Has not worked with cardiomyocytes, but with neurons. Have you considered to use combined oxygen and glucose deprivation - by using  glucose free media?  Add glucose at start of reoxygenation.

http://www.ncbi.nlm.nih.gov/pubmed/11809906

Thank you Runar for your suggestion, I have recently also tried using a glucose free media, and yet did not find reduction in the cell viability. I am wondering if I should change the viability staining method...

Have you tried MTT assay to test viability ?

http://www.ncbi.nlm.nih.gov/pubmed/12603826

Hi Runar, I haven't tried MTT assay. I saw in your paper that you used LDH and MTT assays to detect viability. I will consider the MTT assay as my next step.

Just a quick question about your 2002 paper, at what point did you add the antioxidant treatments, is it before or after the hypoxic conditions? thank you..

Good luck with MTT. I added antioxidants before hypoxia.

Hello. In fact hypoxia by itself has indeed little effect on cardiomyocytes. You must had substrate deletion  (a minimal reproduction of ischemia) to get significant cell injury. Do not add any additional ischemia component such as acidosis, which is protective, or hyperkaliemia. Be aware that disposable, plastic cell culture dish are not gas tight, and therefore oxygen and CO2 can diffuse trough dish wall! It is thus needed to check PO2 in cell culture medium by drawing microsample and analyse them with a blood gas analyser. LDH and MTT test are good way to appreciate cell injury, but there are very delayed markers : functional (electrical) dysfunctions appear far before. Long simulated ischemia (e.g. 18-24 h) is withour pathological pertinency : in these condition, LDH loss is maximal within 1 h, and cells died after 2h30. Reoxigenataion after at most 2 h lead to near normal functions within 30 min. See our bibliogarphy.

I have worked with isolated adult rat myocytes and I produce the hypoxia bubbling the solutions with a gas combination of nitrogen 95% and carbon dioxide 5%, for three minutes 

By adding COs in the gas mixture, you add acidosis, which is cardioprotective and not a pure hypoxia condition. By using hypoxia (via nitrogen flushing), associated to substarte deletion, CO2 production by the cells is sufficient to maintain pH at  7.34 in good air tight condition.

Thank you Pierre and Roxana for your suggestions. I have ordered a new tank of Nitrogen(95%) and CO2. I will try bubbling the media for some minutes before incubation in the hypoxic chamber.

Hi Pierre,

I understand that the plates are not airtight, but the chamber I'm using is sealed with the oxygen level in the chamber being monitored so we are sure we are getting the oxygen down. As for CO2, do you suggest that we exclude CO2 as well and just use pure nitrogen gas??

By bublling medium and thereafter incubate cells, you have a high risk of O2 enrichment. In a hypoxia ptotocol, culture dishes must be permanently in a N2 athmosphere.

About CO2, you need to check pH. You have to maintain CO2 in atmosphere only if the CO2 production by the cells is not suffiscient to buffer medium pH. This depends on the amont of cells, the medium volume and the dish size.

Hello, Bolanle. How have you been? I recently encountered the same conundrum as yours about hypoxia model in neonatal mouse cardiomyocytes. The cells are surely quiet resistant to death caused by hypoxia in my experiment. The protocol as following: 1. Bubbling the culture media in hypoxia chamber 24h before cells hypoxia. 2. Incubate cells in hypoxic chamber filled with 95% Nitrogen and 5% CO2 for 6hours and then reoxygenation in normoxic conditions for 24 hours.

However, the cells still beat happily.That made me crazy. So, have you resolved your problems? Looking forward to your reply.THANKS!

Probably your incubation box is not gas tight enough. Remember that gas diffuse across plastics! To control accuratly your experimental conditions, you must measure medium O2, CO2 and pH . This can be made with a hospital blood gas analyser. A full analysis requires only 50-100 ul of medium sample in rigourously air tight conditions. Analysis must be done within 20 min after sampling.

Thank you Pierre for your timely suggestions. Actually, I conducted my experiment in a specialized anoxic incubator rather than a simple incubation box. Pleased see the attached photo. So, there is less likely that the normoxic gases diffuse into the culture dishes. That is to say, the hypoxia conditions should be accurately under control unless the anoxic incubator can't work well.

Here is my brief protocol. 1. The cardiomyocytes are subjected to hungry pretreatment 24h without glucose and fetal bovine serum before hypoxia and the ischemia-mimetic solution are placed in the anoxic incubator 24h as well. The formula of ischemia-mimetic solution is as follows (in mmol/L, PH 6.6): 20 deoxyglucose, 125 NaCl, 8 KCl, 1.2 KH2PO4, 1.25 MgSO4, 1.2 CaCl2, 6.25 NaHCO3, 5 sodium lactate, 20 HEPES. 2. Place the culture plates within the anoxic incubator equilibrated with 5% CO2, 1% O2 and N2. After 6h of simulated ischemia, reperfusion was initiated 24h by buffer exchange to normoxic culture medium with 10% fetal bovine serum and incubation in 95% room air, 5% CO2.

However, the result isn’t ideal as the cells almost survive. I cannot see any why. Should I extend the time of hypoxia or what？ Thanks!

You must keep in mind that healthy cardiomyocytes are sensitive to substrate-free hypoxia within 15-20 min! 1/ Ca2+ concentration is too low! This is the amount for serum-enriched medium. Without serum, yo must rise Ca2+ to at least 2 mM, otherwise, the beating are weakened, and hypoxia effects delayed. 2/ Hepes is a bad and toxic pH buffer; change to a full bicarbonate buffered medium, and maintain 5 % CO2 in the gas (5 % CO2 is too much with Hepes); the result is that the pH is far too low, and acidosis is protective! 3/ The substrate deprivation is far too early; this may promote the production of heart shock proteins , which are protective! Good luck! Pierre

Hi, Pierre. Thanks for your valuable suggestions. The formula of ischemia-mimetic solution adopted in my experiment is from an article entitled “Histone Deacetylase Inhibition Blunts Ischemia/Reperfusion Injury by Inducing Cardiomyocyte Autophagy”. I just cannot repeat their results，which is so weird. I may need adjust the formula according to your suggestion. Furthermore, there is one more thing I don't understand. Like you said, the substrate deletion is far too early. Is that mean I should reduce the time of hungry pretreatment before hypoxia, like 6h, 12h rather than 24h？ Many thanks.

Substrate deletion must be perfomed just before imposing hypoxia. There is no pathophysiological reason to dissociated substrate fall and hypoxia. But as far a you use low calcium and HEPES, I am afraid that the hypoxic injury might be strongly reduced...

Regards,

Pierre

Thanks, Pierre. What you suggested I think is very of value. I will try again in these days. Best regards!

Good luck!

Hi Qingqi ji

Did you solve the problem? if yes, could you please share the protocol?