IP3R1/GRP75/VDAC1 complex mediates endoplasmic reticulum stress-mitochondrial oxidative stress in diabetic atrial remodeling

Fig. 1. Endoplasmic reticulum stress (ERS) is upregulated in type 2 diabetic rats and diabetic humans with atrial fibrillation (AF). A–H, Rat model of type 2 diabetes by high-fat feeding + low-dose streptozotocin (STZ) injection with or without 4-phenyl butyric acid (4-PBA) for 8 weeks. Representative Western blot results and analysis of the protein expression in the three groups are shown. A, Western blot detection of GRP78 protein levels. B, Quantification of GRP78 protein levels in (A). C, Western blot detection of CHOP protein levels. D, Quantification of CHOP protein levels in (C). E, Western blot detection of XBP1 protein levels. F, Quantification of XBP1 protein levels in (E). Values are the mean ± SEM. n = 6 per group. *P < 0.05, #P < 0.01, ANOVA with Bonferroni post-test. G, Analysis results of left atrial diameter (LAD). H, Quantification of the inducibility of AF. Values are the mean ± SEM. n = 7 in control group, n = 8 in T2DM group, n = 10 in 4-PBA group,. *P < 0.05, Chi-square test with Pearson's.

Fig. 2. The IP3R1–GRP75–VDAC1 complex plays an important role between the ER and the mitochondria in the heart. A, HL-1 cells were cultured with tunicamycin (TM) for 24 h. Mass spectrometry identified GRP75 as a strong interacting protein. B, Conventional immunofluorescence confirming the colocalization of IP3R1, GRP75, and VDAC1 in HL-1 cells. Scale bar = 5 μm. C, D, In situ proximity ligation assay (PLA) monitoring of perturbation of ER–mitochondria interactions linked to genetic modulation of IP3R1–GRP75–VDAC1 complex proteins. Shown are representative PLA images (C) and quantitative analysis (D) of IP3R1–GRP75, GRP75–VDAC1, and IP3R1–VDAC1 interactions in HL-1 cells cultured with control or with TM for 24 h. Scale bar = 20 μm. Values are the mean ± SEM, n = 4 independent experiments, *P < 0.05, two-tailed Student's t-test.

Fig. 3. GRP75 plays an important role in ER–mitochondria calcium exchange. A, The silencing efficiency of GRP75 siRNAs; siRNAs from two companies (Santa Cruz Biotechnology [Santa] and Jima) were used for the experiment. B, Quantification of GRP75 protein levels in (A). C, Quantification of GRP75 mRNA expression. D, Representative confocal microscopy images of fluorescence staining for Fluo-4 AM and DAPI. Scale bar = 20 μm. E, Quantification of Fluo-4 AM fluorescence intensity in (D). F, Representative confocal microscopy images of fluorescence staining for Rhod-2 AM and Mito Tracker Green. Scale bar = 20 μm. G, Quantification of Rhod-2 AM fluorescence intensity in (F). H, Western blot detection of mitochondria calcium protein VDAC1 and MCU levels in atrial myocytes. Quantification of VDAC1 (I) and MCU (J) protein levels in (H). K, Ultrastructural changes in HL-1 cells (green stars depict the mitochondria; white arrowheads depict the ER; red arrowheads depict ER–mitochondria contacts). N = Nucleus. Scale bars, top = 1 μm; middle = 2 μm; bottom = 500 nm. M, Measurements of ER–mitochondria interactions. Data are the mean ± SEM, n = 4 independent experiments, *P < 0.05, #P < 0.01, ANOVA with…

Fig. 4. Inhibiting GRP75 decreases reactive oxygen species (ROS) and improves mitochondrial function. A, Confocal microscopy detection of HL-1 cells stained with 2′,7′-dichlorofluorescein diacetate (DCFH-DA). Scale bar = 100 μm. B, Quantification of ROS by DCFH-DA intensity in (A). C, Confocal microscopy detection of HL-1 cells stained with Mito Sox Red. Scale bar = 100 μm. D, Quantification of mitochondrial ROS by Mito Sox Red in (C). E, Western blot detection of antioxidant stress protein manganese superoxide dismutase (Mn-SOD) levels in atrial myocytes. F, Quantification of protein levels in (E). G, JC-1 staining. Scale bar = 100 μm. H, Quantification of mitochondrial membrane potential (Δψm) of HL-1 cells by JC-1 aggregate–monomer ratio. Data represent the mean ± SEM (n = 4 independent experiments), #P < 0.01, ANOVA with Bonferroni post-test. I, Analysis of oxygen consumption rate (OCR). Oligomycin inhibits ATP synthase (J), FCCP uncouples oxygen consumption from ATP production (K), and AA + Retenone inhibits complexes I and III (L). Data represent the mean ± SEM (n = 6 independent experiments), *P < 0.05, #P < 0.01, ANOVA with Bonferroni post-test. . (For interpretation of the…

Fig. 5. Inhibition of GRP75 protects atrial myocytes from ERS-induced apoptosis. A, HL-1 cells under 24-h TM stimulation. Apoptosis was evaluated by flow cytometry using annexin–V fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. B–E, GRP75 siRNA inhibited caspase-9 (C) and caspase-3 (D) activation and increased the Bcl-2–Bax ratio (E). Data are the mean ± SEM, n = 4 independent experiments, #P < 0.01, ANOVA with Bonferroni post-test.

Fig. 6. Effect of GRP75 deficiency on echocardiography in TM-induced ERS mice. A, Western blot assessment of GRP75 expression in mouse atria. B, Quantification of protein levels in (A). Data are the mean ± SEM, n = 6 per group, #P < 0.01, two-tailed unpaired Student's t-test. C, Representative 2D and M-mode echocardiographic images. Shown are analysis results of LAD (D), left ventricular ejection fraction (LVEF) (E), and fractional shortening (FS) (F). Data are the mean ± SEM, n = 6 per group, ANOVA with Bonferroni post-test.

Fig. 7. Atrial remodeling and AF progression were attenuated in Myh6-Cre+/Hspa9flox/flox+ TM mice. A, Epicardial electrical mapping recording of the left atria. Analysis results of left atrial conduction velocity (LACV) (B), LA absolute inhomogeneity (C), and LA inhomogeneity index (D), B-D data are the mean ± SEM, #P < 0.01, n = 6 per group, ANOVA with Bonferroni post-test. E, Analysis results of EPS parameters. SCL, sinus cycle length; AVWCL, atrioventricular Wenckebach cycle length. F, Quantifications of sinus node recovery time (SNRT). G, Quantification of atrium effective refractory period (AERP) at basic cycle lengths of 120 ms and 100 ms. H, Representative AF episodes induced by right atrial (RA) burst pacing. I, Quantification of AF inducibility. E-I values are the mean ± SEM. *P < 0.05; #P < 0.01; n = 11 per group, Chi-square test with Pearson's. J, Representative images of RA HE and Masson's trichrome staining. Scale bar = 50 μm. K, Analysis results of interstitial fibrosis. Values are the mean ± SEM. #P < 0.01; n = 6 per group, ANOVA with Bonferroni post-test.

Fig. 8. The IP3R1–GRP75–VDAC1 complex mediates ERS–mitochondria-associated ER membranes (MAM)–mitochondrial oxidative stress and plays an important role in diabetic atrial remodeling. Pharmacological prevention of ERS from upstream by the chemical chaperone 4-PBA precludes mitochondrial oxidative stress and improves atrial remodeling in diabetes. Gene silencing or knockout of the key gene GRP75 of the MAMs inhibited ER–mitochondrial calcium transport, antagonizes mitochondrial oxidative stress and calcium overload, and improves diabetic atrial remodeling.