletal muscle weakness. We report that a single session of sprint interval training (SIT), but not of moderate intensity continuous training (MICT), triggers RyR1 protein oxidation and nitrosylation leading to calstabin1 dissociation in healthy human muscle and in in vitro SIT models (simulated SIT or S-SIT). This is accompanied by decreased sarcoplasmic reticulum Ca2+ content, increased levels of mitochondrial oxidative phosphorylation proteins, supercomplex formation and enhanced NADH-linked mitochondrial respiratory capacity. Mechanistically, (S-)SIT increases mitochondrial Ca2+ uptake in mouse myotubes and muscle fibres, and decreases pyruvate dehydrogenase phosphorylation in human muscle and mouse myotubes. Countering Ca2+ leak or preventing mitochondrial Ca2+ uptake blunts S-SIT-induced adaptations, a result supported by proteomic analyses. Here we show that triggering acute transient Ca2+ leak through RyR1 in healthy muscle may contribute to the multiple health promoting benefits of exercise.

RyR1-related myopathy mutations in ATP and calcium binding sites impair channel regulation.

The type 1 ryanodine receptor (RyR1) is an intracellular calcium (Ca2+) release channel on the sarcoplasmic/endoplasmic reticulum that is required for skeletal muscle contraction. RyR1 channel activity is modulated by ligands, including the activators Ca2+ and ATP. Patients with inherited mutations in RyR1 may exhibit muscle weakness as part of a heterogeneous, complex disorder known as RYR1-related myopathy (RYR1-RM) or more recently termed RYR1-related disorders (RYR1-RD). Guided by high-resolution structures of skeletal muscle RyR1, obtained using cryogenic electron microscopy, we introduced mutations into putative Ca2+ and ATP binding sites and studied the function of the resulting mutant channels. These mutations confirmed the functional significance of the Ca2+ and ATP binding sites identified by structural studies based on the effects on channel regulation. Under normal conditions, Ca2+ activates RyR1 at low concentrations (µM) and inhibits it at high concentrations (mM). Mutations in the Ca2+-binding site impaired both activating and inhibitory regulation of the channel, suggesting a single site for both high and low affinity Ca2+-dependent regulation of RyR1 function. Mutation of residues that interact with the adenine ring of ATP abrogated ATP binding to the channel, whereas mutating residues that interact with the triphosphate tail only affected the degree of activation. In addition, patients with mutations at the Ca2+ or ATP binding sites suffer from muscle weakness, therefore impaired RyR1 channel regulation by either Ca2+ or ATP may contribute to the pathophysiology of RYR1-RM in some patients.

Attenuating persistent sodium current-induced atrial myopathy and fibrillation by preventing mitochondrial oxidative stress.

Mechanistically driven therapies for atrial fibrillation (AF), the most common cardiac arrhythmia, are urgently needed, the development of which requires improved understanding of the cellular signaling pathways that facilitate the structural and electrophysiological remodeling that occurs in the atria. Similar to humans, increased persistent Na+ current leads to the development of an atrial myopathy and spontaneous and long-lasting episodes of AF in mice. How increased persistent Na+ current causes both structural and electrophysiological remodeling in the atria is unknown. We crossbred mice expressing human F1759A-NaV1.5 channels with mice expressing human mitochondrial catalase (mCAT). Increased expression of mCAT attenuated mitochondrial and cellular reactive oxygen species (ROS) and the structural remodeling that was induced by persistent F1759A-Na+ current. Despite the heterogeneously prolonged atrial action potential, which was unaffected by the reduction in ROS, the incidences of spontaneous AF, pacing-induced after-depolarizations, and AF were substantially reduced. Expression of mCAT markedly reduced persistent Na+ current-induced ryanodine receptor oxidation and dysfunction. In summary, increased persistent Na+ current in atrial cardiomyocytes, which is observed in patients with AF, induced atrial enlargement, fibrosis, mitochondrial dysmorphology, early after-depolarizations, and AF, all of which can be attenuated by resolving mitochondrial oxidative stress.

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.

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.

Mitochondrial oxidative stress induces leaky ryanodine receptor during mechanical ventilation.

RATIONALE: Ventilator-induced diaphragm dysfunction (VIDD) increases morbidity and mortality in critical care patients. Although VIDD has been associated with mitochondrial oxidative stress and calcium homeostasis impairment, the underling mechanisms are still unknown. We hypothesized that diaphragmatic mitochondrial oxidative stress causes remodeling of the ryanodine receptor (RyR1)/calcium release channel, contributing to sarcoplasmic reticulum (SR) Ca2+ leak, proteolysis and VIDD. METHOD: In mice diaphragms mechanically ventilated for short (6 h) and long (12 h) period, we assessed mitochondrial ROS production, mitochondrial aconitase activity as a marker of mitochondrial oxidative stress, RyR1 remodeling and function, Ca2+ dependent proteolysis, TGFß1 and STAT3 pathway, muscle fibers cross-sectional area, and diaphragm specific force production, with or without the mitochondrial targeted anti-oxidant peptide d-Arg-2', 6'-dimethyltyrosine-Lys-Phe-NH2 (SS31). MEASUREMENTS AND MAIN RESULTS: 6 h of mechanical ventilation (MV) resulted in increased mitochondrial ROS production, reduction of mitochondrial aconitase activity, increased oxidation, S-nitrosylation, S-glutathionylation and Ser-2844 phosphorylation of RyR1, depletion of stabilizing subunit calstabin1 from RyR1, increased SR Ca2+ leak. Preventing mROS production by SS31 treatment does not affect the TGFß1 and STAT3 activation, which suggests that mitochondrial oxidative stress is a downstream pathway to TGFß1 and STAT3, early involved in VIDD. This is further supported by the fact that SS-31 rescue all the other described cellular events and diaphragm contractile dysfunction induced by MV, while SS20, an analog of SS31 lacking antioxidant properties, failed to prevent these cellular events and the contractile dysfunction. Similar results were found in ventilated for 12 h. Moreover, SS31 treatment prevented calpain1 activity and diaphragm atrophy observed after 12 h of MV. This study emphasizes that mitochondrial oxidative stress during 6 h-MV contributes to SR Ca2+ leak via RyR1 remodeling, and diaphragm weakness, while longer periods of MV (12 h) were also associated with increased Ca2+-dependent proteolysis and diaphragm atrophy.

Dietary Saturated Fat Promotes Arrhythmia by Activating NOX2 (NADPH Oxidase 2).

BACKGROUND: Obesity and diets high in saturated fat increase the risk of arrhythmias and sudden cardiac death. However, the molecular mechanisms are not well understood. We hypothesized that an increase in dietary saturated fat could lead to abnormalities of calcium homeostasis and heart rhythm by a NOX2 (NADPH oxidase 2)-dependent mechanism. METHODS: We investigated this hypothesis by feeding mice high-fat diets. In vivo heart rhythm telemetry, optical mapping, and isolated cardiac myocyte imaging were used to quantify arrhythmias, repolarization, calcium transients, and intracellular calcium sparks. RESULTS: We found that saturated fat activates NOX (NADPH oxidase), whereas polyunsaturated fat does not. The high saturated fat diet increased repolarization heterogeneity and ventricular tachycardia inducibility in perfused hearts. Pharmacological inhibition or genetic deletion of NOX2 prevented arrhythmogenic abnormalities in vivo during high statured fat diet and resulted in less inducible ventricular tachycardia. High saturated fat diet activates CaMK (Ca2+/calmodulin-dependent protein kinase) in the heart, which contributes to abnormal calcium handling, promoting arrhythmia. CONCLUSIONS: We conclude that NOX2 deletion or pharmacological inhibition prevents the arrhythmogenic effects of a high saturated fat diet, in part mediated by activation of CaMK. This work reveals a molecular mechanism linking cardiac metabolism to arrhythmia and suggests that NOX2 inhibitors could be a novel therapy for heart rhythm abnormalities caused by cardiac lipid overload.

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.