1) The heart is getting no blood flow after excision before perfusion, so the cold buffer slows down the metabolic demand of the heart and stops the heart from continuing to beat. 2) We usually gas our HEPES buffered media with 100% oxygen. Amount of oxygen can be important depending on how long the perfusion/isolation protocol is. 3) Yes too much spontaneous beating after reintroduction of calcium is suggestive of unhealthy cell. You can try rentroducing the calcium more slowly to avoid the caclium paradox, or being more gentle during any steps of mechanical dissociation (e.g trituration). If you are talking about mouse cardiac myocytes, you can use a contraction inhibitor (BDM, blebbistatin) during isolation and in the culture media which improves cell viability (Kabaeva et al, AJP 2008).

During cardiomyocyte isolation there is a shift in microenviroment from a conducive in vivo setting. This is a cause of a well known phenomenon called 'cell-shock'. To mitigate this and inactivate stress signals leading to apoptosis, hypothermia lowers cell metabolism c9nsiderably. This improves cell viability during in vitro expansion.

Any decrease in temperature is indeed protective for the cardiomyocytes (as long as one does not reach temperatures near freezing inside the cardiomyocytes themselves). This is already true if body temperature is lower (Duncker et al AJP 2006) and it is the reason that cooling of the heart and body are a common practice in an attempt to reduce injuries after infarctions of various organs.

I have not worked on isolated cardiomyocytes but I worked extensively on the isolated perfused heart. I think it is very important to keep the energy resources of cardiac cells by putting them in cold KHB; it allows to stop the heart beat and the different energy transfer process as well as enzymatic reactions. At reperfusion, the heart is doing very well and keeps all its capabilities. I think we can make the same reasoning for isolated heart cells.

I would agree with the above comments keeping any tissue cold during cell preparation is essential to slow down metabolic demand, reduce stress signals/apoptosis and thus preserve cell viability. Of course in the heart it has the added advantage of reducing beating making your life easier and again slowing metabolic demand and resultant damage to cells. It will also reduce the function of enzymes particularly proteolytic enzymes.

Cooling down the tissue reduces the metabolic activity by factor 2 with each 10 degree of Celsius, thus protects the heart in the hypoxic environment (hybernation). You can skip cold media, if rapidly perfuse coronary arteries with Ca free solution or high potassium KB and paralyze the myocardium. Each heartbeat after the removal of the heart reduces the viability of the isolated cells, especially if you use large mammalian heart, like dog or pig. Small hearts (mouse, rat, guinea pig) tolerate hypoxia better, we mount them on Langedorff system while they are still beating and wash out blood with normal calcium media. They pump out blood in a flash, then we switch to calcium free media.

Cooling is required for the above statement. Moreover, cold induces an early HSP production, that is an additional key of protection. But, since oxidative metabolism is extinguished, oxygenation is not necessary, and even may enhance the risk of ROS production. Additionnaly, buffer rinsing dilutes red blood cells, which may unfavor cardiomyocyte developpment.

I agree with the comments above. For heart perfusion at Langendorff apparature we use KHB solution, which is equalibrated /gas bubbled) with 02. For the measurements with isolated cardiomyocytes i use a basis of Hepes- buffer , which is titrated to 7.4 at 37°C and no oxygenation in addition to it is required.

Thank you for all advices, very helpful to me.

We have routinely isolated cardiomyocytes earlier from rat and rabbit, and more recently from mice. We have never utilized a cold solution and never oxygenated any medium prior to isolation. We do however place the heart immediately in a calcium free buffer (Ca2+/Mg2+ free HBSS with 10 mM BDM). If you are able to quickly remove the heart and perfuse it there is no need for cold or oxygenation in my opinion.

As many of the replies posted so far have indicated, the purpose of cooling the heart is to reduce metabolic activity and thereby avoid ischaemia. However, whether for experiments with the Langendorff-perfusion technique or for cell isolation, it is necessary to re-warm the heart. Provided that it is possible to perfuse the heart in Langendorff within a very few (i.e. 2) minutes of excision, it is not necessary to cool the heart below room temperature. We get perfectly adequate viable cell yields (mouse, rabbit, rat) without cooling the heart beyond room temperature. Some colleagues infact excise into ~37 degC Tyrode's and this also works provided the cannulation of the aorta and perfusion of the heart are sufficiently rapid.

Ca2+ tolerant cardiomyocytes are ones that do not contract spontaneously on exposure to quasi-physiological concentrations of Ca2+ (1 - 2 mM). Unless the cells are nodal (sino-atrial or atrio-ventricular) or conductive (Purkinje fibre) in origin, the cells should not show spontaneous activity. Thus, if you have spontaneously beating cells in culture, the cells are either damaged or changing phenotype.

By the way, Suyeol, do you know Professor Shang Jin Kim in Veterinary Pharmacology at Chonbuk National University? He worked with me in Bristol for a few years and has a lot of experience with Langendorff techniques. Please ask him for advice on Langendorff perfusion.