Department of Biology, York University, Toronto, Ontario, Canada.
Revvity, Hopkinton, Massachusetts, USA.
Clin Transl Sci. 2024 Mar;17(3):e13758. doi: 10.1111/cts.13758.
Strategies to enhance autophagy flux have been suggested to improve outcomes in cardiac ischemic models. We explored the role of adiponectin in mediating cardiac autophagy under ischemic conditions induced by permanent coronary artery ligation. We studied the molecular mechanisms underlying adiponectin's cardio-protective effects in adiponectin knockout (Ad-KO) compared with wild-type (WT) mice subjected to ischemia by coronary artery ligation and H9c2 cardiomyocyte cell line exposed to hypoxia. Systemic infusion of a cathepsin-B activatable near-infrared probe as a biomarker for autophagy and detection via noninvasive three-dimensional fluorescence molecular tomography combined with computerized tomography to quantitate temporal changes, indicated increased activity in the myocardium of WT mice after myocardial infarction which was attenuated in Ad-KO. Seven days of ischemia increased myocardial adiponectin accumulation and elevated ULK1/AMPK phosphorylation and autophagy assessed by Western blotting for LC3 and p62, an outcome not observed in Ad-KO mice. Cell death, assessed by TUNEL analysis and the ratio of Bcl-2:Bax, plus cardiac dysfunction, measured using echocardiography with strain analysis, were exacerbated in Ad-KO mice. Using cellular models, we observed that adiponectin stimulated autophagy flux in isolated primary adult cardiomyocytes and increased basal and hypoxia-induced autophagy in H9c2 cells. Real-time temporal analysis of caspase-3/7 activation and caspase-3 Western blot indicated that adiponectin suppressed activation by hypoxia. Hypoxia-induced mitochondrial reactive oxygen species production and cell death were also attenuated by adiponectin. Importantly, the ability of adiponectin to reduce caspase-3/7 activation and cell death was not observed in autophagy-deficient cells generated by CRISPR-mediated deletion of Atg7. Collectively, our data indicate that adiponectin acts in an autophagy-dependent manner to attenuate cardiomyocyte caspase-3/7 activation and cell death in response to hypoxia in vitro and ischemia in mice.
已有研究表明，增强自噬通量的策略可以改善心脏缺血模型的预后。我们研究了脂联素在永久性冠状动脉结扎诱导的缺血条件下介导心脏自噬的作用。我们研究了脂联素在野生型（WT）和脂联素敲除（Ad-KO）小鼠中的心脏保护作用的分子机制，这些小鼠通过冠状动脉结扎和 H9c2 心肌细胞系暴露于缺氧来诱导缺血。通过非侵入性三维荧光分子断层扫描与计算机断层扫描相结合，对脂联素激活的组织蛋白酶 B 近红外探针进行全身输注，作为自噬的生物标志物进行检测，并定量分析时间变化，结果表明，心肌梗死大鼠心肌中 WT 小鼠的自噬活性增加，而 Ad-KO 小鼠的自噬活性则减弱。7 天的缺血导致心肌脂联素积累增加，ULK1/AMPK 磷酸化和自噬增加，用 LC3 和 p62 的 Western blot 进行评估，而 Ad-KO 小鼠则没有观察到这种结果。通过 TUNEL 分析和 Bcl-2:Bax 比值评估细胞死亡，以及通过应变分析进行超声心动图评估心脏功能障碍，结果表明 Ad-KO 小鼠的细胞死亡和心脏功能障碍加重。在细胞模型中，我们观察到脂联素刺激分离的成年心肌细胞中的自噬通量增加，并增加 H9c2 细胞中的基础和缺氧诱导的自噬。实时分析 caspase-3/7 激活和 caspase-3 Western blot 表明，脂联素抑制缺氧诱导的激活。脂联素还减弱了缺氧诱导的线粒体活性氧产生和细胞死亡。重要的是，在由 CRISPR 介导的 Atg7 缺失生成的自噬缺陷细胞中，未观察到脂联素减少 caspase-3/7 激活和细胞死亡的能力。总之，我们的数据表明，脂联素通过自噬依赖性方式在体外和小鼠缺血中减轻缺氧诱导的心肌细胞 caspase-3/7 激活和细胞死亡。
