Cellular Electrophysiology Research Articles

Fibrotic Remodeling during Persistent Atrial Fibrillation: In Silico Investigation of the Role of Calcium for Human Atrial Myofibroblast Electrophysiology.

During atrial fibrillation, cardiac tissue undergoes different remodeling processes at different scales from the molecular level to the tissue level. One central player that contributes to both electrical and structural remodeling is the myofibroblast. Based on recent experimental evidence on myofibroblasts’ ability to contract, we extended a biophysical myofibroblast model with Ca2+ handling components and studied the effect on cellular and tissue electrophysiology. Using genetic algorithms, we fitted the myofibroblast model parameters to the existing in vitro data. In silico experiments showed that Ca2+ currents can explain the experimentally observed variability regarding the myofibroblast resting membrane potential. The presence of an L-type Ca2+ current can trigger automaticity in the myofibroblast with a cycle length of 799.9 ms. Myocyte action potentials were prolonged when coupled to myofibroblasts with Ca2+ handling machinery. Different spatial myofibroblast distribution patterns increased the vulnerable window to induce arrhythmia from 12 ms in non-fibrotic tissue to 22 ± 2.5 ms and altered the reentry dynamics. Our findings suggest that Ca2+ handling can considerably affect myofibroblast electrophysiology and alter the electrical propagation in atrial tissue composed of myocytes coupled with myofibroblasts. These findings can inform experimental validation experiments to further elucidate the role of myofibroblast Ca2+ handling in atrial arrhythmogenesis.

Development an ovine myocardial infarction- induced heart failure model and characterisation of altered calcium homeostasis mechanisms

Abstract Introduction Ventricular arrhythmias (VA) and heart failure (HF) are the major complications following myocardial infarction (MI). In both conditions, there is a key role for perturbed calcium homeostasis of which the underlying mechanisms remain unclear. A preclinical model that faithfully presents most of the features of MI-induced HF has been lacking. The complexity of this syndrome means that animal modelling is difficult. As the hearts of large animals share many electrophysiological similarities to humans, ovine modelling of cardiac diseases could better reflect human pathologies than in small mammals. Question Is it possible to develop a clinically relevant ovine model with moderate cardiac dysfunction following myocardial infarction? Methods MI was induced in sheep by inflating an angioplasty balloon distal to the second diagonal branch of the left anterior descending artery for 90 min. Cardiac function was monitored for 20 weeks using electrocardiography (ECG), echocardiography, blood biochemical analysis, and subjective signs of cardiac deterioration (lethargy, dyspnoea, and cough). 20 weeks post-MI, the animals were humanely killed and single left ventricular myocytes were isolated from the infarct border zone. Changes in cellular electrophysiology and intracellular calcium concentration were monitored using whole-cell patch technique in voltage-clamp mode and the calcium sensitive fluorescent indicator Fura-2 (K5 salt). Results By using minimally invasive procedures, we obtained a survival rate of 80% (n=15). During surgery, our data show clinical features of ischaemia, including changes in the ECG features (elevation of the ST and T segment, left bundle branch block and/or pathological Q waves) and elevation of the cardiac biomarker such as troponin I. Following MI, we observed a decline in ejection fraction (−25±3%, p<0.0001) and an increase in whole animal arrhythmias (incidence of VA 72 hours post-MI, ∼70%). On cardiac removal apico-septal transmural necrosis / scarring was evident. Importantly, the L-type calcium current (ICaL) was decreased in MI cells compared to healthy cells (−1.87±0.73 pA/pF, p<0.05), but isoprenaline had no effet on ICaL (0.48±1.2 pA/pF, p=0.70) in MI cells. Moreover, the amplitude of the systolic calcium transient (−0.33±0.1 F/F0, p<0.05) and the sarcoplasmic reticulum calcium content (−23±7 μmol/L, p<0.01) were also decreased. The shortening velocity of the sarcomere was also decreased in MI cells (−0.55±0.18 μm/s, p<0.01). Conclusion We successfully established an ovine MI model using minimally invasive procedure which displays a moderately impaired cardiac function, reduced contractility, and pro-arrhythmic electrophysiological remodelling. Future analysis will examine the role of the L-type calcium channel with respect to the excitation-contraction coupling process and myocyte contractility and how we can improve therapeutic strategies towards VA and HF. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart Foundation

In silico Identification of Disrupted Myocardial Calcium Homeostasis as Proarrhythmic Trigger in Arrhythmogenic Cardiomyopathy.

Background: Patients with arrhythmogenic cardiomyopathy may suffer from lethal ventricular arrhythmias. Arrhythmogenic cardiomyopathy is predominantly triggered by mutations in plakophilin-2, a key component of cell-to-cell adhesion and calcium cycling regulation in cardiomyocytes. Calcium dysregulation due to plakophilin-2 mutations may lead to arrhythmias but the underlying pro-arrhythmic mechanisms remain unclear.Aim: To unravel the mechanisms by which calcium-handling abnormalities in plakophilin-2 loss-of-function may contribute to proarrhythmic events in arrhythmogenic cardiomyopathy.Methods: We adapted a computer model of mouse ventricular electrophysiology using recent experimental calcium-handling data from plakophilin-2 conditional knock-out (PKP2-cKO) mice. We simulated individual effects of beta-adrenergic stimulation, modifications in connexin43-mediated calcium entry, sodium-calcium exchanger (NCX) activity and ryanodine-receptor 2 (RyR2) calcium affinity on cellular electrophysiology and occurrence of arrhythmogenic events (delayed-afterdepolarizations). A population-of-models approach was used to investigate the generalizability of our findings. Finally, we assessed the potential translation of proposed mechanisms to humans, using a human ventricular cardiomyocyte computational model.Results: The model robustly reproduced the experimental calcium-handling changes in PKP2-cKO cardiomyocytes: an increased calcium transient amplitude (562 vs. 383 nM), increased diastolic calcium (120 vs. 91 nM), reduced L-type calcium current (15.0 vs. 21.4 pA/pF) and an increased free SR calcium (0.69 vs. 0.50 mM). Under beta-adrenergic stimulation, PKP2-cKO models from the population of models (n = 61) showed a higher susceptibility to delayed-afterdepolarizations compared to control (41 vs. 3.3%). Increased connexin43-mediated calcium entry further elevated the number of delayed-afterdepolarizations (78.7%, 2.5-fold increase in background calcium influx). Elevated diastolic cleft calcium appeared responsible for the increased RyR2-mediated calcium leak, promoting delayed-afterdepolarizations occurrence. A reduction in RyR2 calcium affinity prevented delayed-afterdepolarizations in PKP2-cKO models (24.6 vs. 41%). An additional increase in INCX strongly reduced delayed-afterdepolarizations occurrence, by lowering diastolic cleft calcium levels. The human model showed similar outcomes, suggesting a potential translational value of these findings.Conclusion: Beta-adrenergic stimulation and connexin43-mediated calcium entry upon loss of plakophilin-2 function contribute to generation of delayed-afterdepolarizations. RyR2 and NCX dysregulation play a key role in modulating these proarrhythmic events. This work provides insights into potential future antiarrhythmic strategies in arrhythmogenic cardiomyopathy due to plakophilin-2 loss-of-function.

Critical analysis of explanations for cellular homeostasis and electrophysiology from murburn perspective.

Pursuits in modern cellular electrophysiology are fraught with disagreements at a fundamental level. While the membrane theory of homeostasis deems the cell membrane and proteins embedded therein as the chief players, the association-induction (or sorption/bulk-phase) hypothesis considers the aqueous phase of dissolved proteins (cytoplasm/protoplasm) as the key determinant of cellular composition and ionic fluxes. In the first school of thought, trans-membrane potential (TMP) and selective ion pumps/channels are deemed as key operative principles. In the latter theory, sorption-desorption dynamics and rearrangements of bulk phase determine the outcomes. In both these schools of thought, theorists believe that the macroscopic phase electroneutrality holds, TMP (whether in resting or in activated state) results solely due to ionic concentration differentials across the membrane, and the concerned proteins undergo major conformation changes to affect/effect the noted outcomes. The new entry into the field, murburn concept, builds starting from molecular considerations to macroscopic observations. It moots "effective charge separation" and intricate "molecule-ion-radical" electron transfer equilibriums as a rationale for ionic concentration differentials and TMP variation. After making an unbiased appraisal of the two classical schools of thought, the review makes a point-wise analysis of some hitherto unresolved observations/considerations and suggests the need to rethink the mechanistic perspectives.

Calmodulin Interactions with Voltage-Gated Sodium Channels.

Calmodulin (CaM) is a small protein that acts as a ubiquitous signal transducer and regulates neuronal plasticity, muscle contraction, and immune response. It interacts with ion channels and plays regulatory roles in cellular electrophysiology. CaM modulates the voltage-gated sodium channel gating process, alters sodium current density, and regulates sodium channel protein trafficking and expression. Many mutations in the CaM-binding IQ domain give rise to diseases including epilepsy, autism, and arrhythmias by interfering with CaM interaction with the channel. In the present review, we discuss CaM interactions with the voltage-gated sodium channel and modulators involved in CaM regulation, as well as summarize CaM-binding IQ domain mutations associated with human diseases in the voltage-gated sodium channel family.

Mutation location and IKs regulation in the arrhythmic risk of long QT syndrome type 1: the importance of the KCNQ1 S6 region.

Mutation type, location, dominant-negative IKs reduction, and possibly loss of cyclic adenosine monophosphate (cAMP)-dependent IKs stimulation via protein kinase A (PKA) influence the clinical severity of long QT syndrome type 1 (LQT1). Given the malignancy of KCNQ1-p.A341V, we assessed whether mutations neighbouring p.A341V in the S6 channel segment could also increase arrhythmic risk. Clinical and genetic data were obtained from 1316 LQT1 patients [450 families, 166 unique KCNQ1 mutations, including 277 p.A341V-positive subjects, 139 patients with p.A341-neighbouring mutations (91 missense, 48 non-missense), and 900 other LQT1 subjects]. A first cardiac event represented the primary endpoint. S6 segment missense variant characteristics, particularly cAMP stimulation responses, were analysed by cellular electrophysiology. p.A341-neighbouring mutation carriers had a QTc shorter than p.A341V carriers (477 ± 33 vs. 490 ± 44 ms) but longer than the remaining LQT1 patient population (467 ± 41 ms) (P < 0.05 for both). Similarly, the frequency of symptomatic subjects in the p.A341-neighbouring subgroup was intermediate between the other two groups (43% vs. 73% vs. 20%; P < 0.001). These differences in clinical severity can be explained, for p.A341V vs. p.A341-neighbouring mutations, by the p.A341V-specific impairment of IKs regulation. The differences between the p.A341-neighbouring subgroup and the rest of LQT1 mutations may be explained by the functional importance of the S6 segment for channel activation. KCNQ1 S6 segment mutations surrounding p.A341 increase arrhythmic risk. p.A341V-specific loss of PKA-dependent IKs enhancement correlates with its phenotypic severity. Cellular studies providing further insights into IKs-channel regulation and knowledge of structure-function relationships could improve risk stratification. These findings impact on clinical management.

Study on the temperature characteristics of fast capacitance in patch clamp experiments

Patch clamp is a technique that can measure weak current in the level of picoampere (pA). It has been widely used for cellular electrophysiological recording in fundamental medical researches, such as membrane potential and ion channel currents recording, etc. In order to obtain accurate measurement results, both the resistance and capacitance of the pipette are required to be compensated. Capacitance compensations are composed of slow and fast capacitance compensation. The slow compensation is determined by the lipid bilayer of cell membrane, and its magnitude usually ranges from a few picofarads (pF) to a few microfarads (μF), depending on the cell size. The fast capacitance is formed by the distributed capacitance of the glass pipette, wires and solution, mostly ranging in a few picofarads. After the pipette sucks the cells in the solution, the positions of the glass pipette and wire have been determined, and only taking once compensation for slow and fast capacitance will meet the recording requirements. However, when the study needs to deal with the temperature characteristics, it is still necessary to make a recognition on the temperature characteristic of the capacitance. We found that the time constant of fast capacitance discharge changed with increasing temperature of bath solution when we studied the photothermal effect on cell membrane by patch clamp. Based on this phenomenon, we proposed an equivalent circuit to calculate the temperature-dependent parameters. Experimental results showed that the fast capacitance increased in a positive rate of 0.04 pF/℃, while the pipette resistance decreased. The fine data analysis demonstrated that the temperature rises of bath solution determined the kinetics of the fast capacitance mainly by changing the inner solution resistance of the glass pipette. This result will provide a good reference for the fine temperature characteristic study related to cellular electrophysiology based on patch clamp technique.

The murburn precepts for cellular ionic homeostasis and electrophysiology.

Starting from the basic molecular structure and redox properties of its components, we build a macroscopic cellular electrophysiological model. We first present a murburn purview that could explain ion distribution in bulk-milieu/membrane-interface and support the origin of trans-membrane potential (TMP) in cells. In particular, the discussion focuses on how cells achieve disparity in the distribution of monovalent and divalent cations within (K+ > Na+ > Mg2+ > Ca2+ ) and outside (Na+ > K+ > Ca2+ > Mg2+ ). We explore how TMP could vary for resting/graded/action potentials generation and project a model for impulse conduction in neurons. Outcomes based on murburn bioenergetic equilibriums leading to solubilization of ion-pairs, membrane's permittivity, protein channels' fluxes, and proteins' innate ability to bind/adsorb ions selectively are projected as the integral rationale. We also provide experimental modalities to ratify the projections.

Acupuncture ameliorates neurological function in rats with cerebral ischemia-reperfusion by regulating the opening of large-conductance Ca2+ -activated potassium channels.

Acupuncture has a good effect on improving neurological function after cerebral ischemia‐reperfusion, but there are few studies on the neuroprotective effect of acupuncture from the perspective of ion channel cellular electrophysiology. Studies have shown that the over activation of large‐conductance Ca2+‐activated potassium channel （BKCa） after cerebral ischemia‐reperfusion can reduce the excitability of neurons and induce apoptosis. This study intends to establish middle cerebral artery occlusion/reperfusion (MCAO/R) model, with acupuncture at GV26 as the intervention measure, using patch‐clamp technique to record the electrophysiological changes of BKCa channel. The results showed that the neurological function score of MCAO/R rats was significantly decreased, and the conductance, open dwell time and open probability of BKCa channel in hippocampal CA1 neurons of MCAO/R rats were significantly increased. Acupuncture at GV26 could significantly improve the neurological function scores of MCAO/R rats, and reduce the conductance, open dwell time, and open probability of BKCa channel. The effect of acupuncture at GV26 was significantly better than acupuncture at non‐acupuncture point. The neuroprotective effect of acupuncture at GV26 after cerebral ischemia‐reperfusion may be related to regulating the electrophysiological characteristics of BKCa channel opening.

Update on ICH E14/S7B Cardiac Safety Regulations: The Expanded Role of Preclinical Assays and the "Double-Negative" Scenario.

For nearly 2 decades, regulators have adopted a harmonized approach to drug development, which has succeeded in bringing new pharmaceuticals to market without significant cardiac liability. Ushered in by technological advancements and better understanding of cellular electrophysiology, the initial paradigm detailed in the 2005 International Conference for Harmonization E14 and S7B documents has undergone evolutionary changes designed to streamline drug development and improve regulatory decision‐making and product labeling. The intent of this review is to summarize the new US Food and Drug Administration (FDA) Question and Answer update from August 2020 and key messaging from a subsequent FDA webinar describing best practices for preclinical and clinical data integration into a QT risk prediction model.

Edward E. Carmeliet, MD, PhD (January 4, 1930–April 5, 2021): A pioneer in cardiac cellular electrophysiology

The passing of Edward E. Carmeliet entrusts to our memories the life and the work of a brilliant cardiac cellular electrophysiologist, indeed, one of the most brilliant of our time. Not only did he have a major innovative impact on our understanding of cardiac bioelectricity in health and disease, he also was a revered mentor who influenced the work of scholars worldwide with his scientific competence and his unique personality.

ESC working group on cardiac cellular electrophysiology position paper: relevance, opportunities, and limitations of experimental models for cardiac electrophysiology research.

Cardiac arrhythmias are a major cause of death and disability. A large number of experimental cell and animal models have been developed to study arrhythmogenic diseases. These models have provided important insights into the underlying arrhythmia mechanisms and translational options for their therapeutic management. This position paper from the ESC Working Group on Cardiac Cellular Electrophysiology provides an overview of (i) currently available in vitro, ex vivo, and in vivo electrophysiological research methodologies, (ii) the most commonly used experimental (cellular and animal) models for cardiac arrhythmias including relevant species differences, (iii) the use of human cardiac tissue, induced pluripotent stem cell (hiPSC)-derived and in silico models to study cardiac arrhythmias, and (iv) the availability, relevance, limitations, and opportunities of these cellular and animal models to recapitulate specific acquired and inherited arrhythmogenic diseases, including atrial fibrillation, heart failure, cardiomyopathy, myocarditis, sinus node, and conduction disorders and channelopathies. By promoting a better understanding of these models and their limitations, this position paper aims to improve the quality of basic research in cardiac electrophysiology, with the ultimate goal to facilitate the clinical translation and application of basic electrophysiological research findings on arrhythmia mechanisms and therapies.

Electrophysiological Characterization of Human Atria: The Understated Role of Temperature.

Ambient temperature has a profound influence on cellular electrophysiology through direct control over the gating mechanisms of different ion channels. In the heart, low temperature is known to favor prolongation of the action potential. However, not much is known about the influence of temperature on other important characterization parameters such as the resting membrane potential (RMP), excitability, morphology and characteristics of the action potential (AP), restitution properties, conduction velocity (CV) of signal propagation, etc. Here we present the first, detailed, systematic in silico study of the electrophysiological characterization of cardiomyocytes from different regions of the normal human atria, based on the effects of ambient temperature (5−50°C). We observe that RMP decreases with increasing temperature. At ~ 48°C, the cells lose their excitability. Our studies show that different parts of the atria react differently to the same changes in temperature. In tissue simulations a drop in temperature correlated positively with a decrease in CV, but the decrease was region-dependent, as expected. In this article we show how this heterogeneous response can provide an explanation for the development of a proarrhythmic substrate during mild hypothermia. We use the above concept to propose a treatment strategy for atrial fibrillation that involves severe hypothermia in specific regions of the heart for a duration of only ~ 200 ms.

TRPV1 activation and internalization is part of the LPS-induced inflammation in human iPSC-derived cardiomyocytes

The non-selective cation channel transient receptor potential vanilloid 1 (TRPV1) is expressed throughout the cardiovascular system. Recent evidence shows a role for TRPV1 in inflammatory processes. The role of TRPV1 for myocardial inflammation has not been established yet. Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (hiPSC-CM) from 4 healthy donors were incubated with lipopolysaccharides (LPS, 6 h), TRPV1 agonist capsaicin (CAP, 20 min) or the antagonist capsazepine (CPZ, 20 min). TRPV1 expression was studied by PCR and western blotting. TRPV1 internalization was analyzed by immunofluorescence. Interleukin-6 (IL-6) secretion and phosphorylation of JNK, p38 and ERK were determined by ELISA. TRPV1-associated ion channel current was measured by patch clamp. TRPV1-mRNA and -protein were expressed in hiPSC-CM. TRPV1 was localized in the plasma membrane. LPS significantly increased secretion of IL-6 by 2.3-fold, which was prevented by pre-incubation with CPZ. LPS induced TRPV1 internalization. Phosphorylation levels of ERK, p38 or JNK were not altered by TRPV1 stimulation or inhibition. LPS and IL-6 significantly lowered TRPV1-mediated ion channel current. TRPV1 mediates the LPS-induced inflammation in cardiomyocytes, associated with changes of cellular electrophysiology. LPS-induced inflammation results in TRPV1 internalization. Further studies have to examine the underlying pathways and the clinical relevance of these findings.

A computational model of pig ventricular cardiomyocyte electrophysiology and calcium handling: Translation from pig to human electrophysiology.

The pig is commonly used as an experimental model of human heart disease, including for the study of mechanisms of arrhythmia. However, there exist differences between human and porcine cellular electrophysiology: The pig action potential (AP) has a deeper phase-1 notch, a longer duration at 50% repolarization, and higher plateau potentials than human. Ionic differences underlying the AP include larger rapid delayed-rectifier and smaller inward-rectifier K+-currents (IKr and IK1 respectively) in humans. AP steady-state rate-dependence and restitution is steeper in pigs. Porcine Ca2+ transients can have two components, unlike human. Although a reliable computational model for human ventricular myocytes exists, one for pigs is lacking. This hampers translation from results obtained in pigs to human myocardium. Here, we developed a computational model of the pig ventricular cardiomyocyte AP using experimental datasets of the relevant ionic currents, Ca2+-handling, AP shape, AP duration restitution, and inducibility of triggered activity and alternans. To properly capture porcine Ca2+ transients, we introduced a two-step process with a faster release in the t-tubular region, followed by a slower diffusion-induced release from a non t-tubular subcellular region. The pig model behavior was compared with that of a human ventricular cardiomyocyte (O'Hara-Rudy) model. The pig, but not the human model, developed early afterdepolarizations (EADs) under block of IK1, while IKr block led to EADs in the human but not in the pig model. At fast rates (pacing cycle length = 400 ms), the human cell model was more susceptible to spontaneous Ca2+ release-mediated delayed afterdepolarizations (DADs) and triggered activity than pig. Fast pacing led to alternans in human but not pig. Developing species-specific models incorporating electrophysiology and Ca2+-handling provides a tool to aid translating antiarrhythmic and arrhythmogenic assessment from the bench to the clinic.

Remote Optical Modulation of Cellular Electrical Activity Using Two-Dimensional Ti3C2 MXene

The ability to probe and manipulate electrophysiology at the cellular level is crucial for understanding cellular communications and enabling new therapeutics for neurological and psychiatric disorders. Nanomaterial-facilitated photothermal stimulation is a non-invasive technique to manipulate cellular electrophysiology with high spatial precision and rapid response time without the need of genetic modifications. Although recently reported Si-, Au-, and C-based nanomaterials are promising candidates for photothermal stimulation of electrically active cells and tissues, they exhibit limited photothermal conversion efficiency in the near-infrared (NIR) window or have complicated synthesis protocols that prevents direct scale-up. Two-dimensional (2D) Ti3C2 MXene is a promising candidate for optical stimulation due to its high NIR absorption and photothermal energy conversion efficiency. Here, we report an approach for optical modulation of neuronal activity using Ti3C2 MXene flakes. Under illumination with 1 ms pulses of 10 mW NIR laser (λ = 808 nm), the local temperature rise induced by single Ti3C2 MXene flakes was measured to be 3.1 ± 0.7 K. MXene films (25 µg/cm2) and dispersed MXene flakes (100 µg/mL) were evaluated to be biocompatible with dorsal root ganglia (DRG) neurons. Both MXene films and flakes enabled photothermal stimulation of DRG neurons (and DRG networks) with incident energies in the micro-joule regime. Optical stimulation of DRG neurons using Ti3C2 MXene flakes is safe and does not generate cellular stress. Due to its straightforward and large-scale synthesis, MXene can enable neuronal modulation at various scales and dimensions, thus is a powerful tool for future remote, non-genetic biological interfaces.

Treatment of atrial fibrillation with doxapram: TASK-1 potassium channel inhibition as a novel pharmacological strategy.

TASK-1 (K2P3.1) two-pore-domain potassium channels are atrial-specific and significantly up-regulated in atrial fibrillation (AF) patients, contributing to AF-related electrical remodelling. Inhibition of TASK-1 in cardiomyocytes of AF patients was shown to counteract AF-related action potential duration shortening. Doxapram was identified as a potent inhibitor of the TASK-1 channel. In this study, we investigated the antiarrhythmic efficacy of doxapram in a porcine model of AF. Doxapram successfully cardioverted pigs with artificially induced episodes of AF. We established a porcine model of persistent AF in domestic pigs via intermittent atrial burst stimulation using implanted pacemakers. All pigs underwent catheter-based electrophysiological investigations prior to and after 14 days of doxapram treatment. Pigs in the treatment group received intravenous administration of doxapram once per day. In doxapram-treated AF pigs, the AF burden was significantly reduced. After 14 days of treatment with doxapram, TASK-1 currents were still similar to values of sinus rhythm animals. Doxapram significantly suppressed AF episodes and normalized cellular electrophysiology by inhibition of the TASK-1 channel. Patch-clamp experiments on human atrial cardiomyocytes, isolated from patients with and without AF could reproduce the TASK-1 inhibitory effect of doxapram. Repurposing doxapram might yield a promising new antiarrhythmic drug to treat AF in patients.

MicroRNA Biophysically Modulates Cardiac Action Potential by Direct Binding to Ion Channel.

MicroRNAs (miRs) play critical roles in regulation of numerous biological events, including cardiac electrophysiology and arrhythmia, through a canonical RNA interference mechanism. It remains unknown whether endogenous miRs modulate physiologic homeostasis of the heart through noncanonical mechanisms. We focused on the predominant miR of the heart (miR1) and investigated whether miR1 could physically bind with ion channels in cardiomyocytes by electrophoretic mobility shift assay, in situ proximity ligation assay, RNA pull down, and RNA immunoprecipitation assays. The functional modulations of cellular electrophysiology were evaluated by inside-out and whole-cell patch clamp. Mutagenesis of miR1 and the ion channel was used to understand the underlying mechanism. The effect on the heart ex vivo was demonstrated through investigating arrhythmia-associated human single nucleotide polymorphisms with miR1-deficient mice. We found that endogenous miR1 could physically bind with cardiac membrane proteins, including an inward-rectifier potassium channel Kir2.1. The miR1-Kir2.1 physical interaction was observed in mouse, guinea pig, canine, and human cardiomyocytes. miR1 quickly and significantly suppressed IK1 at sub-pmol/L concentration, which is close to endogenous miR expression level. Acute presence of miR1 depolarized resting membrane potential and prolonged final repolarization of the action potential in cardiomyocytes. We identified 3 miR1-binding residues on the C-terminus of Kir2.1. Mechanistically, miR1 binds to the pore-facing G-loop of Kir2.1 through the core sequence AAGAAG, which is outside its RNA interference seed region. This biophysical modulation is involved in the dysregulation of gain-of-function Kir2.1-M301K mutation in short QT or atrial fibrillation. We found that an arrhythmia-associated human single nucleotide polymorphism of miR1 (hSNP14A/G) specifically disrupts the biophysical modulation while retaining the RNA interference function. It is remarkable that miR1 but not hSNP14A/G relieved the hyperpolarized resting membrane potential in miR1-deficient cardiomyocytes, improved the conduction velocity, and eliminated the high inducibility of arrhythmia in miR1-deficient hearts ex vivo. Our study reveals a novel evolutionarily conserved biophysical action of endogenous miRs in modulating cardiac electrophysiology. Our discovery of miRs' biophysical modulation provides a more comprehensive understanding of ion channel dysregulation and may provide new insights into the pathogenesis of cardiac arrhythmias.

Molecular Basis of Coupled Transport and Anion Conduction in Excitatory Amino Acid Transporters

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. After its release from presynaptic nerve terminals, glutamate is quickly removed from the synaptic cleft by excitatory amino acid transporters (EAATs) 1–5, a subfamily of glutamate transporters. The five proteins utilize a complex transport stoichiometry that couples glutamate transport to the symport of three Na+ ions and one H+ in exchange with one K+ to accumulate glutamate against up to 106-fold concentration gradients. They are also anion-selective channels that open and close during transitions along the glutamate transport cycle. EAATs belong to a larger family of secondary-active transporters, the SLC1 family, which also includes purely Na+- or H+-coupled prokaryotic transporters and Na+-dependent neutral amino acid exchangers. In recent years, molecular cloning, heterologous expression, cellular electrophysiology, fluorescence spectroscopy, structural approaches, and molecular simulations have uncovered the molecular mechanisms of coupled transport, substrate selectivity, and anion conduction in EAAT glutamate transporters. Here we review recent findings on EAAT transport mechanisms, with special emphasis on the highly conserved hairpin 2 gate, which has emerged as the central processing unit in many of these functions.

Relationships between serum electrolyte concentrations and ileus: A joint clinical and mathematical modeling study

AimProlonged postoperative ileus (PPOI) occurs in around 15% of patients after major abdominal surgery, posing a significant clinical and economic burden. Significant fluid and electrolyte changes may occur peri‐operatively, potentially contributing to PPOI; however, this association has not been clearly elucidated. A joint clinical‐theoretical study was undertaken to evaluate peri‐operative electrolyte concentration trends, their association with ileus, and predicted impact on bioelectrical slow waves in interstitial cells of Cajal (ICC) and smooth muscle cells (SMC).MethodsData were prospectively collected from 327 patients undergoing elective colorectal surgery. Analyses were performed to determine associations between peri‐operative electrolyte concentrations and prolonged ileus. Biophysically based ICC and SMC mathematical models were adapted to evaluate the theoretical impacts of extracellular electrolyte concentrations on cellular function.ResultsPostoperative day (POD) 1 calcium and POD 3 chloride, sodium were lower in the PPOI group (p < 0.05), and POD3 potassium was higher in the PPOI group (p < 0.05). Deficits beyond the reference range in PPOI patients were most notable for sodium (Day 3: 29.5% ileus vs. 18.5% no ileus, p = 0.04). Models demonstrated an 8.6% reduction in slow‐wave frequency following the measured reduction in extracellular NaCl on POD5, with associated changes in cellular slow‐wave morphology and amplitude.ConclusionLow serum sodium and chloride concentrations are associated with PPOI. Electrolyte abnormalities are unlikely to be a primary mechanism of ileus, but their pronounced effects on cellular electrophysiology predicted by modeling suggest these abnormalities may adversely impact motility recovery. Resolution and correction of electrolyte abnormalities in ileus may be clinically relevant.