Abstract 18111: Flecainide Exerts its Antiarrhythmic Action in CPVT Through Blockade of Neuronal Na+ channel-mediated Arrhythmogenic Diastolic Ca2+ Release

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Przemyslaw Radwanski ◽  
Hsiang-Ting Ho ◽  
Björn Knollmann ◽  
Andriy Belevych ◽  
Sándor Györke
Keyword(s):  
Ventricular Myocytes ◽  
Ryanodine Receptors ◽  
Cellular Level ◽  
Antiarrhythmic Effect ◽  
Na Channel ◽  
Antiarrhythmic Action ◽  
Pharmacological Interventions ◽  
Cellular Excitability

Background: Flecaininde is an effective antiarrhythmic in management of CPVT. Its antiarrhythmic action has been attributed to direct effect on RyR2 and reduced cellular excitability through the inhibition of cardiac-type Na + channels. Recently we demonstrated that neuronal Na + channels (nNa v s) colocalize with the ryanodine receptors (RyR2) Ca 2+ release channels on the sarcoplasmic reticulum. Here we explore a novel mechanism that may contribute to the antiarrhythmic effect of flecainide, mainly uncoupling of aberrant Na + /Ca 2+ signaling through nNa v inhibition. Methods: To study the effects of flecainide on Ca 2+ signaling we used a murine model of cardiac calsequestrin-associated CPVT. We performed confocal microscopy in intact isolated ventricular myocytes to assess Ca 2+ handling and recorded late Na + current (I Na ) during various pharmacological interventions. Surface electrocardiograms were performed during catecholamine challenge to monitor arrhythmic activity in vivo . Results: During catecholamine stimulation with isoproterenol (Iso; 100 nM) disruption of the cross-talk between nNa v s and RyR2 by nNa v blockade with 100nM tetrodotoxin (TTX) and riluzole (10μM) as well as flecainide (2.5μM) reduced Iso-promoted late I Na and DCR in isolated intact CPVT cardiomyocytes. To further examine the role of nNa v -mediated late I Na in genesis of DCR we augmented nNa v channel activity with β-Pompilidotoxin (β-PMTX, 40μM). Effects of β-PMTX in CPVT cardiomyocytes were reversed by nNa v blockade with TTX and riluzole as well as flecainide. This reduction in late I Na and DCR frequency with riluzole and flecainide in the presence of β-PMTX on cellular level translated to decreased ventricular arrhythmias in CPVT mice. Conclusion: These data suggest that disruption of nNa v -mediated late I Na can prevent arrhythmogenic DCR in CPVT. Importantly, the antiarrhythmic effects of flecainide can be attributed, at least in part, to its nNa v blocking properties.

scholarly journals Dual role of junctin in the regulation of ryanodine receptors and calcium release in cardiac ventricular myocytes

2011 ◽  
Vol 589 (24) ◽  
pp. 6063-6080 ◽  
Author(s):  
Beth A. Altschafl ◽  
Demetrios A. Arvanitis ◽  
Oscar Fuentes ◽  
Qunying Yuan ◽  
Evangelia G. Kranias ◽  
...  
Keyword(s):  
Calcium Release ◽  
Ventricular Myocytes ◽  
Ryanodine Receptors ◽  
Dual Role ◽  
Cardiac Ventricular Myocytes

Abstract 17019: Two Distinct mechanisms by which Na+/Ca2+ dysregulation contributes to Arrhythmogenic Diastolic Ca2+ Release

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Przemyslaw Radwanski ◽  
Rengasayee Veeraraghavan ◽  
Björn Knollmann ◽  
Sándor Györke
Keyword(s):  
Ventricular Myocytes ◽  
Cellular Level ◽  
Polymorphic Ventricular Tachycardia ◽  
Cell Periphery ◽  
Individualized Approach ◽  
Triggered Arrhythmias ◽  
Local Contribution ◽  
Dependent Protein ◽  
Camkii Activation

Na + and Ca 2+ imbalance is associated with triggered arrhythmias resulting from diastolic Ca 2+ release (DCR) from sarcoplasmic reticulum (SR). Recent evidence suggests Na + channel blockade to be a promising therapy for pathologies, including catecholaminergic polymorphic ventricular tachycardia (CPVT). However, the specific mechanism(s) as to how Na + /Ca 2+ dysregulation contribute to arrhythmias is unknown. Confocal microscopy of ventricular myocytes isolated from CPVT mice lacking the cardiac calsequestrin was used to assess Ca 2+ handling response to isoproterenol (Iso) and various pharmacological interventions, while electrocardiograms were acquired during catecholamine challenge to assess the roles of various pools of Na + channels in CPVT. We identify two pools of Na + channels: one composed of cardiac-type Na + channels localized to cell periphery, and a ‘ local pool ’ comprised of neuronal Na + channels colocalizing with RyR2 in the T-tubules. Augmenting function of both Na + channel pools with ATX-II in the presence Iso resulted in SR Ca 2+ overload and activation of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII), which precipitated DCR. These, in turn, translated into frequent arrhythmias in CPVT mice. Selectively augmenting function of ‘local pool’ neuronal Na + channels with β-Pompilidotoxin (β-PMTX) precipitated DCR on the cellular level causing frequent arrhythmias during catecholamine challenge in vivo . However, increasing local Na + fluxes reduced SR Ca 2+ load suggesting that local elevation in cytosolic Ca 2+ rather than global SR Ca 2+ overload underlies DCR and arrhythmias under such conditions. These data suggest two distinct mechanisms for Na + /Ca 2+ dysregulation-mediated arrhythmias. The first relies on SR Ca 2+ overload and CaMKII activation and the other on local contribution of Na + -Ca 2+ exchange to DCR. Consideration of these divergent mechanisms may enhance individualized approach to arrhythmia management.

scholarly journals Interaction of the Joining Region in Junctophilin-2 With the L-Type Ca 2+ Channel Is Pivotal for Cardiac Dyad Assembly and Intracellular Ca 2+ Dynamics

2021 ◽  
Vol 128 (1) ◽  
pp. 92-114
Author(s):  
Polina Gross ◽  
Jaslyn Johnson ◽  
Carlos M. Romero ◽  
Deborah M. Eaton ◽  
Claire Poulet ◽  
...  
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Ventricular Myocytes ◽  
Close Association ◽  
Ryanodine Receptors ◽  
Left Ventricular ◽  
Induced Mutations ◽  
Adrenergic Stimulation ◽  
T Tubules ◽  
Junctional Sarcoplasmic Reticulum

Rationale: Ca 2+ -induced Ca 2+ release (CICR) in normal hearts requires close approximation of L-type calcium channels (LTCCs) within the transverse tubules (T-tubules) and RyR (ryanodine receptors) within the junctional sarcoplasmic reticulum. CICR is disrupted in cardiac hypertrophy and heart failure, which is associated with loss of T-tubules and disruption of cardiac dyads. In these conditions, LTCCs are redistributed from the T-tubules to disrupt CICR. The molecular mechanism responsible for LTCCs recruitment to and from the T-tubules is not well known. JPH (junctophilin) 2 enables close association between T-tubules and the junctional sarcoplasmic reticulum to ensure efficient CICR. JPH2 has a so-called joining region that is located near domains that interact with T-tubular plasma membrane, where LTCCs are housed. The idea that this joining region directly interacts with LTCCs and contributes to LTCC recruitment to T-tubules is unknown. Objective: To determine if the joining region in JPH2 recruits LTCCs to T-tubules through direct molecular interaction in cardiomyocytes to enable efficient CICR. Methods and Results: Modified abundance of JPH2 and redistribution of LTCC were studied in left ventricular hypertrophy in vivo and in cultured adult feline and rat ventricular myocytes. Protein-protein interaction studies showed that the joining region in JPH2 interacts with LTCC-α1C subunit and causes LTCCs distribution to the dyads, where they colocalize with RyRs. A JPH2 with induced mutations in the joining region (mut PG1 JPH2) caused T-tubule remodeling and dyad loss, showing that an interaction between LTCC and JPH2 is crucial for T-tubule stabilization. mut PG1 JPH2 caused asynchronous Ca 2+ -release with impaired excitation-contraction coupling after β-adrenergic stimulation. The disturbed Ca 2+ regulation in mut PG1 JPH2 overexpressing myocytes caused calcium/calmodulin-dependent kinase II activation and altered myocyte bioenergetics. Conclusions: The interaction between LTCC and the joining region in JPH2 facilitates dyad assembly and maintains normal CICR in cardiomyocytes.

CaM kinase augments cardiac L-type Ca2+ current: a cellular mechanism for long Q-T arrhythmias

1999 ◽  
Vol 276 (6) ◽  
pp. H2168-H2178 ◽  
Author(s):  
Yuejin Wu ◽  
Leigh B. MacMillan ◽  
R. Blair McNeill ◽  
Roger J. Colbran ◽  
Mark E. Anderson
Keyword(s):  
Ventricular Myocytes ◽  
Ryanodine Receptors ◽  
Cam Kinase ◽  
Early Afterdepolarizations ◽  
Ultrastructural Studies ◽  
Intracellular Ca ◽  
Dependent Protein ◽  
T Tubules ◽  
Rabbit Ventricular Myocytes

Early afterdepolarizations (EAD) caused by L-type Ca2+ current ( I Ca,L) are thought to initiate long Q-T arrhythmias, but the role of intracellular Ca2+ in these arrhythmias is controversial. Rabbit ventricular myocytes were stimulated with a prolonged EAD-containing action potential-clamp waveform to investigate the role of Ca2+/calmodulin-dependent protein kinase II (CaM kinase) in I Ca,L during repolarization. I Ca,L was initially augmented, and augmentation was dependent on Ca2+ from the sarcoplasmic reticulum because the augmentation was prevented by ryanodine or thapsigargin. I Ca,Laugmentation was also dependent on CaM kinase, because it was prevented by dialysis with the inhibitor peptide AC3-I and reconstituted by exogenous constitutively active CaM kinase when Ba2+ was substituted for bath Ca2+. Ultrastructural studies confirmed that endogenous CaM kinase, L-type Ca2+ channels, and ryanodine receptors colocalized near T tubules. EAD induction was significantly reduced in current-clamped cells dialyzed with AC3-I (4/15) compared with cells dialyzed with an inactive control peptide (11/15, P = 0.013). These findings support the hypothesis that EADs are facilitated by CaM kinase.

Abstract 16870: Dissociation of Mitochondrial HK-II Triggers Mitochondria Specific Autophagy

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Shigeki Miyamoto ◽  
David J Roberts ◽  
Valerie P Tan-Sah
Keyword(s):  
Energy Production ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Mouse Heart ◽  
Hexokinase Ii ◽  
Survival Pathway ◽  
Mitochondrial Membrane Depolarization ◽  
Neonatal Rat Ventricular Myocytes

Introduction: There is emerging evidence that the metabolic pathway interplays with the survival pathway to preserve cellular homeostasis. Hexokinases (HKs) catalyze the first step of glucose metabolism and hexokinase-II (HK-II) is the predominant isoform in the heart. Our recent study revealed that HK-II positively regulates general autophagy in the absence of glucose. Mitochondrial HK-II (mitoHK-II) is regulated by Akt and provides cardioprotection while it is decreased in the ischemic heart. Hypothesis: We tested the hypothesis that mitoHK-II dissociation triggers mitochondria specific autophagy (mitophagy). Results: As previously reported, mitoHK-II levels were decreased by ~40% in the perfused mouse heart subjected to global ischemia and in neonatal rat ventricular myocytes (NRVMs) subjected to simulated ischemia. To assess the role of mitoHK-II dissociation, mitoHK-II dissociating peptide (15NG) was expressed in NRVMs. MitoHK-II was decreased by 40% in NRVMs expressing 15NG which was accompanied with Parkin translocation to mitochondria and ubiquitination of mitochondrial proteins. This response was attenuated by Parkin knockdown and reversed by the recovery of mitoHK-II by co-expression of HK-II but not by that of mitochondria binding deficient mutant. 15NG expression did not induce mitochondrial membrane depolarization nor PINK1 stabilization at mitochondria, suggesting that the effects of mitoHK-II dissociation is not dependent on the previously established mitochondria depolarization/PINK1 pathway. This was confirmed by the experiments using PINK1 siRNA. Modest dissociation of mitoHK-II (by 20%) did not induce mitophagic responses but remarkably enhanced FCCP induced mitophagy, indicating that these two pathways are synergetic. We will be analyzing 15NG transgenic mice generated in our lab to determine the mitophagic role of mitoHK-II dissociation in vivo. Conclusions: These results suggest that mitoHK-II dissociation can regulate Parkin dependent mitophagy, in conjunction with depolarization dependent mechanisms and that HK-II could confer cardioprotection by switching the cell from an energy production to an energy conservation mode under ischemia.

Role of the gut proteinases from mosquito larvae in the mechanism of action and the specificity of the Bacillus sphaericus toxin

1990 ◽  
Vol 36 (11) ◽  
pp. 804-807 ◽  
Author(s):  
Luc Nicolas ◽  
Anne Lecroisey ◽  
Jean-François Charles
Keyword(s):  
Cell Cultures ◽  
Spodoptera Littoralis ◽  
Mosquito Species ◽  
Bacillus Sphaericus ◽  
Cellular Level ◽  
Mosquito Cell ◽  
Mosquito Larvae

Gut proteinases from larvae of mosquito species both susceptible and not susceptible to Bacillus sphaericus converted the 43-kDa toxin to a 40-kDa polypeptide exhibiting enhanced cytotoxicity to mosquito cell cultures. The toxin was also activated by gut proteinases from the nonsusceptible Lepidoptera Spodoptera littoralis in vitro and in vivo. Therefore, the specificity of Bacillus sphaericus toxin does not seem to be determined by gut proteinase action. However, susceptibility of mosquito cell cultures did not reflect the specificity of the toxin, which must now be investigated at the cellular level in the larvae. Key words: mosquitoes, Bacillus sphaericus, bacterial toxins, proteinases, specificity.

scholarly journals Involvement of Nitric Oxide in Iodine Deficiency-Induced Microvascular Remodeling in the Thyroid Gland: Role of Nitric Oxide Synthase 3 and Ryanodine Receptors

Endocrinology ◽  
2014 ◽  
Vol 156 (2) ◽  
pp. 707-720 ◽  
Author(s):  
J. Craps ◽  
C. Wilvers ◽  
V. Joris ◽  
B. De Jongh ◽  
J. Vanderstraeten ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Blood Flow ◽  
Thyroid Gland ◽  
Mrna Expression ◽  
Iodine Deficiency ◽  
Ryanodine Receptors ◽  
No Production

Iodine deficiency (ID) induces microvascular changes in the thyroid gland via a TSH-independent reactive oxygen species-hypoxia inducible factor (HIF)-1α-vascular endothelial growth factor (VEGF) pathway. The involvement of nitric oxide (NO) in this pathway and the role of calcium (Ca2+) and of ryanodine receptors (RYRs) in NO synthase 3 (NOS3) activation were investigated in a murine model of goitrogenesis and in 3 in vitro models of ID, including primary cultures of human thyrocytes. ID activated NOS3 and the production of NO in thyrocytes in vitro and increased the thyroid blood flow in vivo. Using bevacizumab (a blocking antibody against VEGF-A) in mice, it appeared that NOS3 is activated upstream of VEGF-A. L-nitroarginine methyl ester (a NOS inhibitor) blocked the ID-induced increase in thyroid blood flow in vivo and NO production in vitro, as well as ID-induced VEGF-A mRNA and HIF-1α expression in vitro, whereas S-nitroso-acetyl-penicillamine (a NO donor) did the opposite. Ca2+ is involved in this pathway as intracellular Ca2+ flux increased after ID, and thapsigargin activated NOS3 and increased VEGF-A mRNA expression. Two of the 3 known mammalian RYR isoforms (RYR1 and RYR2) were shown to be expressed in thyrocytes. RYR inhibition using ryanodine at 10μM decreased ID-induced NOS3 activation, HIF-1α, and VEGF-A expression, whereas RYR activation with ryanodine at 1nM increased NOS3 activation and VEGF-A mRNA expression. In conclusion, during the early phase of TSH-independent ID-induced microvascular activation, ID sequentially activates RYRs and NOS3, thereby supporting ID-induced activation of the NO/HIF-1α/VEGF-A pathway in thyrocytes.

scholarly journals Knockout of vascular smooth muscle EGF receptor in a mouse model prevents obesity-induced vascular dysfunction and renal damage in vivo

Diabetologia ◽  
2020 ◽  
Vol 63 (10) ◽  
pp. 2218-2234
Author(s):  
Christian Stern ◽  
Barbara Schreier ◽  
Alexander Nolze ◽  
Sindy Rabe ◽  
Sigrid Mildenberger ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Smooth Muscle ◽  
Mitochondrial Dysfunction ◽  
Renal Damage ◽  
Egf Receptor ◽  
Vascular Dysfunction ◽  
Cellular Level

Abstract Aims/hypothesis Obesity causes type 2 diabetes leading to vascular dysfunction and finally renal end-organ damage. Vascular smooth muscle (VSM) EGF receptor (EGFR) modulates vascular wall homeostasis in part via serum response factor (SRF), a major regulator of VSM differentiation and a sensor for glucose. We investigated the role of VSM-EGFR during obesity-induced renovascular dysfunction, as well as EGFR–hyperglycaemia crosstalk. Methods The role of VSM-EGFR during high-fat diet (HFD)-induced type 2 diabetes was investigated in a mouse model with inducible, VSM-specific EGFR-knockout (KO). Various structural and functional variables as well as transcriptome changes, in vivo and ex vivo, were assessed. The impact of hyperglycaemia on EGFR-induced signalling and SRF transcriptional activity and the underlying mechanisms were investigated at the cellular level. Results We show that VSM-EGFR mediates obesity/type 2 diabetes-induced vascular dysfunction, remodelling and transcriptome dysregulation preceding renal damage and identify an EGFR–glucose synergism in terms of SRF activation, matrix dysregulation and mitochondrial function. EGFR deletion protects the animals from HFD-induced endothelial dysfunction, creatininaemia and albuminuria. Furthermore, we show that HFD leads to marked changes of the aortic transcriptome in wild-type but not in KO animals, indicative of EGFR-dependent SRF activation, matrix dysregulation and mitochondrial dysfunction, the latter confirmed at the cellular level. Studies at the cellular level revealed that high glucose potentiated EGFR/EGF receptor 2 (ErbB2)-induced stimulation of SRF activity, enhancing the graded signalling responses to EGF, via the EGFR/ErbB2–ROCK–actin–MRTF pathway and promoted mitochondrial dysfunction. Conclusions/interpretation VSM-EGFR contributes to HFD-induced vascular and subsequent renal alterations. We propose that a potentiated EGFR/ErbB2–ROCK–MRTF–SRF signalling axis and mitochondrial dysfunction underlie the role of EGFR. This advanced working hypothesis will be investigated in mechanistic depth in future studies. VSM-EGFR may be a therapeutic target in cases of type 2 diabetes-induced renovascular disease. Data availability The datasets generated during and/or analysed during the current study are available in: (1) share_it, the data repository of the academic libraries of Saxony-Anhalt (10.25673/32049.2); and (2) in the gene expression omnibus database with the study identity GSE144838 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE144838).

scholarly journals Role of Inhibitory Neurotransmission in the Control of Canine Hypoglossal Motoneuron Activity In Vivo

2009 ◽  
Vol 101 (3) ◽  
pp. 1211-1221 ◽  
Author(s):  
Antonio Sanchez ◽  
Sanda Mustapic ◽  
Edward J. Zuperku ◽  
Astrid G. Stucke ◽  
Francis A. Hopp ◽  
...  
Keyword(s):  
Upper Airway ◽  
Cellular Level ◽  
Spontaneous Discharge ◽  
Glycine Receptors ◽  
Active Neuron ◽  
Airway Patency ◽  
Inhibitory Mechanisms ◽  
Premotor Neurons

Hypoglossal motoneurons (HMNs) innervate all tongue muscles and are vital for maintenance of upper airway patency during inspiration. The relative contributions of the various synaptic inputs to the spontaneous discharge of HMNs in vivo are incompletely understood, especially at the cellular level. The purpose of this study was to determine the role of endogenously activated GABAAand glycine receptors in the control of the inspiratory HMN (IHMN) activity in a decerebrate dog model. Multibarrel micropipettes were used to record extracellular unit activity of individual IHMNs during local antagonism of GABAAreceptors with bicuculline and picrotoxin or glycine receptors with strychnine. Only bicuculline had a significant effect on peak and average discharge frequency and on the slope of the augmenting neuronal discharge pattern. These parameters were increased by 30 ± 7% ( P < 0.001), 30 ± 8% ( P < 0.001), and 25 ± 7% ( P < 0.001), respectively. The effects of picrotoxin and strychnine on the spontaneous neuronal discharge and its pattern were negligible. Our data suggest that bicuculline-sensitive GABAergic, but not picrotoxin-sensitive GABAergic or glycinergic, inhibitory mechanisms actively attenuate the activity of IHMNs in vagotomized decerebrate dogs during hyperoxic hypercapnia. The pattern of GABAergic attenuation of IHMN discharge is characteristic of gain modulation similar to that in respiratory bulbospinal premotor neurons, but the degree of attenuation (∼25%) is less than that seen in bulbospinal premotor neurons (∼60%). The current studies only assess effects on active neuron discharge and do not resolve whether the lack of effect of picrotoxin and strychnine on IHMNs also extends to the inactive expiratory phase.

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