Loss of ATP-Dependent Citrate Lyase Drives Left Ventricular Dysfunction by Metabolic Remodeling

Figures

• 

  Figure 1.. Cardiac-specific knockdown of Acly promotes left ventricular dysfunction. A, Volcano plot showing metabolomic changes in left ventricular tissue samples from healthy human donor hearts and from patients (P) with heart failure with reduced ejection fraction (HFrEF) (donor: n=9; HFrEF: n=7). The blue and red circles indicate significantly decreased (blue) or increased (red) metabolite abundances. Statistical significance was assessed using a multiple unpaired t test with a false discovery rate (FDR) and a 2-stage step-up method of Benjamini, Krieger, and Yekutieli (FDR cutoff <0.05). B and C, Protein abundance (B) and enzymatic activity (C) of ACL in left ventricular tissue samples from healthy human hearts (n=5; patients [P] 1–5) and patients with HFrEF (n=5; P6–P10). Statistical significance was assessed using a multiple unpaired t test with a FDR and a 2-stage step-up method of Benjamini, Krieger, and Yekutieli (FDR cutoff <0.05). D, Experimental design for CRISPR/Cas9-mediated cardiac-specific knockdown (KD) of Acly using Myh6-Cas9-2A-tdTomato transgenic mice (age postnatal day 0 to 20 weeks). Guide RNAs (gRNAs) were delivered using AAV9 at postnatal day 6 (P6) to achieve…

• 

  Figure 2.. Loss of Acly promotes increased glucose uptake and glycolytic flux in vivo. A, Representative images of [18F]-fluorodeoxyglucose (FDG)–positron emission tomography analysis in vivo of WT (Myh6-Cas9-) and AclyKD (Myh6-Cas9+) mice at 20 weeks. B and C, Tracer retentions (B) relative to weight-normalized injected dose (standard uptake value [SUV]) in the heart and kidneys of in vivo–injected WT and AclyKD mice at 20 weeks. The top 5 values were used in calculating the average. SUVs were normalized to plasma glucose levels (C); n=6 or 7 male and female mice/group. Statistical significance was calculated using a 2-way ANOVA followed by Šídák multiple comparisons test for post hoc analysis. D, Enzymatic activity for phosphofructokinase (PFK) and lactate dehydrogenase (LDH) was quantified in heart tissue samples from WT and AclyKD mice at 20 weeks using enzymatic assays. n=3 or 4 male and female mice/group. Statistical significance was calculated by a t test. E, Glucose-6 phosphate levels were quantified using spectrophotometric assays in heart tissue samples from WT and AclyKD mice at 20 weeks. n=9 or 10 male and female mice/group. Statistical significance was calculated by a t test.…

• 

  Figure 3.. Functional analysis reveals increased lipid remodeling in AclyKD hearts. A, Gene ontology (GO) enrichment analysis of transcript changes from RNA sequencing of adult mouse ventricular cardiomyocytes isolated from WT (Myh6-Cas9-) and AclyKD (Myh6-Cas9+) mouse hearts at 20 weeks. Genes with significant transcript changes (log-fold changes, logFC) and the top enriched gene ontology pathways are shown in the chord diagram. Left-right connections indicate gene associations to pathways annotated using GO terms. B, Gene expression of critical genes for fatty acid synthesis (Fasn), desaturation (Fads2 and Scd4), and elongation (Elovl5). Expressions were quantified in heart tissue samples from WT and AclyKD at 20 weeks using reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Each biological sample was normalized by Tbp expression. n=3 male and female mice/group. Statistical significance was assessed using a multiple unpaired t test with a false discovery rate (FDR) and a 2-stage step-up method of Benjamini, Krieger, and Yekutieli (FDR cutoff < 0.01). C, Schematic depicting interactions of fatty acid synthesis and modifications from endogenous and dietary sources.…

• 

  Figure 4.. Loss of Acly induces YAP inactivation. A, Schematic of AMPK and PKA regulation of LATS1 and YAP. B and C, Western blotting of total and phosphorylated (P) AMPK (B) and PKA (C), and corresponding ratios of P-AMPK to total AMPK and P-PKA to total PKA in WT (Myh6-Cas9-) and AclyKD hearts. n=6 male and female mice/group. Statistical significance was calculated using a 2-way ANOVA followed by Šídák multiple comparisons test for post hoc analysis. D, PKA activity in heart tissue samples from WT and AclyKD hearts. n=6 male and female mice/group. Statistical significance was calculated by a t test. E, Western blotting of LATS1 expression in WT and AclyKD hearts. n=4 male and female mice/group. Statistical significance was calculated by a t test. F and G, Representative Western blot images of total YAP/TAZ expression (E) and of YAP phosphorylation at serine (S) residues S127 and S397 (F) in WT and AclyKD hearts. n=4 or 5 male and female mice/group. Statistical significance was calculated using a 2-way ANOVA followed by Šídák multiple comparisons test for post hoc analysis. H, Heart weight (HW) normalized by tibia length (TL) in WT and AclyKD treated with PY-60 in vivo. n=3 male and female…

• 

  Figure 5.. AclyKD shifts the contribution of glucose and palmitate to oxidative metabolism in the heart. A, Experimental settings of working heart perfusions. B, Hydraulic power in perfused working mouse hearts from WT (Myh6-Cas9-) or AclyKD male and female mice at 20 weeks. n=5 male and female mice/group. Statistical significance was calculated by a t test. C, Schematic of the theoretical isotopomer distribution in central carbon metabolism using [U-13C]glucose and [U-13C]palmitate tracers. Metabolites are indicated in black circles (representing carbons). The filled red and orange circles denote heavy carbon (13C). D, Fractional enrichment of the 3-phosphoglycerate (3PG) m+3, pyruvate (Pyr) m+3, lactate m+3, citrate (Cit) m+2, malate (Mal) m+2, and fumarate (Fum) m+2 from steady-state [U-13C]glucose perfusions of WT and AclyKD mouse hearts at 20 weeks. n=6 male and female mice/group. Statistical significance was assessed using a Mann-Whitney t test with a false discovery rate (FDR) and a 2-stage step-up method of Benjamini, Krieger, and Yekutieli (FDR cutoff <0.01). E, Comparison of pyruvate carboxylase flux derived from pyruvate m+3 in perfused WT and AclyKD mouse hearts. The fractional…

• 

  Figure 6.. CardioNet simulation reveals metabolic vulnerabilities in AclyKD. A, Regression analysis of computational flux simulations in WT (Myh6-Cas9-) and AclyKD hearts. Experimental data from working mouse heart perfusions were integrated into CardioNet simulations, and metabolic flux rates were calculated using an objective function. Calculated flux rates were compared using a linear regression model and annotated to CardioNet pathways. B, Comparison of critical metabolic fluxes in simulated enzyme knockouts using CardioNet. Theoretical flux distributions from each knockout simulation were compared with WT simulations according to the calculated vIDH1, vOXPHOS, vACC, and vG6PDH. C, Schematic of experimental workflow for in vivo Idh1 knockdown. D, Heart weight to tibia length in WT and AclyKD;Idh1KD mice. n=7 or 8 male and female mice/group. Statistical significance calculated by t test. E, Assessment of cardiac function using echocardiography. Representative M-mode images and quantification of ejection fraction in WT and AclyKD;Idh1KD mice. n=5–7 male and female mice/group. Statistical significance was calculated using a repeated-measures ANOVA followed by Šídák multiple comparisons…