Robert B. Clark

A Defect in the Kv Channel-Interacting Protein 2 (KChIP2) Gene Leads to a Complete Loss of Ito and Confers Susceptibility to Ventricular Tachycardia

KChIP2, a gene encoding three auxiliary subunits of Kv4.2 and Kv4.3, is preferentially expressed in the adult heart, and its expression is downregulated in cardiac hypertrophy. Mice deficient for KChIP2 exhibit normal cardiac structure and function but display a prolonged elevation in the ST segment on the electrocardiogram. The KChIP2(-/-) mice are highly susceptible to the induction of cardiac arrhythmias. Single-cell analysis revealed a substrate for arrhythmogenesis, including a complete absence of transient outward potassium current, I(to), and a marked increase in action potential duration. These studies demonstrate that a defect in KChIP2 is sufficient to confer a marked genetic susceptibility to arrhythmias, establishing a novel genetic pathway for ventricular tachycardia via a loss of the transmural gradient of I(to).

A Mathematical Model of Action Potential Heterogeneity in Adult Rat Left Ventricular Myocytes

Mathematical models were developed to reconstruct the action potentials (AP) recorded in epicardial and endocardial myocytes isolated from the adult rat left ventricle. The main goal was to obtain additional insight into the ionic mechanisms responsible for the transmural AP heterogeneity. The simulation results support the hypothesis that the smaller density and the slower reactivation kinetics of the Ca(2+)-independent transient outward K(+) current (I(t)) in the endocardial myocytes can account for the longer action potential duration (APD), and more prominent rate dependence in that cell type. The larger density of the Na(+) current (I(Na)) in the endocardial myocytes results in a faster upstroke (dV/dt(max)). This, in addition to the smaller magnitude of I(t), is responsible for the larger peak overshoot of the simulated endocardial AP. The prolonged APD in the endocardial cell also leads to an enhanced amplitude of the sustained K(+) current (I(ss)), and a larger influx of Ca(2+) ions via the L-type Ca(2+) current (I(CaL)). The latter results in an increased sarcoplasmic reticulum (SR) load, which is mainly responsible for the higher peak systolic value of the Ca(2+) transient [Ca(2+)](i), and the resultant increase in the Na(+)-Ca(2+) exchanger (I(NaCa)) activity, associated with the simulated endocardial AP. In combination, these calculations provide novel, quantitative insights into the repolarization process and its naturally occurring transmural variations in the rat left ventricle.

Shal‐type channels contribute to the Ca2+‐independent transient outward K+ current in rat ventricle.

1. The hypothesis that Kv4.2 and Kv4.3 are two of the essential K+ channel isoforms underlying the Ca2+‐independent transient outward K+ current (It) in rat ventricle has been tested using a combination of electrophysiological measurements and antisense technology in both native myocytes and a stably transfected mammalian cell line, mouse Ltk‐ cells (L‐cells). 2. The transient outward currents generated by Kv4.2 channels in L‐cells exhibit rapid activation and inactivation properties similar to those produced by It in rat ventricular cells. The current‐voltage relationships and the voltage dependence of steady‐state inactivation are also very similar in these two preparations. However, the recovery from inactivation of Kv4.2 is much slower (time constant, 378 ms) than that of It in rat ventricular cells (58 ms). 3. The K+ current due to Kv4.2 can be blocked by millimolar concentrations of 4‐aminopyridine in L‐cells; a similar pharmacological response has been observed in rat ventricular myocytes. 4. Quinidine inhibits Kv4.2 in L‐cells and It in rat ventricular cells in a similar fashion. In L‐cells quinidine reduced the amplitude of Kv4.2 and accelerated its time course of inactivation, suggesting that quinidine may act as an open channel blocker of Kv4.2, as has been described for It in rat ventricle. 5. To provide further independent evidence that Kv4.2 and Kv4.3 channel isoforms contribute to It in rat ventricular cells, the effects of 20‐mer antisense phosphorothioate oligodeoxynucleotides directed against Kv4.2 and Kv4.3 mRNAs were examined in ventricular myocytes isolated from 14‐ and 20‐day‐old rats, and in L‐cells. In both preparations, Kv4.2 antisense pretreatment significantly reduced the transient outward K+ current (by approximately 55‐60%). Similar reduction of It was produced by the Kv4.3 antisense oligonucleotide on the 14‐day‐old rat myocytes. 6. In 14‐day rat ventricular cells, combination of Kv4.2 and Kv4.3 antisense oligonucleotides did not produce a significantly larger reduction of It than that observed after pretreatment with either antisense oligonucleotide alone. 7. L‐cells stably transfected with Kv4.2 were treated with Kv4.3 antisense oligonucleotide to evaluate the possibility of cross‐reactivity between Kv4.3 antisense and Kv4.2 mRNA. This antisense treatment produced no change in It, verifying the lack of cross‐reactivity. 8. These biophysical and pharmacological results together with the antisense data show that Kv4.2 and Kv4.3 are essential components of the Ca2+‐independent transient outward K+ current, It, in rat ventricular myocytes.

Role of an inwardly rectifying potassium current in rabbit ventricular action potential.

1. Whole‐cell voltage‐clamp measurements were made of the time‐ and voltage‐dependent properties of the inwardly rectifying background potassium current IK1, in single myocytes from rabbit ventricle. The main goal of these experiments was to define the role of IK1 in the plateau and repolarization phases of the action potential (AP). 2. Action potentials from single ventricular myocytes were used as the command signals for voltage‐clamp measurements. In these ‘action potential voltage‐clamp’ experiments, IK1 was isolated from other membrane currents by taking the difference between control currents and currents in K(+)‐free bathing solution. The results show that IK1 is small during the plateau, but then rapidly increases during repolarization and declines in early diastole. 3. Evidence of an important functional role for IK1 in AP repolarization was obtained by comparing the magnitude of IK1 and the rate of change of membrane potential (dVm/dt) in the same cell during the AP. The time courses of IK1 and dVm/dt during the AP were closely correlated, indicating that IK1 was the principal current responsible for final repolarization. 4. Rectangular voltage‐clamp steps were used to study time‐ and voltage‐dependent changes in IK1 at membrane potentials corresponding to the repolarization phase of the AP. ‘Slow’ relaxations or tail currents, lasting 100‐300 ms, were consistently recorded when the cell was repolarized to potentials in the range ‐30 to ‐70 mV, following depolarizations between +10 and ‐10 mV. 5. The close correlation between the magnitude of the steady‐state IK1 (in an external K+ concentration of 5.4 mM), which was outward for membrane potentials in the range ‐30 to ‐70 mV, and the magnitude of the tail currents, suggests that they resulted from a slow increase, or reactivation, of IK1. 6. The component of the slow tails due to reactivation of IK1 can be separated from a previously described component due to Na(+)‐Ca2+ exchange since the IK1 component: (i) does not depend on the presence of the calcium current, ICa; (ii) can be recorded when internal EGTA (5 mM) suppresses large changes in [Ca2+]i; (iii) does not depend on the Na+ electrochemical gradient; (iv) is abolished in K(+)‐free external solution; and (v) is not present in rabbit atrial myocytes, in which IK1 is very small. 7. The time‐ and voltage‐dependent properties of IK1 revealed by these tail current experiments suggest that the measured magnitude of IK1 will be dependent on the voltage‐clamp protocol.(ABSTRACT TRUNCATED AT 400 WORDS)

A rapidly activating sustained K+ current modulates repolarization and excitation-contraction coupling in adult mouse ventricle.

1 The K+ currents which control repolarization in adult mouse ventricle, and the effects of changes in action potential duration on excitation–contraction coupling in this tissue, have been studied with electrophysiological methods using single cell preparations and by recording mechanical parameters from an in vitro working heart preparation. 2 Under conditions where Ca2+‐dependent currents were eliminated by buffering intracellular Ca2+ with EGTA, depolarizing voltage steps elicited two rapidly activating outward K+ currents: (i) a transient outward current, and (ii) a slowly inactivating or ‘sustained’ delayed rectifier. 3 These two currents were separated pharmacologically by the K+ channel blocker 4‐amino‐pyridine (4–AP). 4–AP at concentrations between 3 and 200 μm resulted in (i) a marked increase in action potential duration and a large decrease in the sustained K+ current at plateau potentials, as well as (ii) a significant increase in left ventricular systolic pressure in the working heart preparation. 4 The current‐voltage (I–V) relation, kinetics, and block by low concentrations of 4–AP strongly suggest that the rapid delayed rectifier in adult mouse ventricle is the same K+ current (Kv1.5) that has been characterized in detail in human and canine atria. 5 These results show that the 4–AP–sensitive rapid delayed rectifier is a very important repolarizing current in mouse ventricle. The enhanced contractility produced by 4–AP (50 μm) in the working heart preparation demonstrates that modulation of the action potential duration, by blocking a K+ current, is a very significant inotropic variable.

A Novel Genetic Pathway for Sudden Cardiac Death via Defects in the Transition between Ventricular and Conduction System Cell Lineages

HF-1 b, an SP1 -related transcription factor, is preferentially expressed in the cardiac conduction system and ventricular myocytes in the heart. Mice deficient for HF-1 b survive to term and exhibit normal cardiac structure and function but display sudden cardiac death and a complete penetrance of conduction system defects, including spontaneous ventricular tachycardia and a high incidence of AV block. Continuous electrocardiographic recordings clearly documented cardiac arrhythmogenesis as the cause of death. Single-cell analysis revealed an anatomic substrate for arrhythmogenesis, including a decrease and mislocalization of connexins and a marked increase in action potential heterogeneity. Two independent markers reveal defects in the formation of ventricular Purkinje fibers. These studies identify a novel genetic pathway for sudden cardiac death via defects in the transition between ventricular and conduction system cell lineages.

THYROID HORMONE REGULATES POSTNATAL EXPRESSION OF TRANSIENT K+ CHANNEL ISOFORMS IN RAT VENTRICLE

1. The ability of thyroid hormone to regulate the postnatal changes of the Ca2+‐independent transient outward K+ current (It) was studied in rat ventricular myocytes. 2. In rat ventricle, It is very small at birth and then increases markedly between postnatal days 8 and 20. The time course of this increase in current density is similar to that of a significant rise in plasma thyroid hormone (T3) levels. 3. During early development, the density of expression of It can be altered by changes in thyroid hormone levels. Eight days after birth the density of It measured at +50 mV in control animals is 2.2 +/‐ 0.4 pA pF(‐1). This value is about 3‐fold larger (6.5 +/‐ 0.8 pA pF(‐1)) in myocytes from age‐matched hyperthyroid animals. When the plasma T3 level in newborn rats is not allowed to increase, or is decreased by making animals hypothyroid, this age‐dependent increase in It fails to occur. 4. Using RNase protection assays, Kv4.2 and Kv4.3 mRNA levels were measured in ventricular tissues obtained from age‐matched 8‐day‐old control and hyperthyroid rats. In hyperthyroid animals, where an approximately 3‐fold increase in It was identified, increases in the mRNA levels for Kv4.2 and Kv4.3 were 1.6‐fold and 2.6‐fold, respectively. 5. These results show that thyroid hormone can regulate the development of It in rat ventricle. Direct measurements of It density and mRNA levels as a function of development and thyroid hormone levels also strongly suggest that the Kv4.2 and Kv4.3 channels are essential components of It in rat ventricular cells.

Functional properties of K + currents in adult mouse ventricular myocytes

Although the K+ currents expressed in hearts of adult mice have been studied extensively, detailed information concerning their relative sizes and biophysical properties in ventricle and atrium is lacking. Here we describe and validate pharmacological and biophysical methods that can be used to isolate the three main time‐ and voltage‐dependent outward K+ currents which modulate action potential repolarization. A Ca2+‐independent transient outward K+ current, Ito, can be separated from total outward current using an ‘inactivating prepulse’. The rapidly activating, slowly inactivating delayed rectifier K+ current, IKur, can be isolated using submillimolar concentrations of 4‐aminopyridine (4‐AP). The remaining K+ current, Iss, can be obtained by combining these two procedures: (i) inactivating Ito and (ii) eliminating IKur by application of low concentration of 4‐AP. Iss activates relatively slowly and shows very little inactivation, even during depolarizations lasting several seconds. Our findings also show that the rate of reactivation of Ito is more than 20‐fold faster than that of IKur. These results demonstrate that the outward K+ currents in mouse ventricles can be separated based on their distinct time and voltage dependence, and different sensitivities to 4‐AP. Data obtained at both 22 and 32°C demonstrate that although the duration of the inactivating prepulse has to be adapted for the recording temperature, this approach for separation of K+ current components is also valid at more physiological temperatures. To demonstrate that these methods also allow separation of these K+ currents in other cell types, we have applied this same approach to myocytes from mouse atria. Molecular approaches have been used to compare the expression levels of different K+ channels in mouse atrium and ventricle. These findings provide new insights into the functional roles of IKur, Ito and Iss during action potential repolarization.

Role of sodium-calcium exchange in activation of contraction in rat ventricle

1. The functional role of reverse Na(+)‐Ca2+ exchange in the activation of contraction of rat ventricular myocytes has been studied. Mechanical activity of single cells, measured as unloaded cell shortening, was recorded simultaneously with membrane current and voltage using a single microelectrode voltage clamp and a video edge detection device. 2. The voltage dependence of contraction was studied by applying trains of depolarizations. At test potentials between +20 and +80 mV (under conditions where large outward currents were activated) a plateau on the shortening vs. voltage (S‐V) relationship was observed. Significant cell shortening also occurred at test potentials between ‐70 and ‐40 mV; and these contractions were accompanied by large inward Na+ currents. We have investigated the ionic mechanisms for three components of the S‐V relation in rat ventricle: (i) shortening which occurs between ‐70 and ‐40 mV and is thought to be dependent on the sodium current; (ii) phasic contractions in the voltage range ‐40 to +40 mV where the L‐type Ca2+ current is present; (iii) the plateau of the S‐V relation at strongly depolarized voltages where reverse Na(+)‐Ca2+ exchange may occur. 3. Experiments in which two independent microelectrode impalements were made in a single myocyte showed that during activation of contraction at test potentials between ‐70 and ‐40 mV, and during very large depolarizations (+20 to +80 mV), there were significant deviations of the measured membrane potential from the applied voltages. Activation of cell shortening in these voltage ranges could be eliminated by electronic series resistance compensation, which significantly reduced these voltage errors. Consistent with these findings, when tetrodotoxin (TTX) and 4‐aminopyridine (4‐AP) were used to block inward Na+ and transient outward K+ currents, respectively, no significant voltage errors were present and a bell‐shaped shortening‐voltage (S‐V) relationship was obtained. 4. When Na+ and K+ currents were blocked, depolarizations from holding potentials of either ‐80 or ‐50 mV demonstrated that the threshold for activation of contraction was about ‐30 mV, and that the voltage dependence of peak shortening was very similar to that of the L‐type Ca2+ current (ICa,L). These contractions were suppressed completely by either Cd2+ or ryanodine, showing that activation of cell shortening was due to Ca2+ influx through L‐type channels which induced release of Ca2+ from the sarcoplasmic reticulum (SR). No T‐type calcium currents were observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Properties of the transient outward current in rabbit atrial cells.

1. Whole‐cell and patch clamp techniques have been used to study the steady‐state voltage dependence and the kinetics of a transient outward current, It, in single cells from rabbit atrium. 2. The steady‐state voltage dependence of both activation and inactivation of It are well described by Boltzmann functions. Inactivation is fully removed at potentials negative to ‐70 mV and it is complete near 0 mV. The threshold for activation of It is near ‐30 mV and it is fully activated at +30 mV. The region of overlap between the activation and inactivation curves indicates that a steady non‐inactivating current will be recorded over a membrane potential range from approximately ‐30 to 0 mV. 3. In general, the time course of inactivation at potentials in the range 0 to +50 mV is best described as a sum of two exponential functions. The kinetic parameters controlling these processes exhibit only very weak voltage dependence. 4. Comparison of the time course of the development of inactivation in response to long depolarizing voltage clamp steps with the development of inactivation in response to trains of brief depolarizing pulses indicates that inactivation develops very quickly and decays relatively slowly at potentials near the resting potential (e.g. ‐70 mV). Thus, in response to (i) a train of voltage‐clamp pulses or (ii) a series of action potentials, the magnitude of It decreases due to a progressive increase in the amount of inactivation. 5. A simple model of channel gating is presented: it can account for the major aspects of the voltage dependence and kinetics of It (cf. Aldrich, 1981). 6. Cell‐attached patch clamp recordings have been used to identify the single‐channel or unitary events underlying the current, It. In general, only one active channel is present per patch. The single‐channel conductance in normal Tyrode solution is approximately 14 pS and the current‐voltage relationship is approximately linear between +50 and +150 mV with respect to rest. This information, in combination with the fully activated current‐voltage characteristics from the whole‐cell data, can be used to estimate the number and density of It channels per cell: these are 1600 and one per 3‐4 micron 2, respectively. 7. Ensemble averages obtained from patch recordings are very similar in time course to the macroscopic or whole‐cell current itself: the ensemble current rises to a peak within approximately 5 ms and decays with a biexponential time course in response to depolarizations to approximately +50 mV.(ABSTRACT TRUNCATED AT 400 WORDS)