Mfn2 regulates calcium homeostasis and suppresses PASMCs proliferation via interaction with IP3R3 to mitigate pulmonary arterial hypertension

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  Fig. 1. The MCT-induced PAH rat model exhibits ERS and histopathological changes. A: The mRNA levels of key ER stress-related genes in lung tissue by RT-qPCR (n = 3 per group). The results indicated dysregulation of ER stress-related genes, including a significant reduction in Mfn2 expression and a marked increase in IP3R3, Perk, Eif2α, Chop, and Atf4. B: HE staining of pulmonary arterioles (scale bar = 50 μm) showed that the walls of small pulmonary arteries were normal in the NC group, while they were significantly thickened in the lung tissues of PAH rats. C: TEM analysis of PASMCs mitochondrial structures between groups (n = 5 to 6 images per group, scale bar = 200 nm). In the NC group, mitochondria were structurally intact, with preserved cristae and intact cell membranes. In the PAH group, mitochondria were shortened, and the membranes were damaged and disintegrated, with cristae ruptured or absent. Green arrows indicate mitochondria. NC: Normal control; PAH: MCT-induced PAH rat model. Data are presented as mean ± SD. Data were analyzed using the Student’s t-test (normal distribution) or Mann-Whitney U test (non-normal distribution). **P < 0.01, indicating a significant difference…

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  Fig. 2. Reduced Mfn2 expression and dysregulated ERS-related gene expression in the TNF-α-induced PAH cell model. A: Gradient concentrations of TNF-α stimulation in PASMCs over time. PASMCs were treated with varying concentrations of TNF-α (0-160 ng/mL) for 0, 12, 24, 36, 48, and 60 h, followed by an MTT assay to evaluate cell viability. The results showed that PASMCs proliferation significantly increased in response to TNF-α in a dose-dependent manner, which was more pronounced with extended incubation times. B and C: Immunofluorescence staining to observe Mfn2 in cellular substructures (n = 3 per group, scale bar = 25 μm). The immunofluorescence images showed a distinct red signal when Hoechst staining bound to Mfn2. Mfn2 expression was significantly lower in the TNF-α-induced PAH cell model compared to normal cells. Data were analyzed using the Student’s t-test (normal distribution) or Mann-Whitney U test (non-normal distribution). D and E: Immunofluorescence staining was used to detect Mfn2 distribution and expression among different intervention groups (n = 3 per group, scale bar = 25 μm). The images showed that Mfn2 was expressed at lower levels in the TNF-α-induced PAH cell model…

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  Fig. 3. Mfn2 regulates Ca2+ homeostasis in PAH cells to maintain mitochondrial integrity. A and B: Observed Ca2+ distribution in different cell substructures (n = 3 per group, scale bar = 25 μm). The results showed that Ca2+ concentrations were significantly higher in the cytoplasm and ER and lower in mitochondria in the PAH cell model compared to the normal control group. Data were analyzed using the Student’s t-test (normal distribution) or Mann-Whitney U test (non-normal distribution). C and D: Ca2+ concentrations observed by immunofluorescence staining in different groups (n = 3 per group, scale bar = 25 μm). Ca2+ levels were measured in different cellular compartments, including the ER, cytoplasm, and mitochondria. The result indicated that overexpression of Mfn2 in the PAH cell model led to a significant decrease in cytoplasmic Ca2+ and a significant increase in mitochondrial Ca2+. Conversely, silencing Mfn2 led to increased cytoplasmic Ca2+ and decreased mitochondrial Ca2+. E: Detection of mitochondrial ultrastructure by TEM (n = 5 to 6 images or visual fields per group, scale bar = 2 μm). Green arrows indicate mitochondria. Electron microscopy showed that mitochondria were…

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  Fig. 4. Inhibition of IP3R3 and administration of CPGs reactivates the Mfn2-IP3R3 signaling. A: Immunoprecipitation (IP) analysis of the interaction between Mfn2 and IP3R3 showed that the expression of IP3R3 and Mfn2 was directly verified using antibodies in the Input group. In contrast, no expression of IP3R3 or Mfn2 was detected when IgG antibodies were used, excluding the possibility of non-specific protein binding. Furthermore, immunoprecipitation of IP3R3 with Mfn2 demonstrated co-precipitation of IP3R3 and Mfn2, indicating that IP3R3 and Mfn2 interact and are negatively regulated. B and C: Protein levels of key factors in PAH cells determined by immunoblotting (n = 3 per group). D: RT-qPCR measured the mRNA levels of key genes in PAH cells (n = 3 per group). IP3R3 expression was significantly increased in the PAH cell model, but interventions with the IP3R3 inhibitor XC, application of CPGs, and Mfn2 overexpression inhibited IP3R3 expression. The combination of Mfn2 overexpression and XC further reduced IP3R3 expression. Mfn2 expression was significantly decreased in the PAH cell model, but intervention with XC and CPGs promoted Mfn2 recovery, with CPGs showing a more pronounced…

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  Fig. 5. Mfn2-IP3R3 signaling regulates Ca2+ homeostasis in PAH cells and influences mitochondrial morphology and structure. A and B: Ca2+ concentrations were observed by immunofluorescence staining in different groups (n = 3 per group, scale bar = 25 μm). Cytoplasmic Ca2+ concentrations significantly increased in the PAH cell model, while mitochondrial Ca2+ concentrations significantly decreased. Treatment with the IP3R3 inhibitor XC, application of CPGs, or Mfn2 overexpression reversed these changes, reducing cytoplasmic Ca2+ concentrations and increasing mitochondrial Ca2+ concentrations. Mfn2 overexpression combined with XC had a more pronounced effect, suggesting that the Mfn2-IP3R3 signaling is crucial for regulating Ca2+ homeostasis in PAH cells. Furthermore, CPGs intervention had the most significant impact on restoring intracellular Ca2+ homeostasis. C: TEM observed mitochondrial ultrastructure (n = 5 to 6 images or visual fields per group, scale bar = 1 μm). Green arrows indicate mitochondria. Data are presented as mean ± SD. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (normal distribution) or Kruskal-Wallis test with Dunn’s post hoc test (non-normal…

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  Fig. 6. Mfn2-IP3R3 signaling affects the proliferation and apoptosis of PAH cells. A: CCK-8 assay of cell proliferation in different intervention groups (n = 3 per group). B: Flow cytometry analysis of apoptosis in different intervention groups (n = 3 per group). Data are presented as mean ± SD. Data were analyzed by one-way ANOVA with Tukey’s post hoc test (normal distribution) or Kruskal-Wallis test with Dunn’s post hoc test (non-normal distribution). **P < 0.01 indicates a significant difference compared with NC; ##P < 0.01 indicates a significant difference between intervention groups

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  Fig. 7. Inhibition of ERS regulates Mfn2-IP3R3 signaling to balance Ca2+ homeostasis in mitochondria and thereby maintain mitochondrial integrity. A: RT-qPCR determined the mRNA levels of key genes in lung tissue (n = 3 per group). B and C: Protein levels of key genes in lung tissue determined by immunoblotting (n = 3 per group). NC: Normal control group; PAH: Model group; REV (Remediation group): Saline administered daily at 500 mg/(kg·d) on the first day, followed by 4-PBA at 500 mg/(kg·d) for 2 weeks; PRE (Prevention group): Rats were given 4-PBA at 500 mg/(kg·d) daily for 4 weeks from the start. **P < 0.01, indicating a significant difference compared to the PAH group. D and E: Ca2+ concentration observed by immunofluorescence staining in different groups (n = 3 per group). ER: Ca2+ in endoplasmic reticulum marked in green; Cytoplasm: Ca2+ in cytoplasm marked in blue; Mitochondrion: Ca2+ in mitochondria shown in red. NC: Normal control; TNF-α: TNF-α-induced PAH cell model; 4-PBA: 4-PBA intervention group. F: Mitochondrial ultrastructure observed by TEM in lung tissue of MCT-induced PAH rats (n = 5 to 6 images or fields per group, scale bar = 200 nm). The results indicated that 4-PBA…