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Preparation of viable adult ventricular myocardial slices from large and small mammals

Abstract

This protocol describes the preparation of highly viable adult ventricular myocardial slices from the hearts of small and large mammals, including rodents, pigs, dogs and humans. Adult ventricular myocardial slices are 100- to 400-μm-thick slices of living myocardium that retain the native multicellularity, architecture and physiology of the heart. This protocol provides a list of the equipment and reagents required alongside a detailed description of the methodology for heart explantation, tissue preparation, slicing with a vibratome and handling of myocardial slices. Supplementary videos are included to visually demonstrate these steps. A number of critical steps are addressed that must be followed in order to prepare highly viable myocardial slices. These include identification of myocardial fiber direction and fiber alignment within the tissue block, careful temperature control, use of an excitation–contraction uncoupler, optimal vibratome settings and correct handling of myocardial slices. Many aspects of cardiac structure and function can be studied using myocardial slices in vitro. Typical results obtained with hearts from a small mammal (rat) and a large mammal (human) with heart failure are shown, demonstrating myocardial slice viability, maximum contractility, Ca2+ handling and structure. This protocol can be completed in 4 h.

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Figure 1: Representative images of a rat myocardial slice.
Figure 2: Apparatus required for the production of myocardial slices.
Figure 3: Preparation of a small mammalian (rat) left ventricular tissue block.
Figure 4: Rat myocardial slice on a 1-mm grid visualized using a macroscope.
Figure 5: Myocardial slice viability.
Figure 6: Understanding the viability of myocardial slices.
Figure 7: Contractility of rat and human HF myocardial slices.
Figure 8: Ca2+ handling of rat and human HF myocardial slices was assessed by loading slices with Fluo-4 AM and performing optical mapping.
Figure 9: Conduction velocity of rat and human HF myocardial slices.
Figure 10: Cardiac structure studied using myocardial slices.

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Acknowledgements

We thank the British Heart Foundation for funding our work, particularly the BHF Centre for Regenerative Medicine award at Imperial College London (RM/13/1/30157) and the MBBS PhD studentship to S.A.W. (FS/15/35/31529). We thank The Facility for Imaging by Light Microscopy (FILM) at Imperial College London, in particular S.M. Rothery. Human samples were provided by the NIHR Cardiovascular Biomedical Research Unit at the Royal Brompton and Harefield NHS Foundation Trust and Imperial College London. Canine samples were provided by GlaxoSmithKline. Porcine samples were provided by the Translational Biomedical Research Centre, University of Bristol.

Author information

Authors and Affiliations

Authors

Contributions

S.A.W. wrote the manuscript, collected data and contributed to the optimization of the protocol. M.S. collected data and contributed to the optimization of the protocol. I.B. contributed to the optimization of the protocol. R.A. provided porcine specimens. C.M.T. contributed to the optimization of the protocol. F.P. collected data and contributed to the optimization of the protocol. All authors proof-read the manuscript.

Corresponding authors

Correspondence to Cesare M Terracciano or Filippo Perbellini.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Original vs. optimized protocol for rat (small mammal) & human HF (large mammal) myocardial slices.

Comparison between original and optimized protocols. Original protocol was based on several methods from previously published manuscripts(2,6,13). A) Rat myocardial slices produced using optimized protocol had a significantly higher maximum contractility compared to those produced with original protocol (0.39±0.05mN/mm2 vs. 11.55±1.30mN/mm2, Original: N=24/24, Optimized: N=10/10, Unpaired t-test). B) Human HF myocardial slices produced using optimized protocol had a significantly higher maximum contractility compared to those produced with original protocol (3.44±0.38mN/mm2 vs. 13.77±3.52mN/mm2, Original: N=20/20, Optimized: N=9/9, Unpaired t-test).

Imperial College London provided permission for the use of the animals in this study. All procedures were performed under license by the UK Home Office, in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986. Animals were killed following guidelines established by the European Directive on the protection of animals used for scientific purposes (2010/63/EU).

Data is presented as mean ± standard error

N = number of slices/number of hearts

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1. (PDF 183 kb)

Preparation of left ventricular tissue block from small mammalian heart (Rat) – Part 1 (video depicts Step 2A(i–vii)).

The preparation of the tissue block in this video has been carried out slowly to aid the visualization of the technique. However, tissue block preparation should be carried out as quickly as possible. Tissue should be kept cool throughout. If tissue contracts during block preparation, transfer to holding bath and allow to cool for 30 s. (MP4 24480 kb)

Preparation of left ventricular tissue block from small mammalian heart (Rat) – Part 2 (video depicts Step 2A(viii–x)).

The preparation of the tissue block in this video has been carried out slowly to aid the visualization of the technique. However, tissue block preparation should be carried out as quickly as possible. Tissue should be kept cool throughout. If tissue contracts during block preparation, transfer to holding bath and allow to cool for 30 s. (MP4 24867 kb)

Preparation of left ventricular tissue block from small mammalian heart (Rat) – Part 3 (video depicts Step 2A(xi).

The preparation of the tissue block in this video has been carried out slowly to aid the visualization of the technique. However, tissue block preparation should be carried out as quickly as possible. Tissue should be kept cool throughout. If tissue contracts during block preparation, transfer to holding bath and allow to cool for 30 s. (MP4 24584 kb)

Mounting of left ventricular tissue block on agarose-coated specimen holder.

Video depicts how to handle, dry and mount tissue block correctly. (MP4 18702 kb)

Slicing of left ventricular tissue block.

Video depicts bringing blade to 'starting position' and various stages of slicing. (MP4 26143 kb)

Handling and storage of myocardial slices.

Video depicts how to move a myocardial slice from a vibratome bath to a holding bath. Video also shows how myocardial slices should be kept in a holding bath. (MP4 9422 kb)

Culturing myocardial slices using air–liquid interface method.

Video depicts how to culture myocardial slices on an air–liquid interface using Transwell membranes as described by Brandenburger et al. (ref. 2). (MP4 24327 kb)

Myocardial slice contraction.

Video depicts a human HF myocardial slice contracting (field stimulation, 0.5 Hz, 30 V) and a rat myocardial slice contracting (point stimulation, 1 Hz, 10 V). (MP4 5001 kb)

Ca2+ handling of myocardial slices.

Rat myocardial slice was loaded with Fluo-4 AM (as described in 'Anticipated Results—Ca2+ handling'). Myocardial slice was field stimulated at 1 Hz, 10 V. Cardiomyocytes at the slice surface flash with each calcium transient. (MP4 9282 kb)

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Watson, S., Scigliano, M., Bardi, I. et al. Preparation of viable adult ventricular myocardial slices from large and small mammals. Nat Protoc 12, 2623–2639 (2017). https://doi.org/10.1038/nprot.2017.139

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