Branched-chain amino acids deficiency promotes diabetic cardiomyopathy by activating autophagy of cardiac fibroblasts

Abstract

Rationale: More than half of the patients with type II diabetes mellitus (T2D) develop diabetic cardiomyopathy (DCM). Glycemic control alone cannot effectively prevent or alleviate DCM. Methods: Herein, we concentrated on the variations in levels of metabolites between DCM and T2D patients without cardiomyopathy phenotype. In high-fat diet/low-dose streptozotocin-induced T2D and leptin receptor-deficient diabetic mouse models, we investigated the effect of altering branched-chain amino acids (BCAAs) levels on DCM. Results: We discovered that the levels of plasma BCAAs are notably lower in 15 DCM patients compared to 19 T2D patients who do not exhibit cardiomyopathy phenotype, using nuclear magnetic resonance analysis. This finding was further validated in two additional batches of samples, 123 DCM patients and 129 T2D patients based on the BCAA assay kit, and 30 DCM patients and 30 T2D patients based on the LC-MS/MS method, respectively. Moreover, it is verified that BCAA deficiency aggravated, whereas BCAA supplementation alleviated cardiomyopathy phenotypes in diabetic mice. Furthermore, BCAA deficiency promoted cardiac fibroblast activation by stimulating autophagy in DCM mice. Mechanistically, BCAA deficiency activated autophagy via the AMPK-ULK1 signaling pathway in cardiac fibroblasts. Using pharmacological approaches, we validated our findings that autophagy inhibition relieved, whereas autophagy activation aggravated, DCM phenotypes. Conclusions: Taken together, we describe a novel perspective wherein BCAA supplementation may serve as a potential therapeutic agent to mitigate DCM and fibrosis. Our findings provide insights for the development of preventive measures for DCM.

Keywords: Autophagy; Branched-chain amino acid; Cardiac fibroblasts; Cardiac fibrosis; Diabetic cardiomyopathy.

Figure 1

Decreased plasma BCAA levels are associated with diabetic cardiomyopathy. A, Relative plasma concentrations of metabolites in T2D controls and patients with DCM measured by NMR. B, Plasma concentrations of BCAAs in healthy controls, T2D, and DCM measured using a BCAA assay kit. C-E, Plasma concentrations of valine, leuvine and isoleucine in healthy controls, T2D, and DCM measured using LC-MS/MS. Data are expressed as mean±SEM. The nonparametric two-tailed Student's t-test was used to compare groups. Significance is indicated as ^nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. See also Figure S1.

Figure 2

BCAA deficiency time-dependently impairs cardiac function in STZ/HFD and db/db T2D mice. A, Schematic representation of the establishment of the two T2D models. B,C, M-mode echocardiogram images of STZ/HFD T2D mice (B) and db/db T2D mice (C) at four-week intervals after BCAA-deficient chow feeding (n=6 mice in each group). D,E, Echocardiography analysis illustrating the worsened heart function in STZ/HFD T2D mice (D) and db/db T2D mice (E) after BCAA-deficient chow feeding (n=6 mice in each group). F,G, Ratios of heart weight to body weight (F) and heart weight to tibia length (G) in indicated groups after 16 weeks of BCAA-deficient chow feeding (n=6 mice in each group). H, Hematoxylin and eosin, Masson's trichome, and Sirius Red stainings of heart tissues in indicated groups after 16 weeks of BCAA-deficient chow feeding. The image quantification is shown on the right (n = 5 mice in each group). I, Increased levels of Collagen I and III as detected via immunostaining of heart tissues from STZ/HFD and db/db T2D mice after 16 weeks of BCAA-deficient chow feeding. The image quantification is shown on the right (n = 5 mice per group). Data are expressed as mean±SEM. The nonparametric two-tailed Student's t-test was used to compare groups. Significance is indicated as ^nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. See also Figures S2 and S3.

Figure 3

BCAA supplementation in diabetes prevents the occurrence of cardiomyopathy. A, Schematic representation of the establishment of mice models. B, M-mode echocardiogram images of indicated groups after 16 weeks of high-BCAA chow feeding (n = 6 mice in each group). C,D, Echocardiography analysis showing that high-BCAA chow feeding rescues cardiac function in STZ/HFD (C) and db/db (D) T2D mice (n = 6 mice in each group). E,F, Ratios of heart weight to body weight (E) and heart weight to tibia length (F) in indicated groups after 16 weeks of high-BCAA chow feeding (n=6 mice in each group). G,H, Hematoxylin and eosin, Masson's trichome, and Sirius Red stainings of heart tissues in indicated groups after 16 weeks of high-BCAA chow feeding. The image quantification is shown on the right (n = 5 mice in each group). I,J, Decreased levels of Collagen I and III as detected via immunostaining of heart tissues from STZ/HFD (I) and db/db (J) T2D mice after 16 weeks of high-BCAA chow feeding. The image quantification is shown on the right (n = 5 mice per group). K, Decreased levels of Collagen I and III in heart tissues from T2D mice fed with high-BCAA chow, as detected via western blotting (n = 5 mice in each group). Data are expressed as mean±SEM. The nonparametric two-tailed Student's t-test was used to compare groups. Significance is indicated as ^nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. See also Figure S4.

Figure 4

BCAA deficiency promotes cardiac fibroblast activation by stimulating autophagy. A, Autolysosomes detected by transmission electron microscopy in heart tissues of DCM mice (n = 6 mice in each group). B,C, Expression of the autophagy marker LC3-II in heart tissue samples from STZ/HFD (B) and db/db (C) T2D mice, detected via western blotting (n = 6 mice in each group). D, Strong colocalization of the autophagy marker Beclin 1 and POSTN proteins in heart tissues of DCM mice as detected by immunostaining. The image quantification is shown on the right (n = 3 mice in each group). E, Western blot analysis of AMPKα, p-AMPKα (T172), ULK1, p-ULK1 (S757 and S317), and LC3-II in primary cardiac fibroblasts and MEFs treated with BCAA deficiency (n = 3 in each group). F, EdU assay to detect the proliferation of primary cardiac fibroblasts and MEF treated with BCAA deficiency. The image quantification is shown on the right (n = 6 in each group). G, Western blot analysis of α-SMA expression in primary cardiac fibroblasts and MEF treated with BCAA deficiency (n = 3 in each group). H, EdU assay to detect the proliferation of primary cardiac fibroblasts and MEF treated with BCAA deficiency and chloroquine. The image quantification is shown on the right (n = 6 in each group). I, Western blot analysis of α-SMA expression in primary cardiac fibroblasts and MEF treated with BCAA deficiency and chloroquine (n = 3 in each group). Data are expressed as mean±SEM. The nonparametric two-tailed Student's t-test was used to compare groups. Significance is indicated as ^nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 5

Autophagy inhibition relieves, whereas autophagy activation aggravates, diabetic cardiomyopathy phenotypes. A,B, Echocardiography analysis illustrating the worsened heart function in STZ/HFD (A) and db/db (B) T2D mice after gavage of rapamycin (n = 6 mice in each group). C, Hematoxylin and eosin, Masson's trichome, and Sirius Red staining of heart tissues in T2D mice after gavage of rapamycin. The image quantification is shown on the below (n = 5 mice in each group). D, Western blot analysis of Collagen I expression in T2D mice after gavage of rapamycin (n = 3 in each group). E,F, Echocardiography analysis illustrating the restored heart function in STZ/HFD (E) and db/db (F) T2D mice after gavage of 3-methyladenine (n = 6 mice in each group). G, Hematoxylin and eosin, Masson's trichome, and Sirius Red stainings of heart tissues in T2D mice after gavage of 3-methyladenine. The image quantification is shown on the below (n = 5 mice in each group). H, Western blot analysis of Collagen I expression in T2D mice after gavage of 3-methyladenine (n = 3 in each group). I,J, Echocardiography analysis illustrating the restored heart function in STZ/HFD (I) and db/db (J) BCAA-deficient mice after gavage of 3-methyladenine (n = 6 mice in each group). K, Hematoxylin and eosin, Masson's trichome, and Sirius Red stainings of heart tissues in BCAA-deficient mice after gavage of 3-methyladenine. The image quantification is shown on the below (n = 6 mice in each group). L, Western blot analysis of Collagen I expression in BCAA-deficient mice after gavage of 3-methyladenine (n = 3 in each group). M, Schematic showing that autophagy inhibition relieves, whereas autophagy activation aggravates, DCM phenotypes. Data are expressed as mean±SEM. The nonparametric two-tailed Student's t-test was used to compare groups. Significance is indicated as ^nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.