RNAseq analysis of skeletal muscle transcriptomes from wild-type, LONP1 skeletal muscle specific knockout (LONP1 mKO) and LONP1ATF4 skeletal muscle double-knockout (LONP1ATF4 DmKO) mice.

Studying the molecular basis for the skeletal muscle mitochondrial stress response could potentially be targeted to counteract metabolic disorders such as obesity. We uncovered a crucial role for the mitochondrial protease LONP1 in controlling the muscle mitochondrial proteostasis stress response that governs systemic metabolic homeostasis. Skeletal muscle-specific ablation of LONP1 led to broad genomic reprogramming of muscle and protected the mice from HFD-induced obesity. To thoroughly analyze pathways that are affected by ATF4 deficiency in the context of LONP1 mKO, we performed RNA-Seq transcriptome analysis of skeletal muscles from WT, LONP1 mKO and LONP1ATF4 DmKO mice. We identified a total of 3017 differentially expressed genes with a cutoff of 1.5-fold, and p < 0.05. Surprisingly, we found that ATF4 loss blunted a subset of regulated genes in LONP1 mKO muscles, whereas the majority of differentially regulated genes in LONP1 mKO muscles were not affected by ATF4 ablation. GO analysis of ATF4-dependent genes regulated by LONP1 ablation revealed significant enrichment in amino acid metabolic processes, Interestingly, however, LONP1 deficiency induced activation of muscle UPR and UPR-related RNA processing as well as myokine pathways were not affected by ATF4 ablation. Therefore, our results highlight a ATF4-independent mechanism in mediating the UPRmt in mammals. Gastrocnemius muscle mRNA profiles of 6-week-old WT, LONP1 mKO and LONP1ATF4 DmKO mice were generated by deep sequencing, in duplicate, using Illumina HiSeq 4000.探究骨骼肌线粒体应激应答的分子机制，有望为对抗肥胖等代谢紊乱疾病提供干预靶点。本研究揭示了线粒体蛋白酶LONP1（mitochondrial protease LONP1）在调控肌肉线粒体蛋白稳态应激应答、进而维持系统代谢稳态中的关键作用。骨骼肌特异性敲除LONP1可引发肌肉广泛的基因组重编程，并使小鼠免受高脂饮食（High Fat Diet, HFD）诱导的肥胖。为深入解析LONP1肌肉特异性条件性敲除（LONP1 mKO）背景下ATF4（Activating Transcription Factor 4）缺失所影响的信号通路，本研究对野生型（Wild Type, WT）、LONP1 mKO以及LONP1ATF4双敲除（double knockout, DmKO）小鼠的骨骼肌进行了RNA测序（RNA-Seq）转录组分析。本研究共筛选得到3017个差异表达基因，筛选阈值为1.5倍表达差异且p值<0.05。令人意外的是，本研究发现ATF4缺失会削弱LONP1 mKO小鼠肌肉中部分调控基因的表达，而LONP1 mKO肌肉中绝大多数差异调控基因的表达不受ATF4敲除的影响。对LONP1敲除所调控的ATF4依赖型基因进行基因本体（Gene Ontology, GO）富集分析发现，这些基因显著富集于氨基酸代谢过程。值得注意的是，LONP1缺失诱导的肌肉未折叠蛋白反应（Unfolded Protein Response, UPR）、UPR相关RNA加工过程以及肌因子通路的激活，均不受ATF4敲除的影响。因此，本研究结果表明，哺乳动物中介导线粒体未折叠蛋白反应（mitochondrial Unfolded Protein Response, UPRmt）存在ATF4非依赖型机制。本研究采用Illumina HiSeq 4000平台对6周龄WT、LONP1 mKO以及LONP1ATF4 DmKO小鼠的腓肠肌mRNA进行了深度测序，设置生物学重复两次。