Store-operated calcium entry via ORAI1 regulates doxorubicin-induced apoptosis and prevents cardiotoxicity in cardiac fibroblasts

Abstract

Despite exhibiting cardiotoxicity, doxorubicin (DOX) is widely used for cancer treatments. Cardiac fibroblasts (CFs) are important in the pathogenesis of heart failure. This necessitates the study of the effect of DOX on CFs. The impairment of calcium (Ca2+) homeostasis is a common mechanism of heart failure. Store-operated Ca2+ entry (SOCE) is a receptor-regulated Ca2⁺ entry pathway that maintains calcium balance by sensing reduced calcium stores in the endoplasmic reticulum. ORAI1, a calcium channel protein and the most important component of SOCE, is highly expressed in human cardiac fibroblasts (HCFs). It is upregulated in CFs from failing ventricles. However, whether ORAI1 in HCFs is increased and/or plays a role in DOX-induced cardiotoxicity remains unknown. In this study, we aimed to elucidate the relationship between ORAI1/SOCE and DOX-induced heart failure. Induction of apoptosis by DOX was characterized in HCFs. Apoptosis and cell cycle analyses were performed by fluorescence-activated cell sorting (FACS). Reactive oxygen species (ROS) production was measured using fluorescence. YM-58483 was used as an ORAI1/SOCE inhibitor. ORAI1-knockdown cells were established by RNA interference. In vivo experiments were performed by intraperitoneally injecting YM-58483 and DOX into mice. We first demonstrated that DOX significantly increased the protein expression level of p53 in HCFs by western blotting. FACS analysis revealed that DOX increased early apoptosis and induced cell cycle arrest in the G2 phase in fibroblasts. DOX also increased ROS production. DOX significantly increased the expression level of ORAI1 in CFs. Both YM-58483 and ORAI1 gene knockdown attenuated DOX-induced apoptosis. Similarly, YM-58483 attenuated cell cycle arrest in the G2 phase, and ORAI1 knockdown attenuated DOX-induced ROS production in HCFs. In the animal experiment, YM-58483 attenuated DOX-induced apoptosis. In HCFs, ORAI1/SOCE regulates p53 expression and plays an important role in DOX-induced cardiotoxicity. ORAI1 may serve as a new target for preventing DOX-induced heart failure.

EP4-induced mitochondrial localization and cell migration mediated by CALML6 in human oral squamous cell carcinoma

“…The human oral squamous cell carcinoma cell lines HSC-3 and OSC-19, which are high metastatic potential lines, were purchased from the Health Science Research Resources Bank (Japan Health Sciences Foundation, Tokyo, Japan) 9 , 46 . The human oral squamous cell carcinoma cell lines SAT and HSC-4 were acquired from the Japanese Collection of Research Bioresources (Osaka, Japan).…”

“…Western blot analyses were performed as using the following method 9 , 46 . Briefly, cells were lysed and scraped in RIPA buffer (Thermo Scientific, IL, USA).…”

“…Total RNA from HSC-3 cells was extracted using the ISOSPIN Cell & Tissue RNA Kit (Nippon Gene, Tokyo, Japan) 46 . The quality of the raw paired-end sequencing reads was assessed with FastQC.…”

Progress on role of ion channels of cardiac fibroblasts in fibrosis

“…There are a variety of cells in the heart, they contain cardiomyocytes, fibroblasts, endothelial cells and smooth muscle cells, of which fibroblasts are the most, accounting for about 75% ( Eghbali, 1992 ; Camelliti et al, 2005 ; Nemoto et al, 2022 ). Their main functions are to produce and maintain the homeostasis of myocardial ECM ( Eghbali, 1992 ; Ottaviano and Yee, 2011 ), as well as produce growth factors (GFs), cytokines and intercellular signaling molecules, affecting cell proliferation, angiogenesis, cardiomyocyte hypertrophy and apoptosis ( Ottaviano and Yee, 2011 ; Mitrokhin et al, 2019 ).…”

“…Currents through CRAC channels are critically dependent on the correct function of two proteins-Orai1 and STIM1 ( Humer et al, 2022 ). Orai1 is highly expressed in HCFs ( Nemoto et al, 2022 ). A recent study demonstrated that Orai1 expression was upregulated in CFs from failing ventricles, indicating the pivotal role played by Orai1 in HF ( Ross et al, 2017 ).…”