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Blocking Store‐Operated Ca 2+ Entry to Protect Cardiomyocytes from Epirubicin‐Induced Toxicity
Author(s) -
Liu Xian,
Chang Yan,
Choi Sangyong,
Pan Zui
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.06876
Subject(s) - epirubicin , intracellular , apoptosis , extracellular , chemistry , thapsigargin , microbiology and biotechnology , pharmacology , medicine , endocrinology , biology , biochemistry , cancer , breast cancer
Epirubicin (EPI) is one of widely used anthracycline chemotherapy drugs, yet its cardiotoxicity severely limits its clinical application. Altered intracellular Ca 2+ homeostasis has been linked to epirubicin‐induced cell death and hypertrophy. While store‐operated Ca 2+ entry (SOCE) recently has been linked with cardiac hypertrophy and heart failure, its role in epirubicin‐induced cardiotoxicity remains unknown. METHOD HL‐1, a cardiomyocyte cell line derived from adult mouse atria, was used in this study. Intracellular ROS was measured by dihydroethidium (DHE) fluorescence and intracellular Ca 2+ was monitored by ratiometric dye Fura‐2 on a spectrofluorometer (excitation λ = 350/385nm and emission λ = 510nm; Photon Technology International, NJ). Upon depletion of ER Ca 2+ stores by 10μM thapsigargin (TG) in 0mM Ca 2+ extracellular solution, SOCE was activated when extracellular solution was rapidly exchanged to 2mM Ca 2+ . Apoptosis assay was conducted by nuclei staining, F‐actin phalloidin staining and Western Blotting of caspase‐3. While cell size was compared in HL‐1 cells with different treatments using Image J, the mRNA of Orai1, Orai3, STIM1 and B‐type natriuretic peptide (BNP) were also compared using real time qPCR. RESULTS Incubation of 1uM EPI for 30min induced significant ROS production in cultured HL‐1 cells compared with vehicle control (10410±1492 vs. 2762±939, p<0.0001). EPI induced an enhanced SOCE (0.1214±0.0104 vs. 0.0682±0.0062, n=32, p=0.0008), likely through increased expression of SOCE components Orai1, Orai3, and STIM1 compared with control cells. Apoptosis was observed upon EPI treatment, indicated by the condensed and fragmented nuclei, degradation of F‐actin and increased cleavage of caspase‐3 protein. EPI treatment also resulted in larger cell size (3894±120 μm 2 ) compared with non‐treated control cells (2452±57 μm 2 , n=581, p<0.0001). The hypertrophy marker BNP was up‐regulated upon EPI treatment as well. BTP‐2, a known SOCE blocker, could block this EPI‐induced SOCE and rescue HL‐1 cells from EPI‐induced apoptosis. BTP‐2 also prevented HL‐1 cells from EPI‐induced hypertrophy in terms of cell size (see Figure , 2548±69 μm 2 ) and also reduction of BNP expression. CONCLUSION Epirubicin upregulated SOCE, induced apoptosis and hypertrophy in HL‐1 cardiomyocytes. Blocking SOCE by BTP‐2 could at least partially rescue HL‐1 cells from epirubicin‐induced apoptosis and hypertrophy. Support or Funding Information (1) NIH S10 OD025230 to Dr. Zui Pan(2) University of Texas at Arlington Center for Research and Scholarship Pilot Grant for Trainee to Xian Liu