UNASSIGNED: The docking protein IRS2 (insulin receptor substrate protein-2) is an important mediator of insulin signaling and may also regulate other signaling pathways. Murine hearts with cardiomyocyte-restricted deletion of IRS2 (cIRS2-KO) are more susceptible to pressure overload-induced cardiac dysfunction, implying a critical protective role of IRS2 in cardiac adaptation to stress through mechanisms that are not fully understood. There is limited evidence regarding the function of IRS2 beyond metabolic homeostasis regulation, particularly in the context of cardiac disease.
UNASSIGNED: A retrospective analysis of an electronic medical record database was conducted to identify patients with IRS2 variants and assess their risk of cardiac arrhythmias. Arrhythmia susceptibility was examined in cIRS2-KO mice. The underlying mechanisms were investigated using confocal calcium imaging of ex vivo whole hearts and isolated cardiomyocytes to assess calcium handling, Western blotting to analyze the involved signaling pathways, and pharmacological and genetic interventions to rescue arrhythmias in cIRS2-KO mice.
UNASSIGNED: The retrospective analysis identified patients with IRS2 variants of uncertain significance with a potential association to an increased risk of cardiac arrhythmias compared with matched controls. cIRS2-KO hearts were found to be prone to catecholamine-sensitive ventricular tachycardia and reperfusion ventricular tachycardia. Confocal calcium imaging of ex vivo whole hearts and single isolated cardiomyocytes from cIRS2-KO hearts revealed decreased Ca²+ transient amplitudes, increased spontaneous Ca²+ sparks, and reduced sarcoplasmic reticulum Ca²+ content during sympathetic stress, indicating sarcoplasmic reticulum dysfunction. We identified that overactivation of the AKT1/NOS3 (nitric oxide synthase 3)/CaMKII (Ca2+/calmodulin-dependent protein kinase II)/RyR2 (type 2 ryanodine receptor) signaling pathway led to calcium mishandling and catecholamine-sensitive ventricular tachycardia in cIRS2-KO hearts. Pharmacological AKT inhibition or genetic stabilization of RyR2 rescued catecholamine-sensitive ventricular tachycardia in cIRS2-KO mice.
UNASSIGNED: Cardiac IRS2 inhibits sympathetic stress-induced AKT/NOS3/CaMKII/RyR2 overactivation and calcium-dependent arrhythmogenesis. This novel IRS2 signaling axis, essential for maintaining cardiac calcium homeostasis under stress, presents a promising target for developing new antiarrhythmic therapies.

Micro and nanoscale patterning of surface features and biochemical cues have emerged as tools to precisely direct neurite growth into close proximity with next generation neural prosthesis electrodes. Biophysical cues can exert greater influence on neurite pathfinding compared to the more well studied biochemical cues; yet the signaling events underlying the ability of growth cones to respond to these microfeatures remain obscure. Intracellular Ca2+ signaling plays a critical role in how a growth cone senses and grows in response to various cues (biophysical features, repulsive peptides, chemo-attractive gradients). Here, we investigate the role of inositol triphosphate (IP3) and ryanodine-sensitive receptor (RyR) signaling as sensory neurons (spiral ganglion neurons, SGNs, and dorsal root ganglion neurons, DRGNs) pathfind in response to micropatterned substrates of varied geometries. We find that IP3 and RyR signaling act in the growth cone as they navigate biophysical cues and enable proper guidance to biophysical, chemo-permissive, and chemo-repulsive micropatterns. In response to complex micropatterned geometries, RyR signaling appears to halt growth in response to both topographical features and chemo-repulsive cues. IP3 signaling appears to play a more complex role, as growth cones appear to sense the microfeatures in the presence of xestospongin C but are unable to coordinate turning in response to them. Overall, key Ca2+ signaling elements, IP3 and RyR, are found to be essential for SGNs to pathfind in response to engineered biophysical and biochemical cues. These findings inform efforts to precisely guide neurite regeneration for improved neural prosthesis function, including cochlear implants.

Ca2+ release from the sarcoplasmic reticulum (SR) plays a central role in excitation-contraction coupling (ECC) in skeletal muscles. However, the mechanism by which activation of the voltage-sensors/dihydropyridine receptors (DHPRs) in the membrane of the transverse tubular system leads to activation of the Ca2+-release channels/ryanodine receptors (RyRs) in the SR is not fully understood. Recent observations showing that a very small Ca2+ leak through RyR1s in mammalian skeletal muscle can markedly raise the background [Ca2+] in the junctional space (JS) above the Ca2+ level in the bulk of the cytosol indicate that there is a diffusional barrier between the JS and the cytosol at large. Here, I use a mathematical model to explore the hypothesis that a sudden rise in Ca2+ leak through DHPR-coupled RyR1s, caused by reduced inhibition at the RyR1 Ca2+/Mg2+ inhibitory I1-sites when the associated DHPRs are activated, is sufficient to enable synchronized responses that trigger a regenerative rise of Ca2+ release that remains under voltage control. In this way, the characteristic response to Ca2+ of RyR channels is key not only for the Ca2+ release mechanism in cardiac muscle and other tissues, but also for the DHPR-dependent Ca2+ release in skeletal muscle.

Ventricular fibrillation (VF) is a leading immediate cause of sudden cardiac death. There is a strong association between aging and VF, although the mechanisms are unclear, limiting the availability of targeted therapeutic interventions. Here we found that the stress kinases p38γ and p38δ are activated in the ventricles of old mice and mice with genetic or drug-induced arrhythmogenic conditions. We discovered that, upon activation, p38γ and p38δ cooperatively increase the susceptibility to stress-induced VF. Mechanistically, our data indicate that activated p38γ and p38δ phosphorylate ryanodine receptor 2 (RyR2) disrupt Kv4.3 channel localization, promoting sarcoplasmic reticulum calcium leak, Ito current reduction and action potential duration prolongation. In turn, this led to aberrant intracellular calcium handling, premature ventricular complexes and enhanced susceptibility to VF. Blocking this pathway protected genetically modified animals from VF development and reduced the VF duration in aged animals. These results indicate that p38γ and p38δ are a potential therapeutic target for sustained VF prevention.

Seizures are increasingly being recognized as the hallmark of Alzheimer\'s disease (AD). Neuronal hyperactivity can be a consequence of neuronal damage caused by abnormal amyloid β (Aß) depositions. However, it can also be a cell-autonomous phenomenon causing AD by Aß-independent mechanisms. Various studies using animal models have shown that Ca2+ is released from the endoplasmic reticulum (ER) via type 1 inositol triphosphate receptors (InsP3R1s) and ryanodine receptors (RyRs). To investigate which is the main pathophysiological mechanism in human neurons, we measured Ca2+ signaling in neural cells derived from three early-onset AD patients harboring Presenilin-1 variants (PSEN1 p.A246E, p.L286V, and p.M146L). Of these, it has been reported that PSEN1 p.A246E and p.L286V did not produce a significant amount of abnormal Aß. We found all PSEN1-mutant neurons, but not wild-type, caused abnormal Ca2+-bursts in a manner dependent on the calcium channel, Ryanodine Receptor 2 (RyR2). Indeed, carvedilol, an RyR2 inhibitor, and VK-II-86, an analog of carvedilol without the β-blocking effects, sufficiently eliminated the abnormal Ca2+ bursts. In contrast, Dantrolene, an inhibitor of RyR1 and RyR3, and Xestospongin c, an IP3R inhibitor, did not attenuate the Ca2+-bursts. The Western blotting showed that RyR2 expression was not affected by PSEN1 p.A246E, suggesting that the variant may activate the RyR2. The RNA-Seq data revealed that ER-stress responsive genes were increased, and mitochondrial Ca2+-transporter genes were decreased in PSEN1A246E cells compared to the WT neurons. Thus, we propose that aberrant Ca2+ signaling is a key link between human pathogenic PSEN1 variants and cell-intrinsic hyperactivity prior to deposition of abnormal Aß, offering prospects for the development of targeted prevention strategies for at-risk individuals.

UNASSIGNED: Pathogenic variants of RYR1, the gene encoding the principal sarcoplasmic reticulum calcium release channel (RyR1) with a crucial role in excitation-contraction coupling, are among the most common genetic causes of non-dystrophic neuromuscular disorders. We recently conducted a questionnaire study focusing on functional impairments, fatigue, and quality of life (QoL) in patients with RYR1-related diseases (RYR1-RD) throughout the recognized disease spectrum. In this previous questionnaire study the medical perspective was taken, reflective of a study protocol designed by neurologists and psychologists. With this present study we wanted to specifically address the patient perspective.
UNASSIGNED: Together with affected individuals, family members, and advocates concerned with RYR1-RD, we developed an online patient survey that was completed by 227 patients or their parents/other caretakers (143 females and 84 males, 0-85 years). We invited 12 individuals, representing most of the patient group based on age, sex, race, and type and severity of diagnosis, to share their personal experiences on living with a RYR1-RD during an international workshop in July 2022. Data were analyzed through a mixed-methods approach, employing both a quantitative analysis of the survey results and a qualitative analysis of the testimonials.
UNASSIGNED: Data obtained from the combined quantitative and qualitative analyses provide important insights on six topics: 1) Diagnosis; 2) Symptoms and impact of the condition; 3) Physical activity; 4) Treatment; 5) Clinical research and studies; and 6) Expectations.
UNASSIGNED: Together, this study provides a unique patient perspective on the RYR1-RD spectrum, associated disease impact, suitable physical activities and expectations of future treatments and trials, and thus, offers an essential contribution to future research.

Mutations in the nuclear envelope (NE) protein lamin A/C (encoded by LMNA), cause a severe form of dilated cardiomyopathy (DCM) with early-onset life-threatening arrhythmias. However, molecular mechanisms underlying increased arrhythmogenesis in LMNA-related DCM (LMNA-DCM) remain largely unknown. Here we show that a frameshift mutation in LMNA causes abnormal Ca2+ handling, arrhythmias and disformed NE in LMNA-DCM patient-specific iPSC-derived cardiomyocytes (iPSC-CMs). Mechanistically, lamin A interacts with sirtuin 1 (SIRT1) where mutant lamin A/C accelerates degradation of SIRT1, leading to mitochondrial dysfunction and oxidative stress. Elevated reactive oxygen species (ROS) then activates the Ca2+/calmodulin-dependent protein kinase II (CaMKII)-ryanodine receptor 2 (RYR2) pathway and aggravates the accumulation of SUN1 in mutant iPSC-CMs, contributing to arrhythmias and NE deformation, respectively. Taken together, the lamin A/C deficiency-mediated ROS disorder is revealed as central to LMNA-DCM development. Manipulation of impaired SIRT1 activity and excessive oxidative stress is a potential future therapeutic strategy for LMNA-DCM.

The first symptoms of catecholaminergic polymorphic ventricular tachycardia (CPVT) usually occur in childhood and adolescence. 60% of patients experience syncope before the age of 40. Sudden cardiac death (SCD) is the first symptom of the disease in 30-50% of patients with CPVT. Early diagnosis is therefore crucial for the patient\'s prognosis. The diagnosis of CPVT is confirmed by a normal resting ECG, exclusion of structural heart disease, detection of bidirectional or polymorphic ventricular tachycardia (VT) in the stress ECG and/or detection of a pathogenic mutant in a gene associated with CPVT. Up to 60% of CPVT patients carry changes in the RYR2 gene. This gene encodes the cardiac ryanodine receptor, the most important Ca2+-releasing channel of the sarcoplasmic reticulum, which plays a central role in the contraction and relaxation of the heart muscle. If the function of the ryanodine receptor is impaired, too much calcium enters the cells, which triggers life-threatening arrhythmias. The overactive ryanodine receptor is therefore the main target for gene therapy methods. Even though the development of gene therapy is progressing, there is still no causal therapy available and it is all the more important to make a diagnosis as early as possible, which enables appropriate behavior and adequate symptomatic therapy. The decisive factor here is the evaluation of the genetic analysis in the context of the clinical findings. Based on this, recommendations can be made for preventive measures and the avoidance of specific triggers that could lead to life-threatening arrhythmias.
Die ersten Symptome der katecholaminergen polymorphen ventrikulären Tachykardie (CPVT) treten meist im Kindes- und Jugendalter auf. 60% der Patienten haben Synkopen vor dem 40. Lebensjahr. Der plötzliche Herztod (PHT) ist bei 30-50% der Patienten mit CPVT das erste Symptom der Erkrankung. Die rechtzeitige Diagnosesicherung ist daher entscheidend für die Prognose der Patienten. Gesichert wird die Diagnose CPVT bei unauffälligem Ruhe-EKG, Ausschluss einer strukturellen Herzerkrankung, Nachweis einer bidirektionalen oder polymorphen ventrikulären Tachykardie (VT) im Belastungs-EKG und/oder Nachweis einer pathogenen Variante in einem Gen, das mit einer CPVT assoziiert ist. Bis zu 60% der CPVT-Patienten tragen Veränderungen im RYR2-Gen. Dieses Gen kodiert für den kardialen Ryanodinrezeptor, den wichtigsten Ca2+-freisetzenden Kanal des sarkoplasmatischen Retikulums, der eine zentrale Rolle bei der Kontraktion und Entspannung des Herzmuskels spielt. Ist die Funktion des Ryanodinrezeptors gestört, gelangt zu viel Kalzium in die Zellen, was lebensbedrohliche Arrhythmien auslöst. Der überaktive Ryanodinrezeptor ist daher der Hauptansatzpunkt für die gentherapeutischen Methoden. Auch wenn die Entwicklung der Gentherapie voranschreitet, steht bisher noch keine ursächliche Therapie zur Verfügung und eine möglichst frühzeitige Diagnose, die ein angepasstes Verhalten und eine adäquate symptomatische Therapie ermöglicht, ist umso wichtiger. Entscheidend ist dabei die Bewertung der genetischen Analyse im Kontext mit den klinischen Befunden. Darauf aufbauend können Empfehlungen für präventive Maßnahmen und die Meidung spezifischer Trigger, die zu lebensbedrohlichen Rhythmusstörungen führen könnten, ausgesprochen werden.Schlüsselwörter: Kardiale Ionenkanalerkrankung, plötzlicher Herztod, unklare Synkope, Arrhythmie, Herzgenetik, RyanodinrezeptorEingereicht am 19. März 2024 - Revision akzeptiert am 25. Juni 2024.

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.

Astragaloside IV, a prime active component of Astragalus membranaceus, has potential as a neuroprotectant. We aimed to identify the active ingredients in A. membranaceus and assess if Astragaloside IV can improve cerebral ischemia-reperfusion injury (CIRI) cell apoptosis by reducing P-Src and P-GRK2 via ryanodine receptor (RyR) expression inhibition. We used bioinformatics analysis to examine the effects of A. membranaceus on ischemic stroke. We studied brain samples from middle cerebral artery occlusion (MCAO) mice treated with normal saline, Astragaloside IV, and sham mice for pathology and Western blot tests. We also tested PC12 cells in vitro with or without Astragaloside IV or GSK180736A using Western blotting and fluorescence assays. Our bioinformatics analysis suggested a possible association between A. membranaceus, calcium ion pathways, and apoptosis pathways. Western blot data indicated Astragaloside IV significantly decreased RyR, p-Src, and downstream phosphorylated GRK2, PLC, CaMKII, and IP3R levels in MCAO mice brains. Astragaloside IV also considerably inhibited pro-apoptotic and oxidative stress-associated proteins\' expression while boosting anti-apoptotic protein expression. The results suggest Astragaloside IV can inhibit RyR expression, subsequently reducing brain cell apoptosis.