Metoprolol prevents chronic obstructive sleep apnea-induced atrial fibrillation by inhibiting structural, sympathetic nervous and metabolic remodeling of the atria

Figures

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  Figure 1. Examples of burst pacing-induced AF in sham-treated dogs (A) and OSA dogs (B).

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  Figure 2. AF inducibility, AF duration, AERP, SNTR and intra-atrial conduction velocity in dogs from the Sham, OSA and MET groups. (A) AF inducibility in the Sham, OSA and MET groups. (B) Mean AF duration in the Sham, OSA and MET groups. (C) AERP in the Sham, OSA and MET groups. (D) Sinus node recovery time (SNRT) in the Sham, OSA and MET groups. (E) Intra-atrial conduction velocity in control and chronic OSA dogs. (F) Representative bands and statistical results of connexin (Cx) 40 in atrial tissue. (G) Representative bands and statistical results for Cx43 in the atrial tissue. *p < 0.05, **p < 0.01, ***p < 0.001 vs Sham group, # p < 0.05, ## p < 0.01, ### p < 0.001 vs OSA group, n = 7 per group.

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  Figure 3. Sympathetic nerve sprouting and distribution of nerve fibers in the atria of OSA dogs and metoprolol-treated dogs. (A) Representative images of tyrosine hydroxylase (TH) expression in right atria. The magnification is 200 × (scale bar: 50 μm). (B) Statistical results for the expression of TH. (C) Representative images of GAP-43 expression in right atria. The magnification is 200 × (scale bar: 50 μm). (D) Statistical results for the expression of GAP43. (E–G) Representative bands and statistical results for the protein expression of TH, GAP43 and NGF in right atria. The expression levels of these proteins were normalized to the level of GAPDH or β-actin. **p < 0.01, ***p < 0.001 vs Sham group, # p < 0.05, ## p < 0.01, ### p < 0.001 vs OSA group, n = 6 per group.

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  Figure 4. Structural changes after chronic OSA with or without metoprolol in canine right atrial myocytes. (A) Hematoxylin and eosin (H&E) staining, direct magnification: 200 × (scale bar: 50 μm). (B) Masson’s trichrome staining. The magnification is 200 × (scale bar: 50 μm). (C) TUNEL staining, magnification: 400 × (scale bar: 20 μm). (D) Collagen volume fraction (CVF) per field in the atria of dogs from each group. (E) Apoptotic cells per field in canine atria. (F) Representative bands and statistical results for the protein expression of TGF-β or α-SAM in canine atria. The expression levels of these proteins were normalized to the level of GAPDH. **p < 0.01, ***p < 0.001 vs Sham group, # p < 0.05, ## p < 0.01, ### p < 0.001 vs OSA group, n = 6 per group. (G) Representative bands showing protein expression of caspase-9, BAX and apoptosis-inducing factor (AIF). **p < 0.01, ***p < 0.001 vs Sham group, # p < 0.05, ## p < 0.01, ### p < 0.001 vs OSA group, n = 5 per group.

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  Figure 5. Expression of proteins involved in the Sirt1/AMPK pathway in atria of OSA dogs and metoprolol-treated dogs. (A) Representative bands showing the protein expression of p-Sirt1 and Sirt1 in right atria. (B,C) Statistical results for the expression of p-Sirt1 and Sirt1 in atrial tissue. (D) Representative bands showing the protein expression of p-AMPK and AMPK in right atria. (E,F) Statistical results for the expression of p-AMPK and AMPK in atrial tissue. (G) Representative bands for the protein expression of GLUT4 and FAT/CD36 in right atria. (H,I) Statistical results for the expression of GLUT4 and FAT/CD36 in atrial tissue. The expression levels of these proteins were normalized to the level of GAPDH. **p < 0.01, ***p < 0.001 vs Sham group, # p < 0.05, ## p < 0.01, ### p < 0.001 vs OSA group, n = 6 per group.

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  Figure 6. Expression of proteins involved in the Sirt1/AMPK pathway in hypoxic HL-1 cells. (A) Representative bands and statistical results for the protein expression of p-Sirt1 in HL-1 cells. (B) Representative bands and statistical results for the protein expression of Sirt1 in HL-1 cells. (C) Representative bands and statistical results for the protein expression of p-AMPK in HL-1 cells. (D) Representative bands and statistical results for the protein expression of AMPK in HL-1 cells. (E) Representative bands and statistical results for the protein expression of HIF-1α in HL-1 cells.The expression levels of these proteins were normalized to the level of GAPDH. *p < 0.05, **p < 0.01, ***p < 0.001 vs Normoxia group, # p < 0.05, ## p < 0.01, ### p < 0.001 vs the ISO + MET group, n = 4 each group.

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  Figure 7. Protein levels of mitochondrial respiratory chain complexes and Tfam in atria of OSA dogs and metoprolol-treated dogs. (A) Transmission electron microscopy images in dogs from the Sham group, OSA group and MET group, direct magnification: 8000 × (scale bar: 2 μm). n = 6 each group. (B) Representative immunoblots of Tfam in atrial tissue and statistical results for Tfam. (C) Representative immunoblots of HIF-1α in atrial tissue and statistical results for Tfam. (D) Western blot results for NDUFB8, SDHB, UQCRC2, MTCO1, and ATP5A protein expression and the quantification of NDUFB8, SDHB, UQCRC2, MTCO1, and ATP5A protein levels. NDUFB8 indicates a subunit of nicotinamide-adenine dinucleotide dehydrogenase (complex I); SDHB, 30-kDa subunit of succinate dehydrogenase (complex II); UQCRC2, core protein 2 of ubiquinol-cytochrome c reductase complex (complex III); MTCO1, subunit I of cytochrome c oxidase (complex IV); ATP5A, a subunit of F 0 F 1-ATP synthase (complex V). The expression levels of these proteins were normalized to the level of GAPDH. **p < 0.01, ***p < 0.001 vs Sham group, # p < 0.05, ## p < 0.01, ### p < 0.001 vs OSA group, n = 5 per group. (E) The atrial tissue…

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  Figure 8. Schematic – activation of SIRT1/AMPK pathway by metoprolol is required to inhibit AF induced by chronic OSA. Chronic OSA causes structural, sympathetic nerve and metabolic remodeling of the atria. Metoprolol inhibits sympathetic hyperinnervation, acts as a positive modulator of Sirt1 activity, and initiates an adaptive response. (i) Sirt1/AMPK can act as a potent suppressor of HIF-1α and cause mitochondrial damage; (ii) decreased expression of FAT/CD36 and GLUT4 on the cell membrane was reversed, thus rescuing impaired uptake of glucose and FA; and (iii) metoprolol inhibits atrial apoptosis and fibrosis, perhaps partly through the Sirt1/AMPK pathway.