Fibronectin type III domain containing 5 expression in skeletal muscle in chronic heart failure-relevance of inflammatory cytokines

Abstract

Background: Chronic heart failure (CHF) is commonly associated with muscle atrophy and increased inflammation. Irisin, a myokine proteolytically processed by the fibronectin type III domain containing 5 (FNDC5) gene and suggested to be Peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α activated, modulates the browning of adipocytes and is related to muscle mass. Therefore, we investigated whether skeletal muscle FNDC5 expression in CHF was reduced and if this was mediated by inflammatory cytokines and/or angiotensin II (Ang-II).

Methods: Skeletal muscle FNDC5 mRNA/protein and PGC-1α mRNA expression (arbitrary units) were analysed in: (i) rats with ischemic cardiomyopathy; (ii) mice injected with tumour necrosis factor-α (TNF-α) (24 h); (iii) mice infused with Ang-II (4 weeks); and (iv) C2C12 myotubes exposed to recombinant cytokines or Ang-II. Circulating TNF-α, Ang-II, and irisin was measured by ELISA.

Results: Ischemic cardiomyopathy reduced significantly FNDC5 protein (1.3 ± 0.2 vs. 0.5 ± 0.1) and PGC-1α mRNA expression (8.2 ± 1.5 vs. 4.7 ± 0.7). In vivo TNF-α and Ang-II reduced FNDC5 protein expression by 28% and 45%, respectively. Incubation of myotubes with TNF-α, interleukin-1ß, or TNF-α/interleukin-1ß reduced FNDC5 protein expression by 47%, 37%, or 57%, respectively, whereas Ang-II had no effect. PGC-1α was linearly correlated to FNDC5 in all conditions. In CHF, animals circulating TNF-α and Ang-II were significantly increased, whereas irisin was significantly reduced. A negative correlation between circulating TNF-α and irisin was evident.

Conclusion: A reduced expression of skeletal muscle FNDC5 in ischemic cardiomyopathy is likely modulated by inflammatory cytokines and/or Ang-II via the down-regulation of PGC-1α. This may act as a protective mechanism either by slowing the browning of adipocytes and preserving energy homeostasis or by regulating muscle atrophy.

Keywords: Angiotensin II; Chronics heart failure; Cytokines; FNDC5; Irisin; Skeletal muscle.

Figure 1

Quantitative evaluation of (A, B) fibronectin type III domain containing 5 (FNDC5) and (C) PGC-1α in the skeletal muscle of sham-operated control animals (con) and animals 7 weeks after left anterior descending artery (LAD) ligation (LAD). A linear correlation between PGC-1α and FNDC5 mRNA expression was evident. (B) A representative western blot is shown on top of the figure. (D) Results are presented as mean ± SEM.

Figure 2

Quantitative evaluation of (A, B) fibronectin type III domain containing 5 (FNDC5) and (C) PGC-1α in the skeletal muscle of NaCl-infused control animals (con) and animals receiving angiotensin II (Ang-II) infusion via osmotic mini-pumps for 4 weeks (Ang-II). A linear correlation between PGC-1α and FNDC5 mRNA expression was evident. (B) A representative western blot is shown on top of the figure. (D) Results are presented as mean ± SEM.

Figure 3

Quantitative evaluation of (A, B) fibronectin type III domain containing 5 (FNDC5) and (C) PGC-1α in the skeletal muscle of NaCl-injected control animals (con) and animals injected tumour necrosis factor-α (TNF-α) intraperitoneally for 24 h (TNF-α). A linear correlation between PGC-1α and FNDC5 mRNA expression was evident. (B) A representative western blot is shown on top of the figure. (D) Results are presented as mean ± SEM.

Figure 4

Quantitative evaluation of (A) fibronectin type III domain containing 5 (FNDC5) and (B) PGC-1α in C2C12 myotubes incubated either with angiotensin II (Ang-II), tumour necrosis factor-α (TNF-α), interleukin (IL)-1ß, γ-interferon (IFN-γ), or with a combination of TNF-α/IL-1ß for 24 h. A linear correlation between PGC-1α and FNDC5 mRNA expression was evident. (C) Results are presented as mean ± SEM.

Figure 5

Impact of inhibiting different signal pathways on cytokine-induced fibronectin type III domain containing 5 (FNDC5) expression. C2C12 myotubes were incubated for 2 h with a specific inhibitor for (A) Akt, (B) protein kinase C (PKC), (C) signal transducer and activator of transcription 3 (STAT-3), (D) c-Jun N-terminal kinase (JNK), (E) p42/44 mitogen-activated protein kinase (MAPK), or (F) p38 MAPK before inflammatory cytokines [tumour necrosis factor-α (TNF-α), interleukin (IL)-1ß, or a combination of both] were added to the cells. Expression of FNDC5 mRNA was evaluated by reverse transcription-PCR 24 h later. C2C12 cells only pre-incubated with the inhibitor but without the addition of cytokines served as controls (con). The values were normalized to controls, and results are presented as mean ± SEM. *P < 0.05 vs. con. ERK, extracellular signal-regulated kinase.

Figure 6

Hypothetical working model on how the development of chronic heart failure influences skeletal mass and energy wasting via the involvement of fibronectin type III domain containing 5 (FNDC5). CHF, chronic heart failure; TNF-α, tumour necrosis factor-α; IL, interleukin; Ang-II, angiotensin II.