Effects of Hypertrophy on Regional Action Potential Characteristics in the Rat Left Ventricle
A Cellular Basis for T-Wave Inversion?
Background In cardiac hypertrophy, ECG T-wave changes imply an abnormal sequence of ventricular repolarization. We investigated the hypothesis that this is due to changes in the normal regional differences in action potential duration. We assessed the contribution of potassium- and calcium-dependent currents to these differences. Both the altered sequence of ventricular repolarization and the underlying cellular mechanisms may contribute to the increased incidence of ventricular arrhythmias in hypertrophy.
Methods and Results Rats received daily isoproterenol injections for 7 days. Myocytes were isolated from basal subendocardial (endo), basal midmyocardial (mid), and apical subepicardial (epi) regions of the left ventricular free wall. Action potentials were stimulated with patch pipettes at 37°C. The ratio of heart weight to body weight and mean cell capacitance are increased by 22% and 18%, respectively, in hypertrophy compared with controls (P<.001). Normal regional differences in action potential duration at 25% repolarization (APD25) are reduced in hypertrophy (control: endo, 11.4±0.9 ms; mid, 8.2±0.9 ms; epi, 5.1±0.4 ms; hypertrophy: endo, 11.6±0.9 ms; mid, 10.4±0.8 ms; epi, 7.8±0.6 ms). The regional differences in APD25 are still present in 3 mmol/L 4-aminopyridine. Hypertrophy affects APD75 differently, depending on the region of origin of myocytes (ANOVA P<.05). APD75 is shortened in subendocardial myocytes but is prolonged in subepicardial myocytes (control: endo, 126±7 ms; epi, 96±10 ms; hypertrophy: endo, 91±6 ms; epi, 108±7 ms). These changes in APD75 are altered by intracellular calcium buffering.
Conclusions Normal regional differences in APD and the changes observed in hypertrophy are only partially explained by differences in Ito1. In hypertrophy, the normal endocardial/epicardial gradient in APD75 appears to be reversed. This may explain the T-wave inversion observed and will have implications for arrhythmogenesis.
Regional differences in APD were originally deduced from the observation that the T wave in the ECG of some mammalian species (including humans and rats) is upright.1 Because the T wave is a reflection of ventricular repolarization, the direction of repolarization must normally be opposite to that of depolarization. Therefore, because depolarization normally spreads from the endocardial surface of the ventricle through the myocardium to the epicardial surface,2 it may be predicted that subendocardial myocytes have a longer action potential than subepicardial myocytes. Cardiac hypertrophy is associated with changes in T-wave morphology, implying that the sequence of repolarization is altered. To date, there has been only one study of endocardial/epicardial differences in APD in hypertrophy.3 However, this study (using muscle strip preparations) failed to demonstrate any correlation between endocardial/epicardial differences in APD and T-wave morphology in hypertrophy.
The aims of the present study were therefore (1) to confirm the existence of endocardial/epicardial differences in APD in the normal rat heart and to identify regional differences in the response to hypertrophy and (2) to assess the contribution that calcium-dependent and potassium currents may make to such regional differences and to hypertrophy-associated changes. Previous studies of action potential characteristics of isolated myocytes in hypertrophy have used the whole of the LV, including septum, to obtain myocytes. Our study is therefore the first comparison in isolated myocytes of regional differences in action potential characteristics associated with hypertrophy.
Model of Hypertrophy
A rat model of catecholamine-induced cardiac hypertrophy previously described by Mészáros and Lévai was used.4 Male Wistar rats (weighing 180 to 200 g initially) were injected with isoproterenol 1 mg IP daily for 7 days. Weight-matched controls received the same volume (1 mL) of normal saline. Animals were killed 24 hours after the last injection. Treatment of animals was in accordance with the Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986 (H.M.S.O.).
ECGs were recorded in vivo the day after the last injection from 9 pairs of rats anesthetized with a combination of ketamine 5 mg/kg and fentanyl and fluanisone (Hypnorm) 1 mL/kg. Standard limb electrodes were attached to the legs of the unconscious, prone rats, and a six-lead ECG was recorded at a paper speed of 50 mm/s and a voltage gain of 0.05 mV/mm. These ECGs were subsequently blindly analyzed. Eight pairs of animals were used for measurements of blotted wet weight of organs and 10 pairs for electrophysiological recordings.
Isolation of Myocytes
Rats were killed by cervical dislocation. The heart was quickly excised, suspended from a Langendorff column, and perfused with a nominally calcium-free Tyrode’s solution for 2 minutes, followed by an enzyme solution containing collagenase (0.75 mg/mL, Worthington Biochemical Corp) and protease (0.025 mg/mL, Sigma Chemical Co) for a further 7 minutes. The LV free wall was then removed, and the apical and basal portions were pinned out separately so that thin slices of tissue could be dissected from the basal endocardial and apical epicardial surfaces. Another slice of tissue was excised below the basal subendocardium and discarded before a deep, thicker slice of midmyocardium was retrieved. These three tissue samples (referred to as endo, epi, and mid, respectively) were then separately digested in fresh collagenase solution. Cells were filtered from the solutions, centrifuged, resuspended in DMEM, and stored at 20°C.
Cells were layered onto the coverslip of a perfusion chamber situated on the stage of an inverted microscope (Nikon Diaphot). The chamber was perfused with a modified Tyrode’s solution at 35±1°C. Patch pipettes (resistance, 4 to 7 MΩ when filled with solution) were pulled from filamented glass tubing (GC150TF-15 Clark Electromedical Instruments) on a horizontal puller (BB-CH-PC Mecanex SA). Membrane voltage and currents were measured by the whole-cell patch-clamp technique (Axopatch 2A, Axon Instruments). All analog signals were digitized with a 12-bit A/D converter (1401, C.E.D.) and digitally stored for off-line analysis.
Action potentials were stimulated at 0.2, 1, and 2 Hz in current-clamp mode with a current pulse amplitude ≈50% greater than threshold. Action potential parameters were taken from a mean of five individual action potentials recorded at steady state. Cell membrane capacitance was measured with a standard voltage-ramp protocol.5 After a change to 4-AP perfusion solution, cells were stimulated at 1 Hz until action potentials and contraction had reached a new steady state (≈3 minutes) before the action potential protocols were repeated.
Isolation Tyrode’s solution contained (mmol/L) NaCl 130, KCl 5.4, MgCl2 3.5, NaH2PO4 0.4, glucose 10, HEPES 5, and taurine 20; pH 7.2 adjusted with NaOH. The superfusion solution contained (mmol/L) NaCl 137, KCl 5.4, MgCl 1.2, glucose 10, and HEPES 5; pH 7.4. A stock solution of 3 mol/L 4-AP was diluted in perfusion Tyrode’s solution to give a final concentration of 3 mmol/L. The pipette solution contained (mmol/L) KCl 140, MgCl2 2, Mg-ATP 5, sodium phosphocreatine 5, and HEPES 10; pH 7.4 with KOH. In some experiments, BAPTA 5 mmol/L was included in the pipette to buffer intracellular calcium.
Comparisons of ECG parameters and organ weights between the isoproterenol-treated and control animals were performed by Student’s t test. Electrophysiological parameters were initially assessed with two-way ANOVA using the factors hypertrophy and region of origin of myocytes to define the groups. (A normal distribution for each parameter was confirmed with a Kolmogorov-Smirnov test.) Post hoc analysis was performed with the Student-Newman-Keuls technique, so that multiple comparisons between the data of the six groups were valid. For all tests, P<.05 was taken as statistically significant.
Evidence of Cardiac Hypertrophy
Organ weights. Blotted wet weights of organs from eight pairs of animals are shown in Table 1⇓. LV weight, heart weight, and ratio of heart weight to body weight were increased in the isoproterenol-treated group by 21%, 17%, and 22%, respectively. Liver and lung weights were not significantly different, and there was no macroscopic evidence of edema (pleural or pericardial effusions), indicating that cardiac heart failure was not present in these animals.
Electrocardiograms. The ECGs of control rats had upright T waves, with close concordance between the QRS and T-wave axes and little interanimal variation (see Fig 1⇓).
In the isoproterenol-injected group, the QRS and T-wave axes were divergent and showed wide interanimal variation. The mean QRS amplitude was increased and the mean T-wave amplitude decreased compared with control.
Cell membrane capacitance. Cell membrane capacitance was not significantly different in myocytes from the three regions in either control or hypertrophy groups (mean cell capacitance, control: endo, 117±8 pF, n=22; mid, 110±9 pF, n=17; epi, 110±8 pF, n=21; hypertrophy: endo, 135±10 pF, n=19; mid, 127±11 pF, n=14; epi, 136±8 pF, n=22). Overall mean membrane capacitance was 18% greater in myocytes isolated from isoproterenol-treated rats than from controls.
Action Potential Configuration in Tyrode’s Solution
Representative individual action potentials recorded at 1 Hz from subendocardial and subepicardial myocytes isolated from control rats are shown in Fig 2⇓. The resting potentials are similar, but APA and APD are greater for the subendocardial than the subepicardial myocyte.
Mean data for APA and APD25 show that endocardial/epicardial differences in amplitude and APD25 are significant (see Table 2⇓). The mean APA and APD of midmyocardial myocytes are intermediate between the subendocardial and subepicardial values. Although the regional differences in APD75 of control myocytes do not reach statistical significance (P=.09), they are consistent with the predicted transmural gradient in APD.6
Mean data for myocytes isolated from hypertrophied hearts are shown in Table 2⇑. As in control myocytes, the resting potential does not show significant regional variation in hypertrophy. Action potentials of subendocardial myocytes have greater amplitude and APD25 than subepicardial myocytes, and regional differences are significant, although less so than those seen in controls. The regional differences in APD75 in hypertrophy show the opposite pattern to that seen in control, with APD75 being longest in subepicardial and shortest in subendocardial myocytes in hypertrophy.
Comparison of data for hypertrophied myocytes with those of controls shows that hypertrophy is associated with a depolarization of the resting potential in all three regions. The peak of the action potential is also decreased in hypertrophy, so the APA is reduced compared with control (ANOVA P<.01).
Representative action potentials in Fig 3⇓ show the changes in APD associated with hypertrophy in myocytes isolated from each region. In subendocardial myocytes, APD25 is unchanged and APD75 shortened in hypertrophy compared with control. In subepicardial myocytes, both APD25 and APD75 are prolonged in hypertrophy. In midmyocardial myocytes, APD25 is also prolonged in hypertrophy, but there is no prolongation of APD75. Analysis of the mean data (Table 2⇑) shows a significant increase in APD25 in subepicardial and midmyocardial myocytes (Student-Newman-Keuls P<.05) but not in subendocardial myocytes in hypertrophy. The changes in APD75 associated with hypertrophy are also different depending on the region of origin of the myocytes (ANOVA P<.02; hypertrophy and region interact to influence APD75). The APD75 of subepicardial myocytes is longer in hypertrophy than in control, whereas the APD75 of subendocardial myocytes is shorter in hypertrophy (P<.001). Because the hypertrophy-induced changes in APD75 are discordant with those in APD25, they are not simply a reflection of changes in the early part of the action potential.
APD is dependent on the stimulation frequency for myocytes isolated from both control (Fig 4A⇓) and hypertrophied (Fig 4B⇓) hearts. The pattern of regional differences and hypertrophy-induced changes in APD are the same at all frequencies studied.
Action Potential Configuration in 4-AP
To assess the contribution of Ito1 to the regional differences in action potential characteristics and changes observed in hypertrophy, the same protocols were repeated in the presence of 3 mmol/L 4-AP, a specific inhibitor of Ito1.7 8 9 10 11
Mean data for action potentials recorded after a change to 4-AP are shown in Table 3⇓. The application of 4-AP hyperpolarizes the resting potential (by ≈2 mV) and increases APA and APD in all myocytes. However, the patterns of regional differences in APA and APD25 seen in Tyrode’s solution are preserved; APA and APD25 are greater in subendocardial than subepicardial myocytes in both control and hypertrophy, and regional differences in APA and APD25 remain statistically significant. This suggests that there are regional differences in 4-AP–insensitive currents that affect the early part of the action potential.
After the application of 4-AP, the APD25 of myocytes from the same region was similar in control and hypertrophy, ie, the association of hypertrophy with a prolongation of APD25 is lost. This suggests that the hypertrophy-associated changes in APD25 observed in Tyrode’s solution are due to changes in Ito1.
The influence of Ito1 can also be indirectly assessed by comparison of the relative change in APD25 elicited by 4-AP. The percentage prolongation of APD25 is greatest in subepicardial myocytes and least in subendocardial myocytes in both the control and hypertrophy groups (see Fig 5⇓), although the regional differences in the effects of 4-AP are less in hypertrophy than in control. These data support the hypothesis that Ito1 density is greater in subepicardial than subendocardial myocytes in control myocytes but that this difference is reduced in hypertrophy.
Action Potential Configuration in the Presence of Intracellular BAPTA
Representative action potentials recorded from subepicardial myocytes with patch electrode solutions with and without BAPTA are shown in Fig 6A⇓. The resting potential is depolarized in the presence of BAPTA, but the upstroke and early repolarization phase of the action potential are similar to those recorded when intracellular calcium is not buffered. However, the prominent late plateau phase of the rat action potential, which is dependent on intracellular calcium,12 13 is abolished by intracellular BAPTA.
In Fig 6B⇑, APD75 is plotted against APD25 for action potentials recorded with and without BAPTA from myocytes in Tyrode’s solution. The range of APD25 is similar whether BAPTA is included or not, but in the presence of BAPTA, APD75 is much shorter. Moreover, action potentials recorded without BAPTA show a wide scatter for APD75 and poor correlation between APD25 and APD75, but in the presence of BAPTA APD75 is closely correlated to APD25. Therefore, when BAPTA is included in the patch solution, the regional differences and hypertrophy-associated changes in APD75 mirror those observed in APD25. Because of the close correlation between APD25 and APD75 in the presence of BAPTA, data for the two time points can be summarized together (see Table 4⇓).
In myocytes isolated from control rats, APA and APD in the presence of BAPTA have significant regional variation, being greatest in subendocardial myocytes and least in subepicardial myocytes. In hypertrophy, no regional difference in APA or APD is detected. The changes in APD associated with hypertrophy are different in the three regions (ANOVA hypertrophy and region interaction, P<.05), with APD being prolonged in both midmyocardial and subepicardial myocytes compared with controls (P<.01) but unchanged in the subendocardial myocytes. This is similar to the data for APD25 recorded without BAPTA, which also showed hypertrophy to be associated with a prolongation of APD25 in the midmyocardial and subepicardial myocytes but not in subendocardial myocytes and a reduction in regional differences in APD25 in hypertrophy (cf Table 2⇑).
Under conditions in which intracellular calcium is not buffered, hypertrophy is associated with a significant reduction in APD75 of subendocardial myocytes. However, when BAPTA is included in the patch solution, the APD75 of subendocardial myocytes is unaltered in hypertrophy.
These results demonstrate that there are regional differences in action potential characteristics in the normal rat heart. We have identified, for the first time, regional differences in the electrophysiological changes associated with cardiac hypertrophy. There is prolongation of the early repolarization phase of the action potential (APD25) of subepicardial and midmyocardial myocytes, which may be attributed to a reduction in Ito1 density. In subendocardial myocytes, however, APD25 is unchanged in hypertrophy, and the plateau phase of the action potential is significantly shortened because of changes in calcium-dependent currents (probably Na+-Ca2+ exchange). Midmyocardial myocytes show a combination of the characteristics of both subendocardial and subepicardial myocytes, with prolongation of APD25 but not of APD75 in hypertrophy (see Fig 3⇑).
We have confirmed earlier work showing that catecholamine-induced hypertrophy in rats is associated with T-wave inversion.4 This and the counterclockwise (“left”) shift of the mean QRS axis are similar to ECG changes observed in human cardiac hypertrophy, which suggests that comparable ventricular repolarization abnormalities may be present in cardiac hypertrophy both in the rat model and in humans.
Regional Differences in Action Potential Characteristics of Normal Hearts
Apex/base differences in rat APD have previously been demonstrated by Watanabe et al.14 They found that endocardial action potentials recorded from ventricular wall preparations could be classified by duration into short (found in basal, mid, and apical regions of the right ventricle), intermediate (in mid and apical regions of the LV), and long (basal LV). They also found that myocytes isolated from the whole of both ventricles showed a range of APD that could be divided into three groups and appeared to correspond to the three types recorded from tissue preparations. However, they did not explore possible endocardial/epicardial differences in APD or attempt to isolate myocytes from specific regions.
Differences between action potentials of subendocardial and subepicardial myocytes have previously been demonstrated in rat,15 dog,16 rabbit,17 guinea pig,18 and humans.19 Our results confirm earlier work15 in showing APA and APD of subendocardial myocytes to be greater than those of subepicardial myocytes in normal rat hearts. We have found that regional differences in APA and APD25 are still detectable in the presence of 4-AP, which suggests that although Ito1 density may be greater in subepicardial than in subendocardial myocytes (as in humans,19 cats,20 rabbits,17 and dogs16 ), the magnitude of other rapidly activating currents must also differ between regions.
This article is the first report comparing the electrophysiology of midmyocardial cells to subepicardial and subendocardial myocytes in the rat. The action potential parameters of midmyocardial cells are intermediate between the values for subendocardial and subepicardial cells at all stimulation frequencies studied (0.2, 1.0, and 2.0 Hz; see Fig 4⇑). Sicouri and Antzelevitch21 identified a group of midmyocardial “M cells” with extreme prolongation of the action potential at slow stimulation rates in dogs. We found no evidence of M cells in the rat, because the midmyocardial myocyte action potential characteristics show rate dependence similar to that of myocytes from the other regions.
Increasing stimulation frequency produced an increase in APD25 and a decrease in APD75 in all regions. The prolongation of APD25 and small increase in APA seen with increasing stimulation frequency suggests that the recovery of the Ito from inactivation may be incomplete at the higher frequencies. Human action potentials also show a reduced early repolarization phase as frequency increases, which is also thought to be due to partial inactivation of Ito. Wettwer et al9 directly compared Ito1 in rat and human isolated myocytes and found that the kinetics of inactivation are similar, especially when the faster physiological heart rate of rats is taken into account.
Regional Changes in Action Potential Characteristics Associated With Hypertrophy
The resting potential showed no significant regional variation under any of our experimental conditions. The depolarization of the resting potential in hypertrophy in this model, previously ascribed to a reduction in Na+,K+-ATPase activity,22 is uniform in the different regions.
Previous work in mammalian species has consistently shown a prolongation of APD in hypertrophy, both in isolated myocytes and in tissue preparations (reviewed in Reference 2323 ). However, the whole of the LV, including the septum, has been used to obtain isolated myocytes,24 25 26 despite regional differences in the response to hypertrophy being predictable from the ECG T-wave changes.6
Keung and Aronson3 investigated endocardial/epicardial differences in APD in rats with hypertrophy in LV tissue slices. However, they found regional differences in APD to be insignificant in both sham and hypertrophied rats, and the difference appeared to increase in hypertrophy. This is the opposite of what is expected from the observed T-wave inversion. The only other reported work on regional differences in action potentials in hypertrophy compared endocardial action potentials measured with a floating electrode within the right ventricle of the cat 3 days after pulmonary artery banding.27 The action potential plateau phase was altered in basal (but not apical) areas, and it was suggested that this reflected the local increase in wall stress produced in that model of hypertrophy.
We found significantly different changes in action potential characteristics in response to hypertrophy in myocytes isolated from the three regions. These changes result in a reduction of the normal endocardial/epicardial gradient in APD25 and a reversal of the gradient for APD75, which could explain the observed T-wave inversion. These changes will be considered in two parts, those affecting the early phases of the action potential and those affecting the plateau phase.
Changes in the Early Phase of the Action Potential
APA is reduced in hypertrophy in the three regions whether 4-AP is included in the perfusion solution or not. This suggests that in hypertrophy, net 4-AP–insensitive early inward current is reduced. In hypertrophy, the APD25 of subendocardial myocytes is similar to control but the APD25 of subepicardial and midmyocardial myocytes is prolonged. There is therefore a reduction in regional differences in APD25 in hypertrophy, which may be due to a reduction in Ito1 in midmyocardial and subepicardial myocytes, with Ito1 remaining unchanged in subendocardial myocytes. The reduction in the effect of 4-AP on APD25, especially in the subepicardial and midmyocardial myocytes, supports this hypothesis and suggests that regional differences in Ito1 are reduced in hypertrophy. This finding is in agreement with Cerbai et al,26 who showed a reduction in Ito1 in myocytes isolated from the whole LV. Moreover, Bénitah et al28 showed that hypertrophy was associated with a reduction in Ito1 that was greatest in myocytes isolated from the LV free wall and apex, and that regional variation in Ito1 density was reduced in hypertrophy.
Changes in the Plateau Phase of the Action Potential
These data are the first report of APD being reduced in cardiac hypertrophy in the rat. However, previous studies on isolated rat myocytes have used intracellular calcium buffers, so changes in the plateau phase of the action potential that depend on intracellular calcium will not have been detected.
In this study, we found that hypertrophy was associated with different changes in APD75 in myocytes isolated from the three regions. In subepicardial cells, the plateau phase appears to parallel that seen in control (see Fig 4⇑), so the increase in APD75 may simply follow from the increase in APD25. Surprisingly, however, in subendocardial myocytes, hypertrophy is associated with a significant reduction in APD75. This reduction in APD75 in hypertrophy is not observed when BAPTA is included in the patch solution, suggesting that the decrease in APD75 is due to a reduction in Na+-Ca2+ exchange, which could arise from a reduction in L-type calcium current or in the calcium transient or from reduced expression of the Na+-Ca2+ exchanger protein.
Comparison between our results and previous work on changes in action potentials in LV hypertrophy is complicated by possible effects of species and model differences. There are known species differences in the relative importance of individual currents in the action potential. (Ito is significant in rats and humans but not in guinea pigs.9 It is thought that the L-type calcium current is of less importance in rats than in humans, guinea pigs, and ferrets and conversely, that calcium-dependent currents seem more important in rats than in other species.29 ) Moreover, the effects on different membrane currents may vary depending on the stimulus for hypertrophy (reviewed in Reference 2323 ). Despite these limitations, regional differences in APD and membrane currents in response to pathological stimuli are of increasing interest, especially because endocardial-epicardial differences in Ito1 have been found to be reduced in myocytes isolated from patients with heart failure.19
This study shows that isoproterenol-induced cardiac hypertrophy in rats is associated with a reduction in the regional differences in action potential characteristics, which may explain the T-wave inversion observed in this model. The early repolarization phase (APD25) is prolonged in subepicardial and midmyocardial myocytes in hypertrophy, probably because of a reduction in Ito1. In subendocardial myocytes, early repolarization is unchanged and the plateau phase is shortened in hypertrophy, probably because of a reduction in Na+-Ca2+ exchange. How these changes can be related to the increased incidence of sudden death30 and ventricular arrhythmias31 in LV hypertrophy in humans is less clear. It has previously been argued that there is an increase in APD heterogeneity in hypertrophy (reflected in increased T-wave dispersion) facilitating the development of reentry circuits and increasing the electrical vulnerability of hypertrophied myocardium (reviewed in Reference 3232 ). However, we found a decrease in the differences in APD between regions, and there was no increase in local heterogeneity within each region (ie, the SD of mean APD for each region did not increase in hypertrophy). Nevertheless, functional heterogeneity may exist in the intact heart, because histological studies of this model of hypertrophy have shown localized myocardial fibrosis, myocyte loss, and ischemic foci, and the effects of these features will be lost in the process of myocyte isolation.4
There have been few previous studies examining regional differences in action potentials in pathological conditions. Data obtained from myocytes isolated from the whole of the LV (which will be predominantly midmyocardial) have been extrapolated as though ventricular electrophysiology were homogeneous, despite the known regional differences in cellular electrophysiology within the normal LV. Our findings of discordant changes in APD in subendocardial and subepicardial myocytes from rats with isoproterenol-induced hypertrophy provide the first direct evidence of a disturbance of the normal endocardial-epicardial gradient in APD. The mechanisms giving rise to the different effects of hypertrophy on regional myocyte electrophysiology await further investigation.
Selected Abbreviations and Acronyms
|APA||=||action potential amplitude|
|APD||=||action potential duration|
|APD25||=||APD measured at 25% repolarization|
|APD75||=||APD measured at 75% repolarization|
|I to1||=||4-AP–sensitive transient outward current|
|LV||=||left ventricular, left ventricle|
- Received December 2, 1996.
- Revision received April 3, 1997.
- Accepted April 4, 1997.
- Copyright © 1997 by American Heart Association
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