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. 2018 Feb 2;293(5):1596-1609.
doi: 10.1074/jbc.M117.814996. Epub 2017 Nov 30.

Age-dependent increase in angiopoietin-like protein 2 accelerates skeletal muscle loss in mice

Jiabin Zhao  1 , Zhe Tian  2 , Tsuyoshi Kadomatsu  1 , Peiyu Xie  1 , Keishi Miyata  1   3 , Taichi Sugizaki  1 , Motoyoshi Endo  1 , Shunshun Zhu  1 , Haoqiu Fan  1 , Haruki Horiguchi  1 , Jun Morinaga  1 , Kazutoyo Terada  1 , Tatsuya Yoshizawa  4 , Kazuya Yamagata  4 , Yuichi Oike  5
Affiliations

Age-dependent increase in angiopoietin-like protein 2 accelerates skeletal muscle loss in mice

Jiabin Zhao et al. J Biol Chem. .

Abstract

Skeletal muscle atrophy, or sarcopenia, is commonly observed in older individuals and in those with chronic disease and is associated with decreased quality of life. There is recent medical and broad concern that sarcopenia is rapidly increasing worldwide as populations age. At present, strength training is the only effective intervention for preventing sarcopenia development, but it is not known how this exercise regimen counteracts this condition. Here, we report that expression of the inflammatory mediator angiopoietin-like protein 2 (ANGPTL2) increases in skeletal muscle of aging mice. Moreover, in addition to exhibiting increased inflammation and accumulation of reactive oxygen species (ROS), denervated atrophic skeletal muscles in a mouse model of denervation-induced muscle atrophy had increased ANGPTL2 expression. Interestingly, mice with a skeletal myocyte-specific Angptl2 knockout had attenuated inflammation and ROS accumulation in denervated skeletal muscle, accompanied by increased satellite cell activity and inhibition of muscular atrophy compared with mice harboring wildtype Angptl2 Moreover, consistent with these phenotypes, wildtype mice undergoing exercise training displayed decreased ANGPTL2 expression in skeletal muscle. In conclusion, ANGPTL2 up-regulation in skeletal myocytes accelerates muscle atrophy, and exercise-induced attenuation of ANGPTL2 expression in those tissues may partially explain how exercise training prevents sarcopenia.

Keywords: ANGPTL2; aging; catalase; exercise; inflammation; muscle atrophy; reactive oxygen species (ROS); sarcopenia; satellite cell; senescence.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
ANGPTL2 levels increase in skeletal muscle of aging mice. a, relative transcript levels of cellular senescence-associated (p16, p19, p21, and p57) and pro-inflammatory (Il-1β and Il-6) genes in musculus gastrocnemius and musculus soleus of adult (8-month-old) and aging (18-month-old) female wildtype mice (n = 4 per group). b, absolute muscle mass and body weight as well as tibia length normalized muscle mass in adult and aging mice (n = 7–10 per group). c, representative Western blot of 4-HNE-adducted proteins, a product of oxidative stress, in musculus soleus of adult and aging mice. Asterisks indicate increased levels of 4-HNE-adducted proteins in aging mice. d–f, relative expression of genes encoding antioxidant enzymes (catalase, Sod1, Sod2, and Gpx1) (d) or Cd34, Pax3, and Pax7 (e), or Angptl2 (f) transcripts in musculus gastrocnemius and musculus soleus of adult and aging mice (n = 4 per group). g, representative Western blot and the quantifications of ANGPTL2 and PAX7 proteins in musculus gastrocnemius and musculus soleus of adult and aging mice. h, representative Western blot of ANGPTL2 in SMC-rich or stromal cells-rich fractions isolated from musculus gastrocnemius of adult and aging mice. Myosin light chain (MYL) serves as a skeletal myocyte marker. Relative mRNA expression was normalized to 18S mRNA (a, d, e, and f). Hsc70 and CBB staining serves as an internal loading control (c, g, and h). Values in adult mice were set to 1 (a and d–g). All data are presented as means ± S.D. (a, b, and d–f) or means ± S.E. g, statistical significance was determined by Student's t test. *, p < 0.05; **, p < 0.01; †, p < 0.001.
Figure 2.
Figure 2.
Angptl2 deficiency suppresses inflammation and cellular senescence and increases catalase activity in skeletal myocytes. a–e shows in vivo (skeletal muscles) analysis; f–j shows the in vitro (differentiated C2C12) analysis. a, relative mRNA expression (left) and representative Western blot (middle) and ANGPTL2 quantification in skeletal muscle of Angptl2Flox/Flox;MCK-Cre mice and Angptl2Flox/Flox mice (n = 4 per group). b, relative Angptl2 mRNA expression in skeletal myocyte (SMC)-rich or stromal cell-rich fractions isolated from musculus gastrocnemius of Angptl2Flox/Flox;MCK-Cre mice and Angptl2Flox/Flox mice (n = 6–8 per group). c, relative expression of cellular senescence-associated or pro-inflammatory genes in musculus gastrocnemius and musculus soleus of Angptl2Flox/Flox;MCK-Cre mice and Angptl2Flox/Flox mice (n = 4 per group). d, representative Western blot of 4-HNE-adducted proteins in musculus gastrocnemius of Angptl2Flox/Flox;MCK-Cre and Angptl2Flox/Flox mice. Asterisks indicate decreased levels of these proteins seen in Angptl2Flox/Flox;MCK-Cre mice. e, relative catalase activity in musculus gastrocnemius of Angptl2Flox/Flox;MCK-Cre and Angptl2Flox/Flox mice (n = 6–8 per group). f, representative Western blot and the quantification of ANGPTL2 expression in differentiated C2C12 cells transfected with siRNA control (siControl) or siRNA Angptl2 (siAngptl2) (n = 3 per group for ANGPTL2 quantification). g, relative transcript levels of cellular senescence-associated genes in differentiated C2C12 cells transfected with Angptl2 or control siRNA (UD, undetected) (n = 6 per group). h, representative images and relative fluorescence levels of SPiDER-βgal staining of differentiated C2C12 cells transfected with Angptl2 or control siRNA (scale bar, 50 μm) (n = 10 per group). i, relative fluorescence levels detected by CM-H2DCFDA (general oxidative stress indicator) of differentiated C2C12 cells transfected with Angptl2 or control siRNA (n = 12–13 per group). j, relative catalase transcript levels (left) or activity (right) in differentiated C2C12 cells transfected with Angptl2 or control siRNA (n = 5–6 per group). Relative mRNA expression was normalized to 18S mRNA (a–c, g, and j). Hsc70 and CBB serve as internal loading controls (a, d, and f). Values in Angptl2Flox/Flox mice were set to 1 (a–c, e–g, and i–g). All data are presented as means ± S.D. (a, left, b, c, e, and g–j) or means ± S.E. (a, right, and f); statistical significance was determined by Student's t test. *, p < 0.05; **, p < 0.01; †, p < 0.001.
Figure 3.
Figure 3.
Angptl2 deficiency in skeletal myocytes enhances satellite cell activity. a, relative transcript levels of satellite cell activation-associated genes (Cd34, Pax3, Pax7, Myf5, MyoD, and myogenin) and skeletal muscle myosin subtypes (Myh1, Myh2, Myh4, and Myh7) in musculus gastrocnemius and musculus soleus of Angptl2Flox/Flox;MCK-Cre or Angptl2Flox/Flox mice (n = 4 per group). b, representative immunostaining and quantifications of CD34 (green) and PAX7 (red) in musculus gastrocnemius of Angptl2Flox/Flox;MCK-Cre or Angptl2Flox/Flox mice. Myofibers are co-stained with DAPI (blue) and WGA (gray). Yellow arrowheads in Merge indicate CD34- and PAX7-positive cells. Red arrowheads in Merge indicate PAX7-positive cells. (Scale bar, 50 μm. Six different fields are quantified per mouse, n = 3 per group.) c, representative Western blot and PAX7 quantification in musculus gastrocnemius and musculus soleus of Angptl2Flox/Flox;MCK-Cre and Angptl2Flox/Flox mice. Hsc70 serves as the internal loading control. Values in Angptl2Flox/Flox mice were set to 1 (a and c). All data are presented as means ± S.D. (a and b) or means ± S.E. (c); statistical significance was determined by Student's t test. *, p < 0.05; **, p < 0.01.
Figure 4.
Figure 4.
Angptl2 deficiency in skeletal myocytes prevents skeletal muscle atrophy. a, absolute muscle mass and body weight normalized muscle mass in musculus gastrocnemius and musculus soleus of normal Angptl2Flox/Flox;MCK-Cre or Angptl2Flox/Flox male or female mice (n = 5 per group). b, representative Western blot and ANGPTL2 quantification in musculus gastrocnemius and musculus soleus of wildtype mice after denervation surgery (n = 4 per group). Hsc70 serves as an internal loading control. c, representative images of lower limbs and samples of musculus gastrocnemius and musculus soleus from sham-operated or 2-week-denervated Angptl2Flox/Flox;MCK-Cre or Angptl2Flox/Flox mice. Red arrow indicates atrophied muscle. d, muscle mass is shown as a percentage of the day 0 value of denervated musculus gastrocnemius and musculus soleus in Angptl2Flox/Flox;MCK-Cre or Angptl2Flox/Flox mice (n = 5 per group). e and f, representative HE staining images (left) and cross-sections of myofibers (right) in musculus gastrocnemius (e) and musculus soleus (f) of sham-operated or 2-week-denervated Angptl2Flox/Flox;MCK-Cre or Angptl2Flox/Flox mice. (Scale bar, 50 mm. n = 5, n = 1200–1700 per group for gastrocnemius, n = 800–1000 per group for soleus.) Values in sham mice were set to 1 (b). All data are presented as means ± S.D. (a, e, and f) or means ± S.E. (b and d); statistical significance was determined by Student's t test (a and d–f) or one-way ANOVA (b), *, p < 0.05; †, p < 0.001.
Figure 5.
Figure 5.
Angptl2-deficient skeletal myocytes show decreased inflammation and ROS levels and increased satellite cell activity. a, c, and d, relative expression of Il-1β, Il-6 (a), catalase (c), and satellite cell activation-associated (d) mRNAs in denervated musculus gastrocnemius of Angptl2Flox/Flox;MCK-Cre or Angptl2Flox/Flox mice (n = 5 per group). Relative mRNA expression was normalized to 18S mRNA. b, representative Western blot of 4-HNE-adducted proteins in denervated musculus gastrocnemius of Angptl2Flox/Flox;MCK-Cre or Angptl2Flox/Flox mice. Asterisks indicate decreased levels of these proteins seen in Angptl2Flox/Flox;MCK-Cre mice. e, representative Western blot and PAX7 quantification 14 days after denervation in musculus gastrocnemius of Angptl2Flox/Flox;MCK-Cre or Angptl2Flox/Flox mice. Hsc70 and CBB serve as internal loading controls (b and e). Values at day 0 of denervation in both Angptl2Flox/Flox;MCK-Cre and Angptl2Flox/Flox mice (a, c, and d) and in Angptl2Flox/Flox mice (e) were set to 1. All data are presented as means ± S.E. (a, c, and d) or means ± S.D. (e); statistical significance was determined by Student's t test. *, p < 0.05; **, p < 0.01.
Figure 6.
Figure 6.
Exercise training suppresses Angptl2 expression in skeletal muscles. a–d show in vivo (skeletal muscles) analysis; e and f show in vitro (differentiated C2C12) analysis. a and b, relative transcript levels of markers of senescence and pro-inflammation (a) and satellite cells (b) in musculus gastrocnemius and musculus soleus of wildtype mice, 3 h after an endurance run (n = 7–12 per group). c and d, relative Angptl2 mRNA (c) and protein (d) expression in musculus gastrocnemius and musculus soleus of wildtype C57BL/6NJcl male mice after exercise training. (Sed, sedentary; AR, post-acute run; AR3h, post-acute run plus 3 h; ER3h, post-endurance run plus 3 h.) (n = 6–8 per group for c; n = 3–4 per group for d.) e, relative Angptl2 mRNA expression in differentiated C2C12 cells exposed to mechanical stretch by 5, 10, or 15% extension with 1 Hz sine wave cycle for 15 or 30 min (n = 4–6 per group). f, relative Angptl2 mRNA (left) and protein (right) expression in differentiated C2C12 cells treated with 1 mm AICAR for indicated time points (1, 6, and 12 h) (n = 6 per group). Relative mRNA expression was normalized to 18S mRNA (a–c, e and f). Hsc70 serves as the internal loading control (d and f). Values in sedentary mice (a–d), in the 0-min group (e) and in the vehicle (veh) group (f) were set to 1. All data are presented as means ± S.D. (a–c, e, and f, left) or means ± S.E. (d and f, right); statistical significance was determined by Student's t test (a and b) or one-way ANOVA (c and d–f). *, p < 0.05; **, p < 0.01; †, p < 0.001.
Figure 7.
Figure 7.
Model of ANGPTL2 activity in skeletal muscle. Left, ANGPTL2 expression in skeletal myocytes increases with age. Skeletal myocyte-derived ANGPTL2 promotes inflammation and facilitates ROS accumulation by decreasing catalase expression in skeletal muscle. Increased ANGPTL2 and associated inflammation and ROS accumulation impair satellite cell activity in skeletal muscle, leading to atrophy. Right, exercise training decreases ANGPTL2 expression in skeletal muscle as it suppresses inflammation and ROS accumulation. These activities facilitate satellite cell activation that contribute to maintenance of muscle mass. Thus, exercise training-induced ANGPTL2 down-regulation represents a potential mechanism underlying exercise-induced protection from muscle atrophy.
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