Blocking Store‐Operated Ca2+ Entry to Protect Cardiomyocytes from Epirubicin‐Induced Toxicity

Abstract

Epirubicin (EPI) is one of widely used anthracycline chemotherapy drugs, yet its cardiotoxicity severely limits its clinical application. Altered intracellular Ca2+ homeostasis has been linked to epirubicin‐induced cell death and hypertrophy. While store‐operated Ca2+ entry (SOCE) recently has been linked with cardiac hypertrophy and heart failure, its role in epirubicin‐induced cardiotoxicity remains unknown.METHODHL‐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 Ca2+ 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 Ca2+ stores by 10μM thapsigargin (TG) in 0mM Ca2+ extracellular solution, SOCE was activated when extracellular solution was rapidly exchanged to 2mM Ca2+. 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.RESULTSIncubation 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 μm2) compared with non‐treated control cells (2452±57 μm2, 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 μm2) and also reduction of BNP expression.CONCLUSIONEpirubicin 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 LiuFigure 1