I'm currently working on adult mouse cardiomyocytes and I need to express an artificial protein. Therefore I use adenoviral transfection. This works in general, but for mouse cardiomyocytes I'm facing a time limit because so far I could not keep them in culture for much more than 3 days. As far as I know people working on mouse cardiomyocytes are generally facing this problem. Due to this time limit I cannot just wait for some more days to get a higher protein level. I tried to increase the virus concentration but this did not solve the problem. Currently I'm applying the virus at a concentration of 6 x 10^4 to 6 x 10^5 PFU per cell. In a few cases the cells stayed healthy for 3d and than the expression was ok.
So now I would like to find a way to either elongate the survival of my cells in culture or speed up the protein expression. Is anyone facing similar problems?
For adenovirus infecting primary rat/mouse cardiomyocytes, 6 x 10^4 to 6 x 10^5 PFU /cell is too high to keep the cardiomyocytes healthy, that's incredible high. I usually use a MOI of 50-100 for protein expression with adenovirus in cardiomyocytes, and 48h is enough for a protein expressed to a very considerable quantity.
If you are using ACM from mouse, I would presume you are using BDM. There was a nice paper a few years ago by Kabaeva et al (2008) using blebbistatin instead of BDM for efficient and stable transfection. For longer term culture, blebbistatin works much better than BDM. May be worth a shot.
http://ajpheart.physiology.org/content/294/4/H1667.full#sec-1
Thanks, a lot! Unfortunately i cannot change the system, because for my project i need to work on certain ko animals aswell, there for i need to stay at mouse. Chad, you are right, i'm using BDM. I've never heard of blebbistatin but that is definetly worth a try, thanks alot!
So, now i tried blebbistatin (25 µM) as recommanded by you, Chad. And indeed i could yield much higher/longer viability of adult mouse cardiomyocytes. Today i did experiments on cultures that were allready 5d old and they had a quite good proteinexpression and my FRET-based sensor gave nice signals. So blebbistatin appears to be the better choice to culivate those cells for more than 1 day.
But in case that anyone also wants to transfect cardiomyocytes for imaging experiments, i'd like to mention that blebbistatin shows some autofluorescens, which might be in conflict with your fluorophores (see, beside others: Lucas-Lopez et al., 2005 "Absolute Stereochemical Assignment and Fluorescence Tuning of the Small Molecule Tool, (–)-Blebbistatin"). In my case (im using CFP/YFP FRET) the blebbistatin fluorescenc perfectly overlaps with my FRET signal. So i got a constant drift of that signal (because "YFP signal" (here: YFP and blebbistatin) bleeches faster than CFP signal. To overcome this, before i did start my measurement i perfused cells in blebbistatin-free medium (with BDM instead) for 20 min an than bleeched blebbistatin with 488 nm laser (see: Sakamoto et al., 2005 "Blebbistatin, a Myosin II Inhibitor, Is Photoinactivated by Blue Light") until there was no further bleeching (so bleeching reach a plateau). Than i could start FRET-experiments.
Jan - glad things worked out for you. We have experienced a similar benefit of using blebbistatin for long term culture in our lab. We have also experienced issues with using blebbistatin for imaging with Fura-2 and Fluo-4. We found the same solution - replace with BDM or image without a contraction inhibitor. Best of luck moving forward!
5 days !!! that's great , in my current lab , we're having some problems to maintain mouse adult cardiomyocytes for 24 hours , so can you please send me your protocol for adult mouse cardiomyocytes culture .
and i am looking also for a protocol to transfect adult rat cardiomyocytes because we do not have adenovirus . thanks alot!
Dear Jan, Chad and everyone who faces challenges with blebbistatin fluorescence,
be careful with photo-inactivation as it may lead to phototoxicity (Kolega 2004 BBRC, attached).
To overcome this problem, a new blebbistatin derivative was developed last year: para-nitroblebbistatin (Kepiro et al 2014 Angew Chem, attached). One of its advantages is that para-nitroblebbistatin is not fluorescent, which make its use much more convenient. It was tested on HeLa cells, Dictyostelium cells and zebrafish in detail. Informal preliminary data are promising on cardiomyocytes.
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