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

Bradley Palmer, PhD, answers top questions from a recent webinar on the challenging preparation of cardiac tissue slices and measurement of force and calcium.

The answers to these questions have been provided by:

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

How quickly should an investigator aim to clip and mount the freshly produced slices?

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.

How does one minimize tissue damage during vibratome preparation of the cardiac slices?

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

Does IonWizard have the capability to analyze workloops?

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

Will a dual emission dye like indo-1 minimize motion artifact as effectively as a dual excitation dye like fura-2?

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.

How thick would you cut a cardiac slice for imaging?

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.

Can we use the same method for the mouse heart and would there be any change required?

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.

Can you use agarose to hold the heart during slicing?

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.

How long should one wait for the BDM relaxing solution before one starts force measurements?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.

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.

What source of energy do the myocytes use when they remain in a slice without blood perfusion?

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.

How do you culture the cardiac slices long term?

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.

Is this technique suitable for the procurement of right ventricle (RV) papillary muscles/chordae?

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

Do you think it would be possible to image vasculature in the slice prep?

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

Do you get issues with areas not stimulating in quite the same way as others, perhaps due to variance in excitability or orientation of fibers?

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.

Can you use flash-frozen hearts to section and analyze, or does the tissue have to be fresh?

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.

Will the cutting solution allow protein conformations to stay intact ?

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.

At which temperature the work loop acquisition is possible?

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

What are the most critical steps in slice preparation?

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.

The heart wall consists of 3 layers with different directions, so how do you make the slice consistent and and generate the same consistent force and other parameters?

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.

What material is used to glue the slices?

The adhesive is Histoacryl from Bruan.

For how long are the slices viable during an experiment with electrical stimulation?

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

Can ischemic cardiac tissue slices be used?

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.

Can this prep be used on heart disease preps, and if so, are there additional experimental considerations?

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.

What is the starting tissue tension that you aim for when you hang the tissue on the hooks?

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.

Do you take into account the change in slice thickness (thus in cross-sectional area (CSA)) as a consequence of the applied stretch?

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.

Are there any media components that are very important to add for promoting viability and how is viability typically assessed?

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

Why do the slices need to be 100um thick?

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.

How long can you culture the slices and do they still maintain their contractility?

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.

What are the parameters you use for slicing?

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