damaged cardiomyocyte from adult mouse heart? - (Aug/17/2006 )

Hi, friends
A big problem in my adult mouse cardiomyocyte isolation.
I can have about 90% rod cardiomyocyte after collagenase2 digestion. But the problem is that i have to put cells in KB buffer immediately.
Without high potassium, even in 0-Ca Tyrode's, my cells will beat madly and die. So it is impossible to re-introduce the calcium then.
I guess there's still a damage of the cell by the enzyme, or some other mistakes.
Need some advice badly. Thanks

Hi, friends
A big problem in my adult mouse cardiomyocyte isolation.
I can have about 90% rod cardiomyocyte after collagenase2 digestion. But the problem is that i have to put cells in KB buffer immediately.
Without high potassium, even in 0-Ca Tyrode's, my cells will beat madly and die. So it is impossible to re-introduce the calcium then.
I guess there's still a damage of the cell by the enzyme, or some other mistakes.
Need some advice badly. Thanks

Hi， thanks alot. I've sent you a mail with brief description, please take a look.
The cells will beat even in calcium free tyrode, is this an indication of digestion damage of the cell by the enzyme? I am using single collagenase2, should i use combination of collagenase 1+2??
My cell will only stay quiescent in high potassium buffer, i don't even have a chance to begin the calcium reintroduction:(

God! Hotmail's lost my important mail again.

I don't have a langendorff, so i use a pump with constant flow, and monitor the speed and perfusing temperature which i believe are OK.

CollagenaseII from worthington is used, i don't use combination of collagenase1+2. The working solution is "0.6mg/ml collagenase2 +0.1%BSA" in calcium free tyrode, and i normally perfuse for 10min with speed of 1.6ml/min ( faster speed seems worse for this pump).

The composition of calcium free tyrode is (mM): Nacl 136,Kcl 5.4, Mgcl2 1, NaH2PO4 0.33, Dextrose 10, HEPES 5, Taurine 20. PH 7.35 with NaOH.

1.Oxygenate the solutions. Mouse 5000u/kg heparinized.
2.Cannulated in cold tyrode, initial perfuse with calcium free tyrode for 4min at 37C.
3.Enzyme perfuse 10min at 37C (Found cells beating like crazy)
4.Dissect heart in KB buffer (Soon die if in calcium free tyrode)


I am already using lower enzyme and less time than majority of the publication, what could be the reason then. After in KB for 2h, i think the cell begin to shrink, melt and die . I enclose a pic, it is 2 h after isolation in KB.

I isolate rat myocytes with similar procedure, so I guess procedure should be the same....
your procedure seems fine to me:
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1.Oxygenate the solutions. Mouse 5000u/kg heparinized.
2.Cannulated in cold tyrode, initial perfuse with calcium free tyrode for 4min at 37C.
3.Enzyme perfuse 10min at 37C (Found cells beating like crazy)
4.Dissect heart in KB buffer (Soon die if in calcium free tyrode
------------------
Always make sure that the temp and pH are correct. Most important variables.
what I do is generally perfuse with Ca-tyrode in beginning (5-8min) to clear blood and to see the normal beating of heart....you should be fine as you have heparinized the mouse.
then fwith Ca-free tyrode for 10 mins..... you can increase the time to clear calcium fully from the system.
regarding collagenase I am not sure if the problem is due to non-inclusion of collagenase I. Collagenase II should be enough (and I am sure you make collagenase in Ca-free tyrode). though I use a higher concentration for rat, it also depends on the activity of the collagenase you are getting, so 10 mins is kindda approximate duration... check by yourself if digestion is complete (you can clearly see a colour change and swelling) or by taking a little bit of tissue and look under microscope, if digestion is complete stop perfusing and disperse in KB, and keep in KB for 2 hrs at 4C.
only two reason I can think of for beating are:
either calcium is there when perfusing with collagenase or
digestion is more.