Atypical collective oscillatory activity in cardiac tissue uncovered by optogenetics
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eLife Assessment
This important work provides mechanistic insights into the development of cardiac arrhythmia and establishes a new experimental use case for optogenetics in studying cardiac electrophysiology. The agreement between computational models and experimental observations provides a convincing level of evidence that wave train-induced pacemaker activity can originate in continuously depolarized tissue, with the limitation that there may be differences between depolarization arising from constant optogenetic stimulation, as opposed to pathophysiological tissue depolarization. Future experiments in vivo and in other tissue preparations would extend the generality of these findings.
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Abstract
Abstract
Many biological processes emerge as frequency-dependent responses to trains of external stimuli. Heart rhythm disturbances, i.e. cardiac arrhythmias, are important examples as they are often triggered by specific patterns of preceding stimuli. In this study, we investigated how ectopic arrhythmias can be induced by external stimuli in cardiac tissue containing a localised area of depolarisation. Using optogenetic in vitro experiments and in silico modelling, we systematically explored the dynamics of these arrhythmias, which are characterized by local oscillatory activity, by gradually altering the degree of depolarization in a predefined region. Our findings reveal a bi-stable system, in which transitions between oscillatory ectopic activity and a quiescent state can be precisely controlled, i.e. by adjusting the number and frequency of propagating waves through the depolarized area oscillations could be turned on or off. These frequency-dependent responses arise from collective mechanisms involving stable, non-self-oscillatory cells, contrasting with the typical role of self-oscillations in individual units within biophysical systems. To further generalize these findings, we demonstrated similar frequency selectivity and bi-stability in a simplified reaction-diffusion model. This suggests that complex ionic cell dynamics are not required to reproduce these effects; rather, simpler non-linear systems can replicate similar behaviour, potentially extending beyond the cardiac context.
Article activity feed
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eLife Assessment
This important work provides mechanistic insights into the development of cardiac arrhythmia and establishes a new experimental use case for optogenetics in studying cardiac electrophysiology. The agreement between computational models and experimental observations provides a convincing level of evidence that wave train-induced pacemaker activity can originate in continuously depolarized tissue, with the limitation that there may be differences between depolarization arising from constant optogenetic stimulation, as opposed to pathophysiological tissue depolarization. Future experiments in vivo and in other tissue preparations would extend the generality of these findings.
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Reviewer #1 (Public review):
Summary:
The study by Teplenin and coworkers assesses the combined effects of localized depolarization and excitatory electrical stimulation in myocardial monolayers. They study the electrophysiological behaviour of cultured neonatal rat ventricular cardiomyocytes expressing the light-gated cation channel Cheriff, allowing them to induce local depolarization of varying area and amplitude, the latter titrated by the applied light intensity. In addition, they used computational modeling to screen for critical parameters determining state transitions and to dissect the underlying mechanisms. Two stable states, thus bistability, could be induced upon local depolarization and electrical stimulation, one state characterized by a constant membrane voltage and a second, spontaneously firing, thus oscillatory state. …
Reviewer #1 (Public review):
Summary:
The study by Teplenin and coworkers assesses the combined effects of localized depolarization and excitatory electrical stimulation in myocardial monolayers. They study the electrophysiological behaviour of cultured neonatal rat ventricular cardiomyocytes expressing the light-gated cation channel Cheriff, allowing them to induce local depolarization of varying area and amplitude, the latter titrated by the applied light intensity. In addition, they used computational modeling to screen for critical parameters determining state transitions and to dissect the underlying mechanisms. Two stable states, thus bistability, could be induced upon local depolarization and electrical stimulation, one state characterized by a constant membrane voltage and a second, spontaneously firing, thus oscillatory state. The resulting 'state' of the monolayer was dependent on the duration and frequency of electrical stimuli, as well as the size of the illuminated area and the applied light intensity, determining the degree of depolarization as well as the steepness of the local voltage gradient. In addition to the induction of oscillatory behaviour, they also tested frequency-dependent termination of induced oscillations.
Strengths:
The data from optogenetic experiments and computational modelling provide quantitative insights into the parameter space determining the induction of spontaneous excitation in the monolayer. The most important findings can also be reproduced using a strongly reduced computational model, suggesting that the observed phenomena might be more generally applicable.
Weaknesses:
While the study is thoroughly performed and provides interesting mechanistic insights into scenarios of ventricular arrhythmogenesis in the presence of localized depolarized tissue areas, the translational perspective of the study remains relatively vague. In addition, the chosen theoretical approach and the way the data are presented might make it difficult for the wider community of cardiac researchers to understand the significance of the study.
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Reviewer #2 (Public review):
In the presented manuscript, Teplenin and colleagues use both electrical pacing and optogenetic stimulation to create a reproducible, controllable source of ectopy in cardiomyocyte monolayers. To accomplish this, they use a careful calibration of electrical pacing characteristics (i.e., frequency, number of pulses) and illumination characteristics (i.e., light intensity, surface area) to show that there exists a "sweet spot" where oscillatory excitations can emerge proximal to the optogenetically depolarized region following electrical pacing cessation, akin to pacemaker cells. Furthermore, the authors demonstrate that a high-frequency electrical wave-train can be used to terminate these oscillatory excitations. The authors observed this oscillatory phenomenon both in vitro (using neonatal rat ventricular …
Reviewer #2 (Public review):
In the presented manuscript, Teplenin and colleagues use both electrical pacing and optogenetic stimulation to create a reproducible, controllable source of ectopy in cardiomyocyte monolayers. To accomplish this, they use a careful calibration of electrical pacing characteristics (i.e., frequency, number of pulses) and illumination characteristics (i.e., light intensity, surface area) to show that there exists a "sweet spot" where oscillatory excitations can emerge proximal to the optogenetically depolarized region following electrical pacing cessation, akin to pacemaker cells. Furthermore, the authors demonstrate that a high-frequency electrical wave-train can be used to terminate these oscillatory excitations. The authors observed this oscillatory phenomenon both in vitro (using neonatal rat ventricular cardiomyocyte monolayers) and in silico (using a computational action potential model of the same cell type). These are surprising findings and provide a novel approach for studying triggered activity in cardiac tissue.
The study is extremely thorough and one of the more memorable and grounded applications of cardiac optogenetics in the past decade. One of the benefits of the authors' "two-prong" approach of experimental preps and computational models is that they could probe the number of potential variable combinations much deeper than through in vitro experiments alone. The strong similarities between the real-life and computational findings suggest that these oscillatory excitations are consistent, reproducible, and controllable.
Triggered activity, which can lead to ventricular arrhythmias and cardiac sudden death, has been largely attributed to sub-cellular phenomena, such as early or delayed afterdepolarizations, and thus to date has largely been studied in isolated single cardiomyocytes. However, these findings have been difficult to translate to tissue and organ-scale experiments, as well-coupled cardiac tissue has notably different electrical properties. This underscores the significance of the study's methodological advances: the use of a constant depolarizing current in a subset of (illuminated) cells to reliably result in triggered activity could facilitate the more consistent evaluation of triggered activity at various scales. An experimental prep that is both repeatable and controllable (i.e., both initiated and terminated through the same means).
The authors also substantially explored phase space and single-cell analyses to document how this "hidden" bi-stable phenomenon can be uncovered during emergent collective tissue behavior. Calibration and testing of different aspects (e.g., light intensity, illuminated surface area, electrical pulse frequency, electrical pulse count) and other deeper analyses, as illustrated in Appendix 2, Figures 3-8, are significant and commendable.
Given that the study is computational, it is surprising that the authors did not replicate their findings using well-validated adult ventricular cardiomyocyte action potential models, such as ten Tusscher 2006 or O'Hara 2011. This may have felt out of scope, given the nice alignment of rat cardiomyocyte data between in vitro and in silico experiments. However, it would have been helpful peace-of-mind validation, given the significant ionic current differences between neonatal rat and adult ventricular tissue. It is not fully clear whether the pulse trains could have resulted in the same bi-stable oscillatory behavior, given the longer APD of humans relative to rats. The observed phenomenon certainly would be frequency-dependent and would have required tedious calibration for a new cell type, albeit partially mitigated by the relative ease of in silico experiments.
For all its strengths, there are likely significant mechanistic differences between this optogenetically tied oscillatory behavior and triggered activity observed in other studies. This is because the constant light-elicited depolarizing current is disrupting the typical resting cardiomyocyte state, thereby altering the balance between depolarizing ionic currents (such as Na+ and Ca2+) and repolarizing ionic currents (such as K+ and Ca2+). The oscillatory excitations appear to later emerge at the border of the illuminated region and non-stimulated surrounding tissue, which is likely an area of high source-sink mismatch. The authors appear to acknowledge differences in this oscillatory behavior and previous sub-cellular triggered activity research in their discussion of ectopic pacemaker activity, which is canonically expected more so from genetic or pathological conditions. Regardless, it is exciting to see new ground being broken in this difficult-to-characterize experimental space, even if the method illustrated here may not necessarily be broadly applicable.
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