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Phenotypic differences in transient outward K⁺ current of human and canine ventricular myocytes: insights into molecular composition of ventricular I_to

¹Institute of Molecular Cardiobiology, Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; and ²Cardiovascular Research Group, Department of Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

Submitted 16 July 2003 ; accepted in final form 25 September 2003

	ABSTRACT

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The Ca²⁺-independent transient outward K⁺ current (I_to) plays an important electrophysiological role in normal and diseased hearts. However, its contribution to ventricular repolarization remains controversial because of differences in its phenotypic expression and function across species. The dog, a frequently used model of human cardiac disease, exhibits altered functional expression of I_to. To better understand the relevance of electrical remodeling in dogs to humans, we studied the phenotypic differences in ventricular I_to of both species with electrophysiological, pharmacological, and protein-chemical techniques. Several notable distinctions were elucidated, including slower current decay, more rapid recovery from inactivation, and a depolarizing shift of steady-state inactivation in human vs. canine I_to. Whereas recovery from inactivation of human I_to followed a monoexponential time course, canine I_to recovered with biexponential kinetics. Pharmacological sensitivity to flecainide was markedly greater in human than canine I_to, and exposure to oxidative stress did not alter the inactivation kinetics of I_to in either species. Western blot analysis revealed immunoreactive bands specific for Kv4.3, Kv1.4, and Kv channel-interacting protein (KChIP)2 in dog and human, but with notable differences in band sizes across species. We report for the first time major variations in phenotypic properties of human and canine ventricular I_to despite the presence of the same subunit proteins in both species. These data suggest that differences in electrophysiological and pharmacological properties of I_to between humans and dogs are not caused by differential expression of the K channel subunit genes thought to encode I_to, but rather may arise from differences in molecular structure and/or posttranslational modification of these subunits.

potassium channels; transient outward current; Kv channel-interacting protein; repolarization

Ventricular I_to is highly regulated both temporally and regionally in normal hearts (35) and is significantly modulated in a wide variety of structural heart diseases (23). For instance, action potential duration (APD) prolongation in heart failure is associated with a concomitant reduction of the phase 1 repolarizing notch of the action potential, which is inscribed by I_to (15). In ischemia, I_to is thought to play a critical role in the protection of epicardial cells against metabolic inhibition by contributing to abrupt shortening of epicardial APD (22). Moreover, regional and transmural differences in the balance between I_to and Na⁺ current (I_Na) may underlie ventricular fibrillation and sudden death in patients with inherited arrhythmias such as Brugada syndrome (9, 40). Finally, inhibition of I_to by several antiarrhythmic agents such as flecainide, quinidine, and propafenone contributes to both their therapeutic and proarrhythmic effects (24, 33, 42). Therefore, defining the molecular identity and electrophysiological properties of ventricular I_to is essential for a comprehensive understanding of arrhythmia mechanisms in a wide variety of pathologies and may aid in the proper choice and design of antiarrhythmic drug therapy.

Measurements of human I_to require the availability of explanted hearts from patients with heart failure or nonfailing donor hearts that are deemed technically unsuitable for transplantation. Because of the difficulties associated with obtaining appropriate human cardiac samples and the confounding complexities associated with disease etiology and therapy in humans, animal models of cardiovascular disease, especially in dog, have been widely used. The usefulness of these models is dependent on their mimicry of human physiology and disease. Therefore, the exact identity of human I_to and its variation from the canine counterpart must be investigated in detail.

Kv4.3 has emerged as the leading candidate K⁺ channel gene likely to encode cardiac I_to in large mammals such as dogs and humans (10, 14, 32, 34, 44). Moreover, Kv4 subunits may form heteromeric complexes with a class of Kv channel-interacting proteins, K⁺ channel accessory proteins (KChIPs), in vivo (2, 5, 6, 8, 28, 31). Although KChIP2 significantly modulates Kv4.3 function when expressed in heterologous systems (5, 6, 8, 28), it remains unknown whether KChIP2 or other accessory subunits, such as KChAP (1, 18, 37) or Kv (8, 19, 41), merely act to increase the sarcolemmal expression of Kv4.3 or, in fact, directly participate in the formation of native ventricular I_to.

In the present study, the electrophysiological and pharmacological properties of human and canine I_to were compared. Human ventricular I_to differs appreciably from canine I_to in both steady-state inactivation and recovery from inactivation. Moreover, differences in the pharmacological profiles of human and canine I_to suggest differences in the molecular architecture of the channel pore in these two species. Western blots of human and canine tissues demonstrate expression of Kv4.3, Kv1.4, and KChIP2 in both species but with distinctive variations in apparent molecular weights. Such differences may underlie the heterogeneity of the biophysical fingerprint of I_to across species. Functional differences in human and canine ventricular I_to may be attributable to intrinsic variations in Kv4.3 between dog and human, functional expression of other, yet to be identified - or -subunits, and/or differences in posttranscriptional or translational modification of the two isoforms.

	METHODS

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Human ventricular myocardium was obtained from explanted failing hearts (n = 5) and donor hearts deemed unsuitable for transplantation (n = 2). Canine ventricular myocardium was obtained from adult male mongrel dogs (n = 5) weighing 20–25 kg. Segments of ventricular myocardium adjacent to the region from which myocytes were isolated for electrophysiological recordings were used for protein isolation. The tissue was excised from the left ventricular free wall between the left anterior descending coronary artery (LAD) and the left circumflex artery. The myocyte and protein isolation procedures selected for cells and tissue lysates from the midportion of the ventricular wall. Tissue samples were quick frozen in liquid nitrogen within 10–15 min after organ harvest and stored at –80°C until further processing. All procedures were performed according to the guidelines of the American Physiological Society and were approved by the Animal Care and Use Committee of Johns Hopkins University.

Isolation of human and canine myocytes. Human and canine midventricular myocytes were isolated from the anterior wall of the left ventricle of seven humans (5 failing, 2 normal) and five dogs by perfusion of a diagonal branch of the LAD with a nominally Ca²⁺-free solution containing collagenase and protease, as described previously (13, 15, 26, 27). Myocytes were stored at room temperature (22–23°C) in Tyrode solution consisting of (in mM) 130 NaCl, 4.5 KCl, 5 MgCl₂, 23 HEPES, 21 glucose, 20 taurine, 5 Na pyruvate, and 20 2,3-butanedione monoxime, pH 7.4. The concentration of CaCl₂ was gradually raised from 100 µM to 2 mM. Only rod-shaped cells exhibiting clear cross striations and no spontaneous contractions were selected for electrophysiological study. A total of 22 human and 21 canine myocytes were used in this study.

The objective of this work was to investigate phenotypic differences in I_to across species. Because previously we (14) and others (4) had demonstrated that the biophysical properties of I_to were not altered in human heart failure, we combined the electrophysiological data from cells of normal and failing human hearts because of the limited availability of normal human samples.

Electrophysiological measurements. Current recordings were obtained at 24°C with the patch-clamp technique in whole cell configuration as previously described (6, 7). Glass pipettes were prepared to have a final tip resistance of 2–3 M when filled with internal solution containing (in mM) 120 K⁺-glutamate, 10 KCl, 10 HEPES, 5 EGTA, and 5 MgATP. pH was adjusted to 7.2 with KOH, yielding a final K⁺ concentration of 140 mM. Cells were perfused with Tyrode solution containing (in mM) 140 NaCl, 5 KCl, 1 MgCl₂, 2 CaCl₂, 10 HEPES, and 10 glucose, adjusted to pH 7.4. CdCl₂ (0.3 mM) was added to the bath solution to eliminate voltage-activated L-type Ca²⁺ and Ca²⁺-dependent currents. Cell capacitance was estimated by integrating the area under an uncompensated depolarizing step of 10 mV from a holding potential of –80 mV. Whole cell currents were elicited with a family of depolarizing voltage steps (–40 to +80 mV) from a holding potential of –50 mV. Currents were low-pass filtered at 2 kHz and digitized at 10 kHz for offline analysis. I_to was defined as the difference between the peak transient current and the steady-state current at the end of a 500-ms voltage clamp pulse. Current inhibition by flecainide (Sigma-Aldrich, St. Louis, MO) was measured as the percent decrease in peak current after subtraction of the steady-state current.

Whole cell currents were analyzed with custom-developed software. Pooled data are expressed as means ± SE. Statistical comparisons were performed with the unpaired Student's t-test. Differences in data were considered significant at P < 0.05.

Western blot analysis. Left ventricular samples from normal (n = 2) and failing (n = 5) human hearts and normal canine hearts (n = 5) were rapidly frozen in liquid nitrogen after organ harvest. Proteins were prepared as previously described (7, 14). Samples were run in duplicate or triplicate on 10%, 12.5%, or 15% Tris·HCl precast gels (Bio-Rad, Hercules, CA) in 25 mM Tris, 192 mM glycine, and 0.1% (wt/vol) SDS running buffer. A standard control sample was also run on all gels to allow for comparisons across gels. Primary antibody incubations were performed overnight at 4°C with rabbit anti-KChIP (Wyeth-Ayerst Research, Princeton, NJ), KChIP2S/T (6), Kv1.4 (Alamone Labs, Jerusalem, Israel), and Kv4.3 (Chemicon, Temecula, CA) polyclonal antibodies, as described previously (14). Secondary horseradish peroxidase-conjugated antibodies were obtained from Jackson ImmunoResearch (West Grove, PA). Membranes were exposed and developed with enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer's instructions.

	RESULTS

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Species-dependent differences in activation properties. I_to was activated by a series of depolarizing voltage steps (–40 to +80 mV in 10-mV increments) from a holding potential of –50 mV, followed by a 10-ms I_Na inactivating prepulse to –50 mV. Shown in Fig. 1A are representative whole cell currents recorded from human (top) and canine (bottom) myocytes. Activation of I_to in both species was comparably rapid, as peak current was achieved in <20 ms (Fig. 1A). Average peak current densities in selected myocytes from normal and failing human hearts and canine myocytes are given in Table 1. In general, I_to density was highest in myocytes of normal canine hearts, followed by myocytes of normal and failing human hearts. Importantly, although robust I_to was recorded in all (100%) normal human and canine myocytes studied, only a fraction (40%) of myocytes from failing human hearts expressed appreciable I_to density suitable for detailed electrophysiological analysis. Only failing human myocytes with I_to density of at least 5 pA/pF were studied.

View larger version (31K): [in this window] [in a new window]   Fig. 1. Electrophysiological properties of transient outward K+ current (Ito). A: representative Ito tracings recorded from cells isolated from the left ventricular midwall of human and canine hearts. Currents were elicited by 500-ms depolarizing voltage steps (–40 to +60 mV) from a holding potential of –50 mV. B: inactivation time constants in human and canine myocytes. Time constants were obtained by fitting the current decay phase to a monoexponential function. C: voltage dependence of Ito activation in human and canine myocytes. D: steady-state inactivation curves for human and canine Ito.

As shown in Fig. 1C, the current-voltage relationships of human and canine I_to were linear-positive to the threshold potentials for current activation. The average slope conductances (from 0 to +60 mV) were 7.3 ± 1.8 and 10.9 ± 2.5 pA·pF^–1·mV^–1 (P < 0.01) in human and canine myocytes, respectively. The threshold potential for I_to activation was more depolarized in human (–10 to 0 mV) than canine (–20 to –10 mV) myocytes (Fig. 1C).

Species-dependent differences in inactivation properties. Time constants of I_to decay (_h) were determined by fitting the falling phase of the whole cell current to a monoexponential function. At test potentials >0 mV, _h was independent of voltage (Fig. 1B). Interestingly, _h was significantly shorter (by 40%) in canine (41.1 ± 7.4 ms at +50 mV) than human (61.4 ± 1.7 ms, P < 0.01) myocytes (Fig. 1B, Table 1).

Species-dependent differences in steady-state inactivation of I_to. The steady-state availability of I_to was assessed with a standard two-pulse protocol as shown in Fig. 1D. The half-maximal potential for steady-state inactivation (V_1/2) of human myocytes isolated from normal ventricles (–27.3 ± 1.1 mV) was not significantly different from that of myocytes isolated from failing hearts (–27.7 ± 0.5 mV). However, the steady-state inactivation of I_to in canine myocytes was significantly (P < 0.001) shifted in the hyperpolarized direction compared with human (normal and failing) myocytes (V_1/2 = –37.4 ± 1.1 mV). Finally, similar to our previous findings (14) in canine myocytes isolated from normal and failing hearts, myocytes from normal (4.7 ± 0.3 mV^–1) and failing (4.7 ± 0.4 mV^–1) human hearts also exhibited identical slopes of their steady-state inactivation curves. Importantly, the slope of steady-state inactivation in human myocytes was significantly greater than that in canine myocytes (4.2 ± 0.1 mV^–1).

Species-dependent differences in recovery from inactivation of I_to. Human and canine I_to exhibited distinctive time courses of recovery from inactivation. The recovery kinetics of both human and canine I_to were measured after a 500-ms depolarizing voltage step to +50 mV from a holding potential of –100 mV and a subsequent 10-ms I_Na inactivating prepulse to –40 mV. Myocytes from both normal and failing human ventricles recovered rapidly and almost completely (98.8 ± 1.6%) within 250 ms (Fig. 2). The recovery of human I_to followed a monoexponential time (₁) course with time constants that did not differ in myocytes isolated from normal (₁ = 27.8 ± 6.3 ms at –100 mV) and failing (₁ = 21.3 ± 2.6 ms) human hearts (P = 0.7, not significant). In contrast, canine I_to exhibited a biexponential recovery from inactivation. The fast and slow time constants for canine I_to recovery were 69.1 ± 13.5 ms (40.5 ± 6% of peak I_to) and 316 ± 44 ms (59.4 ± 11% of peak I_to), respectively. As illustrated in Fig. 2C, 90% of the current peak was consistently recovered within 1 s of the initial depolarizing voltage step.

View larger version (24K): [in this window] [in a new window]   Fig. 2. Recovery kinetics of human and canine Ito. Recovery from inactivation is measured after a variable interpulse interval from a holding potential of –100 mV. A: human Ito recovers rapidly with a monoexponential time course. B: canine Ito recovers more slowly and is best fitted with a biexponential function. C: summary data of the time course of recovery of both human and canine currents. P2/P1, ratio of Ito density for second test pulse (P2) over first test pulse (P1).

Species-dependent differences in pharmacological sensitivity of I_to. Flecainide (to block Kv4.3-encoded current) and H₂O₂ (to alter the inactivation kinetics of Kv1.4-encoded current) have been used to determine the contribution of these two K⁺ channel subunits to native atrial I_to (12, 36, 39). We assessed the contribution of Kv4.3 vs. Kv1.4 to native ventricular I_to in dogs and humans by investigating the differential sensitivity of the current in both species to flecainide and H₂O₂. Flecainide (10 µM) produced a significantly larger reduction in peak current density of I_to in human (49%) compared with canine (18%) myocytes (Fig. 3). H₂O₂ (0.01%), on the other hand, produced no measurable effect on the time course of inactivation of human or canine ventricular I_to (Fig. 4). Also shown in Fig. 4B are action potentials recorded from a canine myocyte before (control) and after exposure to H₂O₂. Although H₂O₂ resulted in a trend toward an increase in APD, there was no change in the amplitude of the phase 1 notch. Together, these data suggest that Kv1.4 does not contribute appreciably to ventricular I_to in either species, as it encodes a current that is highly sensitive to H₂O₂.

View larger version (15K): [in this window] [in a new window]   Fig. 3. Effect of flecainide on Ito from myocytes isolated from canine and human hearts. Ito elicited in a representative human myocyte (left) and a representative canine myocyte (right) by a 500-ms voltage step to +50 mV in control conditions or in the presence of 10 µM flecainide.

View larger version (26K): [in this window] [in a new window]   Fig. 4. Effects of H2O2 (0.01%) exposure for 10 min on Ito in human and canine myocytes. A: representative current tracings and summary data of the inactivation time constants of human and canine Ito in control and with 0.01% H2O2. B: effect of 0.01% H2O2 on the action potential in canine myocytes. Effect of H2O2 on Ito steady-state inactivation (C) and recovery from inactivation (D) in canine myocytes are also shown. h, Time constant of Ito decay.

Subunit composition of human and canine I_to. We sought to determine the molecular basis for the differential expression of I_to in human and canine ventricular myocardium. As shown in Fig. 5, immunoreactive Kv4.3, Kv1.4, and KChIP2 proteins are expressed in the left ventricles of both species. Kv4.3 in humans is present at a predominant band of 75 kDa, which is larger than that in canine myocardium (70 kDa). In contrast, the predominant band specific for Kv1.4 in canine ventricles (94 kDa) is appreciably larger than that in humans (86 kDa). Finally, KChIP2, along with several splice variants including KChIP2S and KChIP2T, is also abundantly expressed in both human and canine ventricular myocardium at the nucleotide level (6). In human myocardium, KChIP2 was consistently detected as a single specific band that ran at 35 kDa, whereas in dog two bands were typically observed at 35 and 25 kDa (Fig. 5).

View larger version (50K): [in this window] [in a new window]   Fig. 5. Representative Western blots of Kv4.3, Kv1.4, and Kv channel-interacting protein (KChIP)2 protein expression in canine (C) and human (H) left ventricular muscle. Human and canine KChIP2 runs between 25 and 35 kDa. Human Kv1.4 appears at 86 kDa, compared with canine Kv1.4 at 94 kDa. Kv4.3 in humans is present at a predominant band of 75 kDa, which is larger than that in canine myocardium (70 kDa).

	DISCUSSION

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Species-specific differences in expression of various voltage-gated K⁺ channels have been described among dogs, rabbits, ferrets, rats, and mice (25). I_to exhibits characteristic regional and transmural differences in current density, kinetics, and pharmacological properties across the ventricular free wall in both humans and dogs (16, 17, 20–22, 36). However, electrophysiological properties of ventricular I_to between the two species have not been compared previously under identical experimental conditions.

Human ventricular I_to was found to differ significantly from its canine counterpart in voltage dependence and kinetics of inactivation and rate of current decay. For example, the V_1/2 of human myocytes was significantly more depolarized than canine ventricular I_to. The response of sarcolemmal currents such as I_to to membrane depolarization may have significant implications for a wide variety of human diseases, notably ischemia, in which membrane depolarization is a well-established feature. Because our data suggest that inactivation of human I_to is relatively resistant to membrane depolarization compared with the canine isoform, caution must be exercised when extrapolating the effects of ischemia on I_to in canine experimental models.

I_to recovery from inactivation differs significantly between canine and human myocytes. In canines, recovery from inactivation of I_to is described by two exponential components. The fast component has a time constant of 70 ms, whereas the time constant of the slow component is over fourfold greater (>300 ms). Human ventricular I_to follows a monoexponential time course of recovery, which is significantly (by 61%) faster than the fast component in dogs, consistent with greater availability of human compared with canine I_to under similar recording conditions. Differences in the recovery kinetics of I_to are likely to have strong clinical importance. For example, recent reports have elegantly implicated I_to in the mechanism of arrhythmias associated with Brugada syndrome, whereby phase 2 reentrant excitation could occur between cells with recovered I_to and those with inactive I_to (3, 9).

Both Kv4.3 and Kv1.4 proteins are expressed in the myocardium of large mammals (29). Flecainide and H₂O₂ have been used to determine the contribution of these two K⁺ channel subunits to native I_to (12, 36, 39). Although flecainide (low dose, 10 µM) suppresses peak Kv4.x current (52% mean reduction), it has little effect on Kv1.4 (only 11% mean reduction) when expressed in mammalian cells (30, 43). In our experiments, flecainide reduced the peak current density of human ventricular I_to without affecting the kinetics of its recovery from inactivation. In contrast, flecainide had a relatively diminished effect on canine ventricular I_to density. Unlike flecainide, oxidative stress did not affect the inactivation kinetics of either isoform, suggesting that Kv1.4 is not a significant contributor to human or canine ventricular I_to.

To further investigate differences in the molecular identity of human and canine I_to, Western blots with antibodies recognizing Kv4.3, Kv1.4, and KChIP2 were performed. Interestingly, all three subunits are expressed in the ventricles of humans and dogs. However, despite the presence of Kv1.4 mRNA (not shown) and protein (Fig. 5) in both human and canine ventricular myocardium, the electrophysiological properties of the currents and their sensitivity to flecainide and resistance to H₂O₂ argue against a significant role for this subunit in native ventricular I_to channels. Finally, the differential sensitivity of canine and human I_to to flecainide and the significant variation (by 5 kDa) in the molecular mass of Kv4.3 between dog and human, suggests a difference in the primary structure or posttranslational modification of the subunit, which may underlie the distinct species-specific electrophysiological and pharmacological properties.

It appears most likely that Kv4.3 serves as the major subunit underlying ventricular I_to in both human and canine ventricular myocardium, because Kv4.2 is not present in either species at the mRNA level (data not shown). However, a number of ancillary subunits are known to modify Kv4.3 function. KChIP2 is a Ca²⁺-binding accessory subunit expressed in the heart that increases expression and dramatically slows current decay of all Kv4 isoforms. Moreover, KChIP2 significantly shifts the voltage dependence of inactivation of expressed Kv4.2 (2) but not human Kv4.3 currents (6). The role of this accessory subunit in the formation of native I_to remains incompletely understood. We (6) previously demonstrated protein expression of KChIP2 splice variants in canine and human ventricular myocytes from both normal and failing hearts. However, heterologous expression of Kv4.3 with any combination of the KChIP2 splice variants does not fully reproduce the electrophysiological properties of native I_to (6). Therefore, more investigation is required to determine whether KChIP2 or other, yet to be identified accessory subunits interact with Kv4.x to yield native I_to channels.

Limitations. A reduction of I_to is generally observed in human and animal models of heart failure. Downregulation of I_to may be due, at least in part, to neurohormonal modulation of the current or by direct alterations in - or -subunit expression in the heart. The comparisons in this study are limited by the low availability of normal human myocardium.

Transmural heterogeneity of ion channel expression, including I_to, across the ventricular wall, has been described in many species. Repolarizing K⁺ currents in dogs are also larger in the right than the left ventricle. By design, this study was limited to myocytes obtained from the midwall of the anterior left ventricle of human and canine hearts and did not examine myocytes from epicardial or endocardial layers or other regions. Although our data provide evidence for the lack of involvement of Kv1.4 in the composition of I_to, we cannot definitively rule out the participation of Kv1.4, because an alternative mechanism(s) may protect native I_to against oxidative stress.

Although the use of Cd²⁺ to block L-type Ca²⁺ current shifts the steady-state inactivation of I_to in the depolarizing direction, differences in the voltage dependence and availability of I_to observed in this study are not caused by differences in external divalent cations, because identical recording conditions were used in cells from both species. The tissue and cell isolation methods were also similar across species.

In conclusion, human and canine ventricular isoforms of I_to differ significantly in their voltage dependence and kinetics of inactivation. The variant electrophysiological and pharmacological profiles of human and canine ventricular I_to suggest differences in the molecular constituents of these channels. The relatively rapid recovery kinetics of both currents and the absence of oxidant sensitivity argue against Kv1.4 as a major component of the I_to channel, despite its presence at the immunoreactive protein level. Our data are most consistent with Kv4.3 as the principal subunit in both species, although we cannot conclusively rule out the contribution of other, yet to be identified subunits or factors. The appreciable differences in the function of both isoforms may result from incorporation of different ancillary subunits in the holochannel, differences in posttranslational modification of one or more of the subunits, or even intrinsic differences in human and canine Kv4.3 genes.

	FOOTNOTES

 

Address for reprint requests and other correspondence: G. F. Tomaselli, Inst. of Molecular Cardiobiology, Johns Hopkins Univ. School of Medicine, 720 Rutland Ave., Ross 844, Baltimore, MD 21205 (E-mail: gtomasel{at}jhmi.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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