Hypoxia Inhibits Myogenic Differentiation through Accelerated MyoD Degradation

Abstract

Cells undergo a variety of biological responses when placed in hypoxic conditions, including alterations in metabolic state and growth rate. Here we investigated the effect of hypoxia on the ability of myogenic cells to differentiate in culture. Exposure of myoblasts to hypoxia strongly inhibited multinucleated myotube formation and the expression of differentiation markers. We showed that hypoxia reversibly inhibited MyoD, Myf5, and myogenin expression. One key step in skeletal muscle differentiation involves the up-regulation of the cell cycle-dependent kinase inhibitors p21 and p27 as well as the product of the retinoblastoma gene (pRb). Myoblasts cultured under hypoxic conditions in differentiation medium failed to up-regulate both p21 and pRb despite the G1 cell cycle arrest, as evidenced by p27 accumulation and pRb hypophosphorylation. Hypoxia-dependent of differentiation by the in myoblasts the differentiation by hypoxic hypoxia by accumulation of myogenic differentiation as myogenin and p21 and both cell cycle and a effect by hypoxia in myogenic cells and as a of Cells undergo a variety of biological responses when placed in hypoxic conditions, including alterations in metabolic state and growth rate. Here we investigated the effect of hypoxia on the ability of myogenic cells to differentiate in culture. Exposure of myoblasts to hypoxia strongly inhibited multinucleated myotube formation and the expression of differentiation markers. We showed that hypoxia reversibly inhibited MyoD, Myf5, and myogenin expression. One key step in skeletal muscle differentiation involves the up-regulation of the cell cycle-dependent kinase inhibitors p21 and p27 as well as the product of the retinoblastoma gene (pRb). Myoblasts cultured under hypoxic conditions in differentiation medium failed to up-regulate both p21 and pRb despite the G1 cell cycle arrest, as evidenced by p27 accumulation and pRb hypophosphorylation. Hypoxia-dependent of differentiation by the in myoblasts the differentiation by hypoxic hypoxia by accumulation of myogenic differentiation as myogenin and p21 and both cell cycle and a effect by hypoxia in myogenic cells and as a of muscle differentiation by the cell of muscle gene and cell multinucleated and differentiation that to of muscle muscle retinoblastoma growth differentiation muscle retinoblastoma growth differentiation MyoD, and as of cell of ability to a skeletal muscle to cells to in the of myogenic differentiation myoblasts and the of muscle differentiation and the of myoblasts the cell cycle the expression of cell cycle and differentiation of and kinase inhibitors p21 and the retinoblastoma in the state myogenic p21 expression the G1 of the cell and up-regulation cell cycle of muscle cells One key of the by by accumulation of pRb in the the of the cell cycle in the cell cycle by of the a key both p21 and pRb gene expression muscle to of the p21 in and to p21 and accumulation in muscle cells and to and differentiation on growth and to myogenic cell as in and to multinucleated cells a of to and cell of in conditions, metabolic and biological including to of in growth on the cell of responses by the the expression of in growth arrest, as the as well as the p21 and p27 that growth a and in to of pRb to of myoblasts hypoxia cell and the of myogenic cells to hypoxia as well as the in the we the effect of hypoxia on myogenic differentiation of myogenic differentiation by expression and cell cycle hypoxia the in a cell in hypoxic cells the of we as a of and and cell cultured in medium and medium differentiation cells to medium medium by cells in and conditions cells and a and cells in a of Cells the as by the cells in and to hypoxic and hypoxic cells by and cell in and cells and in of and to of and on a the of cells to Cells and and and as and cell and to in in a by to a of of and on to to of the to and and and in the of of and and of of the MyoD, and and on on and and and inhibitors and by by of by and to myogenin by and by a inhibitors and in and to the cells and and of by of cells the cells and under hypoxic conditions the and by as cells of of as and in hypoxic conditions Cells in and in and in cells and by to and a cells of the the gene under the of the kinase of and of of as a cells in and cultured and under hypoxic cells and in of in and as by the of as the and a hypoxia myogenic myoblasts cultured in under hypoxic conditions, and ability to undergo myogenic myoblasts and expression cells cultured in hypoxia showed myotube formation accumulation and effect of hypoxia on myogenic differentiation in myoblasts cell in conditions in of the myogenic hypoxic conditions inhibited both accumulation and myotube formation the myogenic differentiation inhibited by myoblasts placed in in hypoxic conditions and to conditions in in hypoxic and the cell and of to hypoxic cells in the of myogenic differentiation as evidenced by myotube formation and accumulation that differentiation inhibited by as myoblasts to differentiate when of myogenic differentiation cells cultured in in hypoxia when to in myoblasts to hypoxia medium and cells to and cells and the of hypoxic when to in in hypoxia and to in in by to and to the Myoblasts in G1 cell cycle to muscle differentiation and of gene expression hypoxia both and cell growth and cell cycle in cell we to the effect of hypoxia on the cell cycle of myoblasts cultured in and to G1 in and to cells on and hypoxic and cells as by and by of cell cycle that hypoxia G1 accumulation of cells and the G1 to a of cells in hypoxia cell cycle of cells and both in as well as in hypoxic conditions cells in G1 growth in cell hypoxia Cells of and to and cells and the of as by a as the of and hypoxia G1 accumulation of and cells the of a in Cells and as under the by of hypoxia on cell cycle of myoblasts of cells in and of the cell cycle as in the of and cells G1 and in G1 and in hypoxia in a to the cell cycle to differentiate the G1 to and by the of and the myogenic differentiation the up-regulation of the p21 and p27 growth One of the growth and myogenic the of pRb by the p21 that hypoxia accumulation of myoblasts in G1 and we the expression of cell cycle cells in hypoxic and myoblasts in hypoxia cells and the expression of cell and hypoxia pRb in cells of that of the p27 and p21 of pRb expression of the p27 in cells as the p21 in hypoxic cells by hypoxia in a we in in hypoxic conditions despite accumulation cells to pRb and hypoxia of pRb as conditions both p21 and p27 in p27 in hypoxic conditions that a growth and p27 accumulation in both and hypoxic of pRb and p21 by that cells to the myogenic the G1 of hypoxia on cell cycle of and differentiation involves pRb and p27 cells cultured in and to hypoxia cells and to cells cells in by to and the the skeletal muscle cell differentiation by including MyoD, Myf5, and muscle expression. in the to hypoxia the expression of muscle a and both in and cells myogenin the of differentiation a of MyoD, Myf5, and myogenin and and of expression both and when hypoxic myoblasts cultured in in in in cells in hypoxia that hypoxia reversibly expression in myoblasts cultured under differentiation reversibly hypoxia MyoD, Myf5, and myogenin expression. cells cultured in to and in hypoxia cultured cells by to and the to hypoxia expression. of myoblasts cultured in in hypoxia as and by to of the of expression cells in in hypoxia and to in and as in hypoxia inhibited expression and we to expression as well as myogenic differentiation in cells cultured under hypoxic that in cells myogenic as evidenced by myotube formation and accumulation to hypoxia cells a differentiation as by the of cells multinucleated on hypoxic the of both and accumulation in by a in that in the of myogenic differentiation of of and hypoxic cells Cells and by a a of hypoxia accumulation in cells in to and cultured in hypoxia and by and expression. of the by the hypoxia in of expression in cells and and cells cultured in in and hypoxic conditions and the and that cell hypoxia by accumulation in cells We that of accumulation strongly both in as well as in hypoxic cells and of in hypoxic in conditions and in both in and hypoxic myoblasts of in hypoxic conditions and of a and hypoxia in cells by hypoxia accumulation in cells cultured under hypoxic conditions in the of the of and to to accumulation by the cells cultured under conditions and to hypoxia inhibitors the of by to of the expression by the of conditions conditions by the the of G1 hypoxia inhibited accumulation by cells in hypoxic conditions and and the the of hypoxia that both and in cells and the that of involves by the of cell cycle in a to G1 the in the of the of the of expression the to the myogenic under hypoxic the of the of differentiation in cells expression to cells placed in cultured in hypoxia and by the expression of myogenin and as of myogenic and myogenin and in cells cultured in hypoxic conditions and conditions the of as well as the of the effect of hypoxia on we the ability of to gene expression. cells expression a skeletal the muscle kinase expression of cells in and cultured in hypoxia and of the gene by hypoxic conditions as well as the of the gene strongly by that the expression of to a myogenic differentiation in hypoxic the myogenic differentiation in expression of myogenin and accumulation in hypoxic cultured cells and expression cells to and cultured in in hypoxia Cells and a myogenin and a cells by expression and of the in cells in the of and cells in and cultured in cells of and to to in and the of in cells of skeletal muscle involves the of myoblasts cell cycle and the expression and of myogenic and in and muscle cells a myogenin and and the to differentiation by the accumulation of p21 and pRb of growth and to the of muscle cell growth and the effect of the on differentiation the we showed that hypoxia strongly inhibited skeletal muscle differentiation by a in MyoD, Myf5, and myogenin that myogenic cell in the of in conditions that to the in in conditions as inhibited of myoblasts the cell cells cultured under hypoxic conditions in cell cycle accumulation in the G1 of the cell We that the p27 as well as the of pRb in in the of p21 in a that growth a pRb p21 that growth involves a of p27 by the of of p27 to and pRb in hypoxia p27 p21 pRb accumulation that the of pRb and p21 by of pRb gene by a key in the of skeletal muscle differentiation of pRb cell cycle as well as the differentiation and of we showed that expression of in the of the myogenic differentiation as a of by accumulation of myogenic differentiation as myogenin and p21 as well as pRb and both cell cycle and we that of differentiation cells hypoxia and in ability to differentiate by as well as cell cycle by G1 to when when hypoxic cells in and cultured in the cell that in cells the and by by the that by the a that in G1 of the cell cycle by the and the the hypoxia despite of in and cells a effect to the accumulation of the cells in of of in hypoxic conditions and to a G1 accumulation hypoxia a cell the that a by myogenic to in a that myogenic cells that failed to differentiate under hypoxic conditions that the of a by hypoxia to myogenic differentiation in that as as well as of the state of the cells myogenic differentiation in differentiation and cell we that hypoxia skeletal muscle cells in a growth muscle differentiation by the cell of muscle gene and cell multinucleated and differentiation that to of muscle muscle retinoblastoma growth differentiation muscle retinoblastoma growth differentiation MyoD, and as of cell of ability to a skeletal muscle to cells to in the of myogenic differentiation myoblasts and the of muscle differentiation and the of myoblasts the cell cycle the expression of cell cycle and differentiation of and kinase inhibitors p21 and the retinoblastoma in the state myogenic p21 expression the G1 of the cell and up-regulation cell cycle of muscle cells One key of the by by accumulation of pRb in the the of the cell cycle in the cell cycle by of the a key both p21 and pRb gene expression muscle to of the p21 in and to p21 and accumulation in muscle cells and to and differentiation on growth and to myogenic cell as in and to multinucleated cells a of to and cell of in conditions, metabolic and biological including to of in growth on the cell of responses by the the expression of in growth arrest, as the as well as the p21 and p27 that growth a and in to of pRb to of myoblasts hypoxia cell and the of myogenic cells to hypoxia as well as the in the we the effect of hypoxia on myogenic differentiation of myogenic differentiation by expression and cell cycle hypoxia the in a cell in hypoxic cells the of we as a of and and cell cultured in medium and medium differentiation cells to medium medium by cells in and conditions cells and a and cells in a of Cells the as by the cells in and to hypoxic and hypoxic cells by and cell in and cells and in of and to of and on a the of cells to Cells and and and as and cell and to in in a by to a of of and on to to of the to and and and in the of of and and of of the MyoD, and and on on and and and inhibitors and by by of by and to myogenin by and by a inhibitors and in and to the cells and and of by of cells the cells and under hypoxic conditions the and by as cells of of as and in hypoxic conditions Cells in and in and in cells and by to and a cells of the the gene under the of the kinase of and of of as a cells in and cultured and under hypoxic cells and in of in and as by the of as the and a and and cell cultured in medium and medium differentiation cells to medium medium by cells in and conditions cells and a and cells in a of Cells the as by the cells in and to hypoxic and hypoxic cells by and cell in and cells and in of and to of and on a the of cells to Cells and and and as and cell and to in in a by to a of of and on to to of the to and and and in the of of and and of of the MyoD, and and on on and and and inhibitors and by by of by and to myogenin by and by a inhibitors and in and to the cells and and of by of cells the cells and under hypoxic conditions the and by as cells of of as and in hypoxic conditions Cells in and in and in cells and by to and a cells of the the gene under the of the kinase of and of of as a cells in and cultured and under hypoxic cells and in of in and as by the of as the and a hypoxia myogenic myoblasts cultured in under hypoxic conditions, and ability to undergo myogenic myoblasts and expression cells cultured in hypoxia showed myotube formation accumulation and effect of hypoxia on myogenic differentiation in myoblasts cell in conditions in of the myogenic hypoxic conditions inhibited both accumulation and myotube formation the myogenic differentiation inhibited by myoblasts placed in in hypoxic conditions and to conditions in in hypoxic and the cell and of to hypoxic cells in the of myogenic differentiation as evidenced by myotube formation and accumulation that differentiation inhibited by as myoblasts to differentiate when Myoblasts in G1 cell cycle to muscle differentiation and of gene expression hypoxia both and cell growth and cell cycle in cell we to the effect of hypoxia on the cell cycle of myoblasts cultured in and to G1 in and to cells on and hypoxic and cells as by and by of cell cycle that hypoxia G1 accumulation of cells and the G1 to a of cells in hypoxia cell cycle of cells and both in as well as in hypoxic conditions cells in G1 growth in cell hypoxia Cells of and to and cells and the of as by a as the of and hypoxia G1 accumulation of and cells the of a in Cells and as under the by of hypoxia on cell cycle of myoblasts of cells in and of the cell cycle as in the of and cells G1 and in G1 and in hypoxia in a to the cell cycle to differentiate the G1 to and by the of and the myogenic differentiation the up-regulation of the p21 and p27 growth One of the growth and myogenic the of pRb by the p21 that hypoxia accumulation of myoblasts in G1 and we the expression of cell cycle cells in hypoxic and myoblasts in hypoxia cells and the expression of cell and hypoxia pRb in cells of that of the p27 and p21 of pRb expression of the p27 in cells as the p21 in hypoxic cells by hypoxia in a we in in hypoxic conditions despite accumulation cells to pRb and hypoxia of pRb as conditions both p21 and p27 in p27 in hypoxic conditions that a growth and p27 accumulation in both and hypoxic of pRb and p21 by that cells to the myogenic the G1 of hypoxia on cell cycle of and differentiation involves pRb and p27 cells cultured in and to hypoxia cells and to cells cells in by to and the the skeletal muscle cell differentiation by including MyoD, Myf5, and muscle expression. in the to hypoxia the expression of muscle a and both in and cells myogenin the of differentiation a of MyoD, Myf5, and myogenin and and of expression both and when hypoxic myoblasts cultured in in in in cells in hypoxia that hypoxia reversibly expression in myoblasts cultured under differentiation reversibly hypoxia MyoD, Myf5, and myogenin expression. cells cultured in to and in hypoxia cultured cells by to and the to hypoxia expression. of myoblasts cultured in in hypoxia as and by to of the of expression cells in in hypoxia and to in and as in hypoxia inhibited expression and we to expression as well as myogenic differentiation in cells cultured under hypoxic that in cells myogenic as evidenced by myotube formation and accumulation to hypoxia cells a differentiation as by the of cells multinucleated on hypoxic the of both and accumulation in by a in that in the of myogenic differentiation of of and hypoxic cells Cells and by a a of hypoxia accumulation in cells in to and cultured in hypoxia and by and expression. of the by the hypoxia in of expression in cells and and cells cultured in in and hypoxic conditions and the and that cell hypoxia by accumulation in cells We that of accumulation strongly both in as well as in hypoxic cells and of in hypoxic in conditions and in both in and hypoxic myoblasts of in hypoxic conditions and of a and hypoxia in cells by hypoxia accumulation in cells cultured under hypoxic conditions in the of the of and to to accumulation by the cells cultured under conditions and to hypoxia inhibitors the of by to of the expression by the of conditions conditions by the the of G1 hypoxia inhibited accumulation by cells in hypoxic conditions and and the the of hypoxia that both and in cells and the that of involves by the of cell cycle in a to G1 the in the of the of the of expression the to the myogenic under hypoxic the of the of differentiation in cells expression to cells placed in cultured in hypoxia and by the expression of myogenin and as of myogenic and myogenin and in cells cultured in hypoxic conditions and conditions the of as well as the of the effect of hypoxia on we the ability of to gene expression. cells expression a skeletal the muscle kinase expression of cells in and cultured in hypoxia and of the gene by hypoxic conditions as well as the of the gene strongly by that the expression of to a myogenic differentiation in hypoxic the myogenic differentiation in expression of myogenin and accumulation in hypoxic cultured cells and expression cells to and cultured in in hypoxia Cells and a myogenin and a cells by expression and of the in cells in the of and cells in and cultured in cells of and to to in and the of in cells hypoxia myogenic myoblasts cultured in under hypoxic conditions, and ability to undergo myogenic myoblasts and expression cells cultured in hypoxia showed myotube formation accumulation and effect of hypoxia on myogenic differentiation in myoblasts cell in conditions in of the myogenic hypoxic conditions inhibited both accumulation and myotube formation the myogenic differentiation inhibited by myoblasts placed in in hypoxic conditions and to conditions in in hypoxic and the cell and of to hypoxic cells in the of myogenic differentiation as evidenced by myotube formation and accumulation that differentiation inhibited by as myoblasts to differentiate when Myoblasts in G1 cell cycle to muscle differentiation and of gene expression hypoxia both and cell growth and cell cycle in cell we to the effect of hypoxia on the cell cycle of myoblasts cultured in and to G1 in and to cells on and hypoxic and cells as by and by of cell cycle that hypoxia G1 accumulation of cells and the G1 to a of cells in hypoxia cell cycle of cells and both in as well as in hypoxic conditions cells in G1 to the cell cycle to differentiate the G1 to and by the of and the myogenic differentiation the up-regulation of the p21 and p27 growth One of the growth and myogenic the of pRb by the p21 that hypoxia accumulation of myoblasts in G1 and we the expression of cell cycle cells in hypoxic and myoblasts in hypoxia cells and the expression of cell and hypoxia pRb in cells of that of the p27 and p21 of pRb expression of the p27 in cells as the p21 in hypoxic cells by hypoxia in a we in in hypoxic conditions despite accumulation cells to pRb and hypoxia of pRb as conditions both p21 and p27 in p27 in hypoxic conditions that a growth and p27 accumulation in both and hypoxic of pRb and p21 by that cells to the myogenic the G1 the skeletal muscle cell differentiation by including MyoD, Myf5, and muscle expression. in the to hypoxia the expression of muscle a and both in and cells myogenin the of differentiation a of MyoD, Myf5, and myogenin and and of expression both and when hypoxic myoblasts cultured in in in in cells in hypoxia that hypoxia reversibly expression in myoblasts cultured under differentiation hypoxia inhibited expression and we to expression as well as myogenic differentiation in cells cultured under hypoxic that in cells myogenic as evidenced by myotube formation and accumulation to hypoxia cells a differentiation as by the of cells multinucleated on hypoxic the of both and accumulation in by a in that in the of the and that cell hypoxia by accumulation in cells We that of accumulation strongly both in as well as in hypoxic cells and of in hypoxic in conditions and in both in and hypoxic myoblasts of in hypoxic conditions and of a and hypoxia in cells by by the the of G1 hypoxia inhibited accumulation by cells in hypoxic conditions and and the the of hypoxia that both and in cells and the that of involves by the of cell cycle in a to G1 the in the of the of the of expression the to the myogenic under hypoxic the of the of differentiation in cells expression to cells placed in cultured in hypoxia and by the expression of myogenin and as of myogenic and myogenin and in cells cultured in hypoxic conditions and conditions the of as well as the of the effect of hypoxia on we the ability of to gene expression. cells expression a skeletal the muscle kinase expression of cells in and cultured in hypoxia and of the gene by hypoxic conditions as well as the of the gene strongly by that the expression of to a myogenic differentiation in hypoxic of skeletal muscle involves the of myoblasts cell cycle and the expression and of myogenic and in and muscle cells a myogenin and and the to differentiation by the accumulation of p21 and pRb of growth and to the of muscle cell growth and the effect of the on differentiation the we showed that hypoxia strongly inhibited skeletal muscle differentiation by a in MyoD, Myf5, and myogenin that myogenic cell in the of in conditions that to the in in conditions as inhibited of myoblasts the cell cells cultured under hypoxic conditions in cell cycle accumulation in the G1 of the cell We that the p27 as well as the of pRb in in the of p21 in a that growth a pRb p21 that growth involves a of p27 by the of of p27 to and pRb in hypoxia p27 p21 pRb accumulation that the of pRb and p21 by of pRb gene by a key in the of skeletal muscle differentiation of pRb cell cycle as well as the differentiation and of we showed that expression of in the of the myogenic differentiation as a of by accumulation of myogenic differentiation as myogenin and p21 as well as pRb and both cell cycle and we that of differentiation cells hypoxia and in ability to differentiate by as well as cell cycle by G1 to when when hypoxic cells in and cultured in the cell that in cells the and by by the that by the a that in G1 of the cell cycle by the and the the hypoxia despite of in and cells a effect to the accumulation of the cells in of of in hypoxic conditions and to a G1 accumulation hypoxia a cell the that a by myogenic to in a that myogenic cells that failed to differentiate under hypoxic conditions that the of a by hypoxia to myogenic differentiation in that as as well as of the state of the cells myogenic differentiation in differentiation and cell we that hypoxia skeletal muscle cells in a growth of skeletal muscle involves the of myoblasts cell cycle and the expression and of myogenic and in and muscle cells a myogenin and and the to differentiation by the accumulation of p21 and pRb of growth and to the of muscle cell growth and the effect of the on differentiation the we showed that hypoxia strongly inhibited skeletal muscle differentiation by a in MyoD, Myf5, and myogenin that myogenic cell in the of in conditions that to the in in conditions as inhibited of myoblasts the cell cells cultured under hypoxic conditions in cell cycle accumulation in the G1 of the cell We that the p27 as well as the of pRb in in the of p21 in a that growth a pRb p21 that growth involves a of p27 by the of of p27 to and pRb in hypoxia p27 p21 pRb accumulation that the of pRb and p21 by of pRb gene by a key in the of skeletal muscle differentiation of pRb cell cycle as well as the differentiation and of we showed that expression of in the of the myogenic differentiation as a of by accumulation of myogenic differentiation as myogenin and p21 as well as pRb and both cell cycle and we that of differentiation cells hypoxia and in ability to differentiate by as well as cell cycle by G1 to when when hypoxic cells in and cultured in the cell that in cells the and by by the that by the a that in G1 of the cell cycle by the and the the hypoxia despite of in and cells a effect to the accumulation of the cells in of of in hypoxic conditions and to a G1 accumulation hypoxia a cell the that a by myogenic to in a that myogenic cells that failed to differentiate under hypoxic conditions that the of a by hypoxia to myogenic differentiation in that as as well as of the state of the cells myogenic differentiation in differentiation and cell we that hypoxia skeletal muscle cells in a growth We and We to and of the We and the of