Fig. 3: Mitochondrial function in FGF21 null mice with HFpEF.

A Gene Set Enrichment Analysis (GSEA) - Gene Ontology (GO) analysis of proteomics data from the heart tissue of AAV-Fgf21- or AAV-GFP-treated mice with HFpEF. B, C Top, transmission electron microscopy images of heart tissue in Fgf21 KO and WT mice with or without HFpEF, or AAV-Fgf21-treated mice with HFpEF. Scale bar, 1 μm. Bottom, quantifications of mitochondrial density (n = 5 mice per condition), total area (n = 5 mice per condition), and cristae number (n = 50 mitochondria per condition). D, E Cardiac ATP content in mice mentioned above. n = 5 mice per condition. F Cardiac mitochondrial respiratory electron transport chain (ETC) complex I–V subunits were tested by immunoblot. n =  3 independent experiments. The activity of pyruvate dehydrogenase in the heart tissues (G), cardiac pyruvate (H), and acetyl-CoA (I) levels in Fgf21 KO and WT mice with or without HFpEF. n = 5 mice per condition. The activity of pyruvate dehydrogenase in the heart tissues (J), cardiac pyruvate (K), and acetyl-CoA (L) levels in mice with HFpEF, treated with AAV-Fgf21 or AAV-GFP. n = 5 mice per condition. M–P Neonatal rat ventricular myocytes (NRVMs) were exposed to palmitic acid (PA) with a dosage of 500 μmol/L, then incubated with recombinant mouse FGF21 (rmFGF21) for 24 h. M Representative images of DCFH-DA staining and MitoSOX Green staining (top) and quantification of the DCFH-DA intensity and mitochondrial length (bottom). Scale bars, 50 μm for DCFH-DA staining, 10 μm (top) and 2 μm (bottom) for MitoSOX Green staining. n = 5 biological samples. N Representative images of JC-1 staining (left) and quantification of the red/green fluorescence intensity ratio (right). Scale bars, 50 μm. Aggregates, red; Monomers, green. n = 5 biological samples. O Real-time monitoring of the oxygen consumption rate (OCR) in NRVMs. n = 5 biological samples. P ATP contents tested by spectrophotometric methods. n = 5 biological samples. Violin plots in (B, C) are presented as lines indicating the median and interquartile range; other bar graphs are presented as mean ± SEM. The statistical significance of differences was assessed by two-way ANOVA with Tukey’s multiple comparison.