Phosphorylation of RyR2 simultaneously expands the dyad and rearranges the tetramers.

We have previously demonstrated that type II ryanodine receptors (RyR2) tetramers can be rapidly rearranged in response to a phosphorylation cocktail. The cocktail modified downstream targets indiscriminately, making it impossible to determine whether phosphorylation of RyR2 was an essential element of the response. Here, we used the ß-agonist isoproterenol and mice homozygous for one of the following clinically relevant mutations: S2030A, S2808A, S2814A, or S2814D. We measured the length of the dyad using transmission electron microscopy (TEM) and directly visualized RyR2 distribution using dual-tilt electron tomography. We found that the S2814D mutation, by itself, significantly expanded the dyad and reorganized the tetramers, suggesting a direct link between the phosphorylation state of the tetramer and its microarchitecture. S2808A and S2814A mutant mice, as well as wild types, had significant expansions of their dyads in response to isoproterenol, while S2030A mutants did not. In agreement with functional data from these mutants, S2030 and S2808 were necessary for a complete ß-adrenergic response, unlike S2814 mutants. Additionally, all mutants had unique effects on the organization of their tetramer arrays. Lastly, the correlation of structural with functional changes suggests that tetramer-tetramer contacts play an important functional role. We thus conclude that both the size of the dyad and the arrangement of the tetramers are linked to the state of the channel tetramer and can be dynamically altered by a ß-adrenergic receptor agonist.

Comparison of the structure-function of five newly members of the calcin family.

Calcins are a group of scorpion toxin peptides specifically binding to ryanodine receptors (RyRs) with high affinity, and have the ability to activate and stabilize RyR in a long-lasting subconductance state. Five newly calcins synthesized compounds exhibit typical structural characteristics of a specific family through chemical synthesis and virtual analysis. As the calcins from the same species, Petersiicalcin1 and Petersiicalcin2, Jendekicalcin2 and Jendekicalcin3, have only one residue difference. Both Petersiicalcin1 and Petersiicalcin2 exhibited different affinities in stimulating [3H]ryanodine binding, but the residue mutation resulted in a 2.7 folds difference. Other calcins also exhibited a stimulatory effect on [3H]ryanodine binding to RyR1, however, their affinities were significantly lower than that of Petersiiicalcin1 and Petersiiicalcin2. The channel domain of RyR1 was found to be capable of binding with the basic residues of these calcins, which also exhibited interactions with the S6 helices on RyR1. Dynamic simulations were conducted for Petersiicalcin1 and Petersiicalcin2, which demonstrated their ability to form a highly stable conformation and resulting in an asymmetric tetramer structure of RyR1. The discovery of five newly calcins further enriches the diversity of the natural calcin family, which provides more native peptides for the structure-function analysis between calcin and RyRs.

The Effect of Acidic Residues on the Binding between Opicalcin1 and Ryanodine Receptor from the Structure-Functional Analysis.

Calcin is a group ligand with high affinity and specificity for the ryanodine receptors (RyRs). Little is known about the effect of its acidic residues on the spacial structure as well as the interaction with RyRs. We screened the opicalcin1 acidic mutants and investigated the effect of mutation on activity. The results indicated that all acidic mutants maintained the structural features, but their surface charge distribution underwent significant changes. Molecular docking and dynamics simulations were used to analyze the interaction between opicalcin1 mutants and RyRs, which demonstrated that all opicalcin1 mutants effectively bound to the channel domain of RyR1. This stable binding induced a pronounced asymmetry in the structure of the RyR tetramer, exhibiting a high degree of structural dissimilarity. [3H]Ryanodine binding to RyR1 was enhanced in D2A and D15A, which was similar to opicalcin1, but that effect was suppressed in E12A and E29A and reversed for the DE-4A, thereby inhibiting ryanodine binding. Opicalcin1 and DE-4A also exhibited the ability to form stable docking structures with RyR2. Acidic residues play a crucial role in the structure of calcin and its functional interaction with RyRs that is beneficial for the calcin optimization to develop more active peptide lead compounds for RyR-related diseases.

Barriers to Implementing the ICU Liberation Bundle in a Single-center Pediatric and Cardiac ICUs.

Objectives: The intensive care unit (ICU) Liberation "ABCDEF" Bundle improves outcomes in critically ill adults. We aimed to identify common barriers to Pediatric ICU Liberation Bundle element implementation, to describe differences in barrier perception by ICU staff role, and to describe changes in reported barriers over time. Study Design: A 91-item survey was developed based on existing literature, iteratively revised, and tested by the PICU Liberation Committee at Seattle Children's Hospital, a tertiary free-standing academic children's hospital. Voluntary surveys were administered electronically to all ICU staff twice over 4-week periods in 2017 and 2020. Survey Respondents: 119 (2017) and 163 (2020) pediatric and cardiac ICU staff, including nurses (n = 142, 50%), respiratory therapists (RTs) (n = 46, 16%), attending and fellow physicians, hospitalists, and advanced practice providers (APPs) (n = 62, 22%), physical, occupational, and speech-language pathology therapists (n = 25, 9%), and pharmacists (n = 7, 2%). Measurements and Main Results: Respondents widely agreed that increased workload (78%-100% across roles), communication (53%-84%), and lack of RT-directed ventilator weaning (68%-88%) are barriers to implementation. Other barriers differed by role. In 2020, nurses reported liability (59%) and personal injury (68%) concerns, patient severity of illness (24%), and family discomfort with ICU liberation practices (41%) more frequently than physicians and APPs (16%, 6%, 8%, and 19%, respectively; P < .01 for all). Between 2017 and 2020, some barriers changed: RTs endorsed discomfort with early mobilization less frequently (50% vs 11%, P = .028) and nurses reported concern for patient harm less frequently (51% vs 24%, P = .004). Conclusions: Implementation efforts aimed at addressing known barriers, including educating staff on the safety of early mobility, considering respiratory therapist-directed ventilator weaning, and standardizing interdisciplinary discussion of Pediatric ICU Liberation Bundle elements, will be needed to overcome barriers and improve ICU Liberation Bundle implementation.

OpiCa1-PEG-PLGA nanomicelles antagonize acute heart failure induced by the cocktail of epinephrine and caffeine.

Background: Reducing Ca2+ content in the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs) by calcin is a potential intervention strategy for the SR Ca2+ overload triggered by ß-adrenergic stress in acute heart diseases. Methods: OpiCal-PEG-PLGA nanomicelles were prepared by thin film dispersion, of which the antagonistic effects were observed using an acute heart failure model induced by epinephrine and caffeine in mice. In addition, cardiac targeting, self-stability as well as biotoxicity were determined. Results: The synthesized OpiCa1-PEG-PLGA nanomicelles were elliptical with a particle size of 72.26 nm, a PDI value of 0.3, and a molecular weight of 10.39 kDa. The nanomicelles showed a significant antagonistic effect with 100 % survival rate to the death induced by epinephrine and caffeine, which was supported by echocardiography with significantly recovered heart rate, ejection fraction and left ventricular fractional shortening rate. The FITC labeled nanomicelles had a strong membrance penetrating capacity within 2 h and cardiac targeting within 12 h that was further confirmed by immunohistochemistry with a self-prepared OpiCa1 polyclonal antibody. Meanwhile, the nanomicelles can keep better stability and dispersibility in vitro at 4 °C rather than 20 °C or 37 °C, while maintain a low but stable plasma OpiCa1 concentration in vivo within 72 h. Finally, no obvious biotoxicities were observed by CCK-8, flow cytometry, H&E staining and blood biochemical examinations. Conclusion: Our study also provide a novel nanodelivery pathway for targeting RyRs and antagonizing the SR Ca2+ disordered heart diseases by actively releasing SR Ca2+ through RyRs with calcin.

Sorcin promotes migration in cancer and regulates the EGF-dependent EGFR signaling pathways.

The epidermal growth factor receptor (EGFR) is one of the main tumor drivers and is an important therapeutic target for many cancers. Calcium is important in EGFR signaling pathways. Sorcin is one of the most important calcium sensor proteins, overexpressed in many tumors, that promotes cell proliferation, migration, invasion, epithelial-to-mesenchymal transition, malignant progression and resistance to chemotherapeutic drugs. The present work elucidates a functional mechanism that links calcium homeostasis to EGFR signaling in cancer. Sorcin and EGFR expression are significantly correlated and associated with reduced overall survival in cancer patients. Mechanistically, Sorcin directly binds EGFR protein in a calcium-dependent fashion and regulates calcium (dys)homeostasis linked to EGF-dependent EGFR signaling. Moreover, Sorcin controls EGFR proteostasis and signaling and increases its phosphorylation, leading to increased EGF-dependent migration and invasion. Of note, silencing of Sorcin cooperates with EGFR inhibitors in the regulation of migration, highlighting calcium signaling pathway as an exploitable target to enhance the effectiveness of EGFR-targeting therapies.

PHOSPHORYLATION OF RyR2 SIMULTANEOUSLY EXPANDS THE DYAD AND REARRANGES THE TETRAMERS.

We have previously demonstrated that type II ryanodine receptors (RyR2) tetramers can be rapidly rearranged in response to a phosphorylation cocktail. The cocktail modified downstream targets indiscriminately making it impossible to determine whether phosphorylation of RyR2 was an essential element of the response. We therefore used the ß-agonist isoproterenol and mice with one of the homozygous mutations, S2030A +/+ , S2808A +/+ , S2814A +/+ , or S2814D +/+ , to address this question and to elucidate the role of these clinically relevant mutations. We measured the length of the dyad using transmission electron microscopy (TEM) and directly visualized RyR2 distribution using dual-tilt electron tomography. We found that: 1) The S2814D mutation, by itself, significantly expanded the dyad and reorganized the tetramers suggesting a direct link between the phosphorylation state of the tetramer and the microarchitecture. 2) All of the wild-type, as well as the S2808A and S2814A mice, had significant expansions of their dyads in response to ISO, while S2030A did not. 3) In agreement with functional data from the same mutants, S2030 and S2808 were necessary for a complete ß-adrenergic response, whereas S2814 was not. 4) All the mutated residues had unique effects on the organization of their tetramer arrays. 5) The correlation of structure with function suggests that tetramer-tetramer contacts play an important functional role. We conclude that both the size of the dyad and the arrangement of the tetramers are linked to the state of the channel tetramer and can be dynamically altered by a ß-adrenergic receptor agonist. Summary: Analysis of RyR2 mutants suggests a direct link between the phosphorylation state of the channel tetramer and the microarchitecture of the dyad. All phosphorylation site mutations produced significant and unique effects on the structure of the dyad and its response to isoproterenol.

Caring for Critically Ill Children With the ICU Liberation Bundle (ABCDEF): Results of the Pediatric Collaborative.

OBJECTIVES: Assess clinical outcomes following PICU Liberation ABCDEF Bundle utilization. DESIGN: Prospective, multicenter, cohort study. SETTING: Eight academic PICUs. PATIENTS: Children greater than 2 months with expected PICU stay greater than 2 days and need for mechanical ventilation (MV). INTERVENTIONS: ABCDEF Bundle implementation. MEASUREMENT AND MAIN RESULTS: Over an 11-month period (3-mo baseline, 8-mo implementation), Bundle utilization was measured for 622 patients totaling 5,017 PICU days. Risk of mortality was quantified for 532 patients (4,275 PICU days) for correlation between Bundle utilization and MV duration, PICU length of stay (LOS), delirium incidence, and mortality. Utilization was analyzed as subject-specific (entire PICU stay) and day-specific (single PICU day). Median overall subject-specific utilization increased from 50% during the 3-month baseline to 63.9% during the last four implementation months ( p < 0.001). Subject-specific utilization for elements A and C did not change; utilization improved for B (0-12.5%; p = 0.007), D (22.2-61.1%; p < 0.001), E (17.7-50%; p = 0.003), and F (50-79.2%; p = 0.001). We observed no association between Bundle utilization and MV duration, PICU LOS, or delirium incidence. In contrast, on adjusted analysis, every 10% increase in subject-specific utilization correlated with mortality odds ratio (OR) reduction of 34%, p < 0.001; every 10% increase in day-specific utilization correlated with a mortality OR reduction of 1.4% ( p = 0.006). CONCLUSIONS: ABCDEF Bundle is applicable to children. Although enhanced Bundle utilization correlated with decreased mortality, increased utilization did not correlate with duration of MV, PICU LOS, or delirium incidence. Additional research in the domains of comparative effectiveness, implementation science, and human factors engineering is required to understand this clinical inconsistency and optimize PICU Liberation concept integration into clinical practice.

Cryo-EM analysis of scorpion toxin binding to Ryanodine Receptors reveals subconductance that is abolished by PKA phosphorylation.

Calcins are peptides from scorpion venom with the unique ability to cross cell membranes, gaining access to intracellular targets. Ryanodine Receptors (RyR) are intracellular ion channels that control release of Ca2+ from the endoplasmic and sarcoplasmic reticulum. Calcins target RyRs and induce long-lived subconductance states, whereby single-channel currents are decreased. We used cryo-electron microscopy to reveal the binding and structural effects of imperacalcin, showing that it opens the channel pore and causes large asymmetry throughout the cytosolic assembly of the tetrameric RyR. This also creates multiple extended ion conduction pathways beyond the transmembrane region, resulting in subconductance. Phosphorylation of imperacalcin by protein kinase A prevents its binding to RyR through direct steric hindrance, showing how posttranslational modifications made by the host organism can determine the fate of a natural toxin. The structure provides a direct template for developing calcin analogs that result in full channel block, with potential to treat RyR-related disorders.

Preserved cardiac performance and adrenergic response in a rabbit model with decreased ryanodine receptor 2 expression.

Ryanodine receptor 2 (RyR2) is an ion channel in the heart responsible for releasing into the cytosol most of the Ca2+ required for contraction. Proper regulation of RyR2 is critical, as highlighted by the association between channel dysfunction and cardiac arrhythmia. Lower RyR2 expression is also observed in some forms of heart disease; however, there is limited information on the impact of this change on excitation-contraction (e-c) coupling, Ca2+-dependent arrhythmias, and cardiac performance. We used a constitutive knock-out of RyR2 in rabbits (RyR2-KO) to assess the extent to which a stable decrease in RyR2 expression modulates Ca2+ handling in the heart. We found that homozygous knock-out of RyR2 in rabbits is embryonic lethal. Remarkably, heterozygotes (KO+/-) show ~50% loss of RyR2 protein without developing an overt phenotype at the intact animal and whole heart levels. Instead, we found that KO+/- myocytes show (1) remodeling of RyR2 clusters, favoring smaller groups in which channels are more densely arranged; (2) lower Ca2+ spark frequency and amplitude; (3) slower rate of Ca2+ release and mild but significant desynchronization of the Ca2+ transient; and (4) a significant decrease in the basal phosphorylation of S2031, likely due to increased association between RyR2 and PP2A. Our data show that RyR2 deficiency, although remarkable at the molecular and subcellular level, has only a modest impact on global Ca2+ release and is fully compensated at the whole-heart level. This highlights the redundancy of RyR2 protein expression and the plasticity of the e-c coupling apparatus.

Region-specific distribution of transversal-axial tubule system organization underlies heterogeneity of calcium dynamics in the right atrium.

The atrial myocardium demonstrates the highly heterogeneous organization of the transversal-axial tubule system (TATS), although its anatomical distribution and region-specific impact on Ca2+ dynamics remain unknown. Here, we developed a novel method for high-resolution confocal imaging of TATS in intact live mouse atrial myocardium and applied a custom-developed MATLAB-based computational algorithm for the automated analysis of TATS integrity. We observed a twofold higher (P < 0.01) TATS density in the right atrial appendage (RAA) than in the intercaval regions (ICR, the anatomical region between the superior vena cava and atrioventricular junction and between the crista terminalis and interatrial septum). Whereas RAA predominantly consisted of well-tubulated myocytes, ICR showed partially tubulated/untubulated cells. Similar TATS distribution was also observed in healthy human atrial myocardium sections. In both mouse atrial preparations and isolated mouse atrial myocytes, we observed a strong anatomical correlation between TATS distribution and Ca2+ transient synchronization and rise-up time. This region-specific difference in Ca2+ transient morphology disappeared after formamide-induced detubulation. ICR myocytes showed a prolonged action potential duration at 80% of repolarization as well as a significantly lower expression of RyR2 and Cav1.2 proteins but similar levels of NCX1 and Cav1.3 compared with RAA tissue. Our findings provide a detailed characterization of the region-specific distribution of TATS in mouse and human atrial myocardium, highlighting the structural foundation for anatomical heterogeneity of Ca2+ dynamics and contractility in the atria. These results could indicate different roles of TATS in Ca2+ signaling at distinct anatomical regions of the atria and provide mechanistic insight into pathological atrial remodeling.NEW & NOTEWORTHY Mouse and human atrial myocardium demonstrate high variability in the organization of the transversal-axial tubule system (TATS), with more organized TATS expressed in the right atrial appendage. TATS distribution governs anatomical heterogeneity of Ca2+ dynamics and thus could contribute to integral atrial contractility, mechanics, and arrhythmogenicity.

The V2475F CPVT1 mutation yields distinct RyR2 channel populations that differ in their responses to cytosolic Ca2+ and Mg2.

Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is a lethal genetic disease causing arrhythmias and sudden cardiac death in children and young adults and is linked to mutations in the cardiac ryanodine receptor (RyR2). The effects of CPVT1 mutations on RyR2 ion-channel function are often investigated using purified recombinant RyR2 channels homozygous for the mutation. However, CPVT1 patients are heterozygous for the disease, so this approach does not reveal the true changes to RyR2 function across the entire RyR2 population of channels in the heart. We therefore investigated the native cardiac RyR2 single-channel abnormalities in mice heterozygous for the CPVT1 mutation, V2475F(+/-)-RyR2, and applied molecular modelling techniques to investigate the possible structural changes that could initiate any altered function. We observed that increased sensitivity of cardiac V2475F(+/-)-RyR2 channels to both activating and inactivating levels of cytosolic Ca2+ , plus attenuation of Mg2+ inhibition, were the most marked changes. Severity of abnormality was not uniform across all channels, giving rise to multiple sub-populations with differing functional characteristics. For example, 46% of V2475F(+/-)-RyR2 channels exhibited reduced Mg2+ inhibition and 23% were actually activated by Mg2+ . Using homology modelling, we discovered that V2475 is situated at a hinge between two regions of the RyR2 helical domain 1 (HD1). Our model proposes that detrimental functional changes to RyR2 arise because mutation at this critical site reduces the angle between these regions. Our results demonstrate the necessity of characterising the total heterozygous population of CPVT1-mutated channels in order to understand CPVT1 phenotypes in patients. KEY POINTS: RyR2 mutations can cause type-1 catecholaminergic polymorphic ventricular tachycardia (CPVT1), a lethal, autosomal-dominant arrhythmic disease. However, the changes in RyR2 ion-channel function that result from the many different patient mutations are rarely investigated in detail and often only recombinant RyR2, homozygous for the mutation, is studied. As CPVT1 is a heterozygous disease and the tetrameric RyR2 channels expressed in the heart will contain varying numbers of mutated monomers, we have investigated the range of RyR2 single-channel abnormalities found in the hearts of mice heterozygous for the CPVT1 mutation, V2475F(+/-)-RyR2. Specific alterations to ligand regulation of V2475F(+/-)-RyR2 were observed. Multiple sub-populations of channels exhibited varying degrees of abnormality. In particular, an increased sensitivity to activating and inactivating cytosolic [Ca2+ ], and reduced sensitivity to Mg2+ inhibition were evident. Our results provide mechanistic insight into the changes to RyR2 gating that destabilise sarcoplasmic reticulum Ca2+ -release causing life-threatening arrhythmias in V2475F(+/-)-CPVT1 patients.

Bedside Methods for Transpyloric Feeding Tube Insertion in Hospitalized Children: A Systematic Review of Randomized and non-Randomized Trials.

PROBLEM: Enteral nutrition is a critical component of therapy for many hospitalized children. Some children, especially those with critical illness, require post-pyloric enteral nutrition, but placement of post-pyloric feeding tubes poses challenges, necessitating costly fluoroscopy procedures and delaying initiation of enteral nutrition. There is no established standard method for pediatric transpyloric tube placement at the bedside. ELIGIBILITY CRITERIA: We searched for trials that assessed the efficacy of methods for transpyloric tube placement at the bedside. Studies that evaluated gastric insufflation, prokinetic agents, pH guided devices, and electromagnetic devices with an objective of bedside transpyloric tube placement in children ages one month to 18 years were included. RESULTS: After each author independently reviewed the search results, we agreed on fourteen articles for inclusion, consisting of six randomized controlled trials, five quasi-experimental studies, and three cohort studies. Intervention protocols varied, both within and between studies, with most trials incorporating more than one variable in the intervention. CONCLUSIONS: The heterogeneity of the research does not provide clear direction about best practices. All interventions demonstrated some efficacy, with the exception of erythromycin. Gastric insufflation, the most prevalent intervention studied, is safe and at least moderately effective. The research demonstrates the positive impact of a small, trained team of personnel for the insertion of a transpyloric tube. IMPLICATIONS: High quality studies with clear protocols evaluating a single variable are needed in order to establish a bedside transpyloric tube placement protocol. We recommend studies on the efficacy of a dedicated team for this procedure.

Impaired Binding to Junctophilin-2 and Nanostructural Alteration in CPVT Mutation.

[Figure: see text].

The Ca2+ -binding protein sorcin stimulates transcriptional activity of the unfolded protein response mediator ATF6.

Sorcin is a calcium-binding protein involved in maintaining endoplasmic reticulum (ER) Ca2+ stores. We have previously shown that overexpressing sorcin under the rat insulin promoter was protective against high-fat diet-induced pancreatic beta-cell dysfunction in vivo. Activating transcription factor 6 (ATF6) is a key mediator of the unfolded protein response (UPR) that provides cellular protection during the progression of ER stress. Here, using nonexcitable HEK293 cells, we show that sorcin overexpression increased ATF6 signalling, whereas sorcin knock out caused a reduction in ATF6 transcriptional activity and increased ER stress. Altogether, our data suggest that sorcin downregulation during lipotoxic stress may prevent full ATF6 activation and a normal UPR during the progression of obesity and insulin resistance.

Autonomous activation of CaMKII exacerbates diastolic calcium leak during beta-adrenergic stimulation in cardiomyocytes of metabolic syndrome rats.

Autonomous Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation induces abnormal diastolic Ca2+ leak, which leads to triggered arrhythmias in a wide range of cardiovascular diseases, including diabetic cardiomyopathy. In hyperglycemia, Ca2+ handling alterations can be aggravated under stress conditions via the ß-adrenergic signaling pathway, which also involves CaMKII activation. However, little is known about intracellular Ca2+ handling disturbances under ß-adrenergic stimulation in cardiomyocytes of the prediabetic metabolic syndrome (MetS) model with obesity, and the participation of CaMKII in these alterations. MetS was induced in male Wistar rats by administering 30 % sucrose in drinking water for 16 weeks. Fluo 3-loaded MetS cardiomyocytes exhibited augmented diastolic Ca2+ leak (in the form of spontaneous Ca2+ waves) under basal conditions and that Ca2+ leakage was exacerbated by isoproterenol (ISO, 100 nM). At the molecular level, [3H]-ryanodine binding and basal phosphorylation of cardiac ryanodine receptor (RyR2) at Ser2814, a CaMKII site, were increased in heart homogenates of MetS rats with no changes in RyR2 expression. These alterations were not further augmented by Isoproterenol. SERCA pump activity was augmented 48 % in MetS hearts before ß-adrenergic stimuli, which is associated to augmented PLN phosphorylation at T17, a target of CaMKII. In MetS hearts. CaMKII auto-phosphorylation (T287) was increased by 80 %. The augmented diastolic Ca2+ leak was prevented by CaMKII inhibition with AIP. In conclusion, CaMKII autonomous activation in cardiomyocytes of MetS rats with central obesity significantly contributes to abnormal diastolic Ca2+ leak, increasing the propensity for ß-adrenergic receptor-driven lethal arrhythmias.

Tetrodotoxin-Sensitive Neuronal-Type Na+ Channels: A Novel and Druggable Target for Prevention of Atrial Fibrillation.

Background Atrial fibrillation (AF) is a comorbidity associated with heart failure and catecholaminergic polymorphic ventricular tachycardia. Despite the Ca2+-dependent nature of both of these pathologies, AF often responds to Na+ channel blockers. We investigated how targeting interdependent Na+/Ca2+ dysregulation might prevent focal activity and control AF. Methods and Results We studied AF in 2 models of Ca2+-dependent disorders, a murine model of catecholaminergic polymorphic ventricular tachycardia and a canine model of chronic tachypacing-induced heart failure. Imaging studies revealed close association of neuronal-type Na+ channels (nNav) with ryanodine receptors and Na+/Ca2+ exchanger. Catecholamine stimulation induced cellular and in vivo atrial arrhythmias in wild-type mice only during pharmacological augmentation of nNav activity. In contrast, catecholamine stimulation alone was sufficient to elicit atrial arrhythmias in catecholaminergic polymorphic ventricular tachycardia mice and failing canine atria. Importantly, these were abolished by acute nNav inhibition (tetrodotoxin or riluzole) implicating Na+/Ca2+ dysregulation in AF. These findings were then tested in 2 nonrandomized retrospective cohorts: an amyotrophic lateral sclerosis clinic and an academic medical center. Riluzole-treated patients adjusted for baseline characteristics evidenced significantly lower incidence of arrhythmias including new-onset AF, supporting the preclinical results. Conclusions These data suggest that nNaVs mediate Na+-Ca2+ crosstalk within nanodomains containing Ca2+ release machinery and, thereby, contribute to AF triggers. Disruption of this mechanism by nNav inhibition can effectively prevent AF arising from diverse causes.