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Simultaneous AFM Investigation of the Single Cardiomyocyte Electro-Chemo-Mechanics During Excitation-Contraction Coupling

  • Guido Caluori
  • Roberto Raiteri
  • Mariateresa Tedesco
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1886)

Abstract

The cardiac excitation-contraction coupling is the cellular process through which the heart absolves its blood pumping function, and it is directly affected when cardiac pathologies occur. Cardiomyocytes are the functional units in which this complex biomolecular process takes place: they can be represented as a two-stage electro-chemo and chemo-mechanical transducer, along which each stage can be probed and monitored via appropriate micro/nanotechnology-based tools. Atomic force microscopy (AFM), with its unique nanoresolved force sensitivity and versatile modes of extracting sample properties, can represent a key instrument to study time-dependent heart mechanics and topography at the single cell level. In this work, we show how the integrative possibilities of AFM allowed us to implement an in vitro system which can monitor cardiac electrophysiology, intracellular calcium dynamics, and single cell mechanics. We believe this single cell-sensitive and integrated system will unlock improved, fast, and reliable cardiac in vitro tests in the future.

Key words

Cardiac muscle mechanics Electrophysiology Calcium imaging Cardiomyocyte Atomic force microscopy In vitro models 

Notes

Acknowledgments

We acknowledge the support of A. Cambiaso (Sitem S.r.l., Genova, IT) and G. Carlini (Univ. of Genova) for the development of the acquisition software and the electronic boards, respectively.

References

  1. 1.
    Kenny T (2014) Cardiac conduction system. In: Nuts and bolts of implantable device therapy pacemakers. John Wiley & Sons Ltd, Hoboken, NJ, pp 15–20Google Scholar
  2. 2.
    Bers DM (2002) Cardiac excitation–contraction coupling. Nature 415:198–205CrossRefGoogle Scholar
  3. 3.
    Nichols M, Townsend N, Scarborough P, Rayner M (2015) Cardiovascular disease in Europe—epidemiological update 2015. Eur Heart J 36:2696–2705.  https://doi.org/10.1093/eurheartj/ehv428CrossRefPubMedGoogle Scholar
  4. 4.
    Phenotypic assays for analyses of pluripotent stem cell–derived cardiomyocytesGoogle Scholar
  5. 5.
    Martherus RS, Zeijlemaker VA, Ayoubi TA (2010) Electrical stimulation of primary neonatal rat ventricular cardiomyocytes using pacemakers. Biotechniques 48:65–67.  https://doi.org/10.2144/000113308CrossRefPubMedGoogle Scholar
  6. 6.
    Louch WE, Sheehan KA, Wolska BM (2011) Methods in cardiomyocyte isolation, culture, and gene transfer. J Mol Cell Cardiol 51:288–298.  https://doi.org/10.1016/j.yjmcc.2011.06.012CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Eschenhagen T, Zimmermann WH (2005) Engineering myocardial tissue. Circ Res 97:1220–1231.  https://doi.org/10.1161/01.RES.0000196562.73231.7dCrossRefPubMedGoogle Scholar
  8. 8.
    Stett A, Egert U, Guenther E et al (2003) Biological application of microelectrode arrays in drug discovery and basic research. Anal Bioanal Chem 377:486–495.  https://doi.org/10.1007/s00216-003-2149-xCrossRefPubMedGoogle Scholar
  9. 9.
    Frega M, Tedesco M, Massobrio P et al (2014) Network dynamics of 3D engineered neuronal cultures: a new experimental model for in-vitro electrophysiology. Sci Rep 4:5489.  https://doi.org/10.1038/srep05489CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Simultaneous study of mechanobiology and calcium dynamics on hESC‐derived cardiomyocytes clustersGoogle Scholar
  11. 11.
    Paredes RM, Etzler JC, Watts LT, Lechleiter JD (2009) Chemical Calcium indicators. Methods 46:143–151.  https://doi.org/10.1016/j.ymeth.2008.09.025.ChemicalCrossRefGoogle Scholar
  12. 12.
    Kirmizis D, Logothetidis S (2010) Atomic force microscopy probing in the measurement of cell mechanics. Int J Nanomedicine 5:137–145CrossRefGoogle Scholar
  13. 13.
    Radmacher M (2007) Studying the mechanics of cellular processes by atomic force microscopy. Methods Cell Biol 83:347–372.  https://doi.org/10.1016/S0091-679X(07)83015-9CrossRefPubMedGoogle Scholar
  14. 14.
    Liu J, Sun N, Bruce MA et al (2012) Atomic force mechanobiology of pluripotent stem cell-derived cardiomyocytes. PLoS One 7:e37559.  https://doi.org/10.1371/journal.pone.0037559CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Azeloglu EU, Costa KD (2009) Dynamic AFM elastography reveals phase dependent mechanical heterogeneity of beating cardiac myocytes. In: 31st Annual International Conference of the IEEE EMBS. IEEE Service Center, Minneapolis, MN, p 7180–7183Google Scholar
  16. 16.
    Kiyama NA, Hnuki YO, Unioka YK et al (2006) Transverse stiffness of myofibrils of skeletal and cardiac muscles studied by atomic force microscopy. J Physiol Sci 56:145–151.  https://doi.org/10.2170/physiolsci.RP003205CrossRefGoogle Scholar
  17. 17.
    Cogollo JFS, Tedesco M, Martinoia S et al (2011) A new integrated system combining atomic force microscopy and micro-electrode array for measuring the mechanical properties of living cardiac myocytes. Biomed Microdevices 13:613–621.  https://doi.org/10.1007/s10544-011-9531-9CrossRefGoogle Scholar
  18. 18.
    Ozkan AD, Topal AE, Aykutlu D, et al (2016) Atomic force microscopy for the investigation of molecular and cellular behavior. Micron 89:60–76.  https://doi.org/10.1016/J.MICRON.2016.07.011CrossRefGoogle Scholar
  19. 19.
    Butt H-J, Cappella B, Kappl M (2005) Force measurements with the atomic force microscope: technique, interpretation and applications. Surf Sci Rep 59:1–152.  https://doi.org/10.1016/j.surfrep.2005.08.003CrossRefGoogle Scholar
  20. 20.
    Carl P, Schillers H (2008) Elasticity measurement of living cells with an atomic force microscope: data acquisition and processing. Pflugers Arch 457:551–559.  https://doi.org/10.1007/s00424-008-0524-3CrossRefPubMedGoogle Scholar
  21. 21.
    Haase K, Pelling AE, Haase K (2015) Investigating cell mechanics with atomic force microscopy. J R Soc Interface 12:20140970CrossRefGoogle Scholar
  22. 22.
    Chaturvedi RR, Herron T, Simmons R et al (2010) Passive stiffness of myocardium from congenital heart disease and implications for diastole. Circulation 121:979–988.  https://doi.org/10.1161/CIRCULATIONAHA.109.850677CrossRefPubMedGoogle Scholar
  23. 23.
    Lieber SC, Aubry N, Pain J et al (2004) Aging increases stiffness of cardiac myocytes measured by atomic force microscopy nanoindentation aging increases stiffness of cardiac myocytes measured by atomic force microscopy nanoindentation. Am J Physiol Heart Circ Physiol 287:645–651.  https://doi.org/10.1152/ajpheart.00564.2003CrossRefGoogle Scholar
  24. 24.
    Rapila R, Korhonen T, Tavi P (2008) Excitation–contraction coupling of the mouse embryonic cardiomyocyte. J Gen Physiol 132:397–405.  https://doi.org/10.1085/jgp.200809960CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Chynoweth KM, Wigton M, Cook SM, Schäffer TE (2006) Practical implementation of dynamic methods for measuring atomic force microscope cantilever spring constants. Nanotechnology 17:2135–2145.  https://doi.org/10.1088/0957-4484/17/9/010CrossRefGoogle Scholar
  26. 26.
    Melzak KA, Moreno-Flores S, Yu K et al (2010) Rationalized approach to the determination of contact point in force-distance curves: application to polymer brushes in salt solutions and in water. Microsc Res Tech 73:959–964.  https://doi.org/10.1002/jemt.20851CrossRefPubMedGoogle Scholar
  27. 27.
    Oliver WC, Pharr GM (2011) Measurement of hardness and elastic modulus by instrumented indentation: advances in understanding and refinements to methodology. J Mater Res 19:3–20.  https://doi.org/10.1557/jmr.2004.19.1.3CrossRefGoogle Scholar
  28. 28.
    Azeloglu EU, Costa KD (2010) Cross-bridge cycling gives rise to spatiotemporal heterogeneity of dynamic subcellular mechanics in cardiac myocytes probed with atomic force microscopy. Am J Physiol Heart Circ Physiol 298:853–860.  https://doi.org/10.1152/ajpheart.00427.2009CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Guido Caluori
    • 1
    • 2
    • 3
  • Roberto Raiteri
    • 3
  • Mariateresa Tedesco
    • 3
  1. 1.Fakultni Nemocnice u Sv. Anny v Brne (FNUSA)International Clinical Research Centre (ICRC)BrnoCzech Republic
  2. 2.CEITEC MU, Masaryk UniversityBrnoCzech Republic
  3. 3.Department of Informatics, Bioengineering, Robotics, and Systems Engineering (DIBRIS)Università degli Studi di GenovaGenovaItaly

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