Hypertrophic stimuli upregulate FABP3 expression
First, to gain an overall profile of FABP3 expression, we examined its mRNA and protein level in mouse tissues, including the heart, liver, spleen, muscle, and brown adipose tissue (BAT), using quantitative polymerase chain reaction (qPCR) and western blotting assays. Our results showed that FABP3 protein and Fabp3 mRNA level was exclusively expressed in hearts, BAT and muscles, while its expression was rare in other organs, such as liver and brain, etc (Fig. 1a-c). Intriguingly, hearts and BAT have been recognized for their distinct preference for fatty acids as energy sources13, 14, which underscores the important role of FABP3 in cardiac fatty acid metabolism. Then, TAC operation was used to induce cardiac hypertrophy in vivo. By analyzing the expression of FABP3 at sham, 1-(1W), 2-(2W), 4-(4W), and 8-weeks (8W) after TAC operation using qPCR and western blotting, we observed TAC operation induced FABP3 expression approximately 2-fold at 4W compared with sham groups (Fig. 1d-f). Immunofluorescence co-staining of FABP3 and a-actinin showed a consistent increase of FABP3 expression after TAC treatment (Fig. 1g, h).
Next, to determine the expression of FABP3 in vitro following neurohormonal stimuli, neonatal rat ventricular myocytes (NRVMs) were treated with norepinephrine (NE) or angiotensin (Ang) II for 24h and showed a significant increase of FABP3 levels using western blot. Moreover, Ang II induced higher FABP3 expression than NE (Fig. 1i, j). Therefore, we selected Ang II as the in vitro hypertrophic inducer for further experiments.
Next, we assessed the transcriptional expression of Fabp3 in scRNA-seq datasets, which analyzed the transcriptional differences in mice cardiomyocytes following sham operation or at 3 days (3D), 1W, 2W, 4W, or 8W after TAC surgery, as well as in myocytes from dilated cardiomyopathy (DCM) and normal patients15. Similarly, scRNA-seq data showed significantly increased Fabp3 expression at 4W after TAC surgery compared to the sham group (Fig. 1k) and more than 5-fold higher Fabp3 expression in DCM patients than their normal counterparts (Fig. 1l).
Taken together, these data suggest that FABP3 is expressed in heart tissues, which use fatty acids as a primary fuel substrate, and is upregulated in vivo and in vitro under hypertrophic stimuli.
Loss Of FABP3 Aggravates Continuous Overload-induced Cardiac Hypertrophy
To examine the effects of FABP3 on cardiac hypertrophy, we generated Fabp3 knock-out (F3-KO) mice using CRISPR/Cas9, the Fabp3-null mice were viable. Homozygous, heterozygous allele and wild type mice were identified using PCR (Supplementary Fig. 1a-c). Notably, cardiac FABP3 was completely abolished in Fabp3-null mice which allowed for a direct examination of the role of FABP3 on cardiac hypertrophy (Supplementary Fig. 1d, e).
To explore whether FABP3 contributes to TAC-induced hypertrophy, F3-KO and WT mice were subjected to TAC or sham surgery and observed for 4 weeks (Supplementary Fig. 2a). Firstly, to exclude the systemic differences among TAC-operated WT and F3-KO mice which may exert extra effects on the development of cardiac hypertrophy, organ mass to body weight ratio was measured and similar spleen, kidney, BAT, and white adipose tissue (WAT) ratio was found between WT and F3-KO mice, except for a higher brain weight in F3-KO mice (Supplementary Fig. 2b). Similarly, no difference was observed in liver mass among WT and F3-KO mice (Supplementary Fig. 2c). Moreover, hematoxylin and eosin (H&E) staining of above organs showed no markedly structural differences between WT and F3-KO mice after TAC surgery, demonstrating that FABP3 deletion did not result in systemic abnormity after TAC operations (Supplementary Fig. 2d).
Next, to determine the effects of Fabp3-null on cardiac function, echocardiography (echo) was performed on WT and F3-KO mice at 4W after surgery (Fig. 2a). Compared with WT mice, TAC surgery led to severer cardiac hypertrophy in Fabp3-null mice, revealed as higher thickness of the interventricular septum (IVS; d: 1.56 ± 0.03 versus 1.17 ± 0.04; IVS; s: 2.02 ± 0.05 versus 1.63 ± 0.03; F3-KO versus WT) and left ventricular posterior wall thickness (LVPW; d: 1.38 ± 0.08 versus 1.11 ± 0.06; LVPW; s: 1.82 ± 0.07 versus 1.60 ± 0.07; F3-KO versus WT) in F3-KO mice, meanwhile these parameters were comparable under sham groups (Fig. 2b, c). Although no statistical difference was observed in the lung weight to body weight ratio (LW/BW) between WT and KO mice (Supplementary Fig. 2e), we found increased heart weight to body weight ratio (HW/BW) in F3-KO mice after TAC surgery compared with WT mice (Fig. 2d, 13.65 ± 0.86 in F3-KO, 10.54 ± 0.53 in WT), this result together with the echocardiography data demonstrated a crucial role of FABP3 on cardiac hypertrophy.
Then, histological H&E and WGA staining was conducted to determine the degree of cardiac hypertrophy after echo analysis. A significant increase in left ventricular to right ventricular wall thickness ratio (LV/RV) was found in F3-KO hearts compared to WT littermates after TAC surgery (Fig. 2e, f). Although no difference was observed in the cardiomyocyte area between sham group (139.35 ± 4.86 in WT and 162.29 ± 5.94 in F3-KO), Fabp3-null caused a significantly enlarged cell area (595.64 ± 15.44) compared to the WT counterparts (360.06 ± 12.90) after TAC surgery (Fig. 2g, h). Finally, hypertrophic genes, such as Anp, Bnp, Acta1, and Myh7, were evaluated with qPCR assay. In line with the hypertrophic phenotype in Fabp3-KO hearts, the mRNA levels of Anp and Bnp were upregulated in F3-KO hearts after surgery compared to WT hearts (Fig. 2i). Similarly, ANP levels were increased by nearly 2-fold in F3-KO hearts compared as WT hearts (Fig. 2j, k). Altogether, we concluded that after TAC operation, FABP3 deficiency shows no effects on systemic abnormity, while contributes to the aggravation of cardiac hypertrophy.
Loss Of FABP3 Impairs Cardiac Remodeling After Hypertrophy
Cardiac hypertrophy contributes to heart failure under continuous overload, ultimately leading to adverse cardiovascular events and death. To determine whether Fabp3 deficiency is associated with hypertrophy-induced heart failure, we analyzed the mortality rate of WT and F3-KO mice over 8-weeks post-surgery. At the end of the observation period, a lower survival probability was observed in the F3-KO group compared to the WT group (Supplementary Fig. 3a, p = 0.3). Then, the gross morphology of WT and F3-KO hearts was compared and showed increase of heart size in F3-KO mice after TAC surgery compared to WT hearts (Supplementary Fig. 3b). Consistent with enlarged hearts in Fabp3-defect mice, decline in left ventricular ejection fraction (LVEF, 27.58 ± 1.52 in F3-KO, 43.34 ± 2.65 in WT) and left ventricular fractional shortening (LVFS, 12.79 ± 0.76 in F3-KO, 21.27 ± 1.52 in WT) was found in F3-KO mice compared to their WT group at 8-weeks post-surgery, which was paralleled with higher left ventricular end diastolic volume (LVEDV) and left ventricular end systolic volume (LVESV) in Fabp3-null mice (Supplementary Fig. 3c-e). These results pointed to the important role of FABP3 on TAC induced cardiac hypertrophy and the progress of heart failure.
Then, considering that fibrosis serves as a hallmark of cardiac dysfunction, by measuring the level of fibrosis-related genes via qPCR assay, we found that the mRNA expression of Col3a1 was increased in TAC-operated F3-KO hearts compared to WT hearts. Moreover, Fabp3 ablation reduced the mRNA expression of matrix metallopeptidase 2 (Mmp2) and matrix metallopeptidase (Mmp9) after TAC surgery, suggesting impaired collagen-turnover and homeostasis in Fabp3-KO mice (Supplementary Fig. 3f). In agreement with the increased expression of fibrosis genes in F3-KO hearts, Massons and Sirus red staining confirmed increased left ventricular collagen volume in F3-KO hearts (interstitial: 12.12 ± 1.14, perivascular: 7.28 ± 0.45) compared to WT hearts (interstitial: 4.83 ± 0.57, perivascular: 3.42 ± 0.65) (Supplementary Fig. 3g, h). Cumulatively, these data suggest that the loss of FABP3 contributes to compromised contractility and increased collagen volume following TAC operation.
FABP3 alleviates Ang II-induced cardiomyocyte hypertrophy in vitro
To corroborate the above findings that FABP3 participates in TAC-induced hypertrophy, we manipulated the expression of Fabp3 in vitro using non-targeting small interfering RNA (siRNA) or lentivirus. The siRNA method reduced the mRNA level of Fabp3 by 70% in H9C2 cells (Supplementary Fig. 4a). Firstly, NRVMs were transfected with siRNA-target Fabp3 (Si-F3) or its scrambled control (Si-NC) and the cell sectional area was measured after Ang II or PBS treatment. We showed cell size was comparable in the PBS groups, however, knocking-down Fabp3 significantly enlarged cell area after Ang II treatment compared with Si-NC group (Fig. 3a, b). Moreover, an increase of Anp and Bnp mRNA expression in line with upregulation of ANP protein level was found in Si-F3 group compared with Si-NC group (Fig. 3c-e).
Then, lentivirus vectors containing the full-length Fabp3 transcript (NM_001320996) and green fluorescent protein (GFP) were constructed, transfected and visualized with the fluorescence microscope to confirmed the transfection efficiency (Supplementary Fig. 4b). After transfecting H9C2 cells with optimal multiplicity of infection (MOI) of 10 and 100, lentivirus carrying the Fabp3 transcript (Lenti-F3) markedly increased FABP3 protein levels by more than 4-fold compared to the empty control vector (Lenti-Ctl; Supplementary Fig. 4c, d), which was supported by higher FABP3 fluorescence value in the Lenti-F3 group (Supplementary Fig. 4e), these data confirmed the capability of lentivirus for upregulating the FABP3 expression. In contrast to the pro-hypertrophic effect of Fabp3 ablation, knocking-in expression of FABP3 prevented Ang II-induced cell hypertrophy in vitro. Specifically, Ang II stimulation resulted in enlarged cell sizes in control group, which was markedly blunted when overexpression of FABP3 (Fig. 3f, g). Consistent with the reduction in cell area, knocking in expression of FABP3 curbed the increase of Anp, Bnp, and Myh7 mRNA levels following Ang II treatment (Fig. 3h), as well as the protein expression of ANP (Fig. 3i, j). These in vitro data verified the protective effects of FABP3 on neurohormonal stimuli-induced hypertrophy. Altogether, these FABP3 loss- and gain-of-function results corroborate with in vivo phenotypes and reveal the important role of FABP3 in the development of cardiac hypertrophy.
Ablation of FABP3 leads to defective FA b-oxidation and lipid homeostasis
To explore the mechanism through which FABP3 regulates cardiac hypertrophy, we collected F3-KO or WT hearts at 1-week post-sham or -TAC operation for RNA-seq analysis and liquid chromatography-mass spectrometry (LC-MS) analysis to determine differential genes and metabolites (Fig. 4a). Principal component analysis (PCA) of RNA-seq revealed that the transcriptome of F3-KO hearts was similar to those of WT mice under sham conditions. However, TAC operation induced differentially expressed genes between WT and F3-KO hearts, which separated them on the PCA plot (Supplementary Fig. 5a). Comparing TAC-operated F3-KO hearts with WT hearts, a total of 939 (upregulated: 772, downregulated: 167) differentially expressed genes were identified and analyzed in this study (Fig. 4a). Further, KEGG pathway analysis revealed that these differentially expressed genes were enriched for terms related to “lipid metabolism,” “glycan metabolism,” and “energy metabolism” (Supplementary Fig. 5b).
Next, we performed gene set enrichment analysis (GSEA) based on GO biological process items using all detected genes. We found that “regulation of anatomical structure size,” “wound healing,” and “extracellular structure organization” were positively correlated with TAC-operated F3-KO mice. These pathways, which were consistent with pro-hypertrophic phenotypes in Fabp3-null hearts, suggested that ablation of Fabp3 trigger maladaptive remodeling after TAC operation (Supplementary Fig. 5c).
Intriguingly, GSEA analysis indicated that Fabp3 ablation caused a compromised mitochondrial fatty acid b-oxidation and lipid homeostasis. Specifically, “fatty acid beta oxidation using acyl-CoA dehydrogenase” was negatively correlated with F3-KO hearts (Fig. 4b, and Supplementary Fig. 5c), while positively enriched “lipid biosynthetic process” and “lipid storage” was found in the F3-KO hearts compared with WT hearts (Fig. 4c, and Supplementary Fig. 5d). In line with the aforementioned pathways, the suppressed FAO genes while activated lipid biogenesis genes were observed in F3-KO TAC group than WT TAC group in RNA-seq analysis and in vivo experiments (Fig. 4d, e). Specifically, malonyl-CoA decarboxylase (Mlycd) which increases the rate of fatty acid oxidation, and carnitine palmitoyltransferase 1B (Cpt1b) was downregulated in F3-KO mice, meanwhile, acetyl-CoA carboxylase alpha (Acaca) which synthesis of malonyl-CoA results in immediate inhibition of fatty acid transport into mitochondria, Gpam and Agpat2, genes involve in the committed step in glycerolipid biosynthesis, were increased in F3-KO hearts in RNA-seq analysis and showed a consistent mRNA expression in vivo (Fig. 4d, e). However, these genes were comparable under sham conditions whether in RNA-seq analysis or their mRNA expression in vivo (Fig. 4d and Supplementary Fig. 5e), which manifested that Fabp3 deficiency has no different effect on cardiac energy metabolism under physiological conditions, however, hypertrophic stimulation that occurred with higher energy demand magnified the effects of Fabp3-null on energy homeostasis and resulted to abnormal FAO and lipid biosynthesis.
Furthermore, mitochondrial stress assay was performed in NRVMs with or without knocking-in expression of FABP3 in vitro to measure oxygen consumption rate (OCR). Compared with the negative control, overexpression of FABP3 resulted in significant increases of basal, maximal respiration, and spare respiratory capacity (Fig. 4f, g). Relative to glucose, fatty acid required more oxygen for its b-oxidation, therefore OCR servers as a relative indicator of cell fuel preference. Higher OCR rate in lenti-F3 group was mirrored with the lowed expression of FAO genes in F3-KO hearts, indicating overexpression of FABP3 increased FAO. Then, supplement of etomoxir was used to evaluate the cell dependency on fatty acid as energy substrate. We found that application of etomoxir lead to a markedly drop of respiration in FABP3 knocking-in group compared with its negative control groups, demonstrating NRVMs with FABP3 overexpression have increased reliance on fatty acid as energy fuel (Fig. 4h, i). Moreover, impairing FAO with etomoxir would reverse the protective effects of FABP3 on cardiac hypertrophy and upregulated the mRNA expression of Bnp and Anp (Supplementary Fig. 5f). Taken together, these data points to the dependency of FABP3 on cardiomyocyte FA b-oxidation to meet an effective metabolic demand under hypertrophic stimulations.
FABP3-null Hearts Exhibit Abnormal Lipid Accumulation
As the RNA-seq analysis showed that FABP3 deletion led to abnormal transcriptional profile of FAO and lipid biogenesis (Fig. 4b-d). Lipid-targeted metabolomics analysis was used to determine the differential metabolites in WT and F3-KO hearts. We found that rather than triglycerides (TAG) and Acyl-carnitine (ACar), the fatty acid (16:0) and diglyceride (DAG 18:2–22:5, 18:2–18:2) were significantly increased in F3-KO hearts compared with WT mice (Fig. 4j, k and Supplementary Fig. 6b, c), which was in line with an increase of neutral lipid in Fabp3 knocking-downing cells after Ang II treatment (Supplementary Fig. 6a). It is important to note that previous researches showed that DGA and saturated fatty acid, especially palmitate, was associated with cellular toxicity for their direct actions as signaling lipids16. Therefore, FABP3 deficiency contributed to accumulation of toxic lipids in hearts under hypertrophic stimuli. Consistent with increased toxic lipid species and neutral lipid in vivo and in vitro, we observed a higher level of lipid droplet accumulation in Fabp3-null hearts by using transmission electron microscopy (TEM), which was rare in WT hearts (Fig. 4l and Supplementary Fig. 6d). To determine whether differences in lipid uptake accounted for the abnormal lipid accumulation in F3-KO mice, plasma non-esterified fatty acid (NEFA) was measured at sham, 4W and 8W post-surgery, we found that the plasma concentration of NEFA was higher at 8W compared to the sham group; however, no significant difference was observed between WT and F3-KO mice (Supplementary Fig. 6e), this data in line with comparable mRNA expression of fatty acid transporter Cd36 between WT and F3-KO hearts (Fig. 4d, e) revealed that differences in lipid uptake did not responsible for the greater severity of tissue lipid accumulation in F3-KO mice. Taken together, we showed that Fabp3-null contributes to defective FAO, which triggered the accumulation of long-chain FAs, especially palmitate in F3-KO hearts, and excessive incorporation of DAG and other lipid subspecies, eventually resulted in an increased accumulation of toxic lipid in F3-KO hearts (Supplementary Fig. 6f).
FABP3 Defect Hearts Show Increased Reliance On Glycolysis
In the context of diminished capacity for FAO, we next sorted to determine whether shifted chief energy substrate from fatty acids to glucose was found in F3-KO hearts. Intriguingly, GSEA analysis revealed that the loss of FABP3 triggered abnormally activated glucose metabolic pathways, such as “glucan catabolic process” (Supplementary Fig. 7a) and “regulation of gluconeogenesis” (Supplementary Fig. 7b). In line with activated glucose oxidation and gluconeogenesis pathways, glucokinase (Gck), a gene that participates in the first step in glucose metabolism, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (Pfkfb2), and phosphoenolpyruvate carboxykinase 1 (Pck1) was upregulated in F3-KO TAC hearts (Fig. 5a). A consistent mRNA expression profile in vivo confirmed that increased Gck and Pck1 in Fabp3 deficiency hearts after TAC surgery (Fig. 5b). Meanwhile, “mitochondrial electron transport NADH to ubiquinone” was negatively correlated with F3-KO hearts, which was consistent with markedly dropped expression of TCA cycle genes (Ogdh, Idh2, Aco2) in TAC-operated Fabp3-null hearts than in WT mice, indicating reduced mitochondrial TCA cycle, OXPHOS and ATP production in Fabp3-null hearts under hypertrophic stress (Fig. 5a and Supplementary Fig. 7c). To determine whether Fabp3-defect increases glucose uptake further activates glucose oxidation, we found the glucose transporter, solute carrier family 2 member 1 (Slc2a1) and solute carrier family 2 member 4 (Slc2a4) was downregulated in F3-KO hearts compared with WT mice (Fig. 5a, b), these results were consistent with normal serum glucose, similar glycogen content in small intestine and live, suggesting that Fabp3-defect had no role on the uptake of glucose during cardiac hypertrophy (Supplementary Fig. 7d, e).
Based on activated glucose oxidation genes, we next performed gas chromatography -mass spectrometry (GC-MS) analysis to quantify differential metabolites in WT and F3-KO hearts after TAC surgery. PCA analysis of these metabolites showed that F3-KO hearts were separated from WT samples, which was consistent with the OPLS-DA analysis results (Supplementary Fig. 7f, g). Furthermore, KEGG pathway analysis of differential metabolites indicated that “arginine and proline metabolism” and “citrate cycle” was significantly enriched in F3-KO hearts (Supplementary Fig. 7h). Additionally, we observed significantly increased glycolysis pathway related-metabolites (glucose, glucose-6-phosphate, and fructose 2,6-biphosphate) and considerably reduced TCA cycle metabolites (malic and fumaric acids) in Fabp3-deficient hearts as compared to WT hearts (Fig. 5c). furthermore, by measuring the concentration of glucose-6-phosphate (G6P), an indicator of cellular glycolytic flux, we showed a higher concentration of G6P in Fabp3-knocking down cells, however, overexpression of Fabp3 resulted in dramatic decline in the G6P level (Fig. 5d), which manifesting a glycolytic shift on fuel substrate when knocking-down expression of Fabp3.
Based on the results from the RNA-seq and metabolomics, we performed a glycolytic rate assay to real-time analyze cellular glycolysis by calculating proton efflux rate from glycolysis (glycoPER), a parameter that measures acidification from glycolysis without any contribution from mitochondrial respiration. We observed that NRVMs with Fabp3 knocking-down exhibited a higher basal and compensatory glycolysis compared to its negative control after Ang II stimulation, however, the glycolytic rate showed no significant differences between these two groups under PBS treatment (Fig. 5e, f). In striking contrast to increased glycolysis in Fabp3 knocking-down cells, overexpression of Fabp3 resulted in a marked drop of glycolysis than the control cells after Ang II treatment (Fig. 5g, h). All results combined with multi-omics analysis and glycolytic energetics demonstrated that Fabp3 deficiency resulted in a shift in glycolysis as fuel source and compromised TCA and ATP production (Fig. 5i).
Taken together, these observations confirm that in addition to compromised FAO and ATP production, loss of FABP3 increases glycolysis and toxic lipid accumulation, both of which ultimately aggravate metabolic derangement and heart failure.
FABP3 mediates PPARa level by binding and stabilizing PPARa, further enhances its transcriptional activity under hypertrophic stimuli
As mentioned before, Fabp3 deficiency contributes to deranged metabolic milieu characterized by reduced FAO and increased glycolysis. Next, we sought to explore the mechanism through which Fabp3 mediates metabolic derangement during cardiac hypertrophy. Firstly, our RNA-seq analysis verified that the “PPAR signaling pathway” was one of the top enriched pathways in Fabp3-deficient hearts (Supplementary Fig. 8a). Definitive evidence supports the critical requirement for peroxisome proliferator-activated receptor (PPAR), particularly PPARa, in myocyte metabolism and metabolic reprogramming under cardiac hypertrophy17. Therefore, we postulated that FABP3 participates in cellular metabolism through the PPARa signaling pathway. Firstly, to delineate the mRNA and protein level of PPARa in Fabp3-defect hearts, we found that the mRNA expression of Ppara showed no difference in WT and F3-KO heart (Supplementary Fig. 8b), However, its protein expression was marked downregulation in Fabp3 deficiency mice as compared to WT hearts following TAC surgery. Conversely, in vitro overexpression of Fabp3 using a lentivirus vector rescued the protein level of PPARa following Ang II stimulation (Fig. 6a, b). Immunofluorescence staining of PPARa at 4W post-surgery confirmed that TAC operation resulted in decrease and perinuclear shuttling of PPARa, while Fabp3 ablation accelerated its loss (Fig. 6c, d). These findings indicate that FABP3 might participate in metabolic homeostasis during cardiac hypertrophy via PPARa signaling. However, the mechanism through which FABP3 targets PPARa for metabolic regulation remains elusive.
Next, we transfected NRVMs with Fabp3 or Ppara, co-immunoprecipitated with respective antibodies, and analyzed using SDS-PAGE, the results showed that FABP3 directly bond with PPARa, with or without Ang II stimulation (Fig. 6e, f). As we have shown before, Fabp3 deletion exerted no effect on Ppara mRNA level, suggesting a post-transcriptional modification role of FABP3 on the protein level of PPARa. Then, the NRVMs with Fabp3 knocking-in were treated with cycloheximide (CHX) to eliminate protein translational regulation. We found that Fabp3 overexpression markedly prolonged the half-life of PPARa compared with its negative control, suggesting FABP3 increased PPARa protein level by inhibiting its degradation (Fig. 6g, h). Finally, to determine whether the interaction of FABP3 with PPARa affected its transactivation, we performed luciferase gene transactivation assays in HEK 293T cells. The activation of PPARa was determined based on a reporter plasmid containing firefly luciferase after three PPAR-responsive-element (PPRE3-TK-LUC). Following co-transfection of 293T cells with PPARa, FABP3, and PPRE3-TK-LUC, we found PPARa significantly increased the luciferase expression of PPRE3-TK-LUC; moreover, co-transfection of FABP3 and PPARa induced higher PPRE-driven luciferase activity compared with PPARa alone (Fig. 6i). Together, these findings indicate that FABP3 mediates the protein level of PPARa by interacting with PPARa, inhibiting its degradation, and modulating its transcriptional activity during cardiac hypertrophy.
Required of PPARa on FABP3-modulated FAO/glycolysis balance and cardiac hypertrophy
As the pleiotropic effects of PPARa on inhibiting glucose oxidation, while activating FAO, we aimed to determine whether FABP3 interacts with PPARa and modulates its transcriptional capacities on FAO/glycolysis genes and further involves in the advance of cardiac hypertrophy. Firstly, we curated PPARa target genes in our RNA-seq to reveal their transcriptional profile in sham or TAC operated-WT and F3-KO hearts (Supplementary Table. 1). We observed that the transcriptional level of Gck (participated in glycolysis) and Acaca (participated in lipogenesis) was upregulated in F3-KO hearts, while Mlycd and Cpt1b (participated in mitochondrial FAO) was significantly reduced compared to WT hearts. The western blot assay confirmed a similar protein expressional pattern of MLYCD, CPT1B, ACC, GCK in Fabp3-null hearts. Specifically, Fabp3-ablation led to lower MLYCD and CPT1B protein level while upregulated ACC and GCK, suggesting a direct effect of FABP3 on MLYCD and CPT1B, and an inverse transcriptional effect of FABP3 on ACC and GCK via PPARa (Fig. 6j, k). Therefore, to address the question of whether FABP3 participated in the transcriptional activation of FAO and glycolysis genes via PPARa. We constructed reporter plasmid containing firefly luciferase and Mlycd or Gck promoter (Mlycd-LUC, Gck-LUC, respectively), after transfecting 293T cells with PPARa, FABP3, Mlycd-LUC or Gck-LUC, we showed that PPARa increased Mlycd transcriptional activity; however, higher luciferase activity was observed when 293T cells were co-transfected with FABP3 and PPARa (Fig. 6l). In contrast to Mlycd-LUC, PPARa blunted the transactivation of Gck-LUC, which showed a severer inhibition in the presence of FABP3 (Fig. 6m). These data suggest the metabolic regulatory role of FABP3 in transcriptional activating FAO genes Mlycd, Cpt1b and curbing glycolysis and lipogenesis genes Gck, Acaca via PPARa (Fig. 6n).
Next, to demonstrate the requirement of PPARa in FABP3 mediated cardiac hypertrophy, PPARa was knocked down using siRNA methods as described previously (Supplementary Fig. 8c, d)18. NRVMs with knocking-in expression of FABP3 were transfected Si-PPARa or its scrambled control, we observed that PPARa downregulation abolished the protective effects of FABP3 on cardiomyocyte hypertrophy, resulted in enlarged cell area (Supplementary Fig. 8e, f) and upregulation of Bnp and Anp (Supplementary Fig. 8g). Notably, increased cellular neutral lipid was found after knocking-down PPARa (Supplementary Fig. 8h). Altogether, these data illustrate that FABP3 participates in cardiac hypertrophy by synergistically activating Mlycd, Cpt1b and repressing Gck, Acaca via PPARa.
Activation of PPARa with fenofibrate reverses FABP3-KO induced cardiac hypertrophy
As observed previously, Fabp3 deficiency contributes to hypertrophy and deranged metabolic milieu by impairing PPARa pathway. We next sought to determine whether activating PPARa may rescue the pro-hypertrophic effects of Fabp3-defect after TAC operation and search for clinical benefits on the treatment of cardiac hypertrophy. Firstly, NRVMs were treated with fenofibrate, a PPARa-specific agonist, or vehicle (DMSO) for 24 h, and consistent with previous results, knock-down of Fabp3 with siRNA (Si-F3) resulted in increased cell cross-sectional area compared to its control (Si-NC), while fenofibrate treatment markedly reduced the cell area in both the Si-NC and Si-F3 groups (Fig. 7a, b).
Next, PPAR agonist studies were performed in vivo to investigate the effects of fenofibrate on Fabp3-defect-induced hypertrophy. WT and Fabp3-KO mice were subjected to TAC surgery and randomly treated with fenofibrate (100 mg/kg/d) or vehicle by oral gavage daily for 4W. Interestingly, compared with the vehicle group, fenofibrate treatment significantly rescued cardiac hypertrophy in WT and Fabp3-KO mice. More specifically, IVS and LVPW were markedly decreased following fenofibrate treatment (Fig. 7c-e). Consistent with attenuated cardiac function, hypertrophic and fibrosis-related genes, such as Anp, Bnp, and Col3a1, were inhibited by fenofibrate treatment, but not by the vehicle (Fig. 7f). In addition, histochemical analysis showed that Fabp3 deletion increased left ventricular wall thickness, cardiomyocyte size, and collagen volume, while fenofibrate supplement significantly rescued these effects whether in WT or Fabp3-KO hearts (Fig. 7g-l).
Collectively, these results demonstrate an important role for the PPARa pathway in Fabp3-KO-induced cardiac hypertrophy, while treatment with fenofibrate may reverse hypertrophy, suggesting potential clinical value for fenofibrate in hypertrophic treatment.