5'tiRNA-32-LysCTT-11 ALLEVIATES SEPSIS-INDUCED MYOCARDIAL INJURY BY STABILIZING Mfn2 AND INHIBITING MITOCHONDRIAL...

Transfer RNA-derived small RNAs (tsRNAs) represent a novel class of noncoding RNAs increasingly implicated in cardiovascular regulation. However, their roles in sepsis-induced cardiomyopathy (SICM) remain largely undefined. This study aimed to investigate the function and underlying mechanism of 5'tiRNA-32-LysCTT-11-a highly upregulated tsRNA in sepsis-induced cardiomyopathy-in modulating myocardial injury.

A murine model of sepsis was established via cecal ligation and puncture, and myocardial injury was assessed by serum Creatine Kinase-MB Isoenzyme/lactate dehydrogenase levels, histology, and cardiac function via echocardiography. In vitro , H9C2 cardiomyocytes were exposed to conditioned media from lipopolysaccharide-stimulated macrophages. The expression of 5'tiRNA-32-LysCTT-11 was measured by quantitative reverse transcription polymerase chain reaction. Functional assays, including Cell Counting Kit-8, lactate dehydrogenase release, propidium iodide staining, 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine (JC-1), adenosine triphosphate, reactive oxygen species detection, and MitoTracker staining, were performed. Necroptosis was evaluated via mixed lineage kinase domain-like phosphorylation; mitochondria-associated endoplasmic reticulum membranes (MAMs) formation was assessed by dual-label immunofluorescence and phosphofurin acidic cluster sorting protein 2 expression. Bioinformatics analysis identified mitofusin 2 (Mfn2) as a putative target, validated by western blot, mRNA stability assay (actinomycin D), and rescue experiments.

5'tiRNA-32-LysCTT-11 was significantly upregulated insepsis-induced cardiomyopathy. In vivo , its overexpression improved cardiac function and reduced injury biomarkers. In vitro , 5'tiRNA-32-LysCTT-11 mimics preserved mitochondrial integrity, reduced reactive oxygen species and adenosine triphosphate depletion, suppressed mitochondria-associated endoplasmic reticulum membranes formation and necroptosis. Inhibitor transfection produced opposite effects. Mechanistically, 5'tiRNA-32-LysCTT-11 enhancedmitofusin 2 mRNA stability and protein expression. Silencing mitofusin 2 abrogated the protective effects, confirming its central role in the tsRNA's action.

5'tiRNA-32-LysCTT-11 exerts cardioprotective effects during sepsis by stabilizing Mfn2 mRNA, preserving mitochondrial function, limiting mitochondria-associated endoplasmic reticulum membranes formation, and suppressing necroptosis. These findings uncover a novel regulatory mechanism and suggest 5'tiRNA-32-LysCTT-11 as a promising therapeutic target SICM.