Activation of PKC affects the ventricular restitution properties and arrhythmogenesis through L-type Ca+ current.

OBJECTIVE: To investigate the role of protein kinase C (PKC) in action potential duration (APD) restitution and ventricular tachyarrhythmias (VAs). METHODS AND RESULTS: Rabbits hearts were isolated and prepared for Langendorff perfusion technique. The stimuli-extra-stimulus (S1-S2) method and dynamic S1 pacing protocol were performed to construct APD restitution and to induce APD alternans or VA, respectively, at 10 sites throughout the ventricular chamber. Administration of phorbol-12-myristate-13-acetate (PMA) (100 nM) (n = 15) greatly steepened the restitution curves (Smax > 1) (p < .01) at each site compared to the control group (n = 15). Furthermore, treatment with PMA also induced larger spatial dispersions of Smax (p < .05) and decreased the thresholds of the VA and APD alternans (p < .01). However, perfused with the PKC inhibitor, bisindolylmaleimide (BIM) (500 nM) (n = 10), reversibly flattened the APD restitution curves at each site (Smax < 1), decreased the spatial dispersions of Smax, and increased the thresholds of APD alternans and VA. According to the results of patch-clamp, peak amplitude of L-type Ca2+ current was significantly increased by addition of PMA compared with control (CTL) group (p < .05). Antagonize this current with verapamil (n = 10) can fully inhibited the PMA induced increasing of Smax and inducibility of VA and alternans. CONCLUSION: PKC activation increased the dispersion of APD restitution and thus led to occurrence of VA, which possibly related to the increased Ca2+ influx.

Tricyclic hydrocarbon fluorene attenuates ventricular ionic currents and pressure development in the navaga cod.

The release of polycyclic aromatic hydrocarbons (PAHs) into the environment due to oil and diesel fuel spills is a serious threat to Arctic fish populations. PAHs produce multiple toxic effects in fish, but disturbance of electrical and contractile activity of the heart seems to be the most negative effect. Our study focused on the effects of fluorene, a tricyclic PAH resembling the well-investigated tricyclic phenanthrene, on major ionic currents and action potential (AP) waveform in isolated ventricular myocytes and on contractile activity in isolated whole hearts of polar navaga cod (Eleginus nawaga). Among the studied currents, the repolarizing rapid delayed rectifier K+ current IKr demonstrated the highest sensitivity to fluorene with IC50 of 0.54 µM. The depolarizing inward currents, INa and ICaL, were inhibited with 10 µM fluorene by 20.2 ± 2.8 % and 27.9 ± 8.4 %, respectively, thereby being much less sensitive to fluorene than IKr. Inward rectifier IK1 current was insensitive to fluorene (up to 10 µM). While 3 µM fluorene prolonged APs, 10 µM also slowed the AP upstroke. Resting membrane potential was not affected by any tested concentrations. In isolated heart experiments 10 µM fluorene caused modest depression of ventricular contractile activity. Thus, we have demonstrated that fluorene, a tricyclic PAH present in high quantities in crude oil, strongly impacts electrical activity with only slight effects on contractile activity in the heart of the polar fish, the navaga cod.

Increased in vivo perpetuation of whole-heart ventricular arrhythmia in heterozygous Na+/Ca2+ exchanger knockout mice.

Aims: Na+/Ca2+ exchanger (NCX) upregulation in cardiac diseases like heart failure promotes as an independent proarrhythmic factor early and delayed afterdepolarizations (EADs/DADs) on the single cell level. Consequently, NCX inhibition protects against EADs and DADs in isolated cardiomyocytes. We here investigate, whether these promising cellular in vitro findings likewise apply to an in vivo setup. Methods/Results: Programmed ventricular stimulation (PVS) and isoproterenol were applied to a murine heterozygous NCX-knockout model (KO) to investigate ventricular arrhythmia initiation and perpetuation compared to wild-type (WT). KO displayed a reduced susceptibility towards isoproterenol-induced premature ventricular complexes. During PVS, initiation of single or double ectopic beats was similar between KO and WT. But strikingly, perpetuation of ventricular tachycardia (VT) was significantly increased in KO (animals with VT - KO: 82 %; WT: 47 %; p = 0.0122 / median number of VTs - KO: 4.5 (1.0, 6.25); WT: 0.0 (0.0, 4.0); p = 0.0039). The median VT duration was prolonged in KO (in s; KO: 0.38 (0.19, 0.96); WT: 0.0 (0.0, 0.60); p = 0.0239). The ventricular refractory period (VRP) was shortened in KO (in ms; KO: 15.1 ± 0.7; WT: 18.7 ± 0.7; p = 0.0013). Conclusions: Not the initiation, but the perpetuation of provoked whole-heart in vivo ventricular arrhythmia was increased in KO. As a potential mechanism, we found a significantly reduced VRP, which may promote perpetuation of reentrant ventricular arrhythmia. On a translational perspective, the antiarrhythmic concept of therapeutic NCX inhibition seems to be ambivalent by protecting from initiating afterdepolarizations but favoring arrhythmia perpetuation in vivo at least in a murine model.

Acetylcholine Reduces L-Type Calcium Current without Major Changes in Repolarization of Canine and Human Purkinje and Ventricular Tissue.

Vagal nerve stimulation (VNS) holds a strong basis as a potentially effective treatment modality for chronic heart failure, which explains why a multicenter VNS study in heart failure with reduced ejection fraction is ongoing. However, more detailed information is required on the effect of acetylcholine (ACh) on repolarization in Purkinje and ventricular cardiac preparations to identify the advantages, risks, and underlying cellular mechanisms of VNS. Here, we studied the effect of ACh on the action potential (AP) of canine Purkinje fibers (PFs) and several human ventricular preparations. In addition, we characterized the effects of ACh on the L-type Ca2+ current (ICaL) and AP of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and performed computer simulations to explain the observed effects. Using microelectrode recordings, we found a small but significant AP prolongation in canine PFs. In the human myocardium, ACh slightly prolonged the AP in the midmyocardium but resulted in minor AP shortening in subepicardial tissue. Perforated patch-clamp experiments on hiPSC-CMs demonstrated that 5 µM ACh caused an ≈15% decrease in ICaL density without changes in gating properties. Using dynamic clamp, we found that under blocked K+ currents, 5 µM ACh resulted in an ≈23% decrease in AP duration at 90% of repolarization in hiPSC-CMs. Computer simulations using the O'Hara-Rudy human ventricular cell model revealed that the overall effect of ACh on AP duration is a tight interplay between the ACh-induced reduction in ICaL and ACh-induced changes in K+ currents. In conclusion, ACh results in minor changes in AP repolarization and duration of canine PFs and human ventricular myocardium due to the concomitant inhibition of inward ICaL and outward K+ currents, which limits changes in net repolarizing current and thus prevents major changes in AP repolarization.

T-type Ca2+ channels play a dual role in modulating the excitability of dorsal root ganglia neurons.

A subgroup of low-threshold dorsal root ganglia (DRG) neurons discharge action potentials (APs) with an afterdepolarizing potential (ADP). The ADP is formed by T-type Ca2+ currents. It is known that T-type Ca2+ currents contribute to neuropathic pain. However, the change in ADP-firing of injured DRG neurons has not been widely studied yet. Here we applied patch clamp to record ADP-firing and T-type Ca2+ currents in intact and chronically compressed DRG (CCD) neurons and examined T-type Ca2+ channel proteins expression with western blotting. After CCD injury, the incidences of both ADP firing and non-ADP burst firing increased, and T-type Ca2+ channels contributed to both of these firing patterns. The neurons discharging large-amplitude-ADP firing were TTX-insensitive, implying that high-density T-type Ca2+ channels might cooperate with TTX-insensitive Na+ channels to reduce the AP threshold. By contrast, the neurons displaying non-ADP burst firing were TTX-sensitive, implying that low density T-type Ca2+ channels may cooperate with TTX-sensitive Na+ channels to increase AP number. In DRG neurons, T-type Ca2+ currents density varied widely, ranging between 100 pA/pF and 5 pA/pF. After injury, the proportion of DRG neurons with large T-type Ca2+ currents increased in parallel with the increase in the incidence of large-amplitude-ADP firing. And in addition to Cav3.2, Cav3.3 channels are also likely to contribute to low-threshold firing. The data revealed that T-type Ca2+ channels may play a dual role in modulating the injured neurons' high excitability through a cooperative process with Na+ channels, thereby contributing to neuropathic pain.

Higher Na+-Ca2+ Exchanger Function and Triggered Activity Contribute to Male Predisposition to Atrial Fibrillation.

Male sex is one of the most important risk factors of atrial fibrillation (AF), with the incidence in men being almost double that in women. However, the reasons for this sex difference are unknown. Accordingly, in this study, we sought to determine whether there are sex differences in intracellular Ca2+ homeostasis in mouse atrial myocytes that might help explain male predisposition to AF. AF susceptibility was assessed in male (M) and female (F) mice (4-5 months old) using programmed electrical stimulation (EPS) protocols. Males were 50% more likely to develop AF. The Ca2+ transient amplitude was 28% higher in male atrial myocytes. Spontaneous systolic and diastolic Ca2+ releases, which are known sources of triggered activity, were significantly more frequent in males than females. The time to 90% decay of Ca2+ transient was faster in males. Males had 54% higher Na+-Ca2+ exchanger (NCX1) current density, and its expression was also more abundant. L-type Ca2+ current (ICaL) was recorded with and without BAPTA, a Ca2+ chelator. ICaL density was lower in males only in the absence of BAPTA, suggesting stronger Ca2+-dependent inactivation in males. CaV1.2 expression was similar between sexes. This study reports major sex differences in Ca2+ homeostasis in mouse atria, with larger Ca2+ transients and enhanced NCX1 function and expression in males resulting in more spontaneous Ca2+ releases. These sex differences may contribute to male susceptibility to AF by promoting triggered activity.

5-HT3 Receptors in Rat Dorsal Root Ganglion Neurons: Ca2+ Entry and Modulation of Neurotransmitter Release.

Rat dorsal root ganglion (DRG) neurons express 5-hydroxytryptamine receptors (5-HT3Rs). To elucidate their physiological role in the modulation of sensory signaling, we aimed to quantify their functional expression in newborn and adult rat DRG neurons, as well as their ability to modulate the Ca2+-dependent neurotransmitter release, by means of electrophysiological techniques combined with fluorescence-based Ca2+ imaging. The selective 5-HT3R agonist mCPBG (10 µM) elicited whole-cell currents in 92.5% of adult DRG neurons with a significantly higher density current than in responding newborn cells (52.2%), suggesting an increasing serotoninergic modulation on primary afferent cells during development. Briefly, 5-HT3Rs expressed by adult DRG neurons are permeable to Ca2+ ions, with a measured fractional Ca2+ current (i.e., the percentage of total current carried by Ca2+ ions, Pf) of 1.0%, similar to the value measured for the human heteromeric 5-HT3A/B receptor (Pf = 1.1%), but lower than that of the human homomeric 5-HT3A receptor (Pf = 3.5%). mCPBG applied to co-cultures of newborn DRG and spinal neurons significantly increased the miniature excitatory postsynaptic currents (mEPSCs) frequency in a subset of recorded spinal neurons, even in the presence of Cd2+, a voltage-activated Ca2+ channel blocker. Considered together, our findings indicate that the Ca2+ influx through heteromeric 5-HT3Rs is sufficient to increase the spontaneous neurotransmitter release from DRG to spinal neurons.

Effective Perturbations by Small-Molecule Modulators on Voltage-Dependent Hysteresis of Transmembrane Ionic Currents.

The non-linear voltage-dependent hysteresis (Hys(V)) of voltage-gated ionic currents can be robustly activated by the isosceles-triangular ramp voltage (Vramp) through digital-to-analog conversion. Perturbations on this Hys(V) behavior play a role in regulating membrane excitability in different excitable cells. A variety of small molecules may influence the strength of Hys(V) in different types of ionic currents elicited by long-lasting triangular Vramp. Pirfenidone, an anti-fibrotic drug, decreased the magnitude of Ih's Hys(V) activated by triangular Vramp, while dexmedetomidine, an agonist of α2-adrenoceptors, effectively suppressed Ih as well as diminished the Hys(V) strength of Ih. Oxaliplatin, a platinum-based anti-neoplastic drug, was noted to enhance the Ih's Hys(V) strength, which is thought to be linked to the occurrence of neuropathic pain, while honokiol, a hydroxylated biphenyl compound, decreased Ih's Hys(V). Cell exposure to lutein, a xanthophyll carotenoid, resulted in a reduction of Ih's Hys(V) magnitude. Moreover, with cell exposure to UCL-2077, SM-102, isoplumbagin, or plumbagin, the Hys(V) strength of erg-mediated K+ current activated by triangular Vramp was effectively diminished, whereas the presence of either remdesivir or QO-58 respectively decreased or increased Hys(V) magnitude of M-type K+ current. Zingerone, a methoxyphenol, was found to attenuate Hys(V) (with low- and high-threshold loops) of L-type Ca2+ current induced by long-lasting triangular Vramp. The Hys(V) properties of persistent Na+ current (INa(P)) evoked by triangular Vramp were characterized by a figure-of-eight (i.e., ∞) configuration with two distinct loops (i.e., low- and high-threshold loops). The presence of either tefluthrin, a pyrethroid insecticide, or t-butyl hydroperoxide, an oxidant, enhanced the Hys(V) strength of INa(P). However, further addition of dapagliflozin can reverse their augmenting effects in the Hys(V) magnitude of the current. Furthermore, the addition of esaxerenone, mirogabalin, or dapagliflozin was effective in inhibiting the strength of INa(P). Taken together, the observed perturbations by these small-molecule modulators on Hys(V) strength in different types of ionic currents evoked during triangular Vramp are expected to influence the functional activities (e.g., electrical behaviors) of different excitable cells in vitro or in vivo.

Using Whole-Cell Electrophysiology and Patch-Clamp Photometry to Characterize P2X7 Receptor Currents.

The fundamental property of P2X7 receptor (P2X7R) channels is the transport of cations across the cell surface membrane. Electrophysiology and patch-clamp photometry are readily accessible methods of measuring this flux in a wide range of cell types. They are important tools used to characterize the functional properties of native cells studied in cell culture, in vitro tissue slices, and, in some cases, in situ single cells. Further, they are efficient methods of probing the relation of structure to function of recombinant receptors expressed in heterologous systems. Here, we provide step-by-step procedures for use of two standard recording protocols, broken-patch and perforated-patch voltage clamp. Further, we describe a third technique, called the dye-overload method, that uses simultaneous measurement of membrane current and fura-2 fluorescence to quantify the contribution of Ca2+ flux to the ATP-gated current.

Cation Permeability of Voltage-Gated Hair Cell Ca2+ Channels of the Vertebrate Labyrinth.

Some hearing, vestibular, and vision disorders are imputable to voltage-gated Ca2+ channels of the sensory cells. These channels convey a large Ca2+ influx despite extracellular Na+ being 70-fold more concentrated than Ca2+; such high selectivity is lost in low Ca2+, and Na+ can permeate. Since the permeation properties and molecular identity of sensory Ca2+ channels are debated, in this paper, we examine the Na+ current flowing through the L- and R-type Ca2+ channels of labyrinth hair cells. Ion currents and cytosolic free Ca2+ concentrations were simultaneously monitored in whole-cell recording synchronous to fast fluorescence imaging. L-type and R-type channels were present with different densities at selected sites. In 10 nM Ca2+, the activation and deactivation time constants of the L-type Na+ current were accelerated and its maximal amplitude increased by 6-fold compared to physiological Ca2+. The deactivation of the R-type Na+ current was not accelerated, and its current amplitude increased by 2.3-fold in low Ca2+; moreover, it was partially blocked by nifedipine in a voltage- and time-dependent manner. In conclusion, L channel gating is affected by the ion species permeating the channel, and its selectivity filter binds Ca2+ more strongly than that of R channel; furthermore, external Ca2+ prevents nifedipine from perturbing the R selectivity filter.

Zingerone Modulates Neuronal Voltage-Gated Na+ and L-Type Ca2+ Currents.

Zingerone (ZO), a nontoxic methoxyphenol, has been demonstrated to exert various important biological effects. However, its action on varying types of ionic currents and how they concert in neuronal cells remain incompletely understood. With the aid of patch clamp technology, we investigated the effects of ZO on the amplitude, gating, and hysteresis of plasmalemmal ionic currents from both pituitary tumor (GH3) cells and hippocampal (mHippoE-14) neurons. The exposure of the GH3 cells to ZO differentially diminished the peak and late components of the INa. Using a double ramp pulse, the amplitude of the INa(P) was measured, and the appearance of a hysteresis loop was observed. Moreover, ZO reversed the tefluthrin-mediated augmentation of the hysteretic strength of the INa(P) and led to a reduction in the ICa,L. As a double ramp pulse was applied, two types of voltage-dependent hysteresis loops were identified in the ICa,L, and the replacement with BaCl2-attenuated hysteresis of the ICa,L enhanced the ICa,L amplitude along with the current amplitude (i.e., the IBa). The hysteretic magnitude of the ICa,L activated by the double pulse was attenuated by ZO. The peak and late INa in the hippocampal mHippoE-14 neurons was also differentially inhibited by ZO. In addition to acting on the production of reactive oxygen species, ZO produced effects on multiple ionic currents demonstrated herein that, considered together, may significantly impact the functional activities of neuronal cells.

[8]-Gingerol exerts anti-myocardial ischemic effects in rats via modulation of the MAPK signaling pathway and L-type Ca2+ channels.

Myocardial ischemia (MI) remains the leading cause of mortality worldwide. Therefore, it is urgent to seek the treatment to protect the heart. [8]-Gingerol (8-Gin), one of the most active ingredients in ginger, has antioxidant, cardiotonic, and cardiovascular protective properties. The present study elucidated the cardioprotection effects and underlying mechanisms of 8-Gin in isoproterenol (ISO)-induced MI. ISO (85 mg/kg/d) was subcutaneously injected for 2 consecutive days to induce acute MI model in rats. Electrocardiography, oxidative stress levels, calcium concentrations, and apoptosis degree were observed. The effects of 8-Gin on L-type Ca2+ current (ICa-L ), contraction, and Ca2+ transients were monitored in rat myocytes via patch-clamp and IonOptix detection systems. 8-Gin decreased J-point elevation and heart rate and improved pathological heart damage. Moreover, 8-Gin reduced the levels of CK, LDH, and MDA, ROS production, and calcium concentrations in myocardial tissue, while increased the activities of SOD, CAT, and GSH. In addition, 8-Gin down-regulated Caspase-3 and Bax expressions, while up-regulated Bcl-2 expression. 8-Gin produced a marked decrease in the expression of p38, JNK, and ERK1/2 proteins. 8-Gin inhibited ICa-L , cell contraction, and Ca2+ transients in isolated rat myocytes. The results indicate that 8-Gin could exert anti-myocardial ischemic effects, which may be associated with oxidative stress reduction, cardiomyocytes apoptosis inhibition through MAPK signaling pathway, and Ca2+ homeostasis regulation via ICa-L modulation.

Voltage-dependent calcium channel ß subunit-derived peptides reduce excitatory neurotransmission and arterial blood pressure.

AIMS: Voltage-dependent calcium channels (VDCCs) play an important role in various physiological functions in the nervous system and the cardiovascular system. In L-, N-, P/Q-, and R-type VDCCs, ß subunit assists the channels for membrane targeting and modulates channel properties. In this study, we investigated whether an inhibition of the ß subunit binding to α subunit, the pore-forming main subunit of VDCCs, have any effect on channel activation and physiological functions. MAIN METHODS: Peptides derived from the specific regions of ß subunit that bind to the α-interaction domain in I-II linker of α subunit were manufactured, presuming that the peptides interrupt α-ß subunit interaction in the channel complex. Then, they were tested on voltage-activated Ca2+ currents recorded in acutely isolated trigeminal ganglion (TG) neurons, excitatory postsynaptic currents (EPSCs) in the spinal dorsal horn neurons, and arterial blood pressure (BP) recorded from the rat femoral artery. KEY FINDINGS: When applied internally through patch pipettes, the peptides decreased the peak amplitudes of the voltage-activated Ca2+ currents. After fusing with HIV transactivator of transcription (TAT) sequence to penetrate cell membrane, the peptides significantly decreased the peak amplitudes of Ca2+ currents and the peak amplitudes of EPSCs upon the external application through bath solution. Furthermore, the TAT-fused peptides dose dependently reduced the rat BP when administered intravenously. SIGNIFICANCE: These data suggest that an interruption of α-ß subunit association in VDCC complex inhibits channel activation, thereby reducing VDCC-mediated physiological functions such as excitatory neurotransmission and arterial BP.

Selective changes in cytosolic ß-adrenergic cAMP signals and L-type Calcium Channel regulation by Phosphodiesterases during cardiac hypertrophy.

Background In cardiomyocytes, phosphodiesterases (PDEs) type 3 and 4 are the predominant enzymes that degrade cAMP generated by ß-adrenergic receptors (ß-ARs), impacting notably the regulation of the L-type Ca2+ current (ICa,L). Cardiac hypertrophy (CH) is accompanied by a reduction in PDE3 and PDE4, however, whether this affects the dynamic regulation of cytosolic cAMP and ICa,L is not known. Methods and Results CH was induced in rats by thoracic aortic banding over a time period of five weeks and was confirmed by anatomical measurements. Left ventricular myocytes (LVMs) were isolated from CH and sham-operated (SHAM) rats and transduced with an adenovirus encoding a Förster resonance energy transfer (FRET)-based cAMP biosensor or subjected to the whole-cell configuration of the patch-clamp technique to measure ICa,L. Aortic stenosis resulted in a 46% increase in heart weight to body weight ratio in CH compared to SHAM. In SHAM and CH LVMs, a short isoprenaline stimulation (Iso, 100 nM, 15 s) elicited a similar transient increase in cAMP with a half decay time (t1/2off) of ~50 s. In both groups, PDE4 inhibition with Ro 20-1724 (10 µM) markedly potentiated the amplitude and slowed the decline of the cAMP transient, this latter effect being more pronounced in SHAM (t1/2off ~ 250 s) than in CH (t1/2off ~ 150 s, P < 0.01). In contrast, PDE3 inhibition with cilostamide (1 µM) had no effect on the amplitude of the cAMP transient and a minimal effect on its recovery in SHAM, whereas it potentiated the amplitude and slowed the decay in CH (t1/2off ~ 80 s). Iso pulse stimulation also elicited a similar transient increase in ICa,L in SHAM and CH, although the duration of the rising phase was delayed in CH. Inhibition of PDE3 or PDE4 potentiated ICa,L amplitude in SHAM but not in CH. Besides, while only PDE4 inhibition slowed down the decline of ICa,L in SHAM, both PDE3 and PDE4 contributed in CH. Conclusion These results identify selective alterations in cytosolic cAMP and ICa,L regulation by PDE3 and PDE4 in CH, and show that the balance between PDE3 and PDE4 for the regulation of ß-AR responses is shifted toward PDE3 during CH.

Warmer, faster, stronger: Ca2+ cycling in avian myocardium.

Birds occupy a unique position in the evolution of cardiac design. Their hearts are capable of cardiac performance on par with, or exceeding that of mammals, and yet the structure of their cardiomyocytes resembles those of reptiles. It has been suggested that birds use intracellular Ca2+ stored within the sarcoplasmic reticulum (SR) to power contractile function, but neither SR Ca2+ content nor the cross-talk between channels underlying Ca2+-induced Ca2+ release (CICR) have been studied in adult birds. Here we used voltage clamp to investigate the Ca2+ storage and refilling capacities of the SR and the degree of trans-sarcolemmal and intracellular Ca2+ channel interplay in freshly isolated atrial and ventricular myocytes from the heart of the Japanese quail (Coturnix japonica). A trans-sarcolemmal Ca2+ current (ICa) was detectable in both quail atrial and ventricular myocytes, and was mediated only by L-type Ca2+ channels. The peak density of ICa was larger in ventricular cells than in atrial cells, and exceeded that reported for mammalian myocardium recorded under similar conditions. Steady-state SR Ca2+ content of quail myocardium was also larger than that reported for mammals, and reached 750.6±128.2 µmol l-1 in atrial cells and 423.3±47.2 µmol l-1 in ventricular cells at 24°C. We observed SR Ca2+-dependent inactivation of ICa in ventricular myocytes, indicating cross-talk between sarcolemmal Ca2+ channels and ryanodine receptors in the SR. However, this phenomenon was not observed in atrial myocytes. Taken together, these findings help to explain the high-efficiency avian myocyte excitation-contraction coupling with regard to their reptilian-like cellular ultrastructure.

A low voltage activated Ca2+ current found in a subset of human ventricular myocytes.

Low voltage activated (ICa-LVA) calcium currents including Cav1.3 and T-type calcium current (ICa-T) have not been reported in adult human left ventricular myocytes (HLVMs). We tried to examine their existence and possible correlation with etiology and patient characteristics in a big number of human LVMs isolated from explanted terminally failing (F) hearts, failing hearts with left ventricular assist device (F-LVAD) and nonfailing (NF) human hearts. LVA (ICa-LVA) was determined by subtracting L-type Ca2+ current (ICa-L) recorded with the holding potential of -50 mV from total Ca2+ current recorded with the holding potential of -90 mV or -70 mV. ICa- LVA was further tested with its sensitivity to 100 µM CdCl2 and tetrodotoxin. Three HLVMs (3 of 137 FHLVMs) from 2 (N = 30 hearts) failing human hearts, of which one was idiopathic and the other was due to primary pulmonary hypertension, were found with ICa-LVA. ICa-LVA in one FHLVM was not sensitive to 100 µM CdCl2 while ICa-LVA in another two FHLVMs was not sensitive to tetrodotoxin. It peaked at the voltage of -40~-20 mV and had a time-dependent decay faster than ICa-L but slower than sodium current (INa). ICa-LVA was not found in any HLVMs from NF (75 HLVMs from 17 hearts) or F-LVAD hearts (82 HLVMs from 18 hearts) but a statistically significant correlation could not be established. In conclusion, ICa-LVA was detected in some HLVMs of a small portion of human hearts that happened to be nonischemic failing hearts.

Effective block by pirfenidone, an antifibrotic pyridone compound (5-methyl-1-phenylpyridin-2[H-1]-one), on hyperpolarization-activated cation current: An additional but distinctive target.

Pirfenidone (PFD), a pyridone compound, is well recognized as an antifibrotic agent tailored for the treatment of idiopathic pulmonary fibrosis. Recently, through its anti-inflammatory and anti-oxidant effects, PFD based clinical trial has also been launched for the treatment of coronavirus disease (COVID-19). To what extent this drug can perturb membrane ion currents remains largely unknown. Herein, the exposure to PFD was observed to depress the amplitude of hyperpolarization-activated cation current (Ih) in combination with a considerable slowing in the activation time of the current in pituitary GH3 cells. In the continued presence of ivabradine or zatebradine, subsequent application of PFD decreased Ih amplitude further. The presence of PFD resulted in a leftward shift in Ih activation curve without changes in the gating charge. The addition of this compound also led to a reduction in area of voltage-dependent hysteresis evoked by long-lasting inverted triangular (downsloping and upsloping) ramp pulse. Neither the amplitude of M-type nor erg-mediated K+ current was altered by its presence. In whole-cell potential recordings, addition of PFD reduced the firing frequency, and this effect was accompanied by the depression in the amplitude of sag voltage elicited by hyperpolarizing current stimulus. Overall, this study highlights evidence that PFD is capable of perturbing specific ionic currents, revealing a potential additional impact on functional activities of different excitable cells.

Aqueous Fraction from Hibiscus sabdariffa Relaxes Mesenteric Arteries of Normotensive and Hypertensive Rats through Calcium Current Reduction and Possibly Potassium Channels Modulation.

BACKGROUND/OBJECTIVES: Hibiscus sabdariffa L. (H. sabdariffa (HS)) extract has a vascular relaxant effect on isolated rat thoracic aorta, but data on small resistance arteries, which play an important role on the development of hypertension, are still missing. The purposes of this study were (1) to assess the effect on isolated mesenteric arteries (MA) from normotensive (Wistar and Wistar-Kyoto (WKY)) and spontaneous hypertensive rats (SHR); (2) to elucidate the mechanism(s) of action underling the relaxant effect in light of bioactive components. METHODS: Vascular effects of HS aqueous fraction (AF) on isolated MA rings, as well as its mechanisms of action, were assessed using the contractility and intracellular microelectrode technique. The patch clamp technique was used to evaluate the effect of HS AF on the L-type calcium current. Extraction and enrichment of AF were carried out using liquid-liquid extraction, and the yield was analyzed using HPLC. RESULTS: The HS AF induced a concentration-dependent relaxant effect on MA rings of SHR (EC50 = 0.83 ± 0.08 mg/mL), WKY (EC50 = 0.46 ± 0.04 mg/mL), and Wistar rats (EC50 = 0.44 ± 0.08 mg/mL) pre-contracted with phenylephrine (10 µM). In Wistar rats, the HS AF maximum relaxant effect was not modified after endothelium removal or when a guanylate cyclase inhibitor (ODQ, 10 µM) and a selective ß2-adrenergic receptor antagonist (ICI-118551, 1 µM) were incubated with the preparation. Otherwise, it was reduced by 34.57 ± 10.66% when vascular rings were pre-contracted with an 80 mM [K+] solution (p < 0.001), which suggests an effect on ionic channels. HS AF 2 mg/mL significantly decreased the peak of the L-type calcium current observed in cardiac myocytes by 24.4%. Moreover, though the vasorelaxant effect of HS, AF was reduced by 27% when the nonselective potassium channels blocker (tetraethylammonium (TEA) 20 mM) was added to the bath (p < 0.01). The extract did not induce a membrane hyperpolarization of smooth muscle cells, which might suggest an absence of a direct effect on background potassium current. CONCLUSION: These results highlight that the antihypertensive effect of HS probably involves a vasorelaxant effect on small resistance arteries, which is endothelium independent. L-type calcium current reduction contributes to this effect. The results could also provide a link between the vasorelaxant effect and the bioactive compounds, especially anthocyanins.

Ion currents, action potentials, and noradrenergic responses in rat pulmonary vein and left atrial cardiomyocytes.

The electrophysiological properties of pulmonary vein (PV)-cardiomyocytes, and their responses to the sympathetic neurotransmitter, noradrenaline (NA), are thought to differ from those of the left atrium (LA) and contribute to atrial ectopy. The aim of this study was to examine rat PV cardiomyocyte electrophysiology and responses to NA in comparison with LA cells. LA and PV cardiomyocytes were isolated from adult male Wistar rat hearts, and membrane potentials and ion currents recorded at 36°C using whole-cell patch-clamp techniques. PV and LA cardiomyocytes did not differ in size. In control, there were no differences between the two cell-types in zero-current potential or action potential duration (APD) at 1 Hz, although the incidence of early afterdepolarizations (EADs) was greater in PV than LA cardiomyocytes. The L-type Ca2+ current (ICaL ) was ~×1.5 smaller (p = .0029, Student's t test) and the steady-state K+ current (IKss ) was ~×1.4 larger (p = .0028, Student's t test) in PV than in LA cardiomyocytes. PV cardiomyocyte inward-rectifier current (IK1 ) was slightly smaller than LA cardiomyocyte IK1 . In LA cardiomyocytes, NA significantly prolonged APD30 . In PV cells, APD30 responses to 1 µM NA were heterogeneous: while the mean percentage change in APD30 was not different from 0 (16.5 ± 9.7%, n cells/N animals = 12/10, p = .1177, one-sample t test), three cells showed shortening (-18.8 ± 6.0%) whereas nine showed prolongation (28.3 ± 10.1%, p = .008, Student's t test). NA had no effect on IK1 in either cell-type but inhibited PV IKss by 41.9 ± 4.1% (n/N = 23/11 p < .0001), similar to LA cells. NA increased ICaL in most PV cardiomyocytes (median × 2.2-increase, p < .0001, n/N = 32/14, Wilcoxon-signed-rank test), although in 7/32 PV cells ICaL was decreased following NA. PV cardiomyocytes differ from LA cells and respond heterogeneously to NA.