Obstructive Sleep Apnea Impacts Cardiac Function in Dilated Cardiomyopathy Patients Through Circulating Exosomes

Figures

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  Figure 1. Identification of isolated plasma exosomes. (A) Image of isolated exosomes under a transmission electron microscope. Scale bar = 100 nm. (B) The mean diameter was ~79.2 nm, and the particle concentration was 1.24 × 109 particles/ml as determined by NanoSight analysis. Horizontal axis, particle size (nm); vertical axis, particle concentration (particles/ml). (C) Protein levels of exosomal positive markers CD9, CD81 and TSG101 were significantly increased and negative marker Calnexin was significantly reduced in the exosomes group compared with the plasma group.

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  Figure 2. Exosomes from OSA patients (OSA-Exos) increased the expression of Col1A1, ANP and BNP in H9C2 cells. (A) Col1A1 and BNP protein expression was significantly increased in H9C2 cells after treatment with 5, 10, and 15 μg/ml exosomes from one OSA patient compared with the control (0 μg/ml); ANP protein expression was significantly increased in H9C2 cells after treatment with 10 and 15 μg/ml exosomes compared with the control (0 μg/ml). (B) Densitometric analyses of the Western blot presented in A. (C) Col1A1, ANP and BNP protein expression was significantly increased in H9C2 cells treated with 10 μg/ml OSA-Exos compared with exosomes from healthy controls (Ctrl-Exos) as assessed by Western blot. (D) Densitometric analyses of the Western blot presented in C. GAPDH served as the internal control. (E) H9C2 cells were treated with 10 μg/ml OSA-Exos and Ctrl-Exos. Col1A1 (green) expression was detected by immunofluorescence staining. The bars indicate the mean ± SD of three separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. OSA-Exos 0 group. #p < 0.05, ###p < 0.001, ####p < 0.0001 vs. Ctrl-Exos groups. ns, no significant.

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  Figure 3. OSA-Exos inhibited autophagy via the Akt/mTOR signaling pathway in H9C2 cells. (A) LC3B II/I expression decreased in H9C2 cells treated with 10 and 15 μg/ml exosomes from one OSA patient compared with the 0 (control) and 5 μg/ml. Beclin1 protein expression decreased, p-Akt/Akt and p-mTOR/mTOR expression increased in H9C2 cells treated with 5, 10 and 15 μg/ml exosomes compared with the control (0 μg/ml). (B) Densitometric analyses of the Western blot presented in A. (C) LC3B II/I and beclin1 protein expression decreased, p-Akt/Akt and p-mTOR/mTOR expression increased in H9C2 cells treated with 10 μg/ml OSA-Exos compared with the control as determined by Western blot analysis. (D) Densitometric analyses of the Western blot presented in C. GAPDH served as the internal control. The bars indicate the mean ± SD of three separate experiments. **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. OSA-Exos 0 group. Δp < 0.05, ΔΔp < 0.01, ΔΔΔp < 0.001, ΔΔΔΔp < 0.0001 vs. Ctrl-Exos groups. ns, no significant.

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  Figure 4. Akt/mTOR-mediated autophagy was involved in OSA-Exos induced cardiomyocyte damage. (A) Rapamycin treatment significantly decreased the increased p-mTOR/mTOR, Col1A1, ANP, and BNP protein expression and increased the decreased LC3B II/I and beclin1 expression induced by OSA-Exos treatment. (B) Densitometric analyses of the Western blot presented in A. GAPDH served as the internal control. The bars indicate the mean ± SD of three separate experiments. Δp < 0.05, ΔΔp < 0.01, ΔΔΔp < 0.001, ΔΔΔΔp < 0.0001 vs. OSA-Exos groups. rapa, rapamycin.