Periodic Reporting for period 1 - OPTOCARD (Panoramic optical manipulation of cardiac electrical dynamics: a novel tool to study complex arrhythmias)
Okres sprawozdawczy: 2023-06-01 do 2025-05-31
Podsumowanie kontekstu i ogólnych celów projektu
Cardiovascular diseases are the most common cause of death worldwide, and a substantial number of these deaths are caused by cardiac arrhythmias resulting in sudden cardiac death. Cardiac arrhythmias are caused by disruptions in cardiac electrical activity, leading to a reduction to complete loss of the heart’s ability to maintain blood flow throughout the body. One of the mechanisms underlying these arrhythmias are driven by heterogeneities in repolarisation time (RT), which is the time in which cardiac muscle cells (cardiomyocytes) recover from electrical activation. These RT heterogeneities are caused by action potential (AP) prolongation or abbreviation in certain areas of the heart, and can be caused by inherited disease (i.e. genetic disorders causing ion channel dysfunction), but also occur secondary to acquired heart disease such as myocardial infarction. Current experimental methods aimed to explore the mechanisms causing arrhythmias rely on the infusion of drugs modulating AP duration (APD) in single coronary arteries to generate ventricular RT heterogeneities, limiting versatility of the spatial characteristics of the area with altered APD, as well as restricting studies to larger animal models. Therefore, the conditions facilitating sustained arrhythmia upon the presence of RT heterogeneities remain to be elucidated.
In addition, while optical platforms offer the possibility to study cardiac electrical dynamics in a highly detailed manner, current state-of the-art optical platforms lack the possibilities required for the research objectives of this project. To accurately assess the arrhythmogenic effects of RT heterogeneities, it is vital to be able control optical stimulation across the entire ventricular surface.
In the OptoCARD project, we aimed to develop a novel, state-of-the-art panoramic optical manipulation-and-detection platform to study and manipulate cardiac electrophysiological dynamics in mouse hearts. By using multiple cameras and projectors, and a centralised acquisition system, this platform enables us to perform organ-wide detection of electrical activity. To measure cellular transmembrane potential and thereby map action potentials, we implemented a red-shifted voltage-sensitive dye (VSD) which is excited by red light and emits light in the far-red spectrum. Manipulation of cardiac function is performed via the use of the light-activated ion channel Channelrhodopsin-2 (ChR2), which is activated by blue light. Hence, by using a mouse model with cardiac-specific expression of ChR2 and using the red-shifted VSD, we can perform simultaneous optical mapping and optogenetic stimulation with minimal optical crosstalk.
The scientific goal of this project is to develop a new model to study mechanisms underlying generation and maintenance of lethal ventricular arrhythmias caused by heterogeneities in RT. Previous work by the supervisor’s group demonstrated that sub-threshold optogenetic stimulation, with an intensity insufficient to evoke an AP, induces a depolarization in single cells, translating to conduction slowing and delayed RT in the stimulated area of whole hearts. As such, this approach could allow for specific and fully reversible manipulation of RT and can therefore be used to gain insight in mechanisms driving cardiac arrhythmias in the setting of RT heterogeneities. In addition, patterned supra-threshold allows for the creation of non-conducting areas within the heart and thereby inducing conduction block. Together, these approaches enable advanced investigations of mechanisms driving arrhythmias by mimicking various pathological conditions. Combining this approach with a panoramic aspect allowing to measure and manipulate the entire heart, opens a new avenue of possibilities by gaining control over the entire myocardial surface and simultaneously measuring it.
Apart from the panoramic optical manipulation-and-detection platform, we identified a pharmacological approach to enhance functional gradients induced by sub-threshold optogenetic stimulation. After an extensive literature study, patch-clamp studies were performed on isolated mouse cardiomyocytes to assess the impact of the selected drug, Flecainide. Here, we tested whether the drug Flecainide can be used to enhance heterogeneities in excitability to depolarised resting membrane potential. In addition, we applied the insights gained from these single cell experiments to the entire heart, where we performed sub-threshold optogenetic stimulation in combination with this pharmacological approach to enhance arrhythmia susceptibility. Together, this project contributes to the development of novel approaches to study arrhythmia mechanisms in mouse hearts.
Scientific objectives:
The scientific objectives and their status at the end of the project are as follows:
Objective 1: Develop a panoramic optical platform for measuring and manipulating cardiac activity and employ the system to characterise the spatial properties of repolarisation manipulation.
Objective 2: Establish RMP-dependent drugs and exploit this dependence to enhance RT gradients.
Objective 3: Employ the newly developed panoramic optical platform and apply the established RMP- dependent drugs to understand the mechanisms underlying arrhythmia induction and rotor maintenance.
Training objectives:
Objective 1: Extend technical skills and knowledge in the field of cardiac functional imaging.
Objective 2: Gain programming skills to become a more all-round researcher.
Objective 3: Improve communication skills with general public.
In addition, while optical platforms offer the possibility to study cardiac electrical dynamics in a highly detailed manner, current state-of the-art optical platforms lack the possibilities required for the research objectives of this project. To accurately assess the arrhythmogenic effects of RT heterogeneities, it is vital to be able control optical stimulation across the entire ventricular surface.
In the OptoCARD project, we aimed to develop a novel, state-of-the-art panoramic optical manipulation-and-detection platform to study and manipulate cardiac electrophysiological dynamics in mouse hearts. By using multiple cameras and projectors, and a centralised acquisition system, this platform enables us to perform organ-wide detection of electrical activity. To measure cellular transmembrane potential and thereby map action potentials, we implemented a red-shifted voltage-sensitive dye (VSD) which is excited by red light and emits light in the far-red spectrum. Manipulation of cardiac function is performed via the use of the light-activated ion channel Channelrhodopsin-2 (ChR2), which is activated by blue light. Hence, by using a mouse model with cardiac-specific expression of ChR2 and using the red-shifted VSD, we can perform simultaneous optical mapping and optogenetic stimulation with minimal optical crosstalk.
The scientific goal of this project is to develop a new model to study mechanisms underlying generation and maintenance of lethal ventricular arrhythmias caused by heterogeneities in RT. Previous work by the supervisor’s group demonstrated that sub-threshold optogenetic stimulation, with an intensity insufficient to evoke an AP, induces a depolarization in single cells, translating to conduction slowing and delayed RT in the stimulated area of whole hearts. As such, this approach could allow for specific and fully reversible manipulation of RT and can therefore be used to gain insight in mechanisms driving cardiac arrhythmias in the setting of RT heterogeneities. In addition, patterned supra-threshold allows for the creation of non-conducting areas within the heart and thereby inducing conduction block. Together, these approaches enable advanced investigations of mechanisms driving arrhythmias by mimicking various pathological conditions. Combining this approach with a panoramic aspect allowing to measure and manipulate the entire heart, opens a new avenue of possibilities by gaining control over the entire myocardial surface and simultaneously measuring it.
Apart from the panoramic optical manipulation-and-detection platform, we identified a pharmacological approach to enhance functional gradients induced by sub-threshold optogenetic stimulation. After an extensive literature study, patch-clamp studies were performed on isolated mouse cardiomyocytes to assess the impact of the selected drug, Flecainide. Here, we tested whether the drug Flecainide can be used to enhance heterogeneities in excitability to depolarised resting membrane potential. In addition, we applied the insights gained from these single cell experiments to the entire heart, where we performed sub-threshold optogenetic stimulation in combination with this pharmacological approach to enhance arrhythmia susceptibility. Together, this project contributes to the development of novel approaches to study arrhythmia mechanisms in mouse hearts.
Scientific objectives:
The scientific objectives and their status at the end of the project are as follows:
Objective 1: Develop a panoramic optical platform for measuring and manipulating cardiac activity and employ the system to characterise the spatial properties of repolarisation manipulation.
Objective 2: Establish RMP-dependent drugs and exploit this dependence to enhance RT gradients.
Objective 3: Employ the newly developed panoramic optical platform and apply the established RMP- dependent drugs to understand the mechanisms underlying arrhythmia induction and rotor maintenance.
Training objectives:
Objective 1: Extend technical skills and knowledge in the field of cardiac functional imaging.
Objective 2: Gain programming skills to become a more all-round researcher.
Objective 3: Improve communication skills with general public.
Prace wykonane od początku projektu do końca okresu sprawozdawczego oraz najważniejsze dotychczasowe rezultaty
Objective 1: Develop a panoramic optical platform for measuring and manipulating cardiac activity and employ the system to characterise the spatial properties of repolarisation manipulation.
This objective has been fully achieved, and its associated Work Package 1 has been fully completed. The panoramic optical manipulation-and-detection platform is currently fully operational. Schematic representations of the platform, its optical components per unit, and the connectivity architecture can be observed in Figure 1A. The platform consists of four units each containing a digital light processing (DLP) projector as a light source, series of optical lenses and filters to focus and filter the activation, excitation and emission light, and a high-speed camera with a complementary metal-oxide-semiconductor (CMOS) sensor to detect emission light. The LEDs driver developed during the project to control the DLPs, allow for an illumination strategy where red and blue light are fully controlled, switching at high speed without any oscillations when changing illumination patterns (Figure 1B). During experiments, the DLPs produce red light to excite the voltage-sensitive dye used to measure cardiac activity, while blue light from the same source is used for optogenetic stimulation, manipulating cardiac electrical dynamics. The platform’s components are controlled through the managing software platform developed in-house in the general-purpose and widely used programming language Python (Figure 1C). As illustrated in Figure 1D, the platform allows for the projection of detailed illumination patterns at a very high precision, with the four projectors fully aligned and virtually functioning as one unit.
As illustrated in Figure 2A the system allows for examination of the spread of AP wavefronts in murine hearts. When electrically stimulated with an electrode placed at the apex of the heart, the wavefront moves from the apex to the base, which can be mapped allowing for assessment of conduction in terms of velocity, directionality, and AP wavefront deformation. Importantly, the fluorescent signals recorded in the platform display an impressive signal-to-noise ratio far exceeding our previous optical platforms, allowing in-depth analysis of AP parameters in terms of time to peak and duration across the entire heart. Moreover, less averaging and filtering is required compared to our previous optical mapping setups, thanks to the extremely good quality of the signals. This enables us to create activation and AP parameter maps with a very high resolution, allowing detailed analysis of AP parameters across the heart. To validate the capability of the platform to simultaneously measure and manipulate cardiac electrical dynamics, we projected a line of high-intensity blue light on the cardiac surface. This long-term supra-threshold optogenetic stimulation should bring the tissue in a permanently depolarized state, in which its not excitable anymore. Indeed, our experiments verify that the illumination induced a non-conductive area blocking the activation wavefront. Upon supra-threshold line illumination, conduction from the apex to base induced by electrical stimulation at the apex was blocked (Figure 2B).
In addition to supra-threshold stimulation, the main aim for incorporating optogenetics in this project was to employ sub-threshold optogenetic stimulation to generate gradients in conduction and repolarization time. To assess the capability of the platform in manipulating these parameters, we performed sub-threshold optogenetic stimulation at the apex of the heart and mapped activation time and time-to-peak (as measures for conduction and excitation) and AP duration at 90% of repolarization as a measure for repolarization time. As visualised in Figure 3, sub-threshold illumination induced a localised prolongation in APD90, thereby generating a gradient in repolarization time. While activation and time-to-peak were markedly impacted upon apical sub-threshold illumination, the intracardiac gradients expected as a result of patterned illumination were less evident. Further optimisations aimed at reducing light scattering can potentially improve the spatially specific effect further. Importantly, the impact of the sub-threshold optogenetic stimulation was rapidly and fully reversible, as indicated by the measurements performed directly after optogenetic stimulation. Overall, we were able to construct and commission the panoramic optical manipulation-and-detection platform. This allows us to study electrical dynamics in the entire heart and manipulate these dynamics in a flexible and fully reversible manner.
Objective 2: Establish RMP-dependent drugs and exploit this dependence to enhance RT gradients.
This objective has been fully achieved, and the associated Work Package 2 was fully completed. Our aim for this objective was to identify and characterise a drug which we can implement together with sub-threshold optogenetic stimulation to generate pro-arrhythmogenic ventricular gradients in cardiac electrical characteristics. Through an extensive literature study, we identified Flecainide as a candidate drug. This drug is a widely used anti-arrhythmic drug but is known to cause a pro-arrhythmogenic substrate in specific circumstances, especially in the context of patients with prior myocardial infarction. Interestingly, healed myocardial infarction is associated with the presence of gradients in both resting membrane potential (RMP) and repolarization time (RT), which we can induce in our experimental model by using sub-threshold optogenetic stimulation. Indeed, the impact of RMP on the effectiveness of Flecainide has been confirmed in a previous study performed by another group focussed on atrial disease. As such, Flecainide appeared an ideal candidate the enhance the pro-arrhythmogenic effects of patterned sub-threshold illumination.
To assess the utility of Flecainide for the enhancement of the effects of sub-threshold optogenetic stimulation, we performed specialised single-cell measurements focussed on the time-dependent recovery of the cardiac sodium channel NaV1.5 (Figure 4). This ion channel is the most important modulator of cardiac excitability since the sodium current it generates is the main component driving the AP upstroke. We performed a “recovery from inactivation” protocol to assess the availability of NaV1.5 to open after set intervals, while applying different holding potentials and concentrations of Flecainide. In voltage-clamp protocols -as used in this experiment- the holding potential mimics the RMP. We therefore performed measurements while applying holding potentials of -120 mV (the golden standard used in these type of investigations), -90 mV (the typical RMP in healthy ventricular myocardium), and -80 mV (corresponding to the ~10 mV depolarization caused by sub-threshold illumination). We measured NaV1.5 recovery from inactivation after a pause from 10 to 200 ms, with steps of 10 ms difference to grant us a detailed insight in the dynamics of recovery rate. Measurements were performed in the absence of Flecainide, and upon a 5-minute wash in of 50 nM and 100 nM of the drug. These concentrations are much below the therapeutic concentrations and should therefore only moderately impact NaV1.5 recovery from inactivation under physiological circumstances. Strikingly, the impact of Flecainide was greatly dependent on the holding potential applied. At -90 mV, in line with a physiological RMP, 50 nM Flecainide only affected recovery at extremely short recovery intervals of 10 and 20 ms. These short intervals are not relevant in the entire heart, since the shortest coupling interval in entire hearts is around 40 ms. As such, while 100 nM affected NaV1.5 recovery from activation at a physiological RMP, 50 nM did not have a relevant impact. In contrast, 50 nM Flecainide impacted NaV1.5 recovery from inactivation at the entire range of values when the holding potential was set at -80 mV.
Hence, these findings indicate that 50 nM of Flecainide does not impact excitability in tissue with a RMP of -90 mV (as found in non-illuminated ventricular tissue) but does at the depolarised RMP of -80 mV (corresponding to ventricular tissue upon sub-threshold optogenetic stimulation). Therefore, this concentration can be used to enhance the manipulation of electrophysiological tissue characteristics induced by sub-threshold optogenetic stimulation.
Objective 3: Employ the newly developed panoramic optical platform and apply the established RMP- dependent drugs to understand the mechanisms underlying arrhythmia induction and rotor maintenance.
This objective was partly achieved, and satisfactory progress was achieved in its associated Work Package 3. Due to extensive technical issues encountered during the development of the panoramic optical manipulation-and-detection platform, we could not use the newly developed panoramic platform to perform these analyses. Instead, we used our existing non-panoramic setup to perform experiments applying patterned sub-threshold optogenetic stimulation combined with low-dose Flecainide administration in mouse hearts expressing ChR2. While applying the sub-threshold optogenetic stimulation patterns indicated in Figure 5A, we performed programmed electrical stimulation via an electrode placed at the right-ventricular outflow tract (RVOT) of the hearts. This programmed electrical stimulation protocol was aimed to induce arrhythmias, and consisted of a train of 20 “S1” stimulations with a frequency sufficient to overdrive the sinus rhythm and get the heart in a steady state. After this, a “S2”, “S3”, “S4”, and “S5” stimulus were administered to the heart, which were at decreasing intervals with a difference of 10 ms between every stimulus. The stimulation intervals were modified for every heart, with the S1 interval being as close to the sinus rhythm and the S5 stimulus being as short as possible while still being followed consistently. A total of nine of these challenges were performed per condition.
Cardiac activity was assessed through a pseudo-ECG measured by two electrodes close to the ventricular surface (Figure 5B). An arrhythmic event was classified as irregular spontaneous cardiac activity after the S5 stimulus, with a duration longer than 200 ms. Applying sub-threshold optogenetic stimulation did not result in any arrhythmic events in the absence of Flecainide (Figure 5C). By contrast, in the presence of 50 nM Flecainide, there was a significant increase in arrhythmia incidence, especially when illuminating the entire ventricular surface and the right ventricle. Intriguingly, arrhythmias mainly occurred in the absence of illumination when applying 100 nM Flecainide. These results highlight that 50 nM Flecainide together with patterned sub-threshold optogenetic stimulation creates a pro-arrhythmogenic substrate, which is in line with our findings in WP2.
Overview of activities per Work Package:
Work Package 1- Development of optical manipulation-and-detection platform
The panoramic optical manipulation-and-detection platform has been fully developed and is functional. This includes the “biological” part (i.e. Langendorff perfusion system) and the “technical and optical” part (i.e. projectors, cameras, lenses and filters, acquisition workstation). Additional controller boards were developed to synchronise the timing of the different cameras, as well to gain the necessary to properly operate the projectors. Moreover, we developed an automatic USB switch and implemented other tools to handle the simultaneous acquisition of the four cameras. We also developed a software package to control and manage the platform and acquire data. Experiments and analyses were performed to validate the functionality of the platform. All work within WP1 was performed within the supervisor’s group, by the researcher, and students supervised by the supervisor and researcher.
As a result of the work performed in this WP, we can use the developed platform to perform panoramic optical mapping in a conventional manner, as well as during the application of (sub-threshold) optogenetic stimulation. We are able to reliably induce fully reversible gradients in RT and conduction using patterned sub-threshold stimulation in the panoramic optical manipulation-and-detection platform.
Work Package 2 - Discovery of RMP-dependent APD modulating drugs
An extensive literature study was performed to identify candidate drugs. Based on this literature study, we performed an in-depth patch-clamp on single cardiomyocytes isolated from male and female mice. To better control the experimental conditions and to generate more reproducible results, we chose to perform our experiments in cardiomyocytes obtained from wild-type mice, and controlling the holding potential. All activities in WP2 were performed within the supervisor’s group by the researcher.
From the literature study and the experimental the data obtained as a result of this WP, we were able to select a drug and determine an optimal concentration of the drug to be applied in whole-heart experiments.
Work Package 3 - Cardiac dynamics manipulation and RT gradient generation in whole heart
We have started this activity, applying sub-threshold illumination and a pharmacological intervention in whole hearts from male and female mice in order to induce RT gradients and assess arrhythmogenicity. For the RT gradients, we did not obtain sufficient data to present yet. A pilot dataset has been prepared and presented for the arrhythmia inducibility. We performed these experiments in a non-panoramic optical mapping platform, as we encountered delays in the development panoramic platform. Arrhythmia was assessed through pseudo-ECG. All activities in WP3 were performed within the supervisor’s group by the researcher.
We generated pilot data showing that low-dose (50 nM) Flecainide enhances arrhythmogenicity when combined with sub-threshold optogenetic stimulation. This highlights the potency of the combination of optogenetic manipulation and pharmacological approaches.
This objective has been fully achieved, and its associated Work Package 1 has been fully completed. The panoramic optical manipulation-and-detection platform is currently fully operational. Schematic representations of the platform, its optical components per unit, and the connectivity architecture can be observed in Figure 1A. The platform consists of four units each containing a digital light processing (DLP) projector as a light source, series of optical lenses and filters to focus and filter the activation, excitation and emission light, and a high-speed camera with a complementary metal-oxide-semiconductor (CMOS) sensor to detect emission light. The LEDs driver developed during the project to control the DLPs, allow for an illumination strategy where red and blue light are fully controlled, switching at high speed without any oscillations when changing illumination patterns (Figure 1B). During experiments, the DLPs produce red light to excite the voltage-sensitive dye used to measure cardiac activity, while blue light from the same source is used for optogenetic stimulation, manipulating cardiac electrical dynamics. The platform’s components are controlled through the managing software platform developed in-house in the general-purpose and widely used programming language Python (Figure 1C). As illustrated in Figure 1D, the platform allows for the projection of detailed illumination patterns at a very high precision, with the four projectors fully aligned and virtually functioning as one unit.
As illustrated in Figure 2A the system allows for examination of the spread of AP wavefronts in murine hearts. When electrically stimulated with an electrode placed at the apex of the heart, the wavefront moves from the apex to the base, which can be mapped allowing for assessment of conduction in terms of velocity, directionality, and AP wavefront deformation. Importantly, the fluorescent signals recorded in the platform display an impressive signal-to-noise ratio far exceeding our previous optical platforms, allowing in-depth analysis of AP parameters in terms of time to peak and duration across the entire heart. Moreover, less averaging and filtering is required compared to our previous optical mapping setups, thanks to the extremely good quality of the signals. This enables us to create activation and AP parameter maps with a very high resolution, allowing detailed analysis of AP parameters across the heart. To validate the capability of the platform to simultaneously measure and manipulate cardiac electrical dynamics, we projected a line of high-intensity blue light on the cardiac surface. This long-term supra-threshold optogenetic stimulation should bring the tissue in a permanently depolarized state, in which its not excitable anymore. Indeed, our experiments verify that the illumination induced a non-conductive area blocking the activation wavefront. Upon supra-threshold line illumination, conduction from the apex to base induced by electrical stimulation at the apex was blocked (Figure 2B).
In addition to supra-threshold stimulation, the main aim for incorporating optogenetics in this project was to employ sub-threshold optogenetic stimulation to generate gradients in conduction and repolarization time. To assess the capability of the platform in manipulating these parameters, we performed sub-threshold optogenetic stimulation at the apex of the heart and mapped activation time and time-to-peak (as measures for conduction and excitation) and AP duration at 90% of repolarization as a measure for repolarization time. As visualised in Figure 3, sub-threshold illumination induced a localised prolongation in APD90, thereby generating a gradient in repolarization time. While activation and time-to-peak were markedly impacted upon apical sub-threshold illumination, the intracardiac gradients expected as a result of patterned illumination were less evident. Further optimisations aimed at reducing light scattering can potentially improve the spatially specific effect further. Importantly, the impact of the sub-threshold optogenetic stimulation was rapidly and fully reversible, as indicated by the measurements performed directly after optogenetic stimulation. Overall, we were able to construct and commission the panoramic optical manipulation-and-detection platform. This allows us to study electrical dynamics in the entire heart and manipulate these dynamics in a flexible and fully reversible manner.
Objective 2: Establish RMP-dependent drugs and exploit this dependence to enhance RT gradients.
This objective has been fully achieved, and the associated Work Package 2 was fully completed. Our aim for this objective was to identify and characterise a drug which we can implement together with sub-threshold optogenetic stimulation to generate pro-arrhythmogenic ventricular gradients in cardiac electrical characteristics. Through an extensive literature study, we identified Flecainide as a candidate drug. This drug is a widely used anti-arrhythmic drug but is known to cause a pro-arrhythmogenic substrate in specific circumstances, especially in the context of patients with prior myocardial infarction. Interestingly, healed myocardial infarction is associated with the presence of gradients in both resting membrane potential (RMP) and repolarization time (RT), which we can induce in our experimental model by using sub-threshold optogenetic stimulation. Indeed, the impact of RMP on the effectiveness of Flecainide has been confirmed in a previous study performed by another group focussed on atrial disease. As such, Flecainide appeared an ideal candidate the enhance the pro-arrhythmogenic effects of patterned sub-threshold illumination.
To assess the utility of Flecainide for the enhancement of the effects of sub-threshold optogenetic stimulation, we performed specialised single-cell measurements focussed on the time-dependent recovery of the cardiac sodium channel NaV1.5 (Figure 4). This ion channel is the most important modulator of cardiac excitability since the sodium current it generates is the main component driving the AP upstroke. We performed a “recovery from inactivation” protocol to assess the availability of NaV1.5 to open after set intervals, while applying different holding potentials and concentrations of Flecainide. In voltage-clamp protocols -as used in this experiment- the holding potential mimics the RMP. We therefore performed measurements while applying holding potentials of -120 mV (the golden standard used in these type of investigations), -90 mV (the typical RMP in healthy ventricular myocardium), and -80 mV (corresponding to the ~10 mV depolarization caused by sub-threshold illumination). We measured NaV1.5 recovery from inactivation after a pause from 10 to 200 ms, with steps of 10 ms difference to grant us a detailed insight in the dynamics of recovery rate. Measurements were performed in the absence of Flecainide, and upon a 5-minute wash in of 50 nM and 100 nM of the drug. These concentrations are much below the therapeutic concentrations and should therefore only moderately impact NaV1.5 recovery from inactivation under physiological circumstances. Strikingly, the impact of Flecainide was greatly dependent on the holding potential applied. At -90 mV, in line with a physiological RMP, 50 nM Flecainide only affected recovery at extremely short recovery intervals of 10 and 20 ms. These short intervals are not relevant in the entire heart, since the shortest coupling interval in entire hearts is around 40 ms. As such, while 100 nM affected NaV1.5 recovery from activation at a physiological RMP, 50 nM did not have a relevant impact. In contrast, 50 nM Flecainide impacted NaV1.5 recovery from inactivation at the entire range of values when the holding potential was set at -80 mV.
Hence, these findings indicate that 50 nM of Flecainide does not impact excitability in tissue with a RMP of -90 mV (as found in non-illuminated ventricular tissue) but does at the depolarised RMP of -80 mV (corresponding to ventricular tissue upon sub-threshold optogenetic stimulation). Therefore, this concentration can be used to enhance the manipulation of electrophysiological tissue characteristics induced by sub-threshold optogenetic stimulation.
Objective 3: Employ the newly developed panoramic optical platform and apply the established RMP- dependent drugs to understand the mechanisms underlying arrhythmia induction and rotor maintenance.
This objective was partly achieved, and satisfactory progress was achieved in its associated Work Package 3. Due to extensive technical issues encountered during the development of the panoramic optical manipulation-and-detection platform, we could not use the newly developed panoramic platform to perform these analyses. Instead, we used our existing non-panoramic setup to perform experiments applying patterned sub-threshold optogenetic stimulation combined with low-dose Flecainide administration in mouse hearts expressing ChR2. While applying the sub-threshold optogenetic stimulation patterns indicated in Figure 5A, we performed programmed electrical stimulation via an electrode placed at the right-ventricular outflow tract (RVOT) of the hearts. This programmed electrical stimulation protocol was aimed to induce arrhythmias, and consisted of a train of 20 “S1” stimulations with a frequency sufficient to overdrive the sinus rhythm and get the heart in a steady state. After this, a “S2”, “S3”, “S4”, and “S5” stimulus were administered to the heart, which were at decreasing intervals with a difference of 10 ms between every stimulus. The stimulation intervals were modified for every heart, with the S1 interval being as close to the sinus rhythm and the S5 stimulus being as short as possible while still being followed consistently. A total of nine of these challenges were performed per condition.
Cardiac activity was assessed through a pseudo-ECG measured by two electrodes close to the ventricular surface (Figure 5B). An arrhythmic event was classified as irregular spontaneous cardiac activity after the S5 stimulus, with a duration longer than 200 ms. Applying sub-threshold optogenetic stimulation did not result in any arrhythmic events in the absence of Flecainide (Figure 5C). By contrast, in the presence of 50 nM Flecainide, there was a significant increase in arrhythmia incidence, especially when illuminating the entire ventricular surface and the right ventricle. Intriguingly, arrhythmias mainly occurred in the absence of illumination when applying 100 nM Flecainide. These results highlight that 50 nM Flecainide together with patterned sub-threshold optogenetic stimulation creates a pro-arrhythmogenic substrate, which is in line with our findings in WP2.
Overview of activities per Work Package:
Work Package 1- Development of optical manipulation-and-detection platform
The panoramic optical manipulation-and-detection platform has been fully developed and is functional. This includes the “biological” part (i.e. Langendorff perfusion system) and the “technical and optical” part (i.e. projectors, cameras, lenses and filters, acquisition workstation). Additional controller boards were developed to synchronise the timing of the different cameras, as well to gain the necessary to properly operate the projectors. Moreover, we developed an automatic USB switch and implemented other tools to handle the simultaneous acquisition of the four cameras. We also developed a software package to control and manage the platform and acquire data. Experiments and analyses were performed to validate the functionality of the platform. All work within WP1 was performed within the supervisor’s group, by the researcher, and students supervised by the supervisor and researcher.
As a result of the work performed in this WP, we can use the developed platform to perform panoramic optical mapping in a conventional manner, as well as during the application of (sub-threshold) optogenetic stimulation. We are able to reliably induce fully reversible gradients in RT and conduction using patterned sub-threshold stimulation in the panoramic optical manipulation-and-detection platform.
Work Package 2 - Discovery of RMP-dependent APD modulating drugs
An extensive literature study was performed to identify candidate drugs. Based on this literature study, we performed an in-depth patch-clamp on single cardiomyocytes isolated from male and female mice. To better control the experimental conditions and to generate more reproducible results, we chose to perform our experiments in cardiomyocytes obtained from wild-type mice, and controlling the holding potential. All activities in WP2 were performed within the supervisor’s group by the researcher.
From the literature study and the experimental the data obtained as a result of this WP, we were able to select a drug and determine an optimal concentration of the drug to be applied in whole-heart experiments.
Work Package 3 - Cardiac dynamics manipulation and RT gradient generation in whole heart
We have started this activity, applying sub-threshold illumination and a pharmacological intervention in whole hearts from male and female mice in order to induce RT gradients and assess arrhythmogenicity. For the RT gradients, we did not obtain sufficient data to present yet. A pilot dataset has been prepared and presented for the arrhythmia inducibility. We performed these experiments in a non-panoramic optical mapping platform, as we encountered delays in the development panoramic platform. Arrhythmia was assessed through pseudo-ECG. All activities in WP3 were performed within the supervisor’s group by the researcher.
We generated pilot data showing that low-dose (50 nM) Flecainide enhances arrhythmogenicity when combined with sub-threshold optogenetic stimulation. This highlights the potency of the combination of optogenetic manipulation and pharmacological approaches.
Innowacyjność oraz oczekiwany potencjalny wpływ (w tym dotychczasowe znaczenie społeczno-gospodarcze i szersze implikacje społeczne projektu)
The panoramic optical manipulation-and-detection platform developed during the project is a novel approach to study cardiac arrhythmias in a highly detailed, flexible, and reversible manner. By making our approach and findings fully publicly accessible, this platform will allow for a completely new avenue for researchers to study mechanisms driving and maintaining potentially lethal cardiac arrhythmias.
The way we choose to implement the platform, we can study mechanisms underlying arrhythmias caused by heterogeneities in repolarization time (RT) and conduction velocity (CV), causing gradients in both RT and CV. While heterogeneities in CV is a well-established risk factor for developing arrhythmias, the mechanisms underlying arrhythmias due to gradients in RT remain to be elucidated. Current techniques to study this phenomenon largely rely in the implementation of electrodes physically touching the heart or “zonal” panoramic optical mapping (low resolution), conventional optical mapping (partial coverage) for measuring cardiac electrophysiological characteristics and utilize local infusion of drugs to induce RT gradients (non-reversible, limited spatial control). The platform we developed enables extremely detailed recordings of cardiac electrophysiology across the entire mouse heart using, as well as implementing optogenetics to induce fully reversible RT gradients with unparalleled spatial control. Moreover, given the all-optical nature of the platform, cardiac activity is not affected by the placement of electrodes or surgical interventions. As such, the developed panoramic optical manipulation-and-detection platform is an extremely valuable addition to the field of cardiac electrophysiology research.
Since we are still in the process of optimizing the setup, especially in terms of auxiliary components and software to manage experimental parameters and data acquisition, no manuscripts have been issued for publication yet. We are planning to increase the rigidity of experimental data, as well as further improving software design and stability and optimising user experience to make the platform as user-friendly as possible before publishing. Upon publication, any laboratory or any other interested party is free to build and use the platform, as all necessary software and custom components will be published fully accessible and open source. Moreover, all non-custom components are freely commercially available. Therefore, the platform can be fully reproduced by any interested party in the future.
Since we will be identifiable as the authors of the resources to be published, parties interested in building the platform themselves can contact the supervisor, and members of his research group will provide full support to these parties. These interactions can also harbour collaborations with other academic groups or industrial partners, such as companies with a biotechnology profile. As a result of our dissemination activities, we have already been approached by potential collaborators, which will potentially lead to new projects utilizing the developed platform.
The way we choose to implement the platform, we can study mechanisms underlying arrhythmias caused by heterogeneities in repolarization time (RT) and conduction velocity (CV), causing gradients in both RT and CV. While heterogeneities in CV is a well-established risk factor for developing arrhythmias, the mechanisms underlying arrhythmias due to gradients in RT remain to be elucidated. Current techniques to study this phenomenon largely rely in the implementation of electrodes physically touching the heart or “zonal” panoramic optical mapping (low resolution), conventional optical mapping (partial coverage) for measuring cardiac electrophysiological characteristics and utilize local infusion of drugs to induce RT gradients (non-reversible, limited spatial control). The platform we developed enables extremely detailed recordings of cardiac electrophysiology across the entire mouse heart using, as well as implementing optogenetics to induce fully reversible RT gradients with unparalleled spatial control. Moreover, given the all-optical nature of the platform, cardiac activity is not affected by the placement of electrodes or surgical interventions. As such, the developed panoramic optical manipulation-and-detection platform is an extremely valuable addition to the field of cardiac electrophysiology research.
Since we are still in the process of optimizing the setup, especially in terms of auxiliary components and software to manage experimental parameters and data acquisition, no manuscripts have been issued for publication yet. We are planning to increase the rigidity of experimental data, as well as further improving software design and stability and optimising user experience to make the platform as user-friendly as possible before publishing. Upon publication, any laboratory or any other interested party is free to build and use the platform, as all necessary software and custom components will be published fully accessible and open source. Moreover, all non-custom components are freely commercially available. Therefore, the platform can be fully reproduced by any interested party in the future.
Since we will be identifiable as the authors of the resources to be published, parties interested in building the platform themselves can contact the supervisor, and members of his research group will provide full support to these parties. These interactions can also harbour collaborations with other academic groups or industrial partners, such as companies with a biotechnology profile. As a result of our dissemination activities, we have already been approached by potential collaborators, which will potentially lead to new projects utilizing the developed platform.