Mir221- and Mir222-enriched adsc-exosomes mitigate PM exposure-exacerbated cardiac ischemia-reperfusion injury through the modulation of the BNIP3-MAP1LC3B-BBC3/PUMA pathway

Abstract

Epidemiology has shown a strong relationship between fine particulate matter (PM) exposure and cardiovascular disease. However, it remains unknown whether PM aggravates myocardial ischemia-reperfusion (I/R) injury, and the related mechanisms are unclear. Our previous study has shown that adipose stem cell-derived exosomes (ADSC-Exos) contain high levels of Mir221 and Mir222. The present study investigated the effects of PM exposure on I/R-induced cardiac injury through mitophagy and apoptosis, as well as the potential role of Mir221 and Mir222 in ADSC-Exos. Wild-type, mir221- and mir222-knockout (KO), and Mir221- and Mir222-overexpressing transgenic (TG) mice were intratracheally injected with PM (10 mg/kg). After 24 h, mice underwent left coronary artery ligation for 30 min, followed by 3 h of reperfusion (I/R). H9c2 cardiomyocytes were cultured under 1% O₂ for 6 h, then reoxygenated for 12 h (hypoxia-reoxygenation [H/R]). PM aggravated I/R (or H/R) cardiac injury by increasing ROS levels and causing mitochondrial dysfunction, which increased the expression of mitochondrial fission-related proteins (DNM1L/Drp1 and MFF) and mitophagy-related proteins (BNIP3 and MAP1LC3B/LC3B) in vivo and in vitro. Treatment with ADSC-Exos or Mir221- and Mir222-mimics significantly reduced PM+I/R-induced cardiac injury. Importantly, ADSC-Exos contain Mir221 and Mir222, which directly targets BNIP3, MAP1LC3B/LC3B, and BBC3/PUMA, decreasing their expression and ultimately reducing cardiomyocyte mitophagy and apoptosis. The present data showed that ADSC-Exos treatment regulated mitophagy and apoptosis through the Mir221 and Mir222-BNIP3-MAP1LC3B-BBC3/PUMA pathway and significantly reduced the cardiac damage caused by PM+I/R. The present study revealed the novel therapeutic potential of ADSC-Exos in alleviating PM-induced exacerbation of myocardial I/R injury.Abbreviation: ADSC-Exos: adipose-derived stem cell exosomes; AL: autolysosome; ATP: adenosine triphosphate; BBC3/PUMA: BCL2 binding component 3; BNIP3: BCL2/adenovirus E1B interacting protein 3; CASP3: caspase 3; CASP9: caspase 9; CDKN1B/p27: cyclin dependent kinase inhibitor 1B; CVD: cardiovascular disease; DCFH-DA: 2',7'-dichlorodihydrofluorescein diacetate; DHE: dihydroethidium; DNM1L/Drp1: dynamin 1-like; EF: ejection fraction; FS: fractional shortening; H/R: hypoxia-reoxygenation; I/R: ischemia-reperfusion; LDH: lactate dehydrogenase; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MFF: mitochondrial fission factor; miRNA: microRNA; NAC: N-acetylcysteine; OCR: oxygen consumption rate; PIK3C3/Vps34: phosphatidylinositol 3-kinase catalytic subunit type 3; PM: particulate matter; PRKAA1/AMPK: protein kinase AMP-activated catalytic subunit alpha 1; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TRP53/p53: transformation related protein 53; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling.

Keywords: ADSC-exosomes; Mir221 and Mir222; cardiomyocyte; ischemia/reperfusion injury; mitophagy; particulate matter.

Figure 1.

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.

Figure 2.

PM significantly exacerbates H/R-induced cardiomyocyte damage. H9c2 cells were pretreated with or without different concentrations of PM (10 or 50 μg/mL) for 6 h, followed by treatment with or without hypoxia for 6 h and reoxygenation for 12 h. (A) cell viability was assessed by MTT assay (n = 4). PM (50 μg/mL) was used in the following experiments. (B) a TUNEL assay was used to assess apoptosis. Nuclei were stained with DAPI (blue), and tunel-positive cells were indicated by green fluorescence (scale bar: 50 μm, n = 3). (C) flow cytometry was used to measure the number of apoptotic cells via ANXA5/annexin V-FITC-PI staining (n = 3). (D) the levels of apoptosis-related proteins were measured by western blot analysis (n = 4-7). (E) fluorescence microscopy was used to detect ROS production by MitoSOX red (1 μM) staining (scale bar: 50 μm, n = 4). (F) MitoSOX red (1 μM)/TO-PRO-3 (100 nM) staining was used to identify viable cells producing mitochondrial ROS (MitoSOX red-positive/TO-PRO-3-negative) by flow cytometry (n = 3). (G) by fluorescence microscopy, DCFH-DA (10 μM) was used to detect cytoplasmic H₂O₂ (scale bar: 50 μm, n = 3-5). (H) DCFH-DA/PI staining was used to examine viable cells producing cytoplasmic H₂O₂ (DCFH-DA-positive/PI-negative) by flow cytometry (n = 3). (I) DHE (5 μM) was used to detect cellular superoxide anions by fluorescence microscopy (scale bar: 50 μm, n = 4). (J) DHE/DiOC₆(3) (90 nM) staining was used to examine viable cells producing cellular superoxide anions (DHE-positive/DiOC₆(3)-positive) by flow cytometry (n = 3). (K-M) H9c2 cells were pretreated with MitoTEMPO (10 nM) or NAC (5 mM) for 1 h before exposure to PM+H/R. MitoSOX red was used to detect mitochondrial ROS production through fluorescence microscopy and flow cytometry. Western blot was used to detect the levels of cell apoptosis (scale bar: 50 μm, n = 4-7). The data are expressed as the mean ± SEM; one-way ANOVA.

Figure 3.

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.

Figure 4.

Mir221 and Mir222 regulate the expression of Bnip3 and Lc3b, causing PM to aggravate myocardial H/R injury. H9c2 cells were transfected with Mir221- and Mir222-mimics or inhibitors for 24 h, pretreated with PM for 6 h, and subjected to H/R. (A) Mir221 and Mir222 expression in various treatment groups was assessed using qPCR (n = 3). (B) the impact of ADSC-Exos or Mir221- and Mir222-mimics on Mir221 and Mir222 expression in pm+h/r-treated cells was assessed by qPCR (n = 4). (C) schematic diagram depicting the binding of Mir221 and Mir222 to the 3’ UTRs of Bnip3, Lc3b, and Bbc3/Puma target genes. (D) luciferase activities of the Bnip3 and Lc3b reporters in the Mir221- and Mir222-mimics and scramble groups were measured (n = 4). (E) effects of ADSC-Exos or Mir221- and Mir222-mimics on Bnip3 and Lc3b mRNA expression in pm+h/r-treated cardiomyocytes (n = 3). (F and G) the effects of ADSC-Exos, Mir221- and Mir222-mimics (F), and Mir221- and Mir222-inhibitors (G) on the expression of mitophagy-related proteins were detected by western blot analysis (n = 5-7). (H) effects of ADSC-Exos or Mir221- and Mir222-mimics on the OCR in cardiomyocytes treated with PM+H/R (n = 5). (I) effect of ADSC-Exos, Mir221- and Mir222-mimics, siBNIP3, and siLC3B on the viability of pm+h/r-treated H9c2 cells (n = 5). (J) the impact of ADSC-Exos and Mir221- and Mir222-mimics on apoptosis in pm+h/r-treated cardiomyocytes was assessed via a TUNEL assay (scale bar: 50 μm, n = 6). (K and L) the effects of ADSC-Exos, Mir221- and Mir222-mimics (K), and Mir221- and Mir222-inhibitors (L) on the expression of apoptosis-related proteins were assessed by western blot analysis (n = 4-7). The data are expressed as the mean ± SEM; one-way ANOVA.

Figure 5.

Effects of Bnip3, Lc3b, and Bbc3/Puma downregulation on pm+h/r-induced mitochondrial fission, mitochondrial autophagy, and apoptosis. (A) the effects of the downregulation of Bnip3, Lc3b, and Bbc3/Puma on cell apoptosis in pm+h/r-treated cardiomyocytes were examined by a TUNEL assay (scale bar: 50 μm, n = 4). (B and C) the effects of the downregulation of Bnip3 (B) and Lc3b (C) on the expression of mitochondrial fission-related, mitophagy-related, and apoptosis-related proteins in cells exposed to PM+H/R, was determined by western blot analysis (n = 4-11). (D) western blot analysis of the effects of Bbc3/Puma downregulation on the expression of mitochondrial fission- and mitophagy-related proteins in cells exposed to PM+H/R (n = 5-10). (E) H9c2 cells were transfected with Mir221 and Mir222 or with Bnip3 and Lc3b overexpression, and then exposed to PM+H/R conditions. The expression levels of BBC3/PUMA, MAP1LC3B/LC3B, and BNIP3 proteins were assessed by western blot. The data are expressed as the mean ± SEM; one-way ANOVA.

Figure 6.

Mir221 and Mir222 in ADSC-Exos reduce mitochondrial ROS levels, mitochondrial fission, mitophagy, and apoptosis in cardiomyocytes exposed to PM+H/R. H9c2 cells were treated with ADSC-Exos (2 µg/mL) or transfected with Mir221- and Mir222-mimics for 24 h, followed by treatment with PM (50 µg/mL) and H/R (6 h/12 h). (A and B) the effects of ADSC-Exos, Mir221- and Mir222-mimics, and MitoTEMPO treatment on mitochondrial ROS were evaluated via MitoSOX red staining via fluorescence microscopy and flow cytometry (scale bar: 50 μm, n = 3). (C and D) the effects of ADSC-Exos, Mir221- and Mir222-mimics, MitoTEMPO, and mdivi-1 treatment on δψm were assessed by JC-1 staining under fluorescence microscopy and flow cytometry (scale bar: 50 μm, n = 3). (E) ATP production was assessed using an ATP assay (n = 3). (F) mitochondrial length was assessed by a MitoTracker assay (scale bar: 20 or 5 μm, n = 7). (G) the expression of DNM1L/Drp1 and MFF was assessed by western blot analysis (n = 4-11). (H) the effects of treatment with ADSC-Exos, Mir221- and Mir222-mimics, MitoTEMPO, mdivi-1, and bafilomycin A₁ on AL accumulation was determined by AO staining (scale bar: 20 μm). (I) cardiomyocytes were transduced with RFP-GFP-LC3 lentivirus for 72 h. The lentivirus allowed the distinction of autophagosomes (GFP+ and RFP+; yellow plots) and ALs (GFP− and RFP+; red plots), as GFP fluorescence was quenched in the acidic ALs. The effects of ADSC-Exos, Mir221- and Mir222-mimics, MitoTEMPO, mdivi-1, and bafilomycin A₁ on the formation of autophagosomes and ALs assessed by RFP-GFP-LC3 staining (scale bar: 20 μm, n = 10). (J) the effects of treatment with ADSC-Exos, Mir221- and Mir222-mimics, MitoTEMPO, mdivi-1, and bafilomycin A₁ on the expression of mitophagy-related proteins were examined by western blot analysis (n = 6-17). (K and L) the effects of ADSC-Exos, Mir221- and Mir222-mimics, MitoTEMPO, mdivi-1, and bafilomycin A₁ on cell apoptosis were assessed by ANXA5/annexin V-FITC-PI flow cytometry and western blot analysis (n = 3-13). The data are expressed as the mean ± SEM; one-way ANOVA.

Figure 7.

Mir221 and Mir222 decrease pm+i/r-induced cardiac damage in WT, mir221- and mir222-ko, and Mir221- and Mir222-tg mice. WT or mir221- and mir222-ko mice were preconditioned with PM for 24 h. Then, 25 min after occlusion, ADSC-Exos (100 μg of protein in 50 μL) or Mir221- and Mir222-mimics (100 nM) were uniformly intramuscularly injected into the left ventricular marginal zone. The effects of ADSC-Exos and Mir221- and Mir222-mimics on (A) plasma LDH and TNNI levels (n = 5 mice per group), as well as (B) Mir221 and Mir222 expression (n = 4-7 mice per group), were measured by qPCR. (C) cardiac function was measured via echocardiography (n = 7-9 mice per group). (D and E) ROS levels were measured via MitoSOX red staining and DHE staining (scale bar: 50 μm, n = 3-7). (F) BNIP3 and MAP1LC3B/LC3B expression was measured via western blot analysis (n = 9-10 mice per group). (G) BNIP3 and MAP1LC3B/LC3B expression was measured via immunohistochemistry (scale bar: 50 μm, n = 3-8). (H) the expression of apoptosis-related proteins was evaluated by western blot analysis. (I) the number of apoptotic cells was measured by a TUNEL assay (scale bar: 50 μm, n = 5-11 mice per group). To investigate the impact of Mir221 and Mir222 expression on pm+i/r-induced cardiac damage, Mir221- and Mir222-overexpression TG mice were preconditioned with PM for 24 h, followed by myocardial I/R. The results were compared to those of PM+I/R WT mice and PM+I/R+ADSC-Exos WT mice. (J) qPCR was performed to measure Mir221 and Mir222 expression (n = 5 mice per group). (K) echocardiography was used to assess heart function (n = 5 mice per group). (L) LDH and TNNI levels in plasma were measured (n = 6-7 mice per group). (M) ROS expression was detected using MitoSOX red and DHE staining (scale bar: 50 μm, n = 3-7). (N) western blot analysis was used to evaluate the mitochondrial autophagy-related protein levels (n = 8-9 mice per group). (O) western blot analysis was used to evaluate the apoptosis-related protein levels (n = 6-12 mice per group). (P) a TUNEL assay was used to quantify apoptotic cell numbers (scale bar: 50 μm, n = 7-13 mice per group). The data are expressed as the mean ± SEM; one-way ANOVA.

Figure 8.

Knockdown of Bnip3 and Lc3b expression improves cardiac function and reduces apoptosis-related proteins. (A and B) mir221- and mir222-ko mice were preconditioned with PM for 24 h. Then, 25 min after occlusion, siBNIP3 or siLC3B were uniformly intramuscularly injected into the left ventricular marginal zone. EF and FS results were obtained by echocardiography to assess cardiac function (n = 4 mice per group). The expression levels of BBC3/PUMA, BNIP3, and MAP1LC3B/LC3B were also examined by western blot (n = 3 mice per group). (C) WT mice were intratracheally injected with PM and intraperitoneally injected with bafilomycin A₁ (2.5 mg/kg) for 24 h, followed by 30 min of ischemia and 3 h of reperfusion. The levels of BBC3/PUMA, BNIP3, MAP1LC3B/LC3B, and SQSTM1/p62 were measured by western blot (n = 4-5 mice per group).