Live-Cell Imaging of the Contractile Velocity and Transient Intracellular Ca2+ Fluctuations in Human Stem Cell-Derived Cardiomyocytes

Abstract

Live-cell imaging techniques are essential for acquiring vital physiological and pathophysiological knowledge to understand and treat heart disease. For live-cell imaging of transient alterations of [Ca2+]i in human cardiomyocytes, we engineered human-induced pluripotent stem cells carrying a genetically-encoded Ca2+-indicator (GECI). To monitor sarcomere shortening and relaxation in cardiomyocytes in real-time, we generated a α-cardiac actinin (ACTN2)-copepod (cop) green fluorescent protein (GFP+)-human-induced pluripotent stem cell line by using the CRISPR-Cas9 and a homology directed recombination approach. The engineered human-induced pluripotent stem cells were differentiated in transgenic GECI-enhanced GFP+-cardiomyocytes and ACTN2-copGFP+-cardiomyocytes, allowing real-time imaging of [Ca2+]i transients and live recordings of the sarcomere shortening velocity of ACTN2-copGFP+-cardiomyocytes. We developed a video analysis software tool to quantify various parameters of sarcoplasmic Ca2+ fluctuations recorded during contraction of cardiomyocytes and to calculate the contraction velocity of cardiomyocytes in the presence and absence of different drugs affecting cardiac function. Our cellular and software tool not only proved the positive and negative inotropic and lusitropic effects of the tested cardioactive drugs but also quantified the expected effects precisely. Our platform will offer a human-relevant in vitro alternative for high-throughput drug screenings, as well as a model to explore the underlying mechanisms of cardiac diseases.

Microgravity-induced stress mechanisms in human stem cell-derived cardiomyocytes

“…A brief description is indicated in Figure S3 . The contractile velocity of c-CMs and SMG-CMs was determined by analysing the videos using the VA1.9 software ( Acharya et al., 2022 ).…”

“…To monitor CM sarcomere contraction and relaxation in real-time we generated a α−cardiac actinin (ACTN2)-enhanced green fluorescent protein (eGFP + )-hiPSC line by the CRISPR-Cas9 ( Sachinidis, 2021 ) and the homology-directed recombination (HDR) approach as we described more recently ( Acharya et al., 2022 ). A brief description is indicated in Figure S3 .…”

High-throughput longitudinal electrophysiology screening of mature chamber-specific hiPSC-CMs using optical mapping

“…GCaMP6s (slow) has been used for hiPSC-CM cell functional measurements. 53 , 54 Jiang et al. 53 generated a GCaMP6s knockin hPSC line, differentiated the stem cells to cardiomyocytes, and could detect the CaTs of hPSC-derived CMs.…”

Transcriptome-based prediction of drugs, inhibiting cardiomyogenesis in human induced pluripotent stem cells

“…Among all teratogens tested with the UKK2-CTT, only the retinoids completely inhibited the formation of beating cardiomyocytes at day14, whereas treatment with the non-teratogens had no effects on the beating frequency of the cardiomyocytes at day14. To focus on well-documented teratogens such as VPA and THD on the cardiomyogenesis process we generated a live imaging transgenic IMR90 hiPSCs cell line using the CRISPR-Cas9 and a homology-directed recombination approach as we described previously [2,31]. The ACTN2-cop green uorescent protein (ACTN2-cop-eGFP + -hiPSC line (IMR90 origin) enables the live imaging of sarcomeres after differentiation to ACTN2-cop-eGFP + -CMs, since the α-cardiac speci c actinin (ACTN2) is enriched in the sarcomeres, the smallest contractile unit of cardiomyocytes.…”