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.
