[Overexpression of mitochondrial Lon protease 1 alleviates myocardial injury in septic mice by attenuating mitochondrial oxidative stress].

OBJECTIVE: To investigate the effects of mitochondrial Lon protease 1 (LONP1) overexpression on mitochondrial oxidative stress in a mouse model of septic myocardial injury.

METHODS: Twenty-four SPF-grade male C57BL/6J mice were randomly divided into a normal control group (Control group), a sepsis model group (LPS group), an adeno-associated virus control group (AAV+LPS group), and a LONP1 overexpression group (LONP1+LPS group), with 6 mice in each group. A septic myocardial injury model was established by intraperitoneal injection of lipopolysaccharide (LPS) at 10 mg/kg; the Control group was intraperitoneally injected with an equal volume of normal saline. Three weeks before modeling, the Control group and LPS group were sham-transfected via tail vein injection with an equal volume (100 μL) of normal saline; the AAV+LPS group was injected with adeno-associated virus (AAV) without the Lonp1 gene (100 μL) via tail vein; the LONP1+LPS group was injected with AAV targeting the myocardium and carrying the Lonp1 gene fragment (viral titer: 1×1012 vg/mL, 100 μL) via tail vein. In the LONP1+LPS group, Lonp1 gene overexpression was evaluated by frozen section and Western blotting 3 weeks after viral transfection, and modeling was performed after successful overexpression was confirmed. Twenty-four hours after modeling, the establishment of the septic myocardial injury model was assessed by mouse activity, secretions, urine and feces, combined with cardiac echocardiography, and hematoxylin-eosin (HE) staining of myocardial tissue; reactive oxygen species (ROS) production was detected by dihydroethidium (DHE) method; adenosine triphosphate (ATP) production and mitochondrial membrane potential changes were detected by relevant kits. Changes in mitochondrial oxidative stress indicators [4-hydroxynonenal (4-HNE), malondialdehyde (MDA), mitochondrial aconitase 2 (mt-ACO2)] and cardiac function indicators [left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-systolic volume (LVESV), left ventricular end-diastolic volume (LVEDV)] were also evaluated.

RESULTS: Twenty-four hours after modeling, compared with the Control group, mice in the LPS group exhibited lethargy, piloerection, increased oral and ocular secretions, pyuria, and loose stools; LVEF and LVFS were significantly decreased; HE staining showed degeneration and necrosis of some myocardial fibers in the LPS group, with disordered arrangement and inflammatory cell infiltration. Under fluorescence microscopy after frozen section, successful viral transfection was observed in the LONP1+LPS group; Western blotting showed that, compared with the Control group, LPS group, and AAV+LPS group, protein expression of LONP1 was significantly increased in the LONP1+LPS group (LONP1/GAPDH: 1.47±0.01 vs. 1, 1.11±0.01, 1.12±0.01, all P<0.05). Under fluorescence microscopy, green fluorescent protein (GFP) signals were concentrated in the cytoplasm of cardiomyocytes, showing continuous and striped distribution along the long axis of myocardial fibers, with fluorescence intensity significantly higher than that in the surrounding interstitium, indicating widespread infection of AAV9-cardiac troponin T (cTnT)-LONP1-GFP in myocardial tissue and enrichment of expression products in cardiomyocytes. Compared with the Control group, ROS production was significantly increased in the LPS group and AAV+LPS group; ATP, mitochondrial membrane potential, LVEF, LVFS, and LVESV were significantly decreased; LVEDV, 4-HNE, MDA, and mt-ACO2 contents were significantly increased. Compared with the AAV+LPS group, ROS production was significantly decreased in the LONP1+LPS group (fluorescence intensity: 1.43±0.10 vs. 2.95±0.15); ATP, mitochondrial membrane potential, LVEF, LVFS, and LVESV were significantly increased [ATP (nmol/g): 75.12±6.20 vs. 58.03±4.54, mitochondrial membrane potential: 0.81±0.15 vs. 0.75±0.12, LVEF: 0.69±0.06 vs. 0.39±0.05, LVFS: 0.35±0.04 vs. 0.18±0.03, LVESV (μL): 38.26±4.02 vs. 23.65±5.39, all P<0.05]; LVEDV, 4-HNE, MDA, and mt-ACO2 contents were significantly decreased [LVEDV (μL): 50.26±4.02 vs. 62.82±5.39, 4-HNE (ng/L): 4.70±0.55 vs. 6.01±0.73, MDA (mmol/L): 13.46±1.68 vs. 19.88±2.19, mt-ACO2 (U/g): 528.26±72.41 vs. 1 286.82±151.28, all P<0.05]. No statistically significant differences were observed in the aforementioned indicators between the LPS and AAV+LPS groups.

CONCLUSIONS: LONP1 overexpression reduces ROS production, promote ATP generation, enhance mitochondrial membrane potential, and alleviate mitochondrial oxidative stress in septic mice, thereby improving cardiac function.