Heat Stress Induces Apoptosis through a Ca2+-Mediated Mitochondrial Apoptotic Pathway in Human Umbilical Vein Endothelial Cells

Abstract

BackgroundHeat stress can be acutely cytotoxic, and heat stress-induced apoptosis is a prominent pathological feature of heat-related illnesses, although the precise mechanisms by which heat stress triggers apoptosis are poorly defined.MethodsThe percentages of viability and cell death were assessed by WST-1 and LDH release assays. Apoptosis was assayed by DNA fragmentation and caspase activity. Expression of cleaved PARP, Apaf-1, phospho-PERK, Phospho-eIF2a, ATF4, XBP-1s, ATF6, GRP78, phospho-IP3R, RYR and SERCA was estimated by Western blot. The effect of calcium overload was determined using flow cytometric analysis with the fluorescent probe Fluo-3/AM. The generation of ROS (O2 −, H2O2, NO) was labeled by confocal laser scanning microscopy images of fluorescently and flow cytometry.ResultsIn this study, we found that heat stress in HUVEC cells activated initiators of three major unfolded protein response (UPR) signaling transduction pathways: PERK-eIF2a-ATF4, IRE1-XBP-1S and ATF6 to protect against ER stress, although activation declined over time following cessation of heat stress. Furthermore, we show that intense heat stress may induce apoptosis in HUVEC cells through the calcium-mediated mitochondrial apoptotic pathway, as indicated by elevation of cytoplasmic Ca2+, expression of Apaf-1, activation of caspase-9 and caspase-3, PARP cleavage, and ultimately nucleosomal DNA fragmentation; Reactive oxygen species (ROS) appear to act upstream in this process. In addition, we provide evidence that IP3R upregulation may promote influx of Ca2+ into the cytoplasm after heat stress.ConclusionOur findings describe a novel mechanism for heat stress-induced apoptosis in HUVEC cells: following elevation of cytoplasm Ca2+, activation of the mitochondrial apoptotic pathway via the IP3R upregulation, with ROS acting as an upstream regulator of the process.

Luteolin Modulates SERCA2a Leading to Attenuation of Myocardial Ischemia/ Reperfusion Injury via Sumoylation at Lysine 585 in Mice

“…It has been reported that with the decline of SERCA2a level, myocardial cells lose their ability to transport calcium from the cytosol into the SR. The abnormal high cytoplasmic calcium level could result in mitochondrial permeability transition pore opening, ΔΨm decrease, and finally cause apoptosis of cardiomyocytes [32]. In addition, Lut can regulate cell apoptosis by enhancing autophagy [33, 34].…”

Endoplasmic reticulum stress: a novel mechanism and therapeutic target for cardiovascular diseases

“…In addition, mouse aortic endothelial cells from AMPK2 knockout mice showed higher expression of ERS markers such as XBP1, ATF6, GRP78, p-eIF2α, p-PERK, p-JNK and an increased level of intracellular Ca 2+ . Li et al [92] reported that heat stress activated UPR signaling through the PERK-eIF2α-ATF4, IRE1-XBP1s and ATF6 pathways in human umbilical vein endothelial cells. ERS also mediated aldosterone (Aldo)-induced apoptosis in vascular endothelial cells, as evidenced by increasing expression of GRP78 and CHOP.…”

Mechanism of juglone-induced apoptosis of MCF-7 cells by the mitochondrial pathway

“…In the early phase of apoptosis, mitochondrial Ca 2+ release leads to an increase in intracellular Ca 2+ levels (Akopova et al, 2013). Sustained high levels of ROS likely promote destruction of organelle and cell membranes, as well as redistribution and continuous intracellular accumulation of Ca 2+ (Li et al, 2014). Thus, the generation of ROS and the increase in intracellular Ca 2+ concentration are interconnected during the progression of apoptosis.…”