Extra-cardiac BCAA catabolism lowers blood pressure and protects from heart failure

Abstract

Pharmacologic activation of branched-chain amino acid (BCAA) catabolism is protective in models of heart failure (HF). How protection occurs remains unclear, although a causative block in cardiac BCAA oxidation is widely assumed. Here, we use in vivo isotope infusions to show that cardiac BCAA oxidation in fact increases, rather than decreases, in HF. Moreover, cardiac-specific activation of BCAA oxidation does not protect from HF even though systemic activation does. Lowering plasma and cardiac BCAAs also fails to confer significant protection, suggesting alternative mechanisms of protection. Surprisingly, activation of BCAA catabolism lowers blood pressure (BP), a known cardioprotective mechanism. BP lowering occurred independently of nitric oxide and reflected vascular resistance to adrenergic constriction. Mendelian randomization studies revealed that elevated plasma BCAAs portend higher BP in humans. Together, these data indicate that BCAA oxidation lowers vascular resistance, perhaps in part explaining cardioprotection in HF that is not mediated directly in cardiomyocytes.

Keywords: BCAA; Mendelian randomization; blood pressure; branched-chain amino acid; cardiac metabolism; cardiovascular metabolism; heart failure; hypertension; metabolomics.

Figure 1:. Alterations in the BCAA pathway in failing human myocardium, and increased preference for BCAA oxidation in murine heart failure.

(A) Schematic of BCAA uptake and oxidation pathway, and changes in gene expression or metabolite abundance in failing (HF) vs. non-failing (NF) human myocardium. Asterisks indicate nominal p value by student t-test. (B) Total uptake or release of BCAA carbon by the failing (n = 23) or non-failing (n = 87) human heart. C_CS − C_A = coronary sinus (CS) – artery (A). (C) Contribution of BCAA carbon to myocardial oxygen consumption (mVO₂) (D) Experimental scheme for (E)-(L). (E) Cardiac function measured by echocardiography. LVEF: left ventricular ejection fraction. Sham: n = 4, MI: n = 7. (F) Relative expression of indicated genes in left ventricle (LV) from sham or MI-treated animals. Error bars represent SEM. *p<0.05 by t-test. (G) Western blot and quantification of pBCKDHA and total BCKDHA in LV from sham or MI-treated animals. (H) Relative plasma levels of BCAAs in plasma following a 5-hr fast. (I) Relative tissue levels of BCAAs in LV at the conclusion of infusion. (J) Fractional labeling of plasma BCAAs with ¹³C label during the final 30 minutes of infusion. (K) Total circulatory flux (F_circ) of individual BCAAs (left) and total (right) BCAA carbon. *p<0.05, **p<0.01. (L) Normalized labeling of TCA cycle intermediates in heart and quadriceps muscle at conclusion of infusion.

Figure 2:. Global activation of BCAA catabolism improves cardiac remodeling pre-and post-myocardial infarction without affecting rates of cardiac BCAA oxidation.

(A) Experimental scheme for (B)-(F). (B) Relative abundance of BCAA and BCKA in plasma 4 weeks post-MI in Ctrl or BT2-fed mice. (C) Relative abundance of BCAA in heart 4 weeks post-MI. (D) Left ventricular ejection fraction (LVEF), 4 weeks post-MI. (E) Calculated total BCAA turnover flux (F_circ) of Leu, Ile, Val 4 weeks post-MI. (F) Normalized cardiac ¹³C labeling of TCA cycle intermediates and glutamate. (G) Experimental scheme for (H)-(N). (H) Representative H&E (top) and picrosirius blue (bottom) sections from myocardium 8 weeks post-MI. (I-K) Left ventricular ejection fraction (LVEF), end-diastolic volume (EDV), and end-systolic volume (ESV) assessed up to 8 weeks post-MI. *p<0.05 by repeated-measures ANOVA. (L) Heart weight/tibia length (HW/TL) ratio at 8 weeks post-MI. (M) Relative expression of indicated genes. *p<0.05 by 1-way ANOVA (N) Western blot and quantification of pBCKDHA S293 and total BCKDHa from heart.

Figure 3:. Activation of BCAA oxidation specifically in the heart does not confer cardiac benefit post-MI.

(A) Generation of cardiac-specific BCKDK KO (BCKDK cKO) mice. (B) Relative expression of indicated genes in heart from control vs. BCKDK cKO mice. (C) Immunoblot of BCKDK and BCKDK target pBCKDHA S293. (D) Infusion scheme for (E)-(F). (E) Normalized label of TCA cycle intermediates, glutamate, and aspartate in heart at conclusion of infusion. (F) Relative abundance of BCAA in heart. (G) Experimental scheme for (H)-(I). (H) LV mass/Body Weight ratio at baseline vs. post-isoproterenol. *p<0.05 by 2-way ANOVA. (I) Representative H&E sections from control vs. BCKDK cKO at baseline vs. post-isoproterenol treatment. (J) Representative H&E (left) and picrosirius blue (right) sections from heart from control vs. BCKDK cKO 4 weeks post-MI. (K) Survival post-MI of control vs. BCKDK cKO. (L-M) Left ventricular ejection fraction (LVEF, L), end-diastolic, and end-systolic volume (EDV, ESV, M) from control vs. BCKDK cKO at 1 week and 4 weeks post-MI. (N) Relative expression of indicated genes in LV from control vs. BCKDK cKO at 4 weeks post-MI.

Figure 4:. Activation of BCAA oxidation in skeletal muscle lowers plasma BCAA and confers mild cardiac benefit post-MI

(A) Western blot from quadriceps from control vs. skeletal muscle BCKDK KO (BCKDK iSkM-KO) mice. (B) Infusion schematic. (C) Fractional labeling of TCA intermediates and glutamate in quadriceps muscle. (D-F) Relative abundance of BCAA in quadriceps muscle (D), plasma (E), and heart (F). (G) Fractional labeling of TCA intermediates and glutamate in heart. (H) Experimental scheme for (I)-(N). (I) Representative H&E (top) and picrosirius blue (bottom) sections from heart post-MI. (J) Survival post-MI analyzed by Gehan-Breslow-Wilcoxon test. (K-L) Left ventricular ejection fraction (LVEF), left ventricular end-diastolic volume (EDV) at 2 and 4 weeks post-MI. (M) Heart weight/body weight (HW/BW) at 4 weeks post-MI (N) Relative expression of indicated genes in LV at baseline vs. post-MI. Hearts from infusion experiment were used for baseline comparison. * p<0.05 by t-test

Figure 5:. BT2 requires BCKDK to confer cardioprotection

(A) Experimental scheme. (B) Western blot and quantification of indicated proteins from hearts at 4 weeks post-MI (C) Baseline (pre-MI) left ventricular fractional shortening (%FS) assessed by echo (D) Relative total ion counts (TIC) of BCAA and BCKA in plasma, 4 weeks post-MI (E) Probability of survival following MI (F) Representative H&E (top) and Picrosirius Blue stains from heart sections, 4 weeks post-MI (G-I) Left ventricular EF (LVEF), end-diastolic (LVEDV), and end-systolic (LVESV) volume assessed at 4 weeks post-MI (J-K) Heart weight to body weight (HW/BW) and to tibial length (HW/TL) post-MI

Figure 6:. Global activation of BCAA catabolism promotes vasodilation and lowers blood pressure.

(A) Experimental scheme for (B). n=12/group. (B) Systolic, diastolic, and mean BP averages measured via aortic catheter; 10 minutes recording/mouse. ** p< 0.01, ***<0.001, ****<0.0001 by t-test. (C) Experimental scheme for (D). L-NAME: N^ω-nitro-L-arginine methyl ester (0.5 g/L in drinking water). (D) Systolic, diastolic, and mean BP averages via aortic catheter; 10 minutes recording/mouse. **p<0.01 by 2-way ANOVA. (E) Experimental scheme for (F). (F) BP recording from control vs. BT2 diet and L-NAME+control diet vs. L-NAME + BT2 diet conditions. Each dot represents average data of 1 mouse from indicated treatment period. Data are from dark cycle (8:00 PM – 6:00 AM). *p <0.05, ***p<0.001 by paired t-test. (G) Diameter of carotid artery from mice treated with 7 days control diet + DMSO in buffer (n = 6) or 7 days BT2 diet + BT2 in buffer (n = 6). Following pressure pre-condition, phenylephrine (10⁻⁵ M) was added, followed by indicated amounts of acetylcholine. * p<0.05 by repeated measures ANOVA. (H-I) Response to phenylephrine (H) and acetylcholine (I) in DMSO vs. BT2 condition. *p<0.05, 2-way ANOVA

Figure 7:. Mendelian Randomization studies support a causal relationship between BCAAs and blood pressure in human cohorts

(A) In inverse variance-weighted Mendelian randomization (MR) analyses, elevations in leucine predicted higher systolic, mean, and pulse pressure; elevations in isoleucine predicted higher systolic and pulse pressure. (B) Secondary MR restricted to SNPs within 500kb of one of 14 genes unique to BCAA catabolic pathway.