Cardiac electrophysiological remodeling associated with enhanced arrhythmia susceptibility in a canine model of elite exercise
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eLife assessment
This study presents valuable comprehensive data that underpin the enhanced ventricular arrhythmogenesis in elite trained athletes. The study is logistical challenge and the multiscale approaches used is a strength of the study. The data presented are strong and support most of the authors' claims.
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Abstract
The health benefits of regular physical exercise are well known. Even so, there is increasing evidence that the exercise regimes of elite athletes can evoke cardiac arrhythmias including ventricular fibrillation and even sudden cardiac death (SCD). The mechanism of exercise-induced arrhythmia and SCD is poorly understood. Here, we show that chronic training in a canine model (12 sedentary and 12 trained dogs) that mimics the regime of elite athletes induces electrophysiological remodeling (measured by ECG, patch-clamp, and immunocytochemical techniques) resulting in increases of both the trigger and the substrate for ventricular arrhythmias. Thus, 4 months sustained training lengthened ventricular repolarization (QTc: 237.1±3.4 ms vs. 213.6±2.8 ms, n=12; APD90: 472.8±29.6 ms vs. 370.1±32.7 ms, n=29 vs. 25), decreased transient outward potassium current (6.4±0.5 pA/pF vs. 8.8±0.9 pA/pF at 50 mV, n=54 vs. 42), and increased the short-term variability of repolarization (29.5±3.8 ms vs. 17.5±4.0 ms, n=27 vs. 18). Left ventricular fibrosis and HCN4 protein expression were also enhanced. These changes were associated with enhanced ectopic activity (number of escape beats from 0/hr to 29.7±20.3/hr) in vivo and arrhythmia susceptibility (elicited ventricular fibrillation: 3 of 10 sedentary dogs vs. 6 of 10 trained dogs). Our findings provide in vivo, cellular electrophysiological and molecular biological evidence for the enhanced susceptibility to ventricular arrhythmia in an experimental large animal model of endurance training.
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Author Response
Reviewer #2 (Public Review):
In this manuscript, Polyák et al. report detailed and systematic functional, electrocardiographic, electrophysiologic (both in vivo and in vitro experiments) and histological analysis in a large animal (canine) model of exercise to assess risk of ventricular arrhythmia susceptibility. They find that exercise-trained dogs have a slower heart rate (not accounted by heightened vagal tone alone and consistent with recent work from Denmark), an increased ventricular mass and fibrosis, APD lengthening due to repolarisation abnormality, enhanced HCN4 expression and decreased outward potassium channel density together with increased ventricular ectopic beats and ventricular fibrillation susceptibility (open-chest burst pacing). The authors suggest these changes as underlying the risk of VA in …
Author Response
Reviewer #2 (Public Review):
In this manuscript, Polyák et al. report detailed and systematic functional, electrocardiographic, electrophysiologic (both in vivo and in vitro experiments) and histological analysis in a large animal (canine) model of exercise to assess risk of ventricular arrhythmia susceptibility. They find that exercise-trained dogs have a slower heart rate (not accounted by heightened vagal tone alone and consistent with recent work from Denmark), an increased ventricular mass and fibrosis, APD lengthening due to repolarisation abnormality, enhanced HCN4 expression and decreased outward potassium channel density together with increased ventricular ectopic beats and ventricular fibrillation susceptibility (open-chest burst pacing). The authors suggest these changes as underlying the risk of VA in athletes, and appropriately caution against consigning the beneficial effects of exercise. In general, this study is well done, reasonably well-written, with reasonable conclusions, supported by the data presented and is much needed. There are some methodological, however, given the paucity of experimental data in this area, I think it would still be additive to the literature.
Strengths:
- This is an area with very limited experimental data- this is an area of need.
- The study, in general seems to be well-conducted with two clear groups
- The use of a large animal model is appropriate
- The study findings, in general, support the authors conclusions
- The authors have shown some restraint in their conclusions and the limitations section is detailed and well written.
Weaknesses:
- There are some methodological issues:
a. Authors should explain what the conditioning protocol was and why it was necessary.
In order to cause as little discomfort as possible to the animals, we selected animals that were naturally cooperative with the researchers and not afraid of the noise of the treadmill. This selection period lasted about three weeks, during which the animals were not exercised in a formal setting, but familiarized with the experimental setting and walked on the treadmills for a few minutes. During the conditioning period, both control and trained animals were equally handled.
Following your remarks the corresponding part of the text was extended properly explaining the training protocol in more detail.
b. The rationale for the exercise parameters chosen needs to be presented.
Experimental data on large animal models are very limited. Sled dogs are considered the highest elite of dog exercise. The distances they run are taken as a reference, although this protocol is not exactly the same due to the conditions of training, sledding, and weather. The most widely known races are the Norwegian Finnmarksløp and the Alaskan Iditarod, take place on snow and cover distances ranging from 500–1569 km in a continuous competition lasting for up to 14 days to be completed. (Calogiuri & Weydahl, 2017)
Based on these data, preliminary experiments were conducted to determine the maximum running time and intensity that dogs can sustain without distress, injuries, or severe fatigue. We increased the intensity of exercise in line with the animals' performance. The detailed training protocol and the daily running distances applied are presented in Table 1. Now, a new figure, Figure 1, and a new table, Table 1, illustrate a detailed experimental timeline in the revised manuscript.
Reference:
Calogiuri, G., & Weydahl, A. (2017). Health challenges in long-distance dog sled racing: A systematic review of literature. Int J Circumpolar Health, 76(1), 1396147. https://doi.org/10.1080/22423982.2017.1396147
c. Open chest VF induction was a limitation, and it was unnecessary.
d. A more refined VT/VF induction protocol was required. This is a major limitation to this work.
C, D: Thank you for the reviewer’s comment. For a detailed explanation of the VF induction procedures, please see our responses to question 11 of Reviewer #2.
e. The concept of RV dysfunction has not been considered in the study and its analysis.
Thank you for the suggestion. The complexity of our study and the capacity of our laboratory limited the work that could be carried out, but we are planning to perform additional studies involving the RV.
f. The lack of a quantitative measure for fibrosis is a limitation.
At the Department of Pathology, there was no opportunity to analyze myocardial fibrosis quantitatively. As described by Mustroph et al., quantitative analysis of fibrosis can be based on appropriate software measuring the amount of fibrotic area per total area on digitized slides. Such software was not available during the evaluation. This is a limitation of the study; however, the semi-quantitative assessment in histology reports is widely accepted in human pathology (Mustroph et al., 2021).
Reference:
Mustroph, J., Hupf, J., Baier, M. J., Evert, K., Brochhausen, C., Broeker, K., Meindl, C., Seither, B., Jungbauer, C., Evert, M., Maier, L. S., & Wagner, S. (2021). Cardiac Fibrosis Is a Risk Factor for Severe COVID-19. Front Immunol, 12, 740260. https://doi.org/10.3389/fimmu.2021.740260
- Statistical analysis requires further detail (checking of normality of the data/appropriate statistical test).
Thank you for this comment. This question has been answered in response to question 12 of Reviewer #2 and the statistical part of the methodology in the manuscript has been updated.
- The use of Volders et al. study as a corollary in the discussion does not seem justified given that this study used AV block induced changes as an acquired TdP model.
We agree with the reviewer that the two models involve completely different mechanisms. Therefore, in order to avoid misunderstandings, we have deleted the part of the discussion that made the comparison with the study by Volders et al.(Volders et al., 1998; Volders et al., 1999) Nevertheless, the exercise-induced compensatory adaptive mechanisms of the athlete's heart have been considered as a phenomenon completely distinct from pathological conditions, yet the electrical remodeling observed in our model indicates important similarities with the experimental model of long-term complete AV block. For example, both resulted in profound bradycardia, compensated cardiac hypertrophy, prolonged QTc interval, APD prolongation, and increased spatial and temporal dispersion of repolarization. These changes were attributed to the downregulation of potassium currents and were associated with increased ventricular arrhythmia susceptibility. Therefore, we hypothesized that the mechanisms of increased propensity for ventricular fibrillation in this model may have a similar electrophysiological background to the compensated hypertrophy studies of Volders et al. However, the autonomic changes, the potential impairment of the conduction system of the athlete’s heart, and the electrophysiological background require further, more detailed investigations.
References:
Volders, P. G., Sipido, K. R., Vos, M. A., Kulcsar, A., Verduyn, S. C., & Wellens, H. J. (1998). Cellular basis of biventricular hypertrophy and arrhythmogenesis in dogs with chronic complete atrioventricular block and acquired torsade de pointes. Circulation, 98(11), 1136-1147. https://doi.org/10.1161/01.cir.98.11.1136
Volders, P. G., Sipido, K. R., Vos, M. A., Spatjens, R. L., Leunissen, J. D., Carmeliet, E., & Wellens, H. J. (1999). Downregulation of delayed rectifier K(+) currents in dogs with chronic complete atrioventricular block and acquired torsades de pointes. Circulation, 100(24), 2455-2461. https://doi.org/10.1161/01.cir.100.24.2455
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eLife assessment
This study presents valuable comprehensive data that underpin the enhanced ventricular arrhythmogenesis in elite trained athletes. The study is logistical challenge and the multiscale approaches used is a strength of the study. The data presented are strong and support most of the authors' claims.
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Reviewer #1 (Public Review):
These findings for the first time provide a comprehensive multiscale assessment of the arrhythmogenic potential of elite exercise training.
The authors trained canines using a treadmill over 16 weeks, and compared these animals (n=12) to sedentary animals (n=13). The authors found global evidence of electrophysiologic remodeling ECG indices and heart rate, as well as repolarization variability in trained animals relative to controls.
The authors also demonstrate a range of effects of ventricular cardiomyocyte ion channels and fibrosis. Finally, using an induction protocol, the authors show enhanced risk for ventricular fibrillation as well as spontaneous arrhythmias in trained dogs.
The authors conclude that structural and electrophysiologic remodeling of ventricles in elite trained athletes is associated …
Reviewer #1 (Public Review):
These findings for the first time provide a comprehensive multiscale assessment of the arrhythmogenic potential of elite exercise training.
The authors trained canines using a treadmill over 16 weeks, and compared these animals (n=12) to sedentary animals (n=13). The authors found global evidence of electrophysiologic remodeling ECG indices and heart rate, as well as repolarization variability in trained animals relative to controls.
The authors also demonstrate a range of effects of ventricular cardiomyocyte ion channels and fibrosis. Finally, using an induction protocol, the authors show enhanced risk for ventricular fibrillation as well as spontaneous arrhythmias in trained dogs.
The authors conclude that structural and electrophysiologic remodeling of ventricles in elite trained athletes is associated with ventricular arrhythmogenesis.
First, this is a difficult study to achieve given the logistical challenges of managing a large animal set up as utilized in this study. Further protocols that involve in vivo and subsequently in vitro studies of tissues from large animals are challenging to accomplish. Finally, the multimodal assessments undertaken in this study to achieve these comprehensive objectives are an additional strength.
Weaknesses include the descriptive nature of the work and somewhat low level of rigor in presenting the observed data. The presentation of the data in the text could also be improved. Finally, some of the counterintuitive/conflicting findings e.g. enhanced HCN4 expression with reduced heart rate.
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Reviewer #2 (Public Review):
In this manuscript, Polyák et al. report detailed and systematic functional, electrocardiographic, electrophysiologic (both in vivo and in vitro experiments) and histological analysis in a large animal (canine) model of exercise to assess risk of ventricular arrhythmia susceptibility. They find that exercise-trained dogs have a slower heart rate (not accounted by heightened vagal tone alone and consistent with recent work from Denmark), an increased ventricular mass and fibrosis, APD lengthening due to repolarisation abnormality, enhanced HCN4 expression and decreased outward potassium channel density together with increased ventricular ectopic beats and ventricular fibrillation susceptibility (open-chest burst pacing). The authors suggest these changes as underlying the risk of VA in athletes, and …
Reviewer #2 (Public Review):
In this manuscript, Polyák et al. report detailed and systematic functional, electrocardiographic, electrophysiologic (both in vivo and in vitro experiments) and histological analysis in a large animal (canine) model of exercise to assess risk of ventricular arrhythmia susceptibility. They find that exercise-trained dogs have a slower heart rate (not accounted by heightened vagal tone alone and consistent with recent work from Denmark), an increased ventricular mass and fibrosis, APD lengthening due to repolarisation abnormality, enhanced HCN4 expression and decreased outward potassium channel density together with increased ventricular ectopic beats and ventricular fibrillation susceptibility (open-chest burst pacing). The authors suggest these changes as underlying the risk of VA in athletes, and appropriately caution against consigning the beneficial effects of exercise. In general, this study is well done, reasonably well-written, with reasonable conclusions, supported by the data presented and is much needed. There are some methodological, however, given the paucity of experimental data in this area, I think it would still be additive to the literature.
Strengths
1. This is an area with very limited experimental data- this is an area of need.
2. The study, in general seems to be well-conducted with two clear groups
3. The use of a large animal model is appropriate
4. The study findings, in general, support the authors conclusions
5. The authors have shown some restraint in their conclusions and the limitations section is detailed and well written.Weaknesses
1. There are some methodological issues:
a. Authors should explain what the conditioning protocol was and why it was necessary.
b. The rationale for the exercise parameters chosen needs to be presented.
c. Open chest VF induction was a limitation, and it was unnecessary.
d. A more refined VT/VF induction protocol was required. This is a major limitation to this work.
e. The concept of RV dysfunction has not been considered in the study and its analysis.
f. The lack of a quantitative measure for fibrosis is a limitation.
2. Statistical analysis requires further detail (checking of normality of the data/appropriate statistical test).
3. The use of Volders et al. study as a corollary in the discussion does not seem justified given that this study used AV block induced changes as an acquired TdP model. -
Reviewer #3 (Public Review):
This is a well-designed and well conducted study on the effect of 4 months sustained exercise on atrioventricular function and cardiac remodeling in a clinically relevant large animal (canine) model. All methods are well described with proper controls. The findings support the conclusion. Potential limitations are the study are clearly stated. The findings advance the field and provide clear evidence for the susceptibility of ventricular arrhythmia in the canine model of endurance training.
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Reviewer #4 (Public Review):
In the manuscript the author tried to find the cellular level mechanism that causes sudden cardiac death in elite athletes. They found that there are more ventricular fibrosis, ventricular extrasystole burden, longer action potential duration, higher ventricular fibrillation (VF) inducibility, higher HCN4 expression and decreased Ito in sustained trained dog model.
The author successfully conducted large animal training model, showed bradycardia and ventricular fibrosis as a finding similar in athletes and demonstrated the increased ventricular arrhythmia susceptibility to electrical stimulation. The finding of increased action potential duration can be postulated to be a factor of sudden cardiac death in these athletes. However, the interpretation of these findings should be cautious just like all the …
Reviewer #4 (Public Review):
In the manuscript the author tried to find the cellular level mechanism that causes sudden cardiac death in elite athletes. They found that there are more ventricular fibrosis, ventricular extrasystole burden, longer action potential duration, higher ventricular fibrillation (VF) inducibility, higher HCN4 expression and decreased Ito in sustained trained dog model.
The author successfully conducted large animal training model, showed bradycardia and ventricular fibrosis as a finding similar in athletes and demonstrated the increased ventricular arrhythmia susceptibility to electrical stimulation. The finding of increased action potential duration can be postulated to be a factor of sudden cardiac death in these athletes. However, the interpretation of these findings should be cautious just like all the animal studies. Human has a more complex interaction with the environment and individual variabilities. Will the higher susceptibility of VF to electrical stimulation be the same in athletes is still hard to answer.
Still, it is the first study to provide a large animal model of sustained training mimicking trained athletes and to give insights into the cellular level of change in an athlete's heart. The young death of this special group is a tragedy and the importance of these studies cannot be overemphasized.
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