The Defect of iNKT Cells Mediates Mitochondrial Dysfunction Through IL-6/STAT3 Signaling to Induce Atrial Inflammation and Fibrosis to Aggravate Postoperative Atrial Fibrillation

Figures

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  Fig. 1. Flow chart of experiment

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  Fig. 2. iNKT cells were up-regulated in SP mice. A–B) SP mouse model was established, and the expression of CD1d positive leukocytes (CD1d+CD45+) in atrial tissue was analyzed by flow cytometry, which was significantly increased in SP mice. C–D) The expression of iNKT cells (CD45+CD3+NK1.1+) in atrial tissue was detected using flow cytometry, which was markedly enhanced in SP mice. E–F) The expression of iNKT cell activation marker CD69 in atrial tissue was analyzed by flow cytometry, which was notably enhanced in SP mice. G–H) CD1d protein in atrial tissue was analyzed by western blot, which was significantly increased in SP mice. n=7, ***P < 0.001.

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  Fig. 3. iNKT deficiency increased AF susceptibility in SP mice. A–B) The statistical data of the probabilities and durations of AF induced 2 days after surgery were significantly increased in SP mice. C) The incidence of atrial ectopic or fibrillation per S2 interval was significantly increased in SP mice. D–G) AF-related proteins in atrial tissue were detected using western blot. The levels of Cav1.2, Kv1.5, Nav1.5, CX40, and CX43 proteins were significantly decreased in SP mice. n=7, **P < 0.01, ***P < 0.001.

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  Fig. 4. iNKT deficiency aggravated inflammatory cell infiltration and fibrosis in atrial tissue of SP mice. A–B) HE and Masson staining detected atrial pathological changes. SP tissues exhibited marked inflammatory cell infiltration and cardiomyocyte necrosis. SP tissues exhibited marked blue collagen deposition (reflecting early fibrotic changes) (×20, 100 μm). C–E) Inflammatory cells in atrial tissue were detected by immunohistochemistry. MPO+ cell numbers and CD68+ cell numbers were significantly enhanced in SP mice (×40, 50 μm). F–K) ELISA analyzed inflammatory factors. In SP mice, the contents of IL-1β, IL-6, MCP-1, TGF-β1, and PDGF-AB were also markedly raised, and IL-10 level was notably declined. L–M) α-SMA level in atrial tissue was analyzed by immunohistochemistry, which was significantly increased in SP mice (×20, 100 μm). N–P) Western blot analyzed fibrosis proteins in atrial tissue. α-SMA, Collagen I, Collagen III, MMP9, and TIMP2 levels were significantly raised in SP mice. n=7, *P < 0.05, **P < 0.01, ***P < 0.001.

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  Fig. 5. iNKT deficiency aggravated mitochondrial calcium homeostasis imbalance and mitochondrial damage in SP mice. A–B) The transient parameters of Ca2+ in atrial tissue were calculated. The amplitude and rising rate of Ca2+ were significantly decreased in SP mice. C–D) Western blot detected mitochondrial-endoplasmic reticulum calcium coupling related proteins. p-RyR2, IP3R1, VDAC1, and GRP75 proteins were markedly increased in SP mice. E–G) The morphology of mitochondria in atrial tissue was observed by TEM. The mitochondria of SP mice were swollen, the cristae were reduced or broken, the number of mitochondria was significantly increased, and the surface density of mitochondrial cristae and mitochondrial volume density were significantly decreased (×15.0k, 2.0 μm). H–I) The content of mtProtein and mtDNA in atrial tissue was detected, which was significantly decreased in SP mice. J–M) ATP content and mitochondrial complex enzyme I, II, III activity in atrial tissue were measured, which were significantly decreased in SP mice. N–O) DHE staining assessed atrial ROS levels, which were significantly increased in SP mice (×40, 50 μm). P–Q) The content of oxidation index in atrial tissue…

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  Fig. 6. iNKT deficiency further up-regulated IL-6/STAT3 signaling protein in SP mice. A–C) IL-6 and STAT3 expressions on atrial tissue were assayed by immunohistochemistry, which was significantly increased in SP mice (×40, 50 μm). D–E) Western blot detected IL-6/STAT3 signaling protein. IL-6 and p-STAT3 protein expressions were markedly enhanced in SP mice. n=7, **P < 0.01, ***P < 0.001.

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  Fig. 7. Anti-IL-6 neutralizing antibody improved AF susceptibility in iNKT-deficient SP mice via suppressing IL-6/STAT3 axis. A–B) Western blot detected IL-6/STAT3 signaling protein. Anti-IL-6 significantly reduced IL-6 and p-STAT3 protein levels. C–D) The statistical data of the probabilities and durations of AF induced 2 days after surgery were significantly reduced after anti-IL-6 intervention. E–H) AF-related proteins in atrial tissue were detected using western blot. Anti-IL-6 significantly increased the levels of Cav1.2, Kv1.5, Nav1.5, CX40, and CX43 proteins. n=7, *P < 0.05, **P < 0.01, ***P < 0.001.

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  Fig. 8. Anti-IL-6 neutralizing antibody improved atrial tissue mitochondrial dysfunction, inflammation and fibrosis in iNKT-deficient SP mice by inhibiting IL-6/STAT3 axis. A–B) Transient parameters of Ca2+ in atrial tissue were calculated, and anti-IL-6 significantly increased the amplitude and rate of Ca2+ increase. C–D) The content of mtProtein and mtDNA in atrial tissue was detected, which was significantly increased after anti-IL-6 intervention. E–H) ATP content and mitochondrial complex enzyme I, II, III activity in atrial tissue were measured, which were significantly increased after anti-IL-6 intervention. I–J) DHE staining assessed the level of ROS in atrial tissue, which was significantly reduced after anti-IL-6 intervention (×40, 50 μm). K–O) ELISA detected inflammatory factors. Anti-IL-6 significantly reduced the contents of IL-1β, IL-6, MCP-1, TGF-β1, and PDGF-AB, and increased IL-10 level. P–R) The expression of fibrosis protein in atrial tissue was assessed using western blot. α-SMA, Collagen I, Collagen III, MMP9, and TIMP2 expressions were significantly decreased after anti-IL-6 intervention. n=7, *P < 0.05, **P < 0.01, ***P < 0.001.