Depolarization Phase Research Articles

Downregulation of IL-8 and IL-10 by LRRC8A Inhibition through the NOX2-Nrf2-CEBPB Transcriptional Axis in THP-1-Derived M2 Macrophages.

M2-polarized, tumor-associated macrophages (TAMs) produce pro-tumorigenic and angiogenic mediators, such as interleukin-8 (IL-8) and IL-10. Leucine-rich repeat-containing protein 8 members (LRRC8s) form volume-regulated anion channels and play an important role in macrophage functions by regulating cytokine and chemokine production. We herein examined the role of LRRC8A in IL-8 and IL-10 expression in THP-1-differentiated M2-like macrophages (M2-MACs), which are a useful tool for investigating TAMs. In M2-MACs, the pharmacological inhibition of LRRC8A led to hyperpolarizing responses after a transient depolarization phase, followed by a slight elevation in the intracellular concentration of Ca2+. Both the small interfering RNA-mediated and pharmacological inhibition of LRRC8A repressed the transcriptional expression of IL-8 and IL-10, resulting in a significant reduction in their secretion. The inhibition of LRRC8A decreased the nuclear translocation of phosphorylated nuclear factor-erythroid 2-related factor 2 (Nrf2), while the activation of Nrf2 reversed the LRRC8A inhibition-induced transcriptional repression of IL-8 and IL-10 in M2-MACs. We identified the CCAAT/enhancer-binding protein isoform B, CEBPB, as a downstream target of Nrf2 signaling in M2-MACs. Moreover, among several upstream candidates, the inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) suppressed the Nrf2-CEBPB transcriptional axis in M2-MACs. Collectively, the present results indicate that the inhibition of LRRC8A repressed IL-8 and IL-10 transcription in M2-MACs through the NOX2-Nrf2-CEBPB axis and suggest that LRRC8A inhibitors suppress the IL-10-mediated evasion of tumor immune surveillance and IL-8-mediated metastasis and neovascularization in TAMs.

Long-term changes in transmembrane voltage after electroporation are governed by the interplay between nonselective leak current and ion channel activation

Electroporation causes a temporal increase in cell membrane permeability and leads to prolonged changes in transmembrane voltage (TMV) in both excitable and non-excitable cells. However, the mechanisms of these TMV changes remain to be fully elucidated. To this end, we monitored TMV over 30 min after exposing two different cell lines to a single 100 µs electroporation pulse using the FLIPR Membrane Potential dye. In CHO-K1 cells, which express very low levels of endogenous ion channels, membrane depolarization following pulse exposure could be explained by nonselective leak current, which persists until the membrane reseals, enabling the cells to recover their resting TMV. In U-87 MG cells, which express many different ion channels, we unexpectedly observed membrane hyperpolarization following the initial depolarization phase, but only at 33 °C and not at 25 °C. We developed a theoretical model, supported by experiments with ion channel inhibitors, which indicated that hyperpolarization could largely be attributed to the activation of calcium-activated potassium channels. Ion channel activation, coupled with changes in TMV and intracellular calcium, participates in various physiological processes, including cell proliferation, differentiation, migration, and apoptosis. Therefore, our study suggests that ion channels could present a potential target for influencing the biological response after electroporation.

Caveolar Compartmentalization of Pacemaker Signaling is Required for Stable Rhythmicity of Sinus Nodal Cells and is Disrupted in Heart Failure.

Heart rhythm relies on complex interactions between electrogenic membrane proteins and intracellular Ca2+ signaling in sinoatrial node (SAN) myocytes; however, mechanisms underlying the functional organization of proteins involved in SAN pacemaking and its structural foundation remain elusive. Caveolae are nanoscale, plasma membrane pits that compartmentalize various ion channels and transporters, including those involved in SAN pacemaking, via binding with the caveolin-3 scaffolding protein, but the precise role of caveolae in cardiac pacemaker function is unknown. Our objective was to determine the role of caveolae in SAN pacemaking and dysfunction (SND). Biochemical co-purification, in vivo electrocardiogram monitoring, ex vivo optical mapping, in vitro confocal Ca2+ imaging, and immunofluorescent and electron microscopy analyses were performed in wild type, cardiac-specific caveolin-3 knockout, and 8-weeks post-myocardial infarction heart failure (HF) mice. SAN tissue samples from donor human hearts were used for biochemical studies. We utilized a novel 3-dimensional single SAN cell mathematical model to determine the functional outcomes of protein nanodomain-specific localization and redistribution in SAN pacemaking. In both mouse and human SANs, caveolae compartmentalized HCN4, Cav1.2, Cav1.3, Cav3.1 and NCX1 proteins within discrete pacemaker signalosomes via direct association with caveolin-3. This compartmentalization positioned electrogenic sarcolemmal proteins near the subsarcolemmal sarcoplasmic reticulum (SR) membrane and ensured fast and robust activation of NCX1 by subsarcolemmal local SR Ca2+ release events (LCRs), which diffuse across ~15-nm subsarcolemmal cleft. Disruption of caveolae led to the development of SND via suppression of pacemaker automaticity through a 50% decrease of the L-type Ca2+ current, a negative shift of the HCN current (I f) activation curve, and a 40% reduction of Na+/Ca2+-exchanger function, along with ~2.3-times widening of the sarcolemma-SR distance. These changes significantly decreased the SAN depolarizing force, both during diastolic depolarization and upstroke phase, leading to bradycardia, sinus pauses, recurrent development of SAN quiescence, and significant increase in heart rate lability. Computational modeling, supported by biochemical studies, identified NCX1 redistribution to extra-caveolar membrane as the primary mechanism of SAN pauses and quiescence due to the impaired ability of NCX1 to be effectively activated by LCRs and trigger action potentials. HF remodeling mirrored caveolae disruption leading to NCX1-LCR uncoupling and SND. SAN pacemaking is driven by complex protein interactions within a nanoscale caveolar pacemaker signalosome. Disruption of caveolae leads to SND, potentially demonstrating a new dimension of SAN remodeling and providing a newly recognized target for therapy.

Cardiotoxicity of the diamide insecticide chlorantraniliprole in the intact heart and in isolated cardiomyocytes from the honey bee

In honey bees, circulation of blood (hemolymph) is driven by the peristaltic contraction of the heart vessel located in the dorsal part of the abdomen. Chlorantraniliprole (CHL) is an insecticide of the anthranilic diamide class which main mode of action is to alter the function of intracellular Ca2+ release channels (known as RyRs, for ryanodine receptors). In the honey bee, it was recently found to be more toxic when applied on the dorsal part of the abdomen, suggesting a direct cardiotoxicity. In the present study, a short-term exposure of semi-isolated bee hearts to CHL (0.1–10 µM) induces alterations of cardiac contraction. These alterations range from a slow-down of systole and diastole kinetics, to bradycardia and cardiac arrest. The bees heart wall is made of a single layer of semi-circular cardiomyocytes arranged concentrically all along the long axis of tube lumen. Since the heart tube is suspended to the cuticle through long tubular muscles fibers (so-called alary muscle cells), the CHL effects in ex-vivo heart preparations could result from the modulation of RyRs present in these skeletal muscle fibers as well as cardiomyocytes RyRs themselves. In order to specifically assess effects of CHL on cardiomyocytes, for the first time, intact heart cells were enzymatically dissociated from bees. Exposure of cardiomyocytes to CHL induces an increase in cytoplasmic calcium, cell contraction at the highest concentrations and depletion of intracellular stores. Electrophysiological properties of isolated cardiomyocytes were described, with a focus on voltage-gated Ca2+ channels responsible for the cardiac action potentials depolarization phase. Two types of Ca2+ currents were measured under voltage-clamp. Exposure to CHL was accompanied by a decrease in voltage-activated Ca2+ currents densities. Altogether, these results show that chlorantraniliprole can cause cardiac defects in honey bees.

Optogenetic stimulation of medial septal glutamatergic neurons modulates theta-gamma coupling in the hippocampus

Hippocampal cross-frequency theta-gamma coupling (TGC) is a basic mechanism for information processing, retrieval, and consolidation of long-term and working memory. While the role of entorhinal afferents in the modulation of hippocampal TGC is widely accepted, the influence of other main input to the hippocampus, from the medial septal area (MSA, the pacemaker of the hippocampal theta rhythm) is poorly understood. Optogenetics allows us to explore how different neuronal populations of septohippocampal circuits control neuronal oscillations in vivo. Rhythmic activation of septal glutamatergic neurons has been shown to drive hippocampal theta oscillations, but the role of these neuronal populations in information processing during theta activation has remained unclear. Here we investigated the influence of phasic activation of MSA glutamatergic neurons expressing channelrhodopsin II on theta-gamma coupling in the hippocampus. During the experiment, local field potentials of MSA and hippocampus of freely behaving mice were modulated by 470 nm light flashes with theta frequency (2–10) Hz. It was shown that both the power and the strength of modulation of gamma rhythm nested on hippocampal theta waves depend on the frequency of stimulation. The modulation of the amplitude of slow gamma rhythm (30–50 Hz) prevailed over modulation of fast gamma (55–100 Hz) during flash trains and the observed effects were specific for theta stimulation of MSA. We discuss the possibility that phasic depolarization of septal glutamatergic neurons controls theta-gamma coupling in the hippocampus and plays a role in memory retrieval and consolidation.

The Impact of Potassium Dynamics on Cardiomyocyte Beating in Hemodialysis Treatment.

Background: Observational studies of intermittent hemodialysis therapy have reported that the excess decrease in K+ concentration in plasma (KP) during treatment is associated with the destabilization of cardiac function. Elucidating the mechanism by which the decrease in KP impairs myocardial excitation is indispensable for a deeper understanding of prescription design. Methods: In this study, by using an electrophysiological mathematical model, we investigated the relationship between KP dynamics and cardiomyocyte excitability for the first time. Results: The excess decrease in KP during treatment destabilized cardiomyocyte excitability through the following events: (1) a decrease in KP led to the prolongation of the depolarization phase of ventricular cells due to the reduced potassium efflux rate of the Kr channel, temporarily enhancing contraction force; (2) an excess decrease in KP activated the transport of K+ and Na+ through the funny channel in sinoatrial nodal cells, disrupting automaticity; (3) the excess decrease in KP also resulted in a significant decrease in the resting membrane potential of ventricular cells, causing contractile dysfunction. Avoiding an excess decrease in KP during treatment contributed to the maintenance of cardiomyocyte excitability. Conclusions: The results of these mathematical analyses showed that it is necessary to implement personal prescription or optimal control of K+ concentration in dialysis fluid based on predialysis KP from the perspective of regulatory science in dialysis treatment.

Acute antiarrhythmic effects of SGLT2 inhibitors–dapagliflozin lowers the excitability of atrial cardiomyocytes

In recent years, SGLT2 inhibitors have become an integral part of heart failure therapy, and several mechanisms contributing to cardiorenal protection have been identified. In this study, we place special emphasis on the atria and investigate acute electrophysiological effects of dapagliflozin to assess the antiarrhythmic potential of SGLT2 inhibitors. Direct electrophysiological effects of dapagliflozin were investigated in patch clamp experiments on isolated atrial cardiomyocytes. Acute treatment with elevated-dose dapagliflozin caused a significant reduction of the action potential inducibility, the amplitude and maximum upstroke velocity. The inhibitory effects were reproduced in human induced pluripotent stem cell-derived cardiomyocytes, and were more pronounced in atrial compared to ventricular cells. Hypothesizing that dapagliflozin directly affects the depolarization phase of atrial action potentials, we examined fast inward sodium currents in human atrial cardiomyocytes and found a significant decrease of peak sodium current densities by dapagliflozin, accompanied by a moderate inhibition of the transient outward potassium current. Translating these findings into a porcine large animal model, acute elevated-dose dapagliflozin treatment caused an atrial-dominant reduction of myocardial conduction velocity in vivo. This could be utilized for both, acute cardioversion of paroxysmal atrial fibrillation episodes and rhythm control of persistent atrial fibrillation. In this study, we show that dapagliflozin alters the excitability of atrial cardiomyocytes by direct inhibition of peak sodium currents. In vivo, dapagliflozin exerts antiarrhythmic effects, revealing a potential new additional role of SGLT2 inhibitors in the treatment of atrial arrhythmias.

Interstitial cells of Cajal - pacemakers of the gastrointestinal tract.

Gastrointestinal (GI) organs display spontaneous, non-neurogenic electrical, and mechanical rhythmicity that underlies fundamental motility patterns, such as peristalsis and segmentation. Electrical rhythmicity (aka slow waves) results from pacemaker activity generated by interstitial cells of Cajal (ICC). ICC express a unique set of ionic conductances and Ca2+ handling mechanisms that generate and actively propagate slow waves. GI smooth muscle cells lack these conductances. Slow waves propagate actively within ICC networks and conduct electrotonically to smooth muscle cells via gap junctions. Slow waves depolarize smooth muscle cells and activate voltage-dependent Ca2+ channels (predominantly CaV1.2), causing Ca2+ influx and excitation-contraction coupling. The main conductances responsible for pacemaker activity in ICC are ANO1, a Ca2+ -activated Cl- conductance, and CaV3.2. The pacemaker cycle, as currently understood, begins with spontaneous, localized Ca2+ release events in ICC that activate spontaneous transient inward currents due to activation of ANO1 channels. Depolarization activates CaV 3.2 channels, causing the upstroke depolarization phase of slow waves. The upstroke is transient and followed by a long-duration plateau phase that can last for several seconds. The plateau phase results from Ca2+ -induced Ca2+ release and a temporal cluster of localized Ca2+ transients in ICC that sustains activation of ANO1 channels and clamps membrane potential near the equilibrium potential for Cl- ions. The plateau phase ends, and repolarization occurs, when Ca2+ stores are depleted, Ca2+ release ceases and ANO1 channels deactivate. This review summarizes key mechanisms responsible for electrical rhythmicity in gastrointestinal organs.

Abstract 13327: Mechanistic Basis of Sinoatrial Node Pacemaker Failure in Atrial Cardiomyopathy

Introduction: Sinoatrial node (SAN) dysfunction commonly occurs in atrial cardiomyopathy (ACM: “tachy-brady syndrome”), often requiring pacemaker implantation, but mechanisms are unknown. Atrial-selective myosin light chain-4 (MYL-4) mutations cause familial ACM with a high rate of early SAN dysfunction and pacemaker requirement. We studied rats engineered with a knock-in MYL-4 clinical E11K mutation and addressed mechanisms of SAN dysfunction. Methods: Atrial electrical properties were assessed with in vivo electrophysiology (EPS), ex vivo optical mapping, SAN single-cell voltage and current clamp, Ca 2+ -handling with Ca 2+ -sensitive dye microscopy. Gene expression was assessed by qPCR and fibrosis with Masson-Trichrome stain. Results: E11K rats showed progressive SAN recovery-time increases (Fig. A) and conduction-slowing (Fig. B). SAN cells showed slowed phase-4 depolarization (Fig. C) and reduced pacemaker “funny” current (I f ) (Fig. D). Ca 2+ homeostasis was also affected, showing decreased Ca 2+ -transients (Fig. E). SAN also displayed remodeling with increased collagen deposition (Fig. F). Conclusions: ACM resulting from E11K mutation induces multimodal electrical and structural remodeling of the SAN region, worsening with age. These observations provide new insights into mechanisms of the clinically important tachy-brady syndrome associated with ACM.

Influence of experimental heart failure therapy with different generations of β-adrenergic blockers on Cardiac Electrical Activity (ECG) and Autonomic Regulation of Heart Rhythm (ARHR)

Abstract: In the complex treatment of chronic heart failure (CHF) β-adrenoblockers (carvedilol, nebivolol, bisoprolol, metoprolol) are used, which due to their pharmacological properties increase the survival rate of patients, improve cardio- and haemodynamic parameters, metabolism. However, modern realities in medicine require creation of new more effective and safe β-adrenoblockers. In this regard, the potential drug Hypertirl is of interest. The aim was to evaluate the cardioprotective effect of 1-(b-phenylethyl)-4-amino-1,2,4-triazolium bromide (Hypertril) on cardiac electrical activity and autonomic regulation of heart rhythm in a model of CHF in comparison with β-adrenoblockers of different generations (Nebivolol, Carvedilol, Bisoprolol, Metoprolol). Materials and methods: Chronic heart failure was induced by 14-day administration of doxorubicin in a cumulative dose of 15 mg/kg to white mongrel rats weighing 190–220g (total 85). The investigated drugs were administered after doxorubicin course for 30 days: Hypertril at an experimentally substantiated dose of 3.5 mg/kg, Metoprolol succinate 15 mg/kg, Nebivolol 10 mg/kg, Carvedilol 50 mg/kg, Bisoprolol 10 mg/kg. At the end of drug administration under thiopental anaesthesia (40 mg/kg), electrocardiogram (ECG) and autonomic regulation of heart rhythm (ARHR) were analysed using a computer analyser CardioCom-2000plus (KAI-Medica, Ukraine). The results of the study were calculated using a standard statistical package “STATISTICA for Windows 6.0” (StatSoftInc., №AXXR712D833214FAN5), “SPSS 16.0” and “Microsoft Office Excell 2003”. Results and discussion: Hypertril administration resulted in a negative chronotropic effect, normalisation of atrial (P) and ventricular (R) spike amplitude, ST segment inversion below isoline, increased amplitude of ventricular myocardial repolarisation T. Myocardial repolarisation spike were observed, as well as normalised the duration of atrial (P) and ventricular depolarisation phase (QRS complex) to the intact value and restored the duration of electrical diastole (TR interval). Hypertril reduced systolic and diastolic myocardial dysfunction in animals with CHF. Hypertril restored autonomic mechanisms of heart rhythm regulation and balanced activity of sympathetic and parasympathetic parts of autonomic nervous system in the control of cardiac function. Conclusion: The obtained results demonstrated the undoubted advantage of the new original molecule (Hypertril) over basic β-adrenoblockers (Metoprolol, Nebivolol, Carvedilol and Bisoprolol) and experimentally justify further in-depth study to create on its basis a drug for the treatment of CHF.

IP3R1 underlies diastolic ANO1 activation and pressure-dependent chronotropy in lymphatic collecting vessels.

Pressure-dependent chronotropy of murine lymphatic collecting vessels relies on the activation of the Ca2+-activated chloride channel encoded by Anoctamin 1 (Ano1) in lymphatic muscle cells. Genetic ablation or pharmacological inhibition of ANO1 results in a significant reduction in basal contraction frequency and essentially complete loss of pressure-dependent frequency modulation by decreasing the rate of the diastolic depolarization phase of the ionic pacemaker in lymphatic muscle cells (LMCs). Oscillating Ca2+ release from sarcoendoplasmic reticulum Ca2+ channels has been hypothesized to drive ANO1 activity during diastole, but the source of Ca2+ for ANO1 activation in smooth muscle remains unclear. Here, we investigated the role of the inositol triphosphate receptor 1 (Itpr1; Ip3r1) in this process using pressure myography, Ca2+ imaging, and membrane potential recordings in LMCs of ex vivo pressurized inguinal-axillary lymphatic vessels from control or Myh11CreERT2;Ip3r1fl/fl (Ip3r1ismKO) mice. Ip3r1ismKO vessels had significant reductions in contraction frequency and tone but an increased contraction amplitude. Membrane potential recordings from LMCs of Ip3r1ismKO vessels revealed a depressed diastolic depolarization rate and an elongation of the plateau phase of the action potential (AP). Ca2+ imaging of LMCs using the genetically encoded Ca2+ sensor GCaMP6f demonstrated an elongation of the Ca2+ flash associated with an AP-driven contraction. Critically, diastolic subcellular Ca2+ transients were absent in LMCs of Ip3r1ismKO mice, demonstrating the necessity of IP3R1 activity in controlling ANO1-mediated diastolic depolarization. These findings indicate a critical role for IP3R1 in lymphatic vessel pressure-dependent chronotropy and contractile regulation.

Multi-phase ECG dynamic features for detecting myocardial ischemia and identifying its etiology using deterministic learning

Timely detecting myocardial ischemia and identifying its etiology is of great value to the making of therapeutic regimen. Electrocardiogram (ECG) is a first-line screening tool for myocardial ischemia, but few based-ECG studies into the identification of its etiology have been reported. In this study, we propose a multi-phase ECG dynamic feature-based method for detecting myocardial ischemia and identifying its etiology using deterministic learning and the self-adaptive variational mode decomposition (VMD). Firstly, a self-adaptive approach is designed to produce the VMD components describing the ECG depolarization and repolarization phases, respectively. Then, dynamic modeling of ECG signal and multi-phase VMD components is carried out by deterministic learning, and multiple dynamic features under different phases of ECG activities are extracted from the learned dynamics. Subsequently, we design a hybrid feature reduction scheme to obtain low-dimensional but highly informative features while preserving the physical significance of features. Finally, classifiers are trained and evaluated on 297 ECG records from 297 patients, including 118 patients with non-ischemia chest pain, 41 patients with coronary slow flow (CSF), and 138 patients with coronary artery stenosis (COAS). The results show that logistic regression with a few dynamic features achieves promising performance in detecting myocardial ischemia (CSF and COAS), with the area under the receiver operating characteristic (AUROC) of 0.9126. Especially, it can effectively identify the etiology of myocardial ischemia, with an overall AUROC of 0.9238. The results show that our method provides a powerful tool for clinicians to analyze the etiology of ischemia.

The Action Potential Clamp Technique as a Tool for Risk Stratification of Sinus Bradycardia Due to Loss-of-Function Mutations in HCN4: An In Silico Exploration Based on In Vitro and In Vivo Data.

These days, in vitro functional analysis of gene variants is becoming increasingly important for risk stratification of cardiac ion channelopathies. So far, such risk stratification has been applied to SCN5A, KCNQ1, and KCNH2 gene variants associated with Brugada syndrome and long QT syndrome types 1 and 2, respectively, but risk stratification of HCN4 gene variants related to sick sinus syndrome has not yet been performed. HCN4 is the gene responsible for the hyperpolarization-activated 'funny' current If, which is an important modulator of the spontaneous diastolic depolarization underlying the sinus node pacemaker activity. In the present study, we carried out a risk classification assay on those loss-of-function mutations in HCN4 for which in vivo as well as in vitro data have been published. We used the in vitro data to compute the charge carried by If (Qf) during the diastolic depolarization phase of a prerecorded human sinus node action potential waveform and assessed the extent to which this Qf predicts (1) the beating rate of the comprehensive Fabbri-Severi model of a human sinus node cell with mutation-induced changes in If and (2) the heart rate observed in patients carrying the associated mutation in HCN4. The beating rate of the model cell showed a very strong correlation with Qf from the simulated action potential clamp experiments (R2 = 0.95 under vagal tone). The clinically observed minimum or resting heart rates showed a strong correlation with Qf (R2 = 0.73 and R2 = 0.71, respectively). While a translational perspective remains to be seen, we conclude that action potential clamp on transfected cells, without the need for further voltage clamp experiments and data analysis to determine individual biophysical parameters of If, is a promising tool for risk stratification of sinus bradycardia due to loss-of-function mutations in HCN4. In combination with an If blocker, this tool may also prove useful when applied to human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) obtained from mutation carriers and non-carriers.

Electric Field of the Heart on the Thorax Surface in Highly Trained Athletes during Initial Ventricular Activity

The article is focused on the study of the electrical remodeling of the “sports heart” using multichannel ECG mapping to make a more detailed and informative analysis of the excitation of the myocardium remodeled by sport activity. The study of the heart electrical activity was carried out from 64 unipolar electrodes on the thorax surface synchronously with standard limb leads in highly trained cross-country skiers (n = 15) and young non-trained men (n = 19) at rest. The amplitude-temporal and spatiotemporal characteristics of the electric field of the heart were measured. By the typical distribution of areas of the negative and positive cardiac potentials the durations of depolarization and its individual phases were estimated. ECGII was used to determine the duration of the R-R, PQ (PR), QRS, QT, QTc intervals; in the unipolar lead V5 the R-wave peak time (RWPTV5) was measured. In athletes, the duration of PQII, QTII, QTcII, RWPTV5 intervals were statistically significantly longer than in non-trained men. Analysis of the electrical field of the heart showed a difference in the ratio of the phases of ventricular depolarization in athletes and non-trained individuals at rest. The onset of ventricular depolarization and the first inversion of cardiac potential in athletes were significantly earlier than in non-trained people, while the completion and total duration of the period of ventricular depolarization did not differ. In skiers, the duration of the first inversion was significantly longer than in non-trained individuals; a tendency to shortening of the second period of stable location of potentials on the thorax surface was also shown. The use of surface ECG mapping made it possible to reveal differences in the spatiotemporal organization of heart ventricular depolarization between cross-country skiers and non-trained men at rest. The results of the study may be useful in the analysis of the electrical remodeling of the heart in athletes training the physical quality of endurance.

Resistance of Embryonic Chick Atria to Inhibition of HCN-Channels and Components of the “Ca<sup>2+</sup>-Clock”

Despite its medical importance, the cellular mechanisms activity and the contribution of various ion channels of embryonic heart automatism are not yet fully understood. In this study we investigated the effects of specific ion-channel inhibitors on the generation of action potentials in pacemaker cells of the right atrium in chicken embryos (HH36). We used microelectrode technique and evaluated the sensitivity of pacemaker cells to ivabradine (inhibitor of HCN-channels, through which the hyperpolarization-activated current, If), ryanodine (agonist of ryanodine receptors) and SN6 (inhibitor of Na+/Ca2+-exchange). It was found that the right atrium cells have a phase of slow diastolic depolarization. However, these cells were not sensitive to ivabradine (3 μM). We did not register significant changes in the electrophysiological parameters of action potentials.When ryanodine (1 μM) and SN6 (10 μM) were added to the perfusion solution, we observed similar effects: spontaneous rate the generation of action potential increased by 15%. Disturbance of rhythmic activity or disruption of the generation of electrical impulses were not observed in right atrial samples of chicken embryos. The obtained results allow us to conclude that, at this of embryonic development stage, HCN4-channels, ryanodine receptors, and the Na+/Ca2+-exchange are not decisive for maintaining the automatism of the right atrial cells in the chick embryo. We assume that the ion currents flowing through these channels are important in electrophysiology in adult animals, but they have a modulating function in the embryonic myocardium.

Development of electrophysiological properties of fusiform neurons from the dorsal cochlear nucleus of mice before and after hearing onset.

The dorsal cochlear nucleus (DCN) in the auditory brainstem integrates auditory and somatosensory information. Mature DCN fusiform neurons fall into two qualitatively distinct types: quiet, with no spontaneous regular action potential firing, or active, with regular spontaneous action potential firing. However, how these firing states and other electrophysiological properties of fusiform neurons develop during early postnatal days to adulthood is not known. Thus, we recorded fusiform neurons from mice from P4 to P21 and analyzed their electrophysiological properties. In the prehearing phase (P4-P13), we found that most fusiform neurons are quiet, with active neurons emerging after hearing onset at P14. Subthreshold properties underwent significant changes before hearing onset, whereas changes to the action potential waveform occurred mainly after P14, with the depolarization and repolarization phases becoming markedly faster and half-width significantly decreased. The activity threshold in posthearing neurons was more negative than in prehearing cells. Persistent sodium current (INaP) was increased after P14, coinciding with the emergence of spontaneous firing. Thus, we suggest that posthearing expression of INaP leads to hyperpolarization of the activity threshold and the active state of the fusiform neuron. At the same time, other changes refine the passive membrane properties and increase the speed of action potential firing of fusiform neurons.NEW & NOTEWORTHY Auditory brainstem neurons express unique electrophysiological properties adapted for their complex physiological functions that develop before hearing onset. Fusiform neurons of the DCN present two firing states, quiet and active, but the origin of these states is not known. Here, we showed that the quiet and active states develop after hearing onset at P14, along with changes in action potentials, suggesting an influence of auditory input on the refining of fusiform neuron's excitability.

Aging impairs the clustering and reduces the activity of the L-type calcium channels in the heart's pacemaker

Every heartbeat is initiated by a spontaneous electrical signal generated inside the cardiac pacemaker. In mammals, including humans, aging leads to the slowdown of the pacemaker rate, which can lead to pathological dysfunction and death. However, the mechanisms behind the age-associated slowdown of the pacemaker are not well understood. The automaticity of the pacemaker relies on a finely tuned balance of ionic currents, where L-type calcium channels play a central role. CaV1.2 and CaV1.3 channels are key players involved in the diastolic depolarization phase and action potential upstroke. In this project, we studied the effects of aging on CaV1.2 and CaV1.3 calcium channel function and regulation. Here we recorded the pacemaker activity and calcium currents on isolated spindle pacemaker cells with patch clamp. The clustering of LTCC has been highlighted with Super-resolution microscopy (GSD). We found that in mouse pacemaker cells, aging causes a reduction of calcium entry through L-type calcium channels, which is not accompanied by a reduction of protein expression. In turn, we found that aging disrupts calcium channel function at different levels. First, aging reduces the expression of L-type calcium channels at the plasma membrane. Second, aging reduces the clustering and nano-organization of L-type calcium channels. And finally, aging reduces the open channel probability, a sign of decreased functional coupling. Moreover, aging also alters the association between L-type calcium channels and beta-adrenergic receptors, altering the adrenergic modulation of the pacemaker cells. Next, we tested the hypothesis that reduced open probability of L-type calcium channels was responsible for a slower firing frequency. Interestingly, we found that the application of BayK was enough to restore intrinsic pacemaker rates in old cells to those levels observed in young cells. Overall, our findings provide evidence that the proper organization and function of L-type calcium channels is affected by aging and that this dysfunction contributes to the slowdown of pacemaker cells in old animals.

Action potential firing and sensory transduction are sustained by membrane potential instabilities in peripheral sensory neurons.

Peripheral sensory neurons are fundamental for stimulus detection, but many of their properties remain poorly understood (Velasco et al., 2022). One of these is the marked instability of the resting membrane potential recorded in such neurons, known as Membrane Potential Instabilities (MPIs). MPIs are enhanced in pain states, but their functional significance and molecular bases remain largely unknown. Here, we addressed this using whole-cell current-clamp recordings in primary cultured mouse trigeminal (TG) neurons. MPI detection and morphological analyses were performed semi-automatically, using custom-made scripts. We found that 82.2% of small diameter neurons recorded displayed MPI activity in the shape of Membrane Potential Transients (MPTs), spontaneously or upon depolarizing current injection. The frequency, amplitude and rates of upstroke and repolarization of MPTs were increased by membrane depolarization. Morphological analyses of the MPTs and of the initial depolarizing phase of action potentials (Aps) revealed that larger and faster MPTs precede AP firing. This was confirmed by a regression analysis showing that the maximal rate of depolarization and amplitude of the MPTs strongly predict subsequent AP firing, following a Boltzmann dependency. MPT activity was enhanced by the application of heat, cold, capsaicin and menthol, via their depolarizing effect on the resting membrane potential. MPT activity was reduced by blocking TTX-resistant, but not TTX-sensitive voltage-gated Na+ channels. Accordingly, MPTs were also reduced in neurons isolated from NaV1.8 and NaV1.9 knockout mice. We hereby identify, for the first time, the molecular determinants of the trigger of MPTs and demonstrate a causal relationship between MPTs and AP firing upon exogenous stimulation of sensory neurons. These findings unveil MPTs as crucial regulators of peripheral sensory processing, with possible implications in pathological conditions featuring altered activities of TTX-resistant NaV channels.

Microwave exposure added characteristics to the wounding-induced variation potential of Aloe arborescens leaves

The influence of radiofrequency electromagnetic radiation on the wounding-induced electric potentials (EPs) in Aloe arborescens plants was investigated. Burn wounding-induced electrical potentials of mature A. arborescens plants were observed under the exposure of 2.45 ​GHz, 3.5 ​GHz and 5.5 ​GHz microwaves at incident power density 1.5 ​± ​0.2 ​W ​m−2. Aloe leaves were subjected to flame wounding at the leaf tips and propagation EPs were recorded by inserting a glass Ag/AgCl microelectrode into the leaf pulp. The propagation of electrical potential and a standard deviation of the fluctuations in electrical potential (SDEF) were investigated. The flame wounding generated propagating characteristic electric potential, and the exposure of microwaves added extra characteristics to the signals by reversing the electrical potential temporally for a shorter duration. The characteristics appeared in the repolarization phase of the signal under 2.45 ​GHz and 3.5 ​GHz exposure; for the 5.5 ​GHz exposure, 3 out of 6 characteristics appeared during the depolarization phase. Averaged polarization rates of the characteristics were increased with the increased microwave frequency. Added characteristics to the electric potential may have resulted from a secondary signal triggered due to microwave exposure, which should be further studied. The repolarization and depolarization rates of the wound signals were not different between control and microwave exposures. SDEFs were also not affected by microwave exposure.

ELECTROCARDIOGRAM PATTERN PRE AND 24 HOURS POST THERAPY IN HYPONATREMIA WITH HEART FAILURE PATIENT

Background : Hyponatremia is the most common electrolyte disturbance in hospitalized heart failure patients, with a prevalence of around 19%-25%. The relationship between hyponatremia and cardiac conduction disorders has not been demonstrated yet. When there is a decrease in extracellular Na+ levels, there is a delay in the depolarization phase. This study was conducted to observe the ECG pattern in hyponatremic patients with heart failure (particularly in the P wave, PR interval, and QRS complex) and ECG changes after 24 hours of therapy. Methode: This study used a longitudinal observational descriptive method. Patients who were included in this study were hyponatremic patients with heart failure at the Wahidin Sudirohusodo Hospital Makassar from April to September 2021. The patient's electrocardiogram data when first admitted and after 24 hours of therapy. Statistical test using McNemar and Wilcoxon Signed Rank Test, where the test results are statistically significant when the p value <0.05. Result : This study included 51 subjects with a distribution of 68.6% men with an age range of 18-85 years. Pre-therapy ECG pattern showed 12 patients with prolonged P-R interval, and 4 patients with widening of the QRS complex. After 24 hours of therapy, 12 people became normonatremia and the mean P wave and P-R interval were lower than pre-therapy, while the QRS complex did not show any changes. Conclusion : The ECG pattern in hyponatremia with heart failure are prolonged P-R interval and widening of the QRS complex, but the ECG changes were not significant to Na+ levels changes.