Optical Method to Quantify Mechanical Contraction and Calcium Transients of Human Pluripotent Stem Cell-Derived Cardiomyocytes

Abstract

Differentiation of human pluripotent stem cells into cardiomyocytes (hPS-CMs) holds promise for myocardial regeneration therapies, drug discovery, and models of cardiac disease. Potential cardiotoxicities may affect hPS-CM mechanical contraction independent of calcium signaling. Herein, a method using an image capture system is described to measure hPS-CM contractility and intracellular calcium concurrently, with high spatial and temporal resolution. The image capture system rapidly alternates between brightfield and epifluorescent illumination of contracting cells. Mechanical contraction is quantified by a speckle tracking algorithm applied to brightfield image pairs, whereas calcium transients are measured by a fluorescent calcium reporter. This technique captured changes in contractile strain, calcium transients, and beat frequency of hPS-CMs over 21 days in culture, as well as acute responses to isoproterenol and Cytochalasin D. The technique described above can be applied without the need to alter the culture platform, allowing for determination of hPS-CM behavior over weeks in culture for drug discovery and myocardial regeneration applications.

Keywords: calcium transients; drug testing; high-speed imaging; mechanical contraction; pluripotent stem cell-derived cardiomyocytes.

FIG. 1.

Use of HDM to determine displacement fields of contracting hPS-CMs. HDM was applied by acquiring a high-speed video of contracting hPS-CMs to obtain a stack of images (A) from which an ROI could be selected (B) and subdivided into 16 × 16 pixel windows. Each 16 × 16 pixel window is transformed to the Fourier domain (C) where phase correlation between frames is applied (D) and an inverse Fourier transform is taken, resulting in the peak displacement between the two frames (E). HDM, high-density mapping; hPS-CM, human pluripotent stem cell-derived cardiomyocyte; ROI, region of interest.

FIG. 2.

Image acquisition schematic with temporal control of fluorescent or brightfield illumination. Microscope schematic, a digital microscope controller for image capture of contracting cardiomyocytes using an Orca Flash 4.0 sCMOS camera with alternating pulses of fluorescent and brightfield illumination (A). Timing chart indicating sensor readout and the start of pixel exposure; when all lines are exposed, the controller initiates a pulse of fluorescent (blue) or brightfield (green) illumination (B). The cycle is started over after the total exposure time (7 ms) with alternating brightfield and fluorescent illuminations.

FIG. 3.

The effect of window size and window spacing on reported contractile strain values. The resulting strain magnitude and location was used to generate heat maps (A) with increasing average spatial contractile strain at peak contraction in red. Pixel width for boxes in (A) was 325 wide and 272 pixels in height. Using different window sizes and spacings (1/2 window size) can affect reported contractile strain values (B), area used for contractile strain calculations reported as a change from the original selected ROI (C), and computing times (D). A 16 × 16 pixel window spacing with an 8 pixel window shift and an average contractile strain calculated over five windows was used to report contractile strain (A, B). Mean ± SD.

FIG. 4.

HDM can be used to quantify contraction of hPS-CMs seeded on collagen IV-coated plates over 21 days. hPS-CMs significantly increased beat frequency by 1.5×  between day 7 and 21 (A). A significant decrease between day 7 and 21 was observed for contractile strain (B) and maximum contractile strain (C). Mean ± SD for frequency, mean ± SEM for contractile and maximum strain, n = 6, *p < 0.05.

FIG. 5.

Overlay of mechanical contraction and calcium transients. Alternating brightfield and fluorescent images of contracting induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) loaded with Fluo-4 AM on day 21 resulted in spatial heat maps for peak mechanical contraction (A) and peak calcium flux (B). The location of peak calcium intensity did not correspond with location of peak contractile strains. (A, B, white arrows) The resulting cyclic contractile strain (blue) and calcium signal (black) are presented over 8 s (C) with an isolated cycle indicated by the red box in C (E). Cells treated with Cytochalasin-D exhibit loss of mechanical contraction, while calcium transients are maintained (D).

FIG. 6.

Isoproterenol stimulation of hPS-CMs. Adding isoproterenol to hPS-CMs increased beat frequency (A, B) up to the addition of 10 μM isoproterenol, with representative traces of Fluo-4 AM loaded contracting iPS-CMs (A) and frequency data averaged in (B). Addition of isoproterenol did not affect contractile strain for any condition compared to baseline (C). Mean ± SD for frequency, mean ± SEM for contractile strain, n = 5, *p < 0.05.