Merits of hiPSC-Derived Cardiomyocytes for In Vitro Research and Testing Drug Toxicity

Abstract

The progress of medical technology and scientific advances in the field of anticancer treatment have increased the survival probabilities and duration of life of patients. However, cancer-therapy-induced cardiac dysfunction remains a clinically salient problem. Effective anticancer therapies may eventually induce cardiomyopathy. To date, several studies have focused on the mechanisms underlying cancer-treatment-related cardiotoxicity. Cardiomyocyte cell lines with no contractile physiological characteristics cannot adequately model "true" human cardiomyocytes. However, applying "true" human cardiomyocytes for research is fraught with many obstacles (e.g., invasiveness of the procedure), and there is a proliferative limitation for rodent primary cultures. Human-induced pluripotent stem-cell-differentiated cardiomyocytes (hiPSC-CMs), which can be produced efficiently, are viable candidates for mimicking human cardiomyocytes in vitro. We successfully performed cardiac differentiation of human iPSCs to obtain hiPSC-CMs. These hiPSC-CMs can be used to investigate the pathophysiological basis and molecular mechanism of cancer-treatment-related cardiotoxicity and to develop novel strategies to prevent and rescue such cardiotoxicity. We propose that hiPSC-CMs can be used as an in vitro drug screening platform to study targeted cancer-therapy-related cardiotoxicity.

Keywords: H9c2; cardiac differentiation; cardiomyocyte (CM); cardiotoxicity; hepatocellular carcinoma (HCC); human-induced pluripotent stem cell (hiPSC).

Figure 1

Comparison of the characteristics of hiPSC−differentiated cardiomyocytes and H9c2 cells with drug-induced toxicity by sorafenib in vitro. (a) Schematic of the iPSC−CM differentiation protocol. (b) Establishment and validation of hiPSC and differentiated cardiomyocytes compared with H9c2 cells. The morphology shows the difference between iPSC−CMs and H9c2 cells. (c) Marker confirmation of iPSC−CMs and cardiac myoblast (H9c2 cells) of cardiac active protein Troponin T (cTnT) and ventricular marker Myosin light-chain 2 (MLC2v) by flow cytometry. Blue wave shows isotype control and red shows cardiomyocyte marker. (d) Representative fluorescent images of cardiomyocyte markers: alpha-actinin (α-actinin, green), connexin 43 (Cx43, red), and nucleus staining, DAPI (blue) of hiPSC−differentiated cardiomyocyte and H9c2 cells, scale bar: 50 μM. (e) IC50 of sorafenib treatment in iPSC−CMs, H9c2 cells, and Huh7 after 72 h. (f) Sorafenib-treated dose-dependent cell viability after sorafenib treatment for 72 h. (g) Sorafenib-treated time-dependent cell viability. Each data point represents the mean ± SEM (n = 3), *** p < 0.001 compared with iPSC-CMs, ### p < 0.001 compared with H9c2 cells.

Figure 2

Contractile dysfunction in sorafenib-treated iPSC−CMs. (a–c) Dose-dependent muscle motion of iPSC−CMs and H9c2 cells under 0, 2, 4, 8, and 12 μM sorafenib treatment for 24 h. (a) Contraction amplitude, (b) contraction velocity, and (c) relaxation velocity. (d–f) Time-dependent muscle motion of iPSC−CMs and H9c2 cells treated with 4 μM sorafenib for 72 h. (d) Contraction amplitude, (e) contraction velocity, and (f) relaxation velocity. Each data point represents the mean ± SEM (n = 3).

Figure 3

Bioenergetic parameters in hiPSC-derived cardiomyocytes and H9c2 cells. iPSC-CMs (a–f) and H9c2 cells (g–l) under 0, 2, 4, and 8 μM sorafenib treatment for 24 h. Idealized bioenergetic profiling trace demonstrating oxygen consumption rate (OCR) (a,g), basal respiration (b,h), nonmitochondrial oxygen consumption(c,i), ATP production (d,j), Maximal Respiratory Capacity (e,k), and proton leak (f,l). Each data points represent the mean ± SEM (n = 3). p-values: * p < 0.05, ** p < 0.01 compared with 0 μM sorafenib treatment.

Figure 4

The quantification of ROS generated in sorafenib-treated hiPSC-CMs. Mitochondria-damage-induced reactive oxygen species (ROS) production can be detected in both sorafenib-treated hiPSC-CMs. The imbalance between mitochondrial reactive oxygen species overexpression and removal leads to oxidative damage affecting cellular components. Since the cardiomyocyte is a highly energetic organ, it is vulnerable to damage caused by oxidative stress. p-values, ** p < 0.01, *** p < 0.001, compared with vehicle.

Figure 5

Action current (AC) firing recording in human-induced pluripotent-stem-cell-derived cardiomyocytes (hiPSC−CMs) with or without treatment of sorafenib. Prior to the electrophysiological experiments, hiPSC−CMs were incubated in 4 μM sorafenib for 24 h. This set of experiments were conducted in cell-attached current recordings, and the potential was held at the level of the resting potential (approximately −70 mV). (a) Representative current traces obtained in untreated (a) and treated (b) hiPSC−CMs. The downward deflection shows the occurrence of AC. (b) Summary bar graph showing the firing frequency of untreated and untreated cells (mean ± SEM; n = 7 for each bar). * Significantly different from untreated cells (p < 0.05).

Figure 6

Apoptosis markers of cleaved caspase 9, cleaved caspase 3, and autophagy marker LC3B expressed in hiPSC−CMs and H9c2 cells dose- and time-dependently. (a) hiPSC−CMs and H9c2 cells treated with sorafenib under 2, 4, 8, 10, and 20 μM for 72 h. Apoptosis markers (cleaved caspase 9, cleaved caspase 3) and autophagy marker (LC3B) were analyzed by Western blot. (b) Quantification of cleaved caspase 9, cleaved caspase 3, and LC3B expression from diagram a. p-values, * p < 0.05, ** p < 0.01, *** p < 0.001, compared with 0 μM sorafenib in hiPSC−CMs., ## p < 0.01, compared with 0 μM sorafenib in H9c2 cells. Data are shown as the mean ± SEM. (c) hiPSC−CMs and H9c2 cells treated with sorafenib under 4 μM for 0,12,24,48, and 72 h. Apoptosis markers (cleaved caspase 9, cleaved caspase 3) and autophagy marker (LC3B) were analyzed by Western blot. (d) Quantification of cleaved caspase 9, cleaved caspase 3, and LC3B expression from diagram c. p-values, * p < 0.05, ** p < 0.01, 4 μM sorafenib compared with 0 μM sorafenib in hiPSC−CMs. # p < 0.05, ## p < 0.01, 4 μM sorafenib compared with 0 μM sorafenib in H9c2 cells. Data are shown as the mean ± S.E.M. (e) Schematic representation of the postulated mechanism of action of sorafenib.

Figure 7

Diagrammatic illustration of preponderance of drug screening platform by iPSC-induced cardiomyocytes.