Using cultured canine cardiac slices to model the autophagic flux with doxorubicin.
Using cultured canine cardiac slices to model the autophagic flux with doxorubicin.
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DOI：
10.1371/journal.pone.0282859
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发表时间：
2023
期刊：
影响因子：
3.7
通讯作者：
中科院分区：
文献类型：
--
作者：
化疗诱导的自噬损伤与抗癌药物诱导的心脏毒性有关。来自啮齿动物模型的不完善的翻译和缺乏体外毒性模型限制了自噬通量失调的研究，阻碍了基于自噬控制的新型心脏保护策略的设计。从转化模型发展成人心脏组织培养技术将改善心脏毒性的研究。我们的目的是优化犬心脏切片培养系统，以探索癌症治疗对完整心脏组织的影响，创建一个可翻译的模型，在培养中保持自噬，并适合自噬调节。犬心脏组织切片（350 μm）是从Tufts University的Foster小动物医院对无心血管疾病的客户所有犬（n = 7）实施安乐死后采集的左心室游离壁中制备的。MTT法和TUNEL法检测细胞存活率和凋亡率。用阿霉素和自噬激活剂（雷帕霉素）或抑制剂（氯喹）挑战心脏切片。通过蛋白质印迹定量自噬流组分（LC 3，p62）。心脏切片在培养中保持高细胞活力>7天，自噬标志物的基础水平保持不变。多柔比星处理导致自噬通量的扰动和细胞死亡，而雷帕霉素共处理恢复正常的自噬通量并维持细胞存活。我们开发了一个成年犬心脏切片培养系统，适用于研究自噬通量的影响，可能适用于药物毒性评价。
Chemotherapy-induced impairment of autophagy is implicated in cardiac toxicity induced by anti-cancer drugs. Imperfect translation from rodent models and lack of in vitro models of toxicity has limited investigation of autophagic flux dysregulation, preventing design of novel cardioprotective strategies based on autophagy control. Development of an adult heart tissue culture technique from a translational model will improve investigation of cardiac toxicity. We aimed to optimize a canine cardiac slice culture system for exploration of cancer therapy impact on intact cardiac tissue, creating a translatable model that maintains autophagy in culture and is amenable to autophagy modulation. Canine cardiac tissue slices (350 μm) were generated from left ventricular free wall collected from euthanized client-owned dogs (n = 7) free of cardiovascular disease at the Foster Hospital for Small Animals at Tufts University. Cell viability and apoptosis were quantified with MTT assay and TUNEL staining. Cardiac slices were challenged with doxorubicin and an autophagy activator (rapamycin) or inhibitor (chloroquine). Autophagic flux components (LC3, p62) were quantified by western blot. Cardiac slices retained high cell viability for >7 days in culture and basal levels of autophagic markers remained unchanged. Doxorubicin treatment resulted in perturbation of the autophagic flux and cell death, while rapamycin co-treatment restored normal autophagic flux and maintained cell survival. We developed an adult canine cardiac slice culture system appropriate for studying the effects of autophagic flux that may be applicable to drug toxicity evaluations.
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