Electrical impedance-based contractile stress measurement of human iPSC-Cardiomyocytes
Introduction
Rhythmic contraction of cardiomyocytes is responsible for pumping oxygen-rich blood to the organs and tissues of the body. To accomplish this crucial function, cardiomyocytes utilize structurally ordered force-generating filaments that produce cellular and (through alignment) microscopic organ contractile stress (Gaitas et al., 2015; Brette et al., 2017; Ribeiro et al., 2020; Rudolph et al., 2019). Decline of the contractile function leads to heart failure, the single largest cause of morbidity and mortality in the developed countries (Hassanabad et al., 2019). The continued poor outcomes for heart failure patients, in part, results from a lack of pharmacology that can directly improve cardiomyocyte contractility (Green et al., 2016; Gorski et al., 2015; Sant’Anna et al., 2020). Developing pre-clinical models that emulate human cardiomyocyte and cardiac tissue function is important to understanding heart failure physiology and to evaluate new agents for their ability to improve cardiomyocyte contractility (inotropes) (Liu et al., 2012).
As cardiomyocyte actin is dynamics, with important short-term and long-term variation after drug exposure, end-point analysis (such as α-actinin staining of fixed tissue) is less informative (Jacot et al., 2008; Bildyug and Khaitlina, 2019). End-point analysis is also generally lower throughput, hindering the evaluation of a large number of potential therapeutics. Although label-based measurement using green fluorescent protein (GFP)-tagged proteins and fluorescent calcium dyes can overcome the end-point limitation, photobleaching limits their application to recording dynamic contractility with significant reactive oxygen species generation and cell death overtime. For instance, when GFP was incorporated into β-actin filaments (GFP–β-actin) to examine the contractility of neonatal rat cardiomyocytes, significant photobleaching occurred within 18 s of measurement (Skwarek-Maruszewska et al., 2009).
In comparsion, label-free electrical impedance measurement is commonly used for sensing cell behavior changes (e.g., shape changes) (Bürgel et al., 2015, Fan et al., 2019, Giaever and Keese, 1984, Wegener et al., 2000a, Zhou et al., 2016), and has been applied to the measurement of cardiomyocyte beating (Xiao et al., 2010; Scott et al., 2014; Tsai and Wang, 2016). Impedance-based devices contain micro-scale electrodes as a platform for cell growth. Physiological changes of cells (e.g., cell shape; inflow and outflow of ions) alter the electrical current between electrodes, resulting in impedance changes. Compared to fluorescent labeling, the impedance method enables label-free, long-term recording of cellular signals (Pauwelyn et al., 2015; Qiu et al., 2009). Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) and rat neonatal cardiomyocytes were evaluated by impedance recording including the testing of pharmaceuticals and compounds known to affect cardiomyocyte beating rate, rhythm and contractility, producing results consistent with the known cellular targets of the compounds (Scott et al., 2014). Present impedance-based measurement commonly uses the metric, termed cell index ( −)/ (Wang et al., 2013; Ke et al., 2011; Li et al., 2016), where is the impedance magnitude measured in the presence of cells, and is the baseline cell-free impedance magnitude. While cell index is useful to measure temporal and dynamic changes in impedance, useful for beating rate and rhythms, it is less consistent after changes to cell contractility overtime. Cell index is also strongly electrode geometry dependent, varying with microelectrode number and size and does not genuinely quantify the contractile stress generated by cardiomyocytes. As such, impedance measurement devices have more commercially been used to quantify drug-induced arrhythmia, and less for the evaluation of compounds that alter contractility.
To expand the utility of impedance measurement, capitalizing on the label-free and long-term measurement ability of the technique, we designed and fabricated a microdevice array with interdigitated electrodes (Fig. 1A). In each well, iPSC-CMs were cultured on top of the electrodes, producing a functional, beating 2D tissue monolayer. By recording electrical impedance signals, the device array monitored cell behavior at different phases, including adhesion, initiation of contraction, and plateaued contractility. The contractile stress was measured via atomic force microscopy (AFM), and quantitatively related to the electrical impedance signals. The established relationship was validated by comparing the changes to the AFM-measured and impedance-measured contractile stress after treatment with the L-type calcium channel blocker verapamil. Our quantitative power-law relationship between the contractile stress and impedance per unit electrode area expands the application of impedance measurement to the accurate quantification of cardiomyocyte contractile stress, facilitating the use of this label-free technique in the discovery of potential therapeutics for heart failure.
Section snippets
Impedance and contractile stress measurement
We utilized an impedance spectrometer (HF2IS, Zurich Instruments) and a bio-AFM (AFM, Bioscope Catalyst, Bruker) for impedance and contractile stress measurement on iPSC-CMs. (Fig. 1B). While impedance signals were recorded, the AFM probe was moved onto the surface of a cardiomyocyte for simultaneous measurement of contractile stress.
The AFM measurement requires direct contact between the AFM probe and the cell. Indentation by the AFM probe can mechanically stimulate cardiomyocytes through
Conclusion
Heat failure is the single largest cause of death in the developed countries, and possesses the largest individual treatment cost of any disease. Yet there is a paucity of pharmaceuticals that directly target the contractile defects of the cardiomyocyte, in part because of the difficulty in measuring this quantity accurately and over time in preclinical models. The continuous functional measurement of iPSC cardiomyocyte monolayers by electrical impedance provide an important label-free method
CRediT authorship contribution statement
Xian Wang: Conceptualization, Methodology, Writing - original draft. Li Wang: Conceptualization, Methodology, Writing - original draft. Wenkun Dou: Methodology, Visualization, Validation. Zongjie Huang: Methodology, Visualization. Qili Zhao: Methodology, Visualization. Manpreet Malhi: Methodology. Jason T. Maynes: Supervision, Writing - review & editing. Yu Sun: Conceptualization, Supervision, Writing - original draft.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgment
The authors acknowledge financial support from the Canadian Institutes of Health Research (CIHR) and the Natural Sciences and Engineering Research Council of Canada (NSERC); from the Explore Program of CQDM; and from the University of Toronto via an EMHSeed grant. L.W. acknowledges a postdoctoral fellowship from Ted Rogers Center for Heart Research Education Fund. Y.S. acknowledges the Canada Research Chairs Program. J.T.M. would like to thank the Wasser Family and SickKids Foundation, as the
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