Cardiac p62/Sqstm1 maintains Nrf2 protein levels and protects from oxidative stress

Squestosome 1 (SQSTM1), also known as p62, is a multi-functional adaptor protein known for its pleotropic roles in autophagy, proteostasis, inflammation and cancer. Recently, p62 has emerged as an important modulator of protein quality control and aging. However, its role in the heart is not well understood. Our understanding of the role of p62 in the heart has been limited to the indirect assessment of its function in the setting of autophagy inhibition or proteotoxic stress. However, whether p62 is required to maintain cardiac function at rest or in response to stress has not been explored. Here we investigated the functional consequence of cardiac p62 deletion in the absence of other contributing phenotypic and systemic factors observed in the whole-body p62 deleted mice. Lack of cardiomyocytes p62 precipitated cardiac aging in mice and was associated with reduced contractile function and a progressive development of cardiac hypertrophy and fibrosis. Transcriptomic analysis of p62-deleted heart revealed a selective impairment in Nrf2 transcription, which was confirmed in the hearts of p62cKO mice. We further showed that absence of p62 in adult mice resulted in excessive oxidative stress and cell death when mice were rendered hypoxic. To gain mechanistic insights, we employed loss and gain of p62 function in H9c2 cardiomyoblasts and showed a sustained reduction in Nrf2 protein expression, nuclear translocation and transcriptional activity in p62-deficient cells. Mechanistically, p62-deficient cells exhibited an increase in proteasome-mediated Nrf2 degradation. In contrast, gain of p62 function led to Nrf2 stabilization and transcriptional activity. Whole heart tissue from 2 and 15 months old wildtype and cardiac-specific p62 KO mice (Myh6-Cre-Sqstm1fl/fl) were used to isolate RNA and then sequenced.Squestosome 1（SQSTM1，亦称p62）是一种多功能衔接蛋白，因其在自噬、蛋白质稳态、炎症及癌症进程中发挥多效性作用而为人熟知。近年来，p62已被证实为蛋白质质量控制与衰老过程中的关键调控因子。然而，其在心脏中的生物学功能尚未得到充分阐释。目前学界对p62在心脏中作用的认知，仅局限于在自噬抑制或蛋白毒性应激环境下对其功能的间接评估。但学界尚未明确p62是否为静息状态下或应激反应中维持心脏功能所必需。本研究针对全身p62敲除小鼠中存在的其他干扰表型与系统性混杂因素，探究了心脏特异性p62敲除后的功能结局。心肌细胞p62缺失会诱发小鼠心脏衰老，并伴随收缩功能减退以及心肌肥厚与纤维化的进行性发展。对p62敲除心脏的转录组学分析显示，核因子红细胞2相关因子2（Nrf2）的转录存在选择性损伤，这一结果在p62条件性敲除（p62cKO）小鼠的心脏组织中得到了验证。本研究进一步证实，成年小鼠体内p62缺失会在小鼠遭受缺氧刺激时引发过度氧化应激与细胞死亡。为深入探究其分子机制，本研究在H9c2心肌成肌细胞中分别开展p62功能缺失与功能过表达实验，结果显示，p62缺陷细胞中Nrf2的蛋白表达、核转位及转录活性均持续降低。机制层面，p62缺陷细胞中蛋白酶体介导的Nrf2降解过程显著增强。与之相反，p62过表达则可实现Nrf2的蛋白稳定，并提升其转录活性。本研究采集了2月龄与15月龄的野生型小鼠及心脏特异性p62敲除（Myh6-Cre-Sqstm1fl/fl）小鼠的全心脏组织，用于提取RNA并进行测序。