Blunting effect of hypoxia on the proliferation and differentiation of human primary and rat L6 myoblasts is not counteracted by Epo

Abstract

Objectives: The aim of this study was to evaluate whether hypoxia and/or erythropoietin would be able to modulate proliferation/differentiation processes of rat and human myoblasts.

Materials and methods: Rat L6 and primary human myoblasts were grown in 21% or 1% O(2) in the presence or absence of recombinant human erythropoietin (RhEpo). Presence of erythropoietin receptors (EpoR) was assayed using RT-PCR and Western blotting techniques. Cell proliferation was evaluated by determining the doubling time and kinetics of cultures by counting cells. Cell differentiation was analysed by determining myogenic fusion index using antibodies against the myosin heavy chain. Expression of myogenin and myosin heavy chain (MHC) proteins were evaluated using the Western blotting technique.

Results: After 96 h culture in growth medium for 2.5 and 9 h, doubling time of L6 and human primary myoblasts respectively, had increased in 1% O(2) conditions (P < 0.01). Kinetics of culture showed alteration in proliferation at 72 h in L6 myoblast cultures and at 4 days in human primary myoblasts. The myogenic fusion index had reduced by 30% in L6 myoblasts and by 20% in human myoblasts (P < 0.01). Expression of myogenin and MHC had reduced by around 50%. Despite presence of EpoR mRNA and protein, RhEpo did not counteract the effects of hypoxia either in L6 cells or in human myoblasts.

Conclusions: The data show that exposure to hypoxic conditions (1% O(2)) of rat and human myoblasts altered their proliferation and differentiation processes. They also show that Epo is not an efficient growth factor to counteract this deleterious effect.

Figure 1

 Identification of mRNA encoding EpoR and EpoR protein in rat L6 myoblasts and human myoblasts. L6 and human myoblasts were cultured for 48 h in growth medium and mRNA was extracted for PCR analyses. (a) Amplification products were visualized using agarose gel electropohoresis. Size of the amplification product of EpoR mRNA was 363 bp for rat myoblasts and 485 bp for human myoblasts, H₂O PCR control water, RT control without reverse transcriptase, PM molecular weight. (b) Western blotting of Jurkat cells and C2C12 cells used as positive controls and L6 rat muscle, stained with anti‐mouse EpoR rabbit polyclonal antibody. Western blotting revealed the 59 KDa band in human myoblasts and L6 myoblasts (C2C12 cells used as control).

Figure 2

 Effects of hypoxia and RhEpo on growth of cultured human and rat L6 myoblasts. (a1) Population growth kinetics of L6 myoblasts cultured in both 21% and 1% O₂ conditions in growth medium containing 0, 2 and 5 IU/mL of RhEpo. RhEpo was added to the medium at T0 and T48h. (a2) Determination of doubling time of L6 myoblasts grown for 96 h in both 21% and 1% conditions in growth media containing RhEpo injected at doses ranging from 0 to 10 IU/mL at T0 and T48h. (b1) Population growth kinetics of human primary myoblasts cultured in both 21% and 1% O₂ conditions in growth media containing 0, 2 and 5 IU/mL of RhEpo. RhEpo was added to medium at T0 and T48h. (b2) Determination of doubling time of L6 myoblasts grown for 96 h in both 21% and 1% conditions in growth medium containing RhEpo, injected at doses ranging from 0 to 10 IU/mL at T0 and T48. Values are mean ± SE of three separate experiments in triplicate. *P ≤ 0.01, 21% versus 1% O₂.

Figure 3

 Effects of hypoxia and RhEpo on differentiation of human and rat L6 myoblasts. (a) Immunostaining of L6 myoblasts grown to 80% confluence in growth medium at 21% O₂ containing 0 or 10 IU/mL RhEpo, and incubated for 96 h in differentiation medium for part one in 21% O₂ and part two in 1% O₂ in antibody raised against fast myosin heavy chain; counterstaining was carried out using haematoxylin, bar scale = 2mm. (b) Myogenic fusion index was determined by dividing number of nuclei in multinucleate myotubes by total number of nuclei in a given microscopic field. Ten fields per culture were counted in three independent cultures using MorphoPro software (Explora Nova). Data are presented as mean ± SEM. *P ≤ 0.01, 21% versus 1% O₂.

Figure 4

 Effects of hypoxia and RhEpo on expression of myogenin and fast myosin heavy chain. L6 myoblasts were cultured for 72 h in growth medium containing 0–10 IU/mL RhEpo at 21% O₂ and for 96 h in differentiation medium at 21% or 1% O_2. (a) Western blotting using anti‐mouse myogenin rabbit polyclonal antibody and beta‐actin. (b) Quantification of myogenin expression was carried out as ratio of myogenin/β‐actin. Western blots are representative of those obtained from three different cultures. Data are presented as mean ± SEM. *P ≤ 0.01, 21% versus 1% O₂.

Figure 5

 Expression of fast myosin heavy chain altered by hypoxia: RhEpo did not prevent this. L6 myoblasts were cultured for 72 h in growth medium containing 0–10 IU/mL RhEpo at 21% O₂ and for 96 h in differentiation medium at 21% or 1% O_2. (a) Western blotting shows expression of fast MHC and β‐actin in L6 myoblasts non‐treated (0) or treated, with a range from 1 to 10 IU/mL of RhEpo. (b) Quantification of myosin heavy chain expression was carried out as ratio of myogenin/β‐actin. Western blots are representative of those obtained from three different cultures. Data are presented as mean ± SEM. *P ≤ 0.01, 21% versus 1% O₂.