FGF2 Attenuated Inflammation-Mediated Cardiac Damage: A Novel Mechanistic Insight into the AMPK–FUNDC1–Mitophagy

Figures

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  Fig. 1.. LPS inhibits FGF2 expression in cardiomyocyte. LPS was injected into WT mice to establish the septic cardiomyopathy model. (A and B) Immunohistochemistry of the FGF2 expression decreasing in response to LPS challenge. (C to E) FGF2 expression in different cell types from heart was evaluated using single-cell sequencing analysis. The expression of FGF2 in cardiomyocyte (F and G) and HL-1 (H and I) cells was detected via Western blotting. Data are shown as the means ± SEM (n = 12 per group). *P < 0.05.

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  Fig. 2.. FGF2 deletion aggravates cardiac damage in sepsis. The cardiac injury markers Tn-T, CK-MB, LDH, and BNP (A to D) and the concentrations of inflammatory factors TNFα, IL-6, MCP-1, and IL-β (E to H) in blood were measured via ELISA. (I to L) Following septic cardiomyopathy induced by LPS, the expression levels of mRNAs encoding inflammatory factors, including IL-1β, TNFα, IL-6, and MCP-1, were quantified by qPCR. (M and N) Heart tissues were collected and subsequently subjected to immunofluorescence labeling to examine the aggregation of Gr-1-positive cells in the myocardium. (O and P) Cardiac reactive oxygen species (ROS) content change measured with DHE. Data are shown as the means ± SEM (n = 12 per group). *P < 0.05.

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  Fig. 3.. FGF2 deletion promotes cardiac dysfunction in septic cardiomyopathy. (A) Representative M-mode echocardiographic images. (B) Quantification of left ventricular ejection fraction (LVEF). (C) Fractional shortening (FS). (D) Left ventricular end-diastolic diameter (LVDd). (E) Left ventricular end-systolic diameter. (F) Resting cell length. (G) Maximal velocity of shortening (+dL/dt). (H) Maximal velocity of relengthening (−dL/dt). (I) Peak shortening (PS) (normalized to cell length). (J) Time-to-peak shortening (TPS). (K) Time-to-90% relengthening (TR90). (L) Transmission electron microscopy (TEM) was used to observe the ultrastructural changes after LPS treatment in vivo. Data are shown as the means ± SEM (n = 12 per group). *P < 0.05.

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  Fig. 4.. FGF2 ablation potentiates mitochondrial dysfunction. Comparative analysis of cardiac tissue between FGF2−/− mice and WT mice after LPS administration (n = 4 per group) revealed 447 differentially expressed proteins (|log2FC| > 0.5), with 73 up-regulated and 374 down-regulated proteins. (A to C) Gene Ontology Enrichment Analysis Investigation of the proteins that are expressed at varying levels in heart tissue. (D) KEGG analysis. (E and F) GSEA analysis of differentially expressed genes following FGF2 deletion. (G to I) The activities of the mitochondrial respiratory complexes were quantified using ELISAs. (J) ATP generation. The mitochondrial membrane potential (K and L) and mROS (M and N) were analyzed with an immunofluorescence assay. Data are shown as the means ± SEM (n = 12 per group). *P < 0.05.

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  Fig. 5.. FGF2 deficiency promotes cardiomyocyte apoptosis under septic stress in vivo and in vitro. (A) Representative images of TUNEL staining (green) for apoptotic cells and DAPI (blue) for nuclei in cardiac tissue sections. Scale bar, 100 μm. (B) Quantification of the percentage of TUNEL-positive cells. (C) Representative Western blots showing protein levels of pro-caspase-3, cleaved caspase-3, caspase-9, Bax, and Bcl-2 in cardiac tissue. (D to G) Quantification of the relative protein expression of cleaved caspase-3, Bax, Bcl-2, and caspase-9. (H) mPTP opening was determined by calcein retention. (I) Cell viability assessed by MTT assay. (J) LDH release. (K and L) Representative flow cytometry plots showing Annexin V/PI staining for apoptotic cells. (M to Q) Representative Western blots showing protein levels of pro-caspase-3, cleaved caspase-3, caspase-9, Bax, and Bcl-2 in cell lysates. Data are shown as the means ± SEM (n = 12 per group). *P < 0.05.

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  Fig. 6.. rFGF2 attenuates cardiac damage in sepsis-induced cardiomyopathy in vivo. The cardiac injury markers Tn-T, CK-MB, LDH, and BNP (A to D) and the concentrations of inflammatory factors TNFα, IL-6, and MCP-1 (E to H) in blood were measured via ELISA. (I to L) After LPS-mediated septic cardiomyopathy, the level of mRNAs encoding inflammatory factors, such as IL-1β, TNFα, IL-6, and MCP-1 was measured via qPCR. (M and N) Heart tissues were collected, and then immunofluorescence staining was performed to analyze the accumulation of Gr-1-positive cells in the myocardium. (O and P) Cardiac ROS content change measured with DHE. Data are shown as the means ± SEM (n = 12 per group). *P < 0.05.

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  Fig. 7.. rFGF2 ameliorates LPS-induced mitochondrial damage by activating mitophagy. Comparative analysis of cardiac tissue between rFGF2-treated and WT mice after LPS administration (n = 4 per group) revealed 444 differentially expressed proteins (|log2FC| > 0.5), with 328 up-regulated and 116 down-regulated proteins. (A to C) Gene Ontology Enrichment Analysis of differentially expressed proteins in cardiac tissue. (D to F) KEGG analysis. (G and H) GSEA visual analysis of mitophagy-related pathways and AMPK pathway in cardiac tissue. (I to N) The expression of p-FUNDC1, Beclin1, LC3II, ATG5, and p62 was measured via Western blotting. (O) Co-immunofluorescence of mitochondria and lysosomes in HL-1 cells. Data are shown as the means ± SEM (n = 12 per group). *P < 0.05.

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  Fig. 8.. rFGF2 activated FUNDC1-required mitophagy, which was inhibited in response to LPS. (A) ATP generation. (B and C) HL-1 cells were incubated with the reagents in the MitoProbe Transition Pore Assay Kit and analyzed by flow cytometry. (D and E) mROS were analyzed by flow cytometry. (F and G) The mitochondrial membrane potential was analyzed with an immunofluorescence assay. Cellular viability was measured via MTT assay (H), LDH release (I), and TUNEL assay (J and K). Data are shown as the means ± SEM (n = 12 per group). *P < 0.05.

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  Fig. 9.. FGF2 supplementation promotes FUNDC1-mediated mitophagy by activating AMPK pathway. (A to C) Western blots were used to analyze the expression change of p-AMPK and p-FUNDC1. (D) Co-immunofluorescence of mitochondria and lysosomes in HL-1 cells. (E) ATP generation. (F) mPTP opening time. (G and H) The cellular apoptosis was measured via TUNEL assay. (I to K) Representative M-mode echocardiographic images and quantification of LVEF and FS in mice from different treatment groups. Data are shown as the means ± SEM (n = 12 per group). *P < 0.05.