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Nanomolar ATP binding to single myosin cross-bridges in rigor: a molecular approach to studying myosin ATP kinetics using single human cardiomyocytes

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Abstract

Our knowledge in the field of cardiac muscle and associated cardiomyopathies has been evolving incrementally over the past 60 years and all was possible due to the parallel progress in techniques and methods allowing to take a fresh glimpse at an old problem. Here, we describe an exciting tool used to examine the various states of the human cardiac myosin at the single molecule level. By imaging single Alexa647-ATP binding to permeabilised cardiomyocytes using total internal reflection fluorescence (TIRF) microscopy, we are able to acquire large populations of events in a short timeframe (~ 5000 sites in ~ 10 min) and measure each binding event with high spatio-temporal resolution. The applied algorithm decomposes the point pattern of single molecule binding events into individually distinct binding sites that enables us to recover kinetic parameters, such as bound or free time per site. This single molecule binding approach is a useful tool used to examine muscle contractility. Of particular importance is its application to probing the dynamic lifetimes and proportion of myosins in the super-relaxed state in human cardiomyopathies.

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Authors and Affiliations

  1. Biomedical Imaging Facility, Mark Wainwright Analytical Centre, University of New South Wales, Kensington, 2052, Australia

    Elvis Pandzic & Renee M. Whan

  2. Victor Chang Cardiac Research Institute, Molecular Cardiology Group, Darlinghurst, NSW, 2020, Australia

    Christian A. Morkel & Cristobal G. dos Remedios

  3. Department of Pharmacy & Biomedical Sciences, La Trobe University, Bendigo, 3552, Australia

    Amy Li

  4. Department of Biochemistry, University of California, San Francisco, CA, 94158, USA

    Roger Cooke

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  1. Elvis Pandzic
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  2. Christian A. Morkel
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  3. Amy Li
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  4. Roger Cooke
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  5. Renee M. Whan
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  6. Cristobal G. dos Remedios
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Correspondence to Elvis Pandzic.

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Pandzic, E., Morkel, C.A., Li, A. et al. Nanomolar ATP binding to single myosin cross-bridges in rigor: a molecular approach to studying myosin ATP kinetics using single human cardiomyocytes. Biophys Rev 12, 1031–1040 (2020). https://doi.org/10.1007/s12551-020-00716-2

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  • DOI: https://doi.org/10.1007/s12551-020-00716-2

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