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Cardiac electrophysiology plays a critical role in the development and function of the heart. Studies of early embryonic electrical activity have lacked a viable point stimulation technique to pace in vitro samples. Here, optical pacing by high-precision infrared stimulation is used to pace excised embryonic hearts, allowing electrophysiological parameters to be quantified during pacing at varying rates with optical mapping. Combined optical pacing and optical mapping enables electrophysiological studies in embryos under more physiological conditions and at varying heart rates, allowing detection of abnormal conduction and comparisons between normal and pathological electrical activity during development in various models.
© 2014 Optical Society of America
1. Introduction
Cardiac electrophysiology plays a critical role in both the development and proper functioning of the heart. In embryos, the electrical activity of the heart affects cardiac development, from a linear heart tube to a four-chambered heart. It has been shown that electrical activity can play a role in early development both directly [1] and indirectly via changes to myocardial contraction [2,3]. Additionally, electrical activity can be altered by molecular and mechanical changes [3,4]. Abnormal electrical activity may lead to congenital heart defects [5,6], one of the most common types of birth defects that affects over 32,000 live births in the United States each year, with a quarter of those affected requiring invasive surgery during the first year after birth [7]. Although electrical activity is known to affect and be affected by cardiac development and function, the details of how and when the cardiac conduction system impacts other developmental processes and vice versa are poorly understood. Better technology is required to properly characterize the normal and abnormal development of cardiac conduction, as some of the instrumentation developed for studies in adult hearts cannot be directly implemented for embryonic studies.
In the research setting, cardiac electrophysiology is commonly studied in adult hearts using optical mapping to record and electrodes to stimulate. Optical mapping provides high-resolution, contact-free electrical recordings over large fields of view using a voltage-sensitive dye, with its fluorescence dependent on membrane voltage, and pharmacological intervention to abolish motion [8], For many electrophysiological studies, an electrode is placed in contact with the tissue to provide point stimulation. Point stimulation, which results in action potentials initiating from a small region, is desirable compared to field stimulation, which can result in initiation occurring over the entire sample. This enables maintaining the heart rate of in vitro preparations, which lack a connection to the vasculature and neural innervation, at physiological rates, which is critical for frequency-dependent electrical parameters (e.g. action potential duration (APD) and conduction velocity [9,10]), especially when making direct comparisons between normal and diseased hearts. Point stimulation also allows initiating action potentials at various specific locations to test for arrhythmic substrates [11]. These tools have enabled the quantification of a wide variety of parameters under physiological and pathophysiological conditions, including activation time, APD, conduction velocity, and restitution [8], greatly advancing our knowledge of mechanisms of electrical propagation and aiding to identify and understand disease states.
Optical mapping has also been employed in a limited way for embryonic studies [4,12–18]. These studies have demonstrated activation maps, and one study has recently demonstrated APD maps [17]. The miniscule size of the hearts being imaged (often less than 1 mm in any dimension) makes embryonic studies more difficult than those of adult hearts, as recordings have a much lower signal-to-noise ratio (SNR). Additionally, these studies generally have been conducted in unpaced tissues. The increased difficulty in signal processing due to low SNR and the lack of pacing has limited the parameters that have been quantified through optical mapping and the accuracy of the data obtained.
Electrode pacing, however, presents significant problems in small tissues. First, electrode point stimulation requires contact, which is difficult for small tissues such as the embryonic heart, both due to the tissue being more fragile to damage through contact and the higher precision required to position the electrodes. Second, point stimulation can cause damage through the high charge density in the region around the electrode required for stimulation [19]. Third, the injection of current creates an electrical artifact around the electrode on a millimeter scale [20–22], which significantly obscures electrical recordings in millimeter-sized sample such as the embryonic heart. Finally, current injection also results in stimulation over an area larger than the electrode tip, effectively making electrode-based high-precision point stimulation not feasible in embryonic hearts [23]. With these shortcomings, a different approach is needed for point stimulation in small samples.
Infrared stimulation has recently been developed as an alternate technique for stimulating excitable tissues without genetic modification, generating propagating action potentials using pulsed infrared laser light. Infrared stimulation was first developed in nerves [24] and has been used to stimulate a variety of nerves and neurons [25–30], vestibular hair cells [31], and isolated myocytes [32] using multimode fibers. Additionally, we have recently demonstrated the use of infrared stimulation to optically pace whole intact embryonic [33] and adult hearts [34]. The specific mechanisms for infrared stimulation are still being explored, but it is clearly a thermal effect [25], and heat-induced changes to membrane capacitance seem to play an important role, causing the initial depolarization required to generate an action potential [35]. Optical pacing by infrared stimulation has several advantages over electrode stimulation relevant to electrophysiological studies in embryonic hearts. First, optical pacing is contact-free, avoiding potential physical damage and enabling easy and rapid positioning of the stimulation site. Second, since optical pacing works through a heat deposition mechanism, no areas of potentially damaging high charge densities are generated. While heat can damage tissue, previous studies indicate that the stimulation threshold is below the thermal damage threshold [24–30,33–35]. Third, optical pacing does not inject current, avoiding the electrical artifact created by electrode pacing. It also does not generate a shadow, such as that cast by an electrode that passes between the tissue and the optical mapping camera. Finally, while previous studies using infrared stimulation have used multimode fibers with spot sizes in the hundreds of microns, laser light can be focused to a spot size on the order of microns or even nanometers, providing higher spatial precision. These attributes make infrared stimulation an attractive technique to pace small samples.
Combining optical pacing with optical mapping may be a viable and robust method of studying embryonic cardiac electrophysiology. The goals of this paper are (1) to demonstrate that high-precision infrared point stimulation can successfully optically pace excised embryonic hearts and investigate the effects of varying stimulation parameters, (2) to establish the feasibility of conducting optical mapping experiments while pacing and investigate stimulation artifacts, and (3) to demonstrate acquisition of quantitative electrophysiological information that cannot be obtained in embryonic hearts without pacing.
2. Methods
2.1 Animal model
All animal husbandry and experiments were conducted in compliance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals with the approval of the Institutional Animal Care and Use Committee at Case Western Reserve University.
Quail eggs (Coturnix coturnix; Boyd’s Bird Company, Pullman, WA) were incubated in a humidified, forced draft incubator (G.Q.F Manufacturing, Savannah, GA) at 38°C for 2-7 days, corresponding to Hamburger-Hamilton stages 14-32 [36], during which the heart is transitioning from a looped, tubular heart to a septated four-chambered heart.
Timed development of a mouse embryo (mixed background: sacrificed for an unrelated experiment) was determined at E0.5 days when a vaginal plug was found after overnight mating. The embryo was harvested by a caesarean-driven method at E9.5 days.
For both models, the heart was dissected out and stained in 300-500 µL of 10 µM di-4-ANEPPS (Life Technologies, Carlsbad, CA) in Tyrode’s solution (Sigma-Aldrich, St. Louis, MO) for 12-15 minutes at room temperature. Stained hearts were then transferred to a glass-bottomed imaging chamber in 1 mL of 37 °C Tyrode’s solution with 12-100 µM Cytochalasin D (CytoD; Sigma-Aldrich). Temperature was controlled by enclosing the entire system in a box and heating with a pair of 50 W enclosure heaters (McMaster-Carr, Aurora, OH).
2.2 Stimulation: optical pacing
A 200-mW, 1440-nm diode laser was used to pace younger quail hearts (< 4 days) and the mouse heart while a 400-mW, 1465-nm diode laser (QPhotonics, Ann Arbor, MI) was used for older hearts (> 4 days), with specifications listed in Table 1. The lasers were coupled into single-mode fibers that directed the light through a scanner consisting of a collimating lens, a pair of galvanometer-mounted scanning mirrors (Cambridge Technology, Bedford, MA), and a focusing lens to generate a 12-µm diameter beam waist. An arbitrary waveform generator (Fluke, Everett, WA) provided square-wave pulse trains of the desired frequency, amplitude, and pulse width to a laser diode controller (ILX Lightwave, Bozeman, MT). The scanner was placed below the microscope stage, allowing optical pacing from below the sample while simultaneously recording optical mapping from above.
Table 1. Pacing Laser Parameters
Unless otherwise stated, pulse width was maintained at 20 ms and the beam waist of the laser was positioned just above the upper surface of the imaging chamber’s glass bottom, so that the spot size on the surface of the excised heart was 12 µm. Successful pacing was defined as 1:1 capture during the entire recording, where each stimulus produced a depolarization across the heart and the depolarizations were locked to the stimulation pulses.
2.3 Recording: optical mapping
The imaging apparatus was built around an Axio Scope.A1 microscope with a 10X, 0.45 NA objective for 2-day quail hearts and the mouse heart and a 5X, 0.25 NA objective (Carl Zeiss Microscopy, Thornwood, NY) for older quail hearts. Broadband illumination was provided by a SOLA Light Engine (Lumencor, Beaverton, OR) and images were captured by a 128 × 128 pixel iXon3 860 EMCCD camera (Andor Technology, South Windsor, CT) with a zoom lens set at 0.33X. Optical mapping images were collected at either 500.0 Hz at full resolution for 2-day quail hearts or 1408.5 Hz with 4x4 binning for older hearts via Andor SOLIS software. A custom filter cube (excitation: 510/80 nm, dichroic mirror: 560 nm, emission: 685/80 nm; Chroma Technology, Bellows Falls, VT) was used.
To establish the feasibility of high-precision optical pacing, bright-field images were taken of a 2-day quail heart and the E9.5 mouse heart at 30-60 Hz without di-4-ANEPPS or CytoD while the pacing laser was turned on and off. Illumination was provided from the side by a fiber optic cold light source. The intensity of an arbitrary pixel on the edge of the heart was examined, with traces showing changes when the heart moved during contraction [33]. The quantitative change in intensity and its direction are arbitrary with no physiological meaning.
2.4 Threshold testing
The effects of varying spot size and pulse width on the optical pacing threshold was tested with 2-day (HH 14) quail hearts (n = 7). Each heart was paced at a single point on the atrium with 9 different spot sizes (12 - 480 µm), and a subset of hearts (n = 3) were also paced at 10 different pulse widths (5 - 100 ms). The threshold was found as the minimum setting required to successfully optically pace the heart. The threshold for optical pacing at 12 µm, 20 ms was obtained at the beginning and end of each experiment and verified to be the same to ensure that no physiological changes had occurred over the course of the experiment. Variations in the spot size were accomplished by moving the focus of the laser away from the tissue, with the spot size on the tissue calculated based on a Gaussian beam profile.
Pulse energies corresponding to the arbitrary waveform generator settings at threshold were measured after the experiments using a pyroelectric energy meter (Ophir, North Andover, MA). Pulse amplitudes were calculated by dividing the measured pulse energies by the pulse widths. Radiant exposures per pulse were calculated by dividing the measured pulse energy by the spot size area. When aggregating threshold measurements between hearts, the pulse amplitudes and radiant exposures per pulse were normalized to the value measured at focus (12-µm spot size) and 20-ms pulse width to account for biological variances between hearts.
2.5 Artifact measurement
The optical artifact from optical pacing during optical mapping was quantified on a 2-day (HH 14) heart. The pacing laser was positioned on the atrioventricular junction so that the optical mapping around the pacing site was of a relatively flat and uniform surface of tissue. The action potential traces around the pacing site were examined and the artifact was quantified by the decrease in the signal amplitude from the baseline of the action potential trace during the resting phase. The center of the artifact was defined as the point with the largest change in amplitude and measurements were taken for every pixel in a straight line away from the center. The spatial distribution of the artifact intensity was fit with a Gaussian distribution and the full width half maximum diameter (FWHM) was determined.
2.6 Optical mapping data analysis
Comparisons between the different pacing conditions (unpaced, and pacing at different frequencies) of each heart were performed by examining action potential traces across the heart, the activation maps, and APD maps at 90% repolarization (APD90).
Analysis of optical mapping data was performed with custom software written in MATLAB (MathWorks, Natick, MA). Displayed action potential traces had the baseline (resting phase) slope removed by subtracting the slope from a linear fit of a selection of points at baseline, then each trace was divided by the baseline to produce graphs of the percent change in fluorescence relative to the baseline fluorescence (ΔF/F). Detection of activation and repolarization was done using a double normal cumulative distribution function (CDF) fitting method that has higher accuracy, precision, and robustness in lower SNR recordings compared to traditional signal processing methods. Activation times were taken as the middle of the upstroke normal CDF and APD90s were taken as the time between activation and the point on the repolarization CDF corresponding to 0.9 (90% repolarization). Masking to remove background and poorly fit traces and a 3x3 averaging filter were applied to the maps at the end for display.
3. Results
The feasibility of using high-precision infrared stimulation to optically pace embryonic hearts was established in 2-day quail and E9.5-day mouse hearts. Fig. 1 shows that when the pacing laser was turned on (red bars), the hearts (left (Media 1): quail; right (Media 2): mouse) were successfully paced at the frequency of the stimulation pulses (quail: 2.00 Hz; mouse: 1.00 Hz), above the unpaced rate. After the initial optical pacing, the heart rate immediately decreased and optical pacing was reestablished when the pacing laser was turned on again.
Fig. 1 Demonstration of high-precision optical pacing. Bright-field images of a 2-day quail heart that was paced at 2.00 Hz (left, Media 1) and an E9.5-day mouse heart that was paced at 1.00 Hz (right,Media 2). The hearts were initially unpaced and optical pacing was turned on, off, and then on again. Traces are of pixel intensity at an arbitrary point on the edge of the hearts, providing an indication of contraction, but the degree and direction of the change has no physiological meaning. Red bars indicate when the laser was on. Scale bars are 500 µm.
The effects of varying spot size (n = 7) and the laser pulse width (n = 3) on the optical pacing threshold on the atrium in 2-day (HH 14) hearts were determined (Fig. 2). A subset of the means and standard deviations of the actual pacing threshold energies and radiant exposures per pulse are shown in Table 2. To allow meaningful aggregation, values for each heart were normalized to the value obtained for a 12-µm spot size and a 20-ms pulse width (Fig. 2, red dots). There was no significant difference in the threshold pulse amplitude as the spot size was increased from 12 µm to 240 µm, but at 360 µm and 480 µm, a statistically greater amount of energy was required to establish 1:1 capture (Fig. 2(A)). However, the threshold radiant exposure decreased rapidly and significantly as the spot size increased (Fig. 2(B)). Meanwhile, increasing pulse width decreased the threshold pulse amplitude, but the changes were statistically significant only up to pulse widths of 20 ms (Fig. 2(C)). Threshold radiant exposure increased fairly linearly as pulse width increased (Fig. 2(D)).
Fig. 2 Effect of spot size and pulse width on relative pacing threshold. Spot size (A, B; n = 7) was varied from 12 µm to 480 µm. Pulse width (C, D; n = 3) was varied from 5 ms to 100 ms. The threshold required to achieve 1:1 capture are plotted as pulse amplitude (A, C) and radiant exposure per pulse (B, D) normalized to the value at 12 µm and 20 ms (red points) to allow for aggregation of hearts. * p < 0.05 compared to 12 µm/20 ms (red points).
Table 2. Thresholds per Pulse
An artifact was observed during initial experiments combining optical pacing with optical mapping and was quantified in a 2-day (HH 14) quail embryo heart (Fig. 3). An example fluorescence image after staining with di-4-ANEPPS with contractions stopped by CytoD is shown for a representative heart in Fig. 3(A). Action potentials were optically recorded while unpaced at 0.52 Hz and during pacing at 1.00 Hz, with representative ventricular traces recorded at the green marker shown in Fig. 3(B) and 3(C), respectively. The fluorescence signal with optical pacing near the pacing site displayed a significant decrease in intensity (Fig. 3(D)). This optical artifact manifested as a linear decrease in signal over the duration of the 20-ms pulse of the pacing laser, followed by a return of the signal that was merged with the actual action potential signal over several hundred milliseconds (Fig. 3(E)). Spatially, the intensity of the artifact is greatest at the center of the pacing site and appears to have a Gaussian distribution (Fig. 3(F)), with the artifact becoming undetectable relative to the action potential signal at distances greater than 100-150 µm from the center of the pacing site in the worst recordings. Higher pulse energies resulted in an artifact of greater intensity.
Fig. 3 Quantification of artifact observed in optical mapping during optical pacing. An example excised 2-day (HH 14) quail embryo heart was optically mapped (A) with a 500-µm scale bar. The optical mapping recordings from the single pixel at the green marker on the ventricle are shown while unpaced at 0.52 Hz (B) and during pacing at 1.00 Hz (C). An optical artifact was observed near the pacing site which manifested as a darkening. That area, indicated by the red box, is enlarged in (D) with a portion of the optical mapping recording shown for the pixel on the atrioventricular junction at the blue marker. The area marked by the red arrows in (D) show the darkening between a frame taken before the stimulus (solid red arrow and line) and during the stimulus (dotted red arrow and line). The entire optical mapping recording at the blue marker in (A) is shown in (E) with the sharp downward spikes of the artifact. Spatially, the artifact has a Gaussian distribution as shown for 3 different energies per pulse in (F), with the full width half maximum diameter (FWHM) listed.
Next, quantitative electrophysiological data was obtained from a wide range of cardiac developmental stages while being optically paced. Figure 4 shows representative examples from a 2-day tubular heart (HH 14) and a 5-day chambered heart (HH 26) under both unpaced and paced conditions. Fluorescence images of the hearts are shown in Fig. 4 left. Representative recordings are shown (Fig. 4 center-left) in blue for the atrioventricular junction in the 2-day heart and the atrium in the 5-day heart and in green for the ventricle for both hearts. The paced recordings show that 1:1 capture was achieved (2-day: 1.50 Hz, 5-day: 3.00 Hz) over the unpaced heart rate (2-day: 0.63 Hz, 5-day: 1.35 Hz) with the action potentials propagating through the entire heart. Activation maps (Fig. 4 center-right) show the conduction pattern with 10 ms isochrones for the 2-day heart and 1 ms isochrones for the 5-day heart. The maps show that activation progresses relatively uniformly in the tubular 2-day heart compared to the relatively large delay between atria and ventricles in the septating 5-day heart, but that the activation patterns are similar between unpaced and paced hearts. APD90 maps (Fig. 4 right) show significantly longer APD90 in the ventricles relative to the atrial regions in both hearts and that APD90 decreases in all parts of the heart as it develops.
Fig. 4 Optical mapping of unpaced and optically paced hearts. Representative data from 2-day, with no binning (top rows), and 5-day, with 4x4 binning (bottom rows), excised embryonic quail hearts. Fluorescence images of the hearts (left) have a blue marker on the atrioventricular junction (2-day) or atrium (5-day) and a green marker on the ventricle. The purple marker indicates the position of the pacing laser. Scale bars are 500 µm. Representative recordings of electrical activity (center-left) are shown for the matching colored markers. Activation maps (center-right) are shown with the color map in seconds and the isochrones are 10 ms apart for the 2-day heart and 1 ms apart for the 5-day heart. Action potential duration maps (APD90, right) are shown with the color map in seconds.
Finally, the combination of optical pacing and optical mapping allows detection of rate-dependent effects on cardiac electrophysiology (Fig. 5). In older embryonic hearts, such as the 7-day (HH 32) heart shown in Fig. 5(A), APD90 decreased significantly as the heart rate increased, as can be seen in Fig. 5(B) for both the atria (blue marker and traces) and the ventricles (green marker and traces). Aggregated APD90 of the pixels in the atria and the ventricles of that heart are shown in Fig. 5(C), with APD90 at 3.00 Hz significantly shorter than that at 1.72 Hz in both the atria (59.6 ± 6.5 ms vs. 34.6 ± 2.2 ms, p < 0.001) and the ventricles (147.9 ± 5.1 ms vs. 101.0 ± 16.3 ms, p < 0.001). In a 2-day (HH 15) heart, 2:1 conduction block was observed between the ventricle and the outflow tract. A fluorescence image is shown in Fig. 5(D) and optically recorded electrical traces are shown in Fig. 5(E) for the ventricle (green marker and traces) and the outflow tract (red marker and traces) while optically pacing on the atrium. Successful 1:1 capture through the entire heart tube was achieved at 2.00 Hz optical pacing. However, when the optical pacing rate was increased to 2.50 Hz, there was 1:1 capture in the atrium and ventricle, but there was 2:1 conduction block in the outflow tract, where only every other stimulus resulted in an action potential.
Fig. 5 Frequency-dependent effects. In older embryonic hearts, action potential duration (APD90) was frequency-dependent as illustrated by the example 7-day heart shown in (A), with the blue marker on the atrium and the green marker on the ventricle. Electrical recordings in matching colors from pixels at those marker positions are shown in (B) when unpaced at 1.72 Hz (left) and during optical pacing at 3.00 Hz (right). Aggregate data for the APD90 across the atria and the ventricles are shown in (C), with p < 0.001 between the frequencies in both cases. A 2-day heart that showed frequency-dependent conduction block is shown in (D), with the green marker on the ventricle and the red marker on the outflow tract. Electrical recordings in matching colors from pixels at those marker positions are shown in (E) when optically paced at 2.00 Hz (left) and at 2.50 Hz (right). The purple markers in (A) and (D) indicate the position of the laser.
4. Discussion
Infrared stimulation is an attractive option for pacing small embryonic hearts, as it overcomes many of the limitations of utilizing electrode stimulation in small tissues. Electrodes require contact, inject potentially damaging high currents, create a large electrical artifact, and have a limited spatial precision. Optogenetics is another optical technique that has been developed for stimulating excitable cells by adding expression of exogenous light-sensitive ion channels [37]. While it is a powerful tool for neuromodulation, optogenetics has significant disadvantages as a tool for cardiac electrophysiology. These include the time and cost required to perform genetic manipulations, the potential that the added ion channels may alter the electrical properties of the cells, the decreased responsiveness to light with repeated stimulation [38], and the likelihood of triggering an action potential when the light-sensitive channels are exposed to the optical mapping excitation light due to overlap of the excitation spectra [39,40] and much lower power requirements necessary for exciting the channels [41]. Infrared stimulation does have the potential to damage tissue if too much heat is deposited, but previously published results suggest that infrared stimulation can be used without damage [24–30,33–35]. Similarly, in this study, no functional damage was observed, as the pacing threshold did not increase over the course of an experiment. Optical pacing by infrared stimulation is contact-free, does not inject current or charge, can be focused for high spatial precision, can be performed in models without genetic manipulation, and can use a wavelength widely separated from those used in optical mapping.
Previously, we have demonstrated the feasibility of using infrared stimulation to optically pace embryonic quail [33] and adult rabbit hearts [34] using multimode fiber illumination with fiber core diameters of 400 µm. Most other studies using infrared stimulation have also been conducted with multimode fibers [24–35]. However, it is extremely challenging to focus light from a multimode fiber to a spot size smaller than the fiber core diameter. Smaller spot sizes can be achieved by using smaller diameter fibers, but the coupling efficiency from the laser into the fiber is reduced as the diameter decreases, resulting is less power available at the sample. In this study, diode lasers designed to be coupled to single-mode fiber were used, providing sufficient light to achieve optical pacing (Table 1). Usage of single-mode fiber enabled high-precision infrared point stimulation with a 12-µm diameter beam waist in excised quail and mouse hearts (Fig. 1, Media 1 and Media 2). This technique is also likely to be applicable in cell cultures for high-throughput electrophysiological assays. A pair of galvanometer-mounted mirrors enabled the laser spot to be easily, quickly, and precisely positioned to any location in the field of view at any time during an experiment.
The effect of the infrared stimulation spot size on the optical pacing threshold (Fig. 2(A)-2(B) and Table 2) indicates that there is a trade-off between the spatial precision of the optical stimulus and the radiant exposure per pulse required to pace the tissue, with no significant change in the pulse amplitude needed, up to a 240-µm spot size. This suggests that when a larger number of cells are recruited by the infrared stimulation, the radiant exposure per pulse required to initiate a propagating action potential is lower. This reduction in the pacing threshold may be enabled by gap junctions, which electrically couple the cardiomyocytes to their neighbors, possibly resulting in the stimulated group of cells acting like a single larger cell. Additionally, while the pulse amplitude required for large spot sizes increased, it is likely that a portion of the laser spot was not on the heart, so the actual energy delivered to the heart would have been reduced. For cardiac pacing, high spatial precision for stimulation is often desirable. While higher radiant exposures have a greater risk of damaging tissue over prolonged periods of pacing, there were no signs of functional damage during these pacing experiments even at the maximum power of Laser 1 at a 12-µm spot size (31 J/mm2).
The effect of laser pulse width on the optical pacing threshold (Fig. 2(C)-2(D) and Table 2) shows that shorter pulse widths are better in terms of minimizing radiant exposure per pulse. However, reducing the pulse width below 20 ms significantly increased the required pulse amplitude, requiring a proportionally more powerful laser. Since increasing the pulse width from 5 to 20 ms reduced the pulse amplitude required by about a factor of 3 while increasing radiant exposure by less than a factor of 2, we decided to use a 20-ms pulse width for further experiments. The lowered pulse amplitude requirement to achieve pacing is desirable when performing optical pacing with optical mapping, as the addition of the excitation-contraction uncoupler resulted in a significantly increased optical pacing threshold. It is also for this reason that older hearts were paced using a different laser with double the maximum power output. There is not a significant difference between the water absorption between the two laser wavelengths (1440 nm: 32.2 cm−1 and 1465 nm: ~31.8 cm−1) [42], so there should not be significant effect of this change in wavelength. This increase in pacing threshold may provide some insight into the mechanism of infrared stimulation in the heart. For example, it has been shown that infrared stimulation can cause cardiomyocytes to contract by affecting actin-myosin interactions [43], which CytoD disrupts [44]. It is possible that normal optical pacing of cardiomyocytes works through a combination of contraction and alteration of membrane capacitance [35], and the abolition of motion therefore increases the optical pacing threshold.
Studying electrical activity while using electrode point stimulation is not possible in embryonic hearts, as electrical stimulation has a millimeter-scale space constant [20–22] that would create an electrical artifact across the entire small embryonic heart. This electrical artifact would obscure recording and could cause action potentials to initiate over a larger region. With combined optical pacing and optical mapping, recordings with high-precision infrared stimulation were obtained in a 2-day quail embryonic heart (Fig. 3(A)-3(C)). However, an artifact was also observed in the recordings near the pacing site (Fig. 3(D)-3(F)). This artifact is caused by thermal lensing, where the laser heats the water between the tissue and the camera [45]. This was confirmed by observing the artifact in bright-field imaging when the infrared stimulation was applied to water without tissue. While this thermal artifact obscures optical mapping recordings around the pacing site, compared to electrical stimulation artifacts, it is very limited in size (< 75 µm from the pacing site), does not affect the actual membrane voltage, and can be reduced by a variety of measures. The size of the artifact is larger than the laser spot size because the laser beam diverges away from the focus. The size of the artifact can be reduced by decreasing the laser power to near-threshold levels, by reducing the amount of water between the tissue and the objective, and by increasing the amount of tissue between the laser and the objective lens, allowing more of the energy of the laser to be absorbed by tissue. The impact of the artifact may also be reduced by using signal processing to subtract the artifact where the amplitude is similar to that of the action potential signal, thereby reducing the area obscured by the artifact. Alternatively, a different optical set up with the optical pacing and optical mapping systems both below the sample may eliminate the artifact. Even without any of these measures, the size of the optical artifact is small and allows optical mapping to be performed during optical pacing (Fig. 4).
While optical mapping has previously been used with embryonic hearts by a several groups in rodents [4,12,13,15], zebrafish [17], and avians [12,14,16,18], none have paced early embryonic hearts with point stimulation. Lacking a connection to the vasculature, excised hearts beat slower than hearts in the embryo, and many electrical parameters, such as APD and conduction velocity, vary with heart rate [9,10]. So, pacing is important for obtaining physiologically meaningful measures of cardiac electrophysiology. It also facilitates comparisons between hearts by eliminating heart rate as an uncontrolled variable. Here, we demonstrate that infrared stimulation enables optical mapping of optically paced hearts at a wide range of cardiac developmental stages and in two animal models. We obtained paced recordings from 2-day to 7-day quail hearts (HH 14-32), with examples of 2-day looping heart and 5-day septating hearts shown in Fig. 4. For the 2-day heart, traces were taken from the atrioventricular junction rather than the atrium because the atrium is slightly out of focus as it is not in the same plane as the rest of the heart tube. These results show that while increasing heart rate does not have a significant effect on the activation pattern (Fig. 4, center-right), APD90 (Fig. 4, right) of both hearts was shorter when paced compared to unpaced.
We further demonstrate two examples of rate-dependent parameters that can be studied in embryos with optical pacing. The first example is rate-dependent APD90, shown in Fig. 5(A)-5(C) for a 7-day heart, where APD90 decreases with increasing heart rate, similar to that shown in Fig. 4. This rate-dependent effect on APD has been previously shown in adult cardiac tissues [9], but this is the first time it has been shown in developing hearts. Abnormal changes to APD90 relative to pacing rate can indicate increased susceptibility to arrhythmias or abnormal expression of potassium channels. The second example is rate-dependent conduction block (Fig. 5(D)-5(E)), a condition where action potentials stop propagating past a region of the heart. The 2-day heart shows normal conduction when paced at 2.00 Hz, but when the pacing rate is increased to 2.50 Hz, well above the unpaced rate of 1.45 Hz, there is 2:1 conduction block between the ventricle and the outflow tract, where only every other action potential propagates into the outflow tract, resulting in the frequency in the outflow tract being half that of the ventricle. Clinically, conduction block is usually located between the atria and the ventricles, known as heart block, and can be induced by pacing in both children and adults [46,47]. In the developing heart, maternal autoimmune disease and congenital heart defects can lead to heart block and affects 1 in 22,000 live births in the United States [48]. By pacing, we can screen animal models with congenital heart disease for rate-dependent heart block or possibly create an animal model to study congenital heart block.
The work presented here is of a system that enables a wide range of electrophysiological studies in embryonic hearts using optical techniques. During optical pacing, we did not observe any functional damage from heat deposition. In the future, we will determine how long high-precision optical pacing can be sustained without tissue damage, as we are interested in exploring the possibility of creating abnormal embryonic phenotypes by altering the hemodynamics of the heart with optical pacing. We are also building a system around an inverted microscope where both the imaging and pacing will be performed simultaneously from below the sample to remove the artifact, allowing recordings immediately surrounding the pacing site to be obtained, enabling additional measurements, including conduction anisotropy. Using combined optical mapping and optical pacing, we are studying a variety of disease models to determine when and how the electrical activity of the developing heart differs from that of normal development, and then correlating the electrical changes with that of structure, mechanical function, and molecular markers.
In conclusion, we have developed a combined optical mapping and high-precision optical pacing system that enables physiologically relevant studies of cardiac electrophysiology in excised embryonic hearts. Optical pacing by infrared stimulation enables control of the heart rate in small samples simultaneously with optical mapping recordings that is impractical with either electrode or optogenetic techniques. Optical pacing allows maintenance of in vitro tissues at physiological heart rates and enables studies of frequency-dependent parameters. The combination of optical mapping with optical pacing provides a powerful tool for studying cardiac electrical development and will enable a better understanding of normal and abnormal development when combined with existing methods of studying structure, mechanical function, and molecular markers.
Acknowledgments
This research was supported by the Coulter-Case Translational Research Partnership and the National Heart, Lung, and Blood Institute of the National Institutes of Health (R21-HL115373, R01-HL083048, and R01-HL095717)
References and links
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