L-carnitine attenuated hyperuricemia-associated left ventricular remodeling through ameliorating cardiomyocytic lipid deposition

Abstract

Hyperuricemia (HUA) is associated with left ventricular remodeling (LVR) and thereby causes the initiation and development of a large number of cardiovascular diseases. LVR is typically accompanied by cardiomyocyte energy metabolic disorder. The energy supply of cardiomyocytes is provided by glucose and fatty acid (FA) metabolism. Currently, the effect of HUA on cardiomyocytic FA metabolism is unclear. In this study, we demonstrate that UA-induced cardiomyocyte injury is associated with cytoplasmic lipid deposition, which can be ameliorated by the FA metabolism-promoting drug L-carnitine (LC). UA suppresses carnitine palmitoyl transferase 1B (CPT1B), thereby inhibiting FA transport into the mitochondrial inner matrix for elimination. LC intervention can ameliorate HUA-associated left ventricular anterior wall thickening in mice. This study showed that FA transport dysfunction plays is a critical mechanism in both cardiomyocytic injury and HUA-associated LVR and promoting cytoplasmic FA transportation through pharmacological treatment by LC is a valid strategy to attenuate HUA-associated LVR.

Keywords: L-carnitine; cardiomyocytes; fatty acid; hyperuricemia; ventricular remodeling.

FIGURE 1

FA metabolic agents intervention affected UA induced cardiomyocyte injury. (A) The time-course of cell viability under different concentrations of UA exposure. The results were normalized to the mean value of control (n = 4); (B) Oil red O staining showing cardiomyocytic lipid deposition under different concentrations of UA exposure for 24 h. Dark red indicates deposited lipid, Blue indicated nuclei; (C) The influence of LC intervention on cell viability of cardiomyocytes under UA stimulation. UA concentration is 15 mg/dL. The cells were examined after 48 h treatment. The results were normalized to the mean value of control (n = 4); (D) The influence of TMZ intervention on cell viability of cardiomyocytes under UA stimulation. UA concentration is 15 mg/dL. The cells were examined after 48 h treatment. The results were normalized to the mean value of control (n = 4); (E) Oil red O staining showing UA induced cardiomyocytic lipid deposition under LC or TMZ intervention. UA concentration is 15 mg/dL, LC concentration is 100 μM, TMZ concentration is 1 μM. The cells were fixed for examination after 24 h treatment. Dark red indicates deposited lipid, Blue indicates nuclei.

FIGURE 2

FA metabolic genes response under UA stimulation. (A) Diagram of steps and participated genes in fatty acid metabolism; (B) Heatmap reflect the expression fold change of fatty acid metabolic genes in each step. The expression data were generated from RNAseq; (C) Impact of UA on the gene expression fold change of FABP3, CPT1A and CPT1B. The cells were harvested after 24 h treatment followed by qPCR. The results were normalized to the mean value of control (n = 5); (D) Impact of UA on the protein level of FABP3, CPT1A and CPT1B. The cells were harvested after 1 h treatment followed by western blot analysis. β-actin was used as an internal loading control; (E) The quantification of Figure 2D.

FIGURE 3

Lipidomic profile change of cardiomyocytes under UA stimulation. (A) PLS-DA score scatter plot showing the separation of control group and UA group. Cardiomyocytes were treated with 15 mg/dL UA. The cells were harvested for lipidomic analysis after 24 h treatment; (B) Heatmap showing the global view of the significant metabolites of control and UA treated cardiomyocytes; (C) Pie chart showing the changes in lipid composition under UA treated cardiomyocytes compare with control. P< 0.05 was considered as significance; (D) Bubble plots showing the top 20 significant metabolites based on VIP values; (E) Box plots showing the top five upregulated TGs and DGs. The results were normalized to the mean value of control (n = 5).

FIGURE 4

Lipidomic profile change of hearts under HUA induction. (A) PLS-DA score scatter plot showing the separation of blank group and HUA group; (B) Heatmap showing the global view of the significant metabolites of blank and HUA group; (C) Pie chart showing the changes in lipid composition of HUA compare with blank. P< 0.05 was considered as significance; (D) Bubble plots showing the top 20 significant metabolites based on VIP values; (E) Box plots showing the top five upregulated TGs and DGs. The results were normalized to the mean value of control (n = 8).

FIGURE 5

CPT1B response in early-stage HUA mice. (A) The UA concentration in plasma of blank and HUA group respectively (n = 6); (B) The protein level of CPT1B in the heart samples from blank and HUA group respectively, β-actin was used as an internal loading control; (C) The quantification of Figure 5B; (D) The mRNA folds change of CPT1B in the heart samples from blank and HUA group (n = 6); (E) The immunofluorescence reflects the distribution of CPT1B in the heart samples from blank and HUA group respectively. DAPI indicates nuclei, scale bar = 1000 μm; (F) The protein level of CPT1B in heart samples by ELISA quantification (n = 6); (G) The plasma LC concentrations by different treatment (n = 6); (H) The cardiac plasma LC concentrations by different treatment (n = 6).

FIGURE 6

The influence of fatty acid metabolic agents’ intervention on the cardiac function of chronic HUA mice. (A) Representative photo of echocardiography examination in each group. Echocardiographic examination was performed after 8 weeks experiment. The summarized box plot of left ventricular anterior wall in diastolic-stage (B), left ventricular anterior wall in systolic-stage (C), left ventricular posterior wall in diastolic-stage (D), left ventricular posterior wall in systolic-stage (E), left ventricular internal dimension in diastolic-stage (F), left ventricular internal dimension in systolic-stage (G), left ventricular end-diastolic volume (H), left ventricular end-systolic volume (I), ejection fraction (J), fractional shortening (K), stroke volume (L). blank n = 5, HUA n = 4, LC n = 5, TMZ n = 5, AP n = 6; (M) Masson staining of the heart samples in each group after8-weeks’ treatment. Red indicates muscle, blue indicates fibrous, scale bar = 1000 μm.