Fig 2: PM exacerbates mitochondrial dysfunction in H/R-treated cardiomyocytes. Cardiomyocytes were pretreated with or without 50 μg/mL PM and then exposed to hypoxia for 6 h, followed by reoxygenation for 12 h. (A) ATP levels were quantified (n = 4). (B) the OCR was determined using a seahorse metabolic analyzer. ATP levels and the maximal OCR were quantified (n = 5). (C and D) the δψm was determined by JC-1 staining under a fluorescence microscope and flow cytometry. High δψm and low δψm are shown in red and green, respectively (scale bar: 50 μm, n = 3). (E) mitochondrial length was measured with MitoTracker staining. The photo shown below is an enlargement of the rectangle above. Scale bar: 20 or 5 μm, as indicated in the panel, n = 5). (F) the levels of DNM1L/Drp1 and MFF were detected by western blot analysis (n = 4-5). (G) BNIP3, MAP1LC3B/LC3B, BECN1/Beclin 1, PIK3C3/Vps34, SQSTM1/p62 and ATG14 expression levels were examined by western blot analysis (n = 4-8). (H) the levels of p-DNM1L/DRP1, DNM1L/DRP1, MFF, BECN1/Beclin 1, BNIP3, and MAP1LC3B/LC3B in the cytoplasmic and mitochondrial fractions were examined by western blot analysis (n = 6-8). (I) ALs were observed via AO staining under a fluorescence microscope (scale bar: 100 μm, n = 4-5). Cardiomyocytes were transfected with the mitophagy reporter mitochondria-target Keima (mt-Keima) 48 h before treatment. Control cells showed green fluorescence, while pm+h/r-treated cells showed red fluorescence (scale bar: 20 μm, n = 6). (J) the colocalization of BNIP3 (green) or MAP1LC3B/LC3B (green) and COX4/COXIV (mitochondria, red) was observed by immunofluorescence microscopy (scale bar: 100 μm). (K) the ultrastructures of the PM and mitophagosomes were observed using TEM, as indicated in the panel. Scale bar: 2 μm or 500 nm). (L and M) the effect of mdivi-1 (10 µm) on the δψm was examined by the JC-1 assay (scale bar: 50 μm, n = 4). (N) the impact of mdivi-1 on ALs was examined by AO staining (scale bar: 20 μm). (O) effect of mdivi-1 and bafilomycin A1 (10 µm) on apoptosis according to the TUNEL assay (scale bar: 50 μm, n = 5). (P and Q) the impact of mdivi-1 and bafilomycin A1 treatment on the expression of mitochondrial fission-related, mitophagy-related, and apoptosis-related proteins was determined by western blot analysis (n = 5-7). The data are expressed as the mean ± SEM; one-way ANOVA.

Fig 3: PM significantly impairs cardiac function and increases apoptosis in the hearts of C57BL/6J mice subjected to I/R. C57BL/6J mice were intratracheally injected with PM for 24 h, followed by 30 min of ischemia and 3 h of reperfusion. (A) images of the left ventricular end-systolic diameter (green line) and left ventricular end-diastolic diameter (red line) were obtained by echocardiography. EF and FS percentages were measured in control, PM, I/R, and PM+I/R mice (n = 7 mice per group). (B) cardiac injury was assessed by measuring plasma LDH and TNNI levels (n = 5-7 mice per group). (C) TTC staining was used to detect the ischemic area. The yellow arrows indicate the ischemic area (scale bar: 10 mm, n = 5 mice per group). (D) apoptotic cells were assessed by TUNEL assay (brown). Nuclei were counterstained by hematoxylin staining (blue). Scale bar: 50 μm; n = 3 mice per group. (E) Western blot analysis of the expression of apoptosis-related proteins (BBC3/PUMA, p-TRP53/p53, cl-CASP3, cl-CASP9, and BCL2) (n = 4-7). (F and G) intracellular and mitochondrial ROS were measured using DHE and MitoSOX red, respectively, and nuclei were stained blue with DAPI. Scale bar: 50 μm. (H) ultrastructural morphology observed via TEM. Autophagosome and mitochondrial fission are indicated by arrowheads and arrows, respectively. Scale bar: 2 μm or 500 nm. (I) Western blot analysis of the expression of mitochondrial fission-related proteins (DNM1L/Drp1 and MFF) and mitophagy-related proteins (BNIP3, MAP1LC3B/LC3B, BECN1/Beclin 1, PIK3C3/Vps34, SQSTM1/p62 and ATG14) (n = 5-8). The data are expressed as the mean ± SEM; one-way ANOVA.

Fig 4: NAR reduced the co-localization of Drp1 with TOM20 as well as Drp1 with Fis1 in primary CD4+ T lymphocytes. (A) Co-localization of Drp1 with TOM20. (B) Co-localization of Drp1 with Fis1. * p < 0.05 vs. control group, # p < 0.05 vs. CXCL12 group, by repeated-measures one-way ANOVA followed by post hoc Dunnett’s multiple comparisons test. All scale bars are 5 µm. The data are presented as means ±SEM (n = 3 mice per group).

Fig 5: CMDL-1 regulates apoptosis via Drp1(A and B) CMDL-1 is predicted to interact with Drp1. A positive interaction score indicates the predicted binding between CMDL-1 and Drp1 (A) and maps the CMDL-1 interaction domain to nucleotides 1500–2000 (B). RNA-protein interaction scores were generated using catRAPID software. (C) Drp1 and Trpm-7 expression levels. H9c2 cells were treated with DOX at the indicated time points, and cells were harvested for immunoblot analysis. (D) Immunoblot analysis of Drp1 expression level. Cells were transfected with siDrp1 to knock down Drp1 expression and with scrambled Drp1 (scDrp1) as CT for 24–72 h and harvested for immunoblot analysis (upper panel). Cells were infected with lentiviral overexpressing CMDL-1, and after 24 h they were harvested for immunoblot analysis (low panel). (E and F) CMDL-1 regulates mitochondrial fission via Drp1. Cells were transfected with EV, CMDL-1, and siDrp1 and exposed to DOX, and mitochondrial morphology was analyzed by confocal microscopy. (E) shows the quantification of mitochondrial fission. (F) shows representative mitochondrial morphology stained with MitoTracker red. ∗p < 0.05, ∗∗p < 0.01 versus DOX. Scale bars, 25 μm. (G) The anti-apoptotic effect of CMDL-1 decreases when Drp1 is minimally expressed (analyzed by flow cytometry). ∗p < 0.05, ∗∗p < 0.01 versus the DOX group. (H) Co-immunoprecipitation analysis. The upper panel shows western blot analysis of the specific association of Drp1 and p-Drp1 with CMDL-1. The lower panel shows Drp1 enrichment when CMDL-1 is overexpressed (analyzed by qPCR). IgG served as the negative CT. (I) CMDL-1 overexpression enhances the phosphorylation of Drp1 at the serine 637 residue. Data presented are representative of at least three independent experiments. Data are expressed as the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, one-way ANOVA.