Hyperglycemia Induces Endoplasmic Reticulum Stress in Atrial Cardiomyocytes, and Mitofusin-2 Downregulation Prevents Mitochondrial Dysfunction and Subsequent Cell Death

Abstract

Mitochondrial oxidative stress and dysfunction play an important role of atrial remodeling and atrial fibrillation (AF) in diabetes mellitus. Endoplasmic reticulum (ER) stress has been linked to both physiological and pathological states including diabetes. The aim of this project is to explore the roles of ER stress in hyperglycemia-induced mitochondrial dysfunction and cell death of atrial cardiomyocytes. High glucose upregulated ER stress, mitochondrial oxidative stress, and mitochondria-associated ER membrane (MAM)- enriched proteins (such as glucose-regulated protein 75 (GRP75) and mitofusin-2 (Mfn2)) of primary cardiomyocytes in vitro. Sodium phenylbutyrate (4-PBA) prevented the above changes. Silencing of Mfn2 in HL-1 cells decreased the Ca²⁺ transfer from ER to mitochondria under ER stress conditions, which were induced by the ER stress agonist, tunicamycin (TM). Electron microscopy data suggested that Mfn2 siRNA significantly disrupted ER-mitochondria tethering in ER stress-injured HL-1 cells. Mfn2 silencing attenuated mitochondrial oxidative stress and Ca²⁺ overload, increased mitochondrial membrane potential and mitochondrial oxygen consumption, and protected cells from TM-induced apoptosis. In summary, Mfn2 plays an important role in high glucose-induced ER stress in atrial cardiomyocytes, and Mfn2 silencing prevents mitochondrial Ca²⁺ overload-mediated mitochondrial dysfunction, thereby decreasing ER stress-mediated cardiomyocyte cell death.

Figure 1

High glucose induces ER stress and upregulates intracellular and mitochondrial Ca²⁺ levels. (a) Primary atrial myocytes under normal glucose (NG) or high glucose (HG) with or without 4-PBA for 24 hours. Quantification of GRP78 and CHOP mRNA expression. (b) Primary atrial myocytes under NG or HG with or without 4-PBA for 48 hours. GRP78 and CHOP protein level in atrial myocytes detected by Western blot analysis. (c) Quantification of GRP78 and CHOP protein level in (b). (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 Rhod2 AM and MitoTracker Green. Scale bar, 20 μm. (g) Quantification of Rhod2 AM fluorescence intensity in (f). (h) MAM proteins such as IP₃R1, GRP75, VDAC1, and Mfn2 protein level in atrial myocytes detected by Western blot analysis. (i) Quantification of IP3R1, GRP75, VDAC1, and Mfn2 protein level in (h). Data are mean ± SEM, n = 4 independent experiments, ^∗p < 0.05, ^#p < 0.01, ANOVA with Bonferroni posttest.

Figure 2

High glucose induces mitochondrial oxidative stress and induced apoptosis. (a) Confocal microscopy detected primary atrial myocytes that stained with DCFH-DA. (b) Quantification of ROS by DCFH-DA intensity in (a). Data represent the mean ± SEM (n = 4 independent experiments). (c, d) JC-1 staining. The ratio of red/green fluorescence reflects changes in the Δψm of primary atrial myocytes. Data represent the mean ± SEM (n = 4 independent experiments). Scale bar, 100 μm. (e) Mn-SOD, Bax, and Bcl-2 protein level in atrial myocytes detected by Western blot analysis. (f, g) Quantification of Mn-SOD and Bax/Bcl-2 protein level in (e). Data are mean ± SEM, n = 4 independent experiments, ^∗p < 0.05, ^#p < 0.01, ANOVA with Bonferroni posttest.

Figure 3

Mfn2 siRNA disrupts endoplasmic reticulum- (ER-) mitochondria tethering in tunicamycin- (TM-) injured HL-1 cells. (a) Changes in GRP78 and Mfn2 levels induced by TM were examined using Western blot analysis. The silencing efficiency of Mfn siRNA also shown in (a). (b) Quantification of GRP78 and Mfn2 protein level in (a). (c) Quantification of Mfn2 mRNA expression. (d) Ultrastructural changes in HL-1 cells (green stars depicting the mitochondria, white arrowheads depicting the endoplasmic reticulum). Transmission electron micrograph images (red arrowheads depicting the ER-mitochondria contacts) and measurements of the ER-mitochondria distance (e). Scale bar, top 1 μm, middle 2 μm, and bottom 500 nm. Data are mean ± SEM, n = 4 independent experiments, ^#p < 0.01, ANOVA with Bonferroni posttest.

Figure 4

Mfn2 siRNA inhibited mitochondrial Ca²⁺ overload during ER stress induced by TM. (a) Representative confocal microscopy images of fluorescence staining for Fluo-4 AM and DAPI. Scale bar, 20 μm. (b) Quantification of Fluo-4 AM fluorescence intensity in (a). (c) Representative confocal microscopy images of fluorescence staining for Rhod2 AM and MitoTracker Green. Scale bar, 20 μm. (d) Quantification of Rhod2 AM fluorescence intensity in (c). (e) Mitochondria Ca²⁺ proteins such as VDAC1 and MCU protein level in atrial myocytes detected by Western blot analysis. (f) Quantification of VDAC1 and MCU protein level in (e). Data are mean ± SEM, n = 4 independent experiments, ^#p < 0.01, ANOVA with Bonferroni posttest.

Figure 5

Decreasing ER-mitochondria interactions by genetic downregulation of mitofusin-2 (Mfn2) protects HL-1 cells from mitochondria dysfunction. (a) Flow cytometry detected HL-1 cells that stained with DCFH-DA. (b) Quantification of ROS by DCFH-DA intensity in (a). Data represent the mean ± SEM (n = 4 independent experiments). (c, d) JC-1 staining. The ratio of red/green fluorescence reflects changes in the mitochondrial membrane potential of HL-1 cells. Data represent the mean ± SEM (n = 4 independent experiments). Scale bar, 100 μm. (e, f) Analysis of oxygen consumption rate (OCR) from a Seahorse XF 24 Extracellular Flux Analyzer. Oligomycin inhibits ATP synthase, FCCP uncouples oxygen consumption from ATP production, and AA+Rotenone inhibits complexes I and III, respectively. Data represent the mean ± SEM (n = 6 independent experiments), ^#p < 0.01.

Figure 6

Mfn2 siRNA inhibited endoplasmic reticulum- (ER-) mitochondria-dependent apoptosis. (a) Cell apoptosis was evaluated by flow cytometry using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. (b) HL-1 cells under TM stimulated for 24 hours. Mfn siRNA inhibited the Bax/Bcl-2 ratio and inhibited caspase3 and caspase9 activation (c). Data are mean ± SEM, n = 4 independent experiments, ^∗p < 0.05, ^#p < 0.01, ANOVA with Bonferroni posttest.

Figure 7

Central illustration. Mfn2 deficiency in this study protected cardiomyocytes against mitochondrial depolarization/reactive oxygen species (ROS) and programmed cell death, improving atrial remodeling.