Intracellular Ca2+ release underlies the development of phase 2 in mouse ventricular action potentials

The ventricular action potential (AP) is characterized by a fast depolarizing phase followed by a repolarization that displays a second upstroke known as phase 2. This phase is generally not present in mouse ventricular myocytes. Thus we performed colocalized electrophysiological and optical recordings of APs in Langendorff-perfused mouse hearts founding a noticeable phase 2. Ryanodine as well as nifedipine reduced phase 2. Our hypothesis is that a depolarizing current activated by Ca(2+) released from the sarcoplasmic reticulum (SR) rather than the "electrogenicity" of the L-type Ca(2+) current is crucial in the generation of mouse ventricular phase 2. When Na(+) was partially replaced by Li(+) in the extracellular perfusate or the organ was cooled down, phase 2 was reduced. These results suggest that the Na(+)/Ca(2+) exchanger functioning in the forward mode is driving the depolarizing current that defines phase 2. Phase 2 appears to be an intrinsic characteristic of single isolated myocytes and not an emergent property of the tissue. As in whole heart experiments, ventricular myocytes impaled with microelectrodes displayed a large phase 2 that significantly increases when temperature was raised from 22 to 37°C. We conclude that mouse ventricular APs display a phase 2; however, changes in Ca(2+) dynamics and thermodynamic parameters also diminish phase 2, mostly by impairing the Na(+)/Ca(2+) exchanger. In summary, these results provide important insights about the role of Ca(2+) release in AP ventricular repolarization under physiological and pathological conditions.     

Establishment and characterization of a mouse embryonic heart slice preparation.

BACKGROUND: In contrast to isolated cells, the anatomic and functional integrity of tissue slices remains preserved. Aim of the study was to establish the slice technique in embryonic mouse hearts in order to perform physiological and pharmacological investigations of wild-type mice and genetically engineered mouse models of heart disease. METHODS: Ventricular slices (thickness: 300 mum) were cut from agar-embedded embryonic mouse hearts (ED 16.5-18.5) with a vibratome. Histology, immunostaining with markers for apoptosis induction, intracellular recordings with sharp electrodes and field potential recordings using microelectrode arrays were performed to assess viability. RESULTS: Slices exhibited normal histology without prominent signs of apoptosis for at least 24 hours. Intracellular recordings revealed the typical electrophysiological fingerprint of ventricular cardiomyocytes. Field potential recordings proved that adrenergic and muscarinic signaling was preserved. CONCLUSION: Functionally intact heart slices can be generated from murine embryos.     

A comparative study of collagenase- and trypsin-dissociated embryonic heart cells: reaggregation, electrophysiology, and pharmacology

The electrophysiological and pharmacological properties of aggregates prepared from cells of 7-day-old chick embryo heart ventricles depend on the enzyme used for cell dissociation. The mean beat rate of aggregates formed from trypsin-dissociated cells was about 53 beats/min whereas aggregates formed from collagenase-dissociated cells had a mean beat rate of more than twice this value. Spontaneous activity of most aggregates formed from trypsin-dissociated cells was inhibited by elevating external potassium or by adding tetrodotoxin to the medium. A similar response to potassium was seen in all aggregates formed from collagenase-dissociated cells. However, approximately half of the aggregates formed from collagenase-dissociated cells were tetrodotoxin insensitive. Intracellular microelectrode recordings demonstrated that aggregates formed from collagenase-dissociated cells typically had reduced action potential maximal upstroke velocities and depolarized threshold potentials in comparison to those recorded from aggregates formed from trypsin-dissociated cells. In the presence of tetrodotoxin the maximal upstroke velocity of aggregates formed from either collagenase- or trypsin-dissociated cells decreased markedly. In the case of the collagenase-treated cells, the spontaneous activity which persisted in the presence of tetrodotoxin was abolished by the slow channel blocker D-600. Computer simulation of membrane depolarization supports the view that aggregates formed from collagenase-treated cells have a reduced fast inward sodium current and a significant leakage current. Aggregates prepared from trypsin-dissociated cells display properties which more closely resemble those of intact 7-day embryonic ventricular tissue. We therefore conclude that, contrary to previous reports, collagenase is not the enzyme necessarily best suited for cell dissociation in all tissue culture studies.     

Neonatal murine heart slices. A robust model to study ventricular isometric contractions.

BACKGROUND/AIMS: Cardiac function is increasingly studied using murine models. However, current multicellular preparations to investigate contractile properties have substantial technical and biological limitations and are especially difficult to apply to the developing murine heart. METHODS: Newborn murine hearts were cut with a vibratome into viable tissue slices. The structural and functional integrity of the tissue was shown by histology, ATP content and sharp electrode recordings. RESULTS: Within the first 48 hours after slicing structure remained intact without induction of apoptosis. ATP concentrations and action potential parameters were comparable to those of physiological tissue. Isometric force measurements demonstrated a physiological force-frequency relationship with a ;primary-phase' negative force-frequency relationship up to 1-2 Hz and a ;secondary-phase' positive force-frequency relationship up to 8 Hz. (-)-Isoproterenol (10(-6) mol/l) increased active force to 251 +/- 35% (n=15) of baseline values and shortened relaxation times indicating a preserved beta-adrenergic regulation of contraction. Changes of the force-frequency relationship after application of ryanodine and nifedipine indicated functionality of calcium release from the sarcoplasmic reticulum and of L-type calcium channels. CONCLUSION: Generation of viable, physiological intact ventricular slices from neonatal hearts is feasible and provides a robust model to study loaded contractions.     

Homeostatic plasticity studied using in vivo hippocampal activity-blockade: synaptic scaling, intrinsic plasticity and age-dependence

Homeostatic plasticity is thought to be important in preventing neuronal circuits from becoming hyper- or hypoactive. However, there is little information concerning homeostatic mechanisms following in vivo manipulations of activity levels. We investigated synaptic scaling and intrinsic plasticity in CA1 pyramidal cells following 2 days of activity-blockade in vivo in adult (postnatal day 30; P30) and juvenile (P15) rats. Chronic activity-blockade in vivo was achieved using the sustained release of the sodium channel blocker tetrodotoxin (TTX) from the plastic polymer Elvax 40W implanted directly above the hippocampus, followed by electrophysiological assessment in slices in vitro. Three sets of results were in general agreement with previous studies on homeostatic responses to in vitro manipulations of activity. First, Schaffer collateral stimulation-evoked field responses were enhanced after 2 days of in vivo TTX application. Second, miniature excitatory postsynaptic current (mEPSC) amplitudes were potentiated. However, the increase in mEPSC amplitudes occurred only in juveniles, and not in adults, indicating age-dependent effects. Third, intrinsic neuronal excitability increased. In contrast, three sets of results sharply differed from previous reports on homeostatic responses to in vitro manipulations of activity. First, miniature inhibitory postsynaptic current (mIPSC) amplitudes were invariably enhanced. Second, multiplicative scaling of mEPSC and mIPSC amplitudes was absent. Third, the frequencies of adult and juvenile mEPSCs and adult mIPSCs were increased, indicating presynaptic alterations. These results provide new insights into in vivo homeostatic plasticity mechanisms with relevance to memory storage, activity-dependent development and neurological diseases.     

Functional triads consisting of ryanodine receptors, Ca(2+) channels, and Ca(2+)-activated K(+) channels in bullfrog sympathetic neurons. Plastic modulation of action potential

Fluorescent ryanodine revealed the distribution of ryanodine receptors in the submembrane cytoplasm (less than a few micrometers) of cultured bullfrog sympathetic ganglion cells. Rises in cytosolic Ca(2+) ([Ca(2+)](i)) elicited by single or repetitive action potentials (APs) propagated at a high speed (150 microm/s) in constant amplitude and rate of rise in the cytoplasm bearing ryanodine receptors, and then in the slower, waning manner in the deeper region. Ryanodine (10 microM), a ryanodine receptor blocker (and/or a half opener), or thapsigargin (1-2 microM), a Ca(2+)-pump blocker, or omega-conotoxin GVIA (omega-CgTx, 1 microM), a N-type Ca(2+) channel blocker, blocked the fast propagation, but did not affect the slower spread. Ca(2+) entry thus triggered the regenerative activation of Ca(2+)-induced Ca(2+) release (CICR) in the submembrane region, followed by buffered Ca(2+) diffusion in the deeper cytoplasm. Computer simulation assuming Ca(2+) release in the submembrane region reproduced the Ca(2+) dynamics. Ryanodine or thapsigargin decreased the rate of spike repolarization of an AP to 80%, but not in the presence of iberiotoxin (IbTx, 100 nM), a BK-type Ca(2+)-activated K(+) channel blocker, or omega-CgTx, both of which decreased the rate to 50%. The spike repolarization rate and the amplitude of a single AP-induced rise in [Ca(2+)](i) gradually decreased to a plateau during repetition of APs at 50 Hz, but reduced less in the presence of ryanodine or thapsigargin. The amplitude of each of the [Ca(2+)](i) rise correlated well with the reduction in the IbTx-sensitive component of spike repolarization. The apamin-sensitive SK-type Ca(2+)-activated K(+) current, underlying the afterhyperpolarization of APs, increased during repetitive APs, decayed faster than the accompanying rise in [Ca(2+)](i), and was suppressed by CICR blockers. Thus, ryanodine receptors form a functional triad with N-type Ca(2+) channels and BK channels, and a loose coupling with SK channels in bullfrog sympathetic neurons, plastically modulating AP.     

Firing rhythmicity in the neocortex in vivo and in vitro under sustained iontophoretic excitation

Extracellular recordings were made from the cat intact neocortex and guinea-pig neocortical slices during microiontophoretic application of amino acid neurotransmitters. Spike train autocorrelation analysis showed a high stability of firing patterns in the intact neocortex. When excitation of a cell was increased in a step-wise manner with glutamate iontophoresis only an enhancement of the rate of firing was observed. The rhythmic component, which was mainly due to periodic multiple discharges, remained up to the highest firing frequencies. In contrast to the in vivo observation, glutamate, aspartate or K⁺ iontophoresis in cortical slices resulted in firing pattern alternations (always from bursts or irregular activity to regular spike firing) as well as an increase in firing rate. In slices the periodic component was typically due to single-spike regularity and its frequency rose with an increase of firing rate. The comparison of autocorrelogram alternations in vivo and in vitro suggests that the temporal organization of spike trains in the intact cortex is under tight external control and is defined mainly by neuronal interactions, whereas virtually all the neurons in vitro are very sensitive to the same iontophoretic influences and their individual outputs easily change according to the excitation (depolarization) level. The coincidence of the lowest frequencies of single-spike regularity in the in vitro preparation (5–7 Hz and 8–10 Hz) with theta- and alpha-rhythms in the electroencephalogram (EEG), and with single unit firing rhythmicity in the whole brain, may represent the basis of a unit-circuit resonance and provide a high stability of these EEG-rhythms.Abbreviations ACF autocorrelation function - BFA background firing activity - EEG electroencephalogram     

Rhythmic multiunit neural activity in slices of hamster suprachiasmatic nucleus reflect prior photoperiod

The suprachiasmatic nucleus (SCN) is an endogenous circadian pacemaker, and SCN neurons exhibit circadian rhythms of electrophysiological activity in vitro. In vivo, the functional state of the pacemaker depends on changes in day length (photoperiod), but it is not known if this property persists in SCN tissue isolated in vitro. To address this issue, we prepared brain slices from hamsters previously entrained to light-dark (LD) cycles of different photoperiods and analyzed rhythms of SCN multiunit neuronal activity using single electrodes. Rhythms in SCN slices from hamsters entrained to 8:16-, 12:12-, and 14:10-h LD cycles were characterized by peak discharge rates relatively higher during subjective day than subjective night. The mean duration of high neuronal activity was photoperiod dependent, compressed in slices from the short (8:16 and 12:12 LD) photoperiods, and decompressed (approximately doubled) in slices from the long (14:10 LD) photoperiod. In slices from all photoperiods, the mean phase of onset of high neuronal activity appeared to be anchored to subjective dawn. Our results show that the electrophysiological activity of the SCN pacemaker depends on day length, extending previous in vivo data, and demonstrate that this capacity is sustained in vitro.     

An improved genetically encoded red fluorescent Ca2+ indicator for detecting optically evoked action potentials

Genetically encoded Ca(2+) indicators (GECIs) are powerful tools to image activities of defined cell populations. Here, we developed an improved red fluorescent GECI, termed R-CaMP1.07, by mutagenizing R-GECO1. In HeLa cell assays, R-CaMP1.07 exhibited a 1.5-2-fold greater fluorescence response compared to R-GECO1. In hippocampal pyramidal neurons, R-CaMP1.07 detected Ca(2+) transients triggered by single action potentials (APs) with a probability of 95% and a signal-to-noise ratio >7 at a frame rate of 50 Hz. The amplitudes of Ca(2+) transients linearly correlated with the number of APs. The expression of R-CaMP1.07 did not significantly alter the electrophysiological properties or synaptic activity patterns. The co-expression of R-CaMP1.07 and channelrhodpsin-2 (ChR2), a photosensitive cation channel, in pyramidal neurons demonstrated that R-CaMP1.07 was applicable for the monitoring of Ca(2+) transients in response to optically evoked APs, because the excitation light for R-CaMP1.07 hardly activated ChR2. These technical advancements provide a novel strategy for monitoring and manipulating neuronal activity with single cell resolution.     

Repolarisation and vulnerability to re-entry in the human heart with short QT syndrome arising from KCNQ1 mutation--a simulation study

Idiopathic short QT syndrome (SQTS) is a recently identified, genetically heterogeneous condition characterised by abbreviated QT intervals and an increased susceptibility to arrhythmia and sudden death. This simulation study identifies mechanisms by which cellular electrophysiological changes in the SQT2 (slow delayed rectifier, I_Ks, -linked) SQTS variant increases arrhythmia risk. The channel kinetics of the V307L mutation of the KCNQ1 subunit of the I_Ks channel were incorporated into human ventricular action potential (AP) models and into 1D and 2D transmural tissue simulations. Incorporating the V307L mutation into simulations reproduced defining features of the SQTS: abbreviation of the QT interval, and increases in T wave amplitude and T_peak–T_end duration. In the single-cell model, the V307L mutation abbreviated ventricular cell AP duration at 90% repolarisation (APD₉₀) and increased the maximal transmural voltage heterogeneity (δV) during APs; this resulted in augmented transmural heterogeneity of APD₉₀ and of the effective refractory period (ERP). In the intact tissue model, the vulnerable window for unidirectional conduction block was also increased. In 2D tissue the V307L mutation facilitated and maintained reentrant excitation. Thus, in SQT2 increases in transmural heterogeneity of APD, δV, ERP and an increased vulnerable window for unidirectional conduction block generate an electrical substrate favourable to reentrant arrhythmia.     

GABA excitation in the adult brain: a mechanism for excitation- neurogenesis coupling

The production of new neurons in the adult hippocampus is exquisitely regulated, and alterations in this process may underlie both normal and pathological hippocampal function. In this issue of Neuron, Tozuka et al. describe electrophysiological recordings that target proliferating progenitor cells in adult mouse hippocampal slices. They report that GABAergic synaptic inputs directly depolarize the proliferating progenitors, thereby activating molecular players that favor neuronal differentiation and providing a mechanism for direct excitation-neurogenesis coupling in vivo.     

Pituitary adenylate cyclase-activating peptide enhances electrical coupling in the mouse adrenal medulla

Neuroendocrine adrenal medullary chromaffin cells receive synaptic excitation through the sympathetic splanchnic nerve to elicit catecholamine release into the circulation. Under basal sympathetic tone, splanchnic-released acetylcholine evokes chromaffin cells to fire action potentials, leading to synchronous phasic catecholamine release. Under elevated splanchnic firing, experienced under the sympathoadrenal stress response, chromaffin cells undergo desensitization to cholinergic excitation. Yet, stress evokes a persistent and elevated adrenal catecholamine release. This sustained stress-evoked release has been shown to depend on splanchnic release of a peptide transmitter, pituitary adenylate cyclase-activating peptide (PACAP). PACAP stimulates catecholamine release through a PKC-dependent pathway that is mechanistically independent of cholinergic excitation. Moreover, it has also been reported that shorter term phospho-regulation of existing gap junction channels acts to increase junctional conductance. In this study, we test if PACAP-mediated excitation upregulates cell-cell electrical coupling to enhance chromaffin cell excitability. We utilize electrophysiological recordings conducted in adrenal tissue slices to measure the effects of PACAP stimulation on cell coupling. We report that PACAP excitation increases electrical coupling and the spread of electrical excitation between adrenal chromaffin cells. Thus PACAP acts not only as a secretagogue but also evokes an electrical remodeling of the medulla, presumably to adapt to the organism's needs during acute sympathetic stress.     

Functionally distinct sodium channels in ventricular epicardial and endocardial cells contribute to a greater sensitivity of the epicardium to electrical depression

A greater depression of the action potential (AP) of the ventricular epicardium (Epi) versus endocardium (Endo) is readily observed in experimental models of acute ischemia and Brugada syndrome. Endo and Epi differences in transient outward K(+) current and/or ATP-sensitive K(+) channel current are believed to contribute to the differential response. The present study tested the hypothesis that the greater sensitivity of Epi is due in part to its functionally distinct early fast Na(+) current (I(Na)). APs were recorded from isolated Epi and Endo tissue slices and coronary-perfused wedge preparations before and after exposures to elevated extracellular K(+) concentration ([K(+)](o); 6-12 mM). I(Na) was recorded from Epi and Endo myocytes using whole cell patch-clamp techniques. In tissue slices, increasing [K(+)](o) to 12 mM reduced V(max) to 51.1 +/- 5.3% and 26.8 +/- 9.6% of control in Endo (n = 9) and Epi (n = 14), respectively (P < 0.05). In wedge preparations (n = 12), the increase in [K(+)](o) caused selective depression of Epi APs and transmural conduction slowing and block. I(Na) density was not significantly different between Epi (n = 14) and Endo (n = 15) cells, but Epi cells displayed a more negative half-inactivation voltage [-83.6 +/- 0.1 and -75.5 +/- 0.3 mV for Epi (n = 16) and Endo (n = 16), respectively, P < 0.05]. Our data suggest that reduced I(Na) availability in ventricular Epi may contribute to its greater sensitivity to electrical depression and thus may contribute to the R-ST segment changes observed under a variety of clinical conditions including acute myocardial ischemia, severe hyperkalemia, and Brugada syndrome.     

The feasibility and limitation of patch-clamp recordings from neonatal rat cardiac ventricular slices

Examine the feasibility of whole-cell patch-clamp recordings from the cardiac ventricular slices of newborn (P(3)-P(7)) Sprague-Dawley rats to identify a better substitute for single cardiac myocytes prepared using enzymatic treatment. High resistance seals (>1 G?) were obtained from cardiac ventricle tissues prepared without using enzymatic treatment. Thereafter, cell-attached and whole-cell patch-clamp techniques were used on thin cardiac slices (200 μm thick) in 2009 in the Institute of Molecular Medicine of Peking University. An averaged sodium current (n=11 cells) was recorded in the cell-attached mode, and this displayed features similar to those previously reported for isolated rat ventricular myocytes. The outward potassium current, hyperpolarization-activated cation channel or I (f) channel (HCN channel), and action potential were recorded in the whole-cell mode (n=2 cells), and the identical properties were observed from the cardiac slices. The cell-attached mode is stable and reliable for recording the ion current. The resting potential for cardiac slices measured using current-clamp recording in the whole-cell mode was -50 to -70 mV. The resting potential of cardiac slices has properties similar to those of enzyme-prepared cardiomyocytes, with the exception that it is positive. We achieved whole-cell patch-clamp recordings from cardiac slices and affirmed the feasibility and values of both cell-attached and whole-cell recording modes using this technique. Nevertheless, there remain difficulties and limitations associated with the application of whole-cell patch-clamping to cardiac slices, due primarily to the existence of large amounts of connective tissue even in newborn rats.     

Electrophysiological characterization of murine HL-5 atrial cardiomyocytes

HL-5 cells are cultured murine atrial cardiomyocytes and have been used in studies to address important cellular and molecular questions. However, electrophysiological features of HL-5 cells have not been characterized. In this study, we examined such properties using whole cell patch-clamp techniques. Membrane capacitance of the HL-5 cells was from 8 to 62 pF. The resting membrane potential was –57.8 ± 1.4 mV (n = 51). Intracellular injection of depolarizing currents evoked action potentials (APs) with variable morphologies in 71% of the patched cells. Interestingly, the incidence of successful, current-induced APs positively correlated with the hyperpolarizing degrees of resting membrane potentials (r = 0.99, P < 0.001). Only a few of the patched cells (4 of 51, 7.8%) exhibited spontaneous APs. The muscarinic agonist carbachol activated the acetylcholine-activated K⁺ current and significantly shortened the duration of APs. Immunostaining confirmed the presence of the muscarinic receptor type 2 in HL-5 cells. The hyperpolarization-activated cation current (I_f) was detected in 39% of the patched cells. The voltage to activate 50% of I_f channels was –73.4 ± 1.2 mV (n = 12). Voltage-gated Na⁺, Ca²⁺, and K⁺ currents were observed in the HL-5 cells with variable incidences. Compared with the adult mouse cardiomyocytes, the HL-5 cells had prolonged APs and small outward K⁺ currents. Our data indicate that HL-5 cells display significant electrophysiological heterogeneity of morphological appearance of APs and expression of functional ion channels. Compared with adult murine cardiomyocytes, HL-5 cells show an immature phenotype of cardiac AP morphology. action potential; ion channel; muscarinic receptor     

Evaluation of Excitation Propagation in the Rabbit Heart: Optical Mapping and Transmural Microelectrode Recordings

Background

Because of the optical features of heart tissue, optical and electrical action potentials are only moderately associated, especially when near-infrared dyes are used in optical mapping (OM) studies.

Objective

By simultaneously recording transmural electrical action potentials (APs) and optical action potentials (OAPs), we aimed to evaluate the contributions of both electrical and optical influences to the shape of the OAP upstroke.

Methods and Results

A standard glass microelectrode and OM, using an near-infrared fluorescent dye (di-4-ANBDQBS), were used to simultaneously record transmural APs and OAPs in a Langendorff-perfused rabbit heart during atrial, endocardial, and epicardial pacing. The actual profile of the transmural AP upstroke across the LV wall, together with the OAP upstroke, allowed for calculations of the probing-depth constant (k ~2.1 mm, n = 24) of the fluorescence measurements. In addition, the transmural AP recordings aided the quantitative evaluation of the influences of depth-weighted and lateral-scattering components on the OAP upstroke. These components correspond to the components of the propagating electrical wave that are transmural and parallel to the epicardium. The calculated mean values for the depth-weighted and lateral-scattering components, whose sum comprises the OAP upstroke, were (in ms) 10.18 ± 0.62 and 0.0 ± 0.56 for atrial stimulation, 9.37 ± 1.12 and 3.01 ± 1.30 for endocardial stimulation, and 6.09 ± 0.79 and 8.16 ± 0.98 for epicardial stimulation; (n = 8 for each). For this dye, 90% of the collected fluorescence originated up to 4.83 ± 0.18 mm (n = 24) from the epicardium.

Conclusions

The co-registration of OM and transmural microelectrode APs enabled the probing depth of fluorescence measurements to be calculated and the OAP upstroke to be divided into two components (depth-weighted and lateral-scattering), and it also allowed the relative strengths of their effects on the shape of the OAP upstroke to be evaluated.     

Residual Cx45 and its relationship to Cx43 in murine ventricular myocardium

Gap junction channels in ventricular myocardium are required for electrical and metabolic coupling between cardiac myocytes and for normal cardiac pump function. Although much is known about expression patterns and remodeling of cardiac connexin(Cx)43, little is known about the less abundant Cx45, which is required for embryonic development and viability, is downregulated in adult hearts, and is pathophysiologically upregulated in human end-stage heart failure. We applied quantitative immunoblotting and immunoprecipitation to native myocardial extracts, immunogold electron microscopy to cardiac tissue and membrane sections, electrophysiological recordings to whole hearts, and high-resolution tandem mass spectrometry to Cx45 fusion protein, and developed two new tools, anti-Cx45 antisera and Cre+;Cx45 floxed mice, to facilitate characterization of Cx45 in adult mammalian hearts. We found that Cx45 represents 0.3% of total Cx protein (predominantly 200 fmol Cx43 protein/μg ventricular protein) and colocalizes with Cx43 in native ventricular gap junctions, particularly in the apex and septum. Cre+;Cx45 floxed mice express 85% less Cx45, but do not exhibit overt electrophysiologic abnormalities. Although the basal phosphorylation status of native Cx45 remains unknown, CaMKII phosphorylates 8 Ser/Thr residues in Cx45 in vitro. Thus, although downregulation of Cx45 does not produce notable deficits in electrical conduction in adult, disease-free hearts, Cx45 is a target of the multifunctional kinase CaMKII, and the phosphorylation status of Cx45 and the role of Cx43/Cx45 heteromeric gap junction channels in both normal and diseased hearts merits further investigation.     

Opposing roles of K(+) and Cl(-) channels in maintenance of opossum lower esophageal sphincter tone

The ionic basis underlying the maintenance of myogenic tone of lower esophageal sphincter circular muscle (LES) was investigated in opossum with the use of standard isometric tension and conventional intracellular microelectrode recordings in vitro. In tension recording studies, nifedipine (1 microM) reduced basal tone to 27.7 +/- 3.8% of control. The K(+) channel blockers tetraethylammonium (TEA, 2 mM), charybdotoxin (100 nM), and 4-aminopyridine (4-AP, 2 mM) enhanced resting tone, whereas apamin and glibenclamide were without affect. Cl(-) channel blockers DIDS (500 microM) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (500 microM), as well as niflumic acid (0.1-300 microM), decreased basal tone, but tamoxifen was without effect. Intracellular microelectrode recordings revealed ongoing, spontaneous, spike-like action potentials (APs). Nifedipine abolished APs and depolarized resting membrane potential (RMP). Both TEA and 4-AP significantly depolarized RMP and augmented APs, whereas niflumic acid dose-dependently hyperpolarized RMP and abolished APs. These data suggest that, in the opossum, basal tone is associated with continuous APs and that K(+) and Ca(2+)-activated Cl(-) channels have important opposing roles in the genesis of LES tone.     

Changes in ion channel expression and function associated with cardiac arrhythmogenic remodeling by Sorbs2

The Sorbin and SH3 domain-containing protein 2 (Sorbs2) is an important component of cardiomyocyte sarcomere. It has been recently reported that loss of Sorbs2 is causally associated with arrhythmogenic cardiomyopathy in human. However, the ionic mechanisms leading to cardiac arrhythmogenesis by Sorbs2 deficiency are unknown. In this study, we hypothesized that Sorbs2 plays an important role in regulating cardiac ion channel expression and function. Using electrophysiological and molecular biological approaches, we found that the Sorbs2 knockout (KO) mice progressively developed cardiac structural and electrical remodeling as early as 1 to 2 months of age and died prematurely at 5 to 7 months of age. Electrocardiographic recordings showed that Sorbs2 KO mice had conduction delays, spontaneous ventricular extrasystoles and polymorphic ventricular tachyarrhythmia. Intracellular recordings revealed abnormal action potentials with depolarized resting potential, reduced upstroke velocity, prolonged repolarization, and effective refractory period in the ventricular preparations of Sorbs2 KO mice. Patch clamp experiments demonstrated that Sorbs2 KO mice displayed distinct abnormalities in the expression and function of cardiac ion channels, including those of the voltage-gated Na⁺ channels, L-type Ca²⁺ channels, the voltage-gated K⁺ channels and the inward-rectifier K⁺ channels. Moreover, Sorbs2 physically interacted with the RNAs and/or proteins of important cardiac ion channels and directly regulated their expression in vitro. Our results indicate that Sorbs2 plays a pivotal role in the regulation of cardiac channel physiology. Loss of Sorbs2 promotes cardiac ion channelopathies and life-threatening arrhythmias.