Cholinergic Elicitation Prevents Ventricular Remodeling via Alleviations of Myocardial Mitochondrial Injury Linked to Inflammation in Ischemia‐Induced Chronic Heart Failure Rats

Figures

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  Figure 1. Flow chart of the experiment process: (a) study protocol diagram; (b) grouping criterion.

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  Figure 2. Effects of cholinergic elicitation on cardiac dysfunction and cardiac energy metabolism alterations. Representative M-mode echocardiogram of short-axis views from five groups (a). Comparisons of LVEDd (b), LVEF (c), LVFS (d), and ATP (F) among five groups. Correlation between LVEDd values of the 9th week and ATP contents (g). Differences of BNP (e), INS (h), NEFA (i), and glucose (j) among five groups at baseline, the 6th week, and the 9th week. ∗P < 0.05 vs. Sham group. #P < 0.05 vs. CHF group. $P < 0.05 vs. CHF+Vag group. &P < 0.05 vs. CHF+PNU group. LVEDd: left ventricular end-diastolic dimension; LVEF: left ventricular ejection fraction; LVFS: left ventricular fraction shortening; BNP: brain natriuretic peptide; ATP: adenosine triphosphate; INS: insulin; NEFA: nonesterified fatty acid.

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  Figure 3. Effects of cholinergic elicitation on systemic inflammation and cardiac macrophage infiltration. Typical images of CD68+ staining (a) (×20) and CD163+ staining (b) (×20) among five groups. Quantitative analysis of immunohistochemical results (c, d). Comparisons of IL-1β (e), IL-6 (g), and TNF-α (i) among five groups at three time points at baseline, the 6th week, and the 9th week. Correlations of LVEDd values of the 9th week with IL-1β (f), IL-6 (h), and TNF-α (j) levels of the 9th week. ∗P < 0.05 vs. Sham group. #P < 0.05 vs. CHF group. $P < 0.05 vs. CHF+Vag group. &P < 0.05 vs CHF+PNU group. IL-1β: interlukin-1β; IL-6: interlukin-6; TNF-α: tumor necrosis factor α.

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  Figure 4. Effects of cholinergic elicitation on cardiac fibrosis. Representative images of scar ranges by HE staining in five groups (a) (×10). Masson's trichrome staining and picrosirius red staining of left ventricular noninfarct sections in five groups (b, c) (×20). Quantitative statistics of collagen contents (d, e). ∗P < 0.05 vs. Sham group. #P < 0.05 vs. CHF group. $P < 0.05 vs. CHF+Vag group. &P < 0.05 vs. CHF+PNU group.

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  Figure 5. Effects of cholinergic elicitation on mitochondrial-dependent apoptosis. Representative images and detailed drawings of TUNEL staining among five groups ((a) ×10; (b) ×20). Western blot results for caspase-3, Bcl-2, and Bax of five groups (c). Quantitative statistical results of apoptotic rate (d) and relative expression levels of apoptosis-related proteins (e–g). ∗P < 0.05 vs. Sham group. #P < 0.05 vs. CHF group. $P < 0.05 vs. CHF+Vag group. &P < 0.05 vs. CHF+PNU group. Bcl-2: B cell leukemia/lymphoma 2; Bax: Bcl-2-associated X.

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  Figure 6. Effects of cholinergic elicitation on cardiomyocyte ultrastructural alterations. General observation (a) (×5000) and detailed view (b) (×30000) of transmission electron microscopy among five groups. Red arrow: mitophagy of autolysosome phase; green arrow: mitophagy of phagophore phase; blue arrow: mitophagy of mitophagolysosome phase.

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  Figure 7. Effects of cholinergic elicitation on cardiac ROS production. Typical images of ROS production by flow cytometry among five groups (a) and the related quantitative analysis (b). ∗P < 0.05 vs. Sham group. #P < 0.05 vs. CHF group. $P < 0.05 vs. CHF+Vag group. &P < 0.05 vs. CHF+PNU group.

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  Figure 8. Effects of cholinergic elicitation on myocardial mitochondrial dynamics and the activities of NF-κB and JAK2/STAT3 pathway. Western blot results and quantitative statistics for Drp1, Fis1, Mfn1, Mfn2, Opa1, α7nAChR, NF-κB, P-NF-κB, JAK2, P-JAK2, STAT3, and p-STAT3 (a–m). ∗P < 0.05 vs. Sham group. #P < 0.05 vs. CHF group. $P < 0.05 vs. CHF+Vag group. &P < 0.05 vs. CHF+PNU group. Drp1: dynamin-related protein 1; Fis1: fission, mitochondrial 1; Mfn1: mitofusin 1; Mfn2: mitofusin 2; Opa1: OPA1 mitochondrial dynamin-like GTPase; α7nAChR: α7-nicotinic acetylcholine receptor; NF-κB: nuclear factor kappa B; p-NF-KB: phosphorylated nuclear factor kappa B; JAK2: Janus kinase 2; p-JAK2: phosphorylated Janus kinase 2; STAT3: signal transducer and activator of transcription 3; p-STAT3: phosphorylated signal transducer and activator of transcription 3.

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  Figure 9. Impacts of TNF-α exposure on mitochondrial function of H9C2 cell lines. Evaluation of cell variability by cell growth curve (a). Screening of the optimal TNF-α-disposed concentration by ROS fluorescence intensity detections (b). Detections of cell proliferation by CCK-8 assay (c). Measurements of apoptosis rate, MMP, and mPTP by flow cytometry (d–f). Results of mitochondrial dynamics-related proteins between control and experimental groups by western blot (g). ∗P < 0.05 vs. control group. ROS: reactive oxygen; MMP: mitochondrial membrane potential; mPTP: mitochondrial permeability transition pore.

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  Figure 10. A schematic diagram of the cardioprotection of cholinergic elicitation to heart failure induced by MI. During the period of postinfarction heart advance into heart failure, chronic inflammation response induced by initial proinflammatory cytokines begets a detrimental pathophysiological circle through disturbing the mitochondrial biogenesis and eventually contributes to the ventricular remodeling. Such aforementioned vicious circle may be partly interrupted by cholinergic elicitation.