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Introduction: Atrial fibrillation (AF) is the most common cardiac arrhythmia. Consequently, novel therapies are being developed. Ultimately, the impact of compounds on the action potential (AP) needs to be tested in freshly isolated human atrial myocytes. However, the frequent depolarized state of these cells upon isolation seriously hampers reliable AP recordings. Purpose: We assessed whether AP recordings from single human atrial myocytes could be improved by providing these cells with a proper inward rectifier K + current (I K1 ), and consequently with a regular, non-depolarized resting membrane potential (RMP), through "dynamic clamp". Methods: Single myocytes were enzymatically isolated from left atrial appendage tissue obtained from patients with paroxysmal AF undergoing minimally invasive surgical ablation. APs were elicited at 1 Hz and measured using perforated patch-clamp methodology, injecting a synthetic I K1 to generate a regular RMP. The injected I K1 had strong or moderate rectification. For comparison, a regular RMP was forced through injection of a constant outward current. A wide variety of ion channel blockers was tested to assess their modulatory effects on AP characteristics. Results: Without any current injection, RMPs ranged from -9.6 to -86.2 mV in 58 cells. In depolarized cells (RMP positive to -60 mV), RMP could be set at -80 mV using I K1 or constant current injection and APs could be evoked upon stimulation. AP duration differed significantly between current injection methods ( p < 0.05) and was shortest with constant current injection and longest with injection of I K1 with strong rectification. With moderate rectification, AP duration at 90% repolarization (APD 90 ) was similar to myocytes with regular non-depolarized RMP, suggesting that a synthetic I K1 with moderate rectification is the most appropriate for human atrial myocytes. Importantly, APs evoked using each injection method were still sensitive to all drugs tested (lidocaine, nifedipine, E-4031, low dose 4-aminopyridine, barium, and apamin), suggesting that the major ionic currents of the atrial cells remained functional. However, certain drug effects were quantitatively dependent on the current injection approach used. Conclusion: Injection of a synthetic I K1 with moderate rectification facilitates detailed AP measurements in human atrial myocytes. Therefore, dynamic clamp represents a promising tool for testing novel antiarrhythmic drugs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Verkerk, Marchal, Zegers, Kawasaki, Driessen, Remme, de Groot and Wilders.)

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold great promise for studying inherited cardiac arrhythmias and developing drug therapies to treat such arrhythmias. Unfortunately, until now, action potential (AP) measurements in hiPSC-CMs have been hampered by the virtual absence of the inward rectifier potassium current ( I K1 ) in hiPSC-CMs, resulting in spontaneous activity and altered function of various depolarising and repolarising membrane currents. We assessed whether AP measurements in "ventricular-like" and "atrial-like" hiPSC-CMs could be improved through a simple, highly reproducible dynamic clamp approach to provide these cells with a substantial I K1 (computed in real time according to the actual membrane potential and injected through the patch-clamp pipette). APs were measured at 1 Hz using perforated patch-clamp methodology, both in control cells and in cells treated with all-trans retinoic acid (RA) during the differentiation process to increase the number of cells with atrial-like APs. RA-treated hiPSC-CMs displayed shorter APs than control hiPSC-CMs and this phenotype became more prominent upon addition of synthetic I K1 through dynamic clamp. Furthermore, the variability of several AP parameters decreased upon I K1 injection. Computer simulations with models of ventricular-like and atrial-like hiPSC-CMs demonstrated the importance of selecting an appropriate synthetic I K1 . In conclusion, the dynamic clamp-based approach of I K1 injection has broad applicability for detailed AP measurements in hiPSC-CMs., Competing Interests: The authors declare no conflict of interest.

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are widely used in studying basic mechanisms of cardiac arrhythmias that are caused by ion channelopathies. Unfortunately, the action potential profile of hiPSC-CMs-and consequently the profile of individual membrane currents active during that action potential-differs substantially from that of native human cardiomyocytes, largely due to almost negligible expression of the inward rectifier potassium current (IK1). In the present study, we attempted to "normalize" the action potential profile of our hiPSC-CMs by inserting a voltage dependent in silico IK1 into our hiPSC-CMs, using the dynamic clamp configuration of the patch clamp technique. Recordings were made from single hiPSC-CMs, using the perforated patch clamp technique at physiological temperature. We assessed three different models of IK1, with different degrees of inward rectification, and systematically varied the magnitude of the inserted IK1. Also, we modified the inserted IK1 in order to assess the effects of loss- and gain-of-function mutations in the KCNJ2 gene, which encodes the Kir2.1 protein that is primarily responsible for the IK1 channel in human ventricle. For our experiments, we selected spontaneously beating hiPSC-CMs, with negligible IK1 as demonstrated in separate voltage clamp experiments, which were paced at 1 Hz. Upon addition of in silico IK1 with a peak outward density of 4-6 pA/pF, these hiPSC-CMs showed a ventricular-like action potential morphology with a stable resting membrane potential near -80 mV and a maximum upstroke velocity >150 V/s (n = 9). Proarrhythmic action potential changes were observed upon injection of both loss-of-function and gain-of-function IK1, as associated with Andersen-Tawil syndrome type 1 and short QT syndrome type 3, respectively (n = 6). We conclude that injection of in silico IK1 makes the hiPSC-CM a more reliable model for investigating mechanisms underlying cardiac arrhythmias.

We investigated the contribution of the intracellular calcium (Ca (i) (2+) ) transient to acetylcholine (ACh)-mediated reduction of pacemaker frequency and cAMP content in rabbit sinoatrial nodal (SAN) cells. Action potentials (whole cell perforated patch clamp) and Ca (i) (2+) transients (Indo-1 fluorescence) were recorded from single isolated rabbit SAN cells, whereas intracellular cAMP content was measured in SAN cell suspensions using a cAMP assay (LANCE((R))). Our data show that the Ca (i) (2+) transient, like the hyperpolarization-activated "funny current" (I (f)) and the ACh-sensitive potassium current (I (K,ACh)), is an important determinant of ACh-mediated pacemaker slowing. When I (f) and I (K,ACh) were both inhibited, by cesium (2 mM) and tertiapin (100 nM), respectively, 1 micro M ACh was still able to reduce pacemaker frequency by 72%. In these I (f) and I (K,ACh)-inhibited SAN cells, good correlations were found between the ACh-mediated change in interbeat interval and the ACh-mediated change in Ca (i) (2+) transient decay (r (2) = 0.98) and slow diastolic Ca (i) (2+) rise (r (2) = 0.73). Inhibition of the Ca (i) (2+) transient by ryanodine (3 microM) or BAPTA-AM (5 microM) facilitated ACh-mediated pacemaker slowing. Furthermore, ACh depressed the Ca (i) (2+) transient and reduced the sarcoplasmic reticulum (SR) Ca(2+) content, all in a concentration-dependent fashion. At 1 microM ACh, the spontaneous activity and Ca (i) (2+) transient were abolished, but completely recovered when cAMP production was stimulated by forskolin (10 microM) and I (K,ACh) was inhibited by tertiapin (100 nM). Also, inhibition of the Ca (i) (2+) transient by ryanodine (3 microM) or BAPTA-AM (25 microM) exaggerated the ACh-mediated inhibition of cAMP content, indicating that Ca (i) (2+) affects cAMP production in SAN cells. In conclusion, muscarinic receptor stimulation inhibits the Ca (i) (2+) transient via a cAMP-dependent signaling pathway. Inhibition of the Ca (i) (2+) transient contributes to pacemaker slowing and inhibits Ca (i) (2+) -stimulated cAMP production. Thus, we provide functional evidence for the contribution of the Ca (i) (2+) transient to ACh-induced inhibition of pacemaker activity and cAMP content in rabbit SAN cells.

The development of a genetically engineered 'biological pacemaker', or 'bio-pacemaker', is a rapidly emerging field of research. One of the approaches in this field is to turn intrinsically quiescent myocardial cells, i.e., atrial or ventricular cells, into pacemaker cells by making them express the cardiac hyperpolarization-activated 'pacemaker current' If (known in neurophysiology as Ih), which is encoded by the hyperpolarization-activated cyclic nucleotide-modulated (HCN) gene family. We carried out 'dynamic action potential clamp' (dAPC) experiments in which we record current from a HEK-293 cell transfected with HCN4, which is the dominant HCN isoform in the sinoatrial (SA) node. This HCN4-transfected HEK-293 cell is voltage-clamped by the action potential generated in a real-time simulation of a human atrial cell (Courtemanche-Ramirez-Nattel model). In a continuous feedback loop, this current is injected into the atrial cell, so that this cell effectively expresses an HCN4-based pacemaker current. With sufficiently high 'expression levels' of HCN4 current the atrial cell is turned into a pacemaker cell with an SA nodal like action potential. Lower expression levels are sufficient if the inward rectifier potassium current (IK1), which is largely responsible for the stable resting potential of atrial cells, is 'down-regulated' by 50%, thus mimicking the gene therapy strategy to create a bio-pacemaker by down-regulation of IK1 and (over-)expression of If. Our dAPC experiments provide direct insights into the effects of introducing HCN4 current into an atrial cell, illustrating that dynamic action potential clamp can be a powerful tool in the process of developing a gene-based bio-pacemaker.

In the past decades, various computational models of the pacemaker activity of single sinoatrial (SA) nodal cells have been developed, building on data obtained in patch-clamp experiments on isolated SA nodal myocytes. These models show widely different results regarding the contribution of individual ionic currents to diastolic depolarization and pacemaker activity of the SA nodal myocyte. Because several of these ionic currents are strongly dependent on time, voltage and/or intracellular free calcium concentration ([Ca(2+)](i), one may argue that the apparent differences in the contribution of a particular ionic current to pacemaker activity between SA nodal cell models reflect differences in action potential shape and calcium transient between models rather than intrinsic differences in the ionic current of interest. To better appreciate the contribution of individual ionic currents to pacemaker activity in a computational model of an SA nodal cell, we imposed a realistic action potential shape and calcium transient on the model cell. This was achieved by first simultaneously recording membrane potential and ([Ca(2+)](i) from single isolated SA nodal myocytes and then subjecting the model cell to a combined ;action potential clamp' and ;calcium transient clamp' using a data file with a train of experimentally recorded SA nodal action potentials and associated calcium transients. The thus computed individual ionic currents should then more closely resemble the ;true' ionic currents during pacemaker activity of an SA nodal myocyte. Also, differences between the recorded and the computed net membrane current may prove helpful in identifying shortcomings of the computational model.

The cardiac long QT syndrome (LQTS) is characterized by a delayed repolarization of the ventricular myocytes, resulting in prolongation of the QT interval on the electrocardiogram and increased propensity to cardiac arrhythmias. Congenital LQTS has been linked to mutations in genes encoding ion channel subunits. For a better understanding of LQTS and associated arrhythmias, insight into the nature of ion channel (dys)function is indispensable. Conventionally, voltage-clamp analysis and subsequent mathematical modeling are used to study cardiac channelopathies and to link a certain genetic defect to its cellular phenotype. The recently introduced "dynamic action potential clamp" (dAPC) technique represents an alternative approach, in which a selected native ionic current of the ventricular myocyte can effectively be replaced with wild-type (WT) or mutant current recorded from a human embryonic kidney (HEK)-293 cell that is voltage clamped by the free-running action potential (AP) of the myocyte. Both a computed model of the human ventricular cell and a freshly isolated myocyte can effectively be used in dAPC experiments, resulting in rapid and unambiguous determination of the effect(s) of an ion channel mutation on the ventricular AP. The dAPC technique represents a promising new tool to study various cardiac ion channels and may also prove useful in related fields of research, for example, in neurophysiology.

At the same intracellular pH (pHi) Na+/H+ exchange (NHE-1) fluxes of ventricular myocytes of hypertrophied failing hearts (HFH) are increased. We assessed how NHE-1 affected cell length shortening. pHi was measured fluorimetrically in resting and twitching (1-3 Hz) normal and HFH rabbit myocytes. In HEPES-buffered solutions, increased NHE-1 fluxes (P=0.001, n=14) made HFH resting pHi 0.2+/-0.03 units more alkaline than control (n=27). In CO2/HCO3--buffered solutions, HFH resting pHi was not different (7.05+/-0.02, n=30). Twitching myocytes of both groups shortened 15-16% less per 0.1 pH unit acidification. In HEPES-buffered solutions, cariporide depressed cell length shortening of normal myocytes (1-3 Hz) by 16+/-5.4% (n=9, P=0.005). In HFH myocytes cariporide restored the positive force-frequency relationship (n=7, P=0.009), by depressing twitch amplitudes at 1 Hz (16+/-11%, P=0.047) but not at 2 and 3 Hz. The depressions were all caused by pHi acidification. In CO2/HCO3- buffered solutions the cariporide-induced acidification was too small to explain the cell length shortening depression of normal (19+/-5.0%, n=11, P=0.006) and HFH myocytes (14+/-4.7%, n=11, P=0.001). When compared to HEPES-buffered solutions, HFH myocytes in CO2/HCO3--buffered solutions shortened 12+/-6.8% better than expected given the 0.16+/-0.02 units more acidic pHi's at which they twitched. We conclude that in CO2/HCO3--buffered solutions NHE-1 improved cell length shortening of unstretched normal and HFH myocytes via a pHi-independent mechanism. Although NHE-1 was increased in HFH myocytes, the magnitude of the pHi-independent effect of NHE-1 inhibition on cell length shortening was similar in both groups.

Long-QT3 syndrome (LQT3) is linked to cardiac sodium channel gene (SCN5A) mutations. In this study, we used the 'dynamic action potential clamp' (dAPC) technique to effectively replace the native sodium current (I(Na)) of the Priebe-Beuckelmann human ventricular cell model with wild-type (WT) or mutant I(Na) generated in a human embryonic kidney (HEK)-293 cell that is voltage clamped by the free-running action potential of the ventricular cell. We recorded I(Na) from HEK cells expressing either WT or LQT3-associated Y1795C or A1330P SCN5A at 35 degrees C, and let this current generate and shape the action potential (AP) of subepicardial, mid-myocardial and subendocardial model cells. The HEK cell's endogenous background current was completely removed by a real-time digital subtraction procedure. With WT I(Na), AP duration (APD) was longer than with the original Priebe-Beuckelmann model I(Na), due to a late I(Na) component of approximately 30 pA that could not be revealed with conventional voltage-clamp protocols. With mutant I(Na), this late component was larger ( approximately 100 pA), producing a marked increase in APD ( approximately 70-80 ms at 1 Hz for the subepicardial model cell). The late I(Na) magnitude showed reverse frequency dependence, resulting in a significantly steeper APD-frequency relation in the mutant case. AP prolongation was more pronounced for the mid-myocardial cell type, resulting in increased APD dispersion for each of the mutants. For both mutants, a 2 s pause following rapid (2 Hz) pacing resulted in distorted AP morphology and beat-to-beat fluctuations of I(Na). Our dAPC data directly demonstrate the arrhythmogenic nature of LQT3-associated SCN5A mutations.

The human ether-a-go-go-related gene (HERG) encodes the rapid component of the cardiac delayed rectifier potassium current (I(Kr)). Per-Arnt-Sim domain mutations of the HERG channel are linked to type 2 long-QT syndrome. We studied wild-type and/or type 2 long-QT syndrome-associated mutant (R56Q) HERG current (I(HERG)) in HEK-293 cells, at both 23 and 36 degrees C. Conventional voltage-clamp analysis revealed mutation-induced changes in channel kinetics. To assess functional implication(s) of the mutation, we introduce the dynamic action potential clamp technique. In this study, we effectively replace the native I(Kr) of a ventricular cell (either a human model cell or an isolated rabbit myocyte) with I(HERG) generated in a HEK-293 cell that is voltage-clamped by the free-running action potential of the ventricular cell. Action potential characteristics of the ventricular cells were effectively reproduced with wild-type I(HERG), whereas the R56Q mutation caused a frequency-dependent increase of the action potential duration in accordance with the clinical phenotype. The dynamic action potential clamp approach also revealed a frequency-dependent transient wild-type I(HERG) component, which is absent with R56Q channels. This novel electrophysiological technique allows rapid and unambiguous determination of the effects of an ion channel mutation on the ventricular action potential and can serve as a new tool for investigating cardiac channelopathies.

One variant of the long-QT syndrome (LQT3) is caused by mutations in the human cardiac sodium channel gene. In addition to the characteristic QT prolongation, LQT3 carriers regularly present with bradycardia and sinus pauses. Therefore, we studied the effect of the 1795insD Na+ channel mutation on sinoatrial (SA) pacemaking. The 1795insD channel was previously characterized by the presence of a persistent inward current (Ipst) at -20 mV and a negative shift in voltage dependence of inactivation. In the present study, we first additionally characterized Ipst over the complete voltage range of the SA node action potential (AP) by measuring whole-cell Na+ currents (INa) in HEK-293 cells expressing either wild-type or 1795insD channels. Ipst for 1795insD channels varied between 0.8+/-0.2% and 1.9+/-0.8% of peak INa. Activity of 1795insD channels during SA node pacemaking was confirmed by AP clamp experiments. Next, Ipst and the negative shift were implemented into SA node AP models. The -10-mV shift decreased sinus rate by decreasing diastolic depolarization rate, whereas Ipst decreased sinus rate by AP prolongation, despite a concomitant increase in diastolic depolarization rate. In combination, moderate Ipst (1% to 2%) and the shift reduced sinus rate by approximately 10%. An additional increase in Ipst could result in plateau oscillations and failure to repolarize completely. Thus, Na+ channel mutations displaying an Ipst or a negative shift in inactivation may account for the bradycardia seen in LQT3 patients, whereas SA node pauses or arrest may result from failure of SA node cells to repolarize under conditions of extra net inward current.

The Ca(2+)-activated Cl(-) current (I(Cl(Ca))) has been identified in atrial, Purkinje and ventricular cells, where it plays a substantial role in phase-1 repolarization and delayed after-depolarizations. In sinoatrial (SA) node cells, however, the presence and functional role of I(Cl(Ca)) is unknown. In the present study we address this issue using perforated patch-clamp methodology and computer simulations. Single SA node cells were enzymatically isolated from rabbit hearts. I(Cl(Ca)) was measured, using the perforated patch-clamp technique, as the current sensitive to the anion blocker 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS). Voltage clamp experiments demonstrate the presence of I(Cl(Ca)) in one third of the spontaneously active SA node cells. The current was transient outward with a bell-shaped current-voltage relationship. Adrenoceptor stimulation with 1 microM noradrenaline doubled the I(Cl(Ca)) density. Action potential clamp measurements demonstrate that I(Cl(Ca)) is activate late during the action potential upstroke. Current clamp experiments show, both in the absence and presence of 1 microM noradrenaline, that blockade of I(Cl(Ca)) increases the action potential overshoot and duration, measured at 20 % repolarization. However, intrinsic interbeat interval, upstroke velocity, diastolic depolarization rate and the action potential duration measured at 50 and 90 % repolarization were not affected. Our experimental data are supported by computer simulations, which additionally demonstrate that I(Cl(Ca)) has a limited role in pacemaker synchronization or action potential conduction. In conclusion, I(Cl(Ca)) is present in one third of SA node cells and is activated during the pacemaker cycle. However, I(Cl(Ca)) does not modulate intrinsic interbeat interval, pacemaker synchronization or action potential conduction.

The in vitro motility assay is used to measure speed of actin filaments moving over a glass surface coated with heavy meromyosin. In this paper a new method, the path reconstruction method, is presented to evaluate observed speeds. The method is compared with the commonly used centroid method, in which the centroids of the filaments are followed from frame to frame. Instead, in the path reconstruction method speed is evaluated from determination of perimeters of the filaments in each frame and by reconstruction of the traversed paths of the filaments over a number of frames. Biases in the determination of speed occurring in the centroid method due to curvature of paths and to video noise and Brownian motion are eliminated in the path reconstruction method, allowing measurement over a range of frame rates from 5 to 25 per second. The path reconstruction method leads to a clear separation of motile and nonmotile filaments provided that filaments are analyzed over at least 10 successive frames and allows easier separation of uniform and nonuniform sliding behavior., (Copyright 1999 Academic Press.)