Data_Sheet_1_Isoprenaline modified the lipidomic profile and reduced β-oxidation in HL-1 cardiomyocytes: In vitro model of takotsubo syndrome.docx

Recent studies have suggested a pathogenetic link between impaired mitochondria and Takotsubo syndrome (TTS), which is closely connected with catecholamine overstimulation, poor outcomes, and changes in lipid metabolism. We investigated the changes in lipid metabolism at the level of fatty acid β-oxidation and changes in the intracellular lipidomic spectrum. The immortalized cell line of HL-1 cardiomyocytes was used in this study as an established in vitro model of TTS. The cells were exposed to the non-selective β-agonist isoprenaline (ISO) for acute (2 h) and prolonged (24 h) periods. We investigated the impact on mitochondrial adenosine 5’-triphosphate (ATP) production and β-oxidation using real-time cell metabolic analysis, total lipid content, and changes in the lipidomic spectrum using high-performance liquid chromatography (HPLC) and mass spectrometry. Furthermore, modifications of selected lipid transporters were determined using real-time – polymerase chain reaction (RT-PCR) and/or Western blot techniques. By choosing this wide range of targets, we provide a detailed overview of molecular changes in lipid metabolism during catecholamine overstimulation. The present study demonstrates that acute exposure to ISO decreased ATP production by up to 42.2%, and prolonged exposure to ISO decreased β-oxidation by 86.4%. Prolonged exposure to ISO also increased lipid accumulation by 4%. Lipid spectrum analysis of prolonged exposure to ISO showed a reduced concentration of cardioprotective and an increased concentration of lipotoxic lipid molecules during long-term exposure. Decreased lipid utilization can lead to higher intracellular lipid accumulation and the formation of lipotoxic molecules. Changes in the lipid spectrum can induce pathophysiological signaling pathways leading to cardiomyocyte remodeling or apoptosis. Thus, changes in lipid metabolism induced by excessive doses of catecholamines may cause TTS and contribute to a progression of heart failure, which is at increased risk after a TTS episode.

近期研究证实，线粒体功能受损与Takotsubo综合征（Takotsubo syndrome, TTS）存在致病关联，而该综合征与儿茶酚胺过度刺激、不良预后及脂质代谢紊乱密切相关。本研究围绕脂肪酸β氧化层面的脂质代谢变化，以及细胞内脂质组谱的改变展开探究。本研究采用已被学界广泛验证的TTS体外模型——HL-1心肌细胞永生化细胞系作为实验对象，将细胞暴露于非选择性β受体激动剂异丙肾上腺素（isoprenaline, ISO）中，分别设置2小时急性处理与24小时长期处理两个实验组。本研究通过实时细胞代谢分析、总脂质含量检测，探究其对线粒体腺苷5'-三磷酸（adenosine 5’-triphosphate, ATP）生成及β氧化的影响；并采用高效液相色谱（high-performance liquid chromatography, HPLC）与质谱法，分析细胞脂质组谱的变化。此外，本研究通过实时荧光定量聚合酶链式反应（real-time polymerase chain reaction, RT-PCR）及/或蛋白质印迹（Western blot）技术，检测选定的脂质转运体的表达修饰情况。通过覆盖多维度研究靶点的实验设计，本研究详细阐明了儿茶酚胺过度刺激过程中脂质代谢的分子层面变化。本研究结果显示：急性ISO暴露可使细胞ATP生成最多降低42.2%，长期ISO暴露则使β氧化水平下降86.4%；同时长期ISO暴露还可使细胞内脂质积累增加4%。长期ISO暴露后的脂质谱分析结果显示，心脏保护性脂质分子的浓度显著降低，而脂毒性脂质分子的浓度则明显升高。脂质利用能力下降可导致细胞内脂质过度积累，并促生脂毒性分子。脂质谱的异常改变可激活病理生理信号通路，进而引发心肌细胞重塑或细胞凋亡。综上，过量儿茶酚胺诱导的脂质代谢异常可能参与TTS的发病过程，并加速心力衰竭的进展——而TTS发作后患者的心力衰竭风险本就显著升高。