Small-conductance calcium-activated potassium (SK) channels are expressed in the heart of various species, including humans. The aim of the present study was to address whether SK channels play a ...functional role in human atria.
Quantitative real-time PCR analyses showed higher transcript levels of SK2 and SK3 than that of the SK1 subtype in human atrial tissue. SK2 and SK3 were reduced in chronic atrial fibrillation (AF) compared with sinus rhythm (SR) patients. Immunohistochemistry using confocal microscopy revealed widespread expression of SK2 in atrial myocytes. Two SK channel inhibitors (NS8593 and ICAGEN) were tested in heterologous expression systems revealing ICAGEN as being highly selective for SK channels, while NS8593 showed less selectivity for these channels. In isolated atrial myocytes from SR patients, both inhibitors decreased inwardly rectifying K(+) currents by ∼15% and prolonged action potential duration (APD), but no effect was observed in myocytes from AF patients. In trabeculae muscle strips from right atrial appendages of SR patients, both compounds increased APD and effective refractory period, and depolarized the resting membrane potential, while only NS8593 induced these effects in tissue from AF patients. SK channel inhibition did not alter any electrophysiological parameter in human interventricular septum tissue.
SK channels are present in human atria where they participate in repolarization. SK2 and SK3 were down-regulated and had reduced functional importance in chronic AF. As SK current was not found to contribute substantially to the ventricular AP, pharmacological inhibition of SK channels may be a putative atrial-selective target for future antiarrhythmic drug therapy.
Human atrial electrophysiology exhibits high inter-subject variability in both sinus rhythm (SR) and chronic atrial fibrillation (cAF) patients. Variability is however rarely investigated in ...experimental and theoretical electrophysiological studies, thus hampering the understanding of its underlying causes but also its implications in explaining differences in the response to disease and treatment. In our study, we aim at investigating the ability of populations of human atrial cell models to capture the inter-subject variability in action potential (AP) recorded in 363 patients both under SR and cAF conditions.
Human AP recordings in atrial trabeculae (n = 469) from SR and cAF patients were used to calibrate populations of computational SR and cAF atrial AP models. Three populations of over 2000 sampled models were generated, based on three different human atrial AP models. Experimental calibration selected populations of AP models yielding AP with morphology and duration in range with experimental recordings. Populations using the three original models can mimic variability in experimental AP in both SR and cAF, with median conductance values in SR for most ionic currents deviating less than 30% from their original peak values. All cAF populations show similar variations in G(K1), G(Kur) and G(to), consistent with AF-related remodeling as reported in experiments. In all SR and cAF model populations, inter-subject variability in I(K1) and I(NaK) underlies variability in APD90, variability in I(Kur), I(CaL) and I(NaK) modulates variability in APD50 and combined variability in Ito and I(Kur) determines variability in APD20. The large variability in human atrial AP triangulation is mostly determined by I(K1) and either I(NaK) or I(NaCa) depending on the model.
Experimentally-calibrated human atrial AP models populations mimic AP variability in SR and cAF patient recordings, and identify potential ionic determinants of inter-subject variability in human atrial AP duration and morphology in SR versus cAF.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Human induced pluripotent stem cell (hiPSC) cardiomyocytes (CMs) show less negative resting membrane potential (RMP), which is attributed to small inward rectifier currents (IK1). Here, IK1 was ...measured in hiPSC-CMs (proprietary and commercial cell line) cultured as monolayer (ML) or 3D engineered heart tissue (EHT) and, for direct comparison, in CMs from human right atrial (RA) and left ventricular (LV) tissue. RMP was measured in isolated cells and intact tissues. IK1 density in ML- and EHT-CMs from the proprietary line was similar to LV and RA, respectively. IK1 density in EHT-CMs from the commercial line was 2-fold smaller than in the proprietary line. RMP in EHT of both lines was similar to RA and LV. Repolarization fraction and IK,ACh response discriminated best between RA and LV and indicated predominantly ventricular phenotype in hiPSC-CMs/EHT. The data indicate that IK1 is not necessarily low in hiPSC-CMs, and technical issues may underlie low RMP in hiPSC-CMs.
•Here, we report IK1 current density in hiPSC-CMs as high as in human adult CMs•Physiological RMP was found with sharp microelectrodes in engineered heart tissue•RMP measured by the patch-clamp technique in hiPSC-CMs may be underestimated•RMP and APD90 are not sufficient to categorize CMs as atrial or ventricular
We show here that RMP is systematically underestimated in patch-clamped hiPSC-CMs and, in the 3D EHT format, reaches physiological values of human adult CMs when measured by sharp microelectrodes. This corresponds with IK1 currents as large as in human adult CMs. In human adult preparations, repolarization fraction was more useful than APD and RMP to classify action potentials as atrial or ventricular like.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Vernakalant is a newly developed antiarrhythmic drug against atrial fibrillation (AF). However, its electrophysiological actions on human myocardium are unknown.
Action potentials (APs) and ion ...currents were recorded in right atrial trabeculae and cardiomyocytes from patients in sinus rhythm (SR) and chronic AF. Vernakalant prolonged early repolarization in SR and AF, but late only in AF. AP amplitude (APA) and dV/dtmax were reduced in a concentration- and frequency-dependent manner with IC50 < 10 µM at >3 Hz. Effective refractory period was increased more than action potential duration (APD) in SR and AF. INa was blocked with IC50s of 95 and 84 µM for SR and AF, respectively (0.5 Hz). Vernakalant did not reduce outward potassium currents compared with time-matched controls. However, area under the current-time curve was reduced due to acceleration of current decline with IC50s of 19 and 12 µM for SR and AF, respectively. Vernakalant had less effect on APD than the IKr blocker E-4031, blocked IK,ACh, and had a small inhibitory effect on IK1 at 30 µM. L-Type Ca(2+) currents (SR) were reduced with IC50 of 84 µM.
Rate-dependent block of Na(+) channels represents the main antiarrhythmic mechanism of vernakalant in the fibrillating atrium. Open channel block of early transient outward currents and IK,ACh could also contribute.
Key points
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Cardiac repolarization, through which heart‐cells return to their resting state after having fired, is a delicate process, susceptible to disruption by common drugs and clinical ...conditions.
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Animal models, particularly the dog, are often used to study repolarization properties and responses to drugs, with the assumption that such findings are relevant to humans. However, little is known about the applicability of findings in animals to man.
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Here, we studied the contribution of various ion‐currents to cardiac repolarization in canine and human ventricle.
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Humans showed much greater repolarization‐impairing effects of drugs blocking the rapid delayed‐rectifier current IKr than dogs, because of lower repolarization‐reserve contributions from two other important repolarizing currents (the inward‐rectifier IK1 and slow delayed‐rectifier IKs).
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Our findings clarify differences in cardiac repolarization‐processes among species, highlighting the importance of caution when extrapolating results from animal models to man.
The species‐specific determinants of repolarization are poorly understood. This study compared the contribution of various currents to cardiac repolarization in canine and human ventricle. Conventional microelectrode, whole‐cell patch‐clamp, molecular biological and mathematical modelling techniques were used. Selective IKr block (50–100 nmol l−1 dofetilide) lengthened AP duration at 90% of repolarization (APD90) >3‐fold more in human than dog, suggesting smaller repolarization reserve in humans. Selective IK1 block (10 μmol l−1 BaCl2) and IKs block (1 μmol l−1 HMR‐1556) increased APD90 more in canine than human right ventricular papillary muscle. Ion current measurements in isolated cardiomyocytes showed that IK1 and IKs densities were 3‐ and 4.5‐fold larger in dogs than humans, respectively. IKr density and kinetics were similar in human versus dog. ICa and Ito were respectively ∼30% larger and ∼29% smaller in human, and Na+–Ca2+ exchange current was comparable. Cardiac mRNA levels for the main IK1 ion channel subunit Kir2.1 and the IKs accessory subunit minK were significantly lower, but mRNA expression of ERG and KvLQT1 (IKr and IKsα‐subunits) were not significantly different, in human versus dog. Immunostaining suggested lower Kir2.1 and minK, and higher KvLQT1 protein expression in human versus canine cardiomyocytes. IK1 and IKs inhibition increased the APD‐prolonging effect of IKr block more in dog (by 56% and 49%, respectively) than human (34 and 16%), indicating that both currents contribute to increased repolarization reserve in the dog. A mathematical model incorporating observed human–canine ion current differences confirmed the role of IK1 and IKs in repolarization reserve differences. Thus, humans show greater repolarization‐delaying effects of IKr block than dogs, because of lower repolarization reserve contributions from IK1 and IKs, emphasizing species‐specific determinants of repolarization and the limitations of animal models for human disease.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Selective inhibitors of Kv1.5 channels are being developed for the treatment of atrial fibrillation (AF).
The purpose of this study was to investigate the effects of the highly selective Kv1.5 ...inhibitor XEN-D0103 on human atrial action potentials (APs) at high excitation rates and to assess safety.
Intracellular APs (stimulation rates 1–5 Hz) were measured in right atrial trabeculae from patients in sinus rhythm (SR), chronic AF (cAF; AF of >6 months duration), and paroxysmal AF (pAF). The safety and tolerability of XEN-D0103 were tested in a double-blind, randomized, placebo-controlled phase 1 study.
Depending on its concentration, XEN-D0103 elevated the plateau potential. At 1 Hz, XEN-D0103 (3 µM) shortened action potential duration at 90% repolarization (APD90) and effective refractory period (ERP) in SR preparations, but prolonged these parameters in cAF preparations. In SR and pAF preparations, the shortening effects on APD90 and ERP turned into prolongation at high rates. In cAF trabeculae, XEN-D0103 prolonged APD90 and ERP at 2 and 3 Hz. At high rates, more SR and pAF preparations failed to capture excitation in the presence of the drug than in its absence. XEN-D0103 (10 µM) did not significantly affect human ventricular APs. Even with plasma concentrations reaching 7000 ng/mL, XEN-D0103 did not increase ∆∆QTcF (QT interval corrected by the Fridericia formula) in the analysis of electrocardiograms of healthy volunteers, and no subjects receiving an active treatment had a QT or QTcF interval >450 ms, or increase in QTcF from baseline >30 ms.
APD prolongation and suppression of APs by XEN-D0103 at high stimulation rates in SR and pAF tissue, but not cAF, could be of therapeutic benefit for reducing AF burden. This concept needs to be confirmed in clinical trials.
Human mesenchymal stem cells (hMSC) have gained considerable interest due to their potential use for cell replacement therapy
and tissue engineering. One strategy is to differentiate these bone ...marrow stem cells in vitro into cardiomyocytes prior to implantation. In this context ion channels can be important functional markers of cardiac differentiation.
At present there is little information about the electrophysiological behaviour of the undifferentiated hMSC. We therefore
investigated mRNA expression of 26 ion channel subunits using semiquantitative RT-PCR and recorded transmembrane ion currents
with the whole-cell voltage clamp technique. Bone marrow hMSC were obtained from healthy donors. The cells revealed a distinct
pattern of ion channel mRNA with high expression levels for some channel subunits (e.g. Kv4.2, Kv4.3, MaxiK, HCN2, and α1C
of the L-type calcium channel). Outward currents were recorded in almost all cells. The most abundant outward current rapidly
activated at potentials positive to +20 mV. This current was identified as a large-conductance voltage- and Ca 2+ -activated K + current, conducted by MaxiK channels, due to its high sensitivity to tetraethylammonium (IC 50 = 340 μ m ) and its inhibition by 100 n m iberiotoxin. A large fraction of cells also demonstrated a more slowly activating current at potentials positive to â30 mV.
This current was selectively inhibited by clofilium (IC 50 = 0.8 μ m ). Ba 2+ inward currents, stimulated by 1 μ m BayK 8644 were found in a few cells, indicating the expression of functional L-type Ca 2+ channels. Other inward currents such as sodium currents or inward rectifier currents were absent. We conclude that undifferentiated
hMSC express a distinct pattern of ion channel mRNA and functional ion channels that might contribute to physiological cell
function.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Aim: Isolated papillary muscles and enzymatically dissociated myocytes of guinea-pig hearts are routinely used for experimental cardiac research. The aim of our study is to investigate adult ...mammalian ventricular slices as an alternative preparation. Method: Vibratome cut ventricular slices (350 μm thick) were examined histologically and with 2-photon microscopy for fibre orientation. Intracellular action potentials were recorded with conventional glass microelectrodes, extracellular potentials were measured with tungsten platinum electrodes and multi-electrode arrays (MEA). Results: Dominant direction of fibre orientation was absent in vertical and horizontal transmural slices, but was longitudinal in tangential slices. Control action potential duration (APD90, 169.9 ± 4 ms) and drug effects on this parameter were similar to papillary muscles. The L-type Ca-channel blocker nifedipine shortened APD90 with a half maximal effective concentration (EC50) of 4.5 μM. The IKr blocker E4031 and neuroleptic drug risperidone prolonged APD90 with EC50 values of 31 nM and 0.67 μM, respectively. Mapping field potentials on multi-electrode arrays showed uniform spread of excitation with a mean conduction velocity of 0.47 m ⋅ s-1. Conclusion: Slices from adult mammalian hearts could become a useful routine model for electrophysiological and pharmacological research.
Within the field of new antiarrhythmic compounds, the interesting idea of activating human ether-a-go-go-related gene (HERG1)
potassium channels has recently been introduced. Potentially, drugs that ...increase HERG1 channel activity will augment the
repolarizing current of the cardiac myocytes and stabilize the diastolic interval. This may make the myocardium more resistant
to events that cause arrhythmias. We here present the compound N -(4-bromo-2-(1 H -tetrazol-5-yl)-phenyl)- N â²-(3â²-trifluoromethylphenyl)urea (NS3623), which has the ability to activate HERG1 channels expressed in Xenopus laevis oocytes with an EC 50 value of 79.4 μM. Exposure of HERG1 channels to NS3623 affects the voltage-dependent release from inactivation, resulting
in a half-inactivation voltage that is rightward-shifted by 17.7 mV. Moreover, the compound affects the time constant of inactivation,
leading to a slower onset of inactivation of the macroscopic HERG1 currents. We also characterized the ability of NS3623 to
increase the activity of different mutated HERG1 channels. The mutants S620T and S631A are severely compromised in their ability
to inactivate. Application of NS3623 to any of these two mutants did not result in increased HERG1 current. In contrast, application
of NS3623 to the mutant F656M increased HERG1 current to a larger extent than what was observed with wild-type HERG1 channels.
Because the amino acid F656 is essential for high-affinity inhibition of HERG1 channels, it is concluded that NS3623 has a
dual mode of action, being both an activator and an inhibitor of HERG1 channels. Finally, we show that NS3623 has the ability
to shorten action potential durations in guinea pig papillary muscle.
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