图1 MI与缺氧心肌细胞中miR-503表达上调

Fig.1 MiR-503 expression is upregulated in myocardial tissues of mice with myocardial infarction (MI) and in hypoxic primary neonatal rat cardiomyocytes. A, B: Echocardiography and TTC staining for assessing cardiac function and infarct area ion MI mice (n=5; **P<0.01 vs sham group). C: qRT-PCR showing elevated miR-503 expression in the peri-infarct zone in MI mice (n=10; **P<0.01 vs sham group). D, E: Decreased viability and increased LDH release in hypoxic primary cardiomyocytes (n=15; **P<0.01 vs control group). F: TUNEL staining showing increased apoptotic cells in the hypoxia group (Original magnification: ×100). G: Upregulated miR-503 expression in hypoxic cardiomyocytes (n=10; **P<0.01 vs control group).

图2 AntagomiR-503减轻MI后心肌凋亡

Fig.2 AntagomiR-503 attenuates myocardial apoptosis in MI mice. A-E: TTC staining (n=4) and echocardiography (n=5) showing that antagomiR-503 improvescardiac function and reduces infarct size in MI mice (*P<0.05 vs sham group; #P<0.05 vs MI group). F, G: Western blotting showing that antagomiR-503 reduces myocardial expressions of Bax and BCl-2 in MI mice (n=6; *P<0.05 vs sham group; #P<0.05 vs MI group). H, I: Myocardial expressions of cleaved caspase-3 and cleaved caspase-9 in mice in different groups (n=4; *P<0.05 vs sham group; #P<0.05 vs MI group). J: Transmission electron microscopy showing improved mitochondrial structure after antagomiR-503 treatment in MI mice (Scale bar=2 μm).

图3 AMO-503减轻缺氧诱导的心肌细胞凋亡

Fig.3 AMO-503 alleviates hypoxia-induced cardiomyocyte apoptosis. A, B: AMO-503 reduces LDH release (n=7) and increases cell viability (n=12; *P<0.05, **P<0.01 vs control group; #P<0.05,##P<0.01 vs hypoxia group). C: TUNEL staining showing that AMO-503 decreases cell apoptosis (n=5; **P<0.01 vs control group; ##P<0.01 vs hypoxia group). D-G: Western blotting showing that AMO-503 downregulates the expressions of Bax (n=5) and cleaved caspase-3/9 (n=4; *P<0.05 vs control group; #P<0.05 vs hypoxia group). H: JC-1 staining showing that AMO-503 restores mitochondrial membrane potential in hypoxic cardiomyocytes (n=6; **P<0.01 vs control group; ##P<0.01 vs hypoxia group). I: TEM images showing that AMO-503 improves mitochondrial morphology of the hypoxic cardiomyocytes (Scale bar=2 μm).

图4 AK134630/miR-503调控Apelin表达

Fig.4 Regulation of Apelin expression by AK134630/miR-503. A-D: Western blotting showed that either AK134630 overexpression or miR-503 inhibition (by Antago-503/AMO-503) upregulates Apelin expression (n=5). *P<0.05 vs sham group or hypoxia group; #P<0.05 vs MI group or hypoxia group.

图5 miR-503与AK134630的相互作用

Fig.5 Interaction between miR-503 and AK134630. A: Schematic diagram of the predicted binding site between miR-503 and AK134630. B: Luciferase reporter assay showing that miR-503 inhibits the activity of AK134630 (n=4; *P<0.05 vs blank group; #P<0.05 vs miR-503 group). C: RNA pull-down assay showing that AK134630 bait significantly enriched miR-503 (vs. negative control NC), directly verifying their physical interaction. D-G: qRT-PCR analysis showing a negative correlation between the expression of AK134630 and miR-503 (n=5; *P<0.05 vs control group or sham group; #P<0.05 vs MI group or hypoxia group).

图6 AK134630通过调控miR-503减轻心肌细胞损伤

Fig.6 AK134630 alleviates cardiomyocyte injury by regulating miR-503. A, B: AK134630 overexpression increases cell viability (n=10) and decreases LDH release (n=6; *P<0.05 vs control group; #P<0.05 vs hypoxia group; $P<0.05 vs AK134630 group). C: TUNEL staining showing that AK134630 overexpression reduces hypoxic cardiomyocyte apoptosis (n=6; *P<0.05 vs control group; #P<0.05 vs hypoxia group; $P<0.05 vs AK134630 group). D: JC-1 staining showing that AK134630 overexpression restores mitochondrial membrane potential in hypoxic cardiomyocytes (n=6; *P<0.05 vs control group; #P<0.05 vs hypoxia group; $P<0.05 vs AK134630 group).

图7 机制示意图

Fig.7 Schematic diagram of the proposed mechanism. AK134630 inhibits miR-503 expression, leading to the upregulation of Apelin, which subsequently alleviates cardiomyocyte apoptosis and mitochondrial damage.