Modeling the Transition From Decompensated to Pathological Hypertrophy

Figures

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  Figure 1. Short‐term Acsl1 inactivation induces cardiac hypertrophy before the cardiac dysfunction develops. Ventricular Acsl1 mRNA (A), ACSL1 protein levels (B), ACS activity (C), AMPK (D) and mTORC1 (F) activation 2 weeks after tamoxifen treatment. E, Relative heart weight (heart weight/body weight) in control and Acsl1 H−/− mice treated with vehicle (Veh) or rapamycin (Rapa). Body weights were not significantly affected by Acsl1 knockdown or rapamycin treatment. F, mTOR activation via phosphorylation of downstream targets S6K and 4E‐BP1. G, E/A ratio (ratio between early (E) and late (atrial‐A) ventricular filling velocity) and MPI (mean performance index) in control and Acsl1 H−/− mice. n=3 to 5 per group. Means±SEM (error bars). *P≤0.05, **P≤0.01 compared with littermate controls.

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  Figure 2. Knockdown of ACSL1 results in cardiac‐specific metabolic derangements that do not affect systemic metabolic homeostasis. A, 4‐hour‐fasting plasma non‐esterified fatty acids (NEFA), glycerol, triglycerides (TAG), and glucose levels in control and Acsl1 H−/− mice treated with vehicle (Veh) or rapamycin (Rapa). B, Uptake of 2‐bromo[1‐14C]palmitate in ventricles of control and Acsl1 H−/− knockdown mice treated with vehicle or rapamycin after a 4 hours fast. C, Ventricular 2‐deoxy[1‐14C]‐glucose uptake in control and Acsl1 H−/− hearts. n=3 to 8 per group. The values reported are means±SEM (error bars). *P≤0.05 compared with littermate controls.

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  Figure 3. Microarray gene chip analyses demonstrate that the partial ablation of Acsl1 causes transcriptional derangements that are distinct from those caused by total ACSL1 knockout observed 10‐week after tamoxifen treatment. A, Correlation of 8706 probes identifying 3 distinct groups: 10‐week Acsl1 H−/− knockout, 2‐week Acsl1 H−/− knockout, and a combination of 10‐ and 2‐week Acsl1 flox/flox controls. B, Venn diagram of gene expression overlap with a fold‐change ≥1.5. C, Unsupervised hierarchical clustering analysis of 1244 genes (columns) from 17 samples (rows) using the Euclidian matrix with average linkage (Partek Genomics Suite, v6.6). Three primary clusters of samples comprised solely of Acsl1 flox/flox, 10‐week Acsl1 H−/−, or 2‐week Acsl1 H−/− biological replicates were identified. The genes portioned into 2 groups of higher (yellow) or lower (blue) expression across the 3 sample groups.

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  Figure 4. The early stages of the metabolic switch are accompanied by increased expression of fibrosis markers and no upregulation of the glycolytic program. Expression of rapamycin‐sensitive mTOR genes identified in 10‐week Acsl1 H−/− knockout hearts compared to 2‐week Acsl1 H−/− knockdown and control hearts (A). Expression of 2‐week centric genes suggests increased ECM/fibrosis (B) and is not indicative of a switch towards glycolytic metabolism (C). n=3 to 8 per group. The values reported were determined using microarray gene chips and are represented by the mean±SEM (error bars). *P≤0.05; **P<0.01; † P<0.001; ‡ P<0.0001 compared with littermate controls.

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  Figure 5. Rapamycin treatment reveals novel mTOR targets of cardiac metabolism. A, Partial Acsl1 knockdown resulted in suppression of FA transport and triacylglycerol synthesis, whereas peroxisomal and mitochondrial oxidation gene expression was induced. B, Acsl1 knockdown hearts exhibit decreased glycolytic (Gpi, Alfoa, Gapdh, Ldha) and glycogen degradation (Pygm, Phkg1) genes. C, Glucose is directed towards the pentose phosphate (Rpe, Rpia) and hexosamine biosynthesis (Gfprt1, Oga (protein glycosylation)) alternative metabolic pathways. D, Tricarboxylic acid cycle flux and anaplerosis, and (E) ketogenesis and amino acid metabolism are induced in response to partial Acsl1 ablation. n=5 per group. The values reported were determined using RNAseq and are represented by the mean±SEM (error bars). *P≤0.05; **P<0.01 compared with littermate controls; ▲ denotes genes whose altered expression in Acsl1 H−/− knockdown hearts was reverted by rapamycin treatment; denotes genes whose expression was altered by rapamycin treatment in Acsl1 flox/flox control hearts.

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  Figure 6. Increased cardiac fibrosis is not related to the development of hypertrophy in the early stages of the metabolic switch. A, RNAseq analysis of Acsl1‐dependent alterations in the expression of genes involved in collagen fiber or glycoprotein synthesis (Col14a1, Gpc6, Matn2), cell‐cell and cell‐matrix interactions (Cfh, Adam19, Adamtsl2, Ecm2, Adam11, Thbs2, Fndc3a), ECM degradation (Htra1, Sulf1), and regulation of ECM dynamics (Pdgfrα and Wisp2/CCN5). B, Masson's trichrome staining of control and Acsl1 knockdown hearts in the absence and presence of rapamycin treatment. Original magnification, ×20; scale bar=100 μm. C, Comparison of the relative expression of genes involved in ECM synthesis and degradation determined by microarray analysis in the 2‐week ACSL1 knockdown vs 10‐week knockout models. D, The effects of ACSL1 inactivation on the expression of genes involved in physiological hypertrophy in the early stages of the metabolic switch as measured by RNAseq. n=5 per group. The values reported are means±SEM (error bars). *P≤0.05; **P<0.01; ‡ P<0.0001 compared with littermate controls; ▲ denotes genes whose altered expression in Acsl1 knockdown hearts was reverted by rapamycin…

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  Figure 7. Partial ACSL1 ablation results in cardiac‐specific metabolic derangements that are reverted by rapamycin. A, Unsupervised hierarchical clustering of 64 metabolites identified by 2‐way ANOVA. B, Principal component analysis (PCA) of cardiac metabolite concentrations in control and Acsl1 H−/− mice treated with vehicle (Veh) or rapamycin (Rapa). The first 2 components are shown. Confidence ellipses categorized by genotype×treatment represent 2 standard deviations. n=6 to 8 per group.

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  Figure 8. Acsl1‐dependent changes in metabolic pathways during the early stages of the cardiac metabolic switch. Metabolite concentrations are identified as either increased (yellow) or decreased (blue) in Acsl1 H−/− hearts relative to control hearts. Upregulated (green) and downregulated (red) genes are also shown. Metabolites and genes in gray were not identified in our metabolomic or gene expression analyses. 3‐PG indicates 3‐phosphoglycerate; ACAC‐CoA, acetoacetyl‐CoA; Arg, arginine; Asn, asparagine; Asp, aspartate; Cit, citrate; Cys, cysteine; F‐1,6‐BP, fructose‐1,6‐bisphosphate; Fum, fumarate; G3P, glycerol 3‐phosphate; Glc, glucose; Gln, glutamine; Glu, glutamate; Guo, guanosine; HMG‐CoA, 3‐hydroxy‐3‐methylglutaryl‐CoA; Ino, inosine; LysoPEs, lysophosphatidylethanolamine species; Mal, malate; Met, methionine; OAA, oxaloacetate; Orn, ornithine; PI, phosphatidylinositol species; Ru5P, ribulose‐5‐phosphate; S7P, sedoheptulose‐7‐phosphate; X5P, xylulose‐5‐phosphate; α‐KG, α‐ketoglutarate; β‐HB, β‐hydroxybutyrate. ▲ denotes genes and metabolites whose altered levels in Acsl1 knockdown hearts was reverted by rapamycin treatment.