Erdi Sozen1, Burak Yazgan1, Olgu Enis Tok2, Tugce Demirel1, Feriha Ercan2, Jonathan D Proto3, Nesrin Kartal Ozer4. 1. Department of Biochemistry, Faculty of Medicine, Genetic and Metabolic Diseases Research and Investigation Center (GEMHAM), Marmara University, Maltepe, Istanbul 34854, Turkey. 2. Department of Histology and Embryology, Faculty of Medicine, Marmara University, Maltepe, Istanbul 34854, Turkey. 3. Department of Medicine, Columbia University, New York, NY 10032, USA. 4. Department of Biochemistry, Faculty of Medicine, Genetic and Metabolic Diseases Research and Investigation Center (GEMHAM), Marmara University, Maltepe, Istanbul 34854, Turkey. Electronic address: nkozer@marmara.edu.tr.
Abstract
BACKGROUND: Cardiovascular diseases (CVDs), with highest mortality and morbidity rates, are the major cause of death in the world. Due to the limited information on heart tissue changes, mediated by hypercholesterolemia, we planned to investigate molecular mechanisms of endoplasmic reticulum (ER) stress and related cell death in high cholesterol fed rabbit model and possible beneficial effects of α-tocopherol. METHODS: Molecular changes in rabbit heart tissue and cultured cardiomyocytes (H9c2 cells) were measured by western blotting, qRT-PCR, immunflouresence and flow cytometry experiments. Histological modifications were assessed by light and electron microscopes, while degradation of mitochondria was quantified through confocal microscope. RESULTS: Feeding rabbits 2% cholesterol diet for 8 weeks and treatment of cultured cardiomyocytes with 10 μg/mL cholesterol for 3 h induced excessive autophagic activity via IRE1/JNK pathway. While no change in ER-associated degradation (ERAD) and apoptotic cell death were determined, electron and confocal microscopy analyses in cholesterol supplemented rabbits revealed significant parameters of autophagic cell death, including cytoplasmic autophagosomes, autolysosomes and organelle loss in juxtanuclear area as well as mitochondria engulfment by autophagosome. Either inhibition of ER stress or JNK in cultured cardiomyocytes or α-tocopherol supplementation in rabbits could counteract the effects of cholesterol. CONCLUSION: Our findings underline the essential role of hypercholesterolemia in stimulating IRE1/JNK branch of ER stress response which then leads to autophagic cell death in heart tissue. Results also showed α-tocopherol as a promising regulator of autophagic cell death in cardiomyocytes.
BACKGROUND:Cardiovascular diseases (CVDs), with highest mortality and morbidity rates, are the major cause of death in the world. Due to the limited information on heart tissue changes, mediated by hypercholesterolemia, we planned to investigate molecular mechanisms of endoplasmic reticulum (ER) stress and related cell death in high cholesterol fed rabbit model and possible beneficial effects of α-tocopherol. METHODS: Molecular changes in rabbit heart tissue and cultured cardiomyocytes (H9c2 cells) were measured by western blotting, qRT-PCR, immunflouresence and flow cytometry experiments. Histological modifications were assessed by light and electron microscopes, while degradation of mitochondria was quantified through confocal microscope. RESULTS: Feeding rabbits 2% cholesterol diet for 8 weeks and treatment of cultured cardiomyocytes with 10 μg/mL cholesterol for 3 h induced excessive autophagic activity via IRE1/JNK pathway. While no change in ER-associated degradation (ERAD) and apoptotic cell death were determined, electron and confocal microscopy analyses in cholesterol supplemented rabbits revealed significant parameters of autophagic cell death, including cytoplasmic autophagosomes, autolysosomes and organelle loss in juxtanuclear area as well as mitochondria engulfment by autophagosome. Either inhibition of ER stress or JNK in cultured cardiomyocytes or α-tocopherol supplementation in rabbits could counteract the effects of cholesterol. CONCLUSION: Our findings underline the essential role of hypercholesterolemia in stimulating IRE1/JNK branch of ER stress response which then leads to autophagic cell death in heart tissue. Results also showed α-tocopherol as a promising regulator of autophagic cell death in cardiomyocytes.