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. 2018 Jan;46(1):148-158.
doi: 10.1007/s10439-017-1933-2. Epub 2017 Oct 3.

Simultaneous Measurement of Contraction and Calcium Transients in Stem Cell Derived Cardiomyocytes

A Ahola  1 , R-P Pölönen  2 , K Aalto-Setälä  2   3 , J Hyttinen  4
Affiliations

Simultaneous Measurement of Contraction and Calcium Transients in Stem Cell Derived Cardiomyocytes

A Ahola et al. Ann Biomed Eng. 2018 Jan.

Abstract

Induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) provide a powerful platform for disease modeling and drug development in vitro. Traditionally, electrophysiological methods or fluorescent dyes (e.g. calcium) have been used in their functional characterization. Recently, video microscopy has enabled non-invasive analysis of CM contractile motion. Simultaneous assessments of motion and calcium transients have not been generally conducted, as motion detection methods are affected by changing pixel intensities in calcium imaging. Here, we present for the first time a protocol for simultaneous video-based measurement of contraction and calcium with fluorescent dye Fluo-4 videos without corrections, providing data on both ionic and mechanic activity. The method and its accuracy are assessed by measuring the effect of fluorescence and background light on transient widths and contraction velocity amplitudes. We demonstrate the method by showing the contraction-calcium relation and measuring the transient time intervals in catecholaminergic polymorphic ventricular tachycardia patient specific iPSC-CMs and healthy controls. Our validation shows that the simultaneous method provides comparable data to combined individual measurements, providing a new tool for measuring CM biomechanics and calcium simultaneously. Our results with calcium sensitive dyes suggest the method could be expanded to use with other fluorescent reporters as well.

Keywords: Biomechanics; Calcium-contraction coupling; Fluorescence; Motion analysis.

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Figures

Figure 1
Figure 1
Imaging protocol, processing of the video channels and definition of transient duration parameters. (a) Fluorescent calcium and visible light were recorded in consecutive frames. Midway during the recording, light source was turned on to increase the background light intensity, allowing video signal to be captured. Traditional PIV was measured from the frames with only visible light and CaPIV signal was measured from frames with calcium fluorescence present. Calcium signal was categorized based on the background light intensity to Ca (dark) and Ca (bright). (b) Transient duration parameters are defined as percentages from the peak maximum, similarly as when classifying action potentials.
Figure 2
Figure 2
Representative motion (left side) and displacement (right side) signals plotted over calcium transients for the three cell lines. In the motion signals, the contractile movement velocity is represented with and upward peak and the relaxation movement velocity as a downward peak in red, while calcium transient is shown in blue. In the displacement signals (right side), representing the motion velocity signal integral with respect to time, contraction is shown in red and calcium transient in blue. Representative signals are shown for the three cell lines.
Figure 3
Figure 3
A representative displacement measurement showing overlaid CaPIV (blue) with fluorescence and PIV (shown in red) without fluorescence.
See this image and copyright information in PMC

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