Mitochondrial calcium uptake 1 (micu1) knockout ameliorates cold-induced cell death and inflammatory response by...

Mitochondrial calcium uptake 1 (micu1) plays a critical role in regulating mitochondrial Ca²⁺ (mCa²⁺), maintaining mitochondrial homeostasis, and modulating redox balance. However, the function of micu1 in fish under cold stress remains unclear. In this study, we characterized the micu1 in Takifugu obscurus, which encodes a 489-amino acid protein with the typical EF-hand structure and high sequence identities with other fish species. T. obscurus micu1 was widely expressed in healthy tissues, with the highest expression in the brain. It was markedly activated in wild-type (WT) and cold-tolerant (CT) pufferfish upon cold stress, with different expression patterns. Knockdown of micu1 in T. obscurus gill cell lines could preserve mitochondrial membrane potential (MMP), improve cell viability, and reduce reactive oxygen species (ROS) production under cold stress. Additionally, micu1 knockdown could affect the expression of genes involved in ferroptosis (trf1a, gpx4, aft3, slc7a11, aifm2, acsl4, and hepcidin), apoptosis (bad, bax, p53, fadd, and caspase3) and inflammation (il6, il8, il10, and tnfa) under cold stress. Moreover, micu1 knockout Nile tilapia (micu1^+/-), generated via the CRISPR/Cas9 system, exhibited significantly fewer apoptosis signals under cold stress compared to WT tilapia. The mRNA levels of anti-apoptotic genes (bcl-xl and hsp90) were significantly elevated in the mutant, while pro-apoptotic genes (caspase3, caspase7, bax, and bad) and inflammatory genes (tlr9, nfκb, myd88, il6, il8, and il10) were significantly downregulated under cold stress. Behavioral analysis showed that micu1^+/- tilapia exhibited greater activity at low temperatures than WT tilapia. Taken together, these in vivo and in vitro results indicated that micu1 could enhance cold tolerance trait of fish by maintaining cellular membrane potential, inhibiting cold-induced ROS production, ferroptosis and apoptosis. Our study would help us to better understand the function of micu1 in the response of teleosts to cold stress, and provide a valuable target for breeding cold-resistant aquaculture species.