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

Nature Protocols volume 12, pages 2623–2639 (2017)Cite this article

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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.

References

  1. De Boer, T.P., Camelliti, P., Ravens, U. & Kohl, P. Myocardial tissue slices: organotypic pseudo-2D models for cardiac research & development. Future Cardiol. 5, 425–430 (2009).

    Article  PubMed  Google Scholar 

  2. Brandenburger, M. et al. Organotypic slice culture from human adult ventricular myocardium. Cardiovasc. Res. 93, 50–59 (2012).

    Article  CAS  PubMed  Google Scholar 

  3. Perbellini, F. et al. Investigation of cardiac fibroblasts using myocardial slices. Cardiovasc. Res. 119, 909–920 (2017).

    Google Scholar 

  4. Neri, B., Cini-Neri, G. & D'Alterio, M. Effect of anthracyclines and mitoxantrone on oxygen uptake and ATP intracellular concentration in rat heart slices. Biochem. Biophys. Res. Commun. 125, 954–960 (1984).

    Article  CAS  PubMed  Google Scholar 

  5. Wang, K. et al. Cardiac tissue slices: preparation, handling, and successful optical mapping. Am. J. Physiol. Heart Circ. Physiol. 308, H1112–H1125 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Camelliti, P. et al. Adult human heart slices are a multicellular system suitable for electrophysiological and pharmacological studies. J. Mol. Cell Cardiol. 51, 390–398 (2011).

    Article  CAS  PubMed  Google Scholar 

  7. Kang, C. et al. Human organotypic cultured cardiac slices: new platform for high throughput preclinical human trials. Sci. Rep. 6, 28798 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Claycomb, W.C. Biochemical aspects of cardiac muscle differentiation. Possible control of deoxyribonucleic acid synthesis and cell differentiation by adrenergic innervation and cyclic adenosine 3′:5′-monophosphate. J. Biol. Chem. 251, 6082–6089 (1976).

    Article  CAS  PubMed  Google Scholar 

  9. Yasuhara, S., Takaki, M., Kikuta, A., Ito, H. & Suga, H. Myocardial VO2 of mechanically unloaded contraction of rat ventricular slices measured by a new approach. Am. J. Physiol. 270 (3 Part 2): H1063–H1070 (1996).

    CAS  PubMed  Google Scholar 

  10. Burnashev, N.A., Edwards, F.A. & Verkhratsky, A.N. Patch-clamp recordings on rat cardiac muscle slices. Pflugers Arch. 417, 123–125 (1990).

    Article  CAS  PubMed  Google Scholar 

  11. Katano, Y., Akera, T., Temma, K. & Kennedy, R.H. Enhanced ouabain sensitivity of the heart and myocardial sodium pump in aged rats. Eur. J. Pharmacol. 105, 95–103 (1984).

    Article  CAS  PubMed  Google Scholar 

  12. Bursac, N. et al. Cultivation in rotating bioreactors promotes maintenance of cardiac myocyte electrophysiology and molecular properties. Tissue Eng. 9, 1243–1253 (2003).

    Article  CAS  PubMed  Google Scholar 

  13. Bussek, A. et al. Tissue slices from adult mammalian hearts as a model for pharmacological drug testing. Cell Physiol. Biochem. 24, 527–536 (2015).

    Article  CAS  Google Scholar 

  14. Bussek, A. et al. Cardiac tissue slices with prolonged survival for in vitro drug safety screening. J. Pharmacol. Toxicol. Methods 66, 145–151 (2012).

    Article  CAS  PubMed  Google Scholar 

  15. Pillekamp, F. et al. Neonatal murine heart slices. A robust model to study ventricular isometric contractions. Cell Physiol. Biochem. 20, 837–846 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. Hannes, T. et al. Biological pacemakers: characterization in an in vitro coculture model. J. Electrocardiol. 41, 562–566 (2008).

    Article  PubMed  Google Scholar 

  17. Mawad, D. et al. A conducting polymer with enhanced electronic stability applied in cardiac models. Sci. Adv. 2, e1601007 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Takaki, M. et al. Sarcoplasmic reticulum Ca2+ pump blockade decreases O2 use of unloaded contracting rat heart slices: thapsigargin and cyclopiazonic acid. J. Mol. Cell Cardiol. 30, 649–659 (1998).

    Article  CAS  PubMed  Google Scholar 

  19. Kohzuki, H. et al. Energy expenditure by Ba(2+) contracture in rat ventricular slices derives from cross-bridge cycling. Am. J. Physiol. 277 (1 Partt 2): H74–H79 (1999).

    CAS  PubMed  Google Scholar 

  20. Kohzuki, H., Misawa, H., Sakata, S., Ohga, Y. & Takaki, M. Sustained high O2 use for Ca2+ handling in rat ventricular slices under decreased free shortening after ryanodine. Am. J. Physiol. Heart Circ. Physiol. 281, H566–H572 (2001).

    Article  CAS  PubMed  Google Scholar 

  21. Yamashita, D. et al. O2 consumption of mechanically unloaded contractions of mouse left ventricular myocardial slices. AJP Hear Circ. Physiol. 287, H54–H62 (2004).

    Article  CAS  Google Scholar 

  22. Hattori, H. et al. NHE-1 blockade reversed changes in calcium transient in myocardial slices from isoproterenol-induced hypertrophied rat left ventricle. Biochem. Biophys. Res. Commun. 419, 431–435 (2012).

    Article  CAS  PubMed  Google Scholar 

  23. Takeshita, D. et al. Effects of formaldehyde on cardiovascular system in in situ rat hearts. Basic Clin. Pharmacol. Toxicol. 105, 271–280 (2009).

    Article  CAS  PubMed  Google Scholar 

  24. Shimizu, J. et al. Increased O2 consumption in excitation–contraction coupling in hypertrophied rat heart slices related to increased Na+?Ca2+ exchange activity. J. Physiol. Sci. 59, 63–74 (2009).

    Article  CAS  PubMed  Google Scholar 

  25. Perbellini, F. et al. Free-of-acrylamide SDS-based tissue clearing (FASTClear) for three dimensional visualization of myocardial tissue. Sci. Rep. 7, 5188 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Pillekamp, F. et al. Force measurements of human embryonic stem cell-derived cardiomyocytes in an in vitro transplantation model. Stem Cells 25, 174–180 (2007).

    Article  PubMed  Google Scholar 

  27. Habeler, W., Peschanski, M. & Monville, C. Organotypic heart slices for cell transplantation and physiological studies. Organogenesis 5, 62–66 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Cartledge, J.E. et al. Functional crosstalk between cardiac fibroblasts and adult cardiomyocytes by soluble mediators. Cardiovasc. Res. 105, 260–270 (2015).

    Article  CAS  PubMed  Google Scholar 

  29. Civitarese, R.A., Kapus, A., McCulloch, C.A. & Connelly, K.A. Role of integrins in mediating cardiac fibroblast–cardiomyocyte cross talk: a dynamic relationship in cardiac biology and pathophysiology. Basic Res. Cardiol. 112, 6 (2017).

    Article  PubMed  CAS  Google Scholar 

  30. Louch, W.E., Sheehan, K.A. & Wolska, B.M. Methods in cardiomyocyte isolation, culture, and gene transfer. J. Mol. Cell Cardiol. 51, 288–298 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Zhang, Y. et al. Dedifferentiation and proliferation of mammalian cardiomyocytes. PLoS One 5, e12559 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Mitcheson, J.S., Hancox, J.C. & Levi, A.J. Action potentials, ion channel currents and transverse tubule density in adult rabbit ventricular myocytes maintained for 6 days in cell culture. Pflugers Arch. 431, 814–827 (1996).

    Article  CAS  PubMed  Google Scholar 

  33. Shattock, M.J., Bullock, G., Manning, A.S., Young, M. & Hearse, D.J. Limitations of the isolated papillary muscle as an experimental model: a metabolic, functional and ultrastructural study. Clin. Sci. 64 (2) 4P (1983).

    Article  Google Scholar 

  34. Malan, D. et al. Human iPS cell model of type 3 long QT syndrome recapitulates drug-based phenotype correction. Basic Res. Cardiol. 111, 14 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Robertson, C., Tran, D.D. & George, S.C. Concise review: maturation phases of human pluripotent stem cell-derived cardiomyocytes. Stem Cells 31, 829–837 (2013).

    Article  CAS  PubMed  Google Scholar 

  36. Wu, J., Ocampo, A. & Belmonte, J. Cellular metabolism and induced pluripotency. Cell 166, 1371–1385 (2016).

    Article  CAS  PubMed  Google Scholar 

  37. Kane, C. & Terracciano, C.M. Induced pluripotent stem cell-derived cardiac myocytes to understand and test calcium handling: pie in the sky? J. Mol. Cell. Cardiol. 89, 376–378 (2015).

    Article  CAS  PubMed  Google Scholar 

  38. Frieler, R.A. & Mortensen, R.M. Immune cell and other noncardiomyocyte regulation of cardiac hypertrophy and remodeling. Circulation 131, 1019–1030 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Cordeiro, J.M., Greene, L., Heilmann, C., Antzelevitch, D. & Antzelevitch, C. Transmural heterogeneity of calcium activity and mechanical function in the canine left ventricle. Am. J. Physiol. 286, H1471–H1479 (2004).

    CAS  Google Scholar 

  40. Antzelevitch, C. et al. Heterogeneity within the ventricular wall. Electrophysiology and pharmacology of epicardial, endocardial, and M cells. Circ Res. 69, 1427–1449 (1991).

    Article  CAS  PubMed  Google Scholar 

  41. Bigelow, W.G., Lindsay, W.K. & Greenwood, W.F. Hypothermia; its possible role in cardiac surgery: an investigation of factors governing survival in dogs at low body temperatures. Ann. Surg. 132, 849–866. (1950).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Dae, M.W., Gao, D.W., Sessler, D.I., Chair, K. & Stillson, C.A. Effect of endovascular cooling on myocardial temperature, infarct size, and cardiac output in human-sized pigs. Am. J. Physiol. 282, H1584–H1591 (2002).

    CAS  Google Scholar 

  43. Tissier, R., Ghaleh, B., Cohen, M.V., Downey, J.M. & Berdeaux, A. Myocardial protection with mild hypothermia. Cardiovasc. Res. 94, 217–225 (2012).

    Article  CAS  PubMed  Google Scholar 

  44. Azar, T., Sharp, J. & Lawson, D. Heart rates of male and female Sprague-Dawley and spontaneously hypertensive rats housed singly or in groups. J. Am. Assoc. Lab. Anim. Sci. 50, 175–184 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Bensley, J.G., De Matteo, R., Harding, R., Black, M.J. & Schwalb, H. Three-dimensional direct measurement of cardiomyocyte volume, nuclearity, and ploidy in thick histological sections. Sci. Rep. 6, 23756 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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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.

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Authors and Affiliations

  1. Division of Cardiovascular Sciences, Myocardial Function, National Heart and Lung Institute, Imperial College London, London, UK

    Samuel A Watson, Martina Scigliano, Ifigeneia Bardi, Cesare M Terracciano & Filippo Perbellini

  2. Translational Biomedical Research Centre, University of Bristol, Bristol, UK

    Raimondo Ascione

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  1. Samuel A Watson
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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.

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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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