Therapeutic Effect of Targeting Branched-Chain Amino Acid Catabolic Flux in Pressure-Overload Induced Heart Failure

Abstract

Background Branched-chain amino acid (BCAA) catabolic defect is an emerging metabolic hallmark in failing hearts in human and animal models. The therapeutic impact of targeting BCAA catabolic flux under pathological conditions remains understudied. Methods and Results BT2 (3,6-dichlorobenzo[b]thiophene-2-carboxylic acid), a small-molecule inhibitor of branched-chain ketoacid dehydrogenase kinase, was used to enhance BCAA catabolism. After 2 weeks of transaortic constriction, mice with significant cardiac dysfunctions were treated with vehicle or BT2. Serial echocardiograms showed continuing pathological deterioration in left ventricle of the vehicle-treated mice, whereas the BT2-treated mice showed significantly preserved cardiac function and structure. Moreover, BT2 treatment improved systolic contractility and diastolic mechanics. These therapeutic benefits appeared to be independent of impacts on left ventricle hypertrophy but associated with increased gene expression involved in fatty acid utilization. The BT2 administration showed no signs of apparent toxicity. Conclusions Our data provide the first proof-of-concept evidence for the therapeutic efficacy of restoring BCAA catabolic flux in hearts with preexisting dysfunctions. The BCAA catabolic pathway represents a novel and potentially efficacious target for treatment of heart failure.

Keywords: amino acids; heart failure; hypertrophy/remodeling; metabolism; therapy.

Figure 1

Establishment of a mouse model with preexisting cardiac dysfunction for subsequent pharmacological treatment. A, Schematic timeline and experimental design. C57BL/6N adult male mice (n=21) were subjected to transverse aortic constriction (TAC) to provoke hypertrophy and contractile dysfunction, and echocardiography was performed to confirm left ventricular (LV) contractility and cardiac dysfunction. Four mice died before drug treatment. Mice were then intragastrically administered with BT2 (n=9) or vehicle (n=8) for 6 weeks, and cardiac function was assessed every week. B, A schematic illustration of key enzymes and regulators involved in branched‐chain amino acid (BCAA) catabolism. C through F, Echocardiographic parameters in the first 2 weeks after TAC. Average values of LV ejection fraction (EF; C), LV mass (D), LV internal diameter at systole (LVID;s; E), and LV volume at systole (LV Vol;s; F) obtained from echocardiography are shown. G through J, Analyses of hearts at 8 weeks after TAC procedure (n=5 in vehicle group, n=7 in BT2 group). G, Expression of total, phosphorylated branched‐chain ketoacid dehydrogenase (BCKD) subunit E1α, and BCKD kinase (BCKDK) in mouse heart was determined by Western blotting. H, The relative phosphorylated E1α level and BCKDK level were calculated. Cardiac BCAA (I) and branched‐chain α‐ketoacid (BCKA; J) abundances were measured, as described in Methods. For data of Figure 1 C and 1 F, comparison of indicated time points vs baseline was analyzed by 1‐way ANOVA with Bonferroni's test. For Figure 1 H and 1 J, differences were evaluated by Student's t test. Data were presented as mean±SEM. BCAT indicates branched‐chain amino‐transferase; BCKDH, branched‐chain ketoacid dehydrogenase; KIC, α‐ketoisocaproic; KMV, α‐keto‐β‐methylvaleric; KIV, α‐ketoisovaleric; Suc‐CoA; Succinyl‐CoA. *P<0.05.

Figure 2

Inhibition of branched‐chain α‐ketoacid dehydrogenase kinase (BCKDK) by BT2 preserved systolic function in hearts with preexisting dysfunctions. Mice were subjected to transverse aortic constriction (TAC) procedure for 2 weeks and then were randomized to be treated with BT2 (n=9) or vehicle (n=8) for an additional 6 weeks. Relative changes of left ventricular (LV) fractional shortening (∆FS; A), ejection fraction (∆EF; B), LV internal diameters (∆LVID;s; C), and LV volume (∆LV Vol;s; D) were presented at indicated time points. E, Heart weight/tibia length (HW/TL) ratios at 8 weeks after TAC procedure were presented (n=5 in vehicle group, n=7 in BT2 group). F, Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) expression was quantified by real‐time quantitative reverse transcription–polymerase chain reaction (n=5 in vehicle group, n=7 in BT2 group). For A through D, 2‐way ANOVA, followed by Bonferroni's post hoc test, was performed to evaluate the differences between groups over the treatment period (from week 3 to week 8). For E and F, Student's t test was used for 2‐group comparison. Data were presented as mean±SEM. NS indicates not significant. *P<0.05.

Figure 3

Branched‐chain α‐ketoacid dehydrogenase kinase (BCKDK) inhibitory treatment improved myocardium contractility and wall motion. Mice were subjected to transverse aortic constriction (TAC) procedure for 2 weeks and then were randomized to be treated with BT2 (n=9) or vehicle (n=8) for an additional 6 weeks. Relative global longitudinal strain (GLS; A), global radial strain (GRS; B), and respective global strain rate (GLSR and GRSR; C and D) for both groups at indicated time points were presented. Means of systolic velocity (E and F) and displacement (G and H) at longitudinal and radial planes were presented. Two‐way ANOVA, followed by Bonferroni's post hoc test, was performed to evaluate the differences between groups over the treatment period (from week 4 to week 8). Data were presented as mean±SEM. *P<0.05.

Figure 4

Branched‐chain α‐ketoacid dehydrogenase kinase (BCKDK) inhibitory treatment improved diastolic left ventricular mechanics. Mice were subjected to transverse aortic constriction (TAC) procedure for 2 weeks and then were randomized to be treated with BT2 (n=9) or vehicle (n=8) for an additional 6 weeks. Relative diastolic longitudinal strain rate (A) and diastolic radial strain rate (B) for both groups at indicated time points were presented. Diastolic longitudinal velocity (C) and radial velocity (D) were presented. Two‐way ANOVA, followed by Bonferroni's post hoc test, was performed to evaluate the differences between groups over the treatment period (from week 4 to week 8). Data are presented as mean±SEM. *P<0.05.

Figure 5

BT2 treatment altered the expression of genes involved in substrate metabolism. A, C, and E, Hearts were collected after 6 weeks of treatment with either vehicle or BT2 (8 weeks after surgery). B, D, and F, Neonatal rat ventricular myocytes (NRVMs) were treated with vehicle or phenylephrine (100 μmol/L) for 24 hours, followed by treatments of BT2 (80 μmol/L, dissolved in dimethyl sulfoxide [DMSO]) or 0.1% DMSO as control for an additional 24 hours. Mitochondrial DNA amounts in mouse hearts (A) and NRVMs (B) were analyzed by quantitative polymerase chain reaction (PCR) using primers specific for the mitochondrial cytochrome b (CytB) gene and normalized to genomic DNA by amplification of the large ribosomal protein p0 (36B4) nuclear gene. mRNA expression of genes involved in glucose transportation (Glut1 and Glut4) and oxidation (PDK4) in mouse hearts (C) and NRVMs (D) was determined by quantitative PCR. mRNA expression of genes involved in fatty acid transport (CD36) and β‐oxidation (Acox1, Acadl, and Acadm) in mouse hearts (E) and NRVMs (F) was analyzed by quantitative PCR. For animal studies: n=3 for sham‐vehicle group, n=3 for sham‐BT2 group, n=5 for transverse aortic constriction (TAC)–vehicle group, n=7 for TAC‐BT2 group. For NRVM experiment: n=3 for each group. Two‐way ANOVA, followed by Bonferroni's post hoc test, was performed for multiple comparison. Data were presented as mean±SD. NS indicates not significant. *P<0.05.

Figure 6

BT2 treatment showed no signs of apparent toxicity. Mice were subjected to sham procedure. Two weeks later, animals were randomized into 2 groups to be treated with either BT2 (n=3) or vehicle (n=3). Mean left ventricular ejection fraction (EF; A) and left ventricular internal diameter at systole (LVID;s; B) were obtained from echocardiography. C, Representative M‐mode echocardiographs of mouse hearts after sham procedure, treated with either vehicle or BT2. D, Body weight (BW) from both groups. E, Tissue weight of mice at 8 weeks after sham procedure. F, Expression of total and phosphorylated branched‐chain ketoacid dehydrogenase subunit E1α (BCKDE1α) in mouse heart at 8 weeks after sham procedure was determined by Western blot (left). The relative phosphorylated E1α level was calculated (right). For A through C, statistical analysis was performed using 2‐way ANOVA, followed by Bonferroni's post hoc test. Student's t test was performed for E and F. Data were presented as mean±SEM. NS indicates not significant. *P<0.05.