The mitochondrial citrate carrier SLC25A1 regulates metabolic reprogramming and morphogenesis in the developing heart

Abstract

The developing mammalian heart undergoes an important metabolic shift from glycolysis towards mitochondrial oxidation that is critical to support the increasing energetic demands of the maturing heart. Here, we describe a new mechanistic link between mitochondria and cardiac morphogenesis, uncovered by studying mitochondrial citrate carrier (SLC25A1) knockout mice. Slc25a1 null embryos displayed impaired growth, mitochondrial dysfunction and cardiac malformations that recapitulate the congenital heart defects observed in 22q11.2 deletion syndrome, a microdeletion disorder involving the SLC25A1 locus. Importantly, Slc25a1 heterozygous embryos, while overtly indistinguishable from wild type, exhibited an increased frequency of these defects, suggesting Slc25a1 haploinsuffiency and dose-dependent effects. Mechanistically, SLC25A1 may link mitochondria to transcriptional regulation of metabolism through epigenetic control of gene expression to promote metabolic remodeling in the developing heart. Collectively, this work positions SLC25A1 as a novel mitochondrial regulator of cardiac morphogenesis and metabolic maturation, and suggests a role in congenital heart disease.

Fig. 1. Systemic Slc25a1 deletion impairs mouse embryonic growth and causes perinatal lethality.

A Schematic of the Slc25a1 knockout (KO) first allele. A lacZ-neo KO cassette was inserted between exons 1 and 2 of Slc25a1. B Representative E18.5 Slc25a1^+/+, Slc25a1^+/-, and Slc25a1^-/- embryos obtained from timed intercross of Slc25a1^+/- mice. C Quantification of crown-rump lengths of E18.5 embryos of the indicated genotypes (n = 13–26/group). Values reported as mean ± SEM. One-tailed Student’s t test was used for statistical analysis. ****P < 0.0001. D Western blot of SLC25A1 expression in total protein isolated from E17.5 hearts and brains from the indicated genotypes. GAPDH or TGX protein staining was used as loading controls. E Representative immunofluorescence images of SLC25A1 expression in E14.5 hearts from the indicated genotypes. F Representative image of P0 pups obtained from timed intercross of Slc25a1^+/- mice. G Genotype frequencies of E18.5 embryos and P0 pups obtained from timed intercross of Slc25a1^+/- mice. Illustrations from Clkr.com.

Fig. 2. Partial Slc25a1 deletion is sufficient to produce cardiac structural defects in mice.

A Representative immunofluorescence images of SLC25A1 expression in wild type embryo (E10.5, E12.5, and E14.5) and heart (E18.5) sections (6 μm). DAPI was used to counterstain nuclei, and MF20 was used to counterstain cardiomyocytes. Dashed yellow line outlines myocardium from the epicardium; asterisk indicates epicardium positive for SLC25A1. B Representative images of E10.5 embryos, (C) E12.5 embryos, (D) E14.5 embryos, and (E) E18.5 hearts from the indicated genotypes stained with hematoxylin and eosin. Asterisk indicates noncompaction, arrow indicates ventricular septal defect, and triangle indicates right ventricular hypoplasia. F Quantification of relative compact myocardium thickness or (G) relative trabecular myocardium thickness in E18.5 hearts (n = 8-17/group). H Frequency of cardiac defects observed at E14.5 in embryos with the indicated genotypes (n = 27/group). Values reported as mean ± SEM. One-tailed Student’s t test was used for statistical analysis. *P < 0.05; ***P < 0.0005; ****P < 0.0001.

Fig. 3. Slc25a1 deletion alters metabolic gene expression in mice.

A Volcano plots of Nanostring metabolic transcript profiling comparing gene expression of Slc25a1^-/- and Slc25a1^+/+, Slc25a1^+/- and Slc25a1^+/+, and Slc25a1^-/- and Slc25a1^+/- E17.5 hearts. Blue denotes downregulated transcripts. Yellow denotes upregulated transcripts. See Supplemental Data 3. B Heat map depicting transcripts differentially expressed across genotypes in A. Transcripts in rows were selected by ANOVA multiple comparisons followed by Benjamini-Hochberg correction (q < 0.05) and clustered using Kendall-Tau (see Supplemental Data 3). Each column represents an individual animal. C Gene ontologies identified with Metascape from genes differentially expressed in Slc25a1^-/- and Slc25a1^+/- versus Slc25a1^+/+ hearts. D Geneset enrichment analysis of oxidative phosphorylation, glycolysis, hypoxia, and fatty acid metabolism genes in Slc25a1^+/+ vs. Slc25a1^-/- and Slc25a1^+/+ vs. Slc25a1^+/- E17.5 hearts. E Heatmaps visualizing gene expression of oxidative phosphorylation and glycolysis genes from Nanostring transcript profiling in E17.5 Slc25a1^+/+ vs. Slc25a1^-/- hearts. Blue denotes reduced expression; red denotes increased expression.

Fig. 4. Slc25a1 deletion impairs respiratory chain function in the developing mouse heart.

A Representative electron micrographs of cardiac ultrastructure from the indicated genotypes at E17.5 (n = 3 Slc25a1^+/+; n = 10 Slc25a1^+/-; n = 3 Slc25a1^-/- embryos analyzed). B Coupled and uncoupled mitochondrial oxygen consumption rates measured in lightly permeabilized whole E17.5 hearts of the indicated genotypes (n = 5 Slc25a1^+/+; n = 5 Slc25a1^+/-; n = 3 Slc25a1^-/- hearts analyzed). C Quantification of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA) ratios in E17.5 hearts of the genotypes indicated (n = 6 Slc25a1^+/+; n = 6 Slc25a1^+/-; n = 4 Slc25a1^-/- embryos analyzed). D Western blot of respiratory chain complex subunits (SDHA, ATP5A, UQCRC2, NDUFA9, and COXIV). Coomassie staining of the gel was used as a protein loading control. E Densitometry quantification of NDUFA9, SDHA, UQCRC2, COXIV, and ATP5A expression (n = 3/group). F Respiratory chain complexes in E17.5 heart mitochondria as assessed by BN-PAGE followed by immunoblotting with an OXPHOS antibody cocktail. A western blot of VDAC was used as a protein loading control. Values presented as means ± SEM. One-way ANOVA followed by a post-hoc Dunnett’s test. ns denotes not significant; *P < 0.05; **P < 0.005.

Fig. 5. Targeted metabolomic profiling reveals amino acid dysregulation and impaired Krebs cycle function with SLC25A1 deletion.

A Schematic of the metabolites profiled by GC-MS. B Principal component analysis (PCA) and (C) unbiased hierarchical clustering of profiled analytes showing clear segregation of Slc25a1^+/+(n = 6), Slc25a1^+/- (n = 3), and Slc25a1^-/- (n = 4) hearts. D Normalized abundance of selected metabolites that are significantly altered in HET and KO hearts. Values presented as means ± SEM. One-tailed Student’s t test was used for statistical analysis. P < 0.05 was considered significant, *P < 0.05 and **P < 0.005. E Malate-to-citrate ratios and (F) Fumarate-to-malate ratios in Slc25a1^+/+, Slc25a1^+/- and Slc25a1^-/- hearts. Values presented as means ± SEM. One-way ANOVA followed by a post hoc Dunnett’s test was used for statistical analysis. P < 0.05 was considered significant, ns denotes not significant, *P < 0.05.

Fig. 6. In silico modeling reveals glucose dependency and impaired oxidative phosphorylation with SLC25A1 loss.

A PCA of the CardioNet simulation revealed a clear clustering of Slc25a1^+/+, Slc25a1^+/-, and Slc25a1^-/- hearts by Slc25a1 genotype. B Unsupervised hierarchical clustering of significantly altered flux distributions. Two-way ANOVA with post hoc Tukey’s test followed by multiple comparison analysis (FDR < 1% after Benjamini, Krieger and Yekuteli). C Predicted flux rates for glucose uptake, and D oxidative phosphorylation in response to SLC25A1 deletion. Box plot parameters are defined by the minimum value, the first quartile, the median, the third quartile, and the maximum value. *P < 0.05; ****P < 0.00005.

Fig. 7. Slc25a1 deletion reduces histone acetylation at promoter of metabolism-related genes in developing mouse hearts.

A Representative western blot of histone H3 acetylated at K9 (H3K9ac) and total histone H3 in total heart protein from E17.5 embryos of the indicated genotypes. B Densitometry quantification of H3K9ac to H3 levels in E17.5 hearts (n = 3/group). One-way ANOVA followed by post hoc Dunnett’s test was used for statistical analysis. *P < 0.05. C H3K9ac ChIP qPCR of H3K9ac enrichment at the promoters of Ndufa1, Ndufb2, Ndufb4, Ndufb8, Ndufs8, Cox4i1, Cox5b, Uqcrq, Pdha1 and Pparg in E17.5 hearts (n = 4-5/group). ChIP with anti-rabbit IgG was used as a negative control. Data were normalized using input DNA. Values presented as means ± SE. One-way ANOVA followed by post hoc Dunnett’s test was used for statistical analysis. ns denotes not significant; *P < 0.05, **P < 0.005, and ***P < 0.0005. D Proposed model of SLC25A1-mediated citrate export and its role in regulation of metabolic gene expression in the developing heart. Citrate in the mitochondria is generated from acetyl-CoA and oxaloacetate in the TCA cycle. SLC25A1 mediates mitochondrial citrate export. Citrate in the cytosol is converted to acetyl-CoA, which is used for histone acetylation (H3K9ac) and impacts gene expression necessary for proper heart development. Diagram created using BioRender.