P391Hypoxia aggravates the impact of cardiomyocyte triglyceride accumulation promoted by electronegative LDL

Purpose: The triglyceride (TG) content in the human heart is increased in insulin resistance-related diseases. Plasma lipoproteins are source of lipids for the heart. Electronegative LDL [LDL(-)] is a modified subfraction of LDL which is elevated in different cardiometabolic diseases wih a high cardiovascular component. Our purpose was to assess the effect of LDL(-) on the intracellular neutral lipid accumulation in cardiomyocytes under normoxic and hypoxic conditions. Methods: LDL(-) was isolated from total LDL by anion-exchange chromatography. HL-1 cardiomyocytes were exposed to increasing concentrations of either native LDL (nLDL) or LDL(-) under normoxia (21% O2) or hypoxia (1% O2). Intracellular cholesteryl ester (CE) and TG content was analyzed by TLC after cellular lipid extraction. The distribution of LDL-induced intracellular lipid accumulation was assessed by confocal microscopy experiments in HL-1 cells exposed to DiI-labeled nLDL and DiI-labeled LDL(-). Gene expression and protein level determination was carried out by real time PCR and western blot analysis, respectively. Results: LDL(-), differently to native LDL, induced a strong intracellular TG accumulation in HL-1 cardiomyocytes. The intracellular TG accumulation induced by the exposition to LDL(-) was strongly enhanced under hypoxic conditions. Likewise nLDL, endocytosed DiI-LDL(-) was detected into lipid droplets homogeneously distributed around the nucleus of cells under normoxic conditions. Conversely, under hypoxic conditions, the distribution of internalized lipids from DiI-LDL(-) was spread widely into the whole cytoplasm. Furthermore, this abnormal distribution of intracellular lipids was associated with a reduction in the expression of perilipin 5, a key protein involved in the intracellular lipid storage and metabolism, and an exacerbated downregulatory effect of hypoxia on the gene expression of carnitine palmitoyltransferase I, an enzyme essential for mitochondrial fatty acid β-oxidation. These array of changes were associated with increased cellular levels of the reactive oxygen production (ROS) production in HL-1 cardiomyocytes exposed to LDL(-) under hypoxia. Conclusions: LDL (-), by promoting an altered and detrimental lipid intracellular storage and metabolism, may contribute to exacerbate the effects of ischemia. Therefore, our results highlight a potential relevant and novel mechanism underlying cardiometabolic complications of diabetes.