Ca Currents Research Articles

A Novel Role for UNC119 as an Enhancer of Synaptic Transmission.

Mammalian UNC119 is a ciliary trafficking chaperone highly expressed in the inner segment of retinal photoreceptors. Previous research has shown that UNC119 can bind to transducin, the synaptic ribbon protein RIBEYE, and the calcium-binding protein CaBP4, suggesting that UNC119 may have a role in synaptic transmission. We made patch-clamp recordings from retinal slices in mice with the UNC119 gene deleted and showed that removal of even one gene of UNC119 has no effect on the rod outer segment photocurrent, but acted on bipolar cells much like background light: it depolarized membrane potential, decreased sensitivity, accelerated response decay, and decreased the Hill coefficient of the response-intensity relationship. Similar effects were seen on rod bipolar-cell current and voltage responses, and after exposure to bright light to translocate transducin into the rod inner segment. These findings indicate that UNC119 deletion reduces the steady-state glutamate release rate at rod synapses, though no change in the voltage dependence of the synaptic Ca current was detected. We conclude that UNC119, either by itself or together with transducin, can facilitate the release of glutamate at rod synapses, probably by some interaction with RIBEYE or other synaptic proteins rather than by binding to CaBP4 or calcium channels.

RBM24 controls cardiac QT interval through CaMKIIδ splicing.

Calcium/calmodulin-dependent kinase II delta (CaMKIIδ) is the predominant cardiac isoform and it is alternatively spliced to generate multiple variants. Variable variants allow for distinct localization and potentially different functions in the heart. Dysregulation of CaMKIIδ splicing has been demonstrated to be involved in the pathogenesis of heart diseases, such as cardiac hypertrophy, arrhythmia, and diastolic dysfunction. However, the mechanisms that regulate CaMKIIδ are incompletely understood. Here, we show that RNA binding motif protein 24 (RBM24) is a key splicing regulator of CaMKIIδ. RBM24 ablation leads to the aberrant shift of CaMKIIδ towards the δ-C isoform, which is known to activate the L-type Ca current. In line with this, we found marked alteration in Ca2+ handling followed by prolongation of the ventricular cardiac action potential and QT interval in RBM24 knockout mice, and these changes could be attenuated by treatment with an inhibitor of CaMKIIδ. Importantly, knockdown of RBM24 in human embryonic stem cell-derived cardiomyocytes showed similar electrophysiological abnormalities, suggesting the important role of RBM24 in the human heart. Thus, our data suggest that RBM24 is a critical regulator of CaMKIIδ to control the cardiac QT interval, highlighting the key role of splicing regulation in cardiac rhythm.

Evidence for a Physiological Role of T-Type Ca Channels in Ventricular Cardiomyocytes of Adult Mice.

T-type Ca channels are strongly expressed and important in the developing heart. In the adult heart, these channels play a significant role in pacemaker tissues, but there is uncertainty about their presence and physiological relevance in the working myocardium. Here, we show that the T-type Ca channel isoforms Cav3.1 and Cav3.2 are expressed at a protein level in ventricular cardiomyocytes from healthy adult C57/BL6 mice. Myocytes isolated from adult wild-type and Cav3.2 KO mice showed considerable whole cell T-type Ca currents under beta-adrenergic stimulation with isoprenaline. We further show that the detectability of basal T-type Ca currents in murine wild-type cardiomyocytes depends on the applied experimental conditions. Together, these findings reveal the presence of functional T-type Ca channels in the membrane of ventricular myocytes. In addition, electrically evoked Ca release from the sarcoplasmic reticulum was significantly impaired in Cav3.2 KO compared to wild-type cardiomyocytes. Our work implies a physiological role of T-type Ca channels in the healthy adult murine ventricular working myocardium.

Adrenergic prolongation of action potential duration in rainbow trout myocardium via inhibition of the delayed rectifier potassium current, IKr

Catecholamines mediate the ‘fight or flight’ response in a wide variety of vertebrates. The endogenous catecholamine adrenaline increases heart rate and contractile strength to raise cardiac output. The increase in contractile force is driven in large part by an increase in myocyte Ca2+ influx on the L-type Ca current (ICaL) during the cardiac action potential (AP). Here, we report a K+- based mechanism that prolongs AP duration (APD) in fish hearts following adrenergic stimulation. We show that adrenergic stimulation inhibits the delayed rectifier K+ current (IKr) in rainbow trout (Oncorhynchus mykiss) cardiomyocytes. This slows repolarization and prolongs APD which may contribute to positive inotropy following adrenergic stimulation in fish hearts. The endogenous ligand, adrenaline (1 μM), which activates both α- and β-ARs reduced maximal IKr tail current to 61.4 ± 3.9% of control in atrial and ventricular myocytes resulting in an APD prolongation of ~20% at both 50 and 90% repolarization. This effect was reproduced by the α-specific adrenergic agonist, phenylephrine (1 μM), but not the β-specific adrenergic agonist isoproterenol (1 μM). Adrenaline (1 μM) in the presence of β1 and β2-blockers (1 μM atenolol and 1 μM ICI-118551, respectively) also inhibited IKr. Thus, IKr suppression following α-adrenergic stimulation leads to APD prolongation in the rainbow trout heart. This is the first time this mechanism has been identified in fish and may act in unison with the well-known enhancement of ICaL following adrenergic stimulation to prolong APD and increase cardiac inotropy.

Direct inhibition of CaV2.3 by Gem is dynamin dependent and does not require a direct alfa/beta interaction

The Rad, Rem, Rem2, and Gem/Kir (RGK) sub-family of small GTP-binding proteins are crucial in regulating high voltage-activated (HVA) calcium channels. RGK proteins inhibit calcium current by either promoting endocytosis or reducing channel activity. They all can associate directly with Ca2+ channel β subunit (CaVβ), and the binding between CaVα1/CaVβ appears essential for the endocytic promotion of CaV1.X, CaV2.1, and CaV2.2 channels. In this study, we investigated the inhibition of CaV2.3 channels by RGK proteins in the absence of CaVβ. To this end, Xenopus laevis oocytes expressing CaV2.3 channels devoid of auxiliary subunit were injected with purified Gem and Rem and found that only Gem had an effect. Ca currents and charge movements were reduced by injection of Gem, pointing to a reduction in the number of channels in the plasma membrane. Since this reduction was ablated by co-expression of the dominant-negative mutant of dynamin K44A, enhanced endocytosis appears to mediate this reduction in the number of channels. Thus, Gem inhibition of CaV2.3 channels would be the only example of a CaVβ independent promotion of dynamin-dependent endocytosis.

Enhanced Heart Failure in Redox-Dead Cys17Ser PKARIα Knock-In Mice.

BackgroundPKARIα (protein kinase A type I‐α regulatory subunit) is redox‐active independent of its physiologic agonist cAMP. However, it is unknown whether this alternative mechanism of PKARIα activation may be of relevance to cardiac excitation–contraction coupling.Methods and ResultsWe used a redox‐dead transgenic mouse model with homozygous knock‐in replacement of redox‐sensitive cysteine 17 with serine within the regulatory subunits of PKARIα (KI). Reactive oxygen species were acutely evoked by exposure of isolated cardiac myocytes to AngII (angiotensin II, 1 µmol/L). The long‐term relevance of oxidized PKARIα was investigated in KI mice and their wild‐type (WT) littermates following transverse aortic constriction (TAC). AngII increased reactive oxygen species in both groups but with RIα dimer formation in WT only. AngII induced translocation of PKARI to the cell membrane and resulted in protein kinase A–dependent stimulation of I Ca (L‐type Ca current) in WT with no effect in KI myocytes. Consequently, Ca transients were reduced in KI myocytes as compared with WT cells following acute AngII exposure. Transverse aortic constriction–related reactive oxygen species formation resulted in RIα oxidation in WT but not in KI mice. Within 6 weeks after TAC, KI mice showed an enhanced deterioration of contractile function and impaired survival compared with WT. In accordance, compared with WT, ventricular myocytes from failing KI mice displayed significantly reduced Ca transient amplitudes and lack of I Ca stimulation. Conversely, direct pharmacological stimulation of I Ca using Bay K8644 rescued Ca transients in AngII‐treated KI myocytes and contractile function in failing KI mice in vivo.ConclusionsOxidative activation of PKARIα with subsequent stimulation of I Ca preserves cardiac function in the setting of acute and chronic oxidative stress.

Cardiac Na/Ca Exchange Suppression: A Late‐Breaking Knockout Story Showing That There Is No Free Lunch

Cardiac Na/Ca Exchange Suppression: A Late‐Breaking Knockout Story Showing That There Is No Free Lunch

Serotonergic Modulation of Persistent Inward Currents in Serotonergic Neurons of Medulla in ePet-EYFP Mice.

Serotonergic (5-HT) neurons in the medulla play multiple functional roles associated with many symptoms and motor activities. The descending serotonergic pathway from medulla is essential for initiating locomotion. However, the ionic properties of 5-HT neurons in the medulla remain unclear. Using whole-cell patch-clamp technique, we studied the biophysical and modulatory properties of persistent inward currents (PICs) in 5-HT neurons of medulla in ePet-EYFP transgenic mice (P3–P6). PICs were recorded by a family of voltage bi-ramps (10-s duration, 40-mV peak step), and the ascending and descending PICs were mirrored to analyze the PIC hysteresis. PICs were found in 77% of 5-HT neurons (198/258) with no significant difference between parapyramidal region (n = 107) and midline raphe nuclei (MRN) (n = 91) in either PIC onset (−47.4 ± 10 mV and −48.7 ± 7 mV; P = 0.44) or PIC amplitude (226.9 ± 138 pA and 259.2 ± 141 pA; P = 0.29). Ninety-six percentage (191/198) of the 5-HT neurons displayed counterclockwise hysteresis and four percentage (7/198) exhibited the clockwise hysteresis. The composite PICs could be differentiated as calcium component (Ca_PIC) by bath application of nimodipine (25 μM), sodium component (Na_PIC) by tetrodotoxin (TTX, 2 μM), and TTX- and dihydropyridine-resistance component (TDR_PIC) by TTX and nimodipine. Ca_PIC, Na_PIC and TDR_PIC all contributed to upregulation of excitability of 5-HT neurons. 5-HT (15 μM) enhanced the PICs, including a 26% increase in amplitude of the compound currents of Ca_PIC and TDR_PIC (P < 0.001, n = 9), 3.6 ± 5 mV hyperpolarization of Na_PIC and TDR_PIC onset (P < 0.05, n = 12), 30% increase in amplitude of TDR_PIC (P < 0.01), and 2.0 ± 3 mV hyperpolarization of TDR_PIC onset (P < 0.05, n = 18). 5-HT also facilitated repetitive firing of 5-HT neurons through modulation of composite PIC, Na_PIC and TDR_PIC, and Ca_PIC and TDR_PIC, respectively. In particular, the high voltage-activated TDR_PIC facilitated the repetitive firing in higher membrane potential, and this facilitation could be amplified by 5-HT. Morphological data analysis indicated that the dendrites of 5-HT neurons possessed dense spherical varicosities intensively crossing 5-HT neurons in medulla. We characterized the PICs in 5-HT neurons and unveiled the mechanism underlying upregulation of excitability of 5-HT neurons through serotonergic modulation of PICs. This study provided insight into channel mechanisms responsible for the serotonergic modulation of serotonergic neurons in brainstem.

A model of on/off transitions in neurons of the deep cerebellar nuclei: deciphering the underlying ionic mechanisms

The neurons of the deep cerebellar nuclei (DCNn) represent the main functional link between the cerebellar cortex and the rest of the central nervous system. Therefore, understanding the electrophysiological properties of DCNn is of fundamental importance to understand the overall functioning of the cerebellum. Experimental data suggest that DCNn can reversibly switch between two states: the firing of spikes (F state) and a stable depolarized state (SD state). We introduce a new biophysical model of the DCNn membrane electro-responsiveness to investigate how the interplay between the documented conductances identified in DCNn give rise to these states. In the model, the F state emerges as an isola of limit cycles, i.e. a closed loop of periodic solutions disconnected from the branch of SD fixed points. This bifurcation structure endows the model with the ability to reproduce the text{F}to text{SD} transition triggered by hyperpolarizing current pulses. The model also reproduces the text{F}to text{SD} transition induced by blocking Ca currents and ascribes this transition to the blocking of the high-threshold Ca current. The model suggests that intracellular current injections can trigger fully reversible text{F}leftrightarrow text{SD} transitions. Investigation of low-dimension reduced models suggests that the voltage-dependent Na current is prominent for these dynamical features. Finally, simulations of the model suggest that physiological synaptic inputs may trigger text{F}leftrightarrow text{SD} transitions. These transitions could explain the puzzling observation of positively correlated activities of connected Purkinje cells and DCNn despite the former inhibit the latter.

Calcium-alginate beads as a formulation for the application of entomopathogenic nematodes to control rootworms

Entomopathogenic nematodes (EPN) have great potential as biological control agents against root-feeding insects. They have a rapid and long-lasting mode of action, minimal adverse effects on the environment and can be readily mass-produced. However, they have a relatively short shelf-life and are susceptible to desiccation and UV light. These shortcomings may be overcome by encapsulating EPN in Ca2+-alginate hydrogels, which have been shown to provide a humid and UV protective shelter. Yet, current Ca2+-alginate formulations do not keep EPN vigorous and infectious for a prolonged period of time and do not allow for their controlled release upon application. Here, we introduce solid Ca2+-alginate beads which we supplemented with glycerol to better retain the EPN during storage and to ensure a steady release when applied in soil. Glycerol-induced metabolic arrest in EPN (Heterorhabditis bacteriophora) resulting in quiescence and total retainment of EPN when added to beads made with 0.5% sodium alginate and 2% CaCl2·2H2O solutions. More than 4,000 EPN could be embedded in a single 4–5-mm diameter bead, and quiescence could be broken by adding water, after which the EPN readily emerged from the beads. In a field trial, the EPN beads were as effective in reducing root damage by the western corn rootworm (WCR, Diabrotica virgifera virgifera) as EPN that were applied in water. Although further improvements are desirable, we conclude that Ca2+-alginate beads can provide an effective and practical way to apply EPN for the control of WCR larvae.

Computational modeling of NMDA receptor response in Alzheimer’s disease

N-methyl-d-aspartate receptors (NMDA) located on the postsynaptic terminal of CA1 pyramidal neuron plays an essential role in synaptic plasticity, and the calcium(Ca) current flowing through it drives several intracellular calcium mechanisms. We have developed a single-compartment model involving least mechanisms to study the NMDA receptor response to different input condition and to obtain the voltage-dependent relationship of magnesium (Mg) block in the NMDA receptor. Using this interdependence, we have replicated a pathological condition of Alzheimer’s diseases (AD) to obtain total NMDA receptor current in AD. The results obtained at different Mg concentration suggested that the blocking rate increases linearly with the Mg concentration. In AD there is dysfunction of Mg block which leads to the early opening of the NMDA receptor. So by using a drug which controls the blocking mechanism of Mg, which leads to control of NMDA receptor current. This results in the control of intracellular Ca current.

Co-existence of a fast tetrodotoxin-sensitive low voltage-activated non selective Na/Ca/K channel and of T-type Ca channels in rat isolated pulmonary vein cardiomyocytes

Ectopic foci in pulmonary veins (PV) are involved in the onset of atrial fibrillation. Norepinephrine (NE) induces an automatic activity occurring in bursts in rat PV cardiomyocytes (CM) which depends on Ca (upstroke) and Na (inter-burst) channels. To characterize low voltage-activated (LVA) Ca currents in rat PVCM susceptible to trigger ectopic foci. Whole-cell ICa (5 mM Ca) was recorded from -100 mV with classical Na- and K-free solutions at room temperature in enzymatically isolated PVCM from male Wistar rats. A fast LVA ICa (FLVA-ICa; −1.5 ± 0.2 pA/pF, n = 45; 26% of peak ICa, max recorded at + 20 mV) was activated between −55 and −20 mV in ∼56% of PVCM and blocked by 10 μM tetrodotoxin (TTX). In that, it was similar to the reported TTX-sensitive Ca current. However, it was markedly increased by addition of NaCl (1 or 3 mM) or KCl (5 or 10 mM). Permeability ratios P’Ca/PNa and P’Ca/PK calculated for bi-ionic conditions were respectively 2.25 ± 0.51 (n = 6) and 1.88 ± 0.25 (n = 14), and not different from a value of 2. NE (10 μM, n = 13) increased FLVA-ICa and negatively shifted its activation threshold, an effect partially reversed by 5 μM Atenolol. It was partially blocked by 5 μM Nifedipine (n = 5) and increased by 300 nM BayK8644 (n = 6), but not blocked by Ranolazine (10 μM, n = 6). NiCl2 (40 μM, n = 9) and TTA-A2 (10 or 100 nM, n = 11) increased FLVA-ICa. Neither Ba nor Sr alone could permeate the FLVA channel or block Ca influx but revealed a large slower activating and inactivating LVA Ca current (SLVA-ICa), present in 10 out of 80 PVCM and absent in LACM. SLVA-ICa was partially blocked by 100 nM TTA-A2. The ionic channel underlying FLVA-ICa is likely a fast voltage-gated non-selective channel permeable to Na/Ca/K with a dihydropyridine binding site. SLVA-ICa might correspond to Ca influx through Cav3.1 channels. The respective role of these channels to triggering ectopic foci in the PV myocardial sleeve remained to be determined.

Role of LVA Ca channels in norepinephrine-induced automatic activity in rat pulmonary vein myocardial sleeve

Ectopic foci in pulmonary veins (PV) are involved in the onset of atrial fibrillation. Norepinephrine (NE) induces in rat PV cardiomyocytes (CM) an automatic activity in saw-tooth pattern (catecholaminergic automatic activity or CAA), consisting of bursts of slow action potentials which depends on Ca (upstroke) and Na (inter burst) channels. We have previously shown the co-existence in rat PVCM, of a fast LVA (FLVA) non selective TTX-sensitive cationic channel and of T-type Ca channels. To evaluate in male Wistar rat PV myocardial sleeve the contribution of these channels to triggering NE-induced ectopic foci. Whole-cell currents were studied at room temperature in enzymatically isolated PVCM. Mechanical activity of PV rings was recorded in an organ bath at 37 °C and CAA was induced by application of 10 μM NE. The FLVA Ca current was increased by the Cav3.x blockers TTA-A2 (10-100 nM, n = 11) and NiCl2 (40 μM, n = 9) whereas a large LVA Ca current, present in 10 out of 80 PVCM, was partially reduced by 100 nM TTA-A2. Study of K currents revealed the presence of an inward rectifying current sensitive to TRAM-34 (1 μM, n = 6), a blocker of the Ca-activated K channel SK4. In PV rings, TTA-A2 (1-5 μM) decreased CAA incidence. Total arrhythmia duration, measured over the last 10 min of a 30 min long drug application, was reduced as well as burst duration and the number of spontaneous twitches within a burst. On the other hand, 40 μM NiCl2 or 1-2 μM TRAM-34 increased total arrhythmia duration by decreasing the interval between bursts (no significant effect on the burst characteristics). Only NiCl2 had a significant negative inotropic effect (∼80% inhibition of L-type calcium current). Our results suggest that Ca influx through Cav3.1 channels contribute to triggering bursts of automatic activity and that Cav3.2 Ca channels, through activation of SK4 channels, are involved in the regulation of the interval between bursts.

Autonomic modulation of sinoatrial node: Role of pacemaker current and calcium sensitive adenylyl cyclase isoforms

This article reviews work over the past three decades that is related to the contribution of the pacemaker current, If, to basal and autonomically regulated spontaneous rate in the sinoatrial node. It also addresses how the actions of the pacemaker current relate to those of Ca homeostasis with respect to basal and autonomically regulated rhythm. In this regard, it explores the relative contributions of Ca-sensitive and Ca-insensitive isoforms of adenylyl cyclase to sinoatrial node automaticity. The latter studies include previously unpublished work making use of mice in which both the type 1 and type 8 Ca-sensitive adenylyl cyclase isoforms were knocked out. These studies indicate that the pacemaker current and the L-type Ca current are distinctly influenced by Ca-sensitive and insensitive adenylyl cyclase isoforms.

1884-P: Cardiomyocyte-Specific PKA Inhibition Ameliorates Cardiac Dysfunction Induced by Obesity

Obesity and type 2 diabetic (T2D) cause cardiac dysfunction, which could be related to chronically activated sympathoadrenergic system (SAS). SAS system activation leads to the activation of PKA in cardiomyocytes. In this study, we aimed to test the hypothesis that chronically activated PKA plays an important role in obese induced cardiac dysfunction. Methods and Results: 1. Cardiomyocyte specific PKA inhibition (cPKAi) transgenic (TG) mouse model (n=18) and nontransgenic littermate control (NLC, n=20) mice (3-month old) were fed with high fat food (D12451, Research Diet) or normal chow for 6 months.2. In 3-month old male TG mice, intraperitoneal glucose injection did not increase blood glucose as high as in 3-month old NLC mice. The area under the curve of cPKAi TG mice was significantly less than that of control mice. cPKAi transgenic mice had decreased cardiac function after overnight fasting while control mice did not have cardiac function decrement even though both types of mice had the same extend of glucose reduction after overnight fasting. 3. The BW increase was more in TG mice. 4. After HFD feeding, cPKAi TG group had better cardiac function: (1). The maximal systolic pressure (mmHg) of TG (107.8±4.49) was significantly higher than that of NLC (83.0±8.1) mice. (2). The end diastolic pressure (EDP, mmHg) of TG (2.7 ± 2.2) was significantly lower in TG mice (11.5 ± 3.3); (3). Maximum dp/dt was significantly greater in TG than in NLC mice; (4). The relaxation rate of the heart was significantly better in TG than in NLC mice; 5. The HW/BW (mg/g) was significantly less in TG mice (4.6 ± 0.2) than in control mice (5.2 ± 0.2). 6. HFD reduced myocytes contractions, Ca transients, and Ca currents in both groups but the reduction was less in TG myocytes. 7. TG hearts had less inflammatory cell infiltration and fibrosis than control hearts. Conclusion: cPKAi enhances cardiac glucose uptake and ameliorates cardiac remodeling and preserves cardiac function in HFD-induced obese. Disclosure X. Chen: None. Funding American Diabetes Association (1-19-IBS-210)

Novel role for mineralocorticoid receptors in control of a neuronal phenotype

Mineralocorticoid receptors (MRs) in the brain play a role in learning and memory, neuronal differentiation, and regulation of the stress response. Within the hippocampus, the highest expression of MRs is in area CA2. CA2 pyramidal neurons have a distinct molecular makeup resulting in a plasticity-resistant phenotype, distinguishing them from neurons in CA1 and CA3. Thus, we asked whether MRs regulate CA2 neuron properties and CA2-related behaviors. Using three conditional knockout methods at different stages of development, we found a striking decrease in multiple molecular markers for CA2, an effect mimicked by chronic antagonism of MRs. Furthermore, embryonic deletion of MRs disrupted afferent inputs to CA2 and enabled synaptic potentiation of the normally LTP-resistant synaptic currents in CA2. We also found that CA2-targeted MR knockout was sufficient to disrupt social behavior and alter behavioral responses to novelty. Altogether, these results demonstrate an unappreciated role for MRs in controlling CA2 pyramidal cell identity and in facilitating CA2-dependent behaviors.

Abstract 102: Molecular, Cellular and Systemic Mechanism of Nonlinear Dynamic Patterns of Ventricular Repolarization and Spontaneous Arrhythmic Sudden Death in Non-ischemic Heart Failure

Introduction: Sudden cardiac death (SCD) is the leading cause of death in USA. Heart failure (HF) confers high SCD risk, but most SCD victims do not have HF or well-defined genetic abnormalities. The underlying mechanisms are poorly understood, precluding the design of more effective strategies for risk stratification and therapy. We previously showed that distinct non-linear patterns of cardiac repolarization strongly and independently predict SCD in HF patients [PMID: 27044982]. We also showed in a unique guinea pig HF model that increased levels of mitochondrial reactive oxygen species (mROS) drive spontaneous SCD, even before HF onset [PMID: 29898892]. Herein, we further dissect the underlying mechanisms. Hypothesis: Increased mROS levels in pressure overloaded hearts reduce K+ currents, destabilize the resting membrane potential, increase repolarization lability and cause SCD. Methods: Guinea pigs were randomized to Sham, aortic banding (AC), or AC with daily brief low-dose β-adrenergic stress (ACi) with and without in vivo mROS scavenger MitoTEMPO (ACi+MT). We performed time series analyses on recordings of: (1) 24-hour ECG QT interval in freely ambulating animals before sacrifice at 4 weeks; (2) pressure-volume and electrophysiology (EP) in excised perfused hearts; and (3) duration of action potential (APd), calcium transient (CaTd) and sarcomere shortening (SSd) in isolated LV myocytes. Results: About 50% of ACi animals had SCD by 4 weeks. Increased QT entropy in ACi (at 1 week) predicted SCD over followup, similar to HF patients. Compared to Sham and ACi+MT, LV myocytes isolated from AC and ACi had reduced inward (IK1) and delayed slow (IKs) and rapid (IKr) rectifier currents; and increased beat-to-beat lability in APd, CaTd and SSd. The late Na and L-type Ca currents were similar between models. Acute exposure to isoproterenol (Iso) and carbachol (CCh) increased lability in LV myocytes compared to Iso or CCh alone. Conclusions: High mROS levels reduce repolarization reserve in LV myocytes and contribute, at least in part, to the non-linear dynamics of ventricular repolarization (reflected by high QT entropy), leading to spontaneous arrhythmic SCD. These findings provide important new mechanistic insight with direct clinical implications.

Adenosine A3 receptor activation inhibits pronociceptive N-type Ca2+ currents and cell excitability in dorsal root ganglion neurons.

Recently, studies have focused on the antihyperalgesic activity of the A3 adenosine receptor (A3AR) in several chronic pain models, but the cellular and molecular basis of this effect is still unknown. Here, we investigated the expression and functional effects of A3AR on the excitability of small- to medium-sized, capsaicin-sensitive, dorsal root ganglion (DRG) neurons isolated from 3- to 4-week-old rats. Real-time quantitative polymerase chain reaction experiments and immunofluorescence analysis revealed A3AR expression in DRG neurons. Patch-clamp experiments demonstrated that 2 distinct A3AR agonists, Cl-IB-MECA and the highly selective MRS5980, inhibited Ca-activated K (KCa) currents evoked by a voltage-ramp protocol. This effect was dependent on a reduction in Ca influx via N-type voltage-dependent Ca channels, as Cl-IB-MECA-induced inhibition was sensitive to the N-type blocker PD173212 but not to the L-type blocker, lacidipine. The endogenous agonist adenosine also reduced N-type Ca currents, and its effect was inhibited by 56% in the presence of A3AR antagonist MRS1523, demonstrating that the majority of adenosine's effect is mediated by this receptor subtype. Current-clamp recordings demonstrated that neuronal firing of rat DRG neurons was also significantly reduced by A3AR activation in a MRS1523-sensitive but PD173212-insensitive manner. Intracellular Ca measurements confirmed the inhibitory role of A3AR on DRG neuronal firing. We conclude that pain-relieving effects observed on A3AR activation could be mediated through N-type Ca channel block and action potential inhibition as independent mechanisms in isolated rat DRG neurons. These findings support A3AR-based therapy as a viable approach to alleviate pain in different pathologies.

The necessity of implantable cardioverter defibrillators in patients with Kearns-Sayre syndrome - systematic review of the articles -

The most common cardiac feature of Kearns-Sayre syndrome (KSS) is atrioventricular block (AVB), and pacemaker implantations (PMIs) are recommended for KSS patients with advanced AVB. However, some KSS patients develop fatal arrhythmias such as polymorphic ventricular tachycardia (PMVT) and ventricular fibrillation (VF) and die suddenly even after PMIs. We report a patient with KSS who developed PMVT, VF, and QT prolongation, and was treated with mexiletine and successfully managed with an implantable cardioverter defibrillator (ICD). We reviewed the literature on arrhythmias in KSS published from 1975 to 2018. There were 112 patients with arrhythmia-associated KSS, 10 died, and 6 died suddenly after the PMI. The first manifestation of an arrhythmia was bundle branch block, then it progressed to AVB, and developed into complete AVB (CAVB) in about half the KSS patients. Ventricular arrhythmias were documented in 12 patients, and 8 were implanted with defibrillators afterwards. One patient after the implantation of a cardiac resynchronization therapy defibrillator (CRT-D) was treated for VF by an appropriate shock. This fact suggested that VF occurred even under proper pacing, and that defibrillators were effective. Pacemakers may suppress early afterdepolarizations (EADs) associated with a QT prolongation due to bradycardia. Similarly, mexiletine may suppress EADs by blocking the late sodium and Ca currents. Ventricular arrhythmias observed under suppression of EADs may be caused by delayed afterdepolarization (DADs) via an increasing intracellular Ca concentration due to mitochondrial dysfunction. Therefore, a PMI alone may not be sufficient to prevent sudden death, and an ICD implantation should be necessary.

Pre- and Postsynaptic Effects of Glutamate in the Frog Labyrinth

The role of glutamate in quantal release at the cytoneural junction was examined by measuring mEPSPs and afferent spikes at the posterior canal in the intact frog labyrinth. Release was enhanced by exogenous glutamate, or dl-TBOA, a blocker of glutamate reuptake. Conversely, drugs acting on ionotropic glutamate receptors did not affect release; the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA-R) blocker CNQX decreased mEPSP size in a dose-dependent manner; the NMDA-R blocker d-AP5 at concentrations <200 µM did not affect mEPSP size, either in the presence or absence of Mg and glycine. In isolated hair cells, glutamate did not modify Ca currents. Instead, it systematically reduced the compound delayed potassium current, IKD, whereas the metabotropic glutamate receptor (mGluR)-II inverse agonist, (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl)propanoic acid (LY341495), increased it. Given mGluR-II decrease cAMP production, these finding are consistent with the reported sensitivity of IKD to protein kinase A (PKA)-mediated phosphorylation. LY341495 also enhanced transmitter release, presumably through phosphorylation-mediated facilitation of the release machinery. The observed enhancement of release by glutamate confirms previous literature data, and can be attributed to activation of mGluR-I that promotes Ca release from intracellular stores. Glutamate-induced reduction in the repolarizing IKD may contribute to facilitation of release. Overall, glutamate exerts both a positive feedback action on mGluR-I, through activation of the phospholipase C (PLC)/IP3 path, and the negative feedback, by interfering with substrate phosphorylation through Gi/0-coupled mGluRs-II/III. The positive feedback prevails, which may explain the increase in overall rates of release observed during mechanical stimulation (symmetrical in the excitatory and inhibitory directions). The negative feedback may protect the junction from over-activation.