The goal of the present study was to quantify the levels of cell death, following different hypothermia treatments in a human cardiomyocyte-based model. This model can be used to study the molecular mechanism of hypothermic cardioprotection, which may have important implications for the development of complimentary therapies for use with hypothermia. To culture the HCM cells, thaw the cryopreserved cells from liquid nitrogen in a 37-degree Celsius water bath and maintain them in a humidifier incubator under an atmosphere with 95%air and 5%carbon dioxide.
Once the cells reach 60 to 70%confluence, aspirate the medium from the six-well plate and gently wash the cells three times with PBS. Add two milliliters of fresh sugar-free medium into each well and culture the cells at a constant temperature in a three-gas incubator under a mixture of 95%nitrogen, 5%carbon dioxide, and 0.1%oxygen at 37 degrees Celsius. For the time-temperature protocol, aspirate the medium from the six-well plate and add two milliliters of fresh sugar-free medium.
Culture the cells in a tri-gas incubator for 12 hours to establish hypoxia. Set the temperature program starting with 10 hours of low temperature treatment, followed by a rewarming phase for two hours and 24 hours of normothermia. Remove three Petri dishes at every time point for analysis.
After the low temperature treatment, aspirate the medium from the six-well plate and wash three times with PBS. Add two milliliters of fresh medium to each well, then maintain the cells at 37 degrees Celsius in a humidified incubator under an atmosphere with 95%air and 5%carbon dioxide. Add eight microliters of CCK-8 solution to each well of the plate and leave the plate for one hour in the incubator.
After the incubation, measure the absorbance at 450 nanometers using a microplate reader. For apoptosis analysis, add five microliters of Annexin V-FITC to dye the cells. Then add 10 microliters of propidium iodide to the cell suspension.
Gently mix the cells and incubate them for 20 minutes at room temperature in the dark. In the flow cytometry software, open two dot plot windows and select forward scattered light on the x-axis and side scattered light on the y-axis. Select the PE detection channel and FITC.
Click Record to collect particles from the suspension in the blank sample tube, then gate the cell population for further analysis in the first dot plot. Next, place the single-stained samples on the tube support arm. Click Record to collect particles from the suspension and gate the cell population for further analysis.
For mitochondrial assessment, add 0.5 milliliters of JC-1 working solution to each tube containing the trypsinized cells and incubate them in a 37 degrees Celsius incubator for 20 minutes. After incubation, centrifuge the cells at 600 times g for three minutes at four degrees Celsius and resuspend the HCMs in one milliliter of ice-cold staining buffer in a 1.5-milliliter centrifuge tube. Measure the fluorescence intensity of JC-1 by selecting the PE and FITC detection channels.
For the reactive oxygen species assay, stain the cells and culture medium with 10 micromolar DCFDA and adjust the cell density to around one to 10 million cells. Incubate these cells for 30 minutes at 37 degrees Celsius, then analyze them on a flow cytometer. Add 40 microliters of buffer solution to the enzyme-labeled plate, then add 80 microliters of the sample.
Then add 10 microliters of two millimolar acetyl DEVDp-nitroanilide and incubate the plate at 37 degrees Celsius for 120 minutes. Measure the A405 value on a microplate reader machine using the manufacturer's instructions. The temperature conditions in the time-temperature protocol were created and maintained using a tri-gas incubator, which allows for precise temperature regulation at three time points.
The effect of oxygen and glucose deprivation, or OGD, on the viability of HCMs was determined using the CCK-8 assay. Compared with the control group, cell viability was significantly decreased in a time-dependent manner. The apoptosis rates of HCMs gradually increased between zero and 16 hours after reperfusion, then reached the maximum rate at the 16-hour time point.
Compared with the OGD group, the percentage of viable cells was higher in the three groups treated with hypothermia. In addition, cells in the deep hypothermia group had the highest viability, 2.1-fold higher than that observed in the OGD group. Flow cytometry results confirmed that hypothermia prevented the apoptosis of HCMs under OGD conditions.
Hypothermia treatment also decreased the intracellular ROS levels in HCMs. Mitochondrial membrane potential was detected with JC-1 staining. After OGD treatment, the red-to-green fluorescence ratio was decreased.
Hypothermia significantly inhibited this OGD-induced effect. Moreover, a decrease of caspase-8 and caspase-3 was observed in the cells that were treated with hypothermia. The reliability of this model depends on strict temperature control, controllable experimental procedures, and stable experimental results.
This method can be used to further explore the specific mechanism of hypothermic treatment on cell apoptosis and to study the molecular mechanism of hypothermic cardioprotection. It is of great significance for the development of hypothermic adjuvant therapy.
