Spontaneous Action Potentials Research Articles

Lead exerts a depression of neurotransmitter release through a blockade of voltage dependent calcium channels in chromaffin cells

The present work, using chromaffin cells of bovine adrenal medullae (BCCs), aims to describe what type of ionic current alterations induced by lead (Pb2+) underlies its effects reported on synaptic transmission. We observed that the acute application of Pb2+ lead to a drastic depression of neurotransmitters release in a concentration-dependent manner when the cells were stimulated with both K+ or acetylcholine, with an IC50 of 119,57 μM and of 5,19 μM, respectively. This effect was fully recovered after washout. Pb2+ also blocked calcium channels of BCCs in a time- and concentration-dependent manner with an IC50 of 6,87 μM. This blockade was partially reversed upon washout. This compound inhibited the calcium current at all test potentials and shows a shift of the I-V curve to more negative values of about 8 mV. The sodium current was not blocked by acute application of high Pb2+ concentrations. Voltage-dependent potassium current was also shortly affected by high Pb2+. Nevertheless, the calcium- and voltage-dependent potassium current was drastically depressed in a dose-dependent manner, with an IC50 of 24,49 μM. This blockade was related to the prevention of Ca2+ influx through voltage-dependent calcium channels coupled to Ca2+-activated K+-channels (BK) instead a direct linking to these channels. Under current-clamp conditions, BCCs exhibit a resting potential of −52.7 mV, firing spontaneous APs (1–2 spikes/s) generated by the opening of Na+ and Ca2+-channels, and terminated by the activation of K+ channels. In spite of the effect on ionic channels exerted by Pb2+, we found that Pb2+ didn’t alter cellular excitability, no modification of the membrane potential, and no effect on action potential firing. Taken together, these results point to a neurotoxic action evoked by Pb2+ that is associated with changes in neurotransmitter release by blocking the ionic currents responsible for the calcium influx.

A Mathematical Model of Spontaneous Action Potential Based on Stochastics Synaptic Noise Dynamics in Non-Neural Cells

We developed a mathematical model to simulate the dynamics of background synaptic noise in non-neuronal cells. By employing the stochastic Ornstein–Uhlenbeck process, we represented excitatory synaptic conductance and integrated it into a whole-cell model to generate spontaneous and evoke cellular electrical activities. This single-cell model encompasses numerous biophysically detailed ion channels, depicted by a set of ordinary differential equations in Hodgkin–Huxley and Markov formalisms. Consequently, this approach effectively induced irregular spontaneous depolarizations (SDs) and spontaneous action potentials (sAPs), resembling electrical activity observed in vitro. The input resistance decreased significantly, while the firing rate of spontaneous action potentials increased. Moreover, alterations in the ability to reach the action potential threshold were observed. Background synaptic activity can modify the input/output characteristics of non-neuronal excitatory cells. Hence, suppressing these baseline activities could aid in identifying new pharmaceutical targets for various clinical diseases.

L-Type Calcium Channel Modulates Low-Intensity Pulsed Ultrasound-Induced Excitation in Cultured Hippocampal Neurons.

As a noninvasive technique, ultrasound stimulation is known to modulate neuronal activity both in vitro and in vivo. The latest explanation of this phenomenon is that the acoustic wave can activate the ion channels and further impact the electrophysiological properties of targeted neurons. However, the underlying mechanism of low-intensity pulsed ultrasound (LIPUS)-induced neuro-modulation effects is still unclear. Here, we characterize the excitatory effects of LIPUS on spontaneous activity and the intracellular Ca2+ homeostasis in cultured hippocampal neurons. By whole-cell patch clamp recording, we found that 15min of 1-MHz LIPUS boosts the frequency of both spontaneous action potentials and spontaneous excitatory synaptic currents (sEPSCs) and also increases the amplitude of sEPSCs in hippocampal neurons. This phenomenon lasts for > 10min after LIPUS exposure. Together with Ca2+ imaging, we clarified that LIPUS increases the [Ca2+]cyto level by facilitating L-type Ca2+ channels (LTCCs). In addition, due to the [Ca2+]cyto elevation by LIPUS exposure, the Ca2+-dependent CaMKII-CREB pathway can be activated within 30 min to further regulate the gene transcription and protein expression. Our work suggests that LIPUS regulates neuronal activity in a Ca2+-dependent manner via LTCCs. This may also explain the multi-activation effects of LIPUS beyond neurons. LIPUS stimulation potentiates spontaneous neuronal activity by increasing Ca2+ influx.

Somatodendritic organization of pacemaker activity in midbrain dopamine neurons.

The slow and regular pacemaking activity of midbrain dopamine (DA) neurons requires proper spatial organization of the excitable elements between the soma and dendritic compartments, but the somatodendritic organization is not clear. Here, we show that the dynamic interaction between the soma and multiple proximal dendritic compartments (PDCs) generates the slow pacemaking activity in DA neurons. In multipolar DA neurons, spontaneous action potentials (sAPs) consistently originate from the axon-bearing dendrite. However, when the axon initial segment was disabled, sAPs emerge randomly from various primary PDCs, indicating that multiple PDCs drive pacemaking. Ca2+ measurements and local stimulation/perturbation experiments suggest that the soma serves as a stably-oscillating inertial compartment, while multiple PDCs exhibit stochastic fluctuations and high excitability. Despite the stochastic and excitable nature of PDCs, their activities are balanced by the large centrally-connected inertial soma, resulting in the slow synchronized pacemaking rhythm. Furthermore, our electrophysiological experiments indicate that the soma and PDCs, with distinct characteristics, play different roles in glutamate- induced burst-pause firing patterns. Excitable PDCs mediate excitatory burst responses to glutamate, while the large inertial soma determines inhibitory pause responses to glutamate. Therefore, we could conclude that this somatodendritic organization serves as a common foundation for both pacemaker activity and evoked firing patterns in midbrain DA neurons.

NEDD4-2 and the CLC-2 channel regulate neuronal excitability in the pathogenesis of mesial temporal lobe epilepsy

An increasing number of studies have focused on the role of NEDD4-2 in regulating neuronal excitability and the mechanism of epilepsy. However, the exact mechanism has not yet been elucidated. Here, we explored the roles of NEDD4-2 and the CLC-2 channel in regulating neuronal excitability and mesial temporal lobe epilepsy (MTLE) pathogenesis. First, chronic MTLE models were induced by lithium-pilocarpine in developmental rats. Coimmunoprecipitation analysis revealed that the interaction between CLC-2 and NEDD4-2. Western blot analyses indicated that NEDD4-2 expression was downregulated, while phosphorylated (P-) NEDD4-2 and CLC-2 expression was upregulated in adult MTLE rats. Then, the primary hippocampal neuronal cells were isolated and cultured, and the NEDD4-2 was knocked down by shRNA vector, resulting in decreased protein levels of CLC-2. While CLC-2 absence caused increased NEDD4-2 in cells. Next, in an epileptic cell model induced by a Mg2+-free culture, whole-cell current-clamp recording demonstrated that NEDD4-2 deficiency inhibited the spontaneous action potentials of cells, and CLC-2 absence caused more significant decrease in the spontaneous action potentials of cells. In conclusion, we herein revealed that NEDD4-2 regulates the expression of CLC-2, which is involved in neuronal excitability, and participates in the pathogenesis of MTLE.

Phenotypic Screening of Prospective Analgesics Among FDA-Approved Compounds using an iPSC-Based Model of Acute and Chronic Inflammatory Nociception.

Classical target-based drug screening is low-throughput, largely subjective, and costly. Phenotypic screening based on in vitro models is increasingly being used to identify candidate compounds that modulate complex cell/tissue functions. Chronic inflammatory nociception, and subsequent chronic pain conditions, affect peripheral sensory neuron activity (e.g., firing of action potentials) through myriad pathways, and remain unaddressed in regard to effective, non-addictive management/treatment options. Here, a chronic inflammatory nociception model is demonstrated based on induced pluripotent stem cell (iPSC) sensory neurons and glia, co-cultured on microelectrode arrays (MEAs). iPSC sensory co-cultures exhibit coordinated spontaneous extracellular action potential (EAP) firing, reaching a stable baseline after ≈27 days in vitro (DIV). Spontaneous and evoked EAP metrics are significantly modulated by 24-h incubation with tumor necrosis factor-alpha (TNF-α), representing an inflammatory phenotype. Compared with positive controls (lidocaine), this model is identified as an "excellent" stand-alone assay based on a modified Z' assay quality metric. This model is then used to screen 15 cherry-picked, off-label, Food and Drug Administration (FDA)-approved compounds; 10 of 15 are identified as "hits". Both hits and "misses" are discussed in turn. In total, this data suggests that iPSC sensory co-cultures on MEAs may represent a moderate-to-high-throughput assay for drug discovery targeting inflammatory nociception.

Involvement of NOTCH1-mediated Microglia Activation in Neuromodulation of Chronic Prostatitis-related Pain.

This study aimed to investigate the role of NOTCH receptor 1 (NOTCH1)-mediated activation of microglia in the L5-S2 spinal dorsal horn in chronic prostatitis pain. Rats were divided into chronic prostatitis (CP) group and control group. Complete Freund's adjuvant was injected into the prostate, and prostate pathology and pain-related behavior were monitored to assess the successful establishment of the CP-related pain model. The dorsal horn of the L5-S2 spinal cord was collected for the detection of ionized calcium-binding adapter molecule 1 (IBA-1) and NOTCH1 expression by quantitative real time polymerase chain reaction and the detection of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) by enzyme-linked immunosorbent assay. Electrical excitability was assessed with whole-cell patch clamp. In addition, NOTCH1 receptor inhibitor or inhibitor of microglial cell activation was injected into the subarachnoid space, and the pro-inflammatory cytokines in the spinal cord were detected. In the CP group, the expression of NOTCH1, IBA-1, TNF-α and IL-1β began to increase at 4 days, peaked at 12 days, and began to decline at 24 days, and it was significantly higher than in the control group (p<0.01). Inhibition of microglia or NOTCH1 receptor markedly reduced the content of TNF-α and IL-1β in the spinal cord (p<0.05). At 4, 12 and 24 days, the amplitude and frequency of neuronal action potential increased and the threshold decreased markedly as compared to the control group (p<0.05), and spontaneous action potential was noted. NOTCH1 mediates the activation of microglia in the L5-S2 spinal cord, leading to the secretion of inflammatory factors and enhanced electrical excitability of neurons, which is related to persistent and refractory chronic prostatitis-related pain.

Optogenetic action potentials and intrinsic pacemaker interplay in retrogradely identified midbrain dopamine neurons.

Dissecting the diversity of midbrain dopamine (DA) neurons by optotagging is a promising addition to better identify their functional properties and contribution to motivated behavior. Retrograde molecular targeting of DA neurons with specific axonal projection allows further refinement of this approach. Here, we focus on adult mouse DA neurons in the substantia nigra pars compacta (SNc) projecting to dorsal striatum (DS) by demonstrating the selectivity of a floxed AAV9-based retrograde channelrhodopsin-eYFP (ChR-eYFP) labeling approach in DAT-cre mice. Furthermore, we show the utility of a sparse labeling version for anatomical single-cell reconstruction and demonstrate that ChR-eYFR expressing DA neurons retain intrinsic functional properties indistinguishable from conventionally retrogradely red-beads-labeled neurons. We systematically explore the properties of optogenetically evoked action potentials (oAPs) and their interaction with intrinsic pacemaking in this defined subpopulation of DA neurons. We found that the shape of the oAP and its first derivative, as a proxy for extracellularly recorded APs, is highly distinct from spontaneous APs (sAPs) of the same neurons and systematically varies across the pacemaker duty cycle. The timing of the oAP also affects the backbone oscillator of the intrinsic pacemaker by introducing transient "compensatory pauses". Characterizing this systematic interplay between oAPs and sAPs in defined DA neurons will also facilitate a refinement of DA neuron optotagging in vivo.

Estrogen-induced phenotype in hiPSC-derived cardiomyocytes of a woman affected by both Long QT Syndrome Type 2 and Polycystic Ovary Syndrome

Abstract Introduction Long QT Syndrome (LQTS) is an arrhythmogenic disorder caused either by genetic alteration or by exogenous factors. Polycystic Ovary Syndrome (PCOS) is a disease associated with high levels of testosterone and low levels of estrogen (E2). It has been suggested that hormones can influence the occurrence of malignant cardiac events in LQTS type 2 (LQT2) women, carriers of mutations on the potassium channel (hERG). However, the precise mechanisms governing this process still need to be clarified. Purpose We studied a patient affected by both LQT2 and PCOS who started experiencing cardiac symptoms after initiation of hormonal replacement therapy. We observed a correlation with the occurrence of arrhythmic events and the irregular assumption of the hormonal replacement therapy, thus we aim to dissect the underlying mechanism in patient-in-a-dish cardiac cellular model. Methods Induced Pluripotent Stem Cells (hiPSC) were generated from a woman affected by both LQT2 and PCOS and from a healthy donor, and then differentiated into cardiomyocytes (hiPSC-CMs). hiPSC-CMs were stimulated with E2 10nM for five time points (1, 5, 10, 15 and 30 minutes). The expression of potassium channel hERG, and of the G-coupled estrogen receptor (GPER) regulating the fast non-genomic pathway, were quantified after E2 stimulation at different time points by In Cell Western (ICW). The Unstimulated sample were used to normalize GPER and hERG expression to 100%. Electrophysiological experiments were performed using the whole-cell patch-clamp technique in current-clamp mode to record spontaneous action potentials (APs). AP duration at 30%, 50% and 90% repolarization (APD30, APD50 and APD90) was quantified. During spontaneous activity, AP duration values were rate-corrected with Bazett’s formula, thus obtaining cAPD. Results LQT2 hiPSC-CMs express higher levels of GPER (p&amp;lt;0.05) and hERG (p&amp;lt;0.01) than healthy hiPSC-CMs. A significant decrease in GPER expression was detected during 10nM E2 stimulation in healthy cardiomyocytes at the five time points (p&amp;lt;0.0001), while any difference was detected in hERG expression levels. E2 stimulation at different time points did not affect neither GPER nor hERG expression in LQT2 hiPSC-CMs. In LQT2 hiPSC-CMs, APDC values were significantly increased (p&amp;lt;0.0001) compared to healthy cells; pre-incubation with E2 for 10 minutes significantly shortened cAPD50 and increased spontaneous firing rate. In healthy hiPSC-CMs, E2 had no effect. Conclusions The hiPSC-CM model reproduces the LQT2 clinical phenotype. cAPD50 shortening by E2 was unexpected based on the proarrhythmic effect in the patient. The mechanism of LQT2-specific effects of E2 are under investigation.

ERG K+ channels mediate a major component of action potential repolarization in lymphatic muscle

Smooth muscle cells in the walls of collecting lymphatic vessels fire spontaneous action potentials (APs), which conduct rapidly over the muscle layer to initiate contractions that propel lymph. Several ion channels have been implicated in the currents underlying the AP spike and the preceding diastolic depolarization, but the molecular identities of K+ channels involved in AP repolarization are unknown. Based on previous studies of other rhythmically active smooth muscles, we hypothesized that ether-a-go-go related gene (ERG) K+ channels (Kv11) play an important role in repolarization of the AP in lymphatic muscle. Message for one or more ERG channel isoforms was detected by RT-PCR analysis of lymphatic vessels from mice, rats and humans. Membrane potential recordings in smooth muscle cells of rat and human lymphatics revealed that nanomolar concentrations of ERG-1 inhibitors (E-4031 and BeKm-1) prolonged the duration of the AP plateau (normally ~ 1 s in duration) and induced multiple spikes, whereas ERG-1 activators (ICA-105574 and RPR-260243) shortened the plateau and could completely inhibit spontaneous APs. At relatively high inhibitor concentrations, the AP plateau duration lasted as long as 24 s. ERG activators reversed the effects of ERG inhibitors and vice-versa. In pressure myograph studies, ERG channel inhibition prolonged the diastolic repolarization phase of the contraction cycle and reduced the frequency of spontaneous contractions. This is the first evidence for a specific K+ channel contributing to the AP in lymphatic muscle. Our results imply that lymphatic contractile dysfunction may occur in long QT type II patients with mutations that result in ERG channel loss-of-function or impaired trafficking of the channel to the cell membrane.

PDE4D mediates impaired β-adrenergic receptor signalling in the sinoatrial node in mice with hypertensive heart disease.

The sympathetic nervous system increases HR by activating β-adrenergic receptors (β-ARs) and increasing cAMP in sinoatrial node (SAN) myocytes while phosphodiesterases (PDEs) degrade cAMP. Chronotropic incompetence, the inability to regulate heart rate (HR) in response to sympathetic nervous system activation, is common in hypertensive heart disease; however, the basis for this is poorly understood. The objective of this study was to determine the mechanisms leading to chronotropic incompetence in mice with angiotensin II (AngII)-induced hypertensive heart disease. C57BL/6 mice were infused with saline or AngII (2.5 mg/kg/day for 3 weeks) to induce hypertensive heart disease. HR and SAN function in response to the β-AR agonist isoproterenol (ISO) were studied in vivo using telemetry and electrocardiography, in isolated atrial preparations using optical mapping, in isolated SAN myocytes using patch-clamping, and using molecular biology. AngII-infused mice had smaller increases in HR in response to physical activity and during acute ISO injection. Optical mapping of the SAN in AngII-infused mice demonstrated impaired increases in conduction velocity and altered conduction patterns in response to ISO. Spontaneous AP firing responses to ISO in isolated SAN myocytes from AngII-infused mice were impaired due to smaller increases in diastolic depolarization (DD) slope, hyperpolarization-activated current (If), and L-type Ca2+ current (ICa,L). These changes were due to increased localization of PDE4D surrounding β1- and β2-ARs in the SAN, increased SAN PDE4 activity, and reduced cAMP generation in response to ISO. Knockdown of PDE4D using a virus-delivered shRNA or inhibition of PDE4 with rolipram normalized SAN sensitivity to β-AR stimulation in AngII-infused mice. AngII-induced hypertensive heart disease results in impaired HR responses to β-AR stimulation due to up-regulation of PDE4D and reduced effects of cAMP on spontaneous AP firing in SAN myocytes.

199-OR: Optogenetic Control of Pancreatic Delta Cells Reveals Their Central Role in Hormone Release from Pancreatic Islets

Diabetes mellitus is a metabolic disorder resulting from insufficient insulin release and associated with dysregulation of glucagon secretion. Somatostatin secreted from pancreatic δ-cells is a strong paracrine factor for both insulin and glucagon secretion in islets. Here, we developed an optogenetic approach, through specific expressing light-sensitive channels/pumps in δ-cells, to investigate the paracrine control of hormone release from islets. We generated mouse models that express either a light-sensitive cation channel, channelrhodopsin-2 (ChR2), or a chloride pump, halorhodopsin (HR), specifically in the pancreatic δ-cells (SST-ChR2 and SST-HR mouse). Their specific expression was confirmed by immunostaining. Optoactivation of ChR2 at 1mM glucose rapidly depolarized δ-cells by 37mV (n=5) and stimulated electrical activity and somatostatin release. At high glucose or tolbutamide, light activation of HR repolarized δ-cells by 15 mV (n=3), suppressing action potentials. Optoactivation of SST-ChR2 δ-cells suppressed, after a variable delay depending on the distance from δ-cells, action potential firing in neighbouring α-cells, inhibited exocytosis by 30% (n=5) and glucagon secretion by 55% at 1mM glucose (n=7). In a subset (35%) α-cells with no spontaneous action potentials, opto-inhibition of SST-HR δ-cells caused a 9mV depolarization (n=7) and induced action potential firing, which may account for the stimulation of glucagon secretion. Neither optoactivation, nor opto-inhibition of δ-cells affected insulin secretion at hypo- and hyperglycaemia. However, at normoglycaemia (6 mM glucose), optoactivation of δ-cells depolarized neighbouring β-cells (via gap-junctions) and evoked action potential firing, leading to a paradoxical 160% stimulation of insulin secretion (n=9). We conclude that δ-cells play a central role in the regulation of both insulin and glucagon release but that the effects are more complex than appreciated to date. Disclosure H.Dou: None. C.A.Miranda: None. J.Tolö: None. Q.Zhang: None. P.Rorsman: None. Funding Swedish Research Council

Deep learning-based identification of sinoatrial node-like pacemaker cells from SHOX2/HCN4 double-positive cells differentiated from human iPS cells.

Cardiomyocytes derived from human iPS cells (hiPSCs) include cells showing SAN- and non-SAN-type spontaneous APs. To examine whether the deep learning technology could identify hiPSC-derived SAN-like cells showing SAN-type-APs by their shape. We acquired phase-contrast images for hiPSC-derived SHOX2/HCN4 double-positive SAN-like and non-SAN-like cells and made a VGG16-based CNN model to classify an input image as SAN-like or non-SAN-like cell, compared to human discriminability. All parameter values such as accuracy, recall, specificity, and precision obtained from the trained CNN model were higher than those of human classification. Deep learning technology could identify hiPSC-derived SAN-like cells with considerable accuracy.

Slow ion concentration oscillations and multiple states in neuron-glia interaction-insights gained from reduced mathematical models.

When potassium in the extracellular space separating neurons and glia reaches sufficient levels, neurons may fire spontaneous action potentials or even become inactivated due to membrane depolarisation, which, in turn, may lead to increased extracellular potassium levels. Under certain circumstances, this chain of events may trigger periodic bursts of neuronal activity. In the present study, reduced neuron-glia models are applied to explore the relationship between bursting behaviour and ion concentration dynamics. These reduced models are built based on a previously developed neuron-glia model, in which channel-mediated neuronal sodium and potassium currents are replaced by a function of neuronal sodium and extracellular potassium concentrations. Simulated dynamics of the resulting two reduced models display features that are qualitatively similar to those of the existing neuron-glia model. Bifurcation analyses of the reduced models show rich and interesting dynamics that include the existence of Hopf bifurcations between which the models exhibit slow ion concentration oscillations for a wide range of parameter values. The study demonstrates that even very simple models can provide insights of possible relevance to complex phenomena.

Parkin Maintains Robust Pacemaking in Human Induced Pluripotent Stem Cell-Derived A9 Dopaminergic Neurons.

The degeneration of nigral (A9) dopaminergic (DA) neurons results in cardinal motor symptoms that define Parkinson's disease (PD). Loss-of-function mutations in parkin are linked to a rare form of early-onset PD that is inherited recessively. We generated isogenic human A9 DA neurons with or without parkin mutations to establish the causal relationship between parkin mutations and the dysfunction of human A9 DA neurons. Using TALEN (transcription activator-like effector nuclease)- or CRISPR/Cas9-mediated gene targeting, we produced two isogenic pairs of naivetropic induced pluripotent stem cells (iPSCs) by repairing exon 3 deletions of parkin in iPSCs derived from a PD patient and by introducing the PD-linked A82E mutation into iPSCs from a healthy subject. The four lines of isogenic iPSCs were differentiated to A9 DA neurons, which fired spontaneous pacemaking action potentials (AP) dependent on L-type Ca2+ channels. The frequency of the pacemaking APs was significantly reduced by parkin mutations introduced to normal neurons. Consistent with this, isogenic repair of parkin mutations significantly increased the frequency from that observed in patient-derived neurons. The results show that parkin maintains robust pacemaking in human iPSC-derived A9 DA neurons. The function is critical to normal DA transmission required for controlling voluntary locomotor activities. © 2023 International Parkinson and Movement Disorder Society.

Multicenter clinical and functional evidence reclassifies a recurrent noncanonical filamin C splice-altering variant

Truncating variants in filamin C (FLNC) can cause arrhythmogenic cardiomyopathy (ACM) through haploinsufficiency. Noncanonical splice-altering variants may contribute to this phenotype. The purpose of this study was to investigate the clinical and functional consequences of a recurrent FLNC intronic variant of uncertain significance (VUS), c.970-4A>G. Clinical data in 9 variant heterozygotes from 4 kindreds were obtained from 5 tertiary health care centers. We used in silico predictors and functional studies with peripheral blood and patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Isolated RNA was studied by reverse transcription polymerase chain reaction. iPSC-CMs were further characterized at baseline and after nonsense-mediated decay (NMD) inhibition, using quantitative polymerase chain reaction (qPCR), RNA-sequencing, and cellular electrophysiology. American College of Medical Genetics and Genomics (ACMG) criteria were used to adjudicate variant pathogenicity. Variant heterozygotes displayed a spectrum of disease phenotypes, spanning from mild ventricular dysfunction with palpitations to severe ventricular arrhythmias requiring device shocks or progressive cardiomyopathy requiring heart transplantation. Consistent with in silico predictors, the c.970-4A>G FLNC variant activated a cryptic splice acceptor site, introducing a 3-bp insertion containing a premature termination codon. NMD inhibition upregulated aberrantly spliced transcripts by qPCR and RNA-sequencing. Patch clamp studies revealed irregular spontaneous action potentials, increased action potential duration, and increased sodium late current in proband-derived iPSC-CMs. These findings fulfilled multiple ACMG criteria for pathogenicity. Clinical, in silico, and functional evidence support the prediction that the intronic c.970-4A>G VUS disrupts splicing and drives ACM, enabling reclassification from VUS to pathogenic.

PO-01-209 THE MECHANISMS OF IMPAIRED CALCIUM HANDLING UNDERLYING THE INCREASED SUSCEPTIBILITY TO ATRIAL FIBRILLATION IN METABOLIC SYNDROME

Atrial fibrillation (AF) is becoming increasingly common partly due to the prevalence of metabolic syndrome (MetS). Altered Ca2+-handling proteins on the atrial cellular membrane as well as across the sarcoplasmic reticulum (SR) network and axial tubules due to this condition can precipitate AF through elevation of Ca2+ in junctional clefts, triggering Ca2+ sparks. Through the stochastic recruitment of these sparks, a Ca2+ wave can manifest to activate neighbouring ryanodine receptors (RyRs), stimulating Na+/Ca2+ exchangers (INaCa) into potentially triggering arrhythmogenic spontaneous action potentials (SAPs). However, the mechanisms by which MetS can disrupt Ca2+ handling and trigger SAPs is not well understood. To investigate the mechanisms of impaired calcium handling underlying the increased susceptibility to AF in MetS. We utilized a 3D model of subcellular Ca2+ signalling by Zhang et al. 2022, which compromises of a network of Ca2+-release units (CRUs) and ion channel and exchanger formulations from the Grandi et al. common pool model. The Zhang et al. model far more accurately captures the processes by which Ca2+ sparks can occur and terminate through the inclusion of crucial membrane ion channels/transporters within the extensive network of axial tubules interwoven across the CRUs (Fig A). Based on experimental studies, MetS meditated functional change in Ca2+-handling proteins was replicated by increasing the conductivity of SERCA and RyR and reducing ICaL and INaCa. To evaluate the contribution of each individual remodeled Ca2+-handling component to the membrane potential and Ca2+ sparks, a series of simulations was conducted where we first incorporated each individual ionic remodeling into the control model, then from the MetS model, we reversed each remodeled Ca2+-handling component separately. We have demonstrated that SAPs were attributed to a decrease in INaCa and an increase in SERCA. For the former, this also corresponded to a much greater incidence of Ca2+ sparks compared to other cases. However, for the latter, increasing SERCA surprisingly had a negligible impact on the genesis of these sparks (Fig B). This suggests that an alternate pathway can potentially exist by which upregulated SERCA function can induce SAPs. Increased Ca2+ spark incidence due to functional changes in Ca2+-handling proteins is not the sole mechanism to SAP genesis. Our study therefore highlights that MetS-mediated AF can occur through different pathways.

TRPM4 regulates hilar mossy cell loss in temporal lobe epilepsy

BackgroundMossy cells comprise a large fraction of excitatory neurons in the hippocampal dentate gyrus, and their loss is one of the major hallmarks of temporal lobe epilepsy (TLE). The vulnerability of mossy cells in TLE is well known in animal models as well as in patients; however, the mechanisms leading to cellular death is unclear.ResultsTransient receptor potential melastatin 4 (TRPM4) is a Ca2+-activated non-selective cation channel regulating diverse physiological functions of excitable cells. Here, we identified that TRPM4 is present in hilar mossy cells and regulates their intrinsic electrophysiological properties including spontaneous activity and action potential dynamics. Furthermore, we showed that TRPM4 contributes to mossy cells death following status epilepticus and therefore modulates seizure susceptibility and epilepsy-related memory deficits.ConclusionsOur results provide evidence for the role of TRPM4 in MC excitability both in physiological and pathological conditions.

Pitx2c deficiency confers cellular electrophysiological hallmarks of atrial fibrillation to isolated atrial myocytes

AimsAtrial fibrillation (AF) has been associated with altered expression of the transcription factor Pitx2c and a high incidence of calcium release-induced afterdepolarizations. However, the relationship between Pitx2c expression and defective calcium homeostasis remains unclear and we here aimed to determine how Pitx2c expression affects calcium release from the sarcoplasmic reticulum (SR) and its impact on electrical activity in isolated atrial myocytes. MethodsTo address this issue, we applied confocal calcium imaging and patch-clamp techniques to atrial myocytes isolated from a mouse model with conditional atrial-specific deletion of Pitx2c. ResultsOur findings demonstrate that heterozygous deletion of Pitx2c doubles the calcium spark frequency, increases the frequency of sparks/site 1.5-fold, the calcium spark decay constant from 36 to 42 ms and the wave frequency from none to 3.2 min−1. Additionally, the cell capacitance increased by 30% and both the SR calcium load and the transient inward current (ITI) frequency were doubled. Furthermore, the fraction of cells with spontaneous action potentials increased from none to 44%. These effects of Pitx2c deficiency were comparable in right and left atrial myocytes, and homozygous deletion of Pitx2c did not induce any further effects on sparks, SR calcium load, ITI frequency or spontaneous action potentials. ConclusionOur findings demonstrate that heterozygous Pitx2c deletion induces defects in calcium homeostasis and electrical activity that mimic derangements observed in right atrial myocytes from patients with AF and suggest that Pitx2c deficiency confers cellular electrophysiological hallmarks of AF to isolated atrial myocytes.

In Vivo Photoadduction of Anesthetic Ligands in Mouse Brain Markedly Extends Sedation and Hypnosis.

Photoaffinity ligands are best known as tools used to identify the specific binding sites of drugs to their molecular targets. However, photoaffinity ligands have the potential to further define critical neuroanatomic targets of drug action. In the brains of WT male mice, we demonstrate the feasibility of using photoaffinity ligands in vivo to prolong anesthesia via targeted yet spatially restricted photoadduction of azi-m-propofol (aziPm), a photoreactive analog of the general anesthetic propofol. Systemic administration of aziPm with bilateral near-ultraviolet photoadduction in the rostral pons, at the border of the parabrachial nucleus and locus coeruleus, produced a 20-fold increase in the duration of sedative and hypnotic effects compared with control mice without UV illumination. Photoadduction that missed the parabrachial-coerulean complex also failed to extend the sedative or hypnotic actions of aziPm and was indistinguishable from nonadducted controls. Paralleling the prolonged behavioral and EEG consequences of on target in vivo photoadduction, we conducted electrophysiologic recordings in rostral pontine brain slices. Using neurons within the locus coeruleus to further highlight the cellular consequences of irreversible aziPm binding, we demonstrate transient slowing of spontaneous action potentials with a brief bath application of aziPm that becomes irreversible on photoadduction. Together, these findings suggest that photochemistry-based strategies are a viable new approach for probing CNS physiology and pathophysiology.SIGNIFICANCE STATEMENT Photoaffinity ligands are drugs capable of light-induced irreversible binding, which have unexploited potential to identify the neuroanatomic sites of drug action. We systemically administer a centrally acting anesthetic photoaffinity ligand in mice, conduct localized photoillumination within the brain to covalently adduct the drug at its in vivo sites of action, and successfully enrich irreversible drug binding within a restricted 250 µm radius. When photoadduction encompassed the pontine parabrachial-coerulean complex, anesthetic sedation and hypnosis was prolonged 20-fold, thus illustrating the power of in vivo photochemistry to help unravel neuronal mechanisms of drug action.