Q&A Report: Understanding Advanced Cardiac Tissue Slice Applications

Bradley Palmer, Matthew Caporizzo, and Benjamin Lee answer questions from a recent webinar on how cardiac slices and the IonOptix Cardiac Slice System can be used to complement and improve upon traditional cardiac investigations.

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

Matthew Caporizzo, PhD
Assistant Professor
Molecular Physiology and Biophysics
University of Vermont

Benjamin Lee, MD, PhD
Cardiovascular Medicine
University of Pennsylvania

How long can you keep cardiac slices in vitro? Is it long enough (days) to be able to transfect cardiomyocytes and therefore express a gene of interest before conducting experiments?

The slices remain viable on the microscope for up to 36 hours (at least in our hands) and in the incubator for at least 7 days (in our hands) and weeks/months as reported by others. Yes, 7 days should be long enough for transfection, and we are working on that right now.

What temperature were these studies performed? Is there any temperature dependence of the colchicine treatment?

Colchicine (Colch) at room temperature (~22C) due to the difficulty of maintaining 37C in a continuously perfusing system for 2h. We do think that microtubules (MTs) will be more stable at 37C and colchicine could actually have a greater effect at 37C. We are working towards being able to perform these experiments.

Do you see considerable run down during workloop experiments?

Amazingly, we do not see significant run down in the course of hours of experimentation. We see run down after 5-10 hours on the microscope.

What is the best way to apply preload on (mouse) slices and for the standardization of individual slices?

Preload is often set as the end-diastolic force.

Can you record Calcium (Ca2+) transients with the myocardial slices or the engineered heart tissue (EHTs)?

Yes, although motion artifact is likely to also be recorded. With the use of ratiometric dye, you can minimize the effect, but it is still something to keep in mind.

Could you comment on the strengths and weaknesses of measurements of PV loops collected with the slice compared to those collected by the in vivo technique?

There are at least three differences.

  1. The slice allows for measurement and control of diastolic sarcomere length. With a measure of diastolic sarcomere length, the experimentalist knows exactly where on the force-length curve the muscle is operating.
  2. With the slice, preload and afterload can be precisely established according to the needs of the experiment. Whereas in vivo, the systemic vasculature plays a significant role in establishing preload and afterload and are not really at the command of the experimentalist.
  3. Uncontrolled neurohormonal factors are not at play in the cardiac slice.

Have you compared function of left and right ventricle cardiac slices?

Yes, right ventricle (RV) slices can be prepared. However, from the mouse, we just used the entire RV and didn’t bother making a slice. RV has a slower rate of contraction and relaxation. We have not performed a systematic study of the differences between the ventricle.

What is the significance of measuring diastolic sarcomere length?

With a measure of diastolic sarcomere length, the experimentalist knows exactly where on the force-length curve the muscle is operating.

Can Cholchicine be used in heart failure (HF)?

The dose of Colchicine required to affect microtubule assembly in the heart is so high as to prohibit the use of colchicine in the cardiac clinic.

What heart rates can be maintained? Do you have any sense if oxygen availability is a problem?

We have not observed an hypoxic core in the slices as can occur with some thicker preps, e.g., papillary muscle. We tend to use <200 um thickness, which appears to prevent development of a hypoxic core in rat left ventricle (LV) slice paced at 5 Hz at 37C.

How similar is the generated force to real cardiac tissue? How is the Ca2+ cycling?

The force generated by engineered tissues is lower than in adult living myocardial slices. The EHTs we tested were only 14 days old, and did not receive a dedicated maturation protocol, which we would expect to increase force. We have not looked at calcium cycling in our EHTs yet.

Have you compared normal heart tissue to failing heart (HFpEF versus HFrEF)?

We are in the process of doing this for the ZSF1 Rat. It is too early for us to say much about these differences, but we would anticipate that in failing tissue with contractile dysfunction that this will certainly impact the size of the work-loops obtained.

Which group in Germany did Brad mention during the webinar and what species were these slices from?

Can you maintain sterility of your EHTs (or slices for that matter) whilst measuring? And do you measure in the presence of 5% CO2 when measuring in culture medium?

We are unable to maintain sterility of EHTs or slices while measuring, we solely use them as an endpoint analysis. We currently do not measure in the presence of 5% CO2 but have plans to introduce this to our protocol.

Is PV loop heterogenous transmurally? Is it possible to investigate it with your setup?

It is likely that the slices from different parts of the LV wall will generate different F-L loops. We fully expect that, but have not examined it yet. It is absolutely possible to examine that question with this system.

Can a measure of sarcomere length (SL) in a slice manipulate to whole heart?

The measure of SL in the slice may not translate to whole heart in part because we don’t have a clear idea of the SL is in the whole heart.

Can you comment on the fact that in engineered heart tissue, the force frequency relationship is negative?

There must be some aspects of calcium handling that are not always produced in the EHTs.

How do you get your measurements so that it really showcases the performance of heart muscle in the the physical situation (ie. more than one straight layer of heart muscle/myocytes)?

The slices is only 150 um thick, and therefore we can sample the many different layers of the myocardium. We have not performed such a systematic study, but it would seem called for.

As you know, fatty acids are the preferred fuel for a normal oxygenated cardiomyocyte. Have you measured lactic acid output, considering DMEM has glucose but not necessarily fatty acids?

We have not measured the lactic acid output. It would be interesting to look at the impact of alternative or additional fuel sources on contractility in both EHTs and living myocardial slices.

Are there any technical videos describing the use of mounted tissue slices?

Yes – look for the videos available on the IonOptix website. You can also find videos on my YouTube account: https://www.youtube.com/watch?v=3WcKLTUFPfw.

Do you observe differences between work-loops within the slices from the same heart?

Yes – there are differences between slices even within the same heart. Different levels (base vs apex) and different transmural location (endocardium vs epicardium) will make a difference. We have not yet performed a systematic examination of these differences.

What thickness of slices lends itself to optimal sarcomere visualization?

<200 um. We normally use 150 um for this reason.

How long can you maintain work loop production in the EHTs?

For over an hour with sufficient perfusion.