KO and AC are supported by a grant and a fellowship from the American Heart Association. SIB and RB are supported by grants from NIH.

<a href="https://www.jove.com/files/ftp_upload/1435/SOHA_overview.pdf">Click here to view an overview of the semi automated heartbeat analysis (SOHA) procedure.</a>
Semi-automated optical heartbeat analysis
1. Preprocess -
This step provides information on the diastolic and systolic heart diameters.
Heart edges are identified and marked during maximum diastole and systole. Optical recordings can be advanced at slow speeds and even one frame at a time allowing precise identification of the frames where maximal contraction and relaxation of the heart occur. One pair of marks identifying the diastolic edges and one pair identifying the systolic edges are made. Marks can also be made in duplicate at different horizontal locations along the heart.
The heart diameters obtained in this step are also used to calculate an additional measurement that provides an estimate of the contractility of the heart. This measure is the percent fractional shortening (%FS) and it represents the extent to which the heart edges move toward each other during a contraction. The formula used to calculate %FS is:
(((Diastolic diameter - Systolic diameter)/Diastolic diameter) x 100)
Note - The points identified in this set are used only for diameter and fractional shortening measurements and are not used for movement detection.
Example: In Drosophila, point mutations in myosin heavy chain's transducer domain, a region known to modulate the motor's enzymatic activity, result in hearts that are dilated or restricted depending upon whether they suppress or enhance myosin's ATPase activity (Cammarato, et al. 2008). These types of measurements cannot be made in intact flies as it is usually not possible to identify heart edges along the entire length of the heart tube through the cuticle and the fat, (compare Wildtype, D45 and Mhc5 M-modes in "M-mode Figure" below) which normally surround the heart. Measurements made when visualizing the heart in intact flies with other commonly employed methods are usually restricted to the most anterior portion of the heart, the conical chamber region. It is also not possible to obtain this dynamic measurement in fixed tissue.
2. Movement Detection-
This step is done automatically by the program and uses two different algorithms to analyze movement in each movie. Briefly, the first algorithm, the "Average Frame Darkness" algorithm uses a frame by frame approach. This algorithm averages the darkness intensity for an entire movie frame, normalizes the value to an interval between 0 and 1, and plots the normalized average for every frame in the movie over time. The second approach, the "Pixel by Pixel" algorithm, analyzes the change in darkness of all the individual pixels from one frame to the next. Pixels that exhibit intensity changes above a preset threshold are summed for each frame and plotted for every frame in the movie over time.
3. Check Intervals-
The output from the two movement detection algorithms is displayed in this module with the "Average Darkness" output displayed in a window above the "Pixel by pixel" output window. In order to be sure that the detection intervals agree with movement in the actual movie we display an edge-trace or M-mode made from the frames being analyzed directly below the algorithm output windows. The M-mode is made by digitally cutting slices one pixel wide through the heart from each frame in the movie and aligning them horizontally to provide a snapshot of the heart edge movements over time. In the "Pixel by pixel" window the identified diastolic intervals are indicated by a horizontal green line with the number of frames contributing to that interval directly above. Systolic intervals are identified as the interval between two successive diastoles. The beginning of a systole is indicated in the M-mode by a vertical blue line and the end is indicated by a vertical red line. With this display it is relatively easy to see if the algorithm output agrees with the actual heart movements.
Although movement detection is performed automatically the output must be checked for accuracy. There are several reasons for this; one is that there is often an underlying, slow wave fluctuation in light levels not related to the heart movement. This can usually be eliminated by use of a built in high pass filter. A second reason has to do with the sensitivity of the "Pixel by pixel" approach. The output from this algorithm is typically two movement traces, one for the contraction movement and one for the relaxation movement. In older flies and in some mutants the length of time between these two movements becomes relatively long and the program tries to interpret the two movements as two separate beats. This can be corrected by asking the program to compare the output of the Pixel by Pixel algorithm with that of the Average Frame Darkness algorithm, by clicking on the "Use darkness" box.
Example (refer to Video): In 5 to 7 week old flies the systolic intervals are typically much longer than in young flies. These long intervals are initially detected as two separate contractions separated by a short diastole. This can be corrected by selecting the "Use darkness" check box. In this case the program will compare the diastolic interval identified by the Pixel by Pixel algorithm with the Average Frame Darkness output. If the Average Frame Darkness output has a positive value, then any diastolic interval identified in the Pixel by Pixel algorithm will be ignored and the intervals will be correctly specified. The entire movie can be scanned in 10 second segments and accepted (click "Data OK") or rejected (click "Discard Data"). The Hi and Lo pass filter sensitivities can be manually adjusted to help more accurately define contraction intervals when employing the Darkness algorithm.
4. Output Statistics-
Once the output for a given movie has been accepted the program will use that information to automatically calculate a number of parameters. These include diastolic and systolic interval, heart period and heart rate, direction and velocity of the contraction wave, heart diameters, and %FS. In addition, the program has multiple methods for quantifying arrhythmicity reflecting non-uniform contraction and relaxation cycles (see Fink et al, 2009). Statistical output is provided for each movie individually and for the entire data set in the form of a comma separated value (.csv) file which can be opened in spreadsheet programs such as Microsoft Excel.
Table 1- 
<img src="/files/ftp_upload/1435/1435table1.jpg" alt="table1" /> An example of the output generated by the Semi-automated Optical Heart Analysis program.
5. Update Histograms- 
This portion of the program displays all of the data on heart intervals for a dataset in histogram format. Since heart rates are variable we normalize the histogram output data. However, because the distribution of the intervals is not symmetric around a mean we use the median interval value for our normalization.
Example (refer to Video): Flies that are mutant for the KCNQ potassium channel show extremely irregular diastolic and systolic intervals. Typically the systolic intervals are much longer and more irregular than in age-matched wildtype flies. This can clearly be seen by displaying the data in histogram format (see also Ocorr et al. 2007).
6. M-mode-
This module generates M-modes that can be made from any position along the heart in the movie frame. The length of the M-mode can also be specified. M-modes are useful to show qualitatively what the heart is doing in a movie.
Example (refer to Video): Movie of a 3 day old zebrafish larval heart showing the anterior ventricle that is used to generate an M-mode. The vertical slice of pixels that will be electronically excised from each movie frame is indicated by a red line.
M-mode Figure: Examples of M-modes from Drosophila and zebrafish. One of the myosin mutations discussed in the Preprocess step results in a molecular motor with faster enzyme kinetics resulting in heart restriction and occasionally a conduction block. Both parameters can be shown qualitatively using M-modes. <img src="/files/ftp_upload/1435/1435fig1.jpg" alt="M-modes Examples" /> M-modes made in the same portion of the heart in both wildtype and mutant flies clearly show the restricted phenotype in the Mhc5 mutant and the dilated diameters and arrhythmic contraction cycles of the D45 mutant (which expresses myosin with depressed enzyme kinetics) (modified from Cammarato et. al, 2008).
M-mode made from a Zebrafish larval heart showing the anterior ventricle (same heart movie as shown in the demonstration, Fink et.al. 2009).
7. Red Dot Movie-
This module automatically produces a slowed (1:4), 20 second version of an analyzed movie showing all the pixels that are identified as changing in red. This feature is primarily used for illustration purposes.
Movie 1 - A "red dot movie" showing the pixels that are identified as having darkness intensity changes by the program algorithms in red. The movie speed is slowed from the original by a factor of 4. <a href="https://www.jove.com/files/ftp_upload/1435/redmove.avi">Click here to download Movie 1.</a>
8. Additional Applications-
We have begun to apply this analytical system to high speed movies of other models with small hearts which are often difficult to analyze using traditional methodology. We have successfully applied this analysis to both zebrafish larval hearts and embryonic mouse hearts (Fink et al, 2009).
Movie 2 - Mouse heart movie plus figure with check intervals with data output. <a href="https://www.jove.com/files/ftp_upload/1435/Mouse%20heart%20clip%201.avi">Click here to download Movie 2.</a> <img src="/files/ftp_upload/1435/1435fig2.jpg" alt="Check Mouse Heart Intervals" />

<ol>
	<li>Cammarato, A., Dambacher, C. M., Reedy, M. C., Knowles, A. F., Kronert, W. A., Bodmer, R., Ocorr, K., Bernstein, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+Transducer+mutations+differentially+affect+motor+function,+myofibril+structure,+and+the+performance+of+skeletal+and+cardiac+muscles.">Myosin Transducer mutations differentially affect motor function, myofibril structure, and the performance of skeletal and cardiac muscles.</a> Mol Biol Cell. 19 (2), 553-562 (2008).</li><li>Ocorr, K., Reeves, N., Wessells, R. J., Fink, M., Chen, H. -. S. V., Akasaka, T., Yasuda, S., Metzger, J., Giles, W., Posakony, J. W., Bodmer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KCNQ+potassium+channel+mutations+cause+cardiac+arrhythmias+in+Drosophila+that+mimic+the+effects+of+aging.">KCNQ potassium channel mutations cause cardiac arrhythmias in Drosophila that mimic the effects of aging.</a> Proc Natl Acad Sci U S A. 104, 3943-3948 (2007).</li><li>Fink, M., Callol-Massot, C., Chu, A., Ruiz-Lozano, P., Izpisua Belmonte, J. C., Giles, W., Bodmer, R., Ocorr, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+detection+and+quantification+of+heartbeat+parameters+in+Drosophila,+zebrafish+and+embryonic+mouse+hearts.">A new method for the detection and quantification of heartbeat parameters in Drosophila, zebrafish and embryonic mouse hearts.</a> Biotechniques. 46, 101-113 (2009).</li></ol>

The authors have nothing to disclose.

The Drosophila model has proven to be a powerful genetic tool that has been used to address a variety of scientific questions ranging from embryological development to learning and memory. Recently this versatile model organism has been used to investigate the genetics of heart function. A number of attempts to quantify heart physiology in adult Drosophila have relied on observations made in intact flies through the abdominal cuticle. Most of these approaches depend upon visual observation or recordings of changes in li...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>MatLab software</td>
<td>&nbsp;</td>
<td>Math Works, Inc</td>
<td>&nbsp;</td>
<td>Required environment for the analysis software.</td>
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<td>EM-CCD digital camera</td>
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<td>Hamamatsu Corp.</td>
<td>9100 or 9300</td>
<td>Other high speed digital cameras will also work.</td>
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<td>HC image data capture software</td>
<td>&nbsp;</td>
<td>Hamamatsu Corp.</td>
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<td>Other image capture software that produces movies in avi format will also work.</td>
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<td>Light Microscope with 10x objective </td>
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<td>Leica</td>
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<td>A dipping lens that eliminates the air water interface greatly improves resolution</td>
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semi-automated optical heartbeat analysis of small hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

Watch this Scientific Journal Video about Semi-automated Optical Heartbeat Analysis of Small Hearts at JoVE.com

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

semi-automated-optical-heartbeat-analysis-of-small-hearts

Research

JoVE Journal

Biology

12.1K visualizações.  The Sanford Burnham Institute for Medical Research. Nós desenvolvemos um método semi-automático de análise Optical Heartbeat (SOHA) para analisar as gravações ópticas de alta velocidade a partir de Drosophila, Peixe-zebra e corações embrionárias de camundongos. Nós demonstramos a aplicação de nossa metodologia para a análise da função cardíaca na mosca da fruta e corações embrionárias de camundongos.

Vídeo: Semi-automated Optical Heartbeat Analysis of Small Hearts

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

Bioengineering

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

Developing Drosophila melanogaster Models for Imaging and Optogenetic Control of Cardiac Function

Using Drosophila melanogaster (fruit fly) as a model organism has ensured significant progress in many areas of biological science, from cellular organization and genomic investigations to behavioral studies. Due to the accumulated scientific knowledge, in recent years, Drosophila was brought to the field of modeling human diseases, including heart disorders. The presented work describes the experimental system for monitoring and manipulating the heart function in the context of a whole live organism using red light (617 nm) and without invasive procedures. Control over the heart was achieved using optogenetic tools. Optogenetics combines the expression of light-sensitive transgenic opsins and their optical activation to regulate the biological tissue of interest. In this work, a custom integrated optical coherence tomography (OCT) imaging and optogenetic stimulation system was used to visualize and modulate the functioning D. melanogaster heart at the 3rd instar larval and early pupal developmental stages. The UAS/GAL4 dual genetic system was employed to express halorhodopsin (eNpHR2.0) and red-shifted channelrhodopsin (ReaChR), specifically in the fly heart. Details on preparing D. melanogaster for live OCT imaging and optogenetic pacing are provided. A lab-developed integration software processed the imaging data to create visual presentations and quantitative characteristics of Drosophila heart function. The results demonstrate the feasibility of initiating cardiac arrest and bradycardia caused by eNpHR2.0 activation and performing heart pacing upon ReaChR activation.

Developing Drosophila melanogaster Models for Imaging and Optogenetic Control of Cardiac Function

The present protocol describes the generation of Drosophila melanogaster expressing eNpHR2.0 or ReaChR opsins in the heart for OCT imaging and optogenetic heart pacing. Detailed instructions for Drosophila OCT imaging and heart beating modulation, including the simulation of restorable heart arrest, bradycardia, and tachycardia in live animals at different developmental stages, are reported.

Using Drosophila melanogaster (fruit fly) as a model organism has ensured significant progress in many areas of biological science, from cellular ...

Light-Sheet Microscopy and Tissue Clearing for Comprehensive Cardiac Imaging

Light-Sheet Imaging to Reveal Cardiac Structure in Rodent Hearts

Light-sheet microscopy (LSM) plays a pivotal role in comprehending the intricate three-dimensional (3D) structure of the heart, providing crucial insights into fundamental cardiac physiology and pathologic responses. We hereby delve into the development and implementation of the LSM technique to elucidate the micro-architecture of the heart in mouse models. The methodology integrates a customized LSM system with tissue clearing techniques, mitigating light scattering within cardiac tissues for volumetric imaging. The combination of conventional LSM with image stitching and multiview deconvolution approaches allows for the capture of the entire heart. To address the inherent trade-off between axial resolution and field of view (FOV), we further introduce an axially swept light-sheet microscopy (ASLM) method to minimize out-of-focus light and uniformly illuminate the heart across the propagation direction. In the meanwhile, tissue clearing methods such as iDISCO enhance light penetration, facilitating the visualization of deep structures and ensuring a comprehensive examination of the myocardium throughout the entire heart. The combination of the proposed LSM and tissue clearing methods presents a promising platform for researchers in resolving cardiac structures in rodent hearts, holding great potential for the understanding of cardiac morphogenesis and remodeling.

Author Spotlight: Customized Light-Sheet Imaging for Investigating Myocardial Structures in Rodent Hearts

The protocol utilizes advanced light-sheet microscopy along with adapted tissue clearing methods to investigate intricate cardiac structures in rodent hearts, holding great potential for the understanding of cardiac morphogenesis and remodeling.

Light-sheet microscopy (LSM) plays a pivotal role in comprehending the intricate three-dimensional (3D) structure of the heart, providing crucial insights ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

Medicine

Advanced Cardiac Rhythm Management by Applying Optogenetic Multi-Site Photostimulation in Murine Hearts

Ventricular tachyarrhythmias are a major cause of mortality and morbidity worldwide. Electrical defibrillation using high-energy electric shocks is currently the only treatment for life-threatening ventricular fibrillation. However, defibrillation may have side-effects, including intolerable pain, tissue damage, and worsening of prognosis, indicating a significant medical need for the development of more gentle cardiac rhythm management strategies. Besides energy-reducing electrical approaches, cardiac optogenetics was introduced as a powerful tool to influence cardiac activity using light-sensitive membrane ion channels and light pulses. In the present study, a robust and valid method for successful photostimulation of Langendorff perfused intact murine hearts will be described based on multi-site pacing applying a 3 x 3 array of micro light-emitting diodes (micro-LED). Simultaneous optical mapping of epicardial membrane voltage waves allows the investigation of the effects of region-specific stimulation and evaluates the newly induced cardiac activity directly on-site. The obtained results show that the efficacy of defibrillation is strongly dependent on the parameters chosen for photostimulation during a cardiac arrhythmia. It will be demonstrated that the illuminated area of the heart plays a crucial role for termination success as well as how the targeted control of cardiac activity during illumination for modifying arrhythmia patterns can be achieved. In summary, this technique provides a possibility to optimize the on-site mechanism manipulation on the way to real-time feedback control of cardiac rhythm and, regarding the region specificity, new approaches in reducing the potential harm to the cardiac system compared to the usage of non-specific electrical shock applications.

This work reports a method for controlling the cardiac rhythm of intact murine hearts of transgenic channelrhodopsin-2 (ChR2) mice using local photostimulation with a micro-LED array and simultaneous optical mapping of epicardial membrane potential.

Ventricular tachyarrhythmias are a major cause of mortality and morbidity worldwide. Electrical defibrillation using high-energy electric shocks is ...

Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts

In the heart, molecular signaling studies are usually performed in isolated myocytes. However, many pathological situations such as ischemia and arrhythmias can only be fully understood at the whole organ level. Here, we present the spectroscopic technique of local field fluorescence microscopy (LFFM) that allows the measurement of cellular signals in the intact heart. The technique is based on a combination of a Langendorff perfused heart and optical fibers to record fluorescent signals. LFFM has various applications in the field of cardiovascular physiology to study the heart under normal and pathological conditions. Multiple cardiac variables can be monitored using different fluorescent indicators. These include cytosolic [Ca2+], intra-sarcoplasmic reticulum [Ca2+] and membrane potentials. The exogenous fluorescent probes are excited and the emitted fluorescence detected with three different arrangements of LFFM epifluorescence techniques presented in this paper. The central differences among these techniques are the type of light source used for excitation and on the way the excitation light is modulated. The pulsed LFFM (PLFFM) uses laser light pulses while continuous wave LFFM (CLFFM) uses continuous laser light for excitation. Finally, light-emitting diodes (LEDs) were used as a third light source. This non-coherent arrangement is called pulsed LED fluorescence microscopy (PLEDFM).

In the heart, molecular events coordinate the electrical and contractile function of the organ. A set of local field fluorescence microscopy techniques presented here enables the recording of cellular variables in intact hearts. Identifying mechanisms defining the cardiac function is critical in understanding how the heart works under pathological situations.

In the heart, molecular signaling studies are usually performed in isolated myocytes. However, many pathological situations such as ischemia and ...

Mesoscopic Optical Imaging of Whole Mouse Heart

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition (fibrosis) and cellular misalignment, can affect electrical conduction, introduce electromechanical dysfunctions and, eventually, lead to arrhythmia. Current predictive models of these functional alterations are based on non-integrated and low-resolution structural information. Placing this framework on a different order of magnitude is challenging due to the inefficacy of standard imaging methods in performing high-resolution imaging in massive tissue. In this work, we describe a methodological framework that allows imaging of whole mouse hearts with micrometric resolution. The achievement of this goal has required a technological effort where advances in tissue transformation and imaging methods have been combined. First, we describe an optimized CLARITY protocol capable of transforming an intact heart into a nanoporous, hydrogel-hybridized, lipid-free form that allows high transparency and deep staining. Then, a fluorescence light-sheet microscope able to rapidly acquire images of a mesoscopic field of view (mm-scale) with the micron-scale resolution is described. Following the mesoSPIM project, the conceived microscope allows the reconstruction of the whole mouse heart with micrometric resolution in a single tomographic scan. We believe that this methodological framework will allow clarifying the involvement of the cytoarchitecture disarray in the electrical dysfunctions and pave the way for a comprehensive model that considers both the functional and structural data, thus enabling a unified investigation of the structural causes that lead to electrical and mechanical alterations after the tissue remodeling.

We report a method for mesoscopic reconstruction of the whole mouse heart by combining new advancements in tissue transformation and staining with the development of an axially scanned light-sheet microscope.

Both genetic and non-genetic cardiac diseases can cause severe remodeling processes in the heart. Structural remodeling, such as collagen deposition ...