Cardiac-targeted FGF4 encapsulated nanoliposomes improve acute myocardial injury induced by ischemia-reperfusion and adriamycin in in vitro and in vivo models

Figures

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  Fig. 1. Characterization of FGF4-NANO-IMTP. A. Solution properties of the nanomaterial. B. Nanomaterial morphology identified by transmission electron microscopy. C. Nanomaterial structure identified by scanning electron microscopy. D. Particle size analysis of FGF4-NANO-IMTP. E. Zeta potential analysis. F. Nanomaterial composition identified by NMR. G. Drug release kinetics analysis. H. Fluorescence spectroscopy analysis. I. Structural model of FGF4-NANO-IMTP. J. Interaction between FGF4 and FGFR1. K. Downregulation of FGF4 expression in the I/R group. L. Enrichment analysis of ferroptosis-related genes in the I/R group. p < 0.05 indicates significant difference.

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  Fig. 2. Effect of FGF4-NANO-IMTP on I/R injury. A. TTC staining analysis of FGF4-NANO-IMTP effects on I/R injury. B. FGF4 treatment reduces CK-MB levels. C. FGF4 treatment decreases cTnT concentration. D. FGF4 treatment reduces LDH concentration. E. H&E staining analysis of FGF4-NANO-IMTP-mediated amelioration of I/R injury. F. TUNEL staining analysis of I/R-induced cardiomyocyte death alleviated by FGF4-NANO-IMTP. p < 0.05 indicates significant difference.

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  Fig. 3. Effect of FGF4-NANO-IMTP on myocardial inflammation and oxidative stress. A. FGF4-NANO-IMTP reduces inflammatory cytokine levels by ELISA. B. FGF4-NANO-IMTP alleviates myocardial oxidative stress as measured by ROS assays. p<0.05 indicates significant difference.

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  Fig. 4. FGF4-NANO-IMTP alleviates I/R injury by inhibiting ferroptosis. A. FGF4-NANO-IMTP reduces Fe2+ levels. B. FGF4 increases ATP levels. C. FGF4 upregulates SLC7A11 and GPX4 levels. D. FGF4 reduces ferroptosis marker molecules (COX2 and FTH-1). E. FGF4 decreases ALOX15 expression. F. FGF4-NANO-IMTP alleviates cell death as shown by TUNEL staining. G. FGF4 treatment decreases myocardial lipid peroxidation levels. H. FGF4-NANO-IMTP induces greater AMPK activation. I. FGF4 decreases ALOX15 expression under AMPK inhibition. J. FGF4 fails to inhibit cell death in the presence of AMPK inhibition. K. C11-BODIPY staining shows FGF4 inhibits lipid peroxidation under AMPK inhibition. p<0.05 indicates significant difference.

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  Fig. 5. Effect of FGF4-NANO-IMTP on DOX-induced cardiomyocyte injury. A. FGF4-NANO-IMTP treatment reduces levels of LDH and CK-MB. B. H&E staining analysis of FGF4-NANO-IMTP effects on DOX-induced myocardial injury. C. TUNEL staining analysis of FGF4 effects on DOX-induced cardiomyocyte death. D. Color ultrasound analysis of FGF4-NANO-IMTP effects on cardiac function. E. FGF4 and FGF4-NANO-IMTP improve myocardial fibrosis via Masson's trichrome staining analysis. p<0.05 indicates significant difference. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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  Fig. 6. Effect of FGF4-NANO-IMTP on inflammation and oxidative stress. A. FGF4-NANO-IMTP reduces oxidative stress levels. B. FGF4-NANO-IMTP decreases inflammation levels. C. Immunohistochemical detection of inflammation and oxidative stress markers. p<0.05 indicates significant difference.

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  Fig. 7. FGF4-NANO-IMTP alleviates DOX-induced myocardial injury. A. Differential gene analysis; B. Gene expression heatmap; C. Enrichment analysis of ferroptosis-related signaling pathways. D. FGF4-NANO-IMTP and Fer (positive control) regulate expression of GPX4, SLC7A11, and PTGS2. E. FGF4 activates AMPK. F. PTGS2 expression is increased. G. TUNEL staining analysis of AMPK inhibition effects on FGF4-induced cardiomyocyte death. H. C11-BODIPY analysis of lipid peroxidation in the presence of the AMPK inhibitor. p<0.05 indicates significant difference.

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  Fig. 8. FGF4-NANO-IMTP alleviates DOX-induced cardiomyocyte injury. A. Establishment of a DOX-induced cardiomyocyte injury model (Fig. 8A). B. FGF4 and FGF4-NANO-IMTP enhance cell proliferation. C. FGF4-NANO-IMTP reduces oxidative stress in vitro. D. FGF4-NANO-IMTP improves mitochondrial membrane potential. E. FGF4-NANO-IMTP upregulates NDUFB8 and UQCRC2 levels. F. FGF4-NANO-IMTP alleviates inflammation. G. FGF4-NANO-IMTP reduces cell death as shown by PI staining and LDH release assays.

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  Fig. 9. Analysis of the molecular mechanism by which FGF4-NANO-IMTP inhibits cell death. A. FGF4-NANO-IMTP inhibits cardiomyocyte ferroptosis. B. PI staining analysis of cell death. C. FGF4-NANO-IMTP alleviates ferroptosis via reduced LDH release. D. FGF4-NANO-IMTP upregulates GPX4 and SLC7A11 expression. E. The anti-ferroptotic effect of FGF4 is attenuated when FGFR1 is knocked down. F. FGF4 activates FGFR1. p < 0.05 indicates significant difference.

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  Fig. 10. Analysis of the molecular mechanism of FGF4 inhibiting ferroptosis. A. FGF4 treatment rescues AMPK signaling. B. FGF4-NANO-IMTP and FGF4 fail to alleviate ferroptosis in the presence of an AMPK inhibitor. C. The AMPK activator inhibits DOX-induced ferroptosis. D. FGF4 activates AMPK through CAMKK2. E. Inhibition of CAMKK2 abrogates FGF4-mediated resistance to ferroptosis. F. IP3R inhibitor treatment blocks CAMKK2 and AMPK activation. G. IP3R inhibition exacerbates ferroptosis. H. FGF4-NANO-IMTP activates the AMPK-ACC signaling axis. I. ACC activation inhibition abrogates FGF4's protective effect against DKO- and Erastin-induced cell death. p < 0.05 indicates significant difference.

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  Fig. 11. FGF4-NANO-IMTP alleviates cardiomyocyte damage induced by ischemia and hypoxia. A. Establishment of a glucose/oxygen deprivation (OGD) model in cardiomyocytes. B. FGF4-NANO-IMTP reduces cardiomyocyte ferroptosis. C. FGF4-NANO-IMTP alleviates oxidative stress in cardiomyocytes. D. Mitochondrial membrane potential increases after FGF4-NANO-IMTP treatment. E. FGF4-NANO-IMTP upregulates NDUFB8 and UQCRC2 levels. F. FGF4-NANO-IMTP reduces lipid peroxidation. p < 0.05 indicates a significant difference.

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  Fig. 12. Role of the FGF4-AMPK signaling axis. A. AMPK inhibition blocks FGF4-mediated suppression of ferroptosis. B. FGF4-NANO-IMTP reduces SLC7A11 and GPX4 expression in the presence of AMPK inhibition. C. Lipid peroxidation increases under AMPK inhibition. D. FGF4-NANO-IMTP activates the AMPK-PGC-1α signaling pathway. E. The anti-ferroptotic effect of FGF4-NANO-IMTP is partially attenuated when PGC-1α is inhibited.