The Significance Of Autophagy And UPR Signaling In Myocardial Adaptation To Chronic Hypoxia

Background and Objective:Congenital heart disease, especially cyanotic cardiac defects which result in chronic hypoxia in the neonate until operative correction is achieved, remains a major cause of death in infancy. Myocardial adaptation to chronic hypoxia is critical for the survival and function of cardiomyocytes. Under hypoxic condition, the maturation of protein in the cardiomyocyte was frustrated. Many misfolded or unfolded protein accumulating in the endoplasmic reticulum (ER) triggers ER stress. Cells have developed elaborate protein quality control systems that recognize improperly folded proteins and either refold them or facilitate their degradation. One such quality control system is the unfolded protein response, or the UPR. The UPR is a highly conserved signal transduction system that is activated when cells are subjected to ER stress in ways that impair the folding of nascent proteins in this organelle. Recent observations indicate that in the heart, the UPR is activated during acute stresses, including ischemia/reperfusion. However, the roles of UPR in the longer term stresses such as chronic hypoxia have not been well defined.Autophagy is an evolutionarily conserved process that results in the degradation of cytosolic components inside lysosomes. Under normal conditions, it is a nonstop, reparative, life-sustaining mechanism for recycling cellular components, such as long-lived proteins and damaged organelles. Therefore, autophagy is thought to be involved in many physiological processes, including cellular differentiation, tissue remodeling, growth control, cell defense and adaptation to an adverse environment. Autophagy can be stimulated and this occurs as a cellular response to both extracellular (e.g. nutrient starvation, hypoxia, overcrowding, high temperature) and intracellular (e.g. accumulation of damaged or superfluous organelles) stress conditions. Also, both ER stress and the UPR could trigger autophagy. However, whether autophagy plays a protective role in cardiomyocytes under chronic hypoxia is unclear. So, the aim of this study is to provide an insight into the involvement of autophagy and the UPR in the myocardial adaptation to chronic hypoxia both in vivo and in vitro.Methods:Samples taken from the right ventricular outflow tract were collected from patients with cyanotic(n = 19) or acyanotic (n = 12) congenital heart disease. Autophagosome was studied by electromicroscopy, while the protein level of LC3Ⅰand LC3Ⅱwere evaluated by western blotting. For the activity of UPR, the expression of Bip was examined by immunohistochemistry, while XBP-1 splicing mRNA and Bip mRNA were tested by RT-PCR. The XBP-1s protein, phorspho-eIF2α, Bip, ATF6f and phorspho-PERK were tested by western blotting.To evaluated the effect of chronic hypoxia on each branch of the UPR in the cardiomyocytes in vitro, primary rat cardiomyocytes and embryonic rat-heart-derived H9c2 cells were cultivated and exposed to 1.0% O2, 5.0% CO2 for different durations to establish the chronic hypoxic cell model. Control cells were cultivated in the same conditions except for 21% O2 concentration. After different duration of hypoxic exposure, cells were collected and subjected to RT-PCR and western blot to detecting the mRNA and protein expression of the UPR.Results:1. By electron microscope, we found autophagosome in the cytoplasm.The rate of LC3Ⅱ/LC3Ⅰwas increased (p < 0.001) in the myocardium from patient with cyanotic cardiac defect.2. Immunohistochemistry revealed that GRP78/Bip was expressed in the cytoplasm. The protein levels of GRP78/Bip were significantly elevated (p < 0.001) in patients with cyanotic compared to acyanotic congenital heart disease. Also, the expression of GRP78/Bip mRNA were markedly increased in cyanotic patients (p < 0.05).3. Western blot analysis revealed that the level phospho-PERK, phorspho-eIF2αwas elevated in cyanotic hearts compared with their acyanotic counterparts (p < 0.001); also, the level of ATF6 fragment was increased (p < 0.01). However, there are no significant difference about the XBP-1 splicing mRNA and XBP-1s protein expression in this two groups (p > 0.05).4. The expression of XBP-1 splicing mRNA and XBP-1s protein in hypoxic cultivated H9c2 cells were significantly increased in the first 6h after exposed to hypoxia. And, it decreased sharply to the control level.5. In hypoxia-esposed cells, the levels of phospho-PERK, phorspho-eIF2αwere increased in the first 12h, and stayed at nearly that high level during the period of hypoxia.6. Similarily, ATF6 fragment, Bip mRNA and rotein were all increased in the 24h after exposed to hypoxia, and remained highly expression.Conclusions:Expression of autophagy was increased in patients with cyanotic cardiac defect. Upregulation of endogenous UPR in cardiomyocytes under chronic hypoxia has been observed both in vivo and in vitro. Taken together, the findings here suggested that UPR might plays an important role in myocardial adaptation to chronic hypoxia.