Cardiovascular Theranostics, Istituto Cardiocentro Ticino, Laboratories for Translational Research, Ente Ospedaliero Cantonale, Bellinzona, Switzerland.
Department of Biotechnology and Biosciences, Università degli Studi di Milano-Bicocca, Milano, Italy.
Theranostics. 2022 Jul 4;12(11):5237-5257. doi: 10.7150/thno.70884. eCollection 2022.
Aging in the heart is a gradual process, involving continuous changes in cardiovascular cells, including cardiomyocytes (CMs), namely cellular senescence. These changes finally lead to adverse organ remodeling and resulting in heart failure. This study exploits CMs from human induced pluripotent stem cells (iCMs) as a tool to model and characterize mechanisms involved in aging. Human somatic cells were reprogrammed into human induced pluripotent stem cells and subsequently differentiated in iCMs. A senescent-like phenotype (SenCMs) was induced by short exposure (3 hours) to doxorubicin (Dox) at the sub-lethal concentration of 0.2 µM. Dox treatment induced expression of cyclin-dependent kinase inhibitors p21 and p16, and increased positivity to senescence-associated beta-galactosidase when compared to untreated iCMs. SenCMs showed increased oxidative stress, alteration in mitochondrial morphology and depolarized mitochondrial membrane potential, which resulted in decreased ATP production. Functionally, when compared to iCMs, SenCMs showed, prolonged multicellular QTc and single cell APD, with increased APD variability and delayed afterdepolarizations (DADs) incidence, two well-known arrhythmogenic indexes. These effects were largely ascribable to augmented late sodium current (I) and reduced delayed rectifier potassium current (Ikr). Moreover sarcoplasmic reticulum (SR) Ca content was reduced because of downregulated SERCA2 and increased RyR2-mediated Ca leak. Electrical and intracellular Ca alterations were mostly justified by increased CaMKII activity in SenCMs. Finally, SenCMs phenotype was furtherly confirmed by analyzing physiological aging in CMs isolated from old mice in comparison to young ones. Overall, we showed that SenCMs recapitulate the phenotype of aged primary CMs in terms of senescence markers, electrical and Ca handling properties and metabolic features. Thus, Dox-induced SenCMs can be considered a novel platform to study aging mechanisms and to envision cardiac specific anti-aging approach in humans.
心脏衰老的过程是一个渐进的过程，涉及心血管细胞的持续变化，包括心肌细胞（CMs），即细胞衰老。这些变化最终导致不良的器官重塑，导致心力衰竭。本研究利用来自人诱导多能干细胞（iCMs）的 CMs 作为一种工具来模拟和表征衰老相关机制。人类体细胞被重编程为人诱导多能干细胞，随后分化为 iCMs。通过在亚致死浓度 0.2 μM 的阿霉素（Dox）下短暂暴露（3 小时），诱导出衰老样表型（SenCMs）。与未处理的 iCMs 相比，Dox 处理诱导细胞周期蛋白依赖性激酶抑制剂 p21 和 p16 的表达增加，并增加了衰老相关β-半乳糖苷酶的阳性率。SenCMs 显示氧化应激增加、线粒体形态改变和线粒体膜电位去极化，导致 ATP 产生减少。功能上，与 iCMs 相比，SenCMs 显示出更长的多细胞 QTc 和单细胞 APD，APD 变异性增加，延迟后除极（DAD）发生率增加，这是两种众所周知的致心律失常指数。这些影响主要归因于晚期钠电流（I）的增加和延迟整流钾电流（Ikr）的减少。此外，由于 SERCA2 的下调和 RyR2 介导的 Ca 泄漏增加，肌浆网（SR）Ca 含量减少。SenCMs 中 CaMKII 活性的增加在很大程度上解释了电和细胞内 Ca 的改变。最后，通过比较年轻小鼠和老年小鼠分离的 CMs 中生理衰老的情况，进一步证实了 SenCMs 的表型。总的来说，我们表明 SenCMs 在衰老标志物、电和 Ca 处理特性以及代谢特征方面再现了衰老的原代 CMs 表型。因此，Dox 诱导的 SenCMs 可以被认为是一种研究衰老机制和设想人类心脏特异性抗衰老方法的新平台。
