Supplementary Material for: Engineered Aging Cardiac Tissue Chip Model for Studying Cardiovascular Disease

Due to the rapidly growing number of older people worldwide and the concomitant increase in cardiovascular complications, there is an urgent need for age-related cardiac disease modeling and drug screening platforms. In the present study, we developed a cardiac tissue chip model that incorporates hemodynamic loading and mimics essential aspects of the infarcted aging heart. We induced cellular senescence in H9c2 myoblasts using low-dose doxorubicin treatment. These senescent cells were then used to engineer cardiac tissue fibers, which were subjected to hemodynamic stresses associated with pressure-volume changes in the heart. Myocardial ischemia was modeled in the engineered cardiac tissue via hypoxic treatment. Our results clearly show that acute low-dose doxorubicin treatment-induced senescence, as evidenced by morphological and molecular markers, including enlarged and flattened nuclei, DNA damage response foci, and increased expression of cell cycle inhibitor p16INK4a, p53, and ROS. Under normal hemodynamic load, the engineered cardiac tissues demonstrated cell alignment and retained cardiac cell characteristics. Our senescent cardiac tissue model of hypoxia-induced myocardial infarction recapitulated the pathological disease hallmarks such as increased cell death and upregulated expression of ANP and BNP. In conclusion, the described methodology provides a novel approach to generate stress-induced aging cardiac cell phenotypes and engineer cardiac tissue chip models to study the cardiovascular disease pathologies associated with aging.

鉴于全球老年人口数量快速增长，且伴随而来的心血管并发症发病率持续攀升，当前亟需构建衰老相关心脏疾病建模与药物筛选平台。本研究中，我们开发了一款整合血流动力学加载功能的心肌组织芯片（cardiac tissue chip）模型，可模拟衰老型梗死心脏的核心病理特征。我们采用低剂量阿霉素（doxorubicin）处理H9c2肌母细胞（H9c2 myoblasts），诱导其发生细胞衰老；随后将这些衰老细胞用于构建心肌组织纤维，并使其承受与心脏压力-容积变化相关的血流动力学应力。通过低氧处理，我们在工程化心肌组织中构建了心肌缺血模型。研究结果清晰表明，急性低剂量阿霉素处理可诱导细胞衰老，该现象可通过多项形态学与分子生物学标志物验证：包括细胞核增大变扁、DNA损伤应答灶形成，以及细胞周期抑制因子p16INK4a、p53与活性氧（Reactive Oxygen Species, ROS）的表达水平上调。在正常血流动力学负荷下，工程化心肌组织可呈现细胞排列有序的状态，并维持心肌细胞的固有特性。我们构建的低氧诱导心肌梗死衰老心肌组织模型，可重现疾病的核心病理特征：包括细胞死亡增加，以及心房钠尿肽（Atrial Natriuretic Peptide, ANP）、B型钠尿肽（B-type Natriuretic Peptide, BNP）的表达上调。综上，本研究所述方法为构建应激诱导的衰老心肌细胞表型、制备心肌组织芯片模型以研究衰老相关心血管疾病病理机制提供了全新策略。