Microwave Radiation Induced Human iPSC Derived Cardiomyo-cytes Injury

Research Article

Austin Cardio & Cardiovasc Case Rep. 2023; 8(2): 1058.

Microwave Radiation Induced Human iPSC Derived Cardiomyo-cytes Injury

Liang Yue1#; Jing Zhang2#; Zhi-Min Yun1; Peng-Fei Zhong1,4; Rui-Lin Ma1,3; Xin-Ping Xu2; Hong-Tu Cui1; Bin-Wei Yao2; Lei-Ming Fang1; Qi Liu1; Cheng-Jun Wu3; Rui-Yun Peng2*; Ying-Xia Tan1*

¹Institute of Health Service and Transfusion Medicine, Academy of Military Medical Sciences, Academy of Military Sciences, Beijing, China

²Beijing Institute of Radiation Medicine, Academy of Military Medical Sciences, Academy of Military Sciences, Beijing, China

³School of BME, Faculty of Medicine, Dalian University of Technology, Dalian, China

&sup4;Graduate School, Hebei North University, Zhangjiakou, Hebei Province, China

*Corresponding author: Ying-Xia Tan Institute of Health Service and Transfusion Medicine, Academy of Military Medical Sciences, Academy of Military Sciences, Beijing, China.

Rui-Yun Peng, Beijing Institute of Radiation Medicine, Academy of Military Medical Sciences, Academy of Military Sciences, Beijing, China. Email: ruiyunpeng18@126.com; tanhu333@126.com

#These authors have contributed equally to this article.

Received: July 07, 2023 Accepted: August 14, 2023 Published: August 21, 2023

Abstract

Electromagnetic waves are recognized as the third major source of pollution, which can damage to the heart. However, due to the limitation of human subjects in research, it is difficult to extrapolate the biological effects of electromagnetic waves directly from animals to humans. In this study, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were used to explore the effect of microwave radiation on human heart injury. Human iPSC-CMs were radiated with a single irradiation dose of 30W/Kg for 30 min. After radiation, the content of Reactive Oxygen Species (ROS) in hiPSC-CMs increased significantly, and the Mito-tracker and mitochondrial membrane potential decreased synchronously. Seahorse assay found that the mitochondrial functions in cardiomyocytes were weakened after being exposed irradiation. The electrophysiological functions of hiPSC-CMs were significantly abnormal, with increased proportions of late apoptosis and decreased proportions of viable cells. In addition, the secretion of 10 pro-inflammatory cytokines/chemokines was increased in hiPSC-CMs after radiation. An effector model of microwave irradiation-induced cardiac injury was established using hiPSC-CMs in this study. Based on the fully observation of the structure and functions of iPSC-CMs after radiation, it was speculated that mitochondrial damage might be the dominant cause of delayed intracellular energy transfer, which was synchronously manifested as abnormal electrophysiological functions. The findings of this study provide valuable data for the clinical transformation of basic experimental research, and the damage mechanism will be explored in the future in order to provide effective assistance to the occupational practitioners.

Keywords: Microwave; hiPSC-CMs; Mitochondria; Cytokines; Apoptosis

Introduction

As an important member of the electromagnetic wave family, microwave is widely used in people's lives and work [1,2]. While it brings convenience to people's lives, it also causes certain health problems [3], such as insomnia, chest pain, and other symptoms. Studies have confirmed that microwave radiation has multi-system and multi-target effects on living organisms [4], of which heart is a particularly important target [5]. The cardiovascular system is one of the target systems that is sensitive to microwave radiation [6]. Microwave radiation can disrupt energy metabolism in cardiomyocytes, causing metabolic dysfunction and even apoptosis, resulting in structural and functional abnormalities in the heart [7]. The heart is the crucial organ in the circulatory system that ensures the blood supply to the organs of various organs of the body is maintained [8].

Epidemiological investigations have demonstrated that long-term exposure to low doses of microwave radiation can cause discomfort in the heart area of radiated individuals, and severe cases may exhibit symptoms such as chest tightness and precordial pain. Due to the influence on autonomic neuromodulation, irradiated individual mostly experience bradycardia, and a few with tachycardia [9,10]. Long-term, repeated exposure to microwave radiation with a power density greater than 100mW/cm 2 can lead to organic changes in the rat human cardiovascular system, such as palpitations, chest tightness, precordial pain, hypotension, slow heart rate, atrial and ventricular conduction delays, and ECG waveform changes [11]. At present, Wistar rats are commonly used animals for establishing models, and primary rat cardiomyocytes and murine H9C2 cell lines are frequently used cells in research on cardiac damage caused by microwave radiation. However, there is a big gap between these animal models and the human body, and a lack of objective evaluation indicators that can be converted for use in humans [12].

The depolarization process of human cardiomyocytes differs significantly from that of rodents. For example, under normal physiological conditions, the human heart rate is about 60-90 beats per minute, while the heart rates of rats and mice are 300-400 and 500-700 beats per minute, respectively. Consequently, there are great differences in comparability between different species and between the same species.

The bottleneck problem restricts the transformation of experimental results in this field. At present, iPSC-CMs have been successfully applied in clinical patients with heart failure and can characterize the complex physiological functions of human cardiomyocytes in vitro [13].

In this study, iPSC-CMs were taken as the modeling subject to study the effects of microwave radiation. The S-band was chosen as the simulated source of microwave radiation which was demonstrated in animal studies. And changes in the structure and function of iPSC-CMs were monitored. The purpose of this study was to explore new effect indicators and possible damage mechanisms, in order to provide a better fundamental work for the related research on protection and damage mechanisms [14].

Materials and Methods

hPSC Differentiation into Cardiomyocytes

Cardiomyocyte differentiation was performed on hiPSCs-B1 (blood-derived iPSCs, CA4025106, Cellapy). B1 hiPSC line (CA4025106, Cellapy) was cultured in mTeSR medium (Stemcell, 05850) and incubated at 37°C with 5% CO2. In brief, differentiation medium 1 consisted of RPMI 1640 (Gibco, 1744361) and B-27 (Gibco, A1895601), with extra CHIR-99021 (6 μM, Selleckchem, S2924) on day 0 and day 1 and IWR-1 (5 μM, Sigma, 10161) on day 4 and day 5. The medium was replaced with fresh differentiation medium 2 consisting of RPMI 1640 (Gibco, 1744361), B-27 (Gibco, A1895601), and 2% FBS on day 8.

Immunofluorescence Staining

The hiPSC-CMs were fixed in 4% PFA for 20 min and permeabilized with 0.1% Triton-X 100 for 5 min. The cells were incubated with the following primary antibodies overnight at 4°C: Troponin T 1:100, a- actinin 1:50. Then cells were washed with PBS and incubated with Alexa-conjugated secondary antibodies. Nuclei were stained with DAPI (Life Technologies, P36931). All images were acquired using a Nikon A1 confocal microscope.

Microwave Radiation

The hiPSC-CMs were divided into group C (the normal control group) and group R (the S-band radiation group). The dose was 30W/Kg for a single irradiation of 30min.

Transmission Electron Microscopy

At 24 h after radiation, hiPSC-CMs were scraped off with soft silicone sheets and centrifuged at 10,000 rpm for 10 min. The supernatant was removed; specimens were fixed in 2.5% glutaraldehyde fixation solution for 2h and 1% osmic acid fixation solution for 1h. Specimens were dehydrated in gradient ethanol and embedded in Epon-812 resin. Ultra-thin sections with a thickness of 70 nm were made and stained in uranium acetate and lead nitrate. All images were acquired using a transmission electron microscope (Hi-tachi).

Electrophysiological Detections

At 36 h after radiation, the field potential (beat period, FPD, FPDc and spike amplitude) and cell contractility (beat amplitude, excitation-contraction delay and mean beat width) of hiPSC-CMs were measured using a microelectrode application platform (Axion BioSystems).

Mito-Tracker Green Staining

The hiPSC-CMs were incubated with staining working solution containing 200 nM Mito-Tracker green (M46750, Invitrogen) for 45 min at 37°C. (1) After incubation with 1:1000 dilution of Hoechst 33342 for 10 min, images were acquired using a fluorescence microscope. (2) hiPSC-CMs were digested after staining, and fluorescence intensity was detected by flow cytometry. The average fluorescence intensity was calculated and analyzed using FlowJo software.

Detection of Mitochondrial Membrane Potential

The hiPSC-CMs were incubated with staining working solution containing 150nM TMRE (HY-D0985A, MCE) at room temperature in the dark for 10 min. (1) After incubation with 1:1000 dilution of Hoechst 33342 for 10 min, images were acquired using a fluorescence microscope. (2) hiPSC-CMs were digested after staining, and fluorescence intensity was detected by flow cytometry. The average fluorescence intensity was calculated and analyzed using FlowJo software.

Detection of Reactive Oxygen Species

The hiPSC-CMs were incubated with a staining working solution containing 5μM DCFH-DA (HY-D0940, MCE) at 37°C for 45 min. (1) After incubation with 1:1000 dilution of Hoechst 33342 for 10 min, images were acquired using a fluorescence microscope. (2) hiPSC-CMs were digested after staining, and fluorescence intensity was detected by flow cytometry. The average fluorescence intensity was calculated and analyzed using FlowJo software.

Seahorse Assay

The hiPSC-CMs (105 cells/well) were plated on an XF24 cell culture microplate coated with poly-D lysine (50 μg/mL). After being cultured at 37°C for two days (90% confluency of cells was reached), hiPSC-CMs were radiated and Seahorse assay was performed 24 h after irradiation. Experiments were done in XF assay medium that contained 25 mM glucose, 2 mM L-glutamine and 1 mM Na pyruvate and analyzed using a Seahorse XFe24 Extracellular Flux Analyzers (Agilent Technologies). When indicated, the following were injected: oligomycin (1 μM), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; 1 μM), rotenone (0.5 μM) and antimycin A (0.5 μM), glucose (10mM), oligomycin (1μM) and 2-Deoxy-D-glucose (2-DG; 50mM). Basal OCR and ECAR reports were generated by Wave Desktop software (Agilent Technologies).

Cytokines and Chemokine Assays

The supernatant of hiPSC-CM cultures was collected at 24 h after radiation to detect cytokines using Bio-Rad Bio-Plex Pro human cytokine 48-Plex assay and chemokine panel 40-plex kits (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. A total of 69 cytokines and chemokines were detected. Each sample was incubated in 96-well plates embedded with microbeads for 30 min and then incubated with detection antibody for 30 min. Furthermore, streptavidin-PE was filled in each well for 10 min, and values were read on Bio-Plex MAGPIX System (Bio-Rad, Hercules, CA, USA). The experiments were performed by Wayen Biotechnologies (Shanghai, China).

Apoptosis Assay

Apoptosis was detected using annexin V-FITC apoptosis detection kits (APOAF, Sigma) at 24 h after irradiation. The hiPSC-CMs were washed twice with DPBS and resuspended in a binding buffer at a concentration of 106 cells/mL. 5 μL of annexin V-FITC conjugate and 10 μL of propidium iodide solution were added to each cell suspension and incubated for 10 min at room temperature in the dark. The fluorescence intensity was measured immediately using a flow cytometer. The hiPSC-CMs in the early stages of the apoptotic process were stained with annexin V-FITC conjugate only. Viable hiPSC-CMs were not stained with either propidium iodide solution or annexin V-FITC conjugate. Necrotic hiPSC-CMs were stained with both propidium iodide solution and annexin V-FITC conjugate.

Statistical Analyses

The data in this paper were expressed as mean ± Standard Deviation (SD), the student’s t tests were used to analyze the difference between two groups, and all statistical analyses were two-tailed. The acceptable level of significance for all tests was P<0.05. The marker *represents P<0.05, **represents P<0.01 and ***represents P<0.001.

Results

Human Induced Pluripotent Stem Cells-Derived Cardiomyocytes

A small molecule-based monolayer differentiation protocol was adapted for cardiomyocyte differentiation in vitro using blood derived iPSCs (hiPSCs-B1). Spontaneously contracting Cardiomyocytes (CMs) were observed under light microscopy at days 8-10 after initiation of differentiation (Figure 1A). After 15 days in culture, hiPSC-CMs were immunostained illustrating that cells robustly expressed cardiac Troponin T and cardiac contractile proteins a-actinin (Figure 1B), showing successful differentiation.