Fig 2: Cardiac-specific knockout Klf7 disturbed glycolysis and FAO in adults.a Schematic showing construction of the cardiomyocyte-specific Klf7-knockout mouse model. b, c Klf7 deficiency upregulated Pfkl and downregulated Acadl at the mRNA and protein level, as assessed by qRT-PCR (b, WT, n = 3 biologically independent samples, KO, n = 4 biologically independent samples; c, n = 6 biologically independent samples). d Extracellular acidification rate (ECAR) of isolated cardiomyocytes using the Seahorse XF24 to analyze KO mice glycolytic function (n = 5 biologically independent samples). e KO mice have increased glycolysis and glycolytic reserve activities. (n = 5 biologically independent samples) f Oxygen consumption rate (OCR) was measured by seahorse analysis with the indicated regents in cardiomyocytes isolated from WT and KO adult mice (n = 5 biologically independent samples). g FAO consumption, Basal respiration, ATP production and Maximal respiration were decreased in KO mice compared to WT mice (n = 5 biologically independent samples). The glycolysis and FAO activity were analyzed by the seahorse analyzer as described in the “Methods”. h Heat map showing that medium-, long- chain and very long-chain free fatty acid levels in the myocardial tissue were increased in Klf7KO mice compared to WT mice. (n = 3 biologically independent samples). i Schematic of 13C-labeled glucose metabolic flux analysis in KO and WT mouse left ventricular tissue. j Quantification results of 13C-glucose metabolic flux analysis. F-6-P (m + 6), F-1, 6-BP (m + 6) and PDE (m + 3) were increased by knockout KLF7. Results are shown as fractional changes. (n = 3 biologically independent samples). k The mice intraperitoneal injection stable isotope labeled palmitate was used to calculate the contribution of FAO to acetyl-CoA entering the TAC. The abundance of [U-13C]-Palmitate labeled acetyl-CoA decreased in KO mice compared to WT mice. (n = 3 biologically independent samples). Two-tailed unpaired Student’s t test in (b, c, e, g, j, k). Data are depicted as the mean values ± SEM. FAO, fatty acid oxidation; KO, knockout of klf7 mice. Source data are provided as a Source Data file.

Fig 3: Cardiac-specific overexpression of Acadl protected Klf7-deficient hearts from hypertrophic cardiomyopathy.a Representative echocardiogram of 6-month-old mice treated with AAV-NC or AAV-Acadl for 4 weeks. b–d Cardiac-specific overexpression of Acadl (AAV-Acadl) significantly alleviated the increases in LVvol and LVID during systole and diastole and EF induced by cardiac-specific deficiency of Klf7 in mice (n = 9 mice/group). e AAV-Acadl improved the disordered myocardial tissue arrangement in Klf7KO mice (n = 3 mice/group, 8 sections/mouse); scale bar: 100 µm. f AAV-Acadl decreased HW/BW in Klf7KO mice (n = 6 mice/group). g Masson’s trichrome staining analysis revealed a reduction in myocardial fibrosis in the Klf7KO mice, (n = 3 mice/group, 8 sections/mouse); scale bar: 500 µm. h AAV-Acadl reduced the enlargement of cardiomyocytes induced by Klf7 KO, as shown by WGA staining (n = 3 mice/group, 8 sections/mouse); scale bar: 100 µm. i–m The OCR in cardiomyocytes isolated from mice hearts were measured and quantification of the FAO consumption and mitochondrial basal respiration, ATP production and maximal respiration rate (n = 5 biologically independent samples). n–p ECAR in cardiomyocytes isolated from mice hearts were measured at the indicated time points. Quantification of the glycolysis and glycolytic reserve (n = 5 biologically independent samples). q qRT-PCR analysis showed that AAV-Acadl decreased expression of the hypertrophic genes Anp and Bnp (n = 6 biologically independent experiments). g, h, The boxplot represents the median shown as a line in the center of the box, the boundaries are the first and third quartile, and whiskers represent the minimum and maximum values in the data. One-way ANOVA with Tukey’s multiple comparison test in (b–d, f–h, j–n, p and q). Data are depicted as the mean values ± SEM. AAV adeno-associated virus, AAV-Acadl overexpression Acadl with AAV. Source data are provided as a Source Data file.

Fig 4: PFKL and ACADL function as direct targets of KLF7 in the cardiac metabolism pathway.a–c Quantitative analysis of the mRNA levels of Bnp, Klf7, Pfkl and Acadl in human cardiomyocyte cell line AC16 treated with AngII for 24 h (n = 6 biologically independent samples). d, e GO functional analysis demonstrated enrichment in phosphofructokinase activity and acyl-CoA dehydrogenase activity. KEGG analysis demonstrated enrichment in glycolysis and fatty acid metabolism. f Klf7 positively regulated Acadl and negatively regulated Pfkl expression in a concentration-dependent manner, as quantified by luciferase reporter assay (n = 6 biologically independent experiments). g ChIP-PCR performed upon negative control lentivirus (lenti-NC) transfection or lentivirus-mediated HA-Klf7 expression in NMCMs confirmed that Klf7 binds the promoter regions of the Pfkl and Acadl genes (n = 2 biologically independent experiments, each independent experiment was repeated twice). h–l Cardiac hypertrophic growth induced by TAC downregulated the expression of KLF7, accompanied by upregulated PFKL expression and downregulated ACADL expression at the protein level (h, n = 6 biologically independent samples; i, k, l, sham, n = 4 biologically independent samples, TAC, n = 3 biologically independent samples; j, sham, n = 4 biologically independent samples, TAC, n = 5 biologically independent samples). m–p The protein level of PFKL was upregulated, while that of ACADL was downregulated, in NMCMs 48 h after AngII treatment (m, o, p, PBS, n = 3 biologically independent samples, AngII, n = 4 biologically independent samples; n, n = 6 biologically independent samples). Two-tailed unpaired Student’s t test in a–c and h–p. One-way ANOVA with Tukey’s multiple comparison test in (f). Data are depicted as the mean values ± SEM. KEGG Kyoto Encyclopedia of Genes and Genomes, GO Gene Ontology, NMCMs neonatal mouse cardiomyocytes, AngII angiotensin II, IP immunocoprecipitation, TAC transverse aortic constriction. Source data are provided as a Source Data file.

Fig 5: Cardiac Protein Expression and Regulation of Glucose and Fatty Acid Oxidation Enzymes and Its Regulation by Acetylation in Empagliflozin-Treated db/db MiceCardiac protein expression of glucose oxidation enzyme pyruvate dehydrogenase (PDH) (n = 6–7/group) (A), phosphorylation of PDH at the serine 293 residue (n = 6 to 7/group) (B), protein expression of pyruvate dehydrogenase kinase 4 (PDK4) (n = 6 to 7/group) (C), fatty acid ß-oxidation enzymes long chain acyl CoA dehydrogenase (LCAD) (D), and ß-hydroxyacyl CoA dehydrogenase (ß-HAD) (E) (n = 6 to 7/group) are shown. Total cardiac lysine acetylation blot (F) with quantification (G) (n = 7/group) along with lysine acetylation levels of PDH, LCAD, and ß-HAD normalized to the anti-acetyllysine immunoglobulin heavy chain (n = 5 to 6/group) (H–J) are shown. Data are presented as mean ± SEM. Data were analyzed by 1-way ANOVA followed by LSD post hoc test. *p < 0.05 was considered as a significantly different comparison with C57BL/6J + Vehicle. Abbreviations as Figures 1 and 2.