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. 2016 May 24;133(21):2038-49.
doi: 10.1161/CIRCULATIONAHA.115.020226. Epub 2016 Apr 8.

Catabolic Defect of Branched-Chain Amino Acids Promotes Heart Failure

Haipeng Sun  1 , Kristine C Olson  1 , Chen Gao  1 , Domenick A Prosdocimo  1 , Meiyi Zhou  1 , Zhihua Wang  1 , Darwin Jeyaraj  1 , Ji-Youn Youn  1 , Shuxun Ren  1 , Yunxia Liu  1 , Christoph D Rau  1 , Svati Shah  1 , Olga Ilkayeva  1 , Wen-Jun Gui  1 , Noelle S William  1 , R Max Wynn  1 , Christopher B Newgard  1 , Hua Cai  1 , Xinshu Xiao  1 , David T Chuang  1 , Paul Christian Schulze  1 , Christopher Lynch  1 , Mukesh K Jain  1 , Yibin Wang  2
Affiliations

Catabolic Defect of Branched-Chain Amino Acids Promotes Heart Failure

Haipeng Sun et al. Circulation. .

Abstract

Background: Although metabolic reprogramming is critical in the pathogenesis of heart failure, studies to date have focused principally on fatty acid and glucose metabolism. Contribution of amino acid metabolic regulation in the disease remains understudied.

Methods and results: Transcriptomic and metabolomic analyses were performed in mouse failing heart induced by pressure overload. Suppression of branched-chain amino acid (BCAA) catabolic gene expression along with concomitant tissue accumulation of branched-chain α-keto acids was identified as a significant signature of metabolic reprogramming in mouse failing hearts and validated to be shared in human cardiomyopathy hearts. Molecular and genetic evidence identified the transcription factor Krüppel-like factor 15 as a key upstream regulator of the BCAA catabolic regulation in the heart. Studies using a genetic mouse model revealed that BCAA catabolic defect promoted heart failure associated with induced oxidative stress and metabolic disturbance in response to mechanical overload. Mechanistically, elevated branched-chain α-keto acids directly suppressed respiration and induced superoxide production in isolated mitochondria. Finally, pharmacological enhancement of branched-chain α-keto acid dehydrogenase activity significantly blunted cardiac dysfunction after pressure overload.

Conclusions: BCAA catabolic defect is a metabolic hallmark of failing heart resulting from Krüppel-like factor 15-mediated transcriptional reprogramming. BCAA catabolic defect imposes a previously unappreciated significant contribution to heart failure.

Keywords: amino acids; heart failure; metabolism; oxidant stress; pathogenesis; remodeling.

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Figures

Figure 1
Figure 1
Remodeling of BCAA catabolism in murine failing heart. A, The down-regulated genes in failing heart were mapped into BCAA catabolism pathway by KEGG. B, Real-time RT-PCR result of specific genes using mRNA from myocardium of Neonatal (n=3), normal (Adult Sham, n=3) and failing (Adult Failing, n=3) mouse hearts. Y axis represents relative mRNA level. ANOVA followed by Newman-Keul test was performed*, p<0.05 compared to Neonatal; #, p<0.05 compared to Adult Sham. C, Western blotting result of proteins involved in BCAA catabolism (GAPDH as loading control) using tissue lysates from three individual normal (Sham) or failing mouse hearts (n=3). D, Individual BCKA concentration in tissues from normal (Sham, n=9) and failing (n=7) mouse hearts. Error bars represent standard deviation (B) or SEM (D). *, p<0.05, **, p<0.01.
Figure 1
Figure 1
Remodeling of BCAA catabolism in murine failing heart. A, The down-regulated genes in failing heart were mapped into BCAA catabolism pathway by KEGG. B, Real-time RT-PCR result of specific genes using mRNA from myocardium of Neonatal (n=3), normal (Adult Sham, n=3) and failing (Adult Failing, n=3) mouse hearts. Y axis represents relative mRNA level. ANOVA followed by Newman-Keul test was performed*, p<0.05 compared to Neonatal; #, p<0.05 compared to Adult Sham. C, Western blotting result of proteins involved in BCAA catabolism (GAPDH as loading control) using tissue lysates from three individual normal (Sham) or failing mouse hearts (n=3). D, Individual BCKA concentration in tissues from normal (Sham, n=9) and failing (n=7) mouse hearts. Error bars represent standard deviation (B) or SEM (D). *, p<0.05, **, p<0.01.
Figure 2
Figure 2
Impaired BCAA catabolism in human failing heart. A, Real-time RT-PCR result of specific genes using mRNA from myocardium of control (Ctrl, n=4) and failing (Failure, n=11–15) human hearts. Y axis: relative mRNA level. B, Individual BCKA concentration in tissues from control (n=3) and failing (n=10) human hearts. Error bars represent SEM. *, p<0.05-.
Figure 3
Figure 3
KLF15 regulates BCAA catabolic gene expression. A, Real-time RT-PCR result of specific genes using mRNA from neonatal rat ventricular myocytes with (KLF15) or without (Vector) KLF15 overexpression. (n=6) *, p <0.05 compared to vector control. B, Western blotting result of proteins involved in BCAA catabolism (GAPDH as loading control) using cellular lysates from KLF15 overexpressed Hela cells. C, Illustration of partial mouse PP2Cm promoter fragments with two GC rich sites and Luciferase assay result of PP2Cm promoter-luciferase in HeLa cells co-transfected with either KLF15 or corresponding empty vector. The data represented the average values with standard deviation of triplicate samples from one experiment representative of three independent experiments. *, p <0.05 compared to same promoter without KLF15 overexpression. #, p<0.05 compared to 468bp promoter with KLF15 overexpression (n=3). D, The representative result of chromatin immunoprecipitation-PCR validation for KLF15 binging to PP2Cm gene’s promoter in neonatal rat ventricular myocytes after KLF15 overexpression. The experiment has been repeated twice with similar result.
Figure 4
Figure 4
Ablation of cardiac KLF15 down-regulates BCAA catabolism. A and B, Real-time RT-PCR (A) and Western blotting (B) result of specific genes in wildtype (WT, n=4) and KLF15 deficient (KLF15 KO, n=4) hearts. C. Level of BCKAs in wildtype (WT, n=4) and KLF15 deficient (KLF15 KO, n=5) heart. Error bars represent standard deviation (A) or SEM (C). *, p <0.05 compared to wildtype.
Figure 5
Figure 5
BCAA catabolic defect impairs cardiac function but not structure. A and B, Individual BCKA concentrations in cardiac tissue of PP2Cm knockout (KO) and wildtype (WT) mice. A, Mice on a normal chow (20% protein) were fasted for 6 hours (WT, n=9; KO, n=10). B, Mice were fasted overnight and fed with a high protein diet (40% protein) for 2 hours (WT, n=5; KO, n=5). Error bars represent SEM. **, p<0.01 compared to WT. C and D, Left ventricular ejection fraction (LV%EF) from WT and PP2Cm KO mice at 3 (C, WT n=12 and KO n=15) or 18 (D, n=5 in each group) months of age. E, Morphology of hearts from wild-type (WT) and PP2Cm deficient (PP2Cm KO) mice. F, Longitudinally sectioned heart was stained with hematoxylin and eosin. Magnification, 200×. G, Transmission electron microscopy was performed in hearts from PP2Cm knockout (KO) and wildtype (WT) mice. Magnification, 7400×.
Figure 6
Figure 6
BCAA catabolic defect promotes heart failure progression. A and B, Time course for left ventricular fractional shortening (%FS, A) and left ventricular internal dimension at systole (LVIDs, mm, B) from WT (n=11–15) and PP2Cm KO mice (n=13–19) with TAC surgery. The X-axis shows the time in weeks after surgery. C and D, Representative M-mode echocardiographs (C) or ratio of lung weight (LW) to body weight (BW) (D, WT Sham n=9; KO Sham n=10; WT TAC n=8; KO Sham n=8) from WT and PP2Cm KO mice at 8 weeks after surgery. Error bars represent SEM. Statistical analyses were performed with Student’s t-test (A–B) to compare the values of WT and PP2Cm KO at the same time point (#, p=0.05, *, p<0.05) or Kruskal-Wallis test followed by Dunn’s multiple comparison was performed (D) (*, p<0.05 compared to KO Sham). A repeated measures linear model was also fitted for LVIDs (A) and %FS (B).
Figure 7
Figure 7
Disturbed metabolic and redox homeostasis by BCKAs. A, Oxygen consumption in mitochondria isolated from wildtype hearts in absence or presence of 500μM BCKAs. B, Relative oxygen consumption rate in the absence or presence of BCKAs at different concentrations (n=3–8 in each group; *, p<0.05 vs control). C, Superoxide production in isolated cardiac mitochondria (n=4–7 in each group; *, p<0.05, vs control). D and E, Superoxide production in isolated mitochondria (D, n=5–6 in each group) and myocardium (E, n=3 in each group) from wildtype and PP2Cm deficient mice. F, Immunoblotting of total protein oxidation detected by carbonyl groups (left) from tissue lysates of wildtype (WT) and PP2Cm deficient (KO) mouse hearts. G, Principal component analysis (PCA) of metabolomic profiles revealed a distinct genotype-based separation for the heart samples (WT, n=8; KO, n=7). H, List of the top 30 biochemicals that separated different genotypes based on their importance. Error bars represent standard deviation SEM (B C, D, E).
Figure 8
Figure 8
Inhibition of BCKDK by BT2 promotes BCKA degradation and preserves cardiac function in pressure-overloaded heart. A, Immunoblot for total and phosphorylated BCKD subunit E1α in heart from wildtype mice treated with vehicle (veh, n=4) or with BT2 (n=5). B, The average phosphorylation level of E1α vs. total E1α are presented with SEM. Error bars represent SEM, *, p < 0.05 between Veh and BT2 treated samples. C, BCKD activity in cardiac tissues from wildtype or PP2Cm-KO mice treated with vehicle or BT2 (n=4–5 in each group). *, p < 0.05 between Veh and BT2 treated groups. D, Individual BCKA concentration in plasma from wildtype and PP2Cm-KO (n=4–6) mice treated with vehicle (veh group) or with BT2 (BT2 group).*, p<0.05 between Veh and BT2 treated groups of same genotype. E, Representative M-mode echocardiographs of mouse hearts following sham or post-TAC treated with vehicle (Veh) or BT2. F, Left ventricular Ejection Fraction (%LVEF, n=6–8), and G, left ventricular internal dimension at systole (LVIDs, n=6–8) from mice with sham or TAC surgery for 4 weeks, treated with or without BT2 as indicated. Error bars represent SEM., *, p< 0.05 between designated groups.
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