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(Circulation Research. 2003;93:541.)
© 2003 American Heart Association, Inc.

Cellular Biology

Changes in the Ca²⁺-Activated K⁺ Channels of the Coronary Artery During Left Ventricular Hypertrophy

Nari Kim, Joonyong Chung, Euiyong Kim, Jin Han

From the Departments of Physiology & Biophysics (N.K., E.K., J.H.) and Parasitology (J.C.), College of Medicine, Inje University, Busan, Korea.

Correspondence to Jin Han, MD, PhD, Department of Physiology & Biophysics, College of Medicine, Inje University, 633-165 Gaegeum-Dong, Busanjin-Gu, Busan 613-735, Korea. E-mail phyhanj{at}ijnc.inje.ac.kr

	Abstract

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It has been suggested that impairment of smooth muscle cell (SMC) function by alterations in the Ca²⁺-activated K⁺ (K_Ca) channels accounts for the reduction in coronary reserve during left ventricular hypertrophy (LVH). However, this hypothesis has not been fully investigated. The main goal of this study was to assess whether the properties of K_Ca channels in coronary SMCs were altered during LVH. In patch-clamp experiments, the whole-cell currents of the K_Ca channels were reduced during LVH. The unitary current amplitude and open probability for the K_Ca channels were significantly reduced in LVH patches compared with control patches. The concentration-response curve of the K_Ca channel to [Ca²⁺]_i was shifted to the right. Inhibition of the K_Ca channels by tetraethylammonium (TEA) was more pronounced in LVH cells than in control cells. Western blot analysis indicated no differences in K_Ca channel expression between the control and LVH coronary SM membranes. In contraction experiments, the effect of high K⁺ concentration on the resting tension of the LVH coronary artery was greater than on that of the control. The effect of TEA on the resting tension of the LVH coronary artery was reduced compared with the effect on the control. Our findings imply a novel mechanism for reduced coronary reserve during LVH.

Key Words: left ventricular hypertrophy • reduced coronary reserve • K_Ca channels • smooth muscle cells

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ca²⁺-activated K⁺ (K_Ca) channels are very abundant in smooth muscle cells (SMCs), in which they play an important role in the regulation of arterial tone and vascular resistance. Membrane depolarization and Ca²⁺ influx are proposed to activate the K_Ca channels, which act as a negative-feedback mechanism to limit depolarization, L-type Ca²⁺ channel activation, and contraction.^1,2 Accordingly, the inhibition of K_Ca channels by pharmacological blockers produces vasoconstriction of pressurized arteries and increases the resting tension of isometric arterial preparations, thereby providing strong evidence of channel involvement in the regulation of basal tone.^2–4

During left ventricular hypertrophy (LVH), the reduced coronary reserve enhances the susceptibility of the myocardium to ischemic injury,^5–7 which is a major risk factor for sudden cardiac death. Most of the studies on reduced coronary reserve demonstrate endothelial dysfunction, increased coronary arteriolar tone, structural alterations to the intramyocardial arterioles, increased perivascular fibrosis, increased extravascular compressive forces, and inadequate neoangiogenesis.^8–12 In contrast to these studies, it has been reported that LVH (with the exception of severe LVH) is not always associated with vasculature alterations.^13,14

The goal of this study was to combine patch-clamp and Western blot methods with isometric contraction experiments to compare the levels of K_Ca channel current, protein expression, and the contractility of the coronary arteries in control and LVH specimens.

In the present study, we demonstrate changes in the properties of the K_Ca channels in coronary arterial SMCs during LVH. Our findings indicate that LVH substantially alters the properties of K_Ca currents in coronary arterial SMCs, which may contribute to the reduced coronary reserve in LVH.

	Materials and Methods

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Animals
All of the experimental procedures were approved by the Institutional Animal Care and Use Committee and were conducted in conformity with institutional guidelines. All animals were obtained from Hyochang Sciences (Daegu, Korea). New Zealand White rabbits (0.8 to 1.0 kg) and Sprague-Dawley rats (300 to 400 g) were randomly selected to receive either an injection of isoproterenol (300 µg/kg body weight) or an equal volume of 0.9% saline (1 mL/kg body weight). The animals developed LVH 10 days after injection.^15,16

Patch-Clamp Recording of K_Ca Currents
Enzymatic isolation of single coronary SMCs was performed according to published methods.^17,18 The whole-cell and excised-patch configurations of the patch-clamp recordings were obtained as previously described.¹⁹ Briefly, families of macroscopic K⁺ currents were generated by stepwise 10-mV depolarizing pulses from a holding potential of -60 mV in cells that were dialyzed with 0.1 µmol/L ionized Ca²⁺. The peak current that was elicited at a single membrane potential was defined as the average of 500 sample points encompassing the maximal current point. Single-channel K_Ca currents were obtained in symmetrical 145 mmol/L KCl and subjected to membrane potentials of 0 to +50 mV. Averaged current amplitudes were obtained for the calculation of single-channel conductance. The open state was defined as 50% of the single-channel levels, and NP_O was calculated to obtain the voltage relationships at different [Ca²⁺]_i levels.²⁰

Force Recording of Coronary SM
The specimen (10 mm from the origin of the proximal portions of the left anterior descending arteries) was placed in physiological salt solution (PSS), and excess connective tissue was removed.¹⁸ The preparation was cut into rings of approximately 1 mm and placed in aerated (95% O₂, 5% CO₂) PSS buffer, which was maintained at 37°C. The vessels were loaded with a passive tension of 1 g and subjected to a 90-minute equilibration period. Changes in vascular tone were detected by computer-interfaced force transducers (FT-03) at a sampling rate of 6 per minute (0.1 Hz).

Western Blot Analysis
SDS-PAGE (12% separating gels) and immunoblotting were performed for samples from five control and five LVH rat hearts after routine protocols. For each lane, 10 µg of the total protein was loaded. The membranes were incubated with a 1:100 dilution of either the MaxiK or MaxiKß antibody, followed by incubation with a 1:1000 dilution of the secondary antibody in the wash buffer. The bound antibody was detected by chemiluminescence (ECL; Amersham), and the signals were analyzed by scanning the films with a flatbed office scanner and evaluating the band intensities using NIH Image software.

Statistics
The experimental data are expressed as the mean±SEM of n experiments. Statistically significant differences were estimated using the unpaired Student’s t test. All of the statistical calculations were performed using Origin software. A value of P<0.05 was considered statistically significant.

	Results

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Characteristics of the Experimental Model
The effects of isoproterenol treatment on the body and organ weights of the rabbits and rats are shown in the Table. The body-weight gains were not significantly different between the isoproterenol-treated and control animals. On the other hand, the isoproterenol-treated animals exhibited significant increases in heart weight compared with the control animals. A more detailed analysis of heart weight revealed that the weight of the LV in the isoproterenol-treated animals increased about 1.5-fold in both rabbits and rats compared with their respective control animals. Accordingly, the LV to body weight ratio, as an index of LVH, was significantly increased in the isoproterenol-treated animals compared with the control animals.

View this table: [in this window] [in a new window]   Table 1. Characteristics of Isoproterenol-Induced LVH

Comparison of K_Ca Current Densities
Figure 1A shows the macroscopic outward currents in the control and LVH cells. The peak membrane density of the depolarization-induced K_Ca current, which is defined as the outward current that was sensitive to blockage by 1 mmol/L tetraethylammonium (TEA), was compared in the control and LVH cells. The average data in Figures 1B and 1C show that TEA reduced the maximum current density calculated at +70 mV by 66±4% in the control (n=11) and by 31±4% in the LVH (n=12) cells. Figures 1D and 1E show the effects of iberiotoxin (IBTX) on the current-voltage relationships of the peak macroscopic K⁺ currents in control and LVH cells. IBTX reduced the maximum current density calculated at +70 mV by 61±5% in the control (n=10) and by 30.2±4% in the LVH (n=12) cells. The TEA- or IBTX-sensitive current corresponding to the K_Ca current component (shaded area) was the predominant contributor at positive potentials to the voltage-elicited outward current in the control coronary SMCs but not in the LVH coronary SMCs. Figures 1F and 1G show that the magnitude of the reduction of the current density by IBTX was greater in control cells than in LVH cells.

View larger version (42K): [in this window] [in a new window]   Figure 1. A, Whole-cell K+ currents in control and LVH coronary SMCs. TEA (1 mmol/L) blocked a large component of the outward current in the control cells (left panel), whereas only a small current component was inhibited by TEA in LVH cells (right panel). B and C, I-V relationships showing the effects of TEA on the peak macroscopic K+ currents in 11 control and 12 LVH cells. The shaded areas indicate the current component that is sensitive to blockage by TEA. D and E, I-V relationships showing the effects of IBTX on the peak macroscopic K+ currents in 10 control and 12 LVH cells. Insets, Representative raw current traces. The shaded areas depict the current component that is sensitive to blockage by IBTX. F and G, Comparison of the reductions in current density with IBTX in control (F) and LVH (G). Each bar shows the current density in the presence (closed bar) or absence (open bar) of IBTX. The control current was significantly higher than the residual current after TEA or IBTX at the indicated membrane potential (*P<0.05). The component of the TEA- or IBTX-sensitive current was significantly greater in control cells than in LVH cells at the indicated membrane potential (P<0.05).

Comparison of Single K_Ca Currents
We investigated the single K_Ca current alterations in the LVH coronary SMCs. Representative raw traces of the single K_Ca currents are shown in Figure 2A. The inset in Figure 1A shows the unitary current amplitude versus membrane potential plot. The resulting current-voltage (I-V) relationship yielded a mean single-channel slope conductance of 300.4±4.1 and 247.4±10.1 pS for the control and LVH cells, respectively. In addition, patches from the two groups responded differently to changes in the membrane voltage (Figure 2B). The open probability (P_O) for the LVH cells was less than that for the control cells between +30 and +50 mV. To examine the gating kinetics of the channels, open- and closed-time histograms were calculated at +50 mV (Figures 2C and 2D). Most of the patches contained more than one functional K_Ca channel. However, in some experiments, a single channel was recorded and the open and closed times were analyzed successfully. In the open-time histogram, the open-time constant (_o) did not differ between control (_o=55.73±3.25 ms) and LVH cells (_o=54.30±1.65 ms, P>0.05). In the closed-time histogram, the value of the fast (_c1) component was not influenced by LVH (_c1=0.71±0.04 ms in the control and _c1=0.72±0.05 ms in LVH; P>0.05). The value of the slow (_c2) component was also not influenced by LVH (_c2=76.42±43.58 ms in the control and _c2=44.85±18.50 ms in LVH; P>0.05).

View larger version (45K): [in this window] [in a new window]   Figure 2. Single-channel KCa currents in the inside-out patches. A, Unitary KCa currents were recorded from patches in a solution of 0.1 µmol/L Ca2+ (intracellular). The currents showed reduced amplitude in LVH patches at 5 different membrane potentials. Similar results were obtained for 6 control cells and 8 LVH cells. The dashed line represents the baseline (closed) state. Inset, Single-channel I-V relationship for the KCa channels; the data are averaged and fitted by linear regression to yield single-channel conductances of 300.4±4.1 pS (r2=1; n=6; and 247.4±10.1 pS (r2=0.99; n=8; ) for the control and LVH channels. *P<0.05, LVH vs control. B, Mean±SE of the PO of the KCa channels is plotted as a function of the membrane potential (n=6 for the control, n=8 for LVH, ). *P<0.05 for LVH vs control. C and D, Kinetic properties of the KCa channels. Single-channel currents were recorded at +50 mV in the inside-out patch configuration for control (C) and LVH (D) coronary SMCs. Histograms of open and closed (insets) times within bursts were analyzed from the current records, which were filtered at a cutoff frequency of 10 kHz. The smooth curves were fitted by the single-exponential least-squares method for the open time and using the biexponential least-squares method for the closed time. The bin width was 0.3 ms. None of the time constants was influenced by LVH (P>0.05, n=3).

The calcium dependency of the K_Ca channels is illustrated in Figure 3. With increasing [Ca²⁺]_i, the increases in K_Ca channel activity were less pronounced in LVH than in the control (Figure 3A). The channel activities at five selected concentrations of Ca²⁺ were normalized to the channel activity that was recorded at 0.1 µmol/L Ca²⁺, using recordings that were obtained at +50 mV. As shown in Figure 3B, the concentration-response curve to Ca²⁺ of the K_Ca channels in the LVH cells was shifted to the right compared with the control cells, which indicates that the stimulatory effect of Ca²⁺ on K_Ca channels is reduced in LVH cells. Figure 4A shows the unitary K_Ca currents that were recorded in the patches in the absence and presence of external 100 µmol/L TEA at +40 mV. The reduction in the mean single-channel current and the increase in the open channel noise caused by TEA were more apparent in the LVH cells than in the control cells. When the mean current amplitude was expressed as a function of membrane potential, it was clear that the amplitude was significantly reduced in the LVH cells compared with the control cells, in the presence of TEA (Figure 4B). Figure 4C summarizes the responses to TEA. The magnitude of reduction of the mean current amplitude by TEA was greater in LVH cells than in control cells.

View larger version (20K): [in this window] [in a new window]   Figure 3. Dependence of KCa single-channel opening on intracellular [Ca2+]. A, Representative current traces of chart recordings for the Ca2+ sensitivity of the KCa channels. Similar results were obtained for 5 control cells and 9 LVH cells. B, Isochronal sensitivity curves for the KCa currents. Each channel activity was normalized to the channel activity recorded at 0.1 µmol/L Ca2+. The data points were plotted as a function of [Ca2+]i for KCa channel opening at +50 mV. The smooth curves were best fitted to the Hill equation, such that the relative channel activity=1/{1+(Kd/[Ca2+]i)n}, where Kd is [Ca2+]i at the half-maximal activation of the channel, and n=Hill coefficient (Kd=0.03 µmol/L, n=3.60 for the control, Kd=0.04 µmol/L, n=3.57 for LVH, ). *P<0.05 for LVH vs control.

View larger version (28K): [in this window] [in a new window]   Figure 4. Inhibitory effects of TEA on KCa channels. A, Recording of the KCa channels in control and LVH cells that were held at +40 mV in the absence or presence of TEA. TEA (100 µmol/L) caused profound channel blockage when applied to the external surfaces of the control and LVH patches in the presence of 0.1 µmol/L [Ca2+]i. The designation "C" represents the closed level in each case. B, Unitary current-voltage relationships in the presence of TEA in control cells (n=6, and LVH cells (n=5, ). *P<0.05 for LVH vs control. C, Comparison of the reductions in unitary current brought about by TEA in control (open bar) and LVH (closed bar) cells. Each bar shows the average current in the presence or absence of TEA (ITEA/IControl) measured over 10 to 30 seconds in outside-out patches, such as those shown in panel A.

Comparison of the Levels of K_Ca Channel - and ß-Subunit Expression
The alterations in K_Ca channels that were observed during LVH could have been the result of a change of channel expression. Therefore, we examined the expression levels of the - and ß-subunits of the K_Ca channels in the coronary SM membranes using anti–- and anti–ß-subunit antibodies. Previous studies have established that these antibodies have specific recognition sites on the - and ß-subunits of the K_Ca channel.^19,21 Figure 5 shows a representative experiment, in which adjacent lanes were loaded in duplicate with either control (left) or LVH (right) membrane proteins. The intensities of the 125- and 30-kDa immunoreactive bands were similar for the LVH and control membranes. Four separate experiments with membranes that were obtained from individual rats indicated that the intensities of the 125- and 30-kDa immunoreactive signals were similar for control and LVH membranes.

View larger version (57K): [in this window] [in a new window]   Figure 5. Immunoblot analysis of KCa channel - and ß-subunit expression. Western blotting was performed using the anti–- and anti–ß-subunit antibodies. The samples in the SDS-10% polyacrylamide gel were stained with Coomassie blue to ensure that each lane contained the same amount of protein (A). The intensity of the 125-kDa immunoreactive band, which corresponds to the known molecular size of the -subunit, was similar for the control and LVH samples (B). The intensity of the 30-kDa immunoreactive band, which corresponds to the known molecular size of the ß-subunit, was similar for the control and LVH samples (C). The control and LVH samples are in lanes 1 and 2, respectively.

Comparisons of Coronary Artery Contractility
Figures 6A and 6B show typical experimental tracings of the effects of a high concentration of K⁺ (60 mmol/L) on the resting tension of coronary arterial rings. Figure 6C shows the group data for the contractile responses to high K⁺ concentrations, which are expressed as the absolute increases in tension. Although high K⁺ concentrations produced a significant increase in the resting tension of both the control and LVH rings, the extent of the increase was greater in the LVH rings than in the control rings. To examine the effect of K_Ca channel inhibition (and presumably increased Ca²⁺ flux through the L-type Ca²⁺ channels), we administered TEA at doses of 10^-4 to 10^-2 mol/L. As shown in Figure 7A, in response to increasing TEA dose, the control coronary arterial rings showed typical increases in vascular tension, and the mean half-maximum dose was 2.41±1.53 mmol/L (n=3). Compared with the controls, the mean half-maximum dose for the LVH arterial rings was 5±0.12 mmol/L (n=3).

View larger version (18K): [in this window] [in a new window]   Figure 6. Induction of contractions in coronary arteries by a high concentration of K+. Typical experimental tracings show the effects of 60 mmol/L K+ on arterial ring tension in the control (A) and LVH (B). The arterial rings were stretched to the optimal length and allowed to equilibrate for 60 minutes before the addition of high [K+]. The changes in tension are expressed as the average number of grams of developed tension (C). Tension was significantly higher in the LVH rings (n=6, ) than in the control rings (n=6, . *P<0.05 for LVH versus control.

View larger version (22K): [in this window] [in a new window]   Figure 7. Typical TEA dose-response relationships for coronary arteries. The arterial segments were initially contracted with 120 mmol/L K+ to achieve peak tension. After washing and reequilibration to the basal tension, cumulative doses of TEA were added. A, Vascular tension of the control arterial rings, showing a significant increase in the response to 10-3 mol/L TEA. B, Vascular tension of the LVH arterial rings, showing no significant change in the response to TEA. C, Relative tensions of the control (n=4, and LVH (n=4, ) rings in response to TEA are plotted as a function of [TEA]. The values shown are mean±SE. *P<0.05 for LVH vs control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The main goal of this study was to assess whether K_Ca channel properties are altered in LVH coronary SMCs. The results of our study provide three new findings in this regard. First, the patch-clamp experiments showed that the whole-cell currents of the K_Ca channels were reduced during LVH. The unitary current amplitude and open probability of the K_Ca channels were significantly reduced in LVH compared with the control. The concentration-response curve of the K_Ca channel to [Ca²⁺]_i was shifted to the right, and TEA-mediated inhibition of K_Ca channels was more pronounced in LVH than in the control. Second, Western blot analysis with an antibody that was specific for the - and ß-subunits of the K_Ca channel indicated that there were no differences in K_Ca channel expression between the coronary SM membranes of the control and LVH. Third, the contractility experiments demonstrated that the effect of high [K⁺] on coronary artery resting tension was greater in LVH than in the control. The effect of TEA on the coronary artery resting tension was also greater in LVH than in the control.

It has long been recognized that vascular SMC K⁺ channels are affected by a variety of pathological states that alter the integrity and/or excitability of the vascular SM.^22,23 As is the case with many of the SMCs, the coronary SMCs exhibit substantial outward K⁺ currents, which lead to membrane repolarization and relaxation by closing the L-type Ca²⁺ channels.^3,24 Although several distinct types of K⁺ channels are functional, the K_Ca channels are the predominant type (1000 to 10 000 per cell) in coronary SMCs.² These channels are characterized by their selectivity and large single-channel conductance, as well as their sensitivity to membrane voltage and [Ca²⁺]_i, and they are thought to be key regulators of vascular tone.¹ In the present study, using a multifaceted approach of patch-clamp, Western blotting, and contraction experimental methods, we provide the first comprehensive report on the relationship between reduced coronary reserve and K_Ca channel expression, phenotype, and physiological impact on the coronary circulation. We observed decreases in the single-channel current amplitude and open probability of the K_Ca channels in LVH coronary SMCs compared with the control cells. In addition, we detected no differences in the intensities of the 125-kDa immunoreactive bands between the LVH and control membranes, although the membrane capacitance of the LVH coronary SMCs was higher than that of the control SMCs. Consistent with the above results, we used whole-cell methods to show that macroscopic K_Ca currents were reduced in LVH. Since these channels regulate both [Ca²⁺]_i and membrane potential, their reduction in LVH is likely to produce a deleterious physiological impact, leading to attenuation of the dilatory functions in coronary vascular tone responses to various stimuli. Some studies have suggested that vasoconstriction and the compromised ability of an artery to dilate are consequences of defective K⁺ channel function in blood vessels.^19,20,25 The mechanism underlying the decrease in total membrane current must represent changes in the associated factors (N · i · P_O, where N is channel number, i is unitary current amplitude, and P_O is channel open-state probability).^2,26 Hence, N, i, and P_O represent the three distinct sites of abnormality that could contribute to the reduced K_Ca current in coronary SMCs from LVH. In the present study, the responsiveness of the K_Ca channels to [Ca²⁺]_i was reduced in LVH cells compared with the control cells. The activation of K_Ca channels by Ca²⁺ influx is an important negative-feedback mechanism that regulates the level of vascular tone.² This regulatory pathway is likely to influence arterial tone in many vascular beds, including the coronary circulation.²⁷ Therefore, our results suggest that the negative-feedback action of the K_Ca channels to block the elevation of [Ca²⁺]_i may be attenuated in LVH, which may impair the relaxation of coronary arteries in response to various stimuli. This hypothesis is supported by the results of our contractility experiments. The LVH coronary arteries showed increased contractility in response to high [K⁺] compared with the control coronary arteries. This phenomenon is probably due to the reduced sensitivity to [Ca²⁺]_i of the K_Ca channels in the LVH coronary artery SMCs. In general, cells that are exposed to high [K⁺] depolarize, which increases Ca²⁺ entry through L-type Ca²⁺ channels and leads to vasoconstriction. On the other hand, Ca²⁺ entry activates the K_Ca channels, which participate in a negative-feedback mechanism to limit depolarization, L-type Ca²⁺ channel activation, and contraction. Guia et al²⁸ proposed that a functional local interaction between the L-type Ca²⁺ channels and K_Ca channels probably reflected a more global feedback mechanism, which would exert potent control over Ca²⁺ entry into the coronary arterial SM. Therefore, this result supports the idea that the negative-feedback action of K_Ca channels to block the elevation of [Ca²⁺]_i may be attenuated during LVH. Recent molecular studies suggest that variations in the expression of both the - and ß-subunits, which are thought to make up these channels, can influence the channel responses to changes in the concentration of Ca²⁺. Knaus et al²⁹ have suggested that the conductance and voltage dependencies of K_Ca channels are determined by the N-terminal core of the -subunit, whereas Ca²⁺ sensitivity appears to involve a region of several negatively charged residues at the C-terminal core of the -subunit. Moss et al³⁰ suggested a modular construction for the K_Ca channel, whereby the tail domain modulates the gating kinetics and conductance properties of the voltage-dependent core domain, in addition to mediating most of the high-affinity Ca²⁺ sensitivity. Some studies have reported that the Ca²⁺ sensitivity of K_Ca channels that lack the ß₁-subunit is reduced to such an extent that the channel open probability during a spark is too low to cause a detectable current.^25,31 In these respects, altered Ca²⁺ sensitivity has been suggested as a potential mechanism for the altered P_O of K_Ca channels. However, in the present study, we could not find any differences in the expression levels of the K_Ca channel - and ß-subunits between the control and LVH coronary SM membranes. Multiple K_Ca channel splice-variant isoforms have been expressed heterologously and shown to have differential effects on channel function.^32,33 These isoforms alter the sensitivity to intracellular Ca²⁺ concentrations or voltage, allowing for the fine-tuning of this channel to cellular regulators.^34,35 Variability in channel behavior among different isoforms produced by alternative splicing is a result of the introduction or removal of posttranslational modification sites. The dynamic association of the K_Ca -subunit with accessory ß-subunits is another molecular mechanism to provide channel plasticity. In vascular smooth muscle, the -subunit is associated with the ß₁-subunit via the extracellular terminus of the -subunit, which results in increases in the voltage and calcium sensitivity of the channel.^36,37 Protein kinase A–dependent phosphorylation is affected by ß-subunit binding, which results in a decrease in the open-state probability on ß-subunit association.³⁸ On the other hand, the induction of immediate early genes, such as c-fos and c-jun, contributes to the development of all LVH types.^39–43 In a variety of biological systems, c-fos acts as a transcription factor to modulate the expression of additional (late-response) genes.^44,45 The expression of c-fos has been linked to a number of cellular responses in vascular smooth muscle, including hypertrophy, proliferation, and adaptive changes, such as alterations in ion channel expression.⁴⁵ Therefore, alternative splicing, posttranslational mechanisms, ß-subunit association, and the differential expression of K_Ca channels by alterations in the JNK/AP-1 (c-jun and c-fos) cascades are some of the mechanisms that have been put forward to explain the altered K_Ca channel function during LVH. In these respects, the decreased Ca²⁺ sensitivity of LVH coronary SMCs has been suggested as a potential mechanism for the reduced P_O of K_Ca channels, and the K_Ca channel subunits appear to be functionally damaged in the LVH coronary SMCs. This hypothesis is supported by the results of our contractility experiments. During LVH, the K_Ca channels of the coronary SMCs show altered sensitivity to TEA, which supports the notion of alterations to the K_Ca channel structures in LVH coronary SMCs.

In the control coronary arteries, TEA produced significant increases in the resting tension. This suggests that K_Ca channels are very active at resting tone in the coronary circulation. Our patch-clamp studies of whole-cell currents provide support for this idea. Interestingly, the increase in resting tension produced by TEA was significantly lower in the LVH coronary artery. These data suggest that K_Ca channels are not very active in the coronary circulation during LVH. These observations provide evidence for impaired K_Ca channel function in the regulation of LVH coronary tone, which is corroborated by our whole-cell patch-clamp studies. The lack of activity of K_Ca channels in LVH coronary SMCs may be due to low Ca²⁺ sensitivity.

In summary, the unitary current amplitude, open probability, Ca²⁺ sensitivity, and current density of the K_Ca channel in LVH coronary SMCs were significantly reduced, and the expression levels of the K_Ca channel - and ß-subunits were similar, compared with control coronary SMCs. The effects of TEA and high K⁺ concentrations on the resting tension of the coronary arteries were different in LVH than in the controls. Therefore, our data suggest a novel mechanism for the reduced coronary reserve seen in cases of LVH.

	Acknowledgments

 
This work was supported by Grants R05-2001-000-00413-0 and R05-2002-000-00905-0 and by the BioHealth Products Research Center of the Korea Science and Engineering Foundation.

	Footnotes

 
Original received February 27, 2003; revision received July 22, 2003; accepted July 28, 2003.

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