AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 277: H1403-H1409, 1999;
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Vol. 277, Issue 4, H1403-H1409, October 1999

Molecular and functional distributions of chloride conductances in rabbit ventricle

Kevin R. Wong1, Ann E. O. Trezise1,2, Simon Bryant3, George Hart4, and Jamie I. Vandenberg1

1 Section of Cardiovascular Biology, Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom; 2 Division of Biomolecular and Biomedical Science, Griffith University, Brisbane, Queensland 4111, Australia; 3 Department of Cardiovascular Medicine, University of Oxford, Oxford OX1 2JD United Kingdom; and 4 Department of Medicine, United Kingdom University of Liverpool, Liverpool L69 3BX, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The regulation of cardiac electrical activity is critically dependent on the distribution of ion channels in the heart. For most ion channels, however, the patterns of distribution and what regulates these patterns are not well characterized. The most likely candidates for the genes that encode the cAMP- and swelling-activated chloride conductances in the heart are an alternatively spliced variant of CFTR and ClC-3, respectively. In this study we have 1) measured the density of CFTR and ClC-3 mRNA levels across the left ventricular free wall (LVFW) of the rabbit heart using in situ hybridization and 2) measured the corresponding current density of cAMP- and swelling-activated chloride channels in myocytes isolated from subepicardial, midmyocardial, and subendocardial regions of the LVFW. There was a highly significant gradient in the whole cell slope conductance of cAMP-activated chloride currents; normalized slope conductance at 0 mV was 15.7 ± 1.8 pS/pF (n = 9) in subepicardial myocytes, 7.8 ± 1.5 pS/pF (n = 11) in midmyocardial myocytes, and 4.9 ± 1.1 pS/pF (n = 9) in subendocardial myocytes. The level of CFTR mRNA was closely correlated with the density of cAMP-activated chloride conductances in different regions of the heart, with the level of CFTR mRNA being three times higher in the subepicardium than in the subendocardium. The whole cell slope conductance of swelling-activated chloride channel activity, measured 3-5 min after the commencement of cell swelling, was higher in myocytes isolated from the subepicardium than in myocytes isolated from the midmyocardium or subendocardium. In contrast, there was a uniform expression of ClC-3 mRNA across the LVFW of the rabbit heart. These results suggest that the control of gene expression is an important contributor in regulating the distribution of cAMP-activated chloride channels in the rabbit heart but that it may be less important for the swelling-activated chloride channels.

electrophysiology; in situ hybridization; subepicardium; subendocardium


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SPREAD OF DEPOLARIZATION and repolarization in the heart is determined by the distribution of ion channels in different regions in the heart. In addition to the differences between sinoatrial node, atrial, and ventricular cells, recent work has shown that ion channels are heterogeneously distributed within each tissue (2). For example, in general there are higher densities of repolarizing currents in subepicardial than in subendocardial myocytes (2, 11, 12, 19, 21, 22). Recently, a number of chloride channels, including those activated by cAMP and cell swelling, have been identified in cardiac tissue (1), and it is suggested that their principal function is to modulate cardiac excitation during physiological and pathological stress. For example, activation of cAMP-activated chloride channels (ICl,cAMP) after adrenergic stimulation contributes to depolarization of the resting membrane potential (15, 18) and also influences action potential morphology and duration (18). Similarly, activation of swelling-activated chloride channels (ICl,swell) contributes to depolarization of the resting membrane potential (8, 29) and shortening of action potential duration (APD) during cell swelling (29). The distributions of these chloride channels are also thought to be heterogeneous; for example, the whole cell conductance for ICl,cAMP is twice as high in myocytes isolated from subepicardial compared with subendocardial layers of the left ventricle from rabbit (24) and guinea pig hearts (16). It has also been suggested that the distribution of ICl,swell in the heart may be heterogeneous (31), although there is no direct evidence to support this hypothesis (30).

Previous studies of heterogeneity of cardiac ion channel distribution have predominantly relied on techniques that provide only approximate indexes of ion channel distribution patterns, e.g., patch-clamp studies of cells isolated from different regions (11, 12, 21, 22) or RNase protection assays of RNA extracted from tissue from different regions (7, 33). The documentation of precise cellular distributions requires histological techniques. mRNA in situ hybridization allows the necessary histological correlation as well as providing a direct assessment of the concentration of ion channel mRNAs (4, 6, 10, 25-27). In situ hybridization studies are unable to indicate whether mRNA has been translated into functional ion channels, but such evidence can be obtained using correlative electrophysiology studies.

In this study we have used quantitative mRNA in situ hybridization in combination with patch-clamp analysis of cells isolated from different regions of the left ventricular free wall (LVFW) of rabbit heart to investigate the molecular and functional distributions of ClC-3 and the cystic fibrosis transmembrane conductance regulator (CFTR), the most likely candidates for the genes that respectively encode swelling- and cAMP-activated cardiac chloride channels (9, 14).


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Myocyte preparation. Single cardiac myocytes were isolated from atria and the subepicardial, midwall, and subendocardial regions of rabbit hearts. Male New Zealand White rabbits (n = 5) were killed by intravenous injection of pentobarbitone sodium (200 mg/kg iv). Hearts were rapidly excised and Langendorff perfused with a calcium-free medium for 5 min, followed by perfusion with a collagenase-protease digestion medium for 8-10 min as previously described (20). The basal LVFW was removed and pinned out so that thin tissue slices, ~0.5 × 2 × 5-mm strips, could be dissected from the subepicardial and subendocardial surfaces. A further ~0.5 mm of tissue was dissected from the subendocardial side of the remaining tissue and discarded before tissue samples were taken from the midmyocardium. The subepicardial, subendocardial, midmyocardial, and atrial tissue specimens were then incubated separately with collagenase and protease as previously described (22). Isolated myocytes were stored at room temperature in DMEM and used within 12 h of isolation.

Solutions. The compositions of the internal and external perfusion solutions were designed to block potassium and calcium currents as well as electrogenic transporters (31). The isosmotic external solution contained 70 mM NaCl, 2 mM MgCl2, 2 mM BaCl2, 20 µM ouabain, 2 µM nicardipine, 140 mM sucrose, and 5 mM HEPES, and pH was adjusted to 7.5 (at room temperature) with NaOH or CsOH (osmolality 285-300 mosmol/kg, measured using a freezing point osmometer; Roebling, Camlab, Cambridge, UK). In some experiments BaCl2 was replaced with CaCl2. For anion-substitution experiments NaCl was replaced with equimolar NaI or Na-aspartate; therefore, anion-substituted solutions all contained 8 mM Cl-. The standard hyposmotic solution was identical except that sucrose was omitted (osmolality 150-165 mosmol/kg). The internal (pipette) solution contained (in mM) 58 CsCl, 52 Cs-aspartate, 20 tetraethylammonium chloride, 10 EGTA, 5 MgATP, 0.2 Na3GTP, and 5 HEPES, and pH was adjusted to 7.3 (at room temperature) with CsOH or NaOH. The calculated reversal potential for chloride was 0 mV. Isoproterenol (isoprenaline) was freshly prepared as a 1 mM stock solution in water containing 100 mM ascorbic acid and was added to superfusion solutions to a final concentration of 0.5 µM. DIDS was prepared fresh each day (100 mM in DMSO) and added to superfusion solutions to a final concentration of 0.5 mM. Tamoxifen was prepared as a 10 mM stock solution in DMSO, stored at 20°C, and added to superfusion solutions to a final concentration of 10 µM. All reagents were from Sigma Chemical (Poole, UK).

Electrophysiology. The whole cell tight-seal voltage-clamp technique was used for electrophysiological recording (13). Patch electrodes were fabricated from glass capillaries (GC150TF, Clark Electromedical Instruments, Reading, UK) on a BB-CH-PC electrode puller (Mecanex SA, Geneva, Switzerland). Tip resistance ranged from 1.5 to 4 MOmega when filled with standard internal solution. After the formation of a gigaseal, brief strong suction was applied to the pipette interior to rupture the membrane patch. After membrane rupture, the suction port of the electrode holder was opened to the atmosphere to ensure that pressure was not applied to the back of the pipette. Membrane current and voltage were recorded using a patch-clamp amplifier (Axopatch 1C, Axon Instruments, Foster City, CA) interfaced with a 1401 Plus analog-to-digital converter (Cambridge Electronic Design, Cambridge, UK). The current-voltage (I-V) relation was measured by applying a triangular ramp pulse of 1 V/s, first by depolarizing to +80 mV and then by hyperpolarizing to -80 mV. The I-V curve was measured from the downward limb of the ramp pulse. Alternatively, whole cell currents were recorded during 200-ms rectangular pulses to potentials in the range from -100 to +100 mV. These protocols were repeated at 5-s intervals, with both current and voltage monitored on an oscilloscope and chart recorder. The cell input capacitance was measured from the jump in membrane current recorded at the positive peak of the ramp pulse. The input capacitance was not significantly different in myocytes isolated from different regions of the ventricle (144 ± 8, 155 ± 11, and 157 ± 16 pF in subendocardial, midmyocardial, and subepicardial cells, respectively; n = 11 in all groups). Whole cell currents were not corrected for membrane capacitance currents. In all experiments the patch pipette current was nulled before seal formation with the cells bathed in isotonic external solution. All experiments were performed at 35-37°C. In experiments in which there was no anion substitution, a nonflowing 3 M KCl salt bridge between the Ag-AgCl reference electrode and bath solutions was used. For anion-substitution experiments we used a flowing 3 M KCl bridge between the Ag-AgCl reference electrode and bath solutions, and a freshly chlorided silver reference electrode was used for each experiment.

mRNA in situ hybridization. Male New Zealand White rabbits (n = 5) were killed with an overdose of pentobarbitone (200 mg/kg iv). Hearts were rapidly excised and then retrogradely perfused with phosphate-buffered saline (4°C) containing heparin (4 U/ml), followed by perfusion with 4% paraformaldehyde solution. Hearts were stored in 4% paraformaldehyde solution containing 10% sucrose at 4°C for at least 48 h before they were sectioned. In situ hybridization experiments were carried out essentially as previously described (26). Briefly, 10-µm frozen sections of the LVFW were digested with proteinase K and then hybridized overnight at 50°C with sense or antisense 35S-UTP-labeled riboprobes for ClC-3, CFTR, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (see Riboprobes). Sections were then digested with RNase A and washed in 15 mM NaCl and 1.5 mM Na-citrate at 60°C to remove nonhybridized probe. X-ray film contact sheets (Biomax MR-1; Eastman Kodak, Rochester, NY) were exposed to hybridized slides for 48 h and then developed and fixed.

Riboprobes. A 654-bp fragment of the rabbit cardiac CFTR cDNA (accession no. GB U40227, a generous gift from Dr. Burt Horowitz) spanning exons 4-6 was prepared by introducing an EcoR I site at nucleotide (nt) 274 and a Pst I site at nt 907 using PCR site-directed mutagenesis. The fragment was ligated into pBluescript SK+ (Stratagene, Cambridge, UK), and the identity and orientation of the insert fragment was confirmed by sequencing from both the T7 and T3 promoters of the pBluescript vector.

A 501-bp fragment of the rabbit ClC-3 cDNA and a 454-bp fragment of the rabbit GAPDH cDNA were prepared by PCR amplification of adult rabbit heart cDNA using the following primers: ClC-3, 5'-CCTTTATGCCATGGTTGGTGC-3' and 5'-TGCTGTGCAAAACACACCCGA-3'; GAPDH, 5'-TCTCTCAAGATTGTCAGCAACG-3' and 5'-CTTCACAAAGTGGTCATTGAGG-3'. A touchdown PCR protocol was used to amplify cDNA fragments: denaturation for 1 min at 95°C; annealing for 1 min at 65°C (2 cycles), 60°C (4 cycles), 56°C (7 cycles), 52°C (10 cycles), and 47°C (15 cycles); and extension for 1.5 min at 72°C. cDNA fragments were ligated into the pGEM-T vector, and the identity and orientation were confirmed by sequencing from both the T7 and SP6 promoters of the pGEM-T vector (Promega, Southampton, UK).

Single stranded, 35S-labeled RNA probes were synthesized by linearizing vectors containing the cDNA inserts with the use of appropriate restriction enzymes followed by in vitro runoff RNA transcription using the appropriate RNA polymerase (T3, T7, or SP6 RNA polymerase, Epicentre, CamBio, Cambridge, UK). There was no cold UTP present in the labeling reaction, so all probes had approximately the same specific activity. Labeled probes were separated from unincorporated label by passage through a Sephadex G50 column, and probes were diluted into the hybridization mixture so that the final concentration of all probes was 2 × 107 counts per minute per milliliter.

Densitometry. Autoradiograms were scanned (256 gray scales) at a resolution of 600 dpi using a UMAX Powerlook III scanner. The optical density for each autoradiograph was calibrated using a photographic step tablet (Eastman Kodak). The density of sections across the LVFW was analyzed using NIH Image software (public domain). A minimum of three measurements was made for each section, and a minimum of nine sections per tissue sample was analyzed for each probe in five hearts. The densities of CFTR and ClC-3 mRNA in each heart were calculated by subtracting the value for the density of the sense signal from the value for the antisense signal. The values for each heart were normalized relative to the density of GAPDH mRNA before the mean levels of CFTR and ClC-3 mRNA were calculated for all hearts.

Statistical analysis. All results are reported as means ± SE. Statistical significance was assessed by ANOVA, and t-tests were performed with Fisher's test (3). A P value of <0.05 was regarded as significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Distribution of ICl,cAMP. Superfusion of ventricular myocytes with 0.5 µM isoproterenol resulted in activation of a linear conductance with a reversal potential close to 0 mV (-3.1 ± 1.4 mV, n = 29), the calculated equilibrium potential for chloride (see Fig. 1). DIDS (0.5 mM) had no effect on the isoproterenol-activated current (n = 5, data not shown). The density of current activated by isoproterenol, measured from the slope conductance at 0 mV, was 4.9 ± 1.1, 7.8 ± 1.5, and 15.5 ± 1.8 pS/pF in subendocardial, midmyocardial, and subepicardial cells, respectively. All groups were significantly different from the others.


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  		Fig. 1.   A: time course of changes in whole cell current at +40 () and -40 mV (+) in a midmyocardial ventricular myocyte exposed to 0.5 µM isoproterenol for 1 min. Roman numerals refer to traces depicted in B. B: current traces recorded during downward limb of a triangular voltage-ramp pulse before (i), during (ii), and after (iii) exposure to 0.5 µM isoproterenol. Reversal potential for isoproterenol-sensitive current was close to reversal potential for chloride (0 mV), and current-voltage (I-V) relationship was almost linear. C: summary of current density for cAMP-activated chloride channels (ICl,cAMP) in subepicardial (epi), midmyocardial (mid), and subendocardial (endo) left ventricular myocytes. Current density was measured as slope conductance at 0 mV at 1 min after exposure to isoproterenol. Values are means ± SE for n = 9, 11, and 9 subepicardial, midmyocardial, and subendocardial myocytes, respectively. All groups were significantly different from each other.

Distribution of ICl,swell. Superfusion of ventricular myocytes with a hypotonic solution resulted in activation of an outwardly rectifying conductance after a lag of 1-4 min (see Fig. 2A) in 38 of 43 cells. This time lag is typical for activation of ICl,swell in a wide range of cell types (17, 23). The time lag between exposure to hypotonic solution and activation of the current was slightly longer in subendocardial and midmyocardial cells (134 ± 14 and 142 ± 13 s, respectively) than in subepicardial cells (125 ± 11 s), although this difference was not significant. The swelling-activated current was reversibly inhibited by DIDS in a voltage-dependent manner (see Fig. 2A). In four cells 0.5 mM DIDS caused 92 ± 5% inhibition of the current at +60 mV and 60 ± 6% inhibition of the current at -60 mV. ICl,swell was also reversibly inhibited by tamoxifen (see Fig. 2B). In four cells 10 µM tamoxifen caused 97 ± 2% inhibition of ICl,swell. ICl,swell was more permeable to iodide than to chloride (see Fig. 2C) and less permeable to aspartate. Iodide caused a shift in the reversible potential for the swelling-activated chloride channel of -8 ± 2 mV (n = 3), and aspartate shifted the reversible potential of -42 ± 6 mV (n = 3). The current also showed mild voltage-dependent inactivation at positive potentials (see Fig. 2D). All these features are very similar to those reported for ClC-3-encoded chloride channels (9).


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  		Fig. 2.   A: time course of changes in whole cell current at +40 () and -40 mV () in a midmyocardial ventricular myocyte exposed to hypotonic solution for 5 min. There was a delay of 55 s before whole cell current started to increase. DIDS (0.5 mM) caused rapid and reversible inhibition of swelling-induced current. Roman numerals refer to current traces (right) recorded during downward limb of a triangular voltage-ramp pulse before cell swelling (i), during cell swelling (ii), and during cell swelling in presence of 0.5 mM DIDS (iii). Reversal potential for swelling-induced current was close to reversal potential for chloride (0 mV), and I-V relationship was outwardly rectifying. DIDS caused complete block of swelling-induced current at positive potentials but only partial inhibition of swelling-induced current at negative potentials. B: time course of changes in whole cell current at +40 () and -40 mV () in a midmyocardial ventricular myocyte exposed to hypotonic solution for 5 min, with tamoxifen (Tmx; 10 µM) added for 1 min (3 min after initial exposure to hypotonic solution). Tamoxifen caused a reversible inhibition of swelling-induced current at both positive and negative potentials. C: I-V curves obtained from ramp pulses show sensitivity of swelling-activated chloride current (ICl,swell) to replacement of external NaCl (Cl-) with equimolar NaI (I-). In this cell, reversal potential was shifted from -4.5 mV in presence of NaCl to -12 mV in presence of NaI (arrows). D: during 200-ms voltage steps from 0 to ±100 mV in 20-mV steps ICl,swell exhibited only very mild voltage-dependent inactivation at positive potentials.

In most cells ICl,swell did not reach a clear plateau, although the rate of increase usually declined after 3-5 min (see e.g., Fig. 2B). Furthermore, in cells exposed to hypotonic solution for more than 5-10 min, the cells often died. We therefore estimated the comparative density of ICl,swell whole cell slope conductance by taking measurements at fixed time points after exposure to cell swelling because this presumably would reflect how the different regions of the heart would respond if the whole heart were exposed to a hypotonic insult. The density of ICl,swell whole cell slope conductance, measured at 0 mV at either 3 or 5 min after exposure to hypotonic solutions, showed a gradient of activity between the subepicardial and subendocardial myocytes (see Fig. 3). If the records were corrected for the time lag between exposure to hypotonic solution and activation of the current (see above), then the differences between subepicardial and subendocardial myocytes were not significant. The whole cell slope conductance, measured 2 min after the current started to increase, was 20.4 ± 4.8 (n = 10), 22.7 ± 5.8 (n = 11), and 31.9 ± 6.7 pS/pF (n = 12) in subendocardial, midmyocardial, and subepicardial myocytes, respectively (see Fig. 3C).


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  		Fig. 3.   Summary of current density for ICl,swell in subendocardial (endo; n = 10), midmyocardial (mid; n = 11), and subepicardial (epi; n = 12) left ventricular myocytes. Current density was measured as slope conductance at 0 mV at 3 min after exposure to hypotonic solution (A), 5 min after exposure to hypotonic solution (B), and 2 min after whole cell current had started to increase (C). * Differences between subepicardial myocytes and subendocardial myocytes were significant in A and B (P < 0.05) but not in C.

The properties of the ICl,cAMP observed in this study, i.e., the linear I-V relationship and insensitivity to inhibition by DIDS for ICl,cAMP, are very similar to those reported for currents elicited from Xenopus oocytes injected with cRNAs for rabbit heart CFTR (14). There are likely to be many genes that encode for ICl,swell channels in different tissues (5); however, the most likely candidate for ICl,swell in cardiac tissue is ClC-3 (9). Furthermore, the properties of ICl,swell observed in this study, i.e., outwardly rectifying I-V curve, inhibition by tamoxifen, voltage-dependent inhibition by DIDS, anion permeability of I- > Cl- >> Asp-, and slow deactivation at positive potentials, are very similar to those reported for currents elicited from NIH/3T3 fibroblasts stably transfected with guinea pig heart ClC-3 (9). These data are therefore consistent with rabbit cardiac ICl,cAMP and ICl,swell being encoded by CFTR and ClC-3, respectively. To further test this hypothesis, we next investigated whether the distributions of CFTR and ClC-3 mRNA across the LVFW of the rabbit heart were correlated with the distribution of ICl,cAMP and ICl,swell.

Distribution of CFTR and ClC-3 mRNAs. Typical autoradiographs of slides exposed to sense and antisense riboprobes for CFTR, ClC-3, and GAPDH are shown in Fig. 4. Figure 4 clearly shows that the level of CFTR mRNA in the subepicardium is significantly higher than that in the subendocardium, whereas for ClC-3 and GAPDH there are no significant gradients of mRNA concentration between the subepicardium and subendocardium.


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  		Fig. 4.   Typical examples of autoradiographs obtained from sections of rabbit left ventricular free wall (LVFW) incubated with 35S-labeled sense or antisense riboprobes for cystic fibrosis transmembrane conductance regulator (CFTR) (A), ClC-3 (B), and GAPDH mRNA (C). Scale bar, 2 mm. Dashed boxes indicate regions where densitometry profiles were taken to obtain corresponding density profiles for CFTR (D), ClC-3 (E), and GAPDH mRNA (F). Mean density (±SE; n = 5) are shown for CFTR (G) and ClC-3 mRNA levels (H). CFTR mRNA levels are significantly higher in subepicardium than subendocardium, whereas ClC-3 mRNA levels are uniform across LVFW. For measurement of mean values, optical density measurements have been normalized relative to GAPDH mRNA (antisense - sense) signals in each heart (see MATERIALS AND METHODS).

The mean densities of CFTR and ClC-3 mRNA, normalized to a density of GAPDH mRNA of 1, are shown in Fig. 4, G and H. There was a significant gradient of CFTR mRNA (antisense - sense), with the level in the subepicardial region being 0.106 ± 0.006 units compared with 0.036 ± 0.007 units in the subendocardial region (3:1 ratio). However, there was not a significant gradient of ClC-3 mRNA (antisense - sense) across the LVFW (0.050 ± 0.008 and 0.044 ± 0.005 units in the subepicardium and subendocardium, respectively).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Regional differences in cardiac chloride current density. Two previous studies have shown a gradient of cAMP-activated chloride current density in rabbit (24) and guinea pig hearts (16). The relative level in subepicardial compared with subendocardial myocytes measured in our study, approximately threefold, was slightly higher than that measured by Takano and Noma (24) in rabbit myocytes, approximately twofold. However, sampling errors with regard to the thickness of heart wall taken in the different studies could account for these slight differences. Taking into account that the external chloride concentration used in our studies (78 mM) was lower than that used by Takano and Noma (140 mM), the density of ICl,cAMP in the different regions is very similar between the two studies. These values, however, are approximately two- to threefold lower than those seen in guinea pig heart (16). The most likely explanation for this is species differences.

In previous studies of ICl,swell in cardiac myocytes from guinea pig, it has been suggested (31) that ICl,swell appears to be expressed in only a subset of ventricular myocytes as defined by an increase in chloride-sensitive conductance 3 min after exposure to hypotonic solutions. In this study we found that the time lag between exposure to hypotonic solution and activation of the current was longer in subendocardial compared with subepicardial cells, and in some cells the lag time was longer than 3 min. Consequently, if ICl,swell density was assayed 3 min after exposure to hypotonic solution, there was a significantly higher current density in subepicardial than in subendocardial cells. However, when lag time to activation was taken into account, the differences in current density between subepicardial, midmyocardial, and subendocardial myocytes were not significant. It is possible, however, that there may also be species differences that could account for some of the apparent subsets of distribution in different species.

In cardiac cells the equilibrium potential for chloride ions is approximately -50 mV (32) compared with a resting membrane potential of approximately -80 mV and a plateau potential during the action potential of between 0 and +40 mV, depending on the species and cell type studied. Consequently, activation of a chloride conductance will cause depolarization of the resting membrane potential and decreased APD (1). Thus isoproterenol causes depolarization of the resting membrane potential of isolated ventricular myocytes via stimulation of ICl,cAMP (15, 18). ICl,cAMP also influences action potential morphology and APD after beta -adrenergic stimulation (18). Similarly, activation of ICl,swell contributes to depolarization of the resting membrane potential (8, 29) and shortening of APD during cell swelling (29). The regional distribution of ICl,cAMP, as measured in this study as well as others (16, 24), is therefore consistent with the hypothesis that activation of ICl,cAMP helps to maintain the gradient of APD across the ventricle during physiological stresses such as exercise. The activation of ICl,swell is likely to occur only during pathological stresses, e.g., after ischemia and reperfusion (30), and therefore the more rapid and possibly larger response in subepicardial myocytes would also tend to maintain a gradient of APD across the ventricular wall under these circumstances.

Regional distribution of ClC-3 and CFTR mRNA. The level of CFTR mRNA showed a marked subepicardial-to-subendocardial gradient that closely matched the distribution of ICl,cAMP activity. The gradient of ICl,cAMP density across the LVFW shown in this study as well as others (16, 24) is similar to that reported for transient outward and delayed rectifier potassium currents (2, 11, 12, 19, 21, 22). These gradients are clearly very important for the coordinated spread of repolarization in the heart (2), and therefore the processes that initiate and maintain these gradients of ion channel expression warrant further investigation. The results in this study suggest that, at least with respect to cAMP-activated chloride channels, this gradient of ion channel function may be controlled by regulating the expression of the underlying gene.

The level of ClC-3 mRNA showed a uniform distribution across the LVFW. It is more difficult, however, to make firm conclusions regarding the correlation between the distribution of ClC-3 mRNA and the density of ICl,swell because the levels of ClC-3 mRNA were quite low and, therefore, signals from nonmyoctes could have made a significant contribution to the signals observed. Furthermore, although there appeared to be a gradient for distribution of ICl,swell (see Fig. 3), there was no gradient for ClC-3 mRNA expression across the LVFW of the rabbit heart. Nevertheless, the properties of ICl,swell observed in this study are very similar to those reported for the cloned ClC-3 (9). Therefore, these results suggest that if swelling-activated chloride currents in rabbit heart are encoded by ClC-3, then posttranscriptional and/or posttranslational processes must modify the level of expression of functional channels. Alternatively, factors that regulate the activity of swelling-activated chloride channels, e.g., additional subunits, may be differentially expressed across the wall of the rabbit heart, thereby modifying the response of swelling-activated chloride channels in different regions of the heart.


    ACKNOWLEDGEMENTS

This work was supported by a British Heart Foundation Basic Sciences Award (to J. I. Vandenberg), British Heart Foundation Project Grants (to J. I. Vandenberg and G. Hart), a British Heart Foundation Travelling Research Fellowship (to A. E. O. Trezise), and an Australian Research Council Project Grant (to A. E. O. Trezise).


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests: J. I. Vandenberg, Section of Cardiovascular Biology, Dept. of Biochemistry, University of Cambridge, Tennis Court Rd., Cambridge CB2 1QW, UK (E-mail: j.i.vandenberg{at}mole.bio.cam.ac.uk).

Received 26 January 1999; accepted in final form 27 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ackerman, M. J., and D. E. Clapham. Cardiac chloride channels. Trends Cardiovasc. Med. 3: 23-28, 1993.

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3.   Armitage, P., and G. Berry. Statistical Methods in Medical Research. Oxford, UK: Blackwell Scientific, 1987.

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Am J Physiol Heart Circ Physiol 277(4):H1403-H1409
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society


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