Fig. 5. FGF21 loss in CLPP-deficient mice leads to suppression of different constituents of one-carbon metabolism through ERK1/2 pathway and up-regulation of genes involved in cardiomyopathy.

(A) Western blot analysis of ERK1/2 phosphorylation levels. HSC70 was used as a loading control. Experiments were performed on cardiac lysates of WT, FGF21^KO, CLPP^KO, and DKO^KO mice at 17 weeks of age (±2) (n = 3). (B) Log₂ transcript changes in DKO^WB hearts compared to CLPP^KO, extracted from the RNA sequencing (RNA-seq) data (n = 4). (C) Heatmap of total mRNA fold changes of significantly changed genes significantly up-regulated in CLPP^KO animals and more than twofold down-regulated in DKO^WB (n = 4). Transcripts with CARE/AARE motifs in their promoters [as predicted by Cytoscape plug-in iRegulon (58)] are in bold. (D) Western blot analyses of ATF4 and p-eIF2α levels in cardiac lysates of mice at 17 weeks of age (±2). HSC70 and CANX were used as loading controls (n = 3). (E) mRNA levels (log₂ change) of mitoISR TFs relative to WT level extracted from RNA-seq data (n = 4). (F) Western blot analyses of different proteins involved in 1C metabolism (left) and corresponding quantification in cardiac lysates of mice at 17 weeks of age (±2) (right). Antibodies used were raised against proteins indicated in panels (n = 3). (E and F) Bars represent means ± SD (one-way ANOVA and Tukey’s multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). (G) Heatmap of total mRNA fold changes representing transcripts significantly up-regulated in DKO^WB mice but unchanged or down-regulated in CLPP^KO animals (n = 4). Genes involved in the development of cardiomyopathies are in bold.