This work was funded in part by the National Natural Science Foundation of China (81970265, 81900281,81700288), the China Postdoctoral Science Foundation (2019M651904); and the National Key Research and Development Program of China (2016YFC1101001, 2017YFC1308105).

Information regarding commercial reagents and instruments are listed in the Table of Materials.
The AC16 human cardiomyocyte cell line was derived from the fusion of primary cells from adult ventricular heart tissue with SV40-transformed human fibroblasts17, which were purchased from BLUEFBIO (Shanghai, China). The cell line develops many biochemical and morphological features characteristic of cardiomyocytes. In addition, the cell line is widely used to evaluate myocardial damage and myocardial function in vitro18,19.
1. Cell culture
NOTE: The basal culture medium consists of serum-free Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM), 10% fetal bovine serum (FBS), 1% cardiac myocyte growth supplement, and 1% penicillin/streptomycin solution. Store the medium at 4 &#176;C and prewarm to 37 &#176;C before use.
<ol>
	<li>Remove the cryopreserved human cardiomyocyte (HCM) cells from liquid nitrogen and thaw them in a water bath at 37&#8201;&#176;C.</li>
	<li>Gently shake the vial (&#60;1 minute) in a 37 &#176;C water bath until only a small piece of ice remains in the vial.</li>
	<li>Transfer the vial into a sterile laminar flow hood. Wipe the outside of the vial with a cotton ball dipped in 70% alcohol.</li>
	<li>Transfer 4 mL of prewarmed complete growth medium dropwise into the centrifuge tube containing the thawed cells.</li>
	<li>Centrifuge the cell suspension at 200 &#215; g for 10 minutes. After centrifugation, discard the supernatant and resuspend the pellet in 5 mL of complete medium.</li>
	<li>Maintain the cells at 37&#8201;&#176;C in a humidified incubator under an atmosphere with 95% air and 5% CO2. 
	NOTE: Before harvesting the cells for experiments, the cells are allowed to grow until reaching approximately 60-70% confluency.</li>
</ol>
2. Establishment of an oxygen-glucose deprivation (OGD) model
NOTE: Two hours before the study period, replace the growth medium with serum-free medium, and the cells were reincubated in a humidified incubator for 2 h at 37 &#176;C under an atmosphere with 5% CO2.
<ol>
	<li>Aspirate the medium from a 6-well plate and gently wash the cells three times with phosphate buffered saline (PBS).</li>
	<li>Add 2 mL of fresh sugar-free DMEM per well.</li>
	<li>Culture the cells at a constant temperature in a three-gas incubator under an atmosphere with a mixture of 95% N2, 5% CO2, and 0.1% O2 at 37 &#176;C for 1, 2, 4, 8, 12, or 16 h.</li>
	<li>After the hypoxia treatment, aspirate the medium from the 6-well plate and wash the cells with PBS (pH 7.4) three times.</li>
	<li>Add 2 mL of complete DMEM per well.</li>
	<li>Maintain the cells at 37&#8201;&#176;C in a humidified incubator under an atmosphere with 95% air and 5% CO2.</li>
</ol>
3. Time-temperature protocol
NOTE: A standard time-temperature protocol is used during cardiac surgery, as described previously by others20,21. Treat HCMs according to the following protocol (Figure 1): timepoint 1 (T1) indicates the end of induction, timepoint 2 (T2) indicates the end of maintenance and timepoint 3 (T3) indicates the end of rewarming. Analyze control cells maintained under continuous normothermic conditions (37 &#176;C). The temperature conditions are created using a tri-gas incubator, which allows precise temperature regulation.
<ol>
	<li>Two hours before the experiments, aspirate the culture medium from a 6-well plate and add 2 mL of fresh serum-free DMEM per well.</li>
	<li>Re-incubate the cells in a humidified incubator for 2 h at 37 &#176;C under an atmosphere with 5% CO2.</li>
	<li>After 2 h, aspirate the medium from the 6-well plate and wash the cells with PBS (pH 7.4) three times.</li>
	<li>Add 2 mL of fresh serum-free DMEM per well.</li>
	<li>Reincubate the cells in the tri-gas incubator. 
	NOTE: Replace the medium to remove unattached cells and debris.</li>
	<li>Immediately change the temperature by placing the culture dishes a tri-gas incubator.</li>
	<li>After 1&#8201;h of cooling, quickly aspirate the medium from the 6-well plate and add 2 mL of fresh sugar-free DMEM per well.</li>
	<li>Culture the cells in a tri-gas incubator under an atmosphere comprising 95% N2, 5% CO2, and 0.1% O2 at 37 &#176;C for 12 h to establish hypoxia.</li>
	<li>Set the temperature as described below. 
	NOTE: The protocol starts with 10 h of a low-temperature treatment, followed by a rewarming phase for 2 h up to 37 &#176;C, and 24 h of normothermia (37 &#176;C). At every time point, three Petri dishes are removed for analysis.</li>
	<li>After low-temperature treatment, aspirate the medium from the 6-well plate and wash with PBS (pH 7.4) three times.</li>
	<li>Add 2 mL of complete DMEM per well.</li>
	<li>Maintain the cells at 37&#8201;&#176;C in a humidified incubator under an atmosphere with 95% air and 5% CO2.</li>
</ol>
4. CCK-8 viability assay
<ol>
	<li>Warm the 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) solution and PBS to 37 &#176;C prior to use.</li>
	<li>Aspirate the medium, rinse the cells with 1 mL of PBS and then add 0.5 mL of 0.25% trypsin along the wall of the well. Incubate at 37 &#176;C until almost all HCMs are detached (approximately 1 min).</li>
	<li>Add 1 mL of DMEM complete medium to the wells to neutralize the trypsin.</li>
	<li>Transfer the cell suspension to a 15 mL centrifuge tube and pellet the HCMs by centrifugation at 500 &#215; g for 3 min. Aspirate the supernatant without disturbing the pellet.</li>
	<li>Dispense 100 &#181;L aliquots of the cell suspension (5000 cells/well) into a 96-well plate. Preincubate the plate for 24 h in a humidified incubator (at 37 &#176;C.)</li>
	<li>Use the cultured cells in the 96-well plates to generate different treatment groups.</li>
	<li>Incubate the plate for an appropriate length of time (16 h) in an incubator.</li>
	<li>Add 10 &#181;L of CCK-8 solution to each well of the plate.</li>
	<li>Incubate the plate for 1 hour in the incubator.</li>
	<li>Measure the absorbance at 450 nm using a microplate reader. 
	CAUTION: Be careful not to introduce bubbles to the wells, as they interfere with OD reading measurements.</li>
</ol>
5. Flow cytometry for apoptosis analysis
<ol>
	<li>Perform trypsinization and centrifugation steps by following steps 4.2-4.4. 
	NOTE: Cells are harvested with trypsin without EDTA. To assess apoptosis, both floating and adherent cells are collected.</li>
	<li>Centrifuge the cells for 5&#8201;min at 1000 &#215; g. Discard the supernatant and resuspend the pellet in 1 mL of PBS.</li>
	<li>Count the cells using a hemocytometer. Using a pipette, transfer 100 &#181;L of Trypan Blue-treated cell suspension to a hemocytometer. Using a hand tally counter, count the live, unstained cells in one set of 16 squares and then use PBS to generate a cell suspension at 1&#215;107 cells/ml.</li>
	<li>Obtain 200&#8201;&#181;L of the cell suspension (5&#8201;&#215;&#8201;105 -1&#8201;&#215; 106 cells).</li>
	<li>Centrifuge for 5&#8201;min at 1000 &#215; g and resuspend the pellet in Annexin V-FITC binding solution.</li>
	<li>Add 5&#8201;&#181;L of Annexin V-FITC to dye the cells.</li>
	<li>Add 10 &#181;L of propidium iodide into the cell suspension.</li>
	<li>Gently mix the cells and incubate them for 20 minutes at room temperature in the dark. 
	NOTE: The cells are resuspended 2-3 times during the incubation to improve staining.</li>
	<li>Start the flow cytometer and make sure the software is working appropriately.</li>
	<li>Open two dot plot windows in the flow cytometry software.</li>
	<li>Select forward scattered light (FSC) on the X axis and side scattered light (SSC) on the Y axis to exclude cell debris and/or clumps in terms of their size and granularity, respectively.</li>
	<li>Select the PE (590 mm) detection channel and FITC (530 mm), which is used to measure the fluorescence intensity of the cells.</li>
	<li>Place the blank (HCMs that have not been dyed) sample tube on the flow cytometer.</li>
	<li>Click Record to collect particles from the suspension in the blank sample tube and then gate the cell population for further analysis in the first dot plot.</li>
	<li>Place the single-stained samples on the tube support arm. Click Record to collect particles from the suspension and then gate the cell population for further analysis in the first dot plot.</li>
	<li>Collect the HCMs in other sample tubes and optimize the measurement by adjusting the voltages of the fluorescence channels. 
	NOTE: Unstained and single-stained samples are used as compensation controls during the experiment.</li>
	<li>Display the statistics of each sample tube and calculate the rate of apoptosis of each sample.</li>
</ol>
6. Mitochondrial depolarization assessment
<ol>
	<li>Perform trypsinization and centrifugation by following steps 4.2-4.4.</li>
	<li>Resuspend the cells in 500 &#181;L of complete medium, and then adjust the cell density adjusted to 1&#8201;&#215; 105 - 6&#8201;&#215; 106 cells</li>
	<li>Add 0.5 mL of JC-1 working solution to each tube.</li>
	<li>Incubate the cells in a cell incubator at 37 &#176;C for 20 minutes. 
	NOTE: During the incubation period, add 1 mL of 5&#215; JC-1 staining buffer to 4 mL of distilled water to prepare the JC-1 staining buffer.</li>
	<li>After incubation, centrifuge the cells at 600 &#215; g for 3 min at 4 &#176;C.</li>
	<li>Discard the supernatant and resuspend the pellet in 1 mL of JC-1 staining buffer.</li>
	<li>Repeat steps 6.5 to 6.6 three times.</li>
	<li>Resuspend the HCMs in 1 mL of ice-cold staining buffer in a 1.5 mL centrifuge tube and use the cells for analysis within 30 min.</li>
	<li>Select the PE (590 nm) detection channel and FITC (530 nm) to measure the fluorescence intensity of JC-1 dye in the cells. 
	NOTE: Set up the flow cytometer by following steps 5.10-5.17.</li>
</ol>
7. Reactive oxygen species assay
<ol>
	<li>Perform trypsinization and centrifugation by following steps 4.2-4.4.</li>
	<li>Stain the cells in culture medium with 10 &#181;M DCFDA and adjust the cell density to 1&#8201;&#215;&#8201;106 - 1&#8201;&#215; 107 cells</li>
	<li>Incubate for 30 minutes at 37 &#176;C.</li>
	<li>Gently pipette the cells up/down every 3-5 min.</li>
	<li>After the incubation, wash the cells three times with serum-free cell culture medium.</li>
	<li>Analyze the cells on a flow cytometer by following steps 5.10-5.17. 
	NOTE: DCF is excited at 488 nm and the emission intensity measured at 530 nm.</li>
</ol>
8. Measurement of Caspase 3/ Caspase 8 Activity
<ol>
	<li>Perform trypsinization by following steps 4.2-4.4.</li>
	<li>Collect the cells by centrifugation at 600 &#215; g at 4 &#176;C for 5 minutes.</li>
	<li>Add 100 &#181;L of lysate buffer per 2 &#215; 106 cells.</li>
	<li>Lyse the cells for 15 minutes on ice.</li>
	<li>After incubating for 15 min in an ice bath, centrifuge the sample at 1.6 &#215; 104 x g at 4 &#176;C for 15 minutes.</li>
	<li>Transfer the supernatant to an ice-cold centrifuge tube for use. 
	NOTE: The protein concentration in the sample should be at least 1-3 mg/mL.</li>
	<li>Remove Ac-DEVD-pNA (2 mM) and place it on an ice bath for use.</li>
	<li>Accurately add 40 &#181;L of buffer solution to the enzyme-labeled plate, add 80 &#181;L of the sample, and finally add 10 &#181;L of Ac-DEVD-pNA (2 mM).</li>
	<li>Incubate the sample at 37 &#176;C for 120 minutes.</li>
	<li>Measured the A405 value on a microplate reader according to the manufacturer&#39;s instructions. 
	NOTE: The absorbance produced by the pNA generated by caspase-3/caspase-8 is calculated by subtracting the A405 value of the blank control from that of the sample.</li>
</ol>

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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothermia-induced+spatially+discordant+action+potential+duration+alternans+and+arrhythmogenesis+in+nonhibernating+versus+hibernating+mammals.">Hypothermia-induced spatially discordant action potential duration alternans and arrhythmogenesis in nonhibernating versus hibernating mammals.</a> AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY. 303 (8), 1035-1046 (2012).</li><li>Bobi, 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=Moderate+Hypothermia+Modifies+Coronary+Hemodynamics+and+Endothelium-Dependent+Vasodilation+in+a+Porcine+Model+of+Temperature+Management.">Moderate Hypothermia Modifies Coronary Hemodynamics and Endothelium-Dependent Vasodilation in a Porcine Model of Temperature Management.</a> Journal of the American Heart Association. 9 (3), 014035 (2020).</li><li>Dietrichs, E. S., Tveita, T., Myles, R., Smith, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+ECG-biomarker+for+cardiac+arrest+during+hypothermia.">A novel ECG-biomarker for cardiac arrest during hypothermia.</a> Scandinavian Journal of Trauma Resuscitation &amp; Emergency Medicine. 28 (1), 27 (2020).</li></ol>

The authors have nothing to disclose.

The complexities of intact animals, including the interactions between different types of cells, often prevent detailed studies of specific components of I/R injury. Therefore, it is necessary to establish an in vitro cell model that can accurately reflect the molecular changes after ischemia in vivo. Research on OGD models has been previously reported13,22, and many sophisticated methods have been established23,<sup class="xref...

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery1,2. During nonpulsatile low flow perfusion and periods of total circulatory arrest, damage involving all types of heart cells still occurs. In particular, the sensitivity of cardiomyocytes to ischemic injury is higher than that of other cell populations. At present, therapeutic hypothermia (TH) affords substantial protection against an expected ischemic insult in patients undergoing cardiac surgery3,4. TH is defined as a core body temperature of 14-34 &#176;C, although no consensus exists regarding a definition of cooling during cardiac surgery5,6,7. In 2013, an international panel of experts proposed a standardized reporting system to classify various temperature ranges of systemic hypothermic circulatory arrest8. Based on electroencephalography and metabolism studies of the brain, they divided hypothermia into four levels: profound hypothermia (&#8804; 14 &#176;C), deep hypothermia (14.1-20 &#176;C), moderate hypothermia (20.1-28 &#176;C), and mild hypothermia (28.1-34 &#176;C). The expert consensus provided a clear and uniform classification, allowing studies to be more comparable and provide more clinically relevant outcomes. This protection afforded by TH is based on its capacity to reduce the metabolic activity of cells, further limiting their rate of high-energy phosphates consumption9,10. However, the role of TH in myocardial protection is controversial and may have multiple effects depending on the degree of hypothermia.
Myocardial I/R is well known to be accompanied by increased cell apoptisis11. Recent reports have observed that programmed cardiomyocyte death increases during open-heart surgery, and may coincide with necrosis, thereby increasing the number of dead myocardial cells12. Therefore, reducing cardiomyocyte apoptosis is a useful therapeutic approach in clinical practice. In the mouse atrial HL-1 cardiomyocyte model, therapeutic hypothermia was shown to reduce the mitochondrial release of cytochrome c and apoptosis-inducing factor (AIF) during reperfusion13. However, the effect of temperature in regulating apoptosis is controversial and appears to depend on the degree of hypothermia. Cooper and colleagues observed that compared to a normothermic cardiopulmonary bypass control group, the apoptosis rate of myocardial tissue from pigs with the deep hypothermic circulatory arrest was increased14. In addition, the results of some studies have suggested that deep hypothermia may activate the apoptosis pathway, while less aggressive hypothermia appears to inhibit the pathway12,15,16. The reason for this result may be due to confounding effects associated with ischemic injury and a lack of understanding of the mechanisms by which temperature affects myocardial tissue. Therefore, the temperature limits at which apoptosis is enhanced or attenuated should be accurately defined.
To gain a better understanding of the mechanisms associated with the efficacy of hypothermia and provide a rational basis for its implementation in humans, it is essential to identify a culture condition similar to in vivo conditions that can produce damage similar to that observed for the clinical condition in a reproducible manner. An essential step towards achieving this goal is to establish the optimal conditions for inducing cardiomyocyte apoptosis. Accordingly, in the present study, we explored the methodological details regarding oxygen-glucose deprivation experiments with cultured cells, a facile in vitro model of ischemia-reperfusion. Furthermore, we evaluated the effect of different hypoxic-ischemic times on cardiomyocyte apoptosis, and verified our hypothesis regarding the effect of different temperature conditions on cell apoptosis in vitro.

<table>
	<tbody>
		<tr>
			<td>Annexin V-FITC cell apoptosis detection kit</td>
			<td>Bio-Technology,China</td>
			<td>C1062M</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardiac myocyte growth supplement</td>
			<td>Sciencell,USA</td>
			<td>6252</td>
			<td></td>
		</tr>
		<tr>
			<td>Caspase 3 activity assay kit</td>
			<td>Bio-Technology,China</td>
			<td>C1115</td>
			<td></td>
		</tr>
		<tr>
			<td>Caspase 8 activity assay kit</td>
			<td>Bio-Technology,China</td>
			<td>C1151</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, no glucose</td>
			<td>Gibco,USA</td>
			<td>11966025</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified eagle medium</td>
			<td>Gibco,USA</td>
			<td>11960044</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco,USA</td>
			<td>16140071</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometry</td>
			<td>CytoFLEX,USA</td>
			<td>B49007AF</td>
			<td></td>
		</tr>
		<tr>
			<td>Human myocardial cells</td>
			<td>BLUEFBIO,China</td>
			<td>BFN60808678</td>
			<td></td>
		</tr>
		<tr>
			<td>Mitochondrial membrane potential assay kit with JC-1</td>
			<td>Bio-Technology,China</td>
			<td>C2006</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin solution</td>
			<td>Gibco,USA</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>Reactive oxygen species assay kit</td>
			<td>Bio-Technology,China</td>
			<td>S0033S</td>
			<td></td>
		</tr>
		<tr>
			<td>Three-gas incubator</td>
			<td>Memmert,Germany</td>
			<td>ICO50</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Gibco,USA</td>
			<td>25200056</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro assessment of myocardial protection following hypothermia-preconditioning in a human cardiac myocytes model

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of cardiomyocytes to ischemic injury is higher than that of other cell populations. At present, hypothermia affords considerable protection against an expected ischemic insult. However, investigations into complex hypothermia-induced molecular changes remain limited. Therefore, it is essential to identify a culture condition similar to in vivo conditions that can induce damage similar to that observed in the clinical condition in a reproducible manner. To mimic ischemia-like conditions in vitro, the cells in these models were treated by oxygen/glucose deprivation (OGD). In addition, we applied a standard time-temperature protocol used during cardiac surgery. Furthermore, we propose an approach to use a simple but comprehensive method for the quantitative analysis of myocardial injury. Apoptosis and expression levels of apoptosis-associated proteins were assessed by flow cytometry and using an ELISA kit. In this model, we tested a hypothesis regarding the effects of different temperature conditions on cardiomyocyte apoptosis in vitro. The reliability of this model depends on strict temperature control, controllable experimental procedures, and stable experimental results. Additionally, this model can be used to study the molecular mechanism of hypothermic cardioprotection, which may have important implications for the development of complementary therapies for use with hypothermia.

The effect of OGD exposure on the viability of HCMs was determined by CCK-8 assay. Compared with that observed in the control group, cell viability was significantly decreased in a time-dependent manner (Figure 2A). The apoptosis rates of HCMs at different times after reperfusion showed a specific trend, where from 0 to 16 h, the apoptosis rates gradually increased and reached the maximum rate at the 16 h time point (Figure 2B). As OGD for 12 h reduced cell acti...

Watch this Scientific Journal Video about In vitro Assessment of Myocardial Protection following Hypothermia-Preconditioning in a Human Cardiac Myocytes Model at JoVE.com

In vitro Assessment of Myocardial Protection following Hypothermia-Preconditioning in a Human Cardiac Myocytes Model

The distinct effects of different degrees of hypothermia on myocardial protection have not been thoroughly evaluated. The goal of the present study was to quantify the levels of cell death following different hypothermia treatments in a human cardiomyocyte-based model, laying the foundation for future in-depth molecular research.

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of ...

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2.9K Views.  Children's Hospital of Nanjing Medical University. The distinct effects of different degrees of hypothermia on myocardial protection have not been thoroughly evaluated. The goal of the present study was to quantify the levels of cell death following different hypothermia treatments in a human cardiomyocyte-based model, laying the foundation for future in-depth molecular research.

Video: In vitro Assessment of Myocardial Protection following Hypothermia-Preconditioning in a Human Cardiac Myocytes Model

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery (SALLI) MRI in Rats

Small animal magnetic resonance imaging is an important tool to study cardiac function and changes in myocardial tissue. The high heart rates of small animals (200 to 600 beats/min) have previously limited the role of CMR imaging. Small animal Look-Locker inversion recovery (SALLI) is a T1 mapping sequence for small animals to overcome this problem 1. T1 maps provide quantitative information about tissue alterations and contrast agent kinetics. It is also possible to detect diffuse myocardial processes such as interstitial fibrosis or edema 1-6. Furthermore, from a single set of image data, it is possible to examine heart function and myocardial scarring by generating cine and inversion recovery-prepared late gadolinium enhancement-type MR images 1.
	The presented video shows step-by-step the procedures to perform small animal CMR imaging. Here it is presented with a healthy Sprague-Dawley rat, however naturally it can be extended to different cardiac small animal models.

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery SALLI MRI in Rats

Contrast enhanced cardiac magnetic resonance (CMR) imaging allows comprehensive in vivo assessment of the heart in small animal models of cardiovascular disease. Here we detail the procedures to perform CMR imaging, reconstruction and analysis using Small Animal Lock-Locker Inversion Recovery (SALLI) in rats.

Small animal magnetic resonance  imaging is an important tool to study cardiac function and changes in myocardial  tissue. The high heart rates of small ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment elevated myocardial infarction. However, reperfusion per se can result in further cardiomyocyte death, a phenomenon known as reperfusion injury. The opening of the mitochondrial permeability transition pore (mPTP), with the decrease of the mitochondrial membrane potential (MMP), or mitochondrial depolarization, is universally recognized as the final step of reperfusion injury and is responsible for mitochondrial and cardiomyocyte death. JC-1 is a lipophilic cationic dye that accumulates in mitochondria depending on the value of MMP. The higher the MMP is, the more JC-1 accumulates in the mitochondria. The increasing amounts of JC-1 in mitochondria can be reflected by a fluorescence emission shift from green (~530 nm) to red (~590 nm). Therefore, the reduction of the red/green fluorescence intensity ratio can indicate the depolarization of mitochondria. Here, we take advantage of JC-1 to measure MMP, or the opening of mPTP in human cardiac myocytes after hypoxia/reoxygenation, detected by flow cytometry.

Here, we present a protocol that uses JC-1 dye to assess the mitochondrial membrane potential of cells after being exposed to hypoxia/reoxygenation with or without a protective agent.

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment ...

Implantation of hiPSC-derived Cardiac-muscle Patches after Myocardial Injury in a Guinea Pig Model

Due to the limited regeneration capacity of the heart in adult mammals, myocardial infarction results in an irreversible loss of cardiomyocytes. This loss of relevant amounts of heart muscle mass can lead to the heart failure. Besides heart transplantation, there is no curative treatment option for the end-stage heart failure. In times of organ donor shortage, organ independent treatment modalities are needed. Left-ventricular assist devices are a promising therapy option, however, especially as destination therapy, limited by its side-effects like stroke, infections and bleedings. In recent years, several cardiac repair strategies including stem cell injection, cardiac progenitors or myocardial tissue engineering have been investigated. Recent improvements in cell biology allow for the differentiation of large amounts of cardiomyocytes derived from human induced pluripotent stem cells (iPSC). One of the cardiac repair strategies currently under evaluation is to transplant artificial heart tissue. Engineered heart tissue (EHT) is a three-dimensional in vitro created cardiomyocyte network, with functional properties of native heart tissue. We have created EHT-patches from hiPSC derived cardiomyocytes. Here we present a protocol for the induction of left ventricular myocardial cryoinjury in a guinea pig, followed by implantation of hiPSC derived EHT on the left ventricular wall.

Here we present a protocol for the induction of left ventricular cryoinjury followed by the implantation of a cardiac muscle patch, derived from human iPS-cell cardiomyocytes in a guinea pig model.

Due to the limited regeneration capacity of the heart in adult mammals, myocardial infarction results in an irreversible loss of cardiomyocytes. This loss ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca2+)-containing solutions to determine, amongst others: maximum force development, myofilament Ca2+-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.

This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...