Measurement and analysis of sarcomere length in rat cardiomyocytes in situ and in vitro

G. Bub, P. Camelliti, C. Bollensdorff, D. J. Stuckey, G. Picton, R. A.B. Burton, K. Clarke, P. Kohl

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    Abstract

    Sarcomere length (SL) is an important determinant and indicator of cardiac mechanical function; however, techniques for measuring SL in living, intact tissue are limited. Here, we present a technique that uses two-photon microscopy to directly image striations of living cells in cardioplegic conditions, both in situ (Langendorff-perfused rat hearts and ventricular tissue slices, stained with the fluorescent marker di-4-ANEPPS) and in vitro (acutely isolated rat ventricular myocytes). Software was developed to extract SL from two-photon fluorescence image sets while accounting for measurement errors associated with motion artifact in raster-scanned images and uncertainty of the cell angle relative to the imaging plane. Monte-Carlo simulations were used to guide analysis of SL measurements by determining error bounds as a function of measurement path length. The mode of the distribution of SL measurements in resting Langendorff-perfused heart is 1.95 μm (n = 167 measurements from N = 11 hearts) after correction for tissue orientation, which was significantly greater than that in isolated cells (1.71 μm, n = 346, N = 9 isolations) or ventricular slice preparations (1.79 μm, n = 79, N = 3 hearts) under our experimental conditions. Furthermore, we find that edema in arrested Langendorff-perfused heart is associated with a mean SL increase; this occurs as a function of time ex vivo and correlates with tissue volume changes determined by magnetic resonance imaging. Our results highlight that the proposed method can be used to monitor SL in living cells and that different experimental models from the same species may display significantly different SL values under otherwise comparable conditions, which has implications for experiment design, as well as comparison and interpretation of data.

    Original languageEnglish
    Pages (from-to)H1616-H1625
    JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
    Volume298
    Issue number5
    DOIs
    Publication statusPublished - 1 May 2010

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    Keywords

    • Confocal microscopy
    • Magnetic resonance imaging
    • Monte-carlo simulation
    • Two-photon imaging
    • Ventricular tissue slice

    ASJC Scopus subject areas

    • Physiology
    • Cardiology and Cardiovascular Medicine
    • Physiology (medical)

    Cite this

    Bub, G., Camelliti, P., Bollensdorff, C., Stuckey, D. J., Picton, G., Burton, R. A. B., Clarke, K., & Kohl, P. (2010). Measurement and analysis of sarcomere length in rat cardiomyocytes in situ and in vitro. American Journal of Physiology - Heart and Circulatory Physiology, 298(5), H1616-H1625. https://doi.org/10.1152/ajpheart.00481.2009