Q&A Report: Cardiac Tissue Slices: Preparation, Data Acquisition, and Analysis

Bradley Palmer, PhD
R&D Engineer / Assistant Professor
Molecular Physiology and Biophysics
IonOptix / University of Vermont

BThe cardiac slices in our experience are very robust and remain viable for hours if kept on ice and bathed with a relaxing solution that contains at least 30 mM BDM.

Minimize tissue damage by making sure the blade’s horizontal motion is as flat and as repeatable as possible.

IonOptix is working on this and should have the ability to analyze work loops by end of 2022.

Although I have not tried Indo-1, I would worry that the motion artifact would be different between the two wavelengths, because they are being recorded with different PMTs. Without having compared, I would think fura-2, which uses the same PMT for both excitation wavelengths, would be better at minimizing motion artifact.

I would recommend thin slices like 100 or even 50 micron thick. There would still be several myocytes across this distance and ample extracellular matrix without adding too much bulk of material that might prevent good imaging.

Yes – the mouse heart can be prepared in the same manner. However, because the mouse heart is so much smaller than the rat heart, it is more challenging to get multiple slices. We get 2-4 useable slices from a mouse heart, whereas 5-10 from a rat heart. I would recommend adult sized mice, i.e., older than 12 weeks if possible older than 16/20 weeks.

I have heard of this and have so far been skeptical of its application. That may be due to my lack of experience with it, but my first impression is that it’s an unnecessary step and it adds another barrier for diffusion of oxygen. Other groups, however, have used it successfully.

The BDM will wash out of the tissue within about a minute or two, maybe 5 minutes to be safe. This is assuming perfusion at ~ 2 mL/min.

Most conventional solutions used glucose as the energy source because it’s easy to use. However, the energy source in vivo is mostly free fatty acids, which is more difficult to use because of solubility issues.

We have been able to keep the slices functional up to 7 days in a 5% CO2 incubator. We use a DMEM solution spiked with antibiotics, ITS, FBS, and added HEPES. I am not an expert on this subject and suggest looking for references by Dendorfer, who has had success with this.

Probably, although we have never tried. The RV wall is very thin, so we may only get 1-2 slices.

There is certainly vasculature maintained in these slices. You should be able to image the capillaries, for example. However, we have not yet tried.

Other groups have imaged the propagation of the action potential across these slices. It moves faster along the longitudinal axis of the myocytes compared to the transverse axis. Any discontinuities in myocyte orientation would likely affect this propagation.

The tissue needs to be fresh or nearly fresh for the tissue to remain excitable. Frozen tissue is almost never excitable due to the damage done to the membrane during the freeze-thaw cycle.

Yes and no – part of the appeal of this model system is the maintenance of in vivo architecture including most protein conformations. However, if there are environmental factors that affect proteins, such as temperature (cold will disrupt microtubules for example), then you will have to have keep that in mind. But the cutting itself does not affect protein conformations.

We have been successful between room temperature and 37°C.

Good hands (which most experimentalists have already developed) and solution preparation. If we have a bad day, it’s almost always because the solution was old, not properly pH’d or made incorrectly.

This is a good point. The orientation of the myocytes will vary from slice to slice as the slices are generating across transmural direction. We do like to use only those slices that show a single dominate direction.

The adhesive is Histoacryl from Bruan.

We have kept rat slices going for 36 hours on the microscope. We could probably go longer, but haven’t tried.

Yes – remove oxygen, remove energy supply (glucose), and stop flow. We have done this to mimic ischemia re-perfusion injury. There may be other factors that would improve the model.

Diseased hearts can be used – we have used a model of mouse model of dilated cardiomyopathy (DCM) with success. Be careful of the known properties of the disease state. For example, we found lots of additional collagen in our DCM model. Measuring sarcomere length was more challenging and therefore we enhance our imaging methods.

There are two conventional ways to get your starting muscle length. (i) set a specific sarcomere length, (ii) adjust muscle length until you get the maximum developed tension. For normal muscle, we often start between 1-5 mN.mm^-2.

We do not generally take into account any irregularities in thickness or width. An applied stretch is usually very small and the Poisson ratio of little consequence. On top of that, there’s really no change in the number of force generating molecules (unless you stretch very far), so there’s probably no need to adjust the CSA due to changes in lengthening.

If you mean viability for long term incubation, antibiotics, ITS, Zinc at ~ 10 microM and Mn at ~ 2 microM.

Thin slices minimize diffusion problems. Oxygen and other metabolites exchange more effectively with the thinner slices. 300 microns is probably a good thickness for most functional analyses. We like to use 200 microns to assure visualization of sarcomeres. 100 microns for even better visualization – like for imaging applications.

We have incubated rat slices for up to 7 days and can still record excellent forces. We have not had the same success with mouse slices and are still working on that.

80-200 Hz vibration. 1-2 mm amplitude. 0.01-0.1 mm/min advancing speed.