Sharp Microelectrodes Research Articles

Effects of a new antiarrhythmic drug SS-68 on electrical activity in working atrial and ventricular myocardium of mouse and their ionic mechanisms

SS-68 is a derivative of indole, which demonstrated strong antiarrhythmic effects not associated with significant QT prolongation in dog models of atrial fibrillation. Therefore, SS-68 was proposed as a new antiarrhythmic drug and the present study is the first describing its effects on action potentials (APs) configuration and elucidating the ionic mechanisms of these effects. Sharp microelectrodes were used to record APs in isolated preparations of mouse atrial and ventricular myocardium. In both types of myocardium 10−6 M SS-68 produced reduction of AP duration, 3 × 10−6 M failed to alter AP waveform and 10−5 – 3 × 10−5 M prolonged APs. Sensitivity of main ionic currents to SS-68 was determined using whole-cell patch clamp. Transient potassium current Ito was slightly inhibited by SS-68 with IC50 = 1.43 × 10−4 M. IKur was more sensitive with IC50 = 1.84 × 10−5 M. Background inward rectifier showed very low sensitivity to SS-68 – only 10−4 M SS-68 caused significant reduction of IK1. ICaL was significantly inhibited by 10−6M – 3 × 10−5 M SS-68. The IC50 value for the ICaL was 1.84 × 10−6 M. Thus, main ionic currents of mouse cardiomyocytes are inhibited by SS-68 in the following order of potency: ICaL > IKur > Ito > IK1. While lower concentration of SS-68 shorten APs via suppression of ICaL, higher concentrations inhibit K+-currents leading to APs prolongation.

Late Sodium Current in Human Atrial Cardiomyocytes from Patients in Sinus Rhythm and Atrial Fibrillation.

Slowly inactivating Na+ channels conducting “late” Na+ current (INa,late) contribute to ventricular arrhythmogenesis under pathological conditions. INa,late was also reported to play a role in chronic atrial fibrillation (AF). The objective of this study was to investigate INa,late in human right atrial cardiomyocytes as a putative drug target for treatment of AF. To activate Na+ channels, cardiomyocytes from transgenic mice which exhibit INa,late (ΔKPQ), and right atrial cardiomyocytes from patients in sinus rhythm (SR) and AF were voltage clamped at room temperature by 250-ms long test pulses to -30 mV from a holding potential of -80 mV with a 100-ms pre-pulse to -110 mV (protocol I). INa,late at -30 mV was not discernible as deviation from the extrapolated straight line IV-curve between -110 mV and -80 mV in human atrial cells. Therefore, tetrodotoxin (TTX, 10 μM) was used to define persistent inward current after 250 ms at -30 mV as INa,late. TTX-sensitive current was 0.27±0.06 pA/pF in ventricular cardiomyocytes from ΔKPQ mice, and amounted to 0.04±0.01 pA/pF and 0.09±0.02 pA/pF in SR and AF human atrial cardiomyocytes, respectively. With protocol II (holding potential -120 mV, pre-pulse to -80 mV) TTX-sensitive INa,late was always larger than with protocol I. Ranolazine (30 μM) reduced INa,late by 0.02±0.02 pA/pF in SR and 0.09±0.02 pA/pF in AF cells. At physiological temperature (37°C), however, INa,late became insignificant. Plateau phase and upstroke velocity of action potentials (APs) recorded with sharp microelectrodes in intact human trabeculae were more sensitive to ranolazine in AF than in SR preparations. Sodium channel subunits expression measured with qPCR was high for SCN5A with no difference between SR and AF. Expression of SCN8A and SCN10A was low in general, and lower in AF than in SR. In conclusion, We confirm for the first time a TTX-sensitive current (INa,late) in right atrial cardiomyocytes from SR and AF patients at room temperature, but not at physiological temperature. While our study provides evidence for the presence of INa,late in human atria, the potential of such current as a target for the treatment of AF remains to be demonstrated.

Sympathy for the ganglion.

Sympathy for the ganglion.

Cytosolic calcium changes affect the incidence of early afterdepolarizations in canine ventricular myocytes.

This study was designed to investigate the influence of cytosolic Ca(2+) levels ([Ca(2+)]i) on action potential duration (APD) and on the incidence of early afterdepolarizations (EADs) in canine ventricular cardiomyocytes. Action potentials (AP) of isolated cells were recorded using conventional sharp microelectrodes, and the concomitant [Ca(2+)]i was monitored with the fluorescent dye Fura-2. EADs were evoked at a 0.2 Hz pacing rate by inhibiting the rapid delayed rectifier K(+) current with dofetilide, by activating the late sodium current with veratridine, or by activating the L-type calcium current with BAY K8644. These interventions progressively prolonged the AP and resulted in initiation of EADs. Reducing [Ca(2+)]i by application of the cell-permeant Ca(2+) chelator BAPTA-AM lengthened the AP at 1.0 Hz if it was applied alone, in the presence of veratridine, or in the presence of BAY K8644. However, BAPTA-AM shortened the AP if the cells were pretreated with dofetilide. The incidence of the evoked EADs was strongly reduced by BAPTA-AM in dofetilide, moderately reduced in veratridine, whereas EAD incidence was increased by BAPTA-AM in the presence of BAY K8644. Based on these experimental data, changes in [Ca(2+)]i have marked effects on APD as well as on the incidence of EADs; however, the underlying mechanisms may be different, depending on the mechanism of EAD generation. As a consequence, reduction of [Ca(2+)]i may eliminate EADs under some, but not all, experimental conditions.

Epoxyeicosatrienoic acids act through TRPV4–TRPC1–KCa1.1 complex to induce smooth muscle membrane hyperpolarization and relaxation in human internal mammary arteries

Human left internal mammary arteries (LIMAs) are commonly used as donor grafts for coronary bypass surgery. Previous reports suggested that 11,12-epoxyeicosatrienoic acid (11,12-EET) is an important endothelial-derived hyperpolarizing factor (EDHF) in human LIMAs and that EETs act through large conductance Ca2+-activated K+ channels (KCa1.1) to induce smooth muscle cell hyperpolarization and relaxation in these tissues. In this study, we aimed to explore the role of vanilloid transient receptor potential channel 4 (TRPV4) and canonical transient receptor potential channel 1 (TRPC1) channels in the EET-induced smooth muscle hyperpolarization and vascular relaxation in human LIMAs. Co-immunoprecipitation studies demonstrated that TRPV4, TRPC1, and KCa1.1 physically interacted with each other to form a complex. Sharp microelectrode and vascular tension studies demonstrated that 11,12-EET (300nmol/L) and 4α-phorbol 12,13-didecanoate (5μmol/L) were able to induce smooth muscle membrane hyperpolarization and vascular relaxation in isolated human LIMA segments. The hyperpolarizing and relaxant effects were markedly reduced by treatments that could suppress the expression/activity of TRPV4, TRPC1, or KCa1.1. With the use of human embryonic kidney 293 cells that over-expressed with TRPV4, TRPC1 and KCa1.1, we found that TRPC1 is the linker through which TRPV4 and KCa1.1(α) can interact. The present study revealed that 11,12-EET targets the TRPV4–TRPC1–KCa1.1 complex to induce smooth muscle cell hyperpolarization and vascular relaxation in human LIMAs. This finding provides novel mechanistic insights for the EET action in human LIMAs.

A comparison of responses to raised extracellular potassium and endothelium-derived hyperpolarizing factor (EDHF) in rat pressurised mesenteric arteries.

The present study examined the hypothesis that potassium ions act as an endothelium-derived hyperpolarizing factor (EDHF) released in response to ACh in small mesenteric arteries displaying myogenic tone. Small mesenteric arteries isolated from rats were set up in a pressure myograph at either 60 or 90 mmHg. After developing myogenic tone, responses to raising extracellular potassium were compared to those obtained with ACh (in the presence of nitric oxide synthase and cyclo-oxygenase inhibitors). The effects of barium and oubain, or capsaicin, on responses to raised extracellular potassium or ACh were also determined. The effects of raised extracellular potassium levels and ACh on membrane potential, were measured using sharp microelectrodes in pressurised arteries. Rat small mesenteric arteries developed myogenic tone when pressurised. On the background of vascular tone set by a physiological stimulus (i.e pressure), ACh fully dilated the small arteries in a concentration-dependent manner. This response was relatively insensitive to the combination of barium and ouabain, and insensitive to capsaicin. Raising extracellular potassium produced a more inconsistent and modest vasodilator response in pressurised small mesenteric arteries. Responses to raising extracellular potassium were sensitive to capsaicin, and the combination of barium and ouabain. ACh caused a substantial hyperpolarisation in pressurized arteries, while raising extracellular potassium did not. These data indicate that K+ is not the EDHF released in response to ACh in myogenically active rat mesenteric small arteries. Since the hyperpolarization produced by ACh was sensitive to carbenoxolone, gap junctions are the likely mediator of EDH responses under physiological conditions.

Human lymphatic vessel contractile activity is inhibited in vitro but not in vivo by the calcium channel blocker nifedipine.

Calcium channel blockers (CCB) are widely prescribed anti-hypertensive agents. The commonest side-effect, peripheral oedema, is attributed to a larger arterial than venous dilatation causing increased fluid filtration. Whether CCB treatment is detrimental to human lymphatic vessel function and thereby exacerbates oedema formation is unknown. We observed that spontaneous lymphatic contractions in isolated human vessels (thoracic duct and mesenteric lymphatics) maintained under isometric conditions were inhibited by therapeutic concentrations (nanomolar) of the CCB nifedipine while higher than therapeutic concentrations of verapamil (micromolar) were necessary to inhibit activity. Nifedipine also inhibited spontaneous action potentials measured by sharp microelectrodes. Furthermore, noradrenaline did not elicit normal increases in lymphatic vessel tone when maximal constriction was reduced to 29.4±4.9% of control in the presence of 20nmoll(-1) nifedipine. Transcripts for the L-type calcium channel gene CACNA1C were consistently detected from human thoracic duct samples examined and the CaV1.2 protein was localized by immunoreactivity to lymphatic smooth muscle cells. While human lymphatics ex vivo were highly sensitive to nifedipine, this was not apparent in vivo when nifedipine was compared to placebo in a randomized, double-blinded clinical trial: conversely, lymphatic vessel contraction frequency was increased and refill time was faster despite all subjects achieving target nifedipine plasma concentrations. We conclude that human lymphatic vessels are highly sensitive to nifedipine in vitro but that care must be taken when extrapolating in vitro observations of lymphatic vessel function to the clinical situation, as similar changes in lymphatic function were not evident in our clinical trial comparing nifedipine treatment to placebo.

Application of active electrode compensation to perform continuous voltage-clamp recordings with sharp microelectrodes

Objective. Electrophysiological recordings of single neurons in brain tissues are very common in neuroscience. Glass microelectrodes filled with an electrolyte are used to impale the cell membrane in order to record the membrane potential or to inject current. Their high resistance induces a high voltage drop when passing current and it is essential to correct the voltage measurements. In particular, for voltage clamping, the traditional alternatives are two-electrode voltage-clamp technique or discontinuous single electrode voltage-clamp (dSEVC). Nevertheless, it is generally difficult to impale two electrodes in a same neuron and the switching frequency is limited to low frequencies in the case of dSEVC. We present a novel fully computer-implemented alternative to perform continuous voltage-clamp recordings with a single sharp-electrode. Approach. To reach such voltage-clamp recordings, we combine an active electrode compensation algorithm (AEC) with a digital controller (AECVC). Main results. We applied two types of control-systems: a linear controller (proportional plus integrative controller) and a model-based controller (optimal control). We compared the performance of the two methods to dSEVC using a dynamic model cell and experiments in brain slices. Significance. The AECVC method provides an entirely digital method to perform continuous recording and smooth switching between voltage-clamp, current clamp or dynamic-clamp configurations without introducing artifacts.

Effects of proarrhythmic drugs on relaxation time and beating pattern in rat engineered heart tissue.

The assessment of proarrhythmic risks of drugs remains challenging. To evaluate the suitability of rat engineered heart tissue (EHT) for detecting proarrhythmic effects. We monitored drug effects on spontaneous contractile activity and, in selected cases, on action potentials (sharp microelectrode) and Ca2+ transients (Fura-2) and contraction under electrical pacing. The Ito-blocker inhibitor 4-aminopyridine increased action potential duration and T2 and caused aftercontractions, which were abolished by inhibitors of ryanodine receptors (RyR2; JTV-519) or sodium calcium exchanger (NCX; SEA0400). 77 Drugs were then tested at 1-10-100× free therapeutic plasma concentrations (FTPC): Inhibitors of IKr, IKs, Ito, antiarrhythmics (8), drugs withdrawn from market for torsades des pointes arrhythmias (TdP, 5), drugs with measurable (7) or isolated TdP incidence (13), drugs considered safe (14), 28 new chemical entities (NCE). Inhibitors of IKr or IKs had no effect alone, but substantially prolonged relaxation time (T2) when combined at high concentration. 15/33 drugs associated with TdP and 6/14 drugs considered non-torsadogenic (cibenzoline, diltiazem, ebastine, ketoconazole, moxifloxacin, and phenytoin) induced concentration-dependent T2 prolongations (10-100× FTPC). Bepridil, desipramine, imipramine, thioridazine, and erythromycin induced irregular beating. Three NCE prolonged T2, one reduced force. Drugs inhibiting repolarization prolong relaxation in rat EHTs and cause aftercontractions involving RyR2 and NCX. Insensitivity to IKr inhibitors makes rat EHTs unsuitable as general proarrhythmia screen, but favors detection of effects on Ito, IKs + Ito or IKs + IKr. Screening a large panel of drugs suggests that effects on these currents, in addition to IKr, are more common than anticipated.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-014-0436-7) contains supplementary material, which is available to authorized users.

Asynchronous activation of calcium and potassium currents by isoproterenol in canine ventricular myocytes

Adrenergic activation of L-type Ca(2+) and various K(+) currents is a crucial mechanism of cardiac adaptation; however, it may carry a substantial proarrhythmic risk as well. The aim of the present work was to study the timing of activation of Ca(2+) and K(+) currents in isolated canine ventricular cells in response to exposure to isoproterenol (ISO). Whole cell configuration of the patch-clamp technique in either conventional voltage clamp or action potential voltage clamp modes were used to monitor I(Ca), I(Ks), and I(Kr), while action potentials were recorded using sharp microelectrodes. ISO (10 nM) elevated the plateau potential and shortened action potential duration (APD) in subepicardial and mid-myocardial cells, which effects were associated with multifold enhancement of I(Ca) and I(Ks) and moderate stimulation of I(Kr). The ISO-induced plateau shift and I(Ca) increase developed faster than the shortening of APD and stimulation of I(Ks) and I(Kr). Blockade of β1-adrenoceptors (using 300 nM CGP-20712A) converted the ISO-induced shortening of APD to lengthening, decreased its latency, and reduced the plateau shift. In contrast, blockade of β2-adrenoceptors (by 50 nM ICI 118,551) augmented the APD-shortening effect and increased the latency of plateau shift without altering its magnitude. All effects of ISO were prevented by simultaneous blockade of both receptor types. Inhibition of phosphodiesterases decreased the differences observed in the turn on of the ISO-induced plateau shift and APD shortening. ISO-induced activation of I(Ca) is turned on faster than the stimulation of I(Ks) and I(Kr) in canine ventricular cells due to the involvement of different adrenergic pathways and compartmentalization.

Nitrergic and purinergic mechanisms evoke inhibitory neuromuscular transmission in the human small intestine

Inhibitory neuromuscular transmission in the human colon is due to nitrergic and purinergic (P2Y1 -mediated) inputs. The aim of this study was to determine the mechanisms of neuromuscular transmission in different regions of the human small intestine. Ileal (n = 6) and jejunal (n = 6) samples underwent histological examination and were studied using sharp microelectrodes in smooth muscle cells and conventional muscle bath techniques. Electrical field stimulation (EFS) was used to stimulate inhibitory neurons. No histological abnormalities were found. Resting membrane potential was -39.7 ± 1.5 and -45.5 ± 2.1 mV in the jejunum and ileum, respectively. Slow waves and spontaneous contractions were recorded at a frequency of about 8-9 and 6-7 cpm in the jejunum and ileum, respectively. In non-adrenergic, non-cholinergic conditions, EFS caused an inhibitory junction potential and mechanical relaxation. Both responses were blocked by tissue incubation with the nitric oxide synthase inhibitor (Nω-nitro-l-arginine 1 mM) and the P2Y1 receptor blocker 2'-deoxy-N(6) -methyladenosine 3',5'-bisphosphate tetrasodium salt (MRS2179; 10 μM). Both exogenous addition of sodium nitroprusside (1 μM) and the preferential P2Y1 receptor agonist ADPβS (1 μM) hyperpolarized and relaxed smooth muscle cells. MRS2179 (10 μM) blocked ADPβS-induced responses. Similar to colon, inhibitory neurotransmission in the human small intestine is mainly mediated by purinergic (via P2Y1 receptors) and nitrergic inhibitory neurotransmission. Similar mechanisms of inhibitory neurotransmission are present in different regions of the human intestine.

Abstract 18323: Positive Frequency-Dependent Effects of Highly Selective Kv1.5 Blockers (XEN-D0103 & MK-0448) in Right Atrial Trabeculae From Patients in Sinus Rhythm

Aim: Despite promising effects of Kv1.5 blockers in experimental models of atrial fibrillation, limited data is available for man. Therefore we investigated two selective Kv1.5 channel blockers, XEN-D0103 and MK-0448, on atrial myocardium from patients in sinus rhythm (SR). Method: Kv1.5 currents were measured with in CHO cells expressing hKv1.5. Sharp microelectrodes were used to record action potentials (AP) at various stimulation frequencies (37°C) in right atrial trabeculae from consenting patients in SR. Results: Kv1.5 was activated by voltage-clamp steps from -80 to 0mV (900ms)at 0.2Hz (room temp). XEN-D0103 and MK-0448 inhibited Kv1.5 with IC50 of 25nM and 32nM resp.. Both compounds display >500-fold selectivity over hERG, Kv4.3, Nav1.5, Cav1.2, Kir2.1. The plateau phase of the atrial AP was elevated with XEN-D0103 and MK-0448 in a concentration-dependent manner. At 1Hz this effect was associated with shortening of APD90 and of effective refractory period (ERP) (see Table). At higher stimulation rates the APD90 and ERP shortening effects of XEN-D0103 and MK-0448 (3μM each) diminished and eventually converted to prolongation. With XEN-D0103, significant prolongation of APD90/ERP occurred at 3Hz and higher rates. In control, 10 out of 16 trabeculae followed stimulation at 5Hz, however, in the presence of XEN-D0103 10 out of 10, and with MK-0448 4 out of 6 preparations failed to capture stimulation. Conclusion: This is the first report of robust frequency dependence of selective Kv1.5 channel blockers in human atrial myocardium suggesting that these drugs are more effective at high than at low activation rates. Therefore, XEN-D0103 and MK-0448 should possess potential for suppressing episodes of rapid atrial excitation. Table: Frequency-dependent effects of the Kv1.5 blockers XEN-D0103 and MK-0448 in human right atrial trabeculae. Differences between pre-drug controls and values after 1 hour of drug superfusion (means ± SEM).

Electrophysiologic Characteristics and Pharmacologic Response of Human Cardiomyocytes Isolated from a Patient with Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most common monogenic cardiac disorder encountered in the clinic. Data relative to the electrophysiologic characteristics and pharmacologic responsiveness of human tissues and cells isolated from patients with HCM are rare. As a consequence, cellular mechanisms underlying arrhythmogenicity are poorly understood. Cardiomyocytes were enzymatically dissociated from a septal myectomy surgically removed from a patient with obstructive HCM. Sharp microelectrodes and patch-clamp techniques were used to evaluate action potential and sodium channel current (INa ) characteristics. Action potential morphology recorded was typical of an M cell, but with a longer than normal duration (APD) and a relatively steep APD-rate relationship. APD at all rates was significantly reduced following exposure to ranolazine (10 μM). Whole cell patch-clamp recording yielded robust peak INa and large late INa (1.1% of peak INa vs 0.1-0.2% in healthy controls). A large window current was observed as well. Ranolazine (10 μM) shifted steady-state V0.5 of inactivation by -8 mV, reduced late INa by 82%, and significantly diminished the window current. Our results indicate the presence of cells with M-cell characteristics in the septum of the human heart, as has previously been described in the canine heart. They also point to an ameliorative effect of ranolazine to reduce augmented late INa and thus to reduce the prolonged APD in the setting of HCM. These results suggest a potential therapeutic role for ranolazine in HCM.

TNF-α provokes electrical abnormalities in rat atrial myocardium via a NO-dependent mechanism

Stretch-induced depolarizations of cardiomyocytes, which are related to activity of mechano-gated cation channels (MGCs), can lead to serious arrhythmias. However, signaling pathways leading to activation of mechano-gated channels by stretch remain almost unexplored. Using standard sharp microelectrodes, the present study addresses the hypothesis that tumor necrosis factor-alpha (TNF-α) modulates stretch-induced electrophysiological abnormalities in rat atrial myocardium by a mechanism involving nitric oxide (NO)-dependent pathways. TNF-α (50 ng/ml) produced a marked prolongation of action potential, subsequently transforming into humplike depolarizations and, finally, leading to occurrence of arrhythmias. These effects developed slowly during 25 min of TNF-α application. Similar electrical effects were induced by stretching the preparations. A blocker of MGCs, Gd(3+) (40 μM), completely abolished action potential (AP) prolongations and electrical abnormalities caused by TNF-α or stretch. Further, a donor of exogenous NO, S-nitroso-N-acetylpenicillamine SNAP (300 μM), evoked the same electrical abnormalities as TNF-α and tissue stretch. Both TNF-α and stretch failed to produce their typical effects after pretreatment of the preparations with the NO-synthase inhibitor L-N(G)-nitroarginine methyl ester (L-NAME) (100 μM). Thus, the present study shows (i) that TNF-α and the NO-donor SNAP evoke MGC-mediated electrical abnormalities in rat atrial myocardium in the absence of stretch that is very similar to stretch-evoked electrical events and (ii) that the TNF-α-induced electrical abnormalities are mediated by NO synthase. In conclusion, our data suggest that NO is an endogenous modulator of MGCs and mediates proarrhythmic effects of TNF-α in mammalian organism.

Five types of nonspiking interneurons in local pattern-generating circuits of the crayfish swimmeret system

We conducted a quantitative analysis of the different nonspiking interneurons in the local pattern-generating circuits of the crayfish swimmeret system. Within each local circuit, these interneurons control the firing of the power-stroke and return-stroke motor neurons that drive swimmeret movements. Fifty-four of these interneurons were identified during physiological experiments with sharp microelectrodes and filled with dextran Texas red, Neurobiotin, or both. Five types of neurons were identified on the basis of combinations of physiological and anatomical characteristics. Anatomical categories were based on 16 anatomical parameters measured from stacks of confocal images obtained from each neuron. The results support the recognition of two functional classes: inhibitors of power stroke (IPS) and inhibitors of return stroke (IRS). The IPS class of interneuron has three morphological types with similar physiological properties. The IRS class has two morphological types with physiological properties and anatomical features different from the IPS neurons but similar within the class. Three of these five types have not been previously identified. Reviewing the evidence for dye coupling within each type, we conclude that each type of IPS neuron and one type of IRS neuron occur as a single copy in each local pattern-generating circuit. The last IRS type includes neurons that might occur as a dye-coupled pair in each local circuit. Recognition of these different interneurons in the swimmeret pattern-generating circuits leads to a refined model of the local pattern-generating circuit that includes synaptic connections that encode and decode information required for intersegmental coordination of swimmeret movements.

Identification and characterization of a transient outward K+ current in human induced pluripotent stem cell-derived cardiomyocytes

BackgroundThe ability to recapitulate mature adult phenotypes is critical to the development of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) as models of disease. The present study examines the characteristics of the transient outward current (Ito) and its contribution to the hiPSC-CM action potential (AP). MethodEmbryoid bodies were made from a hiPS cell line reprogrammed with Oct4, Nanog, Lin28 and Sox2. Sharp microelectrodes were used to record APs from beating-clusters (BC) and patch-clamp techniques were used to record Ito in single hiPSC-CM. mRNA levels of Kv1.4, KChIP2 and Kv4.3 were quantified from BCs. ResultsBCs exhibited spontaneous beating (60.5±2.6bpm) and maximum-diastolic-potential (MDP) of 67.8±0.8mV (n=155). A small 4-aminopyridine-sensitive phase-1-repolarization was observed in only 6/155 BCs. A robust Ito was recorded in the majority of cells (13.7±1.9 pA/pF at +40mV; n=14). Recovery of Ito from inactivation (at −80mV) showed slow kinetics (τ1=200±110ms (12%) and τ2=2380±240ms (80%)) accounting for its minimal contribution to the AP. Transcript data revealed relatively high expression of Kv1.4 and low expression of KChIP2 compared to human native ventricular tissues. Mathematical modeling predicted that restoration of IK1 to normal levels would result in a more negative MDP and a prominent phase-1-repolarization. ConclusionThe slow recovery kinetics of Ito coupled with a depolarized MDP account for the lack of an AP notch in the majority of hiPSC-CM. These characteristics reveal a deficiency for the development of in vitro models of inherited cardiac arrhythmia syndromes in which Ito-induced AP notch is central to the disease phenotype.

Hydrogen sulfide dilates rat mesenteric arteries by activating endothelial large-conductance Ca2+-activated K+ channels and smooth muscle Ca2+ sparks

We have previously shown that hydrogen sulfide (H₂S) reduces myogenic tone and causes relaxation of phenylephrine (PE)-constricted mesenteric arteries. This effect of H₂S to cause vasodilation and vascular smooth muscle cell (VSMC) hyperpolarization was mediated by large-conductance Ca(2+)-activated potassium channels (BKCa). Ca(2+) sparks are ryanodine receptor (RyR)-mediated Ca(2+)-release events that activate BKCa channels in VSMCs to cause membrane hyperpolarization and vasodilation. We hypothesized that H₂S activates Ca(2+) sparks in small mesenteric arteries. Ca(2+) sparks were measured using confocal microscopy in rat mesenteric arteries loaded with the Ca(2+) indicator fluo-4. VSMC membrane potential (Em) was measured in isolated arteries using sharp microelectrodes. In PE-constricted arteries, the H₂S donor NaHS caused vasodilation that was inhibited by ryanodine (RyR blocker), abluminal or luminal iberiotoxin (IbTx, BKCa blocker), endothelial cell (EC) disruption, and sulfaphenazole [cytochrome P-450 2C (Cyp2C) inhibitor]. The H₂S donor NaHS (10 μmol/l) increased Ca(2+) sparks but only in the presence of intact EC and this was blocked by sulfaphenazole or luminal IbTx. Inhibiting cystathionine γ-lyase (CSE)-derived H2S with β-cyano-l-alanine (BCA) also reduced VSMC Ca(2+) spark frequency in mesenteric arteries, as did EC disruption. However, excess CSE substrate homocysteine did not affect spark activity. NaHS hyperpolarized VSMC Em in PE-depolarized mesenteric arteries with intact EC and also hyperpolarized EC Em in arteries cut open to expose the lumen. This hyperpolarization was prevented by ryanodine, sulfaphenazole, and abluminal or luminal IbTx. BCA reduced IbTx-sensitive K(+) currents in freshly dispersed mesenteric ECs. These results suggest that H₂S increases Ca(2+) spark activity in mesenteric artery VSMC through activation of endothelial BKCa channels and Cyp2C, a novel vasodilatory pathway for this emerging signaling molecule.

Influence of pressure on adenosine triphosphate function as a sympathetic neurotransmitter in small mesenteric arteries from the spontaneously hypertensive rat

Enhanced sympathetic neurotransmission contributes to hypertension in the spontaneously hypertensive rat (SHR). We recently reported a method for studying sympathetic neurotransmission in pressurized small arteries, demonstrating a major role of adenosine triphosphate (ATP) as a sympathetic neurotransmitter under these physiological conditions. We have now used this methodology to assess the role of ATP as a sympathetic neurotransmitter in small mesenteric arteries isolated from SHRs. Small arteries were mounted in a suction electrode, cannulated and pressurized to either 30 or 90 mmHg. Nerve-evoked alterations in membrane potential were assessed using sharp microelectrodes. Neurally evoked vasoconstrictor responses were measured in the absence and presence of the α1-adrenoceptor antagonist, tamsulosin (0.1 μmol/l), or the P2 purinoceptor antagonist suramin (0.1 mmol/l). At 30 mmHg the P2X-receptor-mediated excitatory junctional potential (EJP) was larger in arteries from SHRs (7.9 ± 0.9 mV) than Wistar-Kyoto (WKY) rats (3.2 ± 0.4 mV, P < 0.05). Increasing pressure increased the amplitude of the EJP, which again, was larger in SHRs. At 90 mmHg, activation of the perivascular nerves produced a larger vasoconstriction in arteries isolated from SHRs compared with WKY rats. The vasoconstrictor response in SHRs was abolished by either suramin or tamsulosin. These data provide electrophysiological evidence for enhanced purinergic function in the SHR and show that ATP is fundamentally important in contributing to the vasoconstriction produced after activation of the perivascular nerves in pressurized arteries from the SHR. This involves a synergistic interaction with noradrenaline to causes enhanced mesenteric arterial vasoconstriction, which may contribute to the hypertension in this model.

Characterization of a Transient Outward K+ Current in Hips-Derived Cardiomyocytes

Background: Human induced pluripotent stem cell (hiPS)-cardiomyocytes can be used to create in vitro models of genetic disease such as Brugada Syndrome (BrS). Central to the development of BrS is the Ca2+-independent transient outward K+ current (Ito). In this study, we characterized Ito in single hiPS -cardiomyocytes and determined its functional role in beating clusters.Methods: Embryoid bodies (EBs) were made from a hiPS cell line reprogrammed with Oct4, Nanog, Lin28 and Sox2. Whole cell patch clamp was used to record Ito in single hiPS cardiomyocytes. Action potential (AP) recordings from spontaneously beating clusters (BCs) were made using sharp microelectrodes. All recordings were done at 36°C.Results: BCs exhibited spontaneous APs with an average rate of 54.9±30.1 bpm and maximum diastolic potential (MDP) of −65.6±9.3 mV (n=122). A small phase 1 repolarization which could be blocked by 4-AP (1 mM) was observed in 6/149 hiPS BCs suggesting the presence of Ito. Interestingly, in single dissociated hiPS cardiomyocytes, patch clamp analysis revealed a robust Ito (13.4±1.79 pA/pF at +40 mV, n=14) in the majority of cells studied. Recovery of Ito (at −80 mV) showed a fast and slow phase as follows: i) 1=271±93 ms and 2= 2697±103 (n=8 cells). These observations demonstrate that Ito is present but the slow recovery suggests minimal contribution during the course of an action potential. Mathematical modeling of APs from hiPSC-CMs confirmed these observations.Conclusion: There is a disconnect between the presence of Ito in cells and the absence of phase 1 repolarization in BCs. In BCs the depolarized MDP and fast spontaneous AP rate suggests negligible contribution of Ito to phase 1 repolarization. Our results point to an important deficiency of hiPSC-CMs in recapitulating the phenotype of adult native myocytes.

The microbiome is essential for normal gut intrinsic primary afferent neuron excitability in the mouse

The role of intestinal microbiota in the development and function of host physiology is of high interest, especially with respect to the nervous system. While strong evidence has accrued that intestinal bacteria alter host nervous system function, mechanisms by which this occurs have remained elusive. For this reason, we have carried out experiments examining the electrophysiological properties of neurons in the myenteric plexus of the enteric nervous system (ENS) in germ-free (GF) mice compared with specific pathogen-free (SPF) control mice and adult germ-free mice that have been conventionalized (CONV-GF) with intestinal bacteria. Segments of jejunum from 8 to 12 week old GF, SPF, and CONV-GF mice were dissected to expose the myenteric plexus. Intracellular recordings in current-clamp mode were made by impaling cells with sharp microelectrodes. Action potential (AP) shapes, firing thresholds, the number of APs fired at 2× threshold, and passive membrane characteristics were measured. In GF mice, excitability was decreased in myenteric afterhyperpolarization (AH) neurons as measured by a lower resting membrane potential and by the number of APs generated at 2× threshold. The post AP slow afterhyperpolarization (sAHP) was prolonged for GF compared with SPF and CONV-GF animals. Passive membrane characteristics were also altered in GF mice by a decrease in input resistance. Here, we report the novel finding that commensal intestinal microbiota are necessary for normal excitability of gut sensory neurons and thus provide a potential mechanism for the transfer of information between the microbiota and nervous system.