Table_10_PERM1 regulates energy metabolism in the heart via ERRα/PGC−1α axis.XLSX

AimsPERM1 is a striated muscle-specific regulator of mitochondrial bioenergetics. We previously demonstrated that PERM1 is downregulated in the failing heart and that PERM1 positively regulates metabolic genes known as targets of the transcription factor ERRα and its coactivator PGC-1α in cultured cardiomyocytes. The aims of this study were to determine the effect of loss of PERM1 on cardiac function and energetics using newly generated Perm1-knockout (Perm1–/–) mice and to investigate the molecular mechanisms of its transcriptional control.
Methods and resultsEchocardiography showed that ejection fraction and fractional shortening were lower in Perm1–/– mice than in wild-type mice (both p < 0.05), and the phosphocreatine-to-ATP ratio was decreased in Perm1–/– hearts (p < 0.05), indicating reduced contractile function and energy reserves of the heart. Integrated proteomic and metabolomic analyses revealed downregulation of oxidative phosphorylation and upregulation of glycolysis and polyol pathways in Perm1–/– hearts. To examine whether PERM1 regulates energy metabolism through ERRα, we performed co-immunoprecipitation assays, which showed that PERM1 bound to ERRα in cardiomyocytes and the mouse heart. DNA binding and reporter gene assays showed that PERM1 was localized to and activated the ERR target promoters partially through ERRα. Mass spectrometry-based screening in cardiomyocytes identified BAG6 and KANK2 as potential PERM1’s binding partners in transcriptional regulation. Mammalian one-hybrid assay, in which PERM1 was fused to Gal4 DNA binding domain, showed that the recruitment of PERM1 to a gene promoter was sufficient to activate transcription, which was blunted by silencing of either PGC-1α, BAG6, or KANK2.
ConclusionThis study demonstrates that PERM1 is an essential regulator of cardiac energetics and function and that PERM1 is a novel transcriptional coactivator in the ERRα/PGC-1α axis that functionally interacts with BAG6 and KANK2.

PERM1是一种横纹肌特异性的线粒体生物能学调控因子。既往研究证实，衰竭心脏中PERM1表达水平下调；在培养的心肌细胞中，PERM1可正向调控以转录因子雌激素相关受体α（ERRα）及其辅激活因子过氧化物酶体增殖物激活受体γ辅激活因子1α（PGC-1α）的靶基因为代表的代谢基因。本研究旨在通过新构建的Perm1基因敲除（Perm1–/–）小鼠，探究PERM1缺失对心脏功能与能量代谢的影响，并解析其转录调控的分子机制。

方法与结果
超声心动图检测结果显示，与野生型小鼠相比，Perm1–/–小鼠的射血分数与短轴缩短率均显著降低（两组均p < 0.05）；Perm1–/–小鼠心脏的磷酸肌酸/ATP比值亦显著下降（p < 0.05），提示心脏收缩功能与能量储备受损。整合蛋白质组学与代谢组学分析发现，Perm1–/–小鼠心脏中氧化磷酸化通路表达下调，而糖酵解与多元醇通路表达上调。
为验证PERM1是否通过ERRα调控能量代谢，我们开展了免疫共沉淀实验，结果证实PERM1可在心肌细胞与小鼠心脏中与ERRα结合。DNA结合与报告基因实验显示，PERM1可定位至ERR靶基因启动子并部分通过ERRα激活其转录。基于质谱的心肌细胞筛选实验鉴定出BAG6与KANK2为PERM1在转录调控中的潜在结合伴侣。将PERM1与Gal4 DNA结合结构域融合的哺乳动物单杂交实验结果表明，PERM1招募至基因启动子即可激活转录，而沉默PGC-1α、BAG6或KANK2均可削弱该转录激活效应。

结论
本研究证实，PERM1是心脏能量代谢与功能的关键调控因子，且PERM1是ERRα/PGC-1α信号轴中的新型转录辅激活因子，可与BAG6及KANK2发生功能性相互作用。