Craig T. January

Early afterdepolarizations: mechanism of induction and block. A role for L-type Ca2+ current.

Early afterdepolarizations (EADs) are a type of triggered activity found in heart muscle. We used voltage-clamped sheep cardiac Purkinje fibers to examine the mechanism underlying EADs induced near action potential plateau voltages with the Ca2+ current agonist Bay K 8644 and the effect of several interventions known to suppress or enhance these EADs. Bay K 8644 produced an inward shift of the steady-state current-voltage relation near plateau voltages. Tetrodotoxin, lidocaine, verapamil, nitrendipine, and raising [K]0 abolish EADs and shift the steady-state current-voltage relations outwardly. Using a two-pulse voltage-clamp protocol, an inward current transient was present at voltages where EADs were induced. The voltage-dependence of availability of the inward current transient and of EAD induction were similar. The time- dependence of recovery from inactivation of the inward current transient and of EAD amplitude were nearly identical. Without recovery of the inward current transient, EADs could not be elicited. The inward current transient was enhanced with Bay K 8644 and blocked by nitrendipine, but was not abolished by tetrodotoxin or replacement of [Na], with an impermeant cation. These results support a hypothesis that the induction of EADs near action potential plateau voltages requires 1) a conditioning phase controlled by the sum of membrane currents present near the action potential plateau and characterized by lengthening and flattening of the plateau within a voltage range where, 2) recovery from inactivation and reactivation of L-type Ca2+channels to carry the depolarizing charge can occur. Our results suggest an essential role for the L-type Ca1+ window current and provide a framework for understanding the role of several membrane currents in the Induction and block of EADs.

High purity human-induced pluripotent stem cell-derived cardiomyocytes: electrophysiological properties of action potentials and ionic currents

Human-induced pluripotent stem cells (hiPSCs) can differentiate into functional cardiomyocytes; however, the electrophysiological properties of hiPSC-derived cardiomyocytes have yet to be fully characterized. We performed detailed electrophysiological characterization of highly pure hiPSC-derived cardiomyocytes. Action potentials (APs) were recorded from spontaneously beating cardiomyocytes using a perforated patch method and had atrial-, nodal-, and ventricular-like properties. Ventricular-like APs were more common and had maximum diastolic potentials close to those of human cardiac myocytes, AP durations were within the range of the normal human electrocardiographic QT interval, and APs showed expected sensitivity to multiple drugs (tetrodotoxin, nifedipine, and E4031). Early afterdepolarizations (EADs) were induced with E4031 and were bradycardia dependent, and EAD peak voltage varied inversely with the EAD take-off potential. Gating properties of seven ionic currents were studied including sodium (I(Na)), L-type calcium (I(Ca)), hyperpolarization-activated pacemaker (I(f)), transient outward potassium (I(to)), inward rectifier potassium (I(K1)), and the rapidly and slowly activating components of delayed rectifier potassium (I(Kr) and I(Ks), respectively) current. The high purity and large cell numbers also enabled automated patch-clamp analysis. We conclude that these hiPSC-derived cardiomyocytes have ionic currents and channel gating properties underlying their APs and EADs that are quantitatively similar to those reported for human cardiac myocytes. These hiPSC-derived cardiomyocytes have the added advantage that they can be used in high-throughput assays, and they have the potential to impact multiple areas of cardiovascular research and therapeutic applications.

Sodium current in voltage clamped internally perfused canine cardiac Purkinje cells

Study of the excitatory sodium current (INa) intact heart muscle has been hampered by the limitations of voltage clamp methods in multicellular preparations that result from the presence of large series resistance and from extracellular ion accumulation and depletion. To minimize these problems we voltage clamped and internally perfused freshly isolated canine cardiac Purkinje cells using a large bore (25-microns diam) double-barreled flow-through glass suction pipette. Control of [Na+]i was demonstrated by the agreement of measured INa reversal potentials with the predictions of the Nernst relation. Series resistance measured by an independent microelectrode was comparable to values obtained in voltage clamp studies of squid axons (less than 3.0 omega-cm2). The rapid capacity transient decays (tau c less than 15 microseconds) and small deviations of membrane potential (less than 4 mV at peak INa) achieved in these experiments represent good conditions for the study of INa. We studied INa in 26 cells (temperature range 13 degrees-24 degrees C) with 120 or 45 mM [Na+]o and 15 mM [Na+]i. Time to peak INa at 18 degrees C ranged from 1.0 ms (-40 mV) to less than 250 microseconds (+ 40 mV), and INa decayed with a time course best described by two time constants in the voltage range -60 to -10 mV. Normalized peak INa in eight cells at 18 degrees C was 2.0 +/- 0.2 mA/cm2 with [Na+]o 45 mM and 4.1 +/- 0.6 mA/cm2 with [Na+]o 120 mM. These large peak current measurements require a high density of Na+ channels. It is estimated that 67 +/- 6 channels/micron 2 are open at peak INa, and from integrated INa as many as 260 Na+ channels/micron2 are available for opening in canine cardiac Purkinje cells.

Cellular Mechanisms of Early Afterdepolarizationsa

Triggered activity is postulated to cause certain cardiac arrhythmias. The term triggered activity was coined to identify “arrhythmias” triggered by one or more preceding action potentials. Triggered activity is easily distinguishable from reentrant mechanisms because it can be elicited in isolated, small multicellular cardiac preparations and in single cells, experimental conditions where reentry is impossible. Triggered activity differs as well from abnormal (enhanced) automaticity, since the latter is thought to occur spontaneously without the requirement of a trigger. Triggered activity is caused by afterdepohrizatwns. These are low frequency (around 5 Hz) depolarizing oscillations in membrane voltage. They can arise before the repolarization of an action potential is completed (early aftwdt.pOlaViuttwns m EADs) or following its complete repolarization (hhyed afterdepolarizations m DADS). EADs occur only at low stimulation frequencies, and are abolished at higher stimulation frequencies, whereas the opposite is true of DADS. EADs have been most closely identified as the cause of torsades de pointes occurring with long syndrome. EADs are postulated to be one proarrhythmic mechanism of some antiarrhythmic drugs. DADS are thought to underlie some tachyarrhythmias of digitalis intoxication. Both EADs and DADS occur in experimental models of myocardd ischemia and/or r e p e h sion. The cellular mechanism for Ca2+ overload postulated to underlie DADS has been reviewed elsewhere.

Direct measurement of L-type Ca2+ window current in heart cells.

The activation and inactivation relations of several ion channel currents overlap, suggesting the existence of a steady-state or window current. We studied L-type Ca2+ channel window current in single cardiac Purkinje cells using a voltage-clamp protocol by which channels were first inactivated nearly completely during a long-duration depolarizing step, and then the recovery of Ca2+ current was observed during repolarizing steps into the L-type Ca2+ window voltage range. With these conditions, a small-amplitude inward Ca2+ current gradually developed after repolarization to voltages within the window but not after steps to voltages positive or negative to it. Window current was suppressed by Cd2+ (50 microM), nifedipine (1 microM), and nicardipine (1 microM), and it was augmented by isoproterenol (5 microM) and Bay K 8644 (1 microM). At voltages at which window current developed, L-type Ca2+ channels also recovered to a closed state from which they could be reopened by an additional depolarizing step. At voltages positive to the window range, channel recovery to a closed state(s) was absent, whereas at voltages negative to the window range, channel recovery to a closed state(s) increased, as expected from the steady-state inactivation relation. Our results provide direct measurement of L-type Ca2+ window current and distinguish it from other processes, such as slow inactivation. Our findings support the postulate that within a window there occur channel transitions from inactivated to closed states, and these channels (re)open, and this process may occur repetitively. Some physiological and pathophysiological roles for L-type Ca2+ window current are discussed.

New studies of the excitatory sodium currents in heart muscle.

After decades of frustration with inadequate methods, cardiac electrophysiologists have developed new techniques for superior control of membrane potential by use of single cells, and they have begun careful study of cardiac Na+ currents. Direct recordings of the behavior of single Na+ channels have been made by the newly developed patch clamp technique. Biochemists have made excellent progress purifying and characterizing the Na+ channel proteins, and there has been some initial success in reconstituting these partially purified channels into lipid bilayers, where their function can be studied. Even at this early stage of development of these new techniques, several conclusions are warranted: The cardiac Na+ currents are not accurately described by the original Hodgkin-Huxley mathematical formulation, making undesirable the further use of this model for study of cardiac excitation and conduction. We need to keep an open mind as to the kinetic behavior of Na+ channels, until the newer experimental techniques provide a more complete picture. Although the cardiac Na+ channel strongly resembles Na+ channels in other excitable tissues, important differences remain, reinforcing the idea that the detailed molecular structure of the cardiac Na+ channel will be different from its close relatives in other excitable cells. The density of Na+ channels in heart cell membranes is much less than in nerve and fast twitch skeletal muscle. The Na+ channels are the focus of action of many drugs and pathological processes. The tools are at hand for a complete description of the Na+ channel, including its gating and its molecular structure. We can expect considerable progress in this decade.

Isolation and characterization of single canine cardiac purkinje cells.

Single cardiac Purkinje cells should permit improved control of membrane potential during voltage clamp studies. We have developed a method for isolation of single canine Purkinje cells and studied their basic elecrrophysiological properties using conventional single and double microelectrode techniques. The single Purkinje cells appeared free of connective tissue, had regular striations, excluded trypan blue vital stain, and remained quiescent in solutions containing 1.8 mM calcium. Elecrrophysiological studies at 22°C showed normal resting membrane potentials, and action potentials could be elicited by extracellular or intracellular stimulation. Plot of the upstroke velocity of the action potential (Vn,) vs. the holding potential showed a sigmoid curve with the peak mean V, of 167 V/sec, and voltage corresponding to half-maximal V−* was about −70 mV. Plot of the overshoot of the action potential vs. the holding potential was similar, with maximal values of about +30 mV. The mean membrane input resistance was 21 Mil and the mean membrane capacitance was 360 pF. These experiments demonstrate that single Purkinje cells have electrical properties similar to intact Purkinje fibers and that they should be useful for more detailed elecrrophysiological experiments.

Early Afterdepolarizations: Newer Insights into Cellular Mechanisms

Early Afterdepolarizations. Early afterdepolarizations (EADs) are a type of bradycardia‐dependent triggered activity postulated to cause certain cardiac arrhythmias. The cellular mechanism(s) that underlie EADs have, until recently, remained obscure. Recent evidence has shown that EADs arise from a cellular mechanism rather than cell‐to‐cell interactions. At least two types of EADs exist that appear to be separable by voltage‐ (and time‐) dependent properties. EADs that arise near action potential plateau voltages depend critically on L‐type Ca2+ current for depolarization. Studies of Ca2+ currents in single heart cells have provided a basis for explaining the potential roles of L‐ and T‐type Ca2+ currents in EADs. (J Cardiovasc Electrophysiol. Vol. 1. pp. 161–169, April 1990)

L- and T-type Ca2+ channels in canine cardiac Purkinje cells. Single-channel demonstration of L-type Ca2+ window current.

Canine cardiac Purkinje cells contain both L- and T-type calcium currents, yet the single Ca2+ channels have not been characterized from these cells. Additionally, previous studies have shown an overlap between the steady-state inactivation and activations curves for L-type Ca2+ currents, suggesting the presence of L-type Ca2+ window current. We used the on-cell, patch-clamp technique to study Ca2+ channels from isolated cardiac Purkinje cells. Patches contained one or more Ca2+ channels 75% of the time. L-type channels were seen in 69% and T-type channels in 73% of these patches. With 110 mM Ba2+ as the charge carrier, the conductances of the L- and T-type Ca2+ channels were 24.2 +/- 0.8 pS (n = 9) and 9.0 +/- 0.5 pS (n = 8), respectively (mean +/- SEM). With 110 mM Ca2+ as the charge carrier, the conductance of the L-type Ca2+ channel decreased to 9.7 +/- 1.2 pS (n = 4), whereas the T-type Ca2+ channel conductance was unchanged. Voltage-dependent inactivation was shown for both L- and T-type Ca2+ channels, although for L-type Ca2+ channel with Ba2+ as the charge carrier, inactivation took at least 30 seconds at a potential of +40 mV. After channel inactivation was complete, L-type Ca2+ channel reopenings were observed following repolarizing steps into the window voltage range. Thus, our data identify both L- and T-type Ca2+ channels in cardiac Purkinje cells and demonstrate, at the single-channel level, L-type channel transitions expected for a window current. Window current may play an important role in shaping the action potential and in arrhythmogenesis.

Chapter 15 – Pharmacology of the Cardiac Sodium Channel

The cardiac sodium (Na) channel is composed of the α subunit Na V 1.5, a product of the gene SCN5A 1 and associated β subunits. This channel is found predominantly in heart but also occurs in parts of the brain and the gastrointestinal tract. As these designations imply, it is just one of a family of genes encoding voltage-dependent Na channels with structural similarity that are found predominantly in other tissues including skeletal muscle (Na V 1.4) and the brain (Na V 1.1). 2 The α subunit by itself has the major characteristics of the channel such as selectivity for Na + ions, voltage-dependent activation and inactivation, and all of the major drug and toxin binding sites. The different Na channels, however, do not have identical pharmacologic and toxicologic properties. For example, a classic functional signature of the cardiac Na channel is relative insensitivity to block by tetrodotoxin. The channel also has β subunits coded for by separate genes that may influence kinetics and pharmacology.