Ryanodine Receptor Type Research Articles

Stable oxidative posttranslational modifications alter the gating properties of RyR1.

The ryanodine receptor type 1 (RyR1) is a Ca2+ release channel that regulates skeletal muscle contraction by controlling Ca2+ release from the sarcoplasmic reticulum (SR). Posttranslational modifications (PTMs) of RyR1, such as phosphorylation, S-nitrosylation, and carbonylation are known to increase RyR1 open probability (Po), contributing to SR Ca2+ leak and skeletal muscle dysfunction. PTMs on RyR1 have been linked to muscle dysfunction in diseases like breast cancer, rheumatoid arthritis, Duchenne muscle dystrophy, and aging. While reactive oxygen species (ROS) and oxidative stress induce PTMs, the impact of stable oxidative modifications like 3-nitrotyrosine (3-NT) and malondialdehyde adducts (MDA) on RyR1 gating remains unclear. Mass spectrometry and single-channel recordings were used to study how 3-NT and MDA modify RyR1 and affect Po. Both modifications increased Po in a dose-dependent manner, with mass spectrometry identifying 30 modified residues out of 5035 amino acids per RyR1 monomer. Key modifications were found in domains critical for protein interaction and channel activation, including Y808/3NT in SPRY1, Y1081/3NT and H1254/MDA in SPRY2&3, and Q2107/MDA and Y2128/3NT in JSol, near the binding site of FKBP12. Though these modifications did not directly overlap with FKBP12 binding residues, they promoted FKBP12 dissociation from RyR1. These findings provide detailed insights into how stable oxidative PTMs on RyR1 residues alter channel gating, advancing our understanding of RyR1-mediated Ca2+ release in conditions associated with oxidative stress and muscle weakness.

Quantification of Sarcoplasmic Reticulum Ca2+ Release in Primary Ventricular Cardiomyocytes.

In the heart, ion channels generate electrical currents that signal muscle contraction through changes in intracellular calcium concentration, i.e., [Ca2+]. The cardiac ryanodine receptor type 2 (RyR2) is the predominant ion channel responsible for increasing intracellular [Ca2+] by releasing Ca2+ from the sarcoplasmic reticulum (SR). Timely Ca2+ release is necessary for appropriate cardiac function, and dysfunction can cause or contribute to life-threatening diseases such as arrhythmia. Quantification of SR-Ca2+ release in the form of sparks and waves can provide valuable insight into RyR2 opening, and factors that influence or regulate channel function. Here, we provide a series of protocols that outline processes for (1) obtaining high-quality isolated cardiomyocytes, (2) preparing samples for experimentally investigating factors that influence RyR2 function, and (3) data acquisition and analysis. Notably, our protocols leverage the potency of the recently developed myosin ATPase inhibitor, Mavacamten. This affords the opportunity to characterize the effects of small molecules or reconstituted proteins/enzymes on RyR2-Ca2+ release events across a range of [Ca2+]. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Cardiomyocyte isolation from mouse Basic Protocol 2: Preparation of cardiomyocytes for Ca2+ imaging Basic Protocol 3: Confocal microscopy and quantitative Ca2+ analysis using SparkMaster 2.

Inhibitors of Intracellular RyR2 Calcium Release Channels as Therapeutic Agents in Arrhythmogenic Heart Diseases.

Ryanodine receptor type 2 (RyR2) is the principal intracellular calcium release channel in the cardiac sarcoplasmic reticulum (SR). Pathological RyR2 hyperactivity generates arrhythmia risk in genetic and structural heart diseases. RYR2 gain-of-function mutations cause catecholaminergic polymorphic ventricular tachycardia. In structural heart diseases (i.e., heart failure), posttranslation modifications render RyR2 channels leaky, resulting in pathologic calcium release during diastole, contributing to arrhythmogenesis and contractile dysfunction. Hence, RyR2 represents a therapeutic target in arrhythmogenic heart diseases. We provide an overview of the structure and function of RyR2, and then review US Food and Drug Administration-approved and investigational RyR2 inhibitors. A therapeutic classification of RyR2 inhibitors is proposed based on their mechanism of action. Class I RyR2 inhibitors (e.g., flecainide) do not change SR calcium content and are primarily antiarrhythmic. Class II RyR2 inhibitors (e.g., dantrolene) increase SR calcium content, making them less effective as antiarrhythmics but preferable in conditions with reduced SR calcium content such as heart failure.

Cryo-EM investigation of ryanodine receptor type 3.

Ryanodine Receptor isoform 3 (RyR3) is a large ion channel found in the endoplasmic reticulum membrane of many different cell types. Within the hippocampal region of the brain, it is found in dendritic spines and regulates synaptic plasticity. It controls myogenic tone in arteries and is upregulated in skeletal muscle in early development. RyR3 has a unique functional profile with a very high sensitivity to activating ligands, enabling high gain in Ca2+-induced Ca2+ release. Here we solve high-resolution cryo-EM structures of RyR3 in non-activating and activating conditions, revealing structural transitions that occur during channel opening. Addition of activating ligands yields only open channels, indicating an intrinsically high open probability under these conditions. RyR3 has reduced binding affinity to the auxiliary protein FKBP12.6 due to several sequence variations in the binding interface. We map disease-associated sequence variants and binding sites for known pharmacological agents. The N-terminal region contains ligand binding sites for a putative chloride anion and ATP, both of which are targeted by sequence variants linked to epileptic encephalopathy.

Nanoscale organization of cardiac calcium channels is dependent on thyroid hormone status.

Thyroid hormone dysfunction is frequently observed in patients with chronic illnesses including heart failure which increases risk of adverse events. This study examined effects of thyroid hormones (TH) on cardiac T-tubule (TT) integrity, Ca2+ sparks, and nanoscale organization of ion channels in excitation-contraction (EC)-coupling, including L-type calcium channel (Cav1.2), ryanodine receptor-type 2 (RyR2), and junctophilin-2 (Jph2). TH deficiency was established in adult female rats by propyl-thiouracil (PTU) ingestion for 8 weeks; followed by randomization to continued PTU without or with oral triiodo-L-thyronine (T3; 10 ug/kg/d) for two additional weeks (PTU+T3). Confocal microscopy of isolated cardiomyocytes (CM) showed significant misalignment of TTs, and increased Ca2+ sparks in thyroid-deficient CMs. Density-Based Spatial Clustering of Applications with Noise (DBSCAN) analysis of STochastic Optical Reconstruction Microscopy (STORM) images showed decreased (p<0.0001) RyR2 cluster number per cell area in PTU CMs compared to euthyroid (EU) control myocytes, and this was normalized by T3-treatment. Cav1.2 channels and Jph2 localized within 210 nm radius of the RyR2 clusters were significantly reduced in PTU myocytes, and these values were increased with T3 treatment. A significant percentage of the RyR2 clusters in the PTU myocytes had neither Cav1.2 or Jph2, suggesting fewer functional clusters in EC-coupling. Nearest neighbor distances between RyR2 clusters were greater (p<0.001) in PTU cells compared to EU and T3-treated CMs that corresponds to disarray of TTs at the sarcomere z-discs. These results support a regulatory role of T3 in the nanoscale organization of RyR2 clusters and co-localization of Cav1.2 and Jph2 in optimizing EC-coupling.

Structure-activity optimization of ryanodine receptor modulators for the treatment of catecholaminergic polymorphic ventricular tachycardia

BackgroundCatecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia disorder associated with potentially lethal arrhythmias. Most CPVT cases are caused by inherited variants in the gene encoding ryanodine receptor type 2 (RYR2). ObjectiveThe goal of this study was to investigate the structure-activity relationship of tetracaine derivatives and to test a lead compound in a mouse model of CPVT. MethodsWe synthesized >200 tetracaine derivatives and characterized 11 of those. The effects of these compounds on Ca2+ handling in cardiomyocytes from R176Q/+ mice was tested with confocal microscopy. The effects of lead compound MSV1302 on arrhythmia inducibility and cardiac contractility were tested by programmed electrical stimulation and echocardiography, respectively. Plasma and microsomal stability and cytotoxicity assays were also performed. ResultsCa2+ imaging revealed that 4 of 11 compounds suppressed sarcoplasmic reticulum Ca2+ leak through mutant RyR2. Two compounds selected for further testing exhibited a half-maximal effective concentration of 146 nM (MSV1302) and 49 nM (MSV1406). Whereas neither compound altered baseline electrocardiogram intervals, only MSV1302 suppressed stress- and pacing-induced ventricular tachycardia in vivo in R176Q/+ mice. Echocardiography revealed that the lead compound MSV1302 did not negatively affect cardiac inotropy and chronotropy. Finally, compound MSV1302 did not block INa, ICa,L, or IKr; it exhibited excellent stability in plasma and microsomes, and it was not cytotoxic. ConclusionStructure-activity relationship studies of second-generation tetracaine derivatives identified lead compound MSV1302 with a favorable pharmacokinetic profile. MSV1302 normalized aberrant RyR2 activity in vitro and in vivo, without altering cardiac inotropy, chronotropy, or off-target effects on other ion channels. This compound may be a strong candidate for future clinical studies to determine its efficacy in CPVT patients.

Structural basis for ryanodine receptor type 2 leak in heart failure and arrhythmogenic disorders

Heart failure, the leading cause of mortality and morbidity in the developed world, is characterized by cardiac ryanodine receptor 2 channels that are hyperphosphorylated, oxidized, and depleted of the stabilizing subunit calstabin-2. This results in a diastolic sarcoplasmic reticulum Ca2+ leak that impairs cardiac contractility and triggers arrhythmias. Genetic mutations in ryanodine receptor 2 can also cause Ca2+ leak, leading to arrhythmias and sudden cardiac death. Here, we solved the cryogenic electron microscopy structures of ryanodine receptor 2 variants linked either to heart failure or inherited sudden cardiac death. All are in the primed state, part way between closed and open. Binding of Rycal drugs to ryanodine receptor 2 channels reverts the primed state back towards the closed state, decreasing Ca2+ leak, improving cardiac function, and preventing arrhythmias. We propose a structural-physiological mechanism whereby the ryanodine receptor 2 channel primed state underlies the arrhythmias in heart failure and arrhythmogenic disorders.

Purinergic agonists increase [Ca2+]i in rat conjunctival goblet cells through ryanodine receptor type 3.

ATP and benzoylbenzoyl-ATP (BzATP) increase free cytosolic Ca2+ concentration ([Ca2+]i) in conjunctival goblet cells (CGCs) resulting in mucin secretion. The purpose of this study was to investigate the source of the Ca2+i mobilized by ATP and BzATP. First-passage cultured rat CGCs were incubated with Fura-2/AM, and [Ca2+]i was measured under several conditions with ATP and BzATP stimulation. The following conditions were used: 1) preincubation with the Ca2+ chelator EGTA, 2) preincubation with the SERCA inhibitor thapsigargin (10-6 M), which depletes ER Ca2+ stores, 3) preincubation with phospholipase C (PLC) or protein kinase A (PKA) inhibitor, or 4) preincubation with the voltage-gated calcium channel antagonist nifedipine (10-5 M) and the ryanodine receptor (RyR) antagonist dantrolene (10-5 M). Immunofluorescence microscopy (IF) and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to investigate RyR presence in rat and human CGCs. ATP-stimulated peak [Ca2+]i was significantly lower after chelating Ca2+i with 2 mM EGTA in Ca2+-free buffer. The peak [Ca2+]i increase in CGCs preincubated with thapsigargin, the PKA inhibitor H89, nifedipine, and dantrolene, but not the PLC inhibitor, was reduced for ATP at 10-5 M and BzATP at 10-4 M. Incubating CGCs with dantrolene alone decreased [Ca2+]i and induced CGC cell death at a high concentration. RyR3 was detected in rat and human CGCs with IF and RT-qPCR. We conclude that ATP- and BzATP-induced Ca2+i increases originate from the ER and that RyR3 may be an essential regulator of CGC [Ca2+]i. This study contributes to the understanding of diseases arising from defective Ca2+ signaling in nonexcitable cells.NEW & NOTEWORTHY ATP and benzoylbenzoyl-ATP (BzATP) induce mucin secretion through an increase in free cytosolic calcium concentration ([Ca2+]i) in conjunctival goblet cells (CGCs). The mechanisms through which ATP and BzATP increase [Ca2+]i in CGCs are unclear. Ryanodine receptors (RyRs) are fundamental in [Ca2+]i regulation in excitable cells. Herein, we find that ATP and BzATP increase [Ca2+]i through the activation of protein kinase A, voltage-gated calcium channels, and RyRs, and that RyRs are crucial for nonexcitable CGCs' Ca2+i homeostasis.

Abstract Mo060: SPEG mediates increased atrial fibrillation incidence in chronic kidney disease

Introduction: Chronic kidney disease (CKD) is a major public health problem. CKD is a risk factor of cardiovascular morbidity and mortality due to the increased incidence of atrial fibrillation (AF). AF onset is known to be the consequence of cardiomyocyte intracellular calcium (Ca 2+ ) mishandling where increased ryanodine receptor type 2 (RyR2) activity causes triggered activity. RyR2 activity is regulated by phosphorylation of serine 2367 (S2367) by the ‘striated muscle preferentially expressed protein’ (SPEG). Reduced RyR2 phosphorylation at S2367 increases the risk of AF. We hypothesized that changes in atrial SPEG underly the increased risk of AF in CKD patients. Methods: The experimental 5/6 nephrectomy model was used to mimic CKD disease in mice. AF inducibility was determined using intracardiac programmed electric stimulation. Echocardiography was used to analyze cardiac structure. RYR2 phosphorylation and SPEG levels were measured by western blotting. Intracellular Ca2+ dynamics were assessed by confocal microscopy. Results: CKD mice showed increased serum phosphate (14.1±1.4 vs 9.3±0.4mg/dL; p&lt;0.01), creatinine (0.4±0.1 vs 0.8±0.3 mg/dL; p&lt;0.001) and urea (30.5±1.1 vs 20.9±0.4 mg/dL; p&lt;0.001). AF incidence was 4-fold higher in CKD respect sham mice (60 vs 14.2%; p&lt;0.05). CKD mice showed longer P waves (14.3±0.4 vs 12.5±0.3; p&lt;0.01) and greater increase of left atrial size than Sham mice (35.0±7.5 vs 13.9±4.6; p&lt;0.05). Ejection fraction was reduced in CKD mice after 6 weeks of CKD progression (57.9±0.6 vs 61.7±0.8%; p&lt;0.001) while no changes were observed in Sham mice. SPEG levels were decreased in atria from CKD mice compared to sham (0.64±0.11 vs 1.19±0.13; p&lt;0.05). RyR2 phosphorylation at S2367 was reduced in CKD mice atria (0.66±0.16 vs 1.25±0.26 in sham; p=0.098). Atrial cardiomyocytes from CKD mice exhibited altered Ca 2+ handling, with decreased SERCA activity (p&lt;0.05), increased spark-mediated diastolic Ca 2+ leak (p&lt;0.01) and increased NCX activity (p&lt;0.05). Finally, CKD-induced AF onset was prevented in a mice with a mutation that mimics RYR2 phosphorylation at SPEG site (S2367D mice) Conclusion: This study shows for the first time that SPEG levels are decreased in atria from CKD mice what induces increased RyR2 activity leading to increased AF incidence. By conserving RYR2 phosphorylation at Ser2367, CKD-induced AF was prevented. Thus, we propose SPEG as a possible therapeutic target to reduce the increased incidence of AF in CKD patients.

Divergent Biochemical Properties and Disparate Impact of Arrhythmogenic Calmodulin Mutations on Zebrafish Cardiac Function.

Calmodulin (CaM) is a ubiquitous, small cytosolic calcium (Ca2+)-binding sensor that plays a vital role in many cellular processes by binding and regulating the activity of over 300 protein targets. In cardiac muscle, CaM modulates directly or indirectly the activity of several proteins that play a key role in excitation-contraction coupling (ECC), such as ryanodine receptor type 2 (RyR2),l-type Ca2+ (Cav1.2), sodium (NaV1.5) and potassium (KV7.1) channels. Many recent clinical and genetic studies have reported a series of CaM mutations in patients with life-threatening arrhythmogenic syndromes, such as long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT). We recently showed that four arrhythmogenic CaM mutations (N98I, D132E, D134H, and Q136P) significantly reduce the binding of CaM to RyR2. Herein, we investigate in vivo functional effects of these CaM mutations on the normal zebrafish embryonic heart function by microinjecting complementary RNA corresponding to CaMN98I, CaMD132E, CaMD134H, and CaMQ136P mutants. Expression of CaMD132E and CaMD134H mutants results in significant reduction of the zebrafish heart rate, mimicking a severe form of human bradycardia, whereas expression of CaMQ136P results in an increased heart rate mimicking human ventricular tachycardia. Moreover, analysis of cardiac ventricular rhythm revealed that the CaMD132E and CaMN98I zebrafish groups display an irregular pattern of heart beating and increased amplitude in comparison to the control groups. Furthermore, circular dichroism spectroscopy experiments using recombinant CaM proteins reveals a decreased structural stability of the four mutants compared to the wild-type CaM protein in the presence of Ca2+. Finally, Ca2+-binding studies indicates that all CaM mutations display reduced CaM Ca2+-binding affinities, with CaMD132E exhibiting the most prominent change. Our data suggest that CaM mutations can trigger different arrhythmogenic phenotypes through multiple and complex molecular mechanisms.

Identifying Key Binding Interactions Between the Cardiac L-Type Calcium Channel and Calmodulin Using Molecular Dynamics Simulations.

Defects in the binding of the calcium sensing protein calmodulin (CaM) to the L-type calcium channel (CaV1.2) or to the ryanodine receptor type 2 (RyR2) can lead to dangerous cardiac arrhythmias with distinct phenotypes, such as long-QT syndrome (LQTS) and catecholaminergic ventricular tachycardia (CPVT). Certain CaM mutations lead to LQTS while other mutations lead to CPVT, but the mechanisms by which a specific mutation can lead to each disease phenotype are not well-understood. In this study, we use long, 2 μs molecular dynamics simulations and a multitrajectory approach to identify the key binding interactions between the IQ domain of CaV1.2 and CaM. Five key interactions are found between CaV1.2 and CaM in the C-lobe, 1 in the central linker, and 2 in the N-lobe. In addition, while 5 key interactions appear between residues 120-149 in the C-lobe of CaM when it interacts with CaV1.2, only 1 key interaction is found within this region of CaM when it interacts with the RyR2. We show that this difference in the distribution of key interactions correlates with the known distribution of CaM mutations that lead to LQTS or CPVT. This correlation suggests that a disruption of key binding interactions is a plausible mechanism that can lead to these two different disease phenotypes.

Cardiovascular and muscular plasticity in an endurance‐master athlete following 12 weeks of detraining and retraining: a case study

AbstractBackgroundThis study examined the cardiorespiratory and muscular adaptations of a 53‐year‐old endurance master athlete following 12 weeks of detraining and retraining.MethodsData were collected before and after detraining, and after retraining. Maximal oxygen uptake (VO2max) was evaluated during maximal cycling exercise. Proteins involved in muscle contraction, mitochondrial function and glycolysis were investigated using western blot analysis.ResultsVO2max decreased by 7% after detraining and was 5% greater than baseline after retraining. Detraining induced an important increase in the ryanodine receptor type 1 protein levels (RyR1, +44%) with a decrease in the protein levels of its stabilizer FKBP12 (−24%). We observed a 138% increase in the sarco‐endoplasmic reticulum ATPase 1 protein and a 42% increase in the myosin heavy chain fast‐twitch protein in response to detraining. This was associated with depressed levels of the mitochondrial biogenesis and oxidative phosphorylation (OXPHOS) proteins, while the expression of the mitochondrial dynamic proteins appeared stimulated. Twelve weeks of retraining reversed almost all the alterations observed in muscle proteins, but specifically increased mitochondrial biogenesis, OXPHOS and antioxidant defence proteins as well as the glucose transporter 4 (Glut‐4, +36%) and hexokinase (+100%) proteins levels above the baseline. The mitochondrial dynamic proteins were further increased with the retraining.ConclusionsThese data provide novel information on cardiorespiratory and muscular plasticity, suggesting that highly endurance‐trained athletes might show substantial muscular adaptations while retrained after a detraining period and call for more extensive clinical trials.

Gene therapy with phosphodiesterases 2A and 4B ameliorates heart failure and arrhythmias by improving subcellular cAMP compartmentation.

Gene therapy with cardiac phosphodiesterases (PDEs), such as phosphodiesterase 4B (PDE4B), has recently been described to effectively prevent heart failure (HF) in mice. However, exact molecular mechanisms of its beneficial effects, apart from general lowering of cardiomyocyte cyclic adenosine monophosphate (cAMP) levels, have not been elucidated. Here, we studied whether gene therapy with two types of PDEs, namely PDE2A and PDE4B, can prevent pressure-overload-induced HF in mice by acting on and restoring altered cAMP compartmentation in distinct subcellular microdomains. HF was induced by transverse aortic constriction followed by tail-vein injection of adeno-associated-virus type 9 vectors to overexpress PDE2A3, PDE4B3, or luciferase for 8 weeks. Heart morphology and function was assessed by echocardiography and histology which showed that PDE2A and especially PDE4B gene therapy could attenuate cardiac hypertrophy, fibrosis, and decline of contractile function. Live cell imaging using targeted cAMP biosensors showed that PDE overexpression restored altered cAMP compartmentation in microdomains associated with ryanodine receptor type 2 (RyR2) and caveolin-rich plasma membrane. This was accompanied by ameliorated caveolin-3 decline after PDE2A3 overexpression, reduced RyR2 phosphorylation in PDE4B3 overexpressing hearts, and antiarrhythmic effects of both PDEs measured under isoproterenol stimulation in single cells. Strong association of overexpressed PDE4B but not PDE2A with RyR2 microdomain could prevent calcium leak and arrhythmias in human-induced pluripotent stem-derived cardiomyocytes with the A2254V mutation in RyR2 causing catecholaminergic polymorphic ventricular tachycardia. Our data indicate that gene therapy with phosphodiesterases can prevent HF including associated cardiac remodelling and arrhythmias by restoring altered cAMP compartmentation in functionally relevant subcellular microdomains.

Significance of Zn2+ in RyR1 for Structural Integrity and Ligand Binding: Insight from Molecular Dynamics.

Ryanodine receptor type 1 (RyR1) is a Ca2+-release channel central to skeletal muscle excitation-contraction (EC) coupling. RyR1's cryo-EM structures reveal a zinc-finger motif positioned within the cytoplasmic C-terminal domain (CTD). Yet, owing to limitations in cryo-EM resolution, RyR1 structures lack precision in detailing the metal coordination structure, prompting the need for an accurate model. In this study, we employed molecular dynamics (MD) simulations and the density functional theory (DFT) method to refine the binding characteristics of Zn2+ in the zinc-finger site of the RyR1 channel. Our findings also highlight substantial conformational changes in simulations conducted in the absence of Zn2+. Notably, we observed a loss of contact at the interface between protein domains proximal to the zinc-finger site, indicating a crucial role of Zn2+ in maintaining structural integrity and interdomain interactions within RyR1. Furthermore, this study provides valuable insights into the modulation of ATP, Ca2+, and caffeine binding, shedding light on the intricate relationship between Zn2+ coordination and the dynamic behavior of RyR1. Our integrative approach combining MD simulations and DFT calculations enhances our understanding of the molecular mechanisms governing ligand binding in RyR1.

Unusual cause of muscle weakness, type II respiratory failure and pulmonary hypertension: a case report of ryanodine receptor type 1(RYR1)-related myopathy

BackgroundPatients with congenital myopathies may experience respiratory involvement, resulting in restrictive ventilatory dysfunction and respiratory failure. Pulmonary hypertension (PH) associated with this condition has never been reported in congenital ryanodine receptor type 1(RYR1)-related myopathy.Case presentation A 47-year-old woman was admitted with progressively exacerbated chest tightness and difficulty in neck flexion. She was born prematurely at week 28. Her bilateral lower extremities were edematous and muscle strength was grade IV−. Arterial blood gas analysis revealed hypoventilation syndrome and type II respiratory failure, while lung function test showed restrictive ventilation dysfunction, which were both worse in the supine position. PH was confirmed by right heart catheterization (RHC), without evidence of left heart disease, congenital heart disease, or pulmonary artery obstruction. Polysomnography indicated nocturnal hypoventilation. The ultrasound revealed reduced mobility of bilateral diaphragm. The level of creatine kinase was mildly elevated. Magnetic resonance imaging showed myositis of bilateral thigh muscle. Muscle biopsy of the left biceps brachii suggested muscle malnutrition and congenital muscle disease. Gene testing revealed a missense mutation in the RYR1 gene (exon33 c.C4816T). Finally, she was diagnosed with RYR1-related myopathy and received long-term non-invasive ventilation (NIV) treatment. Her symptoms and cardiopulmonary function have been greatly improved after 10 months.ConclusionsWe report a case of RYR1-related myopathy exhibiting hypoventilation syndrome, type II respiratory failure and PH associated with restrictive ventilator dysfunction. Pulmonologists should keep congenital myopathies in mind in the differential diagnosis of type II respiratory failure, especially in patients with short stature and muscle weakness.

Phosphodiesterases 4B and 4D Differentially Regulate cAMP Signaling in Calcium Handling Microdomains of Mouse Hearts.

The ubiquitous second messenger 3',5'-cyclic adenosine monophosphate (cAMP) regulates cardiac excitation-contraction coupling (ECC) by signaling in discrete subcellular microdomains. Phosphodiesterase subfamilies 4B and 4D are critically involved in the regulation of cAMP signaling in mammalian cardiomyocytes. Alterations of PDE4 activity in human hearts has been shown to result in arrhythmias and heart failure. Here, we sought to systematically investigate specific roles of PDE4B and PDE4D in the regulation of cAMP dynamics in three distinct subcellular microdomains, one of them located at the caveolin-rich plasma membrane which harbors the L-type calcium channels (LTCCs), as well as at two sarco/endoplasmic reticulum (SR) microdomains centered around SR Ca2+-ATPase (SERCA2a) and cardiac ryanodine receptor type 2 (RyR2). Transgenic mice expressing Förster Resonance Energy Transfer (FRET)-based cAMP-specific biosensors targeted to caveolin-rich plasma membrane, SERCA2a and RyR2 microdomains were crossed to PDE4B-KO and PDE4D-KO mice. Direct analysis of the specific effects of both PDE4 subfamilies on local cAMP dynamics was performed using FRET imaging. Our data demonstrate that all three microdomains are differentially regulated by these PDE4 subfamilies. Whereas both are involved in cAMP regulation at the caveolin-rich plasma membrane, there are clearly two distinct cAMP microdomains at the SR formed around RyR2 and SERCA2a, which are preferentially controlled by PDE4B and PDE4D, respectively. This correlates with local cAMP-dependent protein kinase (PKA) substrate phosphorylation and arrhythmia susceptibility. Immunoprecipitation assays confirmed that PDE4B is associated with RyR2 along with PDE4D. Stimulated Emission Depletion (STED) microscopy of immunostained cardiomyocytes suggested possible co-localization of PDE4B with both sarcolemmal and RyR2 microdomains. In conclusion, our functional approach could show that both PDE4B and PDE4D can differentially regulate cardiac cAMP microdomains associated with calcium homeostasis. PDE4B controls cAMP dynamics in both caveolin-rich plasma membrane and RyR2 vicinity. Interestingly, PDE4B is the major regulator of the RyR2 microdomain, as opposed to SERCA2a vicinity, which is predominantly under PDE4D control, suggesting a more complex regulatory pattern than previously thought, with multiple PDEs acting at the same location.

Cardiovascular and muscular plasticity in an endurance-master athlete following 12 weeks of detraining and retraining: A case study.

Introduction&#x0D; This study aimed at examining the cardiorespiratory and muscular adaptations following 12 weeks of detraining and retraining in a 53-year-old endurance master athlete.&#x0D; Methods&#x0D; Data were collected before and after detraining and after retraining. Maximal oxygen uptake (VO2max) was evaluated during maximal cycling exercise. Proteins involved in muscle contraction, mitochondrial function and glycolysis were investigated using western blot analysis.&#x0D; Results&#x0D; VO2max decreased by 7% after detraining and was 5% greater than baseline after retraining. Detraining induced an important increase in the ryanodine receptor type 1 protein levels (RyR1, +44%) with a decrease in the protein levels of its stabilizer FKBP12 (-24%). We observed a 138% increase in the sarco-endoplasmic reticulum ATPase 1 protein and a 42% increase in the myosin heavy chain fast-twitch protein in response to detraining, suggesting transformation towards a more glycolytic phenotype. This was associated with depressed levels of the mitochondrial biogenesis and oxidative phosphorylation (OXPHOS) proteins, while the expression of the mitochondrial dynamic proteins appeared stimulated. Twelve weeks of retraining reversed almost all the alterations observed in muscle proteins, but specifically increased mitochondrial biogenesis, OXPHOS and antioxidant defense proteins as well as the glucose transporter 4 (Glut -4, +36%) and hexokinase (+100%) proteins levels compared to baseline. The mitochondrial dynamic proteins were further increased with the retraining.&#x0D; Discussion/Conclusion&#x0D; These data provide novel information on cardiorespiratory and muscular plasticity suggesting that highly endurance-trained athletes might show substantial muscular adaptations while retrained after a detraining period and call for more extensive clinical trials.

Cardiac pathophysiology of COVID-19 mediated by ryanodine receptor type 2 Ca2+ leak

Cardiac pathophysiology of COVID-19 mediated by ryanodine receptor type 2 Ca2+ leak

Neuron-targeted exosome therapy: A novel approach for treating cardiogenic dementia via RyR2 inhibition

Cardiogenic dementia (CD), recognized since the late 1970s, manifests as altered consciousness and cognition due to heart conditions. Its elusive molecular mechanisms present diagnostic and management challenges. Emerging research implicates ryanodine receptor type 2 (RyR2) mediated intracellular Ca2 + leaks in myocardial infarction (MI)-linked cognitive impairment. We hypothesize that intravenous delivery of neuron-targeted exosomes loaded with RyR2-targeting siRNA (si-RyR2) to cerebral regions could rectify Ca2 + imbalance, curbing neurotoxic protein generation tied to CD. To investigate, si-RyR2 exosomes will be administered to MI-induced mice, with hippocampal samples analyzed for Ca2 + levels, β-amyloid, and p-tau. Results hold promise for advancing CD treatment insights.

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

Previous cryo-electron micrographs suggested that the skeletal muscle Ca2+ release channel, ryanodine receptor (RyR)1, is regulated by intricate interactions between the EF hand Ca2+ binding domain and the cytosolic loop (S2-S3 loop). However, the precise molecular details of these interactions and functional consequences of the interactions remain elusive. Here, we used molecular dynamics simulations to explore the specific amino acid pairs involved in hydrogen bond interactions within the EF hand-S2-S3 loop interface. Our simulations unveiled two key interactions: (1) K4101 (EF hand) with D4730 (S2-S3 loop) and (2) E4075, Q4078, and D4079 (EF hand) with R4736 (S2-S3 loop). To probe the functional significance of these interactions, we constructed mutant RyR1 complementary DNAs and expressed them in HEK293cells for [3H]ryanodine binding assays. Our results demonstrated that mutations in the EF hand, specifically K4101E and K4101M, resulted in reduced affinities for Ca2+/Mg2+-dependent inhibitions. Interestingly, the K4101E mutation increased the affinity for Ca2+-dependent activation. Conversely, mutations in the S2-S3 loop, D4730K and D4730N, did not significantly change the affinities for Ca2+/Mg2+-dependent inhibitions. Our previous finding that skeletal disease-associated RyR1 mutations, R4736Q and R4736W, impaired Ca2+-dependent inhibition, is consistent with the current results. In silico mutagenesis analysis aligned with our functional data, indicating altered hydrogen bonding patterns upon mutations. Taken together, our findings emphasize the critical role of the EF hand-S2-S3 loop interaction in Ca2+/Mg2+-dependent inhibition of RyR1 and provide insights into potential therapeutic strategies targeting this domain interaction for the treatment of skeletal myopathies.