Skip Navigation

Cardiovascular Research 2003 59(3):705-714; doi:10.1016/S0008-6363(03)00460-7
© 2003 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by de Groot, J. R
Right arrow Articles by Coronel, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by de Groot, J. R
Right arrow Articles by Coronel, R.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2003, European Society of Cardiology

Intrinsic heterogeneity in repolarization is increased in isolated failing rabbit cardiomyocytes during simulated ischemia

Joris R de Groota,*, Cees A Schumachera, Arie O Verkerka,b, Antonius Baartscheera, Jan W.T Fioleta and Ruben Coronela,c

aExperimental and Molecular Cardiology Group, Academic Medical Center, Meibergdreef 9, Room M0-54, 1105 AZ Amsterdam, The Netherlands
bDepartment of Physiology, Academic Medical Center, Amsterdam, The Netherlands
cDepartment of Cardiology, University Medical Center, Utrecht, The Netherlands

j.r.degroot{at}amc.uva.nl

* Corresponding author. Tel.: +31-20-566-3266; fax: +31-20-697-5458.

Received 25 September 2002; accepted 1 May 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 
Objective: Myocardial ischemia and ventricular arrhythmias often complicate congestive heart failure. Ischemia-induced dispersion in repolarization is an important arrhythmogenic factor that might be caused by intrinsic cellular differences in response to simulated ischemia (SI) or by changed coupling of myocytes. We hypothesized that intrinsic heterogeneity in action potential duration (APD) or the occurrence of rigor is larger in failing than in normal rabbit myocytes during SI. Methods: Heart failure (HF) was induced with volume and pressure overload. Left ventricular myocytes from apex, free wall and base were enzymatically isolated and exposed to SI with NaCN. Results: There were no baseline differences in APD before SI. During SI no differences in time to inexcitability occurred but the range in APD increased more in HF than in normal cells. Rigor occurred after 16.8±3.5 and 23.0±7.5 min (P<0.05) in normal and HF myocytes, with no differences between apical, free wall or base cells. Variance in time to rigor was larger in HF than in normal cells (55.7 versus 12.4 min2). Blockade of anaerobic reserve decreased variance in time to rigor, also when normalized to mean, in HF and normal myocytes. In coupled normal and HF cell pairs, no delay in action potential propagation or differences in APD occurred during SI, and time to rigor was synchronized (P<0.05 vs. single cells). Conclusions: Intercellular differences in APD and in time of rigor arise in normal and HF myocytes subjected to SI, and are inhibited by blockade of anaerobic glycolysis. Dispersion in APD and tolerance to SI is increased in HF cells. APD and time to rigor are completely synchronized in coupled cell pairs.

KEYWORDS Ischemia; Hypertrophy; Heart failure; Gap junctions; Arrhythmia (mechanisms)


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 
Patients with heart failure frequently suffer from ventricular arrhythmias [1,2]. They also are susceptible to acute myocardial infarction and have an increased risk for sudden arrhythmic death [3,4].

Dispersion in repolarization is one of the principal factors involved in arrhythmogenesis and may cause unidirectional block and reentry [5,6]. Indeed, heterogeneous repolarization as evidenced from QT dispersion is associated with ventricular arrhythmias and sudden death in patients with heart failure [7]. Under normal conditions, gap junctional coupling nullifies or decreases the occurrence of large heterogeneities. Gap junctional uncoupling can expose intrinsic differences between myocytes and aggravate dispersion in repolarization [8]. In cultured myocytes exposed to a gap junctional uncoupler, intercellular heterogeneities increased to such an extent that microreentry could occur [9]. The formation of interstitial fibrosis and scar tissue insulates myocardial bundles from each other and forms a mode of cellular uncoupling that is independent of gap junctional conductance [10,11]. Indeed, specific types of fibrosis were associated with abnormal conduction curves and increased arrhythmogenesis in end-stage failing human hearts [11].

Whether increased dispersion in repolarization in failing hearts results from decreased intercellular coupling, unmasking a pre-existent intercellular difference in repolarization or from increased intrinsic heterogeneity between cells caused by the process of heart failure itself remains to be established. Moreover, the role of gap junctional communication versus the structural remodeling imposed by the formation of fibrosis in the unmasking of repolarization differences between cells remains unclear. We hypothesized that in failing hearts, intrinsic heterogeneity in action potential duration (APD) is greater than in normal hearts, and that dispersion in repolarization further increases under ischemic conditions. Ischemia is associated with increased dispersion in APD and refractoriness. We recorded action potentials from isolated ventricular myocytes from normal and failing hearts in normal and simulated ischemic conditions. Furthermore, we followed the time course of ischemia-induced rigor, as an indirect measure tolerance to ischemia. Isolated single myocytes were used as a model of cells that are physically disconnected from each other as occurs in hearts with fibrosis or scar tissue. Additionally, we measured action potentials and the occurrence of rigor in coupled cell pairs to investigate the role of equilibration of intrinsic differences by gap junctional coupling during the course of simulated ischemia.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusion
 References
 
All investigations conformed to institutional guidelines and to the Guide for the Care and Use of Laboratory Animals (NIH publication No 85-23, revised 1996).

2.1 Induction of heart failure
Heart failure with hypertrophy was produced by inducing combined volume and pressure overload, as described previously [12,13]. In short, male rabbits of approximately 5 months of age and 3.5 kg were anesthetized and intubated and a fluid filled catheter was advanced through the right carotid artery and moved up and down through the aortic valve until pulse pressure increased by ≥100%. Three weeks later, the suprarenal abdominal aorta was constricted by approximately 50%. The two experimental operations were associated with a postoperative mortality of approximately 30%. Animals were killed 3 months after the second operation.

The severity of heart failure was assessed as described previously with the following parameters: relative heart weight, relative lung weight, and left ventricular end diastolic pressure [13,14].

2.2 Isolation of left ventricular myocytes
Myocytes were isolated from seven failing and nine normal rabbit hearts. Rabbits were anaesthetized with pentobarbital 100 mg/kg i.v. and heparin (1000 I.U., i.v.). The heart was extirpated and immersed in cold Tyrode’s solution (composition see below). Left ventricular myocytes were enzymatically isolated as described previously [15]. In short, the aorta (or the left coronary artery when the aortic valve was destroyed) was cannulated and the heart was perfused with modified Tyrode’s solution of the following composition in mmol/l: Na+ 155.5, K+ 4.7, Ca2+ 1.45, Mg2+ 0.6, PO43– 0.4, Cl– 136.5, HCO3– 27.0, glucose 11.0 at 37°C. After 15 min, perfusion was changed to a nominally calcium free medium containing in mmol/l: HEPES 16.8, Na+ 155, K+ 4.7, Ca2+ 0.01, Mg2+ 2.0, PO43– 1.4, Cl– 149, HCO3– 4.3, glucose 11.0, and perfusion pressure was adjusted to 50 mmHg. After 15 min, collagenase (Boehringer Mannheim), hyaluronidase (Sigma) and trypsin inhibitor (Boehringer Mannheim) were added to the perfusion fluid, and perfusion was maintained until perfusion pressure had decreased to 0 mmHg. After removal of the right ventricle and the intraventricular septum, the epicardium and endocardium were carefully peeled off and from the remaining tissue samples of 3x3 mm were taken from the left ventricular apex, the free wall and the base. In some experiments, all tissue that remained after removal of endocardium and epicardium, irrespective of the region it was derived from, was used for cell isolation. Samples were chopped into pieces and shaken in a gyrotory water bath shaker. Cells were allowed to sediment and were resuspended in HEPES buffer with albumin 1% (w/v) with [Ca2+] of 1.3 mmol/l. Cells were stored in vials containing 3 ml HEPES with albumin at room temperature. Yield of rod shaped myocytes was around 75%. Single cells and cell pairs from samples of the HEPES cell suspension were used for the experiments. Cell size was measured in 50 cells per heart from five failing and four control hearts.

2.3 Action potential recordings
Action potentials were measured from single myocytes with the perforated patch clamp technique at 37°C as described previously [16]. In myocyte pairs a pipette was placed on each cell. Pipette solution consisted of (mmol/l): HEPES 16.8, K+ 140, Na+ 10, Ca2+ 0.01, Mg2+ 2, Cl– 149.7, HCO3– 4.3, PO43– 1.4, EGTA 0.1, glucose 11 and amphotericin B 2.2. pH was adjusted to 7.1 with KOH. The access resistance to the cell rapidly decreased within 10 min of seal formation and remained stable for at least 1.5 h. Bath solutions consisted of the HEPES solution mentioned above with 2.6 mmol/l Ca2+. Simulated ischemia was instituted with this HEPES solution with 2 mmol/l NaCN and no glucose. Action potentials were elicited with a stimulation frequency of 2.0 Hz by 2-ms current pulses (1.5x diastolic threshold). No correction for the liquid junction potential was made. Recordings were filtered on line (1 kHz), digitized at 2 kHz and stored on the hard disk of a personal computer.

2.4 Simulated ischemia and determination of rigor
Cells were attached to a poly-D-lysine coated (0.1 g/l) glass coverslip, placed on a temperature controlled (37°C) stage of an inverted fluorescence microscope (Nikon Diaphot). A temperature controlled perfusion chamber (volume 30 µl) was tightly positioned over the glass coverslip. The chamber content could be changed completely within 0.1 s. Myocytes were superfused with HEPES solution containing 2.6 mmol/l Ca2+. Field stimulation was applied with bipolar square current pulses (2 Hz, 40 V/cm, 200 µs duration) through two parallel thin platinum electrodes at 8 mm distance. Single rod shaped myocytes or myocyte pairs were selected with top illumination.

Simulated ischemia was instituted with normoxic HEPES solution, containing 2.6 mmol/l Ca2+, 2 mmol/l NaCN and no glucose. Myocytes (maximum 10 per field) were monitored continuously on a video screen. Time to rigor was defined as the time that elapsed between the onset of simulated ischemia and the onset of the rapid transition from rod shaped to round shaped, which occurred within seconds. In a separate set of experiments in control and HF cells, glycolysis was blocked with 1 mmol/l iodoacetate added to the HEPES and NaCN solution. This allowed investigation of the contribution of anaerobic reserve (indirectly defined as glycogen content) to the differences in time of rigor among cells.

2.5 Definition of dispersion
Dispersion in time to inexcitability and time to rigor was quantified using the statistical variance of the parameters studied. The range of a parameter under study was defined as the difference between the largest and the smallest value at a certain point in time.

2.6 Statistics
Data are presented as mean±S.D. unless stated otherwise. Differences between groups were tested with an unpaired t-test or a Mann–Whitney test when appropriate. Differences in variability were tested for significance with an F-test. For multiple comparisons ANOVA for repeated measures was used. A P<0.05 indicated statistical significance.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusion
 References
 
Myocytes from failing hearts were longer and wider than normal myocytes (Table 1). Rabbits with combined volume and pressure overload showed characteristics of heart failure, as evident from increased relative heart weight and increased end-diastolic left ventricular pressure indicated in Table 1 [13,14].


View this table:
[in this window]
[in a new window]

  		Table 1 Baseline functional characteristics (mean±S.D.)

 
3.1 Action potentials
Table 2 shows the baseline action potential characteristics in both groups of animals. There were no differences in any of the action potential parameters at a pacing frequency of 2 Hz.


View this table:
[in this window]
[in a new window]

  		Table 2 Baseline action potential characteristics (mean±S.D.)

 
During simulated ischemia, action potentials rapidly shortened and myocytes became inexcitable. Fig. 1A displays a typical example of action potential shortening in a normal and a HF myocyte upon simulated ischemia. Fig. 1B shows the mean action potential duration at 90% repolarization (APD90) of normal and HF myocytes. Mean APD90 decreased during the course of simulated ischemia until inexcitability occurred. There was a trend toward less action potential shortening in the HF cells, however, action potentials were significantly longer only after 17 min of simulated ischemia. Inexcitability occurred in normal cells after 17.0±3.5 min (n=10, range 10–21 min) and in HF cells after 17.7±5.0 min (n=14, range 10–26 min). Because time to inexcitability varied considerably between individual cells, there was no statistical difference in mean time of inexcitability between the groups. The standard deviation of the APD90 values increased (Fig. 1B), but the number of excitable cells decreased during the course of simulated ischemia. Fig. 1C therefore, shows the range in APD90 in 5-min bins. The range in APD90 increased approximately 2.5-fold during the course of simulated ischemia. In normal and HF cells, APD range was significantly higher after 10 min and 15 min of simulated ischemia than before simulated ischemia (ANOVA: P<0.05). This was also the case in HF cells after 20 min of simulated ischemia (ANOVA: P<0.05). In HF cells, APD90 range increased more than in normal cells after 10, 15 and 20 min of simulated ischemia (Mann–Whitney: all P<0.05).


Figure 1
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Action potentials during simulated ischemia (A) Representative series of action potentials from normal and failing myocytes after 0, 1, 5, 10, 15 and 17 min of simulated ischemia. (B) Mean APD (±S.E.M.) in normal (open diamonds, n=10) and failing myocytes (closed circles, n=14). (C) Range in APD (difference between longest and shortest action potential) in normal (open bars, 0 min: n=10, 5 min: n=10, 10 min: n=9, 15 min: n=7, 20 min: n=3) and failing myocytes (closed bars, 0 min: n=14, 5 min: n=14, 10 min: n=13, 15 min: n=9, 20 min: n=5, 25 min: n=2). *, P<0.05 versus before simulated ischemia, {dagger}, P<0.05 normal versus failing cells.

 
3.2 Synchronization of action potentials through gap junctional coupling
Fig. 2A shows action potentials recorded from two paired cells during simulated ischemia. Action potentials arose simultaneously in the paced and the follower cell up to the moment that the former became inexcitable. Fig. 2B shows an enlargement of the action potential upstroke. Even just before inexcitability (very short action potentials), the action potential immediately propagated to the follower cell. Fig. 2C shows the decrease of APD of this myocyte during the course of simulated ischemia. Note that APD in the follower cell exactly equaled that in the paced cell up to the moment of inexcitability. Action potential upstroke occurred simultaneously in all five normal and six HF cell pairs during the course of simulated ischemia. Moreover, APD90 remained equal in paced and follower cell in all experiments up to inexcitability occurred.


Figure 2
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Action potentials in coupled cell pairs. (A) Representative example of APD in two coupled normal myocytes. (B) Enlargement of the upstroke of the action potentials in (A). Upstroke occurs simultaneously in both cells. (C) APD remains equal in the same two cells during simulated ischemia.

 
3.3 Time to rigor
Fig. 3A shows that rigor as a consequence of simulated ischemia occurred later in pooled HF myocytes from left ventricular base, left ventricular free wall and left ventricular apex (23.0±7.5 min, n=331, Mann–Whitney: P<0.05 vs. normal) than in pooled normal myocytes (16.8±3.5 min, n=366). Rigor occurred significantly later than inexcitability in HF (Mann–Whitney: P<0.01) but not in normal myocytes. Statistical variance in time to rigor was larger in HF cells than in normal myocytes (Fig. 3B, 55.7 versus 12.4 min2, F-test: P<0.001). The range in time to rigor, that is the difference between the first and the last cell that underwent rigor, was 18.6 min in normal cells and 36.4 min in HF cells. This indicates an increased intrinsic heterogeneity in tolerance to simulated ischemia in failing hearts.


Figure 3
View larger version (12K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Time to rigor. (A) Mean time to rigor in normal (open bars, n=366) and failing (closed bars, n=331) myocytes. (B) Variance in time to rigor in the same groups as in (A). *, P<0.05.

 
3.4 Regional differences in left ventricular myocytes
Fig. 4A shows the differential time to rigor in normal and HF myocytes selectively isolated from distinct regions of the left ventricle. There were no differences between time to rigor in cells from left ventricular apex (17.6±3.8 min, n=128), free wall (16.2±3.2 min, n=115), or base (16.6±3.4 min, n=123) in normal hearts (ANOVA: all P=NS). In failing hearts, time to rigor was 23.0±7.2 min in cells from the left ventricular apex (n=111), 23.6±7.7 min in cells from the free wall (n=112) and 22.5±7.6 min in cells from the base (n=108). There were no differences between the cells from different regions in failing hearts (ANOVA: all P=NS). However, in all groups of HF cells, time to rigor was larger than in any group of normal cells (ANOVA: P<0.05). Fig. 4B displays the variance in time to rigor in these groups. Statistical variance was larger in failing hearts than in normal hearts in cells from apex (51.4 versus 14.1 min2), from the left ventricular free wall (58.5 versus 10.3 min2) and from the base (57.5 versus 11.6 min2 F-test: all P<0.001). There were no differences in variance between apex, left ventricular free wall and base within the groups of normal and HF myocytes.


Figure 4
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Differential time to rigor. (A) Mean time to rigor in normal cells (open bars) from left ventricular apex (n=128), free wall (n=115) and base (n=123) and failing myocytes (closed bars) from apex (n=111), free wall (n=112) and base (n=108). (B) Variance in time to rigor in the same groups as in (A). *, P<0.05.

 
3.5 Mechanism underlying variability in time to rigor
Variability in time to rigor occurrence was further investigated in 32 normal myocytes and in 53 HF myocytes by blocking anaerobic glycolysis with 1 mM iodoacetate (see Methods). Fig. 5A shows that in normal myocytes rigor developed after 4.2±0.5 min (Mann–Whitney: P<0.0001 versus simulated ischemia without iodoacetate) and that the variance of time to rigor was significantly decreased (Fig. 5B: 0.26 versus 12.4 min2 in myocytes that received NaCN without iodoacetate, F-test: P<0.001), also when variance was normalized to the mean (Fig. 5C, 0.015 versus 0.044, F-test: P<0.01). In HF myocytes subjected to iodoacetate, rigor developed after 3.1±0.2 min (Mann–Whitney: P<0.0001 versus simulated ischemia without iodoacetate), earlier than in normal cells (Mann–Whitney: P<0.001). Variance in time to ischemia-induced rigor was also significantly decreased (Fig. 5B: 0.04 versus 55.7 min2 in HF myocytes with versus without iodoacetate, F-test: P<0.001), also when normalized to the mean (Fig. 5C, 0.004 versus 0.105, F-test: P<0.001). Moreover, variance and normalized variance were smaller in HF than in normal cells with iodoacetate (F-test: both P<0001).


Figure 5
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Effects of blockade of anaerobic glycolysis on variance in time to rigor. (A) Mean time of rigor occurrence in normal cells (open bars, n=366) and failing myocytes (closed bars, n=331) subjected to simulated ischemia and normal (horizontal shading, n=38) and failing cells (vertical shading, n=53) undergoing simulated ischemia with iodoacetate. (B) Statistical variance in time to rigor in the same groups as in (A). (C) Variance in time to rigor normalized to mean time of rigor. {dagger}, P<0.01; {ddagger}, P<0.001.

 
3.6 Synchronization of rigor through gap junctional coupling
To investigate the role of metabolic equilibration between coupled cells, we investigated time to rigor in pairs of myocytes. Fig. 6 shows the difference in time to rigor in paired myocytes compared with the heterogeneity in rigor occurrence in groups of single cells. In order to be able to make such comparison, we assigned each consecutive single cell a number according to the order in which the experiments were performed and we calculated the difference in time to rigor between all consecutive odd and even numbered single cells. These differences in time to rigor between odd and even cells were compared with the differences in time to rigor in the paired myocytes. The pooled data from left ventricular apex, free wall and base are displayed. Difference in time to rigor was larger between HF than between normal single odd/even cells (median 3.1 versus 6.0 min, respectively, Mann–Whitney test: P<0.05). This difference vanished almost completely in cell pairs: median time to rigor between normal paired cells (n=15) decreased to 0.2 min (Mann–Whitney test: P<0.05 vs. single normal cells) and to 0.02 min in HF cell pairs (Mann–Whitney test: n=21, P<0.0001 versus single HF cells). Notably, time to rigor did not differ between normal and HF cell pairs (P=0.20). Thus, despite the larger difference between single HF myocytes than in between normal cells, equilibration occurred to the same extent in paired myocytes. Note that in two normal cell pairs the delay between rigor in subsequent cells exceeded 10 min. Most likely, these cells were initially not coupled despite their physical connection. Nevertheless, these data demonstrate that cells originating from the same region of the heart can exhibit large intrinsic differences in anaerobic reserve.


Figure 6
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Difference in time to rigor between single isolated myocytes and myocyte pairs. Open circles indicate normal myocyte pairs (n=15), open squares indicate differential time to rigor in odd and even normal single cells (n=182). Closed circles show failing myocyte pairs, closed squares indicate differential time to rigor in odd and even failing single myocytes (n=164). Boxplots (range, 25%, median, 75%) show the distribution within the groups. *, P<0.05; {dagger}, P<0.01.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusion
 References
 
4.1 Presence of intercellular heterogeneities in heart failure
We demonstrate a substantial heterogeneity in APD in myocytes during the course of simulated ischemia. We show that, despite the absence of differences in baseline mean values in action potential parameters between the groups (Table 2), APD range is larger in HF than in normal cells during simulated ischemia and that this dispersion is present longer. This observation might explain why arrhythmias during ischemia are more severe in patients with heart failure [1,3]. The absence of baseline action potential prolongation seems to contrast with other models of heart failure, where action potential prolongation is commonly found. Three factors may explain this. Firstly, myocytes were stimulated at 2 Hz. Action potential prolongation is most prominent at low pacing frequencies [13,17]. Indeed, at frequencies of 1 Hz and lower we observed action potential prolongation in HF cells, related to decreased density of the transient outward current (Ito1) in HF cells [A.O. Verkerk, unpublished observation]. Secondly, the pipette sodium concentration was the same in studies on normal and HF cells. In another study in the same model of heart failure, pipette sodium concentration was adjusted to measured values in normal and HF cells. Here, action potential prolongation was observed at pacing frequencies of 2 Hz and lower [18]. Finally, the largest action potential prolongation in heart failure is reported in epi- and endocardium [19,20]. In the present study cells from the midmyocardial wall were used, where these changes might be modest.

Time to rigor was used as an indirect measure of tolerance to ischemia. It occurs later and with more temporal variance in HF cells, indicating an increased tolerance to ischemia. There were no differences in time to rigor in groups of myocytes isolated from left ventricular apex, free wall or base. However, time to rigor and its variance were larger in all groups of HF cells compared to any group of normal cells. Hence, the variance in time to rigor in normal and HF cells is not due to differential origin from the three regions within the left ventricle. Moreover, the increased variance in time to rigor in HF cells compared to normal cells cannot be attributed to a preferential regional effect caused by the remodeling process of heart failure. Our data are in agreement with the previous work by Dekker et al. [14] who showed that the time course of cellular uncoupling is longer in failing than in normal rabbit hearts, although uncoupling starts earlier in the former [14]. These authors also showed a temporal association between rigor and cellular uncoupling [21]. Beardslee et al. [22] showed that dephosphorylation of gap junction proteins and trafficking to intracellular pools is associated with electrical uncoupling, and thus provided a direct relation between ATP depletion during ischemia and cellular uncoupling [22]. A similar mechanism might well underlie our present findings, since rigor directly results from ATP depletion. This is further supported by our observation that blockade of the anaerobic glycolysis significantly decreases variance in time to rigor between normal and HF myocytes, also when normalized to mean time of rigor. Therefore, the dispersion in time to rigor can, at least in part, be attributed to different anaerobic reserve, i.e. glycogen content, of individual myocytes. It is interesting to note that rigor occurred later and with more variance in HF compared to normal cells. This observation suggests that there is more heterogeneity in anaerobic reserve among HF then among normal cells and that it forms a prominent contribution to tolerance to ischemia and to functional dispersion in the failing heart. Despite the difference in time to rigor between normal and HF cells, time to inexcitability was not different. This might relate to different dynamic changes occurring during the process of ischemia-induced ATP depletion. However, the relation between rigor and excitability was not specifically addressed in the current study and remains to be elucidated.

4.2 Synchronization of differences between well-coupled cell pairs
Action potential propagation remains undisturbed and APD remains synchronized in well-coupled cell pairs, also during the course of simulated ischemia. From this, we cannot conclude that coupling conductance remains unchanged during simulated ischemia, but we can infer that it decreases insufficiently to allow the development of heterogeneities between paired cells. Large reductions in gap junctional conductance are required for a delay in action potential propagation. Weingart and Maurer showed that action potentials can still propagate when coupling conductance is as low as 1.3 nS [23]. Jongsma and Wilders calculated that the safety for conduction is so high that a reduction of approximately 90% of open gap junctions is required to achieve 50% reduction in conduction velocity [24]. With the sampling rate used in the present study, we found that the follower cell was activated simultaneously with the paced cell. However, with this sampling rate our detection is limited to conduction velocities less than approximately 7 cm/s.

Rigor too occurred almost simultaneously in paired normal and HF cells. However, in this series of experiments we found that in two out of fifteen normal cell pairs the delay in rigor between the two cells exceeded 10 min. We assume that these cell pairs were initially not coupled but only physically attached during the isolation procedure. Nevertheless, this demonstrates that large intrinsic differences can occur in adjacent cells. This phenomenon was not observed in our experiments on action potential propagation, because here we only used selected cell pairs where action potential propagation was present. The observation that rigor occurs simultaneously in both cells, also in cells from failing hearts, is of particular interest. Although the variance in time to rigor was considerably larger in single HF cells compared to single normal cells, equilibration of metabolism and APD through cellular coupling is as efficient as in normal cell pairs. These data agree with a recent study of Ruiz-Meana et al. [25] who showed that gap junctions remain permeable to luciferrine yellow, even after rigor occurred [25]. We report that not only passive diffusion of a dye can take place, but that gap junctional coupling, also in heart failure, remains functional during simulated ischemia and allows complete synchronization of activation, APD and ATP depletion responsible for the simultaneous occurrence of rigor. Although the intrinsic differences in APD proved larger in isolated HF cells exposed to simulated ischemia, there was still complete equilibration when myocytes were coupled in a cell pair. Hence, ischemia-induced gap junctional changes are insufficient to unmask intercellular dispersion of APD in adjacent cells.

4.3 Implications for arrhythmia mechanism in the intact heart
We observed that inexcitability precedes rigor occurrence during metabolic inhibition in HF but not in normal myocytes. Hence, while cells are still coupled, their inexcitability prevents their contribution to an arrhythmia in the intact heart. Meanwhile, they can exert electrotonic effects on their neighbors. This supplies another mechanism for increased propensity for arrhythmias in failing hearts. Indeed, we previously showed that with critical residual coupling an electrotonic depression of the ischemic subepicardium through the irreversibly damaged ischemic midmyocardium forms an arrhythmogenic substrate that terminates when uncoupling between those two compartments proceeds [26].

Heterogeneities in action potential duration are present in the intact heart despite the absence of observed differences between cell pairs. Electrophysiological gradients are present both under normal and under pathological conditions, as is evident from the presence of a T-wave in the surface EKG, demonstrating the presence of a gradient of repolarization. During acute ischemia, differences in APD and in membrane potential give rise to an intracellular current of injury flowing from the ischemic toward the normal tissue [27,28], and following reperfusion differences in APD up to 100 ms occur [28,29]. Hence, over many cell–cell boundaries, functional differences can and do arise, despite the apparent synchronization between adjacent coupled cells. Our data cannot be immediately extrapolated to the intact heart for another reason also. A cell pair forms a simplification of the complex interaction that cells have in the intact heart where every ventricular cell is connected to approximately nine neighboring cells [30] with different degrees of coupling parallel and perpendicular to the fiber direction. Our experiments in isolated myocytes show that intrinsic differences are larger in HF than in normal myocytes subjected to simulated ischemia. In isolated cells complete uncoupling is evidently present and this model mimics a situation where myocardial bundles are insulated from each other through interstitial fibrosis or scar formation in the heart. Thus, in isolated cells we could measure the intrinsic changes caused by heart failure compared to normal cells. The observation that gap junctional coupling equilibrates these differences between paired cells in combination with the observation that increased dispersion in repolarization is present in hearts from patients with heart failure [7] suggests that exposure of such intrinsic differences relates to structural remodeling of the diseased heart. Such remodeling likely results from the formation of interstitial fibrosis or from the evolution of scar tissue.


   5. Conclusion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
References
 
We show that dispersion in APD and rigor occurrence is larger in HF than normal myocytes during simulated ischemia. This increased heterogeneity is not due to the region cells were isolated from, nor to a preferential regional effect of the process of heart failure. Heterogeneity results, at least in part, from differences in anaerobic reserve. Rigor occurs later than inexcitability in HF but not in normal myocytes, causing a time window where cells are electrically inactive but could exert arrhythmogenic electrotonic interaction. The mechanistic link between rigor and excitability was not studied here and remains to be elucidated. Cellular coupling prevents the exposure of intercellular differences in APD and time to rigor in adjacent cells, and continues to do so during simulated ischemia. Therefore, the circumstances that lead to reentrant arrhythmias seem to be present to a larger extent and during a longer time in heart failure.

Time for primary review 33 days.

{star} E. Cerbai served as Guest Editor.


   Acknowledgements
 
Supported by the Netherlands Heart Foundation, grant 2000T020.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 

  1. Vermeulen J.T. Mechanisms of arrhythmias in heart failure. J Cardiovasc Electrophysiol (1998) 9:208–221.[Web of Science][Medline]
  2. Fu G.-S., Meissner A., Simon R. Repolarization dispersion and sudden cardiac death in patients with impaired left ventricular function. Eur Heart J. (1997) 18:281–289.[Abstract/Free Full Text]
  3. Hallstrom A., Pratt C.M., Greene H.L., et al. Relations between heart failure, ejection fraction, arrhythmia suppression and mortality: Analysis of the Cardiac Arrhythmia Suppression Trial. J Am Coll Cardiol (1995) 25:1250–1257.[Abstract]
  4. Huikuri H.V., Castellanos A., Myerburg R.J. Sudden death due to cardiac arrhythmias. New Eng J Med (2001) 345:1473–1482.[Free Full Text]
  5. Kuo C.-S., Munakata K., Reddy P., Surawicz B. Characteristics and possible mechanism of ventricular arrhythmia dependent on the dispersion of action potential duration. Circulation (1983) 67:1356–1367.[Abstract/Free Full Text]
  6. Janse M.J., Wit A.L. Electrophysiological mechanisms of ventricular arrhythmias resulting from myocardial ischemia and infarction. Physiol Rev (1989) 69:1049–1169.[Free Full Text]
  7. Eckardt L., Haverkamp W., Johna R., et al. Arrhythmias in heart failure: current concepts of mechanisms and therapy. J Cardiovasc Electrophysiol (2000) 11:106–117.[Web of Science][Medline]
  8. Lesh M.D., Pring M., Spear J.F. Cellular uncoupling can unmask dispersion of action potential duration in ventricular myocardium. A computer model study. Circ Res (1989) 65:1426–1440.[Abstract/Free Full Text]
  9. Rohr S., Kucera J.P., Kléber A.G. Slow conduction in cardiac tissue. I. Effects of a reduction of excitability versus a reduction of electrical coupling on microconduction. Circ Res (1998) 83:781–794.[Abstract/Free Full Text]
  10. De Bakker J.M.T., Van Capelle F.J.L., Janse M.J., et al. Slow conduction in the infarcted human heart. ‘Zigzag’ course of activation. Circulation (1993) 88:915–926.[Abstract/Free Full Text]
  11. Kawara T., Derksen R., De Groot J.R., et al. Activation delay after premature stimulation in chronically diseased human myocardium relates to the architecture of interstitial fibrosis. Circulation (2001) 104:3069–3075.[Abstract/Free Full Text]
  12. Bril A., Forest M.-C., Gout B. Ischemia and reperfusion induced arrhythmias in rabbits with chronic heart failure. Am J Physiol (1991) 261:H301–H307.[Web of Science][Medline]
  13. Vermeulen J.T., McGuire M.A., Opthof T., et al. Triggered activity and automaticity in ventricular trabeculae of failing human and rabbit hearts. Cardiovasc Res (1994) 28:1547–1554.[Abstract/Free Full Text]
  14. Dekker L.R.C., Rademaker H., Vermeulen J.T., et al. Cellular uncoupling during ischemia in hypertrophied and failing rabbit ventricular myocardium. Effects of preconditioning. Circulation (1998) 97:1724–1730.[Abstract/Free Full Text]
  15. Ter Welle H.F., Baartscheer A., Fiolet J.W.T., Schumacher C.A. The cytoplasmic free energy of ATP hydrolysis in isolated rod-shaped rat ventricular myocytes. J Mol Cell Cardiol (1988) 20:435–441.[CrossRef][Web of Science][Medline]
  16. Verkerk A.O., Veldkamp M.W., Van Ginneken A.C.G., Bouman L.N. Biphasic response of action potential duration to metabolic inhibition in rabbit and human ventricular myocytes: role of transient outward current and ATP-regulated potassium current. J Mol Cell Cardiol (1996) 28:2443–2456.[CrossRef][Web of Science][Medline]
  17. McIntosh M.A., Cobbe S.M., Kane K.A., Rankin A.C. Action potential prolongation and potassium currents in left-ventricular myocytes isolated from hypertrophied rabbit hearts. J Mol Cell Cardiol (1998) 30:43–53.[CrossRef][Web of Science][Medline]
  18. Baartscheer A., Schumacher C.A., Belterman C.N.W., Coronel R., Fiolet J.W.T. [Na+]i and the driving force of the Na+/Ca2+ exchanger in heart failure. Cardiovasc Res (2003) 57(4):986–995.[Abstract/Free Full Text]
  19. Bryant S.M., Shipsey S.J., Hart G. Regional differences in electrical and mechanical properties of myocytes from guinea-pig hearts with mild ventricular hypertrophy. Cardiovasc Res (1997) 35:315–323.[Abstract/Free Full Text]
  20. Li G.-R., Lau C.-P., Ducharme A., Tardif J.-C., Nattel S. Transmural action potential and ionic current remodeling in ventricles of failing canine hearts. Am J Physiol (2002) 283:H1031–H1041.[Web of Science]
  21. Dekker L.R.C., Fiolet J.W.T., VanBavel E., et al. Intracellular Ca2+, intercellular electrical coupling, and mechanical activity in ischemic rabbit papillary muscle. Effects of preconditioning and metabolic blockade. Circ Res (1996) 79:237–246.[Abstract/Free Full Text]
  22. Beardslee M.A., Lerner D.L., Tadros P.N., et al. Dephosphorylation and intracellular redistribution of ventricular connexin43 during electrical uncoupling induced by ischemia. Circ Res (2000) 87:656–662.[Abstract/Free Full Text]
  23. Weingart R., Maurer P. Action potential transfer in cell pairs isolated from adult rat and guinea pig ventricles. Circ Res (1988) 63:72–80.[Abstract/Free Full Text]
  24. Jongsma H.J., Wilders R. Gap junctions in cardiovascular disease. Circ Res (2000) 86:1193–1197.[Abstract/Free Full Text]
  25. Ruiz-Meana M., Garcia-Dorado D., Lane S., et al. Persistence of gap junction communication during myocardial ischemia. Am J Physiol (2001) 280:H2563–H2571.[Web of Science]
  26. De Groot J.R., Wilms-Schopman F.J.G., Opthof T., Remme C.A., Coronel R. Late ventricular arrhythmias during acute regional ischemia in the isolated blood perfused pig heart. Role of electrical cellular coupling. Cardiovasc Res (2001) 50:362–372.[Abstract/Free Full Text]
  27. Janse M.J., Van Capelle F.J.L., Morsink H., et al. Flow of ‘injury’ current and patterns of excitation during early ventricular arrhythmias in acute regional myocardial ischemia in isolated porcine and canine hearts. Evidence for two different arrhythmogenic mechanisms. Circ Res (1980) 47:151–165.[Free Full Text]
  28. Coronel R., Wilms-Schopman F.J.G., Opthof T., Van Capelle F.J.L., Janse M.J. Injury current and gradients of diastolic stimulation threshold. TQ potential, and extracellular potassium concentration during acute regional ischemia in the isolated perfused pig heart. Circ Res (1991) 68:1241–1249.[Abstract/Free Full Text]
  29. Coronel R., Wilms-Schopman F.J.G., Opthof T., et al. Reperfusion arrhythmias in isolated perfused pig hearts. Inhomogeneities in extracellular potassium, ST and TQ potentials, and transmembrane action potentials. Circ Res (1992) 71:1131–1142.[Abstract/Free Full Text]
  30. Hoyt R.H., Cohen M.L., Saffitz J.E. Distribution and three-dimensional structure of intercellular junctions in canine myocardium. Circ Res (1989) 64:563–574.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
S. Casini, A. O. Verkerk, M. M.G.J. van Borren, A. C.G. van Ginneken, M. W. Veldkamp, J. M.T. de Bakker, and H. L. Tan
Intracellular calcium modulation of voltage-gated sodium channels in ventricular myocytes
Cardiovasc Res, January 1, 2009; 81(1): 72 - 81.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
J. R de Groot and R. Coronel
Acute ischemia-induced gap junctional uncoupling and arrhythmogenesis
Cardiovasc Res, May 1, 2004; 62(2): 323 - 334.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by de Groot, J. R
Right arrow Articles by Coronel, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by de Groot, J. R
Right arrow Articles by Coronel, R.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?