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、炎症与癌症中发挥的多效性作用而被广泛认知。近年来，p62已被证实是蛋白质质量控制与衰老的重要调节因子。然而，其在心脏中的功能尚未得到充分阐明。
此前学界对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条件下对其功能的间接评估。但p62是否为维持静息状态下心脏功能，或是应对应激刺激所必需，这一问题尚未被探索。
本研究在排除全身p62敲除小鼠中观察到的其他表型与系统性干扰因素的前提下，探究了心脏特异性p62敲除的功能后果。研究发现，心肌细胞p62缺失会诱发小鼠心脏衰老，并伴随收缩功能下降，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，该结果在p62心脏特异性敲除（p62cKO）小鼠的心脏组织中得到了验证。
我们进一步证实，成年小鼠心肌p62缺失后，在低氧造模时会出现过度氧化应激与细胞死亡。
为深入探究其分子机制，我们在H9c2心肌成肌细胞中分别开展p62功能敲低与过表达实验，结果显示，p62缺陷细胞中Nrf2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降解水平升高。与之相反，p62过表达可使Nrf2稳定并提升其转录活性。
本研究使用2月龄与15月龄的野生型及心脏特异性p62敲除小鼠（Myh6-Cre-Sqstm1fl/fl）的全心脏组织提取RNA并进行测序。