Age-Independent Cardiac Protection by Pharmacological Activation of Beclin-1 During Endotoxemia and Its Association With Energy Metabolic Reprograming in Myocardium-A Targeted Metabolomics Study

Abstract

Background We showed that Beclin-1-dependent autophagy protects the heart in young and adult mice that underwent endotoxemia. Herein, we compared the potential therapeutic effects of Beclin-1 activating peptide, TB-peptide, on endotoxemia-induced cardiac outcomes in young adult and aged mice. We further evaluated lipopolysaccharide (lipopolysaccharide)-induced and TB-peptide treatment-mediated alterations in myocardial metabolism. Methods and Results C57BL/6J mice that were 10 weeks and 24 months old were challenged by lipopolysaccharide using doses at which cardiac dysfunction occurred. Following the treatment of TB-peptide or control vehicle, heart contractility, circulating cytokines, and myocardial autophagy were evaluated. We detected that TB-peptide boosted autophagy, attenuated cytokines, and improved cardiac performance in both young and aged mice during endotoxemia. A targeted metabolomics assay was designed to detect a pool of 361 known metabolites, of which 156 were detected in at least 1 of the heart tissue samples. Lipopolysaccharide-induced impairments were found in glucose and amino acid metabolisms in mice of all ages, and TB-peptide ameliorated these alterations. However, lipid metabolites were upregulated in the young group but moderately downregulated in the aged by lipopolysaccharide, suggesting an age-dependent response. TB-peptide mitigated lipopolysaccharide-mediated trend of lipids in the young mice but had little effect on the aged. (Study registration: Project DOI: https://doi.org/10.21228/M8K11W). Conclusions Pharmacological activation of Beclin-1 by TB-peptide is cardiac protective in both young and aged population during endotoxemia, suggest a therapeutic potential for sepsis-induced cardiomyopathy. Metabolomics analysis suggests that an age-independent protection by TB-peptide is associated with reprograming of energy production via glucose and amino acid metabolisms.

Keywords: Beclin‐1; autophagy; cardiac function; cardiac metabolism; endotoxemia; sepsis.

Figure 1. Cardiac protective effects of TB‐peptide in young adult and aged mice during endotoxemia.

Mice were given 5 mg kg/lipopolysaccharide intraperitoneally and TB‐peptide, 16 mg/kg, was administered intraperitoneally 30 minutes post lipopolysaccharide challenge. Experiments were performed 18 hours post challenge. Cardiac function was evaluated by echocardiography in the young adult (A, 5/group) and aged (B, 6/group) mice. Circulating cytokine levels were measured in blood serum prepared from the young adult (C, 5/group) and aged (D, 5/group) groups by ELISA. In harvested heart tissue, autophagy marker LC3II was detected by Western blot using the total tissue lysates, and signals were quantified by densitometry (E, 5/group). Levels of lactate were quantified in the heart tissue lysates (F, 5/group). All data were expressed as mean±SEM of at least 3 independent experiments. Data were analyzed by 2‐way ANOVA with post hoc test for comparisons of multiple groups and Student t test for comparisons between 2 groups using GraphPad Prism software. Differences were considered statistically significant as P≤0.05. Significant differences are shown as * for sham vs lipopolysaccharide and ** for with vs without the treatment of TB‐peptide (A through E) or for difference between age groups (F). IFN indicates interferon; IL, interleukin; LPS, lipopolysaccharide; and TNFα, tumor necrosis factor alpha.

Figure 2. Analysis of lipopolysaccharide‐induced changes in myocardial metabolites and the effects of TB‐peptide in young mice.

Mice were given 5 mg/kg lipopolysaccharide intraperitoneally and heart tissues were harvested 18 hours later. A, Volcano plots generated using GraphPad Prism software showing metabolite‐wise fold changes (log2 FC) plotted against false discovery rate (FDR, −log10 FDR). Significantly differentially abundant metabolites were indicated in red for upregulation and blue for downregulation (FDR ≤10%, FDR was determined using Benjamin‐Hochberg procedure). B, Comparison of lipopolysaccharide‐induced changes between groups with and without the treatment of TB‐peptide. Degrees of fold change in abundance were shown in bar graphs. Results obtained from sham and lipopolysaccharide‐challenged groups without the treatment of TB‐peptide were shown in blue, and those from groups given treatment were shown in red. C, Metabolic pathways altered by lipopolysaccharide in the young hearts. D, TB‐peptide‐mediated regulation of metabolic pathways in the young hearts challenged by lipopolysaccharide. In (C and D) pathway analysis was performed by using Shiny GAM (integrated analysis of genes and metabolites) and Cytoscape software. Signaling networks were built on pathway clustering against the small molecule pathway database using MBRole 2.0 software. FC, fold change; FDR, false discovery rate; LPS, lipopolysaccharide; NAD, nicotinamide adenine dinucleotide; and UDP, Uridine diphosphate.

Figure 3. Analysis of lipopolysaccharide‐induced changes in myocardial metabolites and the effects of TB‐peptide in aged mice.

Mice were given 5 mg/kg lipopolysaccharide intraperitoneally and heart tissues were harvested 18 hours later. A, Volcano plots generated using GraphPad Prism software showing metabolite‐wise fold changes (FC; log2 FC) plotted against false discovery rate (FDR; −log10 FDR). Significantly differentially abundant metabolites were indicated in red for upregulation and blue for downregulation (FDR ≤10%, FDR was determined using Benjamin‐Hochberg procedure). B, Comparison of lipopolysaccharide‐induced changes between groups with and without the treatment of TB‐peptide. Degrees of fold change in abundance were shown in bar graphs. Results obtained from sham and lipopolysaccharide‐challenged groups without the treatment of TB‐peptide were shown in blue, and those from groups given treatment were shown in red. C, Metabolic pathways altered by lipopolysaccharide in the aged hearts. D, TB‐peptide‐mediated regulation of metabolic pathways in the aged hearts challenged by lipopolysaccharide. In (C and D) pathway analysis was performed by using Shiny GAM (integrated analysis of genes and metabolites) and Cytoscape software. Signaling networks were built on pathway clustering against the small molecule pathway database using MBRole 2.0 software. LPS indicates lipopolysaccharide.

Figure 4. Comparison of lipopolysaccharide‐ and TB‐peptide‐associated changes in myocardial metabolites between young and aged mice.

Heatmaps and related clustering analysis of metabolites identified in the hearts of young and aged mice given lipopolysaccharide challenge or sham, with or without the treatment of TB‐peptide, were compared. FC indicates fold change; and LPS, lipopolysaccharide.