Sigma1R restores mitochondrial energy metabolism via the IRE1α/XBP1 pathway

Figures

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  Fig. 1. Sigma1R overexpression inhibits tachypacing-induced damage in atrial myocytes. HL-1 cells were transfected with Sigma1R overexpressing lentivirus (OE-Sigma1R) or control lentivirus (OE-NC), followed by electrical stimulation for 24 h. (A) The relative mRNA expression levels of Sigma1R after stimulation (n = 3). (B) The relative protein expression levels of Sigma1R after stimulation by WB analysis(n = 3). (C) Apoptosis detection was performed with flow cytometry using PI and Annexin V-FITC double staining after stimulation (n = 3). (D) CCK-8 assay detected the cell viability after stimulation at 0 h, 24 h, 48 h and 72 h (n = 6). Data are expressed as the mean ± SD. Statistical analysis was performed using one-way ANOVA or two-way ANOVA followed by Tukey’s multiple comparisons test. P* < 0.05, P** < 0.01, P*** < 0.001.

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  Fig. 2. Sigma1R inhibits tachypacing-induced mitochondrial oxidative stress damage in atrial myocytes. HL-1 cells were transfected with Sigma1R overexpressing lentivirus (OE-Sigma1R) or control lentivirus (OE-NC), followed by electrical stimulation for 24 h. (A) Representative transmission electron microscopy images showing mitochondrial morphology in each group (Bar = 500 nm). (B) Quantification of ATP levels across different groups (n = 6). (C) Representative fluorescence microscopy images depicting mitochondrial superoxide production using MitoSOX Red staining (Bar = 100 μm) and quantification of MitoSOX Red fluorescence intensity (n = 3). (D) Representative Mitochondrial membrane potential assessed using fluorescence microscopy (Bar = 100 μm) and quantification of mitochondrial membrane potential across the groups (n = 3). Data are expressed as the mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. P* < 0.05, P** < 0.01.

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  Fig. 3. Sigma1R regulates Ca2+ mobilization in MAMs. (A) Representative transmission electron microscopy images of mitochondrial ultrastructure in each group (Bar = 500 nm). (B) Quantification of mitochondrial number. (C) Analysis of mitochondrial density. (D) Measurement of ER-mitochondrial contact site length. (E) Representative images of immunofluorescence staining using DAPI (nuclei) and Fura-2AM (calcium indicator) (Bar = 100 μm). (F) Quantification of Fura-2AM fluorescence intensity (n = 3) of E. (G) Representative images of immunofluorescence staining using Fura-2AM (cytosolic calcium indicator) and Fura-3AM (mitochondrial calcium indicator) (Bar = 100 μm). (H) Quantification of Fura-2AM or Fura-3AM fluorescence intensity (n = 3). Data are expressed as the mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. P* < 0.05, P** < 0.01, P*** < 0.001.

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  Fig. 4. Sigma1R regulates the Ca2+ transport complex on the MAM by targeting the IRE1α/XBP1 axis. (A) Fluorescence in situ hybridization images showing the localization of IRE1α (red) and Sigma1R (green) in cells, with DAPI staining the nuclei (blue) (Bar = 20 μm). (B and C) Western blot analysis of IRE1α, PIRE1, and XBP1S protein levels in each group (n = 3). (D and E) Western blot analysis of Grp75, IP3R, and VDAC1 in each group (n = 3). Data are expressed as the mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. P* < 0.05, P** < 0.01, P*** < 0.001.

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  Fig. 5. Effects of Sigma1R and IRE1α on mitochondrial integrity and protein expression in atrial fibrillation. (A) Representative Mitochondrial membrane potential assessed using fluorescence microscopy (Bar = 100 μm). (B) Quantification of mitochondrial membrane potential across the groups (n = 3). (C) Representative fluorescence microscopy images depicting mitochondrial superoxide production using MitoSOX Red staining (Bar = 100 μm). (D) Quantification of MitoSOX Red fluorescence intensity (n = 3). (E) Representative transmission electron microscopy images of mitochondrial ultrastructure in each group (Bar = 500 nm), the red arrow indicates the mitochondria. (F) Quantification of mitochondrial number. (G) Analysis of mitochondrial density. (H) Measurement of ER-mitochondrial contact site length. (I and J) Western blot analysis of Grp75, IP3R, and VDAC1 in each group (n = 3). Data are expressed as the mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. P* < 0.05, P** < 0.01, P*** < 0.001.

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  Fig. 6. Regulation of calcium homeostasis by Sigma1R and IRE1α in atrial fibrillation. (A) Representative images of immunofluorescence staining using DAPI (nuclei) and Fura-2AM (calcium indicator) (Bar = 100 μm). (B) Quantification of Fura-2AM fluorescence intensity (n = 3). (C) Quantification of Ca2+ in the endoplasmic reticulum (n = 6). (D) Representative images of immunofluorescence staining using Fura-2AM (cytosolic calcium indicator) and Fura-3AM (mitochondrial calcium indicator) (Bar = 100 μm). (E) Quantification of Fura-2AM or Fura-3AM fluorescence intensity (n = 3). Data are expressed as the mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. P* < 0.05, P** < 0.01, P*** < 0.001.