FABP3 Deficiency Exacerbates Metabolic Derangement in Cardiac Hypertrophy and Heart Failure via PPARα Pathway

Abstract

Background: Cardiac hypertrophy was accompanied by various cardiovascular diseases (CVDs), and due to the high global incidence and mortality of CVDs, it has become increasingly critical to characterize the pathogenesis of cardiac hypertrophy. We aimed to determine the metabolic roles of fatty acid binding protein 3 (FABP3) on transverse aortic constriction (TAC)-induced cardiac hypertrophy. Methods and Results: Transverse aortic constriction or Ang II treatment markedly upregulated Fabp3 expression. Notably, Fabp3 ablation aggravated TAC-induced cardiac hypertrophy and cardiac dysfunction. Multi-omics analysis revealed that Fabp3-deficient hearts exhibited disrupted metabolic signatures characterized by increased glycolysis, toxic lipid accumulation, and compromised fatty acid oxidation and ATP production under hypertrophic stimuli. Mechanistically, FABP3 mediated metabolic reprogramming by directly interacting with PPARα, which prevented its degradation and synergistically modulated its transcriptional activity on Mlycd and Gck. Finally, treatment with the PPARα agonist, fenofibrate, rescued the pro-hypertrophic effects of Fabp3 deficiency. Conclusions: Collectively, these findings reveal the indispensable roles of the FABP3-PPARα axis on metabolic homeostasis and the development of hypertrophy, which sheds new light on the treatment of hypertrophy.

Keywords: FAO; HFABP; PPARα; cardiac hypertrophy; glycolysis; metabolism.

Figure 1

TAC or Ang II upregulates the FABP3 expression in vivo and in vitro. (A–C) Mouse tissues were extracted for qPCR and western blot analyses to determine Fabp3 levels. (A) qPCR analysis of Fabp3 mRNA in the indicated organs; 18s was used as the control. (B) The FABP3 protein level in the corresponding tissue was determined using western blotting. (C) Quantification of FABP3 expression in (B). (D) The mRNA level of Fabp3 after TAC operation, as determined using qPCR. (E) Representative western blot images showing the FABP3 expression. (F) Quantification results for (E). (G) NRVMs treated with NE or Ang II were subjected to western blotting assay to determine the FABP3 protein expression. (H) Quantification results of (G). (I,J) Single cell RNA sequencing of TAC-operated murine hearts or heart samples from DCM and normal patients were recalculated for the transcriptional expression of Fabp3 (GEO accession code: GSE95143). [A, n = 6; C, n = 3; (A,C) Dunnett's post-hoc test; D, n = 6; F, n = 4; (D,F) Games–Howell post-hoc test; H, n = 4; (H,J) Student's t-test; (I) Dunnett's post-hoc test].

Figure 2

Deficiency of FABP3 aggravates TAC-induced cardiac hypertrophy. (A) Representative echo images of the WT and F3-KO mice at 4 weeks after TAC or sham operation. (B) Quantification results for the interventricular septum (IVS) in (A). (C) Quantification results for the left ventricular posterior wall thickness (LVPW) in (A). (D) The ratio of heart weight to body weight from sham or TAC-operated WT or F3-KO mice. (E) Images of H&E stained longitudinal sections of the indicated hearts. (F) Heart sections stained with WGA to compare the size of the cardiomyocyte area. (G) Quantification results in (F); a total of 100 cells/group were calculated. (H) qPCR assays compared the mRNA expression of Anp, Bnp, Acta1, and Myh7 at 4 weeks after sham or TAC operation. NS, not significant, *p < 0.05, **p < 0.01, ***p < 0.001. (I) Representative western blot images of ANP in the indicated groups. (J) Quantification results of (I). [B–D, n = 8, 8, 15, 15, respectively; H, n = 6; J, n = 4; (B–D,G,H,J) Tukey's post-hoc test].

Figure 3

FABP3 participates in Ang II induced cell hypertrophy. (A–E) NRVMs were treated with siRNA-targeting Fabp3 (Si-F3) or its negative control (Si-NC) before PBS or Ang II treatment. (A) Immunofluorescence staining of α-actinin in NRVMs with or without Ang II treatment. (B) Quantification results of the cell area in (A); 50 cells/group were calculated. (C) mRNA expression of hypertrophic genes (Anp, Bnp, and Myh7) in indicated groups. NS, not significant, ^*p < 0.05, **p < 0.01. (D) Representative western blot images of ANP after Fabp3 knocking-down. (E) Quantification of (D). (F–J) NRVMs were transfected with lentivirus-encoding Fabp3 (Lenti-F3) or its negative control (Lenti-Ctl) before PBS or Ang II treatment. (F) The cell area was determined by α-actinin staining in NRVMs. (G) Quantification results of the cell area in (F); 50 cells/group were calculated. (H) The mRNA levels of Anp, Bnp, and Myh7 were compared in the aforementioned groups. NS, not significant, *p < 0.05, **p < 0.01, ***p < 0.001. (I) Representative western blot images revealed reduced ANP protein levels in the Lenti-F3 group. (J) Quantification results of (I). [C,E,J, n = 4; H, n = 6; (B,G): Games–Howell post-hoc test; (C,E,H,J): Tukey's post-hoc test].

Figure 4

FABP3-defect results to compromised fatty acid oxidation (FAO) and toxic lipid accumulation. (A) Experimental schematic of RNA-seq analysis and metabolomics analysis in WT and F3-KO hearts. (B,C) GSEA revealing that F3-KO negatively correlates with fatty acid beta oxidation using acyl-CoA dehydrogenase (B), while positively correlating with the lipid biosynthetic process (C). (D) Heatmap showed scaled expression of FAO and lipid biogenesis genes in indicated samples from RNA-seq analysis. (E) The mRNA expression of FAO and lipid biogenesis genes in TAC-operated WT and F3-KO hearts. NS, not significant, *p < 0.05, **p < 0.01. (F) Mitochondrial stress assay was performed in NRVMs transfected with Fabp3 (Lenti-F3) or its negative control (Lenti-Ctl) after Ang II treatment to measure the oxygen consumption rates (OCR); data were presented as mean ± SD from individual experiments. (G) The parameters of basal respiration, maximal respiration, and spare respiration capacity were calculated from (F); data were presented as mean ± SD from individual experiments. (H) LCFA oxidation stress assay was conducted in NRVMs with or without knocking-in expression of Fabp3 after Ang II treatment; etomoxir (Eto) was applied to inhibit the mitochondrial FAO. Data were presented as mean ± SEM from individual experiments. (I) The parameters of basal respiration and maximal respiration were calculated from (H); data were presented as mean ± SEM from individual experiments. (J and K) The level of fatty acid (FA) and diacylglycerol (DAG) in WT and F3-KO hearts determined by LC-MS analysis; six biological replicates per group. *p < 0.05, **p < 0.01. (L) Representative electron micrographs of WT and F3-KO hearts after TAC operation. (Bottom) Higher magnification images of the dashed rectangle from (L). [E, n = 3, Student's t-test; G, n = 5, 4, respectively, Student's t-test; I, n = 11, 10, 9, 7, respectively, Tukey's post-hoc test; (J,K) Student's t-test].

Figure 5

FABP3-null contributes to increased glycolysis and reduced ATP production under hypertrophic stimulation. (A) Heatmap of glycolysis and TCA cycle genes from RNA-seq analysis. (B) The mRNA expression of Gck, Pck1, Slc2a1, and Slc2a4 in indicated groups. NS, not significant, *p < 0.05, **p < 0.01. (C) Heatmap of differential metabolites from WT and F3-KO hearts. *p < 0.05, **p < 0.01, ***p < 0.001. (D) The intracellular concentration of glucose-6-phosphate (G6P) in NRVMs was measured after manipulating the expression of Fabp3. (E) Glycolytic rate assay was performed in NRVMs after knocking down the expression of Fabp3 with siRNA to measure the OCR and extracellular acidification rates (ECAR) and converted to glycolytic proton efflux rate (glycoPER) in the Seahorse Report Generator. (F) The parameters of basal glycolysis and compensatory glycolysis were calculated from (E). Data were presented as mean ± SEM from individual experiments. (G) Glycolytic rate assay was performed in NRVMs with or without Fabp3 overexpression to measure glycoPER. (H) The parameters of basal glycolysis and compensatory glycolysis were calculated from (G). Data were presented as mean ± SD from individual experiments. [B, n = 3, Tukey's post-hoc test; D, n = 4, 4, 3, 3, respectively, Student's t-test; F, n = 6, 5, 6, 5, respectively; H, n = 12, 10, 8, 11, respectively; (F,H) Tukey's post-hoc test].

Figure 6

FABP3 mediates metabolic reprogramming by directly interacting with PPARα, preventing its degradation and enhancing its transactivation. (A) Representative western blot images of PPARα from TAC-operated WT and F3-KO hearts, or from NRVMs transfected with Fabp3 or its control virus with or without Ang II treatment. (B) Quantification of (A). (C) Immunofluorescence double-staining of PPARα (green) and cTnT (red) in WT and F3-KO hearts, with or without TAC surgery. (Bottom) Higher magnification of dashed rectangle in the upper panel. (D) Quantification results in (C). (E) NRVMs with Fabp3 overexpression were treated with or without Ang II for 24 h and then co-immunoprecipitated (co-IP) with FABP3. Western blotting assay was conducted with indicated antibodies. (F) NRVMs with Pparα overexpression were treated with or without Ang II for 24 h and then co-immunoprecipitated (co-IP) with PPARα. (G) NRVMs transfected with Lenti-F3 or Lenti-Ctl were treated with CHX for 0, 12, 24, or 48 h and the protein expression of PPARα was measured by western blotting. (H) Quantification of PPARα intensities by normalizing to those of GAPDH in (G). (I) HEK 293T cells were transfected with a PPRE-driven luciferase reporter (PPRE₃-TK-LUC) and PPARα or FABP3 for 24 h. Relative activation of PPRE₃-TK-LUC was measured by normalizing its luminescence value to the renilla activity. (J) Representative western blot images of MLYCD, CPT1B, ACC, and GCK in WT and F3-KO mice after sham or TAC operation. (K) The quantification results of (J). (L,M) HEK 293T cells were transfected with a Mlycd-promoter luciferase reporter (Mlycd-LUC, L) or a Gck-promoter luciferase reporter (Gck-Luc, M), PPARα, or FABP3 for 24 h. The relative expression of the Mlycd-promoter and Gck-promoter was measured by normalizing their luminescence value to the corresponding renilla luminescence value. (N) The schematic diagram shows FABP3 binds to PPARα and increases its transcriptional activity on Mlycd and Cpt1b while repressing Acaca and Gck to participate in cardiac FAO/glycolysis shift. [B, n = 4, Games–Howell post-hoc test; D, n = 4, Tukey's post-hoc test; H, n = 2; I, n = 6, Dunnett's post-hoc test; K, n = 4, Tukey's post-hoc test; L, n = 4; M, n = 5; (L,M) Dunnett's post-hoc test].

Figure 7

Fenofibrate represses FABP3-null induced cardiac hypertrophy in vivo and in vitro. (A) NRVMs were treated with siRNA targeted Fabp3 (Si-F3) or its negative control before fenofibrate or vehicle treatment. Immunofluorescence staining of α-actinin used to compare the myocyte area. (B) Quantification of (A). (C) Representative echo images in WT or F3-KO mice with or without fenofibrate treatment (Feno or Vehicle, respectively) at 4 weeks after TAC surgery. (D) Quantification of IVS in (C). (E) Quantification of LVPW in (C). (F) Relative mRNA levels of Anp, Bnp, and Col3a1, as determined by qPCR in the indicated groups. NS, not significant, *p < 0.05, **p < 0.01, ***p < 0.001. (G) Images of H&E-stained longitudinal heart sections from WT or F3-KO mice with or without fenofibrate treatment. (H) Representative immunofluorescence images of WGA staining from WT or F3-KO mice with or without fenofibrate treatment. (I) Quantification of the cardiomyocyte cross-sectional area in (I) (n = 100). (J) Masson staining to determine cardiac fibrosis in the aforementioned groups. (K) Quantification of collagen volume in (L). [D, E, n = 10, 9, 10, 9, respectively; (B, D-IVS; s): Games–Howell post-hoc test; (D-IVS; d, E): Tukey's post-hoc test; F, n = 6, (F-Anp, F-Col3a1, and I): Games–Howell post-hoc test; K, n = 6, 5, 6, 5; (F-Bnp, and K): Tukey's post-hoc test].

Figure 8

Model of the FABP3-mediated PPARα pathway in cardiac metabolic reprogramming and progression of cardiac hypertrophy and heart failure. The schematic demonstrates that the FABP3-mediated PPARα pathway regulates cardiac metabolic reprogramming by directly interacting with PPARα, thereby stabilizing and inducing its transactivation, which triggers downstream Mlycd and Cpt1b gene expression, while suppressing Acaca and Gck gene expression. This results in increased FAO levels and mitochondrial ATP production, ultimately resulting in adaptive cardiac function. However, in mice lacking FABP3, the response of FABP3-mediated PPARα on FAO/Glycolysis balance is abolished, and leads to the degradation of PPARα, lower transcriptional activity on FAO genes accompanied by increased lipogenesis, and glycolysis gene expression, which contributes to the increase in the glycolysis rate, accumulation of toxic lipid species, and compromised mitochondrial ATP production, thereby aggravating the progression of cardiac hypertrophy and heart failure.