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High spatial and temporal resolution Ca2+ imaging of myocardial strips from human, pig and rat

Nature Protocols volume 16, pages 4650–4675 (2021)Cite this article

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Abstract

Ca2+ handling within cardiac myocytes underpins coordinated contractile function within the beating heart. This protocol enables high spatial and temporal Ca2+ imaging of ex vivo multicellular myocardial strips. The endocardial surface is retained, and strips of 150–300-µm thickness are dissected, loaded with Ca2+ indicators and mounted within 1.5 h. A list of the equipment and reagents used and the key methodological aspects allowing the use of this technique on strips from any chamber of the mammalian heart are described. We have successfully used this protocol on human, pig and rat biopsy samples. On use of this protocol with intact endocardial endothelium, we demonstrated that the myocytes develop asynchronous spontaneous Ca2+ events, which can be ablated by electrically evoked Ca2+ transients, and subsequently redevelop spontaneously after cessation of stimulation. This protocol thus offers a rapid and reliable method for studying the Ca2+ signaling underpinning cardiomyocyte contraction, in both healthy and diseased tissue.

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Fig. 1: Images of the three imaging chambers and mounted myocardial strips.
Fig. 2: Dissection, mounting and imaging myocardial strips.
Fig. 3: Spontaneous Ca2+ events in human atrial cardiomyocytes.
Fig. 4: Spontaneous Ca2+ events in porcine atrial cardiomyocytes.
Fig. 5: Spontaneous Ca2+ events in rat atrial cardiomyocytes.
Fig. 6: Ca2+ transients (CaTs) in rat atrial cardiomyocytes.
Fig. 7: Structure and viability of rat myocardial strips.
Fig. 8: Further applications.
Fig. 9: Translation to rat left ventricle myocardial strips.

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Acknowledgements

We thank the British Heart Foundation (BHF) and Medical Research Council (MRC) for grant awards to R.A. enhancing this work (grant numbers BHF PG/18/49/33833, BHF IG/14/2/30991, BHF/PG/16/104/32652 and MRC MR/L012723/1). Collection of human biopsy specimens in Bristol was supported by the NIHR Bristol Biomedical Research Centre. In addition, this work was supported by British Heart Foundation grants to K.A.D. (grant numbers FS/08/033/25111, FS/13/16/30199, IG/13/5/30431 and PG/18/11/33552) and by the Oxford BHF Centre of Research Excellence (RE/13/1/30181). We thank Theodore Burdyga at the University of Liverpool for use of the custom-built chambers and general protocols for using them and Carsten Thorndahl and Rene Hemmel at DMT for the development and supply of the Confocal Cardiac Myograph. We thank the research nurses, laboratory technicians and surgeons at the Bristol Heart Institute, University Hospital Bristol-Weston NHS Foundation Trust and the Bristol Trials Centre (Clinical Trials and Evaluation Unit) at the University of Bristol. We also thank the staff and researchers at the University of Bristol Translational Biomedical Research Centre, a national research facility for large animals co-funded by the BHF and MRC. Finally, we express our full gratitude to all the patients taking part in this study.

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Authors and Affiliations

  1. Vascular Pharmacology Group, Department of Pharmacology, University of Oxford, Oxford, UK

    Lyudmyla Borysova, Y. Y. Hanson Ng, Edward S. Wragg, Lillian E. Wallis, Emily Fay & Kim A. Dora

  2. Bristol Heart Institute, Bristol Royal Infirmary, University of Bristol, Bristol, UK

    Raimondo Ascione

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Contributions

K.A.D. wrote the manuscript, collected and analyzed data and contributed to the optimization of the protocol. L.B. and Y.Y.H.N. contributed to writing the manuscript. L.B., Y.Y.H.N., E.S.W. and L.E.W. collected and analyzed data and contributed to the optimization of the protocol. E.F. collected data. R.A. provided all human and porcine specimens and contributed to the manuscript. All authors proofread the manuscript.

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Correspondence to Kim A. Dora.

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The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Filippo Perbellini, Thomas Seidel and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Burdyga, T. et al. Cell Calcium 34, 27–33 (2003): https://doi.org/10.1016/s0143-4160(03)00019-8

Borisova, L. et al. Circ. Res. 105, 803–810 (2009): https://doi.org/10.1161/CIRCRESAHA.109.202960

Borysova, L. et al. Cell Calcium 75, 21–29 (2018): https://doi.org/10.1016/j.ceca.2018.08.001

Supplementary information

Supplementary Video 1

Cutting and pinning of porcine RAA

Supplementary Video 2

Cutting of porcine RAA into myocardial strips

Supplementary Video 3

Isolation of rat RAA from a whole heart

Supplementary Video 4

Cutting and pinning of rat RAA

Supplementary Video 5

Cutting of rat RAA into myocardial strips

Supplementary Video 6

Mounting of a rat RAA myocardial strip into three types of imaging chambers (#1, #2 and #3)

Supplementary Video 7

Spontaneous Ca2+ waves in a human RAA myocardial strip

Supplementary Video 8

Spontaneous Ca2+ sparks in a human RAA myocardial strip

Supplementary Video 9

Spontaneous Ca2+ waves in a porcine RAA myocardial strip

Supplementary Video 10

Spontaneous Ca2+ waves in a rat RAA myocardial strip

Supplementary Video 11

Electrically evoked CaTs in a rat RAA myocardial strip

Supplementary Video 12

Simultaneous imaging of cardiac and endocardial endothelial cell Ca2+ in a rat RAA myocardial strip

Supplementary Video 13

Isolation, cutting, pinning and dissection of rat ventricles followed by cutting of myocardial strips from a rat left ventricle

Supplementary Video 14

Spontaneous Ca2+ waves in a rat left ventricle myocardial strip

Supplementary Video 15

Electrically evoked CaTs in a rat left ventricle myocardial strip

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Borysova, L., Ng, Y.Y.H., Wragg, E.S. et al. High spatial and temporal resolution Ca2+ imaging of myocardial strips from human, pig and rat. Nat Protoc 16, 4650–4675 (2021). https://doi.org/10.1038/s41596-021-00590-6

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