Myogenic Vector Expression of Insulin-like Growth Factor I Stimulates Muscle Cell Differentiation and Myofiber Hypertrophy in Transgenic Mice

The avian skeletal α-actin gene was used as a template for construction of a myogenic expression vector that was utilized to direct expression of a human IGF-I cDNA in cultured muscle cells and in striated muscle of transgenic mice. The proximal promoter region, together with the first intron and 1.8 kilobases of 3’-noncoding flanking sequence of the avian skeletal α-actin gene directed high level expression of human insulin-like growth factor I (IGF-I) in stably transfected C2C12 myoblasts and transgenic mice. Expression of the actin/IGF-I hybrid gene in C2C12 muscle cells increased levels of myogenic basic helix-loop-helix factor and contractile protein mRNAs and enhanced myotube formation. Expression of the actin/IGF-I hybrid gene in mice elevated IGF-I concentrations in skeletal muscle 47-fold resulting in myofiber hypertrophy. IGF-I concentrations in serum and body weight were not increased by transgene expression, suggesting that the effects of transgene expression were localized. These results indicate that sustained overexpression of IGF-I in skeletal muscle elicits myofiber hypertrophy and provides the basis for manipulation of muscle physiology utilizing skeletal α-actin-based vectors. The avian skeletal α-actin gene was used as a template for construction of a myogenic expression vector that was utilized to direct expression of a human IGF-I cDNA in cultured muscle cells and in striated muscle of transgenic mice. The proximal promoter region, together with the first intron and 1.8 kilobases of 3’-noncoding flanking sequence of the avian skeletal α-actin gene directed high level expression of human insulin-like growth factor I (IGF-I) in stably transfected C2C12 myoblasts and transgenic mice. Expression of the actin/IGF-I hybrid gene in C2C12 muscle cells increased levels of myogenic basic helix-loop-helix factor and contractile protein mRNAs and enhanced myotube formation. Expression of the actin/IGF-I hybrid gene in mice elevated IGF-I concentrations in skeletal muscle 47-fold resulting in myofiber hypertrophy. IGF-I concentrations in serum and body weight were not increased by transgene expression, suggesting that the effects of transgene expression were localized. These results indicate that sustained overexpression of IGF-I in skeletal muscle elicits myofiber hypertrophy and provides the basis for manipulation of muscle physiology utilizing skeletal α-actin-based vectors. INTRODUCTIONInsulin-like growth factor I (IGF-I),1 1The abbreviations used are: IGFinsulin-like growth factorhIGFhuman IGFbpbase pair(s)kbkilobase(s)IGFBPinsulin-like growth factor-binding protein(s). a peptide growth factor that is structurally related to proinsulin (1Daughaday W.H. Rotwein P. Endocrinol. Rev. 1989; 10: 68-91Crossref PubMed Scopus (1606) Google Scholar, 2Sara V.R. Hall K. Physiol. Rev. 1990; 70: 591-614Crossref PubMed Scopus (741) Google Scholar, 3Cohick W.S. Clemmons D.R. Annu. Rev. Physiol. 1993; 55: 131-153Crossref PubMed Scopus (573) Google Scholar, 4D'Ercole A.J. Stiles A.D. Underwood L.E. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 935-939Crossref PubMed Scopus (1067) Google Scholar), has a primary role in promoting the differentiation and growth of skeletal muscle. The effects of IGF-I on myogenic cells include stimulation of myoblast replication, myogenic differentiation, and myotube hypertrophy (see Refs. 5Florini J.R. Muscle 10: 577-598Crossref PubMed Scopus (254) Google Scholar and 6Florini J.R. Magri K.A. Am. J. Physiol. 1989; 256: C701-C711Crossref PubMed Google Scholar for review). In vivo, up-regulation of IGF-I expression in skeletal muscle is coincident with myotube formation in the developing embryo (7Han V.K.M. D'Ercole A.J. Lund P.K. Science. 1987; 236: 193-197Crossref PubMed Scopus (552) Google Scholar), stretch-induced myofiber hypertrophy (8DeVol D.L. Rotwein P. Sadow J.L. Novakofski J. Bechtel P.J. Am. J. Physiol. 1990; 259: E89-E95PubMed Google Scholar) and muscle regeneration following injury (9Levinovitz A. Jennische E. Oldfors A. Edwall D. Norstedt G. Mol. Endocrinol. 1992; 6: 1227-1234Crossref PubMed Scopus (59) Google Scholar), suggesting that IGF-I serves as an autocrine/paracrine mediator of these processes in skeletal muscle. Increased biosynthesis and extracellular secretion of IGF-I from cultured mammalian myoblasts has been shown to be coincident with myoblast alignment, withdrawal from the cell cycle, and fusion (5Florini J.R. Muscle 10: 577-598Crossref PubMed Scopus (254) Google Scholar, 6Florini J.R. Magri K.A. Am. J. Physiol. 1989; 256: C701-C711Crossref PubMed Google Scholar). In addition, inclusion of IGF-I in the media of primary cultures of avian myofibers has been shown to elicit larger fiber diameters, a near doubling in myosin content and substantial increases in protein stability and synthesis in comparison to untreated cultures (10Vandenburgh H.H. Karlisch P. Shansky J. Feldstein R. Am. J. Physiol. 1991; 260: C475-C484Crossref PubMed Google ScholarThe effects of IGF-I overexpression have previously been studied in cultured cells and in transgenic mice, but these earlier studies did not address the effects of IGF-I expression on skeletal muscle specifically (11Mathews L.S. Hammer R.E. Behringer R.R. D'Ercole A.J. Bell G.I. Brinster R.L. Palmiter R.D. Endocrinology. 1988; 123: 2827-2833Crossref PubMed Scopus (378) Google Scholar, 12Behringer R.R. Lewin T.M. Quaife C.J. Palmiter R.D. Brinster R.L. D'Ercole A.J. Endocrinology. 1990; 127: 1033-1040Crossref PubMed Scopus (174) Google Scholar). Mathews et al.(11Mathews L.S. Hammer R.E. Behringer R.R. D'Ercole A.J. Bell G.I. Brinster R.L. Palmiter R.D. Endocrinology. 1988; 123: 2827-2833Crossref PubMed Scopus (378) Google Scholar) utilized the metallothionein promoter to drive expression of an hIGF-I cDNA in transgenic mice resulting in IGF-I overexpression in a broad range of visceral internal organs and increased concentrations of IGF-I in serum. These transgenic mice exhibited an increase in body weight and organomegaly, but only a modest improvement in muscle mass. Thus, in order to test the effects of IGF-I overexpression on muscle growth and physiology in vivo, we reasoned that it would be necessary to target its overexpression specifically to striated muscle.The α-skeletal actin gene is a member of the actin multigene family which, in vertebrates, is made up of three distinct classes of actin isoforms termed as “cytoplasmic,”“striated,” and “smooth muscle” on the basis of their cellular distribution and pattern of expression in adult tissues (13Vandekerckhove J. Weber K. J. Mol. Biol. 1984; 179: PubMed Scopus (174) Google Scholar, G. 10: PubMed Scopus Google Scholar, K. J.L. Biol. 1991; PubMed Scopus Google Scholar). The striated and in and skeletal and of the of actin gene expression studied of and that and skeletal α-actin muscle only skeletal α-actin is high levels in adult skeletal muscle but in (13Vandekerckhove J. Weber K. J. Mol. Biol. 1984; 179: PubMed Scopus (174) Google Scholar, K. J.L. Biol. 1991; PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google Scholar). skeletal α-actin for of the in avian skeletal muscle J. Biol. PubMed Scopus Google Scholar) and is in classes of myofibers R. Annu. Rev. Physiol. 1989; PubMed Google Scholar). the avian skeletal α-actin gene was striated muscle differentiation, Mol. Biol. 6: PubMed Scopus Google Scholar) and Proc. Natl. Acad. Sci. U. S. A. 1990; PubMed Scopus Google Scholar) the that skeletal α-actin and from in skeletal muscle cells and transgenic studies by et C.J. Mol. Biol. 1989; PubMed Scopus Google Scholar) that the in the proximal of the promoter were for the avian skeletal α-actin expression pattern in and skeletal muscle. these studies a high of expression suggesting that from the skeletal for striated the of the human skeletal α-actin gene in and expression of skeletal in mice J. Biol. 1993; PubMed Google Scholar), and we have in in that of the promoter in the to of the Am. J. Physiol. the construction of a myogenic expression vector from the and of the avian skeletal α-actin gene and its for overexpression of an hIGF-I cDNA in cultured muscle cells and transgenic mice. that IGF-I overexpression in cultured muscle cells and fusion of myoblasts and elevated the levels of myogenic basic helix-loop-helix and contractile protein mice a of hybrid skeletal transgene hIGF-I levels that were of the skeletal α-actin gene on a expression In transgenic mice we that elevated levels of IGF-I in skeletal muscle muscle hypertrophy increases in body weight IGF-I of of the avian skeletal α-actin gene G. 10: PubMed Scopus Google Scholar, 1990; PubMed Scopus Google Scholar) and of have been previously Mol. Biol. 6: PubMed Scopus Google Scholar, Mol. Biol. 1990; 10: PubMed Google Scholar). In order to hybrid skeletal the to first and of up to the were from of Scopus Google Scholar). was utilized to the the of and the hIGF-I cDNA J.L. PubMed Scopus Google Scholar) with an on the of Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) utilizing from the with were that a by on to the by the The were to the template and with the of were with and the the were of a The were to the and the of IGF-I was by the hIGF-I cDNA J.L. PubMed Scopus Google Scholar), the IGF-I and construction on an to the skeletal α-actin the skeletal α-actin and 3’-noncoding was from the avian skeletal α-actin and to IGF-I was by the actin promoter and hIGF-I the and from IGF-I on a the of myoblast Science. PubMed Scopus Google Scholar) was utilized for of of myoblasts was by of skeletal with the The vector was by the gene J. PubMed Scopus Google Scholar) the expression R.L. 1987; PubMed Scopus Google Scholar). and with were as previously 1988; 6: Scopus Google Scholar). myoblasts were in and were to serum to myogenic differentiation and of the expression of the skeletal hybrid of resulting from of mice were with of the gene of as previously E. the Scholar). were to mice and were for the of by of J. Mol. Biol. Scopus Google Scholar). was by the for of to that of of in of was a and was from cells and tissues by from as previously P. 1987; PubMed Scopus Google Scholar). were by of on and to was to the by to and the were for and were in serum were with as for (see with were for with were in and in were to with and IGF-I concentrations in media were by as previously D.R. R.L. J. Endocrinol. PubMed Scopus Google Scholar). of C2C12 myoblasts was a of D.R. R.L. J. Endocrinol. PubMed Scopus Google Scholar) as the primary and a IGF-I concentrations in of skeletal muscle and of serum were a of skeletal muscle were as previously A.J. Stiles A.D. Underwood L.E. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 935-939Crossref PubMed Scopus (1067) Google Scholar). muscle was and in a were on for and for The was to a and the was by of and on for The was for were and The was in of and by to of for and were and to a with media with the of a for Muscle were by a of in for muscle were in of in the were to up to in the to and were for was as previously and Scholar). for the were in in and an of myofibers was with the of a were in the muscle exhibited three distinct for and were as for R. Clemmons D.R. Rotwein P. J. Biol. 1989; PubMed Google the of IGF-I order to these we IGF-I expression on the avian skeletal α-actin gene is withdrawal from the cell and myoblast fusion J. Biol. PubMed Scopus Google Scholar). of the hybrid that were utilized for the studies shown in These IGF-I expression were for expression, IGF-I and their on in stably transfected myogenic C2C12 of IGF-I content was by with a human IGF-I cDNA to from myoblasts that were from growth to differentiation media as in to of increase in IGF-I was in C2C12 IGF-I in comparison to the expression of The avian skeletal α-actin promoter expression with the IGF-I but with the skeletal α-actin and 3’-noncoding to increased expression of IGF-I myogenic levels of mRNAs the the of synthesis of and the of of the of the skeletal α-actin is to was to myogenic cultures in order to by content were for as shown in the IGF-I were to of in to skeletal α-actin stability with a to be with a (see J. Biol. PubMed Google These indicate that the avian skeletal α-actin increased stability to a avian skeletal α-actin increased the of hIGF-I in C2C12 myoblasts transfected with skeletal hybrid of C2C12 myoblasts stably transfected with the actin/IGF-I in were to and to differentiation was by of and of were with a hIGF-I cDNA J.L. PubMed Scopus Google Scholar). was by and to the hIGF-I levels the of for to of hIGF-I of IGF-I in and studies indicate that the of myoblasts the differentiation be directed by the autocrine/paracrine of J.R. Magri K. K. Rotwein J. Biol. 1991; PubMed Google Scholar, J.R. Mol. Endocrinol. 1991; PubMed Scopus Google Scholar). in cultures that myoblasts IGF-I and IGF-I exhibited a of fusion to differentiation media as to myoblasts of myogenic cultures increased of IGF-I in of stably transfected but not in C2C12 myoblasts stably transfected with an gene The with the expression of the IGF-I These that increased IGF-I expression with enhanced myotube formation. that IGF-I and IGF-I were in IGF-I expression in muscle we the biosynthesis of factor by of media of IGF-I in cultures were In cultures stably transfected with the vector IGF-I levels of IGF-I in media that were myoblasts in stably transfected with IGF-I IGF-I the level of cultures the IGF-I These results that of the skeletal α-actin and for the IGF-I enhanced IGF-I concentrations in media by muscle cells in a we to IGF-I myogenic gene of from myoblasts cultured in growth media with IGF-I enhanced expression of the myogenic basic helix-loop-helix as and in with and J.R. Mol. Endocrinol. 1991; PubMed Scopus Google Scholar). shown in we a elevated IGF-I expression and increased expression of myogenic gene as the protein and skeletal of IGF-I in muscle myogenic gene C2C12 myoblasts and myoblasts stably transfected with the actin/IGF-I hybrid gene were to and to differentiation media of C2C12 myoblasts were to differentiation media with and was and to differentiation of were for human IGF-I J.L. PubMed Scopus Google Scholar), Mol. Biol. PubMed Scopus Google Scholar), PubMed Scopus Google Scholar), J. Biol. PubMed Scopus Google Scholar), and skeletal α-actin Mol. Biol. 6: PubMed Google Scholar) mRNAs cDNA serum Expression in in a in IGF-I and of mice a of the skeletal transgene IGF-I was shown to be in cultures of myogenic C2C12 cells (see was utilized for of the effects of overexpression of IGF-I in skeletal muscle. The level and of transgene expression in of mice was to that of the skeletal α-actin gene to striated muscle and in skeletal muscle in hIGF-I was on of from skeletal muscle of transgenic mice that was not in mice Expression of the transgene in of mice was that of the skeletal α-actin gene in muscle of adult mice level transgene expression in the in concentrations of IGF-I in of skeletal muscle from transgenic mice 47-fold in mice IGF-I concentrations in serum of transgenic mice were not elevated to mice to that the hIGF-I resulting from transgene expression did not the in is by the of an of transgene expression on body weight IGF-I expression in IGF-I hybrid gene is specifically in striated muscle of transgenic mice. of from of and as IGF-I transgenic mice was to a from the of the avian skeletal α-actin gene J. Biol. PubMed Scopus Google Scholar). for tissues are: skeletal and and and of the IGF-I hybrid gene in transgenic mice is of the level of the skeletal α-actin from muscle and of transgenic and mice was to a hIGF-I cDNA J.L. PubMed Scopus Google Scholar). and of from and skeletal muscle and from and The in expression of the the in was and with a cDNA to from skeletal muscle of mice and adult transgenic from and and and mice was to skeletal α-actin Mol. Biol. 6: PubMed Google Scholar) and hIGF-I J.L. PubMed Scopus Google Scholar) cDNA to the by of the by direct on a that expression of the hybrid gene in skeletal muscle was that of the skeletal α-actin gene on a and and weight and IGF-I concentrations in serum and skeletal muscle of IGF-I and for adult transgenic and mice. in a was on of the muscle for the were on their as as has been previously R. Clemmons D.R. Rotwein P. J. Biol. 1989; PubMed Google Scholar). indicate that overexpression of IGF-I in skeletal muscle in hypertrophy of myofibers of was by increases in the of myofibers that from for high in a transgenic to for in transgenic mice as to In to an increased of a a of high myofibers in and a of myofibers in in transgenic mice. R. Clemmons D.R. Rotwein P. J. Biol. 1989; PubMed Google Scholar) has that high of myofibers with as on myosin with and to These indicate that IGF-I overexpression a fiber and in to the of classes of of myofibers in the muscle of IGF-I and for IGF-I and mice and for IGF-I and in a of the IGF-I hybrid gene in transgenic muscle hypertrophy. from adult of transgenic and mice were and with a in to were from the of the and for and Scholar), and the (see in for of in the muscle in IGF-I Expression to vertebrates, skeletal α-actin is the striated actin in adult skeletal it is a level in muscle in the (5Florini J.R. Muscle 10: 577-598Crossref PubMed Scopus (254) Google Scholar). from studies utilizing have that the for cell and expression the proximal of the avian skeletal α-actin gene promoter Mol. Biol. 6: PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1990; PubMed Scopus Google Scholar), in these were in earlier transgenic studies C.J. Mol. Biol. 1989; PubMed Scopus Google Scholar, Mol. Biol. 6: PubMed Scopus Google Scholar, G. PubMed Scopus Google Scholar). et C.J. Mol. Biol. 1989; PubMed Scopus Google Scholar) that by the avian skeletal α-actin proximal promoter to sequence to exhibited expression in transgenic in to increased expression in adult to skeletal muscle. in the that inclusion of the skeletal α-actin with of sequence in a actin/IGF-I transgene increased its expression in cultured and in expression to striated muscle in transgenic mice. level of expression in skeletal muscle was to that of the skeletal α-actin these that the to the level and of expression for the skeletal α-actin gene the IGF-I of the skeletal α-actin and sequence in a actin/IGF-I increased and protein levels in cultured and that a primary the skeletal α-actin increased expression of the actin/IGF-I hybrid gene in was was not we the of the of striated α-actin mRNAs in primary myogenic and sequence these actin isoforms in their have been in the and cellular content of the mRNAs J. Biol. 1991; PubMed Scopus Google Scholar). the to the skeletal α-actin the level of transgene expression in is and J. Biol. 1993; PubMed Google Scholar) that a with for the human skeletal α-actin and flanking expression by that In to effects of the on that the of the human striated actin that expression of by the human skeletal α-actin promoter to striated muscle as as their pattern of expression J. Biol. 1993; PubMed Google Scholar, J. Biol. PubMed Google Scholar). have that the expression of by the avian skeletal α-actin proximal promoter to be by the skeletal α-actin and of sequence for the not Thus, in to the promoter and region, the 3’-noncoding of the striated actin necessary for and expression of skeletal α-actin-based and we that avian skeletal α-actin-based expression of the skeletal α-actin proximal and sequence be utilized to target high level expression of specifically to striated of IGF-I Muscle studies in have that IGF-I elicits effects on myogenic cells stimulation of myoblast and myogenic differentiation (see Refs. 5Florini J.R. Muscle 10: 577-598Crossref PubMed Scopus (254) Google Scholar and 6Florini J.R. Magri K.A. Am. J. Physiol. 1989; 256: C701-C711Crossref PubMed Google Scholar for review). that myoblasts transfected with IGF-I levels of mRNAs did myoblasts with IGF-I the of IGF-I in media by these cells for was that the IGF-I a sustained autocrine/paracrine was as a myogenic IGF-I as a is that a insulin-like growth factor-binding the effects of In we did that expression of an that is specifically differentiation of C2C12 myoblasts W.H. Clemmons D. Rotwein P. J. Biol. 1993; PubMed Google Scholar), was with overexpression of IGF-I in not the of these that the of overexpression of IGF-I in myoblasts the role of in myoblast and of the IGF-I gene P. S. D. PubMed Scopus Google Scholar, J. A. 1993; PubMed Scopus Google Scholar) and of the I J. A. 1993; PubMed Scopus Google Scholar) have direct in for the of IGF-I in skeletal muscle et P. S. D. PubMed Scopus Google Scholar) that IGF-I mice and in and skeletal muscle. that the of IGF-I mice to by of the and et J. A. 1993; PubMed Scopus Google Scholar) that mice a for the I exhibited cell and specifically a of and In to from gene of the role of IGF-I in the of skeletal up-regulation of IGF-I expression has been as a mediator of stretch-induced myofiber hypertrophy and muscle regeneration (8DeVol D.L. Rotwein P. Sadow J.L. Novakofski J. Bechtel P.J. Am. J. Physiol. 1990; 259: E89-E95PubMed Google Scholar, A. Jennische E. Oldfors A. Edwall D. Norstedt G. Mol. Endocrinol. 1992; 6: 1227-1234Crossref PubMed Scopus (59) Google Scholar). these to a in IGF-I is a growth factor for the of the of myogenic differentiation, and growth and hypertrophy of In addition, on the overexpression of IGF-I in skeletal muscle in elicit effects of skeletal muscle expression of the skeletal α-actin gene is not in muscle myoblast fusion K. J.L. Biol. 1991; PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google overexpression of IGF-I in a skeletal vector is not to elicit effects on myoblast in the that transgenic overexpression of IGF-I hypertrophy of classes of In addition, results that overexpression of IGF-I a in myofiber fiber is with from a that growth of increased expression of an increase in the of I myofibers D. J. Endocrinol. 1989; 123: PubMed Scopus (59) Google it is not the myofiber hypertrophy in IGF-I transgenic mice, but on results from it is that overexpression of IGF-I hypertrophy a of IGF-I is to elicit effects on the of skeletal muscle that in as stimulation of and and of protein effects on protein synthesis and (see Refs. 2Sara V.R. Hall K. Physiol. Rev. 1990; 70: 591-614Crossref PubMed Scopus (741) Google Scholar and 3Cohick W.S. Clemmons D.R. Annu. Rev. Physiol. 1993; 55: 131-153Crossref PubMed Scopus (573) Google Scholar for review). Thus, it be that the myofiber hypertrophy in the was to the effects of is that overexpression of IGF-I processes that in muscle regeneration and stretch-induced hypertrophy. has that expression of IGF-I is increased in muscle (9Levinovitz A. Jennische E. Oldfors A. Edwall D. Norstedt G. Mol. Endocrinol. 1992; 6: 1227-1234Crossref PubMed Scopus (59) Google Scholar) and in muscle stretch-induced hypertrophy (8DeVol D.L. Rotwein P. Sadow J.L. Novakofski J. Bechtel P.J. Am. J. Physiol. 1990; 259: E89-E95PubMed Google Scholar), and it is that increase in IGF-I the the of on regeneration and hypertrophy of skeletal muscle the of and R. J. Biol. 1990; PubMed Scopus Google Scholar) have in studies of muscle with cells in that cell is to an IGF-I the cells first to an as factor in muscle that in myofiber hypertrophy the with overexpression of IGF-I in striated muscle. studies to address these INTRODUCTIONInsulin-like growth factor I (IGF-I),1 1The abbreviations used are: IGFinsulin-like growth factorhIGFhuman IGFbpbase pair(s)kbkilobase(s)IGFBPinsulin-like growth factor-binding protein(s). a peptide growth factor that is structurally related to proinsulin (1Daughaday W.H. Rotwein P. Endocrinol. Rev. 1989; 10: 68-91Crossref PubMed Scopus (1606) Google Scholar, 2Sara V.R. Hall K. Physiol. Rev. 1990; 70: 591-614Crossref PubMed Scopus (741) Google Scholar, 3Cohick W.S. Clemmons D.R. Annu. Rev. Physiol. 1993; 55: 131-153Crossref PubMed Scopus (573) Google Scholar, 4D'Ercole A.J. Stiles A.D. Underwood L.E. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 935-939Crossref PubMed Scopus (1067) Google Scholar), has a primary role in promoting the differentiation and growth of skeletal muscle. The effects of IGF-I on myogenic cells include stimulation of myoblast replication, myogenic differentiation, and myotube hypertrophy (see Refs. 5Florini J.R. Muscle 10: 577-598Crossref PubMed Scopus (254) Google Scholar and 6Florini J.R. Magri K.A. Am. J. Physiol. 1989; 256: C701-C711Crossref PubMed Google Scholar for review). In vivo, up-regulation of IGF-I expression in skeletal muscle is coincident with myotube formation in the developing embryo (7Han V.K.M. D'Ercole A.J. Lund P.K. Science. 1987; 236: 193-197Crossref PubMed Scopus (552) Google Scholar), stretch-induced myofiber hypertrophy (8DeVol D.L. Rotwein P. Sadow J.L. Novakofski J. Bechtel P.J. Am. J. Physiol. 1990; 259: E89-E95PubMed Google Scholar) and muscle regeneration following injury (9Levinovitz A. Jennische E. Oldfors A. Edwall D. Norstedt G. Mol. Endocrinol. 1992; 6: 1227-1234Crossref PubMed Scopus (59) Google Scholar), suggesting that IGF-I serves as an autocrine/paracrine mediator of these processes in skeletal muscle. Increased biosynthesis and extracellular secretion of IGF-I from cultured mammalian myoblasts has been shown to be coincident with myoblast alignment, withdrawal from the cell cycle, and fusion (5Florini J.R. Muscle 10: 577-598Crossref PubMed Scopus (254) Google Scholar, 6Florini J.R. Magri K.A. Am. J. Physiol. 1989; 256: C701-C711Crossref PubMed Google Scholar). In addition, inclusion of IGF-I in the media of primary cultures of avian myofibers has been shown to elicit larger fiber diameters, a near doubling in myosin content and substantial increases in protein stability and synthesis in comparison to untreated cultures (10Vandenburgh H.H. Karlisch P. Shansky J. Feldstein R. Am. J. Physiol. 1991; 260: C475-C484Crossref PubMed Google ScholarThe effects of IGF-I overexpression have previously been studied in cultured cells and in transgenic mice, but these earlier studies did not address the effects of IGF-I expression on skeletal muscle specifically (11Mathews L.S. Hammer R.E. Behringer R.R. D'Ercole A.J. Bell G.I. Brinster R.L. Palmiter R.D. Endocrinology. 1988; 123: 2827-2833Crossref PubMed Scopus (378) Google Scholar, 12Behringer R.R. Lewin T.M. Quaife C.J. Palmiter R.D. Brinster R.L. D'Ercole A.J. Endocrinology. 1990; 127: 1033-1040Crossref PubMed Scopus (174) Google Scholar). Mathews et al.(11Mathews L.S. Hammer R.E. Behringer R.R. D'Ercole A.J. Bell G.I. Brinster R.L. Palmiter R.D. Endocrinology. 1988; 123: 2827-2833Crossref PubMed Scopus (378) Google Scholar) utilized the metallothionein promoter to drive expression of an hIGF-I cDNA in transgenic mice resulting in IGF-I overexpression in a broad range of visceral internal organs and increased concentrations of IGF-I in serum. These transgenic mice exhibited an increase in body weight and organomegaly, but only a modest improvement in muscle mass. Thus, in order to test the effects of IGF-I overexpression on muscle growth and physiology in vivo, we reasoned that it would be necessary to target its overexpression specifically to striated muscle.The α-skeletal actin gene is a member of the actin multigene family which, in vertebrates, is made up of three distinct classes of actin isoforms termed as “cytoplasmic,”“striated,” and “smooth muscle” on the basis of their cellular distribution and pattern of expression in adult tissues (13Vandekerckhove J. Weber K. J. Mol. Biol. 1984; 179: PubMed Scopus (174) Google Scholar, G. 10: PubMed Scopus Google Scholar, K. J.L. Biol. 1991; PubMed Scopus Google Scholar). The striated and in and skeletal and of the of actin gene expression studied of and that and skeletal α-actin muscle only skeletal α-actin is high levels in adult skeletal muscle but in (13Vandekerckhove J. Weber K. J. Mol. Biol. 1984; 179: PubMed Scopus (174) Google Scholar, K. J.L. Biol. 1991; PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google Scholar). skeletal α-actin for of the in avian skeletal muscle J. Biol. PubMed Scopus Google Scholar) and is in classes of myofibers R. Annu. Rev. Physiol. 1989; PubMed Google Scholar). the avian skeletal α-actin gene was striated muscle differentiation, Mol. Biol. 6: PubMed Scopus Google Scholar) and Proc. Natl. Acad. Sci. U. S. A. 1990; PubMed Scopus Google Scholar) the that skeletal α-actin and from in skeletal muscle cells and transgenic studies by et C.J. Mol. Biol. 1989; PubMed Scopus Google Scholar) that the in the proximal of the promoter were for the avian skeletal α-actin expression pattern in and skeletal muscle. these studies a high of expression suggesting that from the skeletal for striated the of the human skeletal α-actin gene in and expression of skeletal in mice J. Biol. 1993; PubMed Google Scholar), and we have in in that of the promoter in the to of the Am. J. Physiol. the construction of a myogenic expression vector from the and of the avian skeletal α-actin gene and its for overexpression of an hIGF-I cDNA in cultured muscle cells and transgenic mice. that IGF-I overexpression in cultured muscle cells and fusion of myoblasts and elevated the levels of myogenic basic helix-loop-helix and contractile protein mice a of hybrid skeletal transgene hIGF-I levels that were of the skeletal α-actin gene on a expression In transgenic mice we that elevated levels of IGF-I in skeletal muscle muscle hypertrophy increases in body weight IGF-I