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Am J Physiol Heart Circ Physiol 292: H475-H482, 2007. First published May 26, 2006; doi:10.1152/ajpheart.00774.2005
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Heterogeneous Kv1 function and expression in coronary myocytes from right and left ventricles in rats

Mathieu Gautier,* Jean-Marc Hyvelin,* Valérie de Crescenzo, Véronique Eder, and Pierre Bonnet

Laboratoire de Physiopathologie de la Paroi Artérielle, Université François Rabelais, Tours, France

Submitted 21 July 2005 ; accepted in final form 19 May 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Coronary blood flow control is not uniform along the vascular tree and particularly between the right coronary artery and the left anterior descending artery. Resting membrane potential that contributes largely to the vascular tone is mainly regulated by K+ channels in coronary myocytes. In the present study, we hypothesized that right coronary artery (RCA) and left coronary artery (LCA) exhibited a cell-specific function of K+ channels. The net outward current was markedly greater in RCA compared with LCA cells, and this difference was due to a larger 4-aminopyridine (4-AP)-sensitive voltage-gated potssium (Kv) current in RCA cells, whereas the iberiotoxin (IbTx)-sensitive, large conductance Ca2+-dependent potassium (BKCa) current was smaller in RCA cells. To go further in the molecular identity of this Kv current, we used 50 nM correolide, which specifically blocked Kv1 family {alpha}-subunits. Outward currents generated by ramp depolarization protocols were highly sensitive to correolide in both RCA and LCA cells, suggesting that Kv1 contributed for a large part to the net outward current. 4-AP-induced contractions in isolated RCA, and LCA were greater than IbTx-induced contraction. Furthermore, the 4-AP-induced contraction in RCA was significantly greater than that in LCA, which is in agreement with the electrophysiological data. Finally, the Kv1.2 {alpha}-subunit but not the Kv1.5 was detected in both RCA and LCA using primary specific antibody in Western blotting and immunofluorescence assay, and expression of Kv1.2 {alpha}-subunit was markedly higher in RCA compared with LCA. In summary, we reported for the first time a heterogeneous function and expression of Kv1 {alpha}-subunits in rat coronary myocytes isolated from RCA or LCA.

coronary circulation; potassium channel; perfused coronary; membrane currents

THE FUNCTIONAL CHARACTERISTICS of the coronary circulation may be described in terms of the relationship between steady-state perfusion pressure and the amount of blood flow. There are functional hemodynamic differences among coronary vascular beds that are linked to pressure differences along the vascular bed. These differences in compressive forces result in almost continuous flow in the right coronary bed, whereas flow in the anterior left descending bed occurs mainly during diastole (24, 30). Furthermore, when aortic pressure rises and the ventricle is loaded, the right coronary bed exhibits reflex increase of blood flow, whereas the anterior left descending coronary artery vasodilates poorly (29). Such differences in the hemodynamic profile between the left (LCA) and right coronary arteries (RCA) may underline differences in the regulation of basal tone between these two vascular beds.

The coronary vascular tone is modulated by several factors, including neural, hormonal, metabolic, and myogenic, which act, in part, on smooth muscle (10, 11, 17). Interestingly, it has been shown that the myogenic response, which contributes significantly to flow regulation (7), is not uniform within the coronary vascular tree (10). In the arterial circulation, it is well known that tone is mainly determined by the membrane potential of vascular myocytes. Under physiological conditions, resting membrane potential is mainly regulated by voltage-gated K+ (Kv) channels, particularly at lower levels of Ca2+, when large conductance Ca2+-dependent K+ (BKCa) are less active (33). Similarly, in the coronary vascular bed, it is assumed that the resting membrane potential under physiological conditions is regulated mainly by Kv with a minor contribution from the BKCa (3, 21, 23, 31). Chemical and mechanical stimuli, such as transmural pressure, can influence the open-state probability of these channels to regulate the level of membrane resting potential (Em) in arterial myocytes and thereby can alter the coronary vascular tone (9).

Thus it is conceivable that the functional hemodynamic differences between the left and right coronary vascular beds result in different K+ channels expression and consequently in different tone regulation among these arteries. It is noteworthy that different types of K+ channels have been described in coronary myocytes from several animal models (8, 22, 23, 35, 37), supporting that coronary vascular tone may reflect the integrated contribution of a diverse population of K+ channels. Nevertheless, to the best of our knowledge no study has so far compared the contribution of the K+ channels in both LCA and RCA from the same species.

Kv channels may have a heterotetrameric structure. Recently, it has been shown that heteromultimeric Kv1 channels contributed to myogenic control of arterial diameters in rats (27). Moreover, in rat renal microvasculature, K+ current was thought to be heteromultimeric delayed-rectifier Kv1.2 and A-type Kv1.4 subunits that contribute to blood pressure regulation (12), whereas Kv1.2/Kv1.5 heteromultimeric channel was preferentially expressed in rat cerebral myocytes and contribute to Em and diameter of small cerebral arteries (1). Then, in these two vascular types, the Kv1.2 {alpha}-subunit has been identified to contribute to the delayed rectifier K+ current (KDR), which strongly regulates Em.

In the present study, we tested the hypothesis that the functional hemodynamic difference between the RCA and the LCA could be linked to differential properties of K+ channels, and more particularly, the Kv1 channel family, in the membranes of the RCA and LCA myocytes. We assessed the electrophysiological profile RCA and LCA myocytes, and more particularly, the contribution of both IbTx-sensitive BKCa- and 4-aminopyridine (4-AP)-sensitive KV currents. We confirmed differences in these profiles by performing functional studies, Western blot, and immunohistochemistry.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
All animal experiments were conducted according to the ethical standards of the Ministère Français de l'Agriculture for the Care and the Use of Laboratory Animals (authorization no. 006121).

Tissue preparation. Adult (10–12 wk old, 300–350 g) male Wistar rats (Charles River Laboratories, L'Arbresle, France) were anesthetized with intraperitoneal pentobarbital sodium (100 mg/kg). After a thoracotomy was peformed, hearts were quickly excised and immersed in cold (4°C) physiological saline solution (PSS) containing (in mM) 138.6 NaCl, 5.4 KCl, 1.8 CaCl2, 1.2 MgCl2, 0.33 NaH2PO4, 10 HEPES, and 11 glucose (pH was adjusted to 7.4 with NaOH). The heart was cannulated and retrogradely perfused through the coronary arteries using cold PSS to remove blood from the circulation. Coronary arteries were then carefully dissected in cold PSS. The left anterior descending branch of the main LCA and the right marginal branch of the RCA were removed. Vessels were placed in cold PSS and carefully cleaned of fat and adventitia.

Isolation of coronary myocytes. The enzymatic isolation of single coronary myocytes was performed according to published dissociation methods for rat microvessels (6, 18) and modified for coronary arteries (3). Briefly, coronary arteries were cut into small rings that were placed successively in a (Ca2+ free) dissociation solution containing (in mM) 138.6 NaCl, 5.4 KCl, 1.2 MgCl2, 10 HEPES, and 11 glucose (pH was adjusted to 7.4 with NaOH) for 10 min at room temperature and then placed in a second dissociation solution at 37°C containing 1 mg/ml papain and 1 mg/ml dithioerythritol (both from Sigma, St. Louis, MO) for 12 min and in a third dissociation solution at 37°C containing 1.6 mg/ml collagenase (type H, Sigma) and 1.6 mg/ml trypsin inhibitor (type I-S, Sigma) for 9 min. Tissues were then replaced twice in the first dissociation solution for 10 min and were gently agitated using a polished wide-bore Pasteur pipette to release the cells. Cells were stored at 4°C and used on the day of experiment. Only relaxed, long, smooth and optically refractive myocytes were used for patch-clamp experiments.

Patch-clamp experiments. Electrophysiological recordings were obtained by using the conventional patch-clamp technique (15) in the whole cell configuration. Myocytes were placed in a 0.5-ml volume bath and continuously superfused by gravity with PSS at the rate of 1 ml/min. Different test solutions were applied to the cell at 100 µl/min by microcapillaries, and <10 s was needed to completely change the perfusing solution around the cell. Cell membrane currents were recorded with an Axopatch 200B patch-clamp amplifier (Axon Instruments). Patch pipettes were pulled from borosilicate glass capillaries and had a resistance of 5–7 M{Omega}. Pipette potential and capacitance were electronically compensated. Intracellular pipette solution contained (in mM) 125 glutamic acid, 20 KCl, 1 Na2ATP, 0.37 CaCl2, 1 MgCl2, 10 HEPES, and 1 EGTA (pH was adjusted to 7.2 with KOH). pCa (~7) was calculated by a computer program developed by Godt and Lindley (14), and the evaluated concentration of K+ was 120 mM. The membrane capacitance was determined by dividing integration of capacitive currents by amplitude of 10-mV voltage steps. Membrane resistance was estimated as the slope of the current-voltage curves between –90 and –60 mV, where no dynamic currents were activated. Cell membrane currents were recorded with a patch-clamp amplifier (Axopatch 200B, Axon Instruments). Signals were filtered at 1 kHz and digitized at 5 kHz. Peak current elicited at a single membrane potential was defined as the average of the latest 100 ms of the pulse encompassing the maximal current point. Trials were performed in triplicate in the same cell and averaged to estimate peak current amplitude. Currents were normalized to cell capacitance and were expressed as picoamperes per picofarad (pA/pF).

Net macroscopic currents were generated by stepwise 8-mV depolarizing pulses (400 ms duration, 5-s intervals) with a constant holding potential of –60 mV to +60 mV. Based on their pharmacological and electrophysiological properties, the following K+ current subtypes were identified. Response to K+ channels blockers was recorded after the addition of iberiotoxin (IbTx) for a final concentration of 100 nM in the bath and 4-AP (3 mM) to block BKCa channel type (20) and Kv channel type (32), respectively. Correolide (50 nM), a selective inhibitor of the Kv1.X gene family (16), was used to examine the molecular basis of Kv current. Both IbTx and 4-AP were provided from Sigma, whereas correolide was a kind gift from Dr. Gregory Kazcorowski and the Merck Research Laboratories.

The IbTx-sensitive current was defined as the difference between outward current recorded in drug-free bath solution and in the presence of 100 nM IbTx. The 4-AP-sensitive current was defined as the difference between outward current recorded in the presence of 100 nM IbTx and that in the presence of 100 nM IbTx plus 3 mM 4-AP.

Voltage-clamp protocols were generated, and the data were captured with a computer using a Digidata 1200 interface (Axon Instruments) and pClamp8 software (Axon Instruments). The analysis was carried out using Clampfit 8.1 and Origin 6.0 software (Microcal Software, Northampton, MA).

Coronary contractility measurements. Rats were anesthetized with intraperitoneal pentobarbital sodium (100 mg/kg) and heparinized (100 UI/kg). After a thoracotomy was performed, the heart was quickly excised and immersed in cold cardioplegic solution. Myocardium was dissected, under binocular control, to expose the left anterior descending branch of the main LCA or the right marginal branch of the RCA. Basically, a small window (5 x 5 mm) was dissected to expose a small segment of the coronary artery (5 mm length). Thus the vascular segment was carefully cleaned off the myocardium, and a thin dark plastic film was placed under the vessels. The heart was placed into a 3-ml chamber filled with warmed PSS. The pulmonary artery was transected to facilitate coronary venous drainage. Hearts were perfused at constant flow by an aortic cannula delivering warm PSS. The perfusion pressure was set to 60 mmHg and allowed to stabilize over 1 h before the vessel entered into the protocol. Coronary arteries were successively perfused with PSS, PSS plus 4-AP (3 mM), and PSS + IbTx (100 nM). A 30-min rinsing period was allowed between each experimental condition. The diameter of the coronary artery was continuously visualized on a monitor using a CCD black and white camera fitted to a binocular. For each experimental condition, once steady state was achieved, a snapshot of the vessel was taken and stored into a computer for offline analysis.

Coronary protein preparation and Western blot. Coronary proteins were prepared as previously published with modifications (38). Briefly, coronary arteries were homogenized with a PowerGen 125 homogenizer (Fisher Scientific) in 1 ml of Tris-buffered saline (10 mM Tris·HCl, pH = 7.4, 1 mM EDTA, 0.3 mM sucrose) containing protease inhibitor mixture (1 µg/ml antipain hydrochloride, 1 µg/ml leupeptin hemisulfate, 1 mM benzamidine hydrochloride hydrate, 1 mM iodoacetamide, 1 mM 1,10-phenanthroline monohydrate, 0.1 mM phenylmethylsulfonyl fluoride, and 0.001 mM pepstatin A). Homogenates were clarified by centrifugation at 8,500 rpm at 4°C for 10 min. Protein concentration was determined using Bradford Protein Assay with BSA as standard. Each sample was then analyzed by SDS-7.5% polyacrylamide gel. Resolved proteins were transferred onto nitrocellulose membrane. Primary polyclonal antibodies against Kv1.2 or Kv1.5 (Alomone Labs, Jerusalem, Israel) were used at 1:200 dilution; primary monoclonal antibody against {alpha}-smooth muscle actin (Sigma) was used at 1:400. Horseradish peroxidase-conjugated secondary goat anti-rabbit IgG for Kv1.2 and Kv1.5 was used at 1:5,000 dilution, and goat anti-mouse IgG for {alpha}-smooth muscle actin was used at 1:10,000. Membranes were incubated overnight at 4°C with primary antibodies and 1 h at room temperature with secondary antibodies. The immunoreactive bands were detected by Enhanced Chemiluminescence Western Blotting Detection Kit (ECL, Amersham Biosciences, Little Chalfont Buckinghamshire, UK). Signal intensity of the immunoreactive Kv bands was normalized to the expression of {alpha}-smooth muscle actin.

Immunofluorescence. Coronary arteries were carefully dissected as previously described. A small part of thoracic aorta was also collected and served as positive control. Vascular tissues were embedded in OCT compound and snap frozen. Thin (10-µm thick) sections were washed twice for 5 min in solution containing 70% PBS and 30% fetal bovine serum. Slides were incubated with 1:200 polyclonal anti-Kv1.2 {alpha}-subunit (Alomone) in PBS during 30 min. Slides were then washed with PBS twice for 5 min and incubated with 1:5,000 secondary labeled goat anti-rabbit IgG antibody with Alexa Fluor 594 fluorescent dye conjugated (Molecular Probes) in PBS during 30 min. Finally, they were washed with PBS twice for 5 min and allowed to dry. Slides were cover-slipped in Fluoromount-G. Images were acquired on a fluorescence microscope (Leica, DMR, Leica Microsystems Wetzlar), where approximate absorption was 590 nm and fluorescence maximal emission was 617 nm for conjugates.

Statistical analysis. Results are expressed as means ± SE. For electrophysiological data, statistical analyses were made with the unpaired Student's t-test or the Mann-Whitney's test when normality test failed (Anderson-Darling test). The number of experiments (n) refers to the number of cells or to the number of Western blots. Differences were considered significant for P < 0.05. For coronary contractility measurements, internal coronary artery diameter was measured using Optimas 6.5 (Media Cybernetics) software. Internal diameter of coronary artery was measured by two different blind observers. No statistical differences were observed between the two observers. The decrease of the diameter induced by each drug 4-AP and IbTx was expressed as a percentage of the diameter recorded before the addition of the drug. Comparisons intravascular bed were made with the paired Student's t-test, whereas intervascular bed comparison (i.e., LCA vs. RCA) were made with the unpaired Student's t-test. The number of experiments (n) refers to the number of coronary arteries tested. All statistical analysis was realized using Minitab software (Minitab) and SigmaStat 3.0 (Systat Software).


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
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 REFERENCES
 
Effect of K+ channel blockers on whole cell currents in LCA and RCA. Typical effects of IbTx and 4-AP are shown on the whole cell current recorded in LCA (Fig. 1A) and RCA (Fig. 1B) myocytes. In these two examples, rapidly activating outward currents were observed at test potentials positive to –20 mV, which showed little inactivation during the 400-ms test pulse as already described in rabbit, guinea pig, and human coronary myocytes (35). These currents seemed smaller in the LCA cell compared with the RCA cell. Outward currents recorded in both LCA and RCA cells exhibited little sensibility to superfusion with 100 nM IbTx, a specific calcium-dependent K+ channels inhibitor, whereas a subsequent addition of 3 mM 4-AP, a Kv channel inhibitor, in the superfusion solution containing 100 nM IbTx blocked a large component of these currents. This latter component appeared lower in the LCA cell compared with the RCA cell.


Figure 1
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  		Fig. 1. Whole cell K+ currents in left coronary artery (LCA) and right coronary artery (RCA) myocytes. A and B: typical currents elicited by incremental 8-mV depolarizing steps from –60 mV to +60 mV in myocytes isolated from RCA and LCA. Currents were recorded in the same cell, in the control condition, in the presence of iberiotoxin (IbTx, 100 nM), a large conductance Ca2+ potassium (BKCa) channels blocker, or 4-aminopyridine (4-AP, 3 mM), a KV channel blockers. IbTx blocked a small component of outward current in both cell types in the same extent. Addition of 4-AP blocked a small component of residual outward current in LCA cell, whereas a larger current component is inhibited by addition of 4-AP in the RCA cell. B and D: current-voltage relationship during voltage steps in isolated RCA and LCA cells. B: shows the total outward current density [IK(total)] in cells from RCA and LCA. C: indicates the BKCa IbTx-sensitive current density component of total outward current [IK(IbTx)], which was significantly smaller in RCA cells compared with LCA cells. D: Kv 4-AP-sensitive current density component of total current [IK(4-AP)], which was significantly greater in RCA cells compared with LCA cells. Each symbol represents the mean ± SE. *P <0.05 significantly different.

 
To take into account the cell membrane area, we divided the mean current amplitude by the respective membrane capacitance (Fig. 1C). The current densities in LCA cells were markedly and significantly lower (P < 0.05) than in RCA cells (16.4 ± 1.3 pA/pF, n = 26 and 22.3 ± 1.6 pA/pF, n = 30, at +60 mV, respectively).

The IbTx-sensitive current density due to the activity of BKCa was obtained by subtracting the outward current recorded in the presence of 100 nM IbTx from the net outward current recorded in control PSS superfusion (Fig. 1D). This IbTx-sensitive current density was greater in LCA compared with RCA cells (9.0 ± 1.5 pA/pF and 4.7 ± 0.7 pA/pF, respectively, at +60 mV; P < 0.05).

The 4-AP-sensitive current density due to the activity of Kv was obtained by subtracting the outward current recorded in the presence of 100 nM IbTx plus 3 mM 4-AP from the resulting outward current recorded in the presence of 100 nM IbTx (Fig. 1E). This current density was significantly lower in LCA (n = 26) than in RCA (n = 30) cells (P < 0.05). At +60 mV, this 4-AP-sensitive current density was 4.3 ± 0.5 pA/pF in LCA and 13.0 ± 1.2 pA/pF in RCA. Then the net outward current was lower in LCA than in RCA cells, and this difference was due to a greater 4-AP-sensitive current density in RCA cells, whereas the IbTx-sensitive current was lower in RCA compared with that in LCA cells.

Effect of correolide on whole cell current in LCA and RCA. To further investigate the nature of the Kv channel involved in the repolarizing net outward current, we tested the effect of correolide, a selective blocker of the Kv1 channels family. Using coronary myocytes preincubated with 100 nM IbTx to eliminate BKCa current, we tested the effect of 50 nM of correolide on the remaining current. In the presence of IbTx, the outward remaining current was higher in RCA than in LCA myocytes (Fig. 2A). This remaining current was highly sensitive to correolide. The correolide-sensitive Kv-family current densities averaged 12.6 ± 2.8 pA/pF in LCA cells (n = 6) and 22.0 ± 3.4 pA/pF in RCA cells (n = 7) (Fig. 2B). Finally, after pretreatment with correolide, we observed a small residual current in both LCA and RCA, which was sensitive to 3 mM 4-AP (Fig. 2C). This residual current, after application of correolide, was equal in the two cell types, which suggests that the differential Kv current in LCA and RCA cells could be attributed to a Kv1 current family.


Figure 2
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  		Fig. 2. Effect of correolide on outward current-voltage relationship in LCA and RCA. A: outward current-density [IK(Total)] in both LCA and RCA. B: KV correolide-sensitive current-density [IK(Corr)] was significantly greater in RCA compared with LCA cells. C: residual 4-AP-sensitive current [IK(4-AP)] was the same for RCA and LCA cells after inhibition of a KV1.X-sensitive current by correolide (50 nM). Each symbol represents the mean ± SE. *P < 0.05 significantly different.

 
Effect of 4-AP and IbTx on basal tone in LCA and RCA. To ensure that the difference in the electrophysiological profile has a functional role in the regulation of basal tone of coronary bed, we assessed the effect of the K+ channels blockers on perfused coronary artery. Both coronary arteries LCA and RCA developed a contractile force in response to K+-rich solution (KCl 80 mM), as indicated by the decrease in the internal diameter. The response to 80 mM KCl was similar in both coronary arteries (Fig. 3).


Figure 3
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  		Fig. 3. Effect of IbTx and 4-AP on basal tone in RCA and LCA. Left: typical snapshot of a LCA and a RCA artery perfused successively with physiological saline solution (A), KCl (80 mM, B), 4-AP (3 mM, Cc), and IbTx (100 nM, D). A 30-min rinsing period was allowed among KCl, 4-AP, and IbTx. Right: change in the internal diameter of the RCA and LCA in presence of KCl, 4-AP, or IbTx. Changes in diameter are expressed as percentage of reduction of diameter compared with the diameter of the vessel prior addition of the drug. *, **Significant difference between two experimental conditions within the same vessel (*P < 0.01 and **P < 0.005, paired Student's t-test). #Significant difference between the RCA and LCA under similar experimental condition (#P < 0.05, unpaired Student's t-test).

 
In the RCA, the addition of 4-AP (3 mM) induced a contraction that was about twofold higher than that elicited by IbTx (100 nM) (P < 0.001), whereas in LCA, the 4-AP-induced contraction was slightly higher than that elicited by IbTx (Fig. 3).

Interestingly, the comparison of the RCA to the LCA revealed that the 4-AP induced a greater contraction in the RCA compared with the LCA, whereas there was no significant difference in the response induced by IbTx between the two vascular beds (Fig. 3). These data are in agreement with the difference in the electrophysiological profile between the RCA and LCA.

Expression of Kv1.5 and Kv1.2 {alpha}-subunits in coronary arteries. It was reported that the delayed-rectifier K+ current was due to a heterotetrameric assembly of Kv1.2/Kv1.5 {alpha}-subunits in various vascular types, including cerebral (1), mesenteric (25, 38), and coronary (22) in the rat as well as in the rabbit portal vein (19, 34). We tested the polyclonal antibody targeted to the Kv1.5 pore-forming {alpha}-subunit in coronary arteries as well as in the mesenteric artery. Whereas incubation of mesenteric vascular proteins with anti-Kv1.5 revealed two intense immunoreactive bands at ~50 and ~100 kDa, no signals were detected for coronary arteries (Fig. 4B). On the other hand, we tested the polyclonal antibody targeted to the Kv1.2 pore-forming {alpha}-subunit. Figure 4A showed that incubation of coronary vascular proteins with anti-Kv1.2 antibody revealed one intense immunoreactive band at ~75 kDa in both RCA and LCA as previously shown in other vascular tissues as in the mesenteric artery (38) and in the pulmonary artery (36). Kv1.2 immunoreactive bands were normalized to {alpha}-smooth muscle-actin immunoreactive band and indicated that the amount of Kv1.2 protein was significantly lower in LCA (n = 3) than that in RCA (n = 3, P < 0.05; Fig. 4C). In agreement with the Western blot and the functional studies, immunofluorescence on coronary rings showed that LCA expressed a reduced amount of Kv1.2 {alpha}-subunit protein compared with the RCA (Fig. 5).


Figure 4
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  		Fig. 4. Western blots of vascular tissue homogenates from RCA and LCA (30 µg/lane); molecular mass markers (high-range prestained SDS-PAGE standards, Bio-Rad) are shown on left. A: incubation of proteins with affinity-purified Kv1.2 antibody revealed one intense immunoreactive band at the expected molecular mass (~75 kDa) in the two coronary arteries. B: no signal was apparent in coronary arteries following incubation of proteins with affinity-purified Kv1.5 antibody, whereas two intense immunoreactive bands were revealed in mesenteric artery (MA) tissue. C: normalization of Kv1.2 signal to {alpha}-sm-actin signal indicated that the pore-forming Kv1.2 {alpha}-subunit is more expressed in RCA (n = 3) than in LCA (n = 3).

 

Figure 5
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  		Fig. 5. Arterial rings (10-µm thick section) of (A) and (B) labeled with anti-Kv1.2 antibody and visualized with Alexa Fluor 594-conjugated secondary antibody. Negative control for RCA and LCA, i.e., staining in absence of the first antibody, are showed in C and D, respectively.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 DISCLOSURES
 REFERENCES
 
The present study reveals differences in the repolarizing net outward currents recorded in left and right coronary myocytes of the adult rat, which contribute in a greater role of Kv+ channels in the control of resting tone of the RCA. Specifically, we have identified marked differences in the Kv current between the LCA and RCA. The delayed-rectifier Kv1 current is larger in the RCA. The Kv1.2 pore-forming {alpha}-subunit, but not the Kv1.5, was more expressed in the RCA than in the LCA. Finally, Kv+ channels play a greater role in basal tone of the RCA compared with the LCA.

Net outward current is greater in RCA than in LCA cells. Membrane currents elicited by membrane depolarization are outwardly rectifier. These currents displayed fast activation from about –20 mV and slow or no inactivation during the 400-ms pulse (Fig. 1A). Our records are consistent with previous studies performed in guinea pigs, humans, rabbits (35), and rats (23). Most studies related to the characterization of membrane currents in coronary smooth muscle cells were performed on cells isolated from the LCA (8, 21, 26, 35, 37). In the present study, macroscopic outward currents were comparatively measured in myocytes from both LCA and RCA, and for the first time, we reported that macroscopic outward current elicited by membrane depolarization were greater in RCA cells than in LCA cells (Fig. 1C).

KV1 currents are greater in RCA than in LCA cells. Our data showed that 100 nM IbTx superfusion resulted in a small decrease in the macroscopic outward current, whereas the subsequent addition of 3 mM 4-AP blocked a large part of the residual outward current in both LCA and RCA cells. Our results demonstrated that Kv and BKCa currents coexist in both populations of cells (LCA and RCA) and contribute to the majority of the net outward current in coronary myocytes as previously shown (21). We observed that BKCa current density was lower in RCA cells compared with LCA and that its contribution in the net outward current is small. On the other hand, the Kv current density is greater in RCA than in LCA cells. Our results demonstrated that this Kv current is highly sensitive to correolide, suggesting that the coronary Kv channel is mainly composed of pore-forming Kv1 {alpha}-subunits (Fig. 2).

Myocardium does not have energetic reserve, thus an adequate oxygen delivery is crucial to ensure a proper functionality of the cardiomyocytes. In this way coronary circulation needs to adapt the blood flow (thus the diameter of coronary) to the variation of myocardial oxygen consumption. Among Kv1 channels, the Kv1.2 and Kv1.5 are known to be sensitive to oxygen levels. Also, we specifically looked at their expression in the LCA and RCA. It has been shown that Kv1.2 and Kv1.5 coassemble to form heteromultimeric delayed rectifier K+ current in the rabbit portal vein (19, 34). Moreover protein expression of these two Kv1 {alpha}-subunits has been reported in various vascular types (1, 2). In the present study, Western blot data provide the first evidence that the pore-forming Kv1.2 {alpha}-subunit is expressed in coronary tissues, which is confirmed by immunofluorescence. Kv1.2 is more expressed in the RCA than in the LCA artery, which could explain the electrophysiological heterogeneity between these two vascular sisters (Fig. 4). Surprisingly, we did not detect the pore-forming Kv1.5 {alpha}-subunit in rat coronary arteries, whereas our anti-Kv1.5 antibody was able to detect protein of the predicted mass in mesenteric artery samples. Similar negative findings for Kv1.5 expression in vascular tissue have been reported and extensively discussed by Cheong et al. (5). However, Li et al. (22) recently observed Kv1.5 expression in left coronary myocytes. Although our present results differ from those reported by Li et al., in this previous study it is noteworthy that Western blot was performed from myocytes isolated from LCA and incubated in medium culture at 37°C for 24 h. Furthermore, in this previous study, myocytes were isolated from smaller coronary artery (internal diameter 150–200 µm) than that in our present study (300–350 µm). Such differences in the experimental protocol could explain the differences in the results. Finally, protein expression of additional Kv1 subunits, including Kv1.1 (13, 28), Kv1.3 (5, 13, 38), and Kv1.6 (4, 5, 27), has been also reported in vascular tissues, and a complete molecular characterization of the coronary delayed rectifier K+ current needs further investigations.

Physiological relevance. At the physiological level of intracellular Ca2+ concentration, the Kv channels importantly regulates the resting membrane potential of coronary myocytes (3, 21, 23), which is the major determinant of coronary arterial tone (9), whereas the BKCa channel is enhanced by elevation of intracellular Ca2+ and induces membrane hyperpolarization to limit Ca2+ entry through voltage-gated Ca2+ channels (21). There is much evidence that Kv1 channels contribute to arterial tone and vessel diameter regulation. In rat cerebral small arteries and arterioles as well as in rabbit portal vein, both resting membrane potential and arterial diameter were shown to be sensitive to specific Kv1 blockers that induce depolarization and vasoconstriction (1, 4, 5, 27). In the present study we observed a similar role of the Kv1 channels. Indeed, in pressurized arteries, we clearly showed that 4-AP induced greater contraction than IbTx in both RCA and LCA, suggesting that the Kv1 channels play important role in regulating basal tone. Our finding that 4-AP-induced contraction was greater in RCA than in LCA supported our electrophysiological data (Figs. 1B and 3). The greater contribution of Kv channels in RCA could explain the heterogeneity in autoregulatory mechanisms along the coronary vascular tree (10, 29) and could be linked to the difference in the transmural pressure between the LCA and the RCA. Particularly, the left anterior descending branch appears to be less protected than the RCA and vasodilates poorly when aortic pressure rises and the left ventricle is loaded (29).

In summary, the present study is the first to demonstrate regional electrophysiological heterogeneity at the single cell level in the rat coronary circulation. These differences in repolarizing currents between myocytes from the LCA and the RCA are due to a greater activity and protein expression of Kv1 channels in the RCA.


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 MATERIALS AND METHODS
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This work was supported by grant from la Fondation Simone and Cino del Duca. M. Gautier's fellowship is supported by l'Agence de l'Environnement et de la Maîtrise de l'Energie (ADEME) and by Pfizer Global Research and Development, Amboise, France.


   DISCLOSURES
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 MATERIALS AND METHODS
 RESULTS
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REFERENCES
 
Present address of V. de Crescenzo: Dept. of Physiology, University of Massachusetts Medical School, Worcester, MA 01655.


   ACKNOWLEDGMENTS
 
We thank Dr. Nancy Rusch for providing us correolide and for all scientifical advice. We also thank Manuel Rebocho for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Bonnet, LABPART-EA 3852, UFR Médecine, Université François Rabelais, 10 Bld Tonnellé-BP 3223, 37032 Tours Cedex-France (e-mail: bonnet{at}med.univ-tours.fr)

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.

* M. Gautier and J.-M. Hyvelin contributed equally to this work. Back


   REFERENCES
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