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

Lyudmyla Borysova; Y Y Hanson Ng; Edward S Wragg; Lillian E Wallis; Emily Fay; Raimondo Ascione; Kim A Dora

doi:10.1038/s41596-021-00590-6

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

Lyudmyla Borysova, Y Y Hanson Ng, Edward S Wragg, Lillian E Wallis, Emily Fay, Raimondo Ascione, Kim A Dora^*

^*Corresponding author for this work

Bristol Medical School (THS)

Research output: Contribution to journal › Article (Academic Journal) › peer-review

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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- to 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 biopsies. 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
following cessation of stimulation. This protocol thus offers a rapid and reliable method for studying the Ca2+ signaling underpinning cardiomyocyte contraction, both in healthy and diseased tissue.

Original language	English
Pages (from-to)	4650-4675
Number of pages	26
Journal	Nature Protocols
Volume	16
Issue number	10
Early online date	16 Aug 2021
DOIs	https://doi.org/10.1038/s41596-021-00590-6
Publication status	Published - Oct 2021

Bibliographical note

Funding Information:
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.

Publisher Copyright:
© 2021, The Author(s), under exclusive licence to Springer Nature Limited.

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

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- to 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 biopsies. 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 spontaneouslyfollowing cessation of stimulation. This protocol thus offers a rapid and reliable method for studying the Ca2+ signaling underpinning cardiomyocyte contraction, both in healthy and diseased tissue. ",

author = "Lyudmyla Borysova and {Hanson Ng}, {Y Y} and Wragg, {Edward S} and Wallis, {Lillian E} and Emily Fay and Raimondo Ascione and Dora, {Kim A}",

note = "Funding Information: 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. Publisher Copyright: {\textcopyright} 2021, The Author(s), under exclusive licence to Springer Nature Limited.",

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AU - Borysova, Lyudmyla

AU - Hanson Ng, Y Y

AU - Wragg, Edward S

AU - Wallis, Lillian E

AU - Fay, Emily

AU - Ascione, Raimondo

AU - Dora, Kim A

N1 - Funding Information: 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. Publisher Copyright: © 2021, The Author(s), under exclusive licence to Springer Nature Limited.

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N2 - 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- to 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 biopsies. 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 spontaneouslyfollowing cessation of stimulation. This protocol thus offers a rapid and reliable method for studying the Ca2+ signaling underpinning cardiomyocyte contraction, both in healthy and diseased tissue.

AB - 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- to 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 biopsies. 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 spontaneouslyfollowing cessation of stimulation. This protocol thus offers a rapid and reliable method for studying the Ca2+ signaling underpinning cardiomyocyte contraction, both in healthy and diseased tissue.

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DO - 10.1038/s41596-021-00590-6

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JO - Nature Protocols

JF - Nature Protocols

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ER -

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

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