Contractile properties of EDL and soleus muscles of myostatin-deficient mice

Abstract

Myostatin is a negative regulator of muscle mass. The impact of myostatin deficiency on the contractile properties of healthy muscles has not been determined. We hypothesized that myostatin deficiency would increase the maximum tetanic force (P(o)), but decrease the specific P(o) (sP(o)) of muscles and increase the susceptibility to contraction-induced injury. The in vitro contractile properties of extensor digitorum longus (EDL) and soleus muscles from wild-type (MSTN(+/+)), heterozygous-null (MSTN(+/-)), and homozygous-null (MSTN(-/-)) adult male mice were determined. For EDL muscles, the P(o) of both MSTN(+/-) and MSTN(-/-) mice were greater than the P(o) of MSTN(+/+) mice. For soleus muscles, the P(o) of MSTN(-/-) mice was greater than that of MSTN(+/+) mice. The sP(o) of EDL muscles of MSTN(-/-) mice was less than that of MSTN(+/+) mice. For soleus muscles, however, no difference in sP(o) was observed. Following two lengthening contractions, EDL muscles from MSTN(-/-) mice had a greater force deficit than that of MSTN(+/+) or MSTN(+/-) mice, whereas no differences were observed for the force deficits of soleus muscles. Myostatin-deficient EDL muscles had less hydroxyproline, and myostatin directly increased type I collagen mRNA expression and protein content. The difference in the response of EDL and soleus muscles to myostatin may arise from differences in the levels of a myostatin receptor, activin type IIB. Compared with the soleus, the amount of activin type IIB receptor was approximately twofold greater in EDL muscles. The results support a significant role for myostatin not only in the mass of muscles but also in the contractility and the composition of the extracellular matrix of muscles.

Figure 1

Relative hydroxyproline content of EDL (A) and soleus (B) muscles from MSTN^+/+, MSTN^+/− and MSTN^−/− mice. (A) Compared with MSTN^+/+ mice, the amount of hydroxyproline per mg of dry EDL muscle mass was less for MSTN^+/− and MSTN^−/− mice. (B) There was no difference in the amount of hydroxyproline per mg of dry soleus muscle mass between MSTN^+/+, MSTN^+/− and MSTN^−/− mice. Values are means ± SE. N = 5 muscles per genotype. *Significantly different from MSTN^+/+ at P < 0.05.

Figure 2

Myostatin increases (A) col1α2 expression and (B) procollagen I content of primary skeletal muscle myotubes. (A) RT-qPCR: Myostatin increases the expression of col1α2 normalized to β2m in a dose-dependant fashion. Values are means ± SE. *Significantly different from the 0 ng/mL group at P < 0.05. #Significantly different from the 10ng/mL group at P < 0.05. (B) Immunoblot: Myostatin increases the procollagen I protein content of myotubes in a dose-dependant fashion. β-tubulin is shown as a loading control.

Figure 3

Force values for EDL muscles (A, B) and soleus muscles (C,D) from MSTN^+/+, MSTN^+/− and MSTN^−/− mice. (A) The P_o of EDL muscles of MSTN^−/− mice is greater than the P_o of MSTN^+/+ and MSTN^+/− mice. (B) When P_o is normalized to CSA, the sP_o of EDL muscles of MSTN^−/− mice is less than the sP_o of MSTN^+/+ and MSTN^+/− mice. (C) The P_o of soleus muscles of MSTN^−/− mice is greater than the P_o of MSTN^+/+ and MSTN^+/− mice. (D) When P_o is normalized to CSA, there is no difference in sP_o of soleus muscles. Values are means ± SE. N = 6 muscles per genotype. *Significantly different from MSTN^+/+ at P < 0.05. #Significantly different from MSTN^+/− at P < 0.05.

Figure 4

Force deficits following contraction-induced injury to EDL muscles (A) and soleus muscles (B). (A) Muscles from MSTN^−/− mice had a force deficit that was greater than MSTN^+/+ mice after the first lengthening contraction, and a force deficit that was greater than MSTN^+/+ and MSTN^+/− mice after the second lengthening contraction. (B) There was no difference in the force deficits between soleus muscles following the lengthening contractions. LC = Lengthening contraction. Values are means ± SE. N = 6 muscles per genotype. *Significantly different from MSTN^+/+ at P < 0.05. #Significantly different from MSTN^+/− at P < 0.05.

Figure 5

ActRIIB protein content of EDL and soleus muscles from MSTN^+/+ mice. Compared with soleus muscles, the amount of ActRIIB protein is greater in EDL muscles (immunoblot). Sarcomeric myosin proteins are shown as loading controls (Coomassie Brilliant Blue staining).