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The patch clamp technique underwent continual advancement and developed numerous variants in cardiac electrophysiology since its introduction in the late 1970s. In the beginning, the capability of the technique was limited to recording one single current from one cell stimulated with a rectangular command pulse. Since that time, the technique has been extended to record multiple currents under various command pulses including action potential. The current review summarizes the development of the patch clamp technique in cardiac electrophysiology with special focus on the potential applications in integrative physiology.

The role of Ca2+-activated Cl- current (ICl(Ca)) in cardiac arrhythmias is still controversial. It can generate delayed afterdepolarizations in Ca2+-overloaded cells while in other studies incidence of early afterdepolarization (EAD) was reduced by ICl(Ca). Therefore our goal was to examine the role of ICl(Ca) in spatial and temporal heterogeneity of cardiac repolarization and EAD formation. Experiments were performed on isolated canine cardiomyocytes originating from various regions of the left ventricle; subepicardial, midmyocardial and subendocardial cells, as well as apical and basal cells of the midmyocardium. ICl(Ca) was blocked by 0.5mmol/L 9-anthracene carboxylic acid (9-AC). Action potential (AP) changes were tested with sharp microelectrode recording. Whole-cell 9-AC-sensitive current was measured with either square pulse voltage-clamp or AP voltage-clamp (APVC). Protein expression of TMEM16A and Bestrophin-3, ion channel proteins mediating ICl(Ca), was detected by Western blot. 9-AC reduced phase-1 repolarization in every tested cell. 9-AC also increased AP duration in a reverse rate-dependent manner in all cell types except for subepicardial cells. Neither ICl(Ca) density recorded with square pulses nor the normalized expressions of TMEM16A and Bestrophin-3 proteins differed significantly among the examined groups of cells. The early outward component of ICl(Ca) was significantly larger in subepicardial than in subendocardial cells in APVC setting. Applying a typical subepicardial AP as a command pulse resulted in a significantly larger early outward component in both subepicardial and subendocardial cells, compared to experiments when a typical subendocardial AP was applied. Inhibiting ICl(Ca) by 9-AC generated EADs at low stimulation rates and their incidence increased upon beta-adrenergic stimulation. 9-AC increased the short-term variability of repolarization also. We suggest a protective role for ICl(Ca) against risk of arrhythmias by reducing spatial and temporal heterogeneity of cardiac repolarization and EAD formation.

Several ion currents in the mammalian ventricular myocardium are substantially regulated by the sympathetic nervous system via β-adrenergic receptor activation, including the slow delayed rectifier K+ current and the L-type calcium current. This study investigated the downstream mechanisms of β-adrenergic receptor stimulation by isoproterenol (ISO) on the inward rectifier (IK1) and the rapid delayed rectifier (IKr) K+ currents using action potential voltage clamp (APVC) and conventional voltage clamp techniques in isolated canine left ventricular cardiomyocytes. IK1 and IKr were dissected by 50 µM BaCl2 and 1 µM E-4031, respectively. Acute application of 10 nM ISO significantly increased IK1 under the plateau phase of the action potential (0–+20 mV) using APVC, and similar results were obtained with conventional voltage clamp. However, β-adrenergic receptor stimulation did not affect the peak current density flowing during terminal repolarization or the overall IK1 integral. The ISO-induced enhancement of IK1 was blocked by the calcium/calmodulin kinase II (CaMKII) inhibitor KN-93 (1 µM) but not by the protein kinase A inhibitor H-89 (3 µM). Neither KN-93 nor H-89 affected the IK1 density under baseline conditions (in the absence of ISO). In contrast, parameters of the IKr current were not affected by β-adrenergic receptor stimulation with ISO. These findings suggest that sympathetic activation enhances IK1 in canine left ventricular cells through the CaMKII pathway, while IKr remains unaffected under the experimental conditions used. [ABSTRACT FROM AUTHOR]

The present study was designed to test the hypothesis that the selectivity of blocking the late Na+ current (INaL) over the peak Na+ current (INaP) is related to the fast offset kinetics of the Na+ channel inhibitor. Therefore, the effects of 1 µM GS967 (INaL inhibitor), 20 µM mexiletine (I/B antiarrhythmic) and 10 µM quinidine (I/A antiarrhythmic) on INaL and INaP were compared in canine ventricular myocardium. INaP was estimated as the maximum velocity of action potential upstroke (V+max). Equal amounts of INaL were dissected by the applied drug concentrations under APVC conditions. The inhibition of INaL by mexiletine and quinidine was comparable under a conventional voltage clamp, while both were smaller than the inhibitory effect of GS967. Under steady-state conditions, the V+max block at the physiological cycle length of 700 ms was 2.3% for GS967, 11.4% for mexiletine and 26.2% for quinidine. The respective offset time constants were 110 ± 6 ms, 456 ± 284 ms and 7.2 ± 0.9 s. These results reveal an inverse relationship between the offset time constant and the selectivity of INaL over INaP inhibition without any influence of the onset rate constant. It is concluded that the selective inhibition of INaL over INaP is related to the fast offset kinetics of the Na+ channel inhibitor. [ABSTRACT FROM AUTHOR]

A correction has been issued for an article titled "ABT-333 (Dasabuvir) Increases Action Potential Duration and Provokes Early Afterdepolarizations in Canine Left Ventricular Cells via Inhibition of I Kr." The correction states that there was an error in the original publication regarding the calculation of the therapeutic ABT-333 concentration. The correction clarifies that ABT-333 can indeed lead to cardiac side effects, as its peak therapeutic plasma concentration is approximately 1 µM, not 1 nM as previously stated. The correction also provides additional information about the plasma concentration of ABT-333 in different scenarios and highlights the potential risks of AP prolongation and arrhythmias, especially in cases of overdose or coadministration with drugs that inhibit CYP2C8. The authors state that the scientific conclusions of the article remain unaffected. [Extracted from the article]

Late sodium current (INa,late) is an important inward current contributing to the plateau phase of the action potential (AP) in the mammalian heart. Although INa,late is considered as a possible target for antiarrhythmic agents, several aspects of this current remained hidden. In this work, the profile of INa,late, together with the respective conductance changes (GNa,late), were studied and compared in rabbit, canine, and guinea pig ventricular myocytes using the action potential voltage clamp (APVC) technique. In canine and rabbit myocytes, the density of INa,late was relatively stable during the plateau and decreased only along terminal repolarization of the AP, while GNa,late decreased monotonically. In contrast, INa,late increased monotonically, while GNa,late remained largely unchanged during the AP in guinea pig. The estimated slow inactivation of Na+ channels was much slower in guinea pig than in canine or rabbit myocytes. The characteristics of canine INa,late and GNa,late were not altered by using command APs recorded from rabbit or guinea pig myocytes, indicating that the different shapes of the current profiles are related to genuine interspecies differences in the gating of INa,late. Both INa,late and GNa,late decreased in canine myocytes when the intracellular Ca2+ concentration was reduced either by the extracellular application of 1 µM nisoldipine or by the intracellular application of BAPTA. Finally, a comparison of the INa,late and GNa,late profiles induced by the toxin of Anemonia sulcata (ATX-II) in canine and guinea pig myocytes revealed profound differences between the two species: in dog, the ATX-II induced INa,late and GNa,late showed kinetics similar to those observed with the native current, while in guinea pig, the ATX-II induced GNa,late increased during the AP. Our results show that there are notable interspecies differences in the gating kinetics of INa,late that cannot be explained by differences in AP morphology. These differences must be considered when interpreting the INa,late results obtained in guinea pig. [ABSTRACT FROM AUTHOR]

Aims: Heart failure with reduced ejection fraction (HFrEF) is a disease with high mortality and morbidity. Recent positive inotropic drug developments focused on cardiac myofilaments, that is, direct activators of the myosin molecule and Ca2+ sensitizers for patients with advanced HFrEF. Omecamtiv mecarbil (OM) is the first direct myosin activator with promising results in clinical studies. Here, we aimed to elucidate the cellular mechanisms of the positive inotropic effect of OM in a comparative in vitro investigation where Ca2+‐sensitizing positive inotropic agents with distinct mechanisms of action [EMD 53998 (EMD), which also docks on the myosin molecule, and levosimendan (Levo), which binds to troponin C] were included. Methods: Enzymatically isolated canine cardiomyocytes with intact cell membranes were loaded with Fura‐2AM, a Ca2+‐sensitive, ratiometric, fluorescent dye. Changes in sarcomere length (SL) and intracellular Ca2+ concentration were recorded in parallel at room temperature, whereas cardiomyocyte contractions were evoked by field stimulation at 0.1 Hz in the presence of different OM, EMD, or Levo concentrations. Results: SL was reduced by about 23% or 9% in the presence of 1 μM OM or 1 μM EMD in the absence of electrical stimulation, whereas 1 μM Levo had no effect on resting SL. Fractional sarcomere shortening was increased by 1 μM EMD or 1 μM Levo to about 152%, but only to about 128% in the presence of 0.03 μM OM. At higher OM concentrations, no significant increase in fractional sarcomere shortening could be recorded. Contraction durations largely increased, whereas the kinetics of contractions and relaxations decreased with increasing OM concentrations. One‐micromole EMD or 1 μM Levo had no effects on contraction durations. One‐micromole Levo, but not 1 μM EMD, accelerated the kinetics of cardiomyocyte contractions and relaxations. Ca2+ transient amplitudes were unaffected by all treatments. Conclusions: Our data revealed major distinctions between the cellular effects of myofilament targeted agents (OM, EMD, or Levo) depending on their target proteins and binding sites, although they were compatible with the involvement of Ca2+‐sensitizing mechanisms for all three drugs. Significant part of the cardiotonic effect of OM relates to the prolongation of systolic contraction in combination with its Ca2+‐sensitizing effect. [ABSTRACT FROM AUTHOR]

Recent cardiotropic drug developments have focused on cardiac myofilaments. Danicamtiv, the second direct myosin activator, has achieved encouraging results in preclinical and clinical studies, thus implicating its potential applicability in the treatment of heart failure with reduced ejection fraction (HFrEF). Here, we analyzed the inotropic effects of danicamtiv in detail. To this end, changes in sarcomere length and intracellular Ca2+ levels were monitored in parallel, in enzymatically isolated canine cardiomyocytes, and detailed echocardiographic examinations were performed in anesthetized rats in the absence or presence of danicamtiv. The systolic and diastolic sarcomere lengths decreased; contraction and relaxation kinetics slowed down with increasing danicamtiv concentrations without changes in intracellular Ca2+ transients in vitro. Danicamtiv evoked remarkable increases in left ventricular ejection fraction and fractional shortening, also reflected by changes in systolic strain. Nevertheless, the systolic ejection time was significantly prolonged, the ratio of diastolic to systolic duration was reduced, and signs of diastolic dysfunction were also observed upon danicamtiv treatment in vivo. Taken together, danicamtiv improves cardiac systolic function, but it can also limit diastolic performance, especially at high drug concentrations. [ABSTRACT FROM AUTHOR]