The authors would like to thank Marion Kunze and Tina Althaus for their excellent technical support during experiments. The research leading to the results has received funding from the European Community&#39;s Seventh Framework Programme FP7/2007-2013 under grant agreement number HEALTH-F2-2009-241526. Support was also provided by the German Center for Cardiovascular Research, DZHK e.V. (Project MD28), partner site Goettingen, the German Research Foundation CRC 1002 (project C03), and the Max Planck Society. This work was partly supported by BrainLinks-BrainTools, Cluster of Excellence funded by the German Research Foundation (DFG, grant number EXC 1086).

All experiments strictly followed the animal welfare regulation, in agreement with German legislation, local stipulations, and in accordance with recommendations of the Federation of European Laboratory Animal Science Associations (FELASA). The application for approval of animal experiments has been approved by the responsible animal welfare authority, and all experiments were reported to our animal welfare representatives.
1. Experiment preparation and materials
<ol>
	<li>Optical mapping se...

<ol>
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A., Luther, S., Diaz-Maue, L., Richter, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy-reduced+arrhythmia+termination+using+global+photostimulation+in+optogenetic+murine+hearts.">Energy-reduced arrhythmia termination using global photostimulation in optogenetic murine hearts.</a> Frontiers in Physiology. 9 (1651), (2018).</li><li>Moreno, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LED+irradiance+pattern+at+short+distances.">LED irradiance pattern at short distances.</a> Applied Optics. 59 (1), 190-195 (2020).</li><li>Behrend, A., Bittihn, P., Luther, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+unpinning+success+rates+for+a+pinned+spiral+in+an+excitable+medium.">Predicting unpinning success rates for a pinned spiral in an excitable medium.</a> , 345-348 (2010).</li><li>Kappadan, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+optical+measurement+of+cardiac+restitution,+contraction,+and+fibrillation+dynamics+in+beating+vs.+blebbistatin-uncoupled+isolated+rabbit+hearts.">High-resolution optical measurement of cardiac restitution, contraction, and fibrillation dynamics in beating vs. blebbistatin-uncoupled isolated rabbit hearts.</a> Frontiers in Physiology. 11 (464), (2020).</li><li>Christoph, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electromechanical+vortex+filaments+during+cardiac+fibrillation.">Electromechanical vortex filaments during cardiac fibrillation.</a> Nature. 555, 667-672 (2018).</li><li>O'Shea, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+optogenetics+and+optical+mapping+-+Overcoming+spectral+congestion+in+all-optical+cardiac+electrophysiology.">Cardiac optogenetics and optical mapping - Overcoming spectral congestion in all-optical cardiac electrophysiology.</a> Frontiers in Physiology. 10 (182), (2019).</li><li>Aras, K. K., Faye, N. R., Cathey, B., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+volume+of+human+myocardium+necessary+to+maintain+ventricular+fibrillation.">Critical volume of human myocardium necessary to maintain ventricular fibrillation.</a> Circulation: Arrhythmia and Electrophysiology. 11 (11), e006692 (2018).</li><li>Trayanova, N., Doshi, A. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+panoramic+in+situ+mapping+of+action+potential+propagation+in+transgenic+hearts+to+investigate+initiation+and+therapeutic+control+of+arrhythmias.">Toward panoramic in situ mapping of action potential propagation in transgenic hearts to investigate initiation and therapeutic control of arrhythmias.</a> Frontiers in Physiology. 5, 337 (2014).</li><li>Crocini, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetics+design+of+mechanistically-based+stimulation+patterns+for+cardiac+defibrillation.">Optogenetics design of mechanistically-based stimulation patterns for cardiac defibrillation.</a> Science Reports. 6 (35628), (2016).</li><li>Nyns, E. C. 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A successful treatment of cardiac tachyarrhythmias is key to cardiac therapy. However, the biophysical mechanisms underlying arrhythmia initiation, perpetuation and termination are not fully understood. Therefore, cardiac research aims to optimize electrical shock therapy towards a more gentle termination of arrhythmias, thereby increasing the quality of life of patients28,29,30,31. Low energy ...

Early investigations of the spatial-temporal dynamics during arrhythmia revealed that the complex electrical patterns during cardiac fibrillation are driven by vortex-like rotating excitation waves1. This finding gave new insights into the underlying mechanisms of arrhythmias, which then led to the development of novel electrical termination therapies based on multi-site excitation of the myocardium2,3,4. However, treatments using electric field stimulation are non-local and may innervate all surrounding excitable cells, including muscle tissue, causing cellular and tissue damage, as well as intolerable pain. In contrast to electrical therapies, optogenetic approaches provide a specific and tissue-protective technique for evoking cardiomyocyte action potentials with high spatial and temporal precision. Therefore, optogenetic stimulation has the potential for minimal invasive control of the chaotic activation patterns during cardiac fibrillation.
The introduction of the light-sensitive ion channel channelrhodopsin-2 (ChR2) into excitable cells via genetic manipulation5,6,7, enabled the depolarization of the membrane potential of excitable cells using photostimulation. Several medical applications, including the activation of neuronal networks, the control of cardiac activity, the restoration of vision and hearing, the treatment of spinal cord injuries, and others8,9,10,11,12,13,14 have been developed. The application of ChR2 in cardiology has significant potential due to its millisecond response time15, making it well suited for the targeted control of arrhythmic cardiac dynamics.
In this study, multi-site photostimulation of intact hearts of a transgenic mouse model is shown. In summary, a transgenic alpha-MHC-ChR2 mouse line was established within the scope of the European Community&#39;s Seventh Framework Programme FP7/2007-2013 (HEALTH-F2-2009-241526) and kindly provided by Prof. S. E. Lehnart. In general, transgenic adult male C57/B6/J, expressing Cre-recombinase under control of alpha-MHC were paired to mate with female B6.Cg-Gt(ROSA)26Sortm27.1(CAG-COP4*H134R/tdTomato)Hye/J. Since the cardiac STOP cassette was deleted in the second-generation, the offspring showed a stable MHC-ChR2 expression and was used to maintain cardiac photosensitive colonies. All experiments were done with adult mice of both genders at an age of 36 - 48 weeks. The illumination is achieved using a 3 x 3 micro-LED array, fabricated as described in16,17 except that the silicon-based housing and the short optical glass fibers are not implemented. Its first usage in a cardiac application is found in18. A linear micro-LED array based on a similar fabrication technology has been applied as a penetrating probe for heart pacing19. The micro-LEDs are arranged in a 3 x 3 array at a pitch of 550 &#181;m, providing both a high spatial resolution and a high radiant power on a very small area. The authors demonstrate in this work a versatile local multi-site photostimulation that may open the path for developing novel anti-arrhythmic therapy methods.
The following experimental protocol involves a retrograde Langendorff perfusion ex vivo, for which the cannulated aorta functions as perfusion inlet. Due to the applied perfusion pressure and the cardiac contraction the perfusate is flowing through the coronary arteries, which branch off the aorta. In the presented work, the heart is perfused using a constant pressure setup achieved by elevating the perfusate reservoirs to 1 m height, equivalent to 73.2 mmHg, which yields to a flow rate of 2.633 &#177; 0.583 mL/min. Two kinds of Tyrode&#39;s solution are used as perfusate during the experiment. Regular Tyrode&#39;s solution supports a stable sinus rhythm, whereas Low-K+ Tyrode&#39;s solution is mixed with Pinacidil to enable the induction of arrhythmia in murine hearts. The usage of a hexagonal water bath permits the observation of the heart through six different planar windows, allowing the coupling of several optical components with less distortion by refraction.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemical Components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blebbistatin</td>
			<td>TargetMol</td>
			<td>T6038</td>
			<td>10 mM stock solution</td>
		</tr>
		<tr>
			<td>BSA/Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A4919</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C1016</td>
			<td>CaCl2</td>
		</tr>
		<tr>
			<td>Carbogen</td>
			<td>Westfalen</td>
			<td></td>
			<td>50 l bottle</td>
		</tr>
		<tr>
			<td>DI-4-ANBDQPQ</td>
			<td>AAT Bioquest</td>
			<td>21499</td>
			<td>Dye for Optical Mapping</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D9434</td>
			<td>C6H12O6</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>LEO Pharma</td>
			<td></td>
			<td>Heparin-Natrium Leo 25.000 I.E./5 ml, available only on prescription</td>
		</tr>
		<tr>
			<td>Hydrochlorid Acid</td>
			<td>Merck</td>
			<td>1.09057.1000</td>
			<td>HCl, 1 M stock solution</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>CP Pharma</td>
			<td></td>
			<td>1 ml/ml, available only on prescription</td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Merck</td>
			<td>8.14733.0500</td>
			<td>MgCl2</td>
		</tr>
		<tr>
			<td>Monopotassium Phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>30407</td>
			<td>KH2PO4</td>
		</tr>
		<tr>
			<td>Pinacidil monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>P154-500mg</td>
			<td>10 mM stock solution</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P5405</td>
			<td>KCl</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td>NaHCO3</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td>NaCl</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Merck</td>
			<td>1.09137.1000</td>
			<td>NaOH, 1 M stock solution</td>
		</tr>
		<tr>
			<td>Electrical Setup</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biopac MP150</td>
			<td>Biopac Systems</td>
			<td>MP150WSW</td>
			<td>data acquisition and analysis system</td>
		</tr>
		<tr>
			<td>Custom-built ECG, alternative ECG100C</td>
			<td>Biopac Systems</td>
			<td>ECG100C</td>
			<td>Electrocardiogram Amplifier</td>
		</tr>
		<tr>
			<td>Custom-built water bath heater using heating cable</td>
			<td>RMS Heating System</td>
			<td>HK-5,0-12</td>
			<td>Heating cable 120W</td>
		</tr>
		<tr>
			<td>Hexagonal water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LED Driver Power supply</td>
			<td>Thorlabs</td>
			<td>KPS101</td>
			<td>15 V, 2.4 A Power Supply Unit with 3.5 mm Jack Connector for One K- or T-Cube.</td>
		</tr>
		<tr>
			<td>LEDD1B LED Driver</td>
			<td>Thorlabs</td>
			<td>LEDD1B</td>
			<td>T-Cube LED Driver, 1200 mA Max Drive Current</td>
		</tr>
		<tr>
			<td>MAP, ECG Electrode</td>
			<td>Hugo Sachs Elektronik</td>
			<td>BS4&#160;73-0200</td>
			<td>Mini-ECG Electrode for isoalted hearts</td>
		</tr>
		<tr>
			<td>micro-LED Driver e.g. AFG</td>
			<td>Agilent Instruments</td>
			<td>A-2230</td>
			<td>Arbitrary function generator (AFG)</td>
		</tr>
		<tr>
			<td>Signal Generator</td>
			<td>Agilent Instruments</td>
			<td>A-2230</td>
			<td>AFG</td>
		</tr>
		<tr>
			<td>micro-LED Array Components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxid glue</td>
			<td>Epoxy Technology</td>
			<td>EPO-TEK 353ND</td>
			<td>Two component epoxy</td>
		</tr>
		<tr>
			<td>Fluoropolymer</td>
			<td>&#160;Asahi Glass Co. Ltd.</td>
			<td>Cytop 809M</td>
			<td>Fluoropolymer with high transparency</td>
		</tr>
		<tr>
			<td>Image reversal photoresist</td>
			<td>Merck KGaA</td>
			<td>AZ 5214E</td>
			<td>Image Reversal Resist for High Resolution</td>
		</tr>
		<tr>
			<td>LED chip</td>
			<td>&#160;Cree Inc.</td>
			<td>C460TR2227-S2100</td>
			<td>Blue micro-LED</td>
		</tr>
		<tr>
			<td>Photoresist</td>
			<td>Merck KGaA</td>
			<td>AZ 9260</td>
			<td>Thick Positive Photoresists</td>
		</tr>
		<tr>
			<td>Polyimide</td>
			<td>UBE Industries Ltd.</td>
			<td>U-Varnish S</td>
			<td>Polyimide Solution</td>
		</tr>
		<tr>
			<td>Silicone</td>
			<td>NuSil Technology LLC</td>
			<td>MED-6215</td>
			<td>Low viscosity silicone elastomer</td>
		</tr>
		<tr>
			<td>Solvent free adhesive</td>
			<td>John P. Kummer GmbH</td>
			<td>Epo-Tek 301-2</td>
			<td>Epoxy resin with low viscosity</td>
		</tr>
		<tr>
			<td>Optical Mapping</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blue Filter</td>
			<td>Chroma Technology Corporation</td>
			<td>ET470/40x</td>
			<td>Blue excitation filter</td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Photometrics</td>
			<td>Cascade 128+</td>
			<td>High performance EMCCD Camera</td>
		</tr>
		<tr>
			<td>Camera Objective</td>
			<td>Navitar</td>
			<td>DO-5095</td>
			<td>Navitar high speed fixed focal length lenses work with CCD and CMOS cameras</td>
		</tr>
		<tr>
			<td>Dichroic Mirror</td>
			<td>Semrock</td>
			<td>FF685-Di02-25x36</td>
			<td>685 nm edge BrightLine&#174; single-edge standard epi-fluorescence dichroic beamsplitter</td>
		</tr>
		<tr>
			<td>Emmision Filter</td>
			<td>Semrock</td>
			<td>FF01-775/140-25</td>
			<td>775/140 nm BrightLine&#174; single-band bandpass filter</td>
		</tr>
		<tr>
			<td>Heatsink</td>
			<td>Advanced Thermal Solutions</td>
			<td>ATSEU-077A-C3-R0</td>
			<td>Heat Sinks - LED STAR LED Heatsink, 45mm dia., 68mm, Black/Silver, Unthreaded Baseplate Hardware</td>
		</tr>
		<tr>
			<td>LED 1 and LED 2</td>
			<td>LED Engin Osram</td>
			<td>LZ4-00B208</td>
			<td>High Power LEDs - Single Colour Blue, 460 nm 130 lm, 700mA</td>
		</tr>
		<tr>
			<td>LED 3</td>
			<td>Thorlabs</td>
			<td>M625L3</td>
			<td>625 nm, 700 mW (Min) Mounted LED, 1000 mA</td>
		</tr>
		<tr>
			<td>Lenses</td>
			<td>LED Engin Osram</td>
			<td>LLNF-2T06-H</td>
			<td>LED Lighting Lenses Assemblies LZ4 LENS NARROW FLOOD BEAM</td>
		</tr>
		<tr>
			<td>Photodiode for power meter</td>
			<td>Thorlabs</td>
			<td>S120VC</td>
			<td>Standard Photodiode Power Sensor</td>
		</tr>
		<tr>
			<td>Power Meter</td>
			<td>Thorlabs</td>
			<td>PM100D</td>
			<td>Compact Power and Energy Meter</td>
		</tr>
		<tr>
			<td>Red Filter</td>
			<td>Semrock</td>
			<td>FF02-628/40-25</td>
			<td>BrightLine&#174; single-band bandpass filter</td>
		</tr>
	</tbody>
</table>

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.

The protocol allows the induction of ventricular arrhythmias in intact murine hearts using photostimulation pulses generated by LED 1 and LED 2 (Figure 1) with a frequency find between 25 Hz and 35 Hz and a pulse duration Wind between 2 ms and 10 ms. Please notice that the aim of such rapid light pulses is not to capture the cardiac rhythm but rather to unbalance the cardiac activity so that erratic electrical waves can be generated, which then facilitate an arrhythmia....

Watch this Scientific Journal Video about Advanced Cardiac Rhythm Management by Applying Optogenetic Multi-Site Photostimulation in Murine Hearts at JoVE.com

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

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 ...

The physical most underlying cardiac fibrillation and defibrillation remain not completely understood. Optogenetics can offer a way to gain more insight into cardiac phenomena in a well-controlled environment. Local light stimulation using micro LED's permits the innovation of specific cardiac tissue areas.This targeted stimulation is a new tool to develop heart gentle anti arithmetic therapy methods. The shown protocol can be used to study heart behavior during cardiac arrhythmia and especially during termination. In prospective, the developed techniques can be used to study more clinically relevant large animal models.Before every experiment, clean all tubes with fully de-mineralized water. Aerate thyroid solutions with carbogen for 30 minutes at room temperature and adjust the pH to 7.3 with sodium hydroxide and hydrochloric acid. Heat the perfusion system to 37 degrees Celsius with a water heat pump.Keep the perfusate temperature constant within the water bath by using an additional heating element such as a waterproof heating cable. Add 500 milliliters of each thyroid solution to the corresponding reservoir and de-aerate the tubes as well as the bubble trap by running thyroid solution through the perfusion system until no more trapped air bubbles are seen in the tubes or in the bubble trap. Continue aerating the thyroid solutions during the whole experiment in the reservoirs with carbogen to ensure that the pH of the perfusate remains stable later during perfusion.After removing the heart, perform the fine preparation under a stereoscopic microscope. Attach the aorta onto the blunt needle and fix the vessel with suture material. As a control, inject ice cold thyroid solution through the needle into the heart, and check that the heart is tightly mounted.Transfer the mounted heart to the perfusion system and ensure that the perfusate is flowing to prevent air from entering the heart while connecting the needle with the bubble trap. Check that the heart is covered with thyroid solution in the water bath and that it is beating within a few minutes. Place one of the ECG electrodes as close as possible to the heart surface to ensure good signal quality.Suspend the second ECG electrode in the thyroid solution, making sure that the ECG is being recorded. Place the micro LED array on the area of interest. Change the perfusion to low potassium thyroid with penecidle and peruse the heart for 15 to 30 minutes.To induce arrhythmia, illuminate the heart with LED one and LED two with a train of 20 to 50 light pulses with a frequency of 25 to 35 hertz, pulse duration of two to 15 milliseconds, and light intensity of 2.8 milli watts per square millimeters. Repeat the process until arrhythmia is induced. Once a sustained arrhythmia is visually detected, apply a burst of pulses with different widths and frequencies using three, six, or nine micro LEDs of the array at a pulsing current of 15 milliamp piers.If the arrhythmia keeps going after five micro LED array based defibrillation trials, classify the attempt as unsuccessful and start backup defibrillation, using LED one and LED two with same timing parameters. Perfuse the heart with the blebbistatin solution and wait until mechanical uncoupling occurs, which is accomplished when the heart stops beating, but an ECG signal is still measurable. Give the one milliliter voltage dye, dye 4-A-N-B-D-Q-P-Q as a bolus in the bubble trap of the Langendorff perfusion and wait for five to 10 minutes to allow the dye to peruse the heart uniformly.Turn on LED three. Focus the camera on the heart's surface and apply 1.27 milli watts per square millimeter optical power. Turn off the laboratory lights and start recording, making sure that an optical signal is being acquired by comparing the frequency of the obtained signal to the frequency of the recorded ECG.A series of experiments with different frequencies, number of micro LED and pulse durations were tested for 11 mice, demonstrating that pulses of 1 to 20 milliseconds can defibrillate with different success rates. There is a significant increase in success rate of the nine LEDs with pulse durations of one millisecond and 20 milliseconds at a defibrillation frequency of 18 and 20 hertz, which is closer to the analyzed mean arrhythmia frequency of 22.55 of all the arrhythmias with an error interval of plus or minus four point zero three hertz. Two different defibrillation attempts with 14 hertz frequency with their ECG recordings and their respective spectrograms are shown here.In an example of defibrillation with 30 millisecond pulses, the dominant frequency of the arrhythmia slightly increases until photo stimulation begins. The VF is turned into a VT where the dominant frequency drops to 14 hertz followed by unsuccessful termination and return to arrhythmic behavior with a dominant frequency of 24 hertz. In the second example, segment one shows a VT with dominant frequency of 23 hertz and it's harmonic components until photo stimulation begins, with a pulse width of 20 milliseconds.Segment three displays a successful termination which leads to a normal sinus rhythm with a fundamental frequency of 3.5 hertz and the resulting harmonics. Optical mapping with high speed cameras showed a change of fluorescence intensity during a single beat of the heart during a normal sinus rhythm. The length of perfusion must maintain all conditions to keep the heart healthy, namely the temperature, the pH value, and the oxidation need to remain constant.It is particularly important that the tyroid solution do not run empty as air bubbles in the system may damage the heart. The micro reality array offers great flexibility. You can target different locations of the heart or even combine multiple arrays in one experiment in order to properly terminate arrhythmia.

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Research

JoVE Journal

Medicine

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of cardiovascular disease. Although molecular biology is necessary in identifying key changes in the signaling pathway, it is not a surrogate for functional significance. While physiology can provide answers to the question of function, combining physiology with biochemical assessment of metabolites in the intact, beating heart allows for a complete picture of cardiac function and energetics. For years, our laboratory has utilized isolated heart perfusions combined with nuclear magnetic resonance (NMR) spectroscopy to accomplish this task. Left ventricular function is assessed by Langendorff-mode isolated heart perfusions while cardiac energetics is measured by performing 31P magnetic resonance spectroscopy of the perfused hearts. With these techniques, indices of cardiac function in combination with levels of phosphocreatine and ATP can be measured simultaneously in beating hearts. Furthermore, these parameters can be monitored while physiologic or pathologic stressors are instituted. For example, ischemia/reperfusion or high workload challenge protocols can be adopted. The use of aortic banding or other models of cardiac pathology are apt as well. Regardless of the variants within the protocol, the functional and energetic significance of molecular modifications of transgenic mouse models can be adequately described, leading to new insights into the associated enzymatic and metabolic pathways. Therefore, 31P NMR spectroscopy in the isolated perfused heart is a valuable research technique in animal models of cardiovascular disease.

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of ...

Cardiac Stress Test Induced by Dobutamine and Monitored by Cardiac Catheterization in Mice

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the arteries (&beta;2). When systemically administered, it increases cardiac demand. Thus, dobutamine unmasks abnormal rhythm or ischemic areas potentially at risk of infarction. 
Monitoring of heart function during a cardiac stress test can be performed by either ecocardiography or cardiac catheterization. The latter is an invasive but more accurate and informative technique that the former.
Cardiac stress test induced by dobutamine and monitored by cardiac catheterization accomplished as described here allows, in a single experiment, the measurement of the following hemodynamic parameters: heart rate (HR), systolic pressure, diastolic pressure, end-diastolic pressure, maximal positive pressure development (dP/dtmax) and maximal negative pressure development (dP/dtmin), at baseline conditions and under increasing doses of dobutamine.
As expected, in normal mice we observed a dobutamine dose-related increase in HR, dP/dtmax and dP/dtmin. Moreover, at the highest dose tested (12 ng/g/min) the cardiac decompensation of high fat diet-induced obese mice was unmasked.

We describe the protocol to perform a cardiac stress test induced by dobutamine and monitored by cardiac catheterization in normal mice. Also we show its application to unmask subclinical cardiac disease in high fat diet-induced obese mice.

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the ...

Direct Pressure Monitoring Accurately Predicts Pulmonary Vein Occlusion During Cryoballoon Ablation

Cryoballoon ablation (CBA) is an established therapy for atrial fibrillation (AF). Pulmonary vein (PV) occlusion is essential for achieving antral contact and PV isolation and is typically assessed by contrast injection. We present a novel method of direct pressure monitoring for assessment of PV occlusion.
Transcatheter pressure is monitored during balloon advancement to the PV antrum. Pressure is recorded via a single pressure transducer connected to the inner lumen of the cryoballoon. Pressure curve characteristics are used to assess occlusion in conjunction with fluoroscopic or intracardiac echocardiography (ICE) guidance. PV occlusion is confirmed when loss of typical left atrial (LA) pressure waveform is observed with recordings of PA pressure characteristics (no A wave and rapid V wave upstroke). Complete pulmonary vein occlusion as assessed with this technique has been confirmed with concurrent contrast utilization during the initial testing of the technique and has been shown to be highly accurate and readily reproducible.
We evaluated the efficacy of this novel technique in 35 patients. A total of 128 veins were assessed for occlusion with the cryoballoon utilizing the pressure monitoring technique; occlusive pressure was demonstrated in 113 veins with resultant successful pulmonary vein isolation in 111 veins (98.2%). Occlusion was confirmed with subsequent contrast injection during the initial ten procedures, after which contrast utilization was rapidly reduced or eliminated given the highly accurate identification of occlusive pressure waveform with limited initial training.
Verification of PV occlusive pressure during CBA is a novel approach to assessing effective PV occlusion and it accurately predicts electrical isolation. Utilization of this method results in significant decrease in fluoroscopy time and volume of contrast.

Effective pulmonary vein isolation utilizing a cryoballoon depends on complete pulmonary vein occlusion. The point of occlusion can be effectively predicted by direct analysis of pulmonary vein pressure waveform analysis during balloon inflation using a simple and reproducible technique.

Cryoballoon  ablation (CBA) is an established therapy for atrial fibrillation (AF).  Pulmonary vein (PV) occlusion is essential for achieving antral ...

Intramyocardial Cell Delivery: Observations in Murine Hearts

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue revascularization and cell survival. In addition, further observations revealed that specific stem cells, such as cardiac stem cells, mesenchymal stem cells and cardiospheres have the ability to integrate within the surrounding myocardium by differentiating into cardiomyocytes, smooth muscle cells and endothelial cells.
Here, we present the materials and methods to reliably deliver noncontractile cells into the left ventricular wall of immunodepleted mice. The salient steps of this microsurgical procedure involve anesthesia and analgesia injection, intratracheal intubation, incision to open the chest and expose the heart and delivery of cells by a sterile 30-gauge needle and a precision microliter syringe.
Tissue processing consisting of heart harvesting, embedding, sectioning and histological staining showed that intramyocardial cell injection produced a small damage in the epicardial area, as well as in the ventricular wall. Noncontractile cells were retained into the myocardial wall of immunocompromised mice and were surrounded by a layer of fibrotic tissue, likely to protect from cardiac pressure and mechanical load.

Intramyocardial cell delivery in murine models of cardiovascular diseases, such as hypertension or myocardial infarction, is widely used to test the therapeutic potential of different cell types in regenerative studies. Therefore, a detailed description and a clear visualization of this surgical procedure will help to define the limits and advantages of cardiovascular cell therapeutic analyses in small rodents.

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue ...

EEG Mu Rhythm in Typical and Atypical Development

Electroencephalography (EEG) is an effective, efficient, and noninvasive method of assessing and recording brain activity. Given the excellent temporal resolution, EEG can be used to examine the neural response related to specific behaviors, states, or external stimuli. An example of this utility is the assessment of the mirror neuron system (MNS) in humans through the examination of the EEG mu rhythm. The EEG mu rhythm, oscillatory activity in the 8-12 Hz frequency range recorded from centrally located electrodes, is suppressed when an individual executes, or simply observes, goal directed actions. As such, it has been proposed to reflect activity of the MNS. It has been theorized that dysfunction in the mirror neuron system (MNS) plays a contributing role in the social deficits of autism spectrum disorder (ASD). The MNS can then be noninvasively examined in clinical populations by using EEG mu rhythm attenuation as an index for its activity. The described protocol provides an avenue to examine social cognitive functions theoretically linked to the MNS in individuals with typical and atypical development, such as ASD.&#160;

Assessment of the EEG mu rhythm provides a unique methodology for examining brain activity and when combined with behaviorally based assays, can be a powerful tool for elucidating aspects of social cognition, such as imitation, in clinical populations.

Electroencephalography (EEG) is an effective, efficient, and noninvasive method of assessing and recording brain activity. Given the excellent temporal ...

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and fluid retention. Changes in myocardial structure, electrical conduction, and energy metabolism develop with heart failure, leading to contractile dysfunction, increased risk of arrhythmias, and sudden death. Hypertensive heart disease is one of the key contributing factors of cardiac remodeling associated with heart failure. The most commonly-used animal model mimicking hypertensive heart disease is created via surgical interventions, such as by narrowing the aorta. Abdominal aortic constriction is a useful experimental technique to induce a pressure overload, which leads to heart failure. The surgery can be easily performed, without the need for chest opening or mechanical ventilation. Abdominal aortic constriction-induced cardiac pathology progresses gradually, making this model relevant to clinical hypertensive heart failure. Cardiac injury and remodeling can be observed 10 weeks after the surgery. The method described here provides a simple and effective approach to produce a hypertensive heart disease animal model that is suitable for studying disease mechanisms and for testing novel therapeutics.

A rat model of abdominal aortic constriction that induces cardiac hypertrophy and remodeling is described. An efficient, highly-reproducible, and minimally-invasive method is used to provide a simple yet useful platform for research in myocardial hypertrophy and dysfunction.

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and ...

Cardiac Magnetic Resonance Imaging at 7 Tesla

CMR at an ultra-high field (magnetic field strength B0&#160;&#8805; 7 Tesla) benefits from the signal-to-noise ratio (SNR) advantage inherent at higher magnetic field strengths and potentially provides improved signal contrast and spatial resolution. While promising results have been achieved, ultra-high field CMR is challenging due to energy deposition constraints and physical phenomena such as transmission field non-uniformities and magnetic field inhomogeneities. In addition, the magneto-hydrodynamic effect renders the synchronization of the data acquisition with the cardiac motion difficult. The challenges are currently addressed by explorations into novel magnetic resonance technology. If all impediments can be overcome, ultra-high field CMR may generate new opportunities for functional CMR, myocardial tissue characterization, microstructure imaging or metabolic imaging. Recognizing this potential, we show that multi-channel radio frequency (RF) coil technology tailored for CMR at 7 Tesla together with higher order B0 shimming and a backup signal for cardiac triggering facilitates high fidelity functional CMR. With the proposed setup, cardiac chamber quantification can be accomplished in examination times similar to those achieved at lower field strengths. To share this experience and to support the dissemination of this expertise, this work describes our setup and protocol tailored for functional CMR at 7 Tesla.

The sensitivity gain inherent to ultrahigh field magnetic resonance holds promise for high spatial resolution imaging of the heart. Here, we describe a protocol customized for functional cardiovascular magnetic resonance (CMR) at 7 Tesla using an advanced multi-channel radio-frequency coil, magnetic field shimming and a triggering concept.

CMR at an ultra-high field (magnetic field strength B0&#160;&#8805; 7 Tesla) benefits from the signal-to-noise ratio (SNR) advantage inherent at higher ...

Esophageal Heat Transfer for Patient Temperature Control and Targeted Temperature Management

Controlling patient temperature is important for a wide variety of clinical conditions. Cooling to normal or below normal body temperature is often performed for neuroprotection after ischemic insult (e.g. hemorrhagic stroke, subarachnoid hemorrhage, cardiac arrest, or other hypoxic injury). Cooling from febrile states treats fever and reduces the negative effects of hyperthermia on injured neurons. Patients are warmed in the operating room to prevent inadvertent perioperative hypothermia, which is known to cause increased blood loss, wound infections, and myocardial injury, while also prolonging recovery time. There are many reported approaches for temperature management, including improvised methods that repurpose standard supplies (e.g., ice, chilled saline, fans, blankets) but more sophisticated technologies designed for temperature management are typically more successful in delivering an optimized protocol. Over the last decade, advanced technologies have developed around two heat transfer methods: surface devices (water blankets, forced-air warmers) or intravascular devices (sterile catheters requiring vascular placement). Recently, a novel device became available that is placed in the esophagus, analogous to a standard orogastric tube, that provides efficient heat transfer through the patient&#39;s core. The device connects to existing heat exchange units to allow automatic patient temperature management via a servo mechanism, using patient temperature from standard temperature sensors (rectal, Foley, or other core temperature sensors) as the input variable. This approach eliminates vascular placement complications (deep venous thrombosis, central line associated bloodstream infection), reduces obstruction to patient access, and causes less shivering when compared to surface approaches. Published data have also shown a high degree of accuracy and maintenance of target temperature using the esophageal approach to temperature management. Therefore, the purpose of this method is to provide a low-risk alternative method for controlling patient temperature in critical care settings.

This study presents a novel method to provide efficient patient temperature control for cooling or warming patients. A single use, triple lumen device is placed into the esophagus, analogous to a standard orogastric tube, and connects to existing heat exchange units to perform automatic patient temperature management.

Controlling patient temperature is important for a wide variety of clinical conditions. Cooling to normal or below normal body temperature is often ...

Isolation and Purification of Murine Cardiac Pericytes

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier integrity. However, their tissue-specific functions in the heart are not well understood. Moreover, there is currently no protocol utilizing readily accessible materials to isolate and purify pericytes of cardiac origin. Our protocol focuses on using the widely used mammalian model, the mouse, as our source of cells. Using the enzymatic digestion and mechanical dissociation of heart tissue, we obtained a crude cell mixture that was further purified by fluorescence activating cell sorting (FACS) by a plethora of markers. Because there is no single unequivocal marker for pericytes, we gated for cells that were CD31-CD34-CD45-CD140b+NG2+CD146+. Following purification, these primary cells were cultured and passaged multiple times without any changes in morphology and marker expression. With the ability to regularly obtain primary murine cardiac pericytes using our protocol, we hope to further understand the role of pericytes in cardiovascular physiology and their therapeutic potential.

We have optimized a protocol to isolate and purify murine cardiac pericytes for basic research and investigation of their biology and therapeutic potential.

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier ...