Department of Internal Medicine I, Division of Cardiology, University Hospital Jena, FSU, Jena, Germany.
Department of Internal Medicine I, Division of Cardiology, University Hospital Jena, FSU, Jena, Germany.
Free Radic Biol Med. 2021 May 1;167:66-80. doi: 10.1016/j.freeradbiomed.2021.02.016. Epub 2021 Mar 9.
Oversupply of fatty acids (FAs) to cardiomyocytes (CMs) is associated with increased ceramide content and elevated the risk of lipotoxic cardiomyopathy. Here we investigate the role of ceramide accumulation on mitochondrial function and mitophagy in cardiac lipotoxicity using CMs derived from human induced pluripotent stem cell (hiPSC). Mature CMs derived from hiPSC exposed to the diabetic-like environment or transfected with plasmids overexpressing serine-palmitoyltransferase long chain base subunit 1 (SPTLC1), a subunit of the serine-palmitoyltransferase (SPT) complex, resulted in increased intracellular ceramide levels. Accumulation of ceramides impaired insulin-dependent phosphorylation of Akt through activating protein phosphatase 2A (PP2A) and disturbed gene and protein levels of key metabolic enzymes including GLUT4, AMPK, PGC-1α, PPARα, CD36, PDK4, and PPARγ compared to controls. Analysis of CMs oxidative metabolism using a Seahorse analyzer showed a significant reduction in ATP synthesis-related O consumption, mitochondrial β-oxidation and respiratory capacity, indicating an impaired mitochondrial function under diabetic-like conditions or SPTLC1-overexpression. Further, ceramide accumulation increased mitochondrial fission regulators such as dynamin-related protein 1 (DRP1) and mitochondrial fission factor (MFF) as well as auto/mitophagic proteins LC3B and PINK-1 compared to control. Incubation of CMs with the specific SPT inhibitor (myriocin) showed a significant increase in mitochondrial fusion regulators the mitofusin 2 (MFN2) and optic atrophy 1 (OPA1) as well as p-Akt, PGC-1 α, GLUT-4, and ATP production. In addition, a significant decrease in auto/mitophagy and apoptosis was found in CMs treated with myriocin. Our results suggest that ceramide accumulation has important implications in driving insulin resistance, oxidative stress, increased auto/mitophagy, and mitochondrial dysfunction in the setting of lipotoxic cardiomyopathy. Therefore, modulation of the de novo ceramide synthesis pathway may serve as a novel therapeutic target to treat metabolic cardiomyopathy.
脂肪酸（FAs）供应过盛与细胞内神经酰胺含量增加以及发生脂毒性心肌病的风险升高有关。在此，我们通过源自人诱导多能干细胞（hiPSC）的心肌细胞（CM）来研究神经酰胺积累对心脏脂毒性中线粒体功能和自噬的作用。在糖尿病样环境中暴露或转染过表达丝氨酸棕榈酰转移酶长链碱基亚基 1（SPTLC1）的质粒的 hiPSC 衍生的成熟 CM 导致细胞内神经酰胺水平升高。神经酰胺的积累通过激活蛋白磷酸酶 2A（PP2A）来抑制胰岛素依赖性 Akt 的磷酸化，并扰乱包括 GLUT4、AMPK、PGC-1α、PPARα、CD36、PDK4 和 PPARγ 在内的关键代谢酶的基因和蛋白水平，与对照组相比，这种情况更为明显。使用 Seahorse 分析仪对 CMs 氧化代谢进行分析显示，与对照相比，与 ATP 合成相关的 O 消耗、线粒体β-氧化和呼吸能力显著降低，表明在糖尿病样条件下或 SPTLC1 过表达时存在受损的线粒体功能。此外，与对照组相比，神经酰胺的积累增加了线粒体分裂调节剂，如 dynamin-related protein 1（DRP1）和 mitochondrial fission factor（MFF）以及自噬/线粒体自噬蛋白 LC3B 和 PINK-1。与对照相比，用特异性 SPT 抑制剂（曲古抑菌素）孵育 CMs 后，线粒体融合调节剂 mitofusin 2（MFN2）和 optic atrophy 1（OPA1）以及 p-Akt、PGC-1α、GLUT-4 和 ATP 产量显著增加。此外，在用曲古抑菌素处理的 CMs 中，自噬/线粒体自噬和细胞凋亡显著减少。我们的结果表明，神经酰胺的积累在脂毒性心肌病中对驱动胰岛素抵抗、氧化应激、增加自噬/线粒体自噬和线粒体功能障碍具有重要意义。因此，调节从头合成神经酰胺的途径可能成为治疗代谢性心肌病的新治疗靶点。
