Sarcopenia associated with portosystemic shunting is reversed by follistatin

Abstract

Background & aims: The distinct role of portosystemic shunting (PSS) in the pathogenesis of sarcopenia (skeletal muscle loss) that occurs commonly in cirrhosis is unclear. We have previously shown increased expression of myostatin (inhibitor of skeletal muscle mass) in the portacaval anastamosis (PCA) rat model of sarcopenia of PSS. The present study was performed to examine the mechanisms of sarcopenia following PCA.

Methods: In PCA and sham operated pair fed control rats, the phenylalanine flooding dose method was used to quantify the fractional and absolute protein synthesis rates in the skeletal muscle over time and in response to follistatin, a myostatin antagonist. The expression of myostatin and markers of satellite cell (myocyte precursors) proliferation and differentiation were quantified by real-time PCR and Western blot analyses.

Results: The absolute synthesis rate (ASR) was lower at 2, 4, and 6 weeks (p<0.05) and the fractional synthesis rate (FSR) of skeletal muscle protein was significantly lower (p<0.05) at week 2 in the PCA rats compared to control rats. Expression of myostatin was elevated while markers of satellite cell proliferation and differentiation were lower at 4 and 6 weeks after PCA. Follistatin increased skeletal muscle mass, muscle FSR and ASR, decreased expression of myostatin protein, and increased expression of markers of satellite cell function.

Conclusions: Sarcopenia associated with PSS is caused by impaired protein synthesis and reduced satellite cell function due to increased myostatin expression. Confirming these alterations in human patients with cirrhosis will provide novel therapeutic targets for sarcopenia of liver disease.

Fig. 1. Histograms (A–C) showing relative expression of mRNA by real-time PCR of genes regulating skeletal muscle protein synthesis, expressed as fold change in PCA compared to control rats

Bars extending above the baseline show an increased expression and bars extending below the baseline show a lower expression in PCA compared with sham operated control rats. (A) Expression of myostatin, its receptor activin 2br, and intracellular signaling protein, p21 was significantly elevated (p <0.01) while that of insulin like growth factor 1 was lower in the PCA compared to that in the control rats at weeks 4 and 6 (p <0.01). (B) Relative expression of mRNA by real-time PCR of the markers of satellite cell function – the proliferating cell nuclear antigen (satellite cell proliferation) and myoD, myf 5, and myogenin (satellite cell differentiation), was significantly lower in PCA compared to control rats at weeks 4 and 6 (p <0.01). (C) The expression of proteasome C3, C5, C9, and atrogin was significantly higher at week 2 and subsequently at weeks 4 and 6, their expression was lower in PCA rats compared to the control rats (p <0.01). (D) Proteasome 20S activity assay showed higher activity at 2 weeks after PCA (p <0.05) while subsequently the activity was lower (p < 0.05) than that in the control rats.

Fig. 2. Histogram showing relative expression of mRNA by real-time PCR of genes regulating skeletal muscle protein synthesis and satellite cell function expressed as fold change in PCA rats at the end of treatment with follistatin compared with those treated with vehicle alone

The expression of myostatin was unchanged, while its receptor activin 2br, and its intracellular signal, cyclin dependant kinase p21 were significantly lower (p <0.01) in the PCA rats treated with follistatin compared to those treated with vehicle. In contrast, expression of markers of satellite cell function – proliferating cell nuclear antigen, myoD and myogenin were significantly higher in the PCA rats administered follistatin (p <0.01).

Fig. 3

Follistatin did not result in an alteration in proteasome activity in either PCA or control rats (p > 0.1).

Fig. 4. (A) Representative Western blots of proteins regulating skeletal muscle mass, markers of protein synthesis and satellite cell function

β-Actin was used as a loading control in all experiments. (B) Densitometry (n = 8 each) showed that the expression of myostatin, its receptor activin 2br and intracellular signal, CDKI p21 were significantly lower in PCA rats administered follistatin. The expression of markers of satellite cell function – proliferating cell nuclear antigen (PCNA), and markers of differentiation (myoD and myogenin) were significantly higher in the PCA rats administered follistatin. Follistatin did not have any significant effect in control rats.

Fig. 5. (A) Representative Western blots of IGF1, phospho-Akt ( ), phospho-mTOR, phospho-AMPKα, total and phospho-p70s6k in the PCA and sham rats treated with vehicle and follistatin

(B) Densitometry of these blots showed lower expression of IGF1 in the PCA rats compared to control animals and did not change in response to follistatin in either group. Elevated phospho AMPkinase, a direct inhibitor of mTOR, resulted in lower expression of both phospho mTOR and phospho-p70s6k in the PCA rats treated with vehicle and were reversed in response to blocking myostatin. Follistatin did not have any significant effect in control rats.