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Journal of Physiology (2001), 533.1, pp. 145-154
© Copyright 2001 The Physiological Society
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ABSTRACT |
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) were compared in epicardial and endocardial regions of the rat ventricle.
with a diacylglycerol analogue (di-octanoyl-glycerol (DiC8), 20 µM) leads to differential effects in epicardial and endocardial cells. In epicardial cells (n = 20) It and Iss are attenuated by 17.7 ± 2.1 % and 11.9 ± 3.1 %, respectively (means ± S.E.M.). In endocardial cells It attenuation was significantly smaller (4.6 ± 1.6 %, n = 14, P < 0.0005). Iss attenuation was similar to that in epicardial cells (10.5 ± 3.8 %).
expression was measured by Western blotting. Calculated endocardial/epicardial ratios showed no regional differences in total protein extracts (1.04 ± 0.11, mean ± S.E.M, n = 4), but PKC distribution in the cytosolic fraction showed a marked difference, with significantly (P < 0.05) higher levels in endocardial extracts. The cytosolic endocardial/epicardial PKC ratio was 2.64 ± 0.24 (n = 4), indicating a reduced amount of PKC in the membrane fraction of the endocardium. This could account for the reduced effect of DiC8 on It in endocardial myocytes.
levels was absent (ratios of 0.86 ± 0.21 (n = 4) and 1.09 ± 0.16 (n = 3), respectively; means ± S.E.M.). Ratios in the total protein extracts were not significantly different from those in control conditions.
activation on a cardiac K+ current, and in the subcellular distribution of PKC. These differences are absent in diabetic and hypothyroid conditions.
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INTRODUCTION |
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It has long been recognised that the distribution of several potassium currents is not homogeneous across the wall of the mammalian ventricle (Fedida & Giles, 1991; Furukawa et al. 1991, 1992; Brahmajothi et al. 1997; Xu et al. 1999). The epicardial-endocardial gradient in K+ currents underlies the transmural differences in the configuration of the ventricular action potential (Litovski & Antzelevitch, 1988; Fedida & Giles, 1991). This difference, which underlies the upright T wave of the electrocardiogram (Burgess, 1979), provides protection against retrograde propagation of the action potential during each cycle of activation, thus preventing abnormal conduction pathways which may lead to 'reentrant' arrhythmias (Burgess, 1979; Surawicz, 1997).
A prominent current with heterogeneous distribution across the myocardial wall is the calcium-independent transient outward current, It, which is significantly larger in epicardial cells. In human, rat and canine ventricle there is also a gradient in the recovery kinetics of this current, which is slower in endocardial cells (Wettwer et al. 1994; Shimoni et al. 1995; Nabauer et al. 1996; Brahmajothi et al. 1999; Yu et al. 2000). The cloning of channel isoforms has made it possible to demonstrate that Kv4.2, a major component of this current in the rat and mouse (Fiset et al. 1997; Guo et al. 1999), exhibits a transmural gradient at the level of both mRNA (Dixon & McKinnon, 1994) and protein expression (Guo et al. 1999; Wickenden et al. 1999) in these species. In several pathological conditions the epicardial-endocardial transmural gradient in It is diminished. This was found to occur following hypertrophy (Bryant et al. 1999) and under hypothyroid or insulin-deficient conditions (Shimoni et al. 1995). The diminished gradient can compromise the normal protective mechanisms and predispose the heart to development of arrhythmias.
Not all K+ currents are distributed unevenly across the myocardial wall. An additional repolarising current in the rat ventricle is a delayed rectifier, quasi-steady-state current labelled Iss (Apkon & Nerbonne, 1991). In contrast to It, this current has similar density and characteristics in epicardial and endocardial cells (Shimoni et al. 1995).
In addition to the transmural gradient in potassium currents, there are also reports demonstrating epicardial- endocardial differences in the sensitivity of potassium currents to ischaemia or hypoxia (Gilmour & Zipes, 1980; Kimura et al. 1986, 1990). There are also epicardial- endocardial differences in calcium handling (Figueredo et al. 1993) and in calcium currents during ischaemia or metabolic inhibition (Kimura et al. 1991). These may be related to transmural gradients in the distribution of glycolytic enzymes (Lundsgaard-Hansen et al. 1967) and/or metabolites (Allison et al. 1977). Recently, it has been shown that there are differential responses of canine epicardial and endocardial cells to the chronic activation or blockade of the local renin-angiotensin system (Yu et al. 2000). This may be due to regional heterogeneity in the cardiac components of the system, such as the angiotensin converting enzyme (Yamada et al. 1991). In combination, these results suggest that there are fundamental differences in cellular properties and functions across the myocardial wall.
Numerous cellular functions are regulated by the various isoforms of protein kinase C (PKC) (Steinberg et al. 1995). For example, the acute activation of specific isoforms of PKC can directly modulate potassium currents (Nakamura et al. 1997; Lo & Numann, 1998; Shimoni, 1999). A major isoform of PKC expressed in cardiac cells is PKC (Bogoyevitch et al. 1993; Disatnik et al. 1994). Our recent work showed that under hypothyroid and diabetic conditions in which PKC is chronically up-regulated (Xiang & McNeill, 1992; Rybin & Steinberg, 1996; Malhotra et al. 1997), the acute modulation of It by the activation of PKC is absent (Shimoni, 1999). Furthermore, under hypothyroid and diabetic conditions there is also a chronic attenuation of It (Shimoni et al. 1995). Similar changes in It and PKC are present in cardiac hypertrophy (Gu & Bishop, 1994; Meszaros et al. 1996) and in heart failure (Rouet-Benzineh et al. 1996; Nabauer & Kaab, 1998). Since PKC has been shown to modulate the expression of a variety of genes (Harrington & Ware, 1995), the correlations between changes in PKC and It suggest that PKC may regulate the expression of Kv channel subunits underlying It, as well as modulating It in an acute manner.
The present experiments were designed to address the following questions: (1) are there differences in the response of It and Iss in epicardial and endocardial cells to acute activation of PKC?; (2) are there baseline differences in the expression and/or subcellular distribution of PKC between epicardial and endocardial cells?; (3) if the expression or subcellular distribution of PKC varies across the myocardial wall, is this altered by the same chronic hormonal changes which have been shown to reduce the baseline gradient of It, as well as abolishing the acute effects (on It) of PKC activation?
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METHODS |
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All experiments were done in accordance with the guidelines of the Animal Care Committee of the University of Calgary. Rats were anaesthetised by methoxyflurane inhalation and killed by cervical dislocation. Hearts were removed and the aortas cannulated for coronary perfusion using a Langendorff apparatus.
Cell isolation
Electrophysiological recordings were performed in enzymatically isolated single rat cardiac myocytes. In these experiments, cells were obtained separately from epicardial and endocardial layers of the left ventricular wall. Details of the method of cell isolation and solution composition are described in our earlier publications (Shimoni et al. 1995; Shimoni, 1999). After the initial perfusion of the whole heart in an enzyme-containing perfusion solution, thin layers of epicardial and endocardial tissue were removed and incubated separately in an enzyme-containing solution, yielding epicardial or endocardial single myocytes.
Current recordings
Currents were recorded using the whole cell suction electrode method. Low resistance electrodes (2-3 M
) and electronic compensation minimised series resistance. Data were discarded when series resistance changed by more than 10 % during recordings. Two currents were measured: the calcium-independent transient out-ward K+ current, It, and the sustained quasi-steady-state current, Iss. These were elicited by 500 ms pulses from -80 to +50 mV (at which the fast Na+ current is negligible). In some endocardial cells the transient current component was very small. A more accurate measure of this current could be obtained by introducing short prepulses (100 ms, to voltages between -10 and +10 mV) before the test pulse (to +50 mV). The (first) prepulse eliminated the transient current elicited by the (second) test pulse, so that a subtraction of the currents elicited with and without a prepulse gave the net transient current (see Fig. 2). The L-type calcium current was blocked by 0.3 mM CdCl2. It was measured as the peak current (since Iss activates much more slowly than It at +50 mV), whereas Iss was measured at the end of the pulse. The experiments were done at 21-22 °C.
Western blotting
Three experimental groups were used: control rats; rats made diabetic by a single I.V. injection of 100 mg kg-1 streptozotocin (STZ) 6-10 days before experiments; rats made hypothyroid by thyroidectomy, 3-4 weeks before experiments.
Thin sheets of tissue were dissected from the endocardial and epicardial walls of the ventricle. Cytosolic protein extractions were prepared by homogenisation in buffer containing (mM): Tris-HCl 20, DTT 10, EDTA 2, EGTA 5; pH 7.5 and a protease inhibitor cocktail of 0.5 mM phenylmethylsulphonylfluoride 10 µg ml-1 pepstatin A and 50 µg ml-1 leupeptin (10 ml (g tissue)-1). This was followed by centrifugation at 100 000 g for 30 min. The supernatant was taken as the cytosolic fraction and protein concentration was determined by the bicinchoninic acid assay (Pierce, Rockford, IL, USA). Total protein extracts were prepared by direct homogenisation in sodium dodecyl sulphate-polyacrylamide gel electropheresis (SDS-PAGE) sample buffer (50 mM Tris-HCl, 10 % SDS, 0.1 % bromophenol blue, 10 % glycerol, and 5 % 
-mercaptoethanol). Cytosolic or total endocardial and epicardial extracts were separated by 10 % acrylamide SDS-PAGE and transferred to 0.45 µm nitrocellulose membranes prior to blocking with 5 % non-fat dried milk (NFDM) in Tris-buffered solution (NFDM-TBST) containing (mM): Tris-HCl 20 NaCl 500 and 0.05 % Tween 20; pH 7.5. Membranes were labelled with rabbit polyclonal anti-PKC (Gibco BRL) at 5 µg ml-1 (demonstrated previously to be specific for PKC; Clement-Chomienne & Walsh, 1996) in 1 % NFDM-TBST, washed with TBST and probed with 1:4000 horseradish peroxidase conjugated goat anti-rabbit IgG secondary antibody (New England Biolabs, Mississauga, Ontario, Canada). Detection was achieved with SuperSignal West Pico Chemiluminescent Substrate (Pierce) and BioMax film (Kodak). Rat brain PKC purified free of other known PKC isoforms (by sequential chromatography on DEAE Sephacel, Phenyl Sepharose, Mono Q, hydroxyapatite and Superose 12) was included as a positive control on immunoblots. Preabsorption of the PKC antibody with an equivalent weight of peptide antigen blocked PKC immunoreactivity of ~85 kDa in purified rat brain and ventricular extracts (data not shown).
Quantification of PKC
SDS-PAGE gels for transfer to nitrocellulose and Western blotting were run simultaneously with equivalently loaded gels, stained with Coomassie Brilliant Blue R-250 (BioRad, Mississauga, Ontario, Canada) and destained with 10 % acetic acid. Densitometry of PKC immunoreactivity on immunoblots, and of an arbitrary band on stained protein gels to normalise loading levels, was performed using a Sharp JX-330 scanner and ImageMaster 1D software (Pharmacia Biotech, Piscataway, NJ, USA). PKC of endocardial and epicardial Western blots was quantified and normalised to protein loading level. The endocardial/epicardial protein ratio was then determined for each Western blot.
Statistics
A comparison between groups (electrophysiology and densitometry results) was done using Student's t test (paired or unpaired, according to the comparisons made). Differences with P < 0.05 were considered significant.
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RESULTS |
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PKC activation and K+ currents
Earlier work (Shimoni, 1999) demonstrated that It and Iss are inhibited by acute activation of a specific PKC isoform, PKC, in rat ventricular myocytes. The present work examined whether the effects of PKC activation on either of these currents are region specific.
In these experiments, as in our earlier work (Shimoni, 1999), PKC was activated by exposing cells to 20 µM di-octanoyl-glycerol (DiC8), a synthetic analogue of diacylglycerol, the physiological activator of most PKC isoforms. The effects of DiC8 on these currents were blocked by specific PKC inhibitors, including a specific PKC translocation inhibitory peptide (Shimoni, 1999). This demonstrated the role of PKC in It and Iss inhibition. In earlier and present experiments PKC activation often had independent effects on It and Iss, with either one or both currents attenuated in different cells. In the present experiments, the effects of DiC8 were examined separately in epicardial and endocardial cells.
The addition of DiC8 led to several patterns of response to PKC activation in the two cell types, as illustrated in Fig. 1. The left column shows current traces from two epicardial cells, one with an attenuation of both peak and steady-state currents (top) and one with only the peak current attenuated (bottom). The right-hand column shows current traces from two endocardial cells. In one case (top) DiC8 (*) had no effect, whereas in the other cell the steady-state current was attenuated, with no effect on the peak current. The downward deflections are current traces obtained in response to pulses to -110 mV, eliciting the background inward rectifier K+ current IK1. This current is unaffected by DiC8, and is used as a sensitive indicator of changes in series resistance.
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Figure 1. Effects of PKC activation by DiC8 on epicardial and endocardial K+ currents Superimposed current traces are shown before and 5-10 min after addition of 20 µM DiC8 (*) for two epicardial cells (left) and two endocardial cells (right). The upward deflections are in response to pulses from -80 to +50 mV, whereas the downward traces are currents elicited by pulses to -110 mV. These are used as controls for stable series resistance. DiC8 attenuates the peak transient current in both epicardial cells, with an attenuation of the steady-state current in one case (upper left). In endocardial cells, in contrast, the peak current is unaffected. Iss is attenuated variably, as with epicardial cells, with such a cell shown on the bottom right.
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The bottom right trace of Fig. 1 illustrates the fact that some endocardial cells have very little or no evident transient current component at all. Therefore, to enable a more reliable comparison of the effects of DiC8 on the transient current in the two cell types, an additional protocol was employed. By inserting a brief prepulse before the test depolarising pulse, it is possible to abolish the transient component, without affecting the slower steady-state component, since the latter is much slower (Apkon & Nerbonne, 1991). The subtraction of currents elicited with a prepulse (no transient current) from currents obtained without prepulses (in which a transient current is elicited) produces difference currents, which reflect the uncontaminated transient current. Thus, the measurement of very small transient currents is possible even when they are obscured by the steady-state current. This was done before and after exposure to DiC8, allowing an accurate determination of the effects of PKC activation on both currents. The left-hand column of Fig. 2 shows the voltage protocol used (prepulse denoted by *), as well as the currents elicited with (*) and without prepulses. The right-hand column shows the difference currents obtained by subtraction. Superimposed traces are shown of the difference currents before and after (*) DiC8 treatment. Examples are given from one epicardial cell (top current traces) and from two endocardial cells, one of which had a very small transient current component (bottom traces). In the latter case, the subtraction procedure enabled the measurement of the net transient component, despite its small magnitude. DiC8 was without effect in both endocardial cells.
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Figure 2. Effects of DiC8 on the difference currents Difference currents were obtained using a prepulse protocol (see text for details). The left-hand column shows the voltage protocol (top), with the prepulse (*). Sample traces of currents elicited with and without the prepulses are illustrated below, with one epicardial and two endocardial cells shown. One of these endocardial cells (bottom traces) had a very small transient component. The right-hand column shows the difference currents obtained by subtraction, before and after addition of 20 µM DiC8 (*). Whereas the transient component is attenuated in the epicardial cell, there is no attenuation in either of the endocardial cells.
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The time course and reversibility of current changes in response to DiC8 are shown in Fig. 3A. It is shown as open circles (
) and Iss as filled circles (
), with an epicardial cell shown on the left and an endocardial cell on the right. Partial reversal of the DiC8 effect was obtained in the epicardial cell, with no effect in the endocardial cell.
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Figure 3. Effect of DiC8 on time course of It and Iss A, time course of changes in It (
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Figure 3B shows the summary data for the effects of DiC8 on It and Iss in epicardial and endocardial cells. The mean (± S.E.M.) attenuation of It (peak current) was 17.7 ± 2.1 % in epicardial cells (n = 20), whereas the attenuation in endocardial cells (n = 14) was only 4.6 ± 1.6 %. The effect of DiC8 was significantly (P < 0.0005) larger in the epicardial cells. The attenuation of Iss was not significantly different in the two cell types, 11.9 ± 3.1 % in epicardial and 10.5 ± 3.8 % in endocardial cells.
Although the effects on the two currents are small, they can lead to significant prolongation of the action potential, since all the currents flowing during the plateau phase of the action potential are small. The accompanying large input resistance enables small current changes to have large effects on the membrane potential (Noble, 1987).
The results so far indicate that the responsiveness of the transient K+ current to PKC activation is larger in epicardial than in endocardial cells, in which It attenuation is barely detectable. In earlier work (Shimoni, 1999), we showed that in diabetic and hypothyroid conditions, in which PKC expression is elevated (Xiang & McNeill, 1992; Rybin & Steinberg, 1996), there was no It attenuation following the activation of PKC. It was thus considered possible that endocardial cells had higher levels of PKC expression, so that further activation by DiC8 would no longer affect It. An alternative interpretation is that a different distribution of PKC between membrane and cytosolic compartments accounts for epicardial-endocardial differences in It attenuation, with no differences in total PKC levels. This was addressed in the following experiments.
PKC expression in epicardial and endocardial tissue
In these experiments we compared the expression of PKC in epicardial and endocardial tissue. Using thin strips isolated separately from epicardial and endocardial layers of the left ventricle, PKC expression was compared using Western blotting with a specific antibody to PKC. In the first set of these experiments, the expression of PKC in total protein extracts of epicardial and endocardial tissue was compared. Figure 4A shows a Western blot obtained using anti-PKC, and total protein extracts from endocardial and epicardial tissues, at two loading levels. Purified rat brain PKC (left lane) was used as a positive control. Quantification of the PKC immunoreactivity by densitometry enabled calculation of the ratio of endocardial to epicardial PKC. As shown in Fig. 4B, the amount of total PKC in endocardial tissue is not significantly different from that found in epicardial tissue. This is shown as the calculated endocardial/epicardial ratios, obtained from epicardial and endocardial samples from four rats. The mean (± S.E.M., n = 4) ratio of endocardial to epicardial total PKC (
in Fig. 4B) was 1.04 ± 0.11.
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Figure 4. Immunoblotting for PKC A, representative immunoblot with two loading levels for endocardial (Endo) and epicardial (Epi) tissues. The left lane shows PKC
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Cytosolic fractions were also prepared from epicardial and endocardial tissues, and immunoblotting for PKC was performed to determine cytoplasmic PKC levels. In this case, a clear difference was found, with endocardial PKC levels being significantly (P < 0.05) higher than epicardial levels, as shown in Fig. 5. Figure 5A shows a representative blot with two loading levels, whereas Fig. 5B shows the individual calculated endocardial/ epicardial ratios (based on densitometry). The mean endocardial/epicardial cytosolic PKC ratio ( in Fig. 5B) was 2.64 ± 0.24, n = 4.
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Figure 5. Immunoblotting for PKC A, representative immunoblot with two loading levels for endocardial (Endo) and epicardial (Epi) tissues, with purified rat brain PKC
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These results are consistent with the electrophysiological data. The similarity in total PKC expression in the epicardial and endocardial tissue, together with the higher cytosolic levels in endocardial tissue, indicate that there is less membrane-associated PKC in endocardial than in epicardial cells. This is a likely mechanism for the marked reduction in the attenuation of It by PKC activation in endocardial cells, compared to epicardial cells. Less membrane-associated PKC in endocardial cells presumably leads to less PKC localised in close proximity to It channels, thus precluding or diminishing the (attenuating) effects on current flow.
PKC distribution in hypothyroid and diabetic conditions
In recent work (Shimoni, 1999), we showed that the acute effect of DiC8 on It and Iss is absent in myocytes from diabetic and hypothyroid rats. In earlier work (Shimoni et al. 1995), we showed that the transmural gradient in the transient outward K+ current could be greatly reduced under experimental conditions involving hormonal alterations, such as insulin or thyroid deficiency. Both of these conditions have been shown to be associated with an elevation of PKC (Xiang & McNeill, 1992; Rybin & Steinberg, 1996), or a change in its subcellular distribution (Malhotra et al. 1997). Thus, it was of great relevance to establish whether the epicardial-endocardial differences in PKC subcellular distribution are dissipated under conditions in which It attenuation is eliminated. Measurements of PKC levels in total and cytosolic protein extracts were repeated using rats made either diabetic (with STZ, 6-10 days before experiments) or hypothyroid (3-4 weeks before the experiments). Figure 6 shows the results obtained in four hypothyroid rats. Figure 6A shows a representative blot obtained from a total extract, with B showing the individual calculated endocardial/epicardial ratios, as well as the mean ratio, 0.98 ± 0.10 (n = 4). Figure 6C and D shows the results from the cytosolic fractions, illustrating that the normal epicardial-endocardial difference in cytosolic PKC is dissipated. The mean endocardial-epicardial ratio of cytosolic PKC under hypothyroid conditions was 0.86 ± 0.21 (n = 4). Figure 7 shows the same pattern in total and cytosolic fractions obtained from a diabetic rat. The cytosolic endocardial/epicardial PKC ratio was 1.09 ± 0.18 (n = 3). The endocardial/epicardial ratio for total extract PKC was 1.25 ± 0.10 (n = 4) in the samples from diabetic rats. These results show that the normal endocardial-epicardial differences in cytosolic PKC are absent under insulin or thyroid hormone-deficient conditions. This, along with an increase in total PKC expression under these conditions (Xiang & McNeil, 1992; Rybin & Steinberg, 1996), may explain the lack of effect of acute PKC activation on It and Iss under these conditions (Shimoni, 1999).
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Figure 6. Immunoblotting for PKC A and C, representative immunoblots with two loading levels of total (A) and cytosolic (C) extracts of endocardial (Endo) and epicardial (Epi) tissues. B and D show PKC
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DISCUSSION |
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Summary of results
The findings presented here show two novel aspects of transmural heterogeneity in the myocardial wall. This is the first report demonstrating that the cardiac ventricular wall exhibits a transmural difference in the subcellular distribution of a specific PKC isoform, PKC (Fig 5). This epicardial-endocardial difference is reflected in a regional functional disparity in which PKC activation by a diacylglycerol analogue (DiC8) attenuates a transient K+ current (It) only in epicardial, but not in endocardial cells (Figs 1-3). This presumably reflects the fact that in endocardial cells there is less membrane-associated PKC to mediate inhibitory phosphorylation of the channel. Moreover, the epicardial-endocardial differences in cytosolic PKC are dissipated under STZ-induced diabetic and hypothyroid conditions (Fig. 6 and Fig. 7), in which acute PKC activation is without effect on the K+ currents. This is in addition to the dissipation of the epicardial-endocardial gradient in It, shown previously (Shimoni et al. 1995) to occur under these conditions, and which may also relate to the subcellular differences in PKC distribution shown here.
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Figure 7. Immunoblotting for PKC A and C show representative immunoblots with two loading levels of total (A) and cytosolic (C) extracts of endocardial (Endo) and epicardial (Epi) tissues. B and D show PKC
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Significance and implications
It attenuation by PKC activation is greatly reduced in endocardial cells, in which membrane-associated PKC levels are lower than in epicardial cells. This result suggests that signalling mediated through PKC will have different effects on It (and possibly on other PKC substrates) in epicardial and endocardial cells in the mammalian ventricle. The differences in PKC distribution shown here could therefore account for the differential response of epicardial and endocardial regions to hypoxia and ischaemia (Gilmour & Zipes, 1980; Kimura et al. 1990; Furukawa et al. 1991), which are suggested to be mediated by It (Lukas & Antzelevitch, 1993). Agonists which activate PKC, such as angiotensin II (Clement-Chomienne et al. 1998) may have region specific effects, including effects of angiotensin II on the transient K+ current, as shown recently (Yu et al. 2000). The region specific effects of PKC on It are absent in diabetes or hypothyroid conditions, following the subcellular redistribution of PKC and the coincident dissipation of the endocardial-epicardial difference in It. This may have detrimental implications, since a concomitant dissipation of the It gradient and a reduction in the acute regulatory effects of PKC (Shimoni et al. 1995; Shimoni, 1999) could be arrhythmogenic.
Earlier, we showed that PKC activation does not attenuate It or Iss in diabetic or hypothyroid conditions (Shimoni, 1999). This may be due to the fact that overall PKC expression is augmented under these conditions (Xiang & McNeill, 1992, in which no regional distinctions were made; Rybin & Steinberg, 1996), and/or to the fact that PKC distribution is altered (Fig. 6 and Fig. 7). This may have wider implications, since changes in subcellular distribution reportedly occur in response to hypoxia, ischaemia, hypertrophy and heart failure (Gu & Bishop, 1994; Steinberg et al. 1995; Rouet-Benzineh et al. 1996; Bryant et al. 1999), as well as in hypothyroid and diabetic conditions (present work). This would suggest that under these conditions the acute regulation of K+ channel activity could be compromised. In addition, many other cellular functions mediated by PKC may be altered or diminished in these situations. The present results add another dimension of complexity, since changes in PKC isoform distribution in response to pathological conditions should now also be considered on a regional basis.
It and Iss are both attenuated through PKC activation, since the effect of DiC8 on both currents is blocked by the specific inhibition of PKC translocation (Shimoni, 1999). However, our results suggest that the PKC regulation of the two K+ currents It and Iss differs. Thus, in endocardial cells, in which It is unresponsive to PKC activation by DiC8, Iss is still attenuated (Figs 1-3). It is not clear at present what underlies this difference. It is possible that different pools of PKC are associated with the two channels, conferring differential regulatory mechanisms. However, no evidence for this is available at present.
Limitations
A limitation of this work lies in the fact that the PKC measurements were done on pieces of tissue containing several cell types, whereas the electrophysiology was done using single myocardial cells. The use of tissue was necessary in order to obtain the required amount of protein for Western blotting. However, since the measurements of the effects of PKC activation in single epicardial and endocardial cells correlate with the PKC measurements in the epicardial and endocardial tissue, this limitation was not considered to be a hindrance to the major conclusion reached. This is particularly so since the PKC differences were dissipated under the same conditions in which It attenuation by PKC was lost. A further limitation is the fact that a third important cell type, the M cell, which has been identified in the mid-myocardium of several larger mammalian species (Yan et al. 1998), could not be isolated from the smaller rat heart. M cells have a different electrophysiological profile, and a study of PKC in these cells would be of great interest.
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Acknowledgements
This work was supported by grants to Y.S. and M.P.W. from the Heart and Stroke Foundation of Alberta, the North West Territories and Nunavut. K.S.T. is the recipient of Studentships from the Canadian Institutes of Health Research and the Alberta Heritage Foundation for Medical Research (AHFMR). M.P.W. is an AHFMR Medical Scientist.
Corresponding author
Y. Shimoni: Department of Physiology and Biophysics, 3330 Hospital Dr. NW, Calgary, AB, Canada, T2N 4N1.
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), as well as the mean (± S.E.M,