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&#039;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...

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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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Medicine

3.0K Views.  Children's Hospital of Nanjing Medical University. De verschillende effecten van verschillende graden van onderkoeling op myocardbescherming zijn niet grondig geëvalueerd. Het doel van deze studie was om de niveaus van celdood na verschillende hypothermiebehandelingen te kwantificeren in een op humane cardiomyocyten gebaseerd model, dat de basis legde voor toekomstig diepgaand moleculair onderzoek.

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

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

Murine Isolated Heart Model of Myocardial Stunning Associated with Cardioplegic Arrest

The following protocol is of use to evaluate impaired cardiac function or myocardial stunning following moderate ischemic insults. The technique is useful for modeling ischemic injury associated with numerous clinically relevant phenomenon including cardiac surgery with cardioplegic arrest and cardiopulmonary bypass, off-pump CABG, transplant, angina, brief ischemia, etc. The protocol presents a general method to model hypothermic hyperkalemic cardioplegic arrest and reperfusion in rodent hearts focusing on measurement of myocardial contractile function. In brief, a mouse heart is perfused in langendorff mode, instrumented with an intraventricular balloon, and baseline cardiac functional parameters are recorded. Following stabilization, the heart is then subject to brief infusion of a cardioprotective hypothermic cardioplegia solution to initiate diastolic arrest. Cardioplegia is delivered intermittently over 2 hr. The heart is then reperfused and warmed to normothermic temperatures and recovery of myocardial function is monitored. Use of this protocol results in reliable depressed cardiac contractile function free from gross myocardial tissue damage in rodents.

The goal of this protocol to assess myocardial stunning following ischemic cardioplegic arrest in rodents.

The following protocol is of use to evaluate impaired cardiac function or myocardial stunning following moderate ischemic insults. The technique is useful ...

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 Recovery Cardiopulmonary Bypass Model Without Transfusion or Inotropic Agents in Rats

Cardiopulmonary bypass (CPB) is indispensable in cardiovascular surgery. Despite the dramatic refinement of CPB technique and devices, multi-organ complications related to prolonged CPB still compromise the outcome of cardiovascular surgeries, and may worsen postoperative morbidity and mortality. Animal models recapitulating the clinical usage of CPB enable the clarification of the pathophysiological processes that occur during CPB, and facilitate pre-clinical studies to develop strategies protecting against these complications. Rat CPB models are advantageous because of their greater cost-effectiveness, convenient experimental processes, abundant testing methods at the genetic or protein levels, and genetic consistency. They can be used for investigating the immune system activation and synthesis of proinflammatory cytokines, compliment activation, and production of oxygen free radicals. The rat models have been refined and have gradually taken the place of large-animal models. Here, we describe a simple CPB model without transfusion and/or inotropic agents in a rat. This recovery model allows the study of the long-term multiple organ sequelae of CPB.

Here, we present a protocol to describe a simple recovery cardiopulmonary bypass model without transfusion or inotropic agents in a rat. This model allows the study of the long-term multiple organ sequelae of cardiopulmonary bypass.

Cardiopulmonary bypass (CPB) is indispensable in cardiovascular surgery. Despite the dramatic refinement of CPB technique and devices, multi-organ ...

Immunology and Infection

Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering is crucial for basic science cardiac research; in vitro human cell culture eliminates the interspecies differences of animal models, while a more tissue-like 3D environment (e.g., with extracellular matrix and heterocellular coupling) simulates in vivo conditions to a greater extent than traditional two-dimensional culture on plastic Petri dishes. However, each model system requires specialized equipment, for example, custom-designed bioreactors and functional assessment devices. Additionally, these protocols are often complicated, labor-intensive, and plagued by the failure of the small, delicate tissues.
This paper describes a process for generating a robust human engineered cardiac tissue (hECT) model system using induced pluripotent stem-cell-derived cardiomyocytes for the longitudinal measurement of tissue function. Six hECTs with linear strip geometry are cultured in parallel, with each hECT&#160;suspended from a pair of force-sensing polydimethylsiloxane (PDMS) posts attached to PDMS racks. Each post is capped with a black PDMS stable post tracker (SPoT), a new feature that improves the ease of use, throughput, tissue retention, and data quality. The shape allows for the reliable optical tracking of post deflections, yielding improved twitch force tracings with absolute active and passive tension. The cap geometry eliminates tissue failure due to hECTs slipping off the posts, and as they involve a second step after PDMS rack fabrication, the SPoTs can be added to existing PDMS post-based designs without major changes to the bioreactor fabrication process.
The system is used to demonstrate the importance of measuring hECT function at physiological temperatures and shows stable tissue function during data acquisition. In summary, we describe a state-of-the-art model system that reproduces key physiological conditions to advance the biofidelity, efficiency, and rigor of engineered cardiac tissues for in vitro applications.

Three-dimensional cardiac tissues bioengineered using stem-cell-derived cardiomyocytes have emerged as promising models for studying healthy and diseased human myocardium in vitro while recapitulating key aspects of the native cardiac niche. This manuscript describes a protocol for fabricating and analyzing high-content engineered cardiac tissues generated from human induced pluripotent stem-cell-derived cardiomyocytes.

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering ...

Bioengineering

Pre-clinical Model of Cardiac Donation after Circulatory Death

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after circulatory death (DCD) is a solution to address this limited availability, but due to a period of prolonged warm ischemia and the risk of tissue injury, its routine use in cardiac transplantation is seldom seen. In this manuscript we provide a detailed protocol closely mimicking current clinical practices in the context of DCD with continuous monitoring of heart function, allowing for the evaluation of novel cardioprotective strategies and interventions to decrease ischemia-reperfusion injury.
In this model, the DCD protocol is initiated in anesthetized Lewis rats by stopping ventilation to induce circulatory death. When systolic blood pressure drops below 30 mmHg, the warm ischemic time is initiated. After a pre-set warm ischemic period, hearts are flushed with a normothermic cardioplegic solution, procured, and mounted onto a Langendorff ex vivo heart perfusion system. Following 10 min of initial reperfusion and stabilization, cardiac reconditioning is continuously evaluated for 60 min using intraventricular pressure monitoring. A heart injury is assessed by measuring cardiac troponin T and the infarct size is quantified by histological staining. The warm ischemic time can be modulated and tailored to develop the desired amount of structural and functional damage. This simple protocol allows for the evaluation of different cardioprotective conditioning strategies introduced at the moment of cardioplegia, initial reperfusion and/or during ex vivo perfusion. Findings obtained from this protocol can be reproduced in large models, facilitating clinical translation.

This protocol shows a simple and flexible approach for the evaluation of new conditioning agents or strategies to increase the feasibility of cardiac donation after circulatory death.

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

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 ...

Assessment of Ex Vivo Murine Biventricular Function in a Langendorff Model

Primary graft dysfunction (PGD) remains the leading cause of early death following heart transplantation. Prolonged ischemic time during cold preservation is an important risk factor for PGD, and reliable evaluation of cardiac function is essential to study the functional responses of the donor heart after cold preservation. The accompanying video describes a technique to assess murine right and left ventricular function using ex vivo perfusion based in a Langendorff model after cold preservation for different durations. In brief, the heart is isolated and stored in a cold histidine-tryptophan-ketoglutarate (HTK) solution. Then, the heart is perfused with a Kreb buffer in a Langendorff model for 60 min. A silicone balloon is inserted into the left and right ventricle, and cardiac functional parameters are recorded (dP/dt, pressure-volume relationships). This protocol allows the reliable evaluation of cardiac function after different heart preservation protocols. Importantly, this technique allows the study of cardiac preservation responses specifically in native cardiac cells. The use of very small murine hearts allows access to an enormous array of transgenic mice to investigate the mechanisms of PGD.

Assessment of Ex Vivo Murine Biventricular Function in a Langendorff Model

Presented here is a protocol to reliably quantify the right and left ventricular function of donor hearts after cold preservation using an ex vivo perfusion system.

Primary graft dysfunction (PGD) remains the leading cause of early death following heart transplantation. Prolonged ischemic time during cold preservation ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

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

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