Dietary methionine restriction in mice elicits an adaptive cardiovascular response to hyperhomocysteinemia

Abstract

Dietary methionine restriction (MR) in rodents increased lifespan despite higher heart-to-body weight ratio (w/w) and hyperhomocysteinemia, which are symptoms associated with increased risk for cardiovascular disease. We investigated this paradoxical effect of MR on cardiac function using young, old, and apolipoprotein E-deficient (ApoE-KO) mice. Indeed, MR animals exhibited higher heart-to-body weight ratio (w/w) and hyperhomocysteinemia with a molecular pattern consistent with cardiac stress while maintaining the integrity of cardiac structure. Baseline cardiac function, which was measured by non-invasive electrocardiography (ECG), showed that young MR mice had prolonged QRS intervals compared with control-fed (CF) mice, whereas old and ApoE-KO mice showed similar results for both groups. Following β-adrenergic challenge, responses of MR mice were either similar or attenuated compared with CF mice. Cardiac contractility, which was measured by isolated heart retrograde perfusion, was similar in both groups of old mice. Finally, the MR diet induced secretion of cardioprotective hormones, adiponectin and fibroblast growth factor 21 (FGF21), in MR mice with concomitant alterations in cardiac metabolic molecular signatures. Our findings demonstrate that MR diet does not alter cardiac function in mice despite the presence of hyperhomocysteinemia because of the adaptive responses of increased adiponectin and FGF21 levels.

Figure 1. Young MR mice exhibit a predisposition to cardiovascular disease.

(A). Body weight of 8-week-old mice under the CF (0.84% methionine) or MR (0.12% methionine) diets for 12 weeks. (B). Food intake of the mice per gram body weight. (C). and (D). Heart weight and heart-to-body weight ratios of the mice upon sacrifice. (E). Fold changes of sulfur amino acids in the plasma of MR mice relative to CF mice based on HPLC measurements. (F). Cardiac gene expression as measured by quantitative real-time PCR using TaqMan primers for Nppa and Nppb. (G). Non-invasive ECGs performed on conscious mice at basal conditions and following daily repeated 100 nM isoproterenol injections. Data were analyzed using a 2-way (A) or 1-way (G) ANOVA with Bonferroni post-tests or Student's unpaired t-tests (B–F) (n = 7–8 per group, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 2. The MR diet predisposed old mice to cardiovascular disease but did not alter cardiac function.

(A). Body weight of the 60-week-old mice under the CF (0.84% methionine) or MR (0.12% methionine) diets for 14 weeks. (B). Food intake of the mice per gram body weight. (C). and (D). Heart weight and heart-to-body weight ratios of the mice upon sacrifice. (E). Plasma homocysteine levels as measured by immunoassay. (F). Cardiac gene expression as measured by quantitative real-time PCR using TaqMan primers for Nppa and Nppb. (G). Non-invasive ECG performed on conscious mice at basal conditions and following daily repeated 100 nM isoproterenol injections. Data were analyzed by 2-way (A) or 1-way (G) ANOVA with Bonferroni post-tests or Student's unpaired t-tests (B–F) (n = 7 per group, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 3. MR in ApoE-KO mice attenuated the effects of β-adrenergic stimulation on cardiac function.

(A). Body weight of 8-week-old ApoE-KO mice given CF (0.84% methionine) or MR (0.12% methionine) diets for 12 weeks. (B). Food intake of the mice per gram body weight. (C). and (D). Heart weight and heart-to-body weight ratios of the mice upon sacrifice. (E). Plasma homocysteine levels as measured by immunoassay. (F). Cardiac gene expression as measured by quantitative real-time PCR using TaqMan primers for Nppa and Nppb. (G). Non-invasive ECG performed on conscious mice at basal conditions and following daily repeated 100 nM isoproterenol injections. Data were analyzed by 2-way (A) or 1-way (G) ANOVA with Bonferroni post-tests or Student's unpaired t-tests (B–F) (n = 7 per group, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 4. Dietary MR increased the adiponectin and FGF21 levels and altered cardiac metabolic signaling.

Plasma adiponectin (A) and FGF21 (B) levels in young, old, and ApoE-KO mice as determined by ELISA. (C). Gene set enrichment analysis of canonical pathways affected in the hearts of the MR mice compared with their CF counterparts (n = 4 per group) based on the Kyoto Encyclopedia of Genes and Genomes database. Upregulated (black bars) and downregulated (clear bars) pathways were altered by ≥1.2-fold and had a false discovery rate of q < 0.01 and significance of P < 0.001. (D). Cardiac gene expression analysis in CF and MR mice using quantitative real-time PCR with TaqMan primers. Data were analyzed by Student's unpaired t-test relative to the control groups in (A), (B), and (D) (n = 7–8 per group, ^aP < 0.05, ^bP < 0.01, ^cP < 0.001).