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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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.
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 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)
Video depicts how to handle, dry and mount tissue block correctly. (MP4 18702 kb)
Video depicts bringing blade to 'starting position' and various stages of slicing. (MP4 26143 kb)
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)
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)
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)
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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