Activation of cardiac AMPK-FGF21 feed-forward loop in acute myocardial infarction: Role of adrenergic overdrive and lipolysis byproducts

Abstract

Fibroblast growth factor 21 (FGF21) is a metabolic hormone having anti-oxidative and anti-hypertrophic effects. However, the regulation of FGF21 expression during acute myocardial infarction (AMI) remains unclear. We tested blood samples from 50 patients with AMI and 43 patients with stable angina pectoris (sAP) for FGF21, fatty acid binding protein 4 (FABP4), a protein secreted from adipocytes in response to adrenergic lipolytic signal, and total and individual fatty acids. Compared with sAP patients, AMI patients had higher serum FGF21 levels on admission, which were significantly correlated with peak FABP4 and saturated fatty acids (SFAs) but not with peak levels of cardiac troponin T. In mice, myocardial ischemia rapidly induced FGF21 production by the heart, which accompanied activation of AMP-activated protein kinase (AMPK)-dependent pathway. Like AICAR, an activator of AMPK, catecholamines (norepinephrine and isoproterenol) and SFAs (palmitate and stearate) significantly increased FGF21 production and release by cardiac myocytes via AMPK activation. Recombinant FGF21 induced its own expression as well as members of down-stream targets of AMPK involved in metabolic homeostasis and mitochondrial biogenesis in cardiac myocytes. These findings suggest that adrenergic overdrive and resultant adipose tissue lipolysis induce cardiac AMPK-FGF21 feed-forward loop that potentially provides cardioprotection against ischemic damage.

Figure 1

Serum concentrations of FGF21, FABP4 and FFA in patients with stable AP and AMI. (A) Serum concentrations of FGF21, FABP4 and FFA in the stable AP patients (n = 43) and AMI patients (n = 50) on admission. Results are represented as the means ± SEM. ***p < 0.001, as measured by Mann-Whitney U-test, or student t-test. (B) Time course of serum FGF21, FABP4, FFA and cTnT and FFA in patients with AMI Data are presented as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 compared with samples on admission (adm). PCI: percutaneous coronary intervention.

Figure 2

Serum FGF21 and cardiac FGF21 expression in myocardial ischemia in mice. (A) Results of ELISA assay of serum and heart lysate at 1 h (n = 6) and 24 h (n = 6) after myocardial ischemia or sham-operated mice (n = 6). (B) Representative western blots are shown on the left. Protein levels were normalized by GAPDH, and phosphorylation of proteins were normalized by total proteins in the same samples. Full-length blots are presented in Supplementary Fig. S1. Data are presented as mean ± SEM from 2 different sets of experiments on the right. Expression level of sham is expressed as 1.0. *p < 0.05, **p < 0.01, as measured by Tukey-Kramer test (A) or Steel-Dwass test (B).

Figure 3

Effects of 5-aminoimidazole-4-carboxyamide ribofuranoside (AICAR) on FGF21 production in cultured neonatal rat cardiac myocytes. (A) FGF21 protein levels in cultured medium and cell lysates were measured by ELISA after stimulation of cultured neonatal rat cardiac myocytes with AICAR (1 mM) in the presence or absence of compound C (10 μM). (B) Representative Western blots are shown on the left. Full-length blots are presented in Supplementary Fig. S2. (C) Cultured neonatal rat cardiac myocytes were transfected with the luciferase reporter plasmid pGL3-mFgf21pro1-luc, which contains 1.5 kb of murine FGF21 promoter region in front of the luciferase gene or control plasmid (thymidine kinase-luc (TK-luciferase)). Cells were then treated with either vehicle or 1 mM AICAR for 24 h before harvest. Data are presented as mean ± SEM from 3 different sets of experiments. Expression level of control is expressed as 1.0. *p < 0.05, **p < 0.01, as measured by Tukey-Kramer test (A), Steel-Dwass test (B) or unpaired Student’s t-test (C).

Figure 4

Induction of FGF21 production by catecholamines or SFAs in cultured neonatal rat cardiac myocytes. (A–D) Cultured neonatal rat cardiac myocytes were stimulated with a pan-β-adrenergic receptor agonist isoproterenol (ISO) (10 μM), norepinephrine (NE) (10 μM) or fatty acid-free bovine serum albumin (BSA)-conjugated palmitic acid (PA), stearic acid (SA), and oleic acid (OA) (250 μM) in the presence or absence of compound C (10 μM) for 24 h. (A,B) FGF21 concentrations in culture medium or cell lysates were measured by ELISA. Data are resented as the means ± SEM from 3 different sets of experiments. (C,D) Representative Western blots are shown on the left. Full-length blots are presented in Supplementary Figs S3 and S4. (E) Cultured neonatal rat cardiac myocytes were transfected with pGL3-mFgf21pro1-luc or TK-luc. Cells were then treated with either vehicle or ISO (10 μM), NE (10 μM), PA (250 μM) or SA (250 μM) before being harvested. Data are presented as mean ± SEM from 3 different sets of experiments on the right. Expression level of control is expressed as 1.0. *p < 0.05, **p < 0.01 compared with vehicle control (Con) or BSA, as measured by Tukey-Kramer test (A,B), Steel-Dwass test (C,D) or unpaired Student’s t-test (E).

Figure 5

Activation of AMPK by recombinant FGF21 in the cardiac myocytes. (A) Representative Western blots in neonatal rat cardiac myocytes exposed to recombinant FGF21 (10 ng/mL) for 24 h are shown on the left. Full-length blots are presented in Supplementary Fig. S5. Data are presented as the means ± SEM from 2 different sets of experiments. Protein levels were normalized by GAPDH, and phosphorylation of proteins were normalized by total proteins in the same samples. Expression level of control is expressed as 1.0. *p < 0.05, **p < 0.01, as measured by Steel-Dwass test. (B) siRNA-mediated silencing of the expression of the AMPKα1 or FGF21 genes in neonatal rat cardiac myocytes and representative Western blots. Full-length blots are presented in Supplementary Fig. S6. (C) Proposed scheme for the feed-forward regulation of AMPK-FGF21 pathways in cardiac myocytes in response to acute ischemia. Upon onset of myocardial ischemia, circulating norepinephrine and SFAs concentrations rise following adrenergic hyper-activation and adipose tissue lipolysis. Elevated levels of norepinephrine and SFAs induce AMPK phosphorylation, which in turn activates down-stream targets of AMPK, such as SIRT1, PGC1α, ERK, p38 MAPK and FGF21. FGF21 then activates AMPK in the cardiac myocytes in an autocrine manner, and may contribute to mitochondrial homeostasis and cardioprotection.