Cardiac Ischemia On-a-Chip: Antiarrhythmic Effect of Levosimendan on Ischemic Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Abstract

Ischemic heart disease (IHD) is one of the leading causes of mortality worldwide. Preserving functionality and preventing arrhythmias of the heart are key principles in the management of patients with IHD. Levosimendan, a unique calcium (Ca²⁺) enhancer with inotropic activity, has been introduced into clinical usage for heart failure treatment. Human-induced pluripotent cell-derived cardiomyocytes (hiPSC-CMs) offer an opportunity to better understand the pathophysiological mechanisms of the disease as well as to serve as a platform for drug screening. Here, we developed an in vitro IHD model using hiPSC-CMs in hypoxic conditions and defined the effects of the subsequent hypoxic stress on CMs functionality. Furthermore, the effect of levosimendan on hiPSC-CMs functionality was evaluated during and after hypoxic stress. The morphology, contractile, Ca²⁺-handling, and gene expression properties of hiPSC-CMs were investigated in response to hypoxia. Hypoxia resulted in significant cardiac arrhythmia and decreased Ca²⁺ transient amplitude. In addition, disorganization of sarcomere structure was observed after hypoxia induction. Interestingly, levosimendan presented significant antiarrhythmic properties, as the arrhythmia was abolished or markedly reduced with levosimendan treatment either during or after the hypoxic stress. Moreover, levosimendan presented significant protection from the sarcomere alterations induced by hypoxia. In conclusion, this chip model appears to be a suitable preclinical representation of IHD. With this hypoxia platform, detailed knowledge of the disease pathophysiology can be obtained. The antiarrhythmic effect of levosimendan was clearly observed, suggesting a possible new clinical use for the drug.

Keywords: antiarrhythmic effect; calcium transient; cardiac ischemia on-a-chip; human-induced pluripotent stem cell-derived cardiomyocytes; ischemic heart disease; levosimendan.

Figure 1

Structure and function of the OxyGenie mini-incubator: 1-well assembly includes a 1-well chamber on a glass plate, a lid and lid lock, and a cover and cover lock to maintain the required gas environment.

Figure 2

Schematic diagram of the experimental workflow in (A) control, (B) hypoxia without levosimendan (levo.), and (C) hypoxia with levosimendan groups.

Figure 3

Representative Ca²⁺ transient categories of hiPSC-CMs. (a) Normal. Abnormal Ca²⁺ traces showing (b) irregular phase, (c) double peaks, (d) multiple peaks, (e) prolonged rise, (f) plateau abnormality, (g) low peaks, and (h) high peaks.

Figure 4

Incidence percent of (a) normal and abnormal Ca²⁺ transients of hiPSC-CMs. (b) Different Ca²⁺ transient abnormality categories in control, hypoxia without levosimendan, and hypoxia with levosimendan groups. The sample sizes in control group were 0 h (baseline) n = 38, 4 h n = 34, 7 h n = 36, and 10 h n = 37; in the hypoxia without levosimendan group, the sample size was 0 h (baseline) n = 46, 4 h n = 57, 7 h n = 56, 10 h n = 53 and following 10 h n = 20; and in the hypoxia with levosimendan group, the sample size was 0 h (baseline) n = 19, 4 h n = 24, 7 h n = 19, and 10 h n = 18.

Figure 5

Levosimendan addition abolished or significantly reduced ischemia-induced arrhythmia either during or after hypoxic stress.

Figure 6

Parameters of the Ca²⁺ transient in the control and hypoxic conditions with or without levosimendan until 10 h and after levosimendan addition following 10 h of hypoxia: (a) frequency: the beating frequency of CMs; (b) ΔF/F0: Ca²⁺ peak amplitude; (c) beat-to-beat interval: time from the beginning of one Ca²⁺ transient until the beginning of the next transient; (d) peak duration: Ca²⁺ peak duration; (e) half-width: duration at half-maximum amplitude; (f) rise time 10% to 90%: rise time of the Ca²⁺ transient starting from 10% until 90% of the transient rise; (g) decay time 90% to 10%: decay time of the Ca²⁺ transient starting from 90% until 10% of the transient decay. Data are shown as averages. Statistical significance: * p < 0.05; ** p < 0.01; and *** p < 0.001. Error bars represent the standard error of the mean (SEM).

Figure 6

Parameters of the Ca²⁺ transient in the control and hypoxic conditions with or without levosimendan until 10 h and after levosimendan addition following 10 h of hypoxia: (a) frequency: the beating frequency of CMs; (b) ΔF/F0: Ca²⁺ peak amplitude; (c) beat-to-beat interval: time from the beginning of one Ca²⁺ transient until the beginning of the next transient; (d) peak duration: Ca²⁺ peak duration; (e) half-width: duration at half-maximum amplitude; (f) rise time 10% to 90%: rise time of the Ca²⁺ transient starting from 10% until 90% of the transient rise; (g) decay time 90% to 10%: decay time of the Ca²⁺ transient starting from 90% until 10% of the transient decay. Data are shown as averages. Statistical significance: * p < 0.05; ** p < 0.01; and *** p < 0.001. Error bars represent the standard error of the mean (SEM).

Figure 7

(a) Incidence percent of normal and abnormal rhythm of hiPSC-CMs and (b) incidence percent of different rhythm abnormality categories. (c) Beating frequency of hiPSC-CMs in control and hypoxia without levosimendan groups. The sample sizes in the control group were 0 h (baseline) n = 38, 4 h n = 36, 7 h n = 35, and 10 h n = 38 and under the hypoxic condition were 0 h (baseline) n = 20, 4 h n = 76, 7 h n = 112, and 10 h n = 110. Data are shown as averages. Statistical significance: * p < 0.05 and *** p < 0.001. Error bars represent the standard error of the mean (SEM).

Figure 8

Representative images of immunocytochemical staining of hiPSC-CMs by cardiac alpha-actinin (red), HIF1α (green), and dapi (blue). Hypoxia changed the structure, sarcomere organization, and nucleus size of hiPSC-CMs. Levosimendan addition abolished or significantly reduced the structure and sarcomere alteration either during or after hypoxic stress. Scale bar corresponds to 20 µm.

Figure 9

Relative gene expression levels of cardiac-related genes: (a) Ca²⁺-handling (CACNA1C and SLC8A1) gene expression; (b) hypoxia marker (HIF1A) gene expression; (c) RYR2, ATP2A2, TNNT2, ABCC9, KCNJ8, and KCNJ11 gene expression. Samples from qPCR analysis presented as mean + standard deviation. * Statistical significance (p < 0.05).