Differential patterns of transcript accumulation during human myogenesis.

Abstract

We evaluated the extent to which muscle-specific genes display identical patterns of mRNA accumulation during human myogenesis. Cloned satellite cells isolated from adult human skeletal muscle were expanded in culture, and RNA was isolated from low-and high-confluence cells and from fusing cultures over a 15-day time course. The accumulation of over 20 different transcripts was compared in these samples with that in fetal and adult human skeletal muscle. The expression of carbonic anhydrase 3, myoglobin, HSP83, and mRNAs encoding eight unknown proteins were examined in human myogenic cultures. In general, the expression of most of the mRNAs was induced after fusion to form myotubes. However, several exceptions, including carbonic anhydrase and myoglobin, showed no detectable expression in early myotubes. Comparison of all transcripts demonstrated little, if any, identity of mRNA accumulation patterns. Similar variability was also seen for mRNAs which were also expressed in nonmuscle cells. Accumulation of mRNAs encoding oa-skeletal, ft-cardiac, I-and y-actin, total myosin heavy chain, and ot-and I-tubulin also displayed discordant regulation, which has important implications for sarcomere assembly. Cardiac actin was the only muscle-specific transcript that was detected in low-confluency cells and was the major ft-actin mRNA at all times in fusing cultures. Skeletal actin was transiently induced in fusing cultures and then reduced by an order of magnitude. Total myosin heavy-chain mRNA accumulation lagged behind that of at-actin. Whereas ,B-and -y-actin displayed a sharp decrease after initiation of fusion and thereafter did not change, at-and I-tubulin were transiently induced to a high level during the time course in culture. We conclude that each gene may have its own unique determinants of transcript accumulation and that the phenotype of a muscle may not be determined so much by which genes are active or silent but rather by the extent to which their transcript levels are modulated. Finally, we observed that patterns of transcript accumulation established within the myotube cultures were consistent with the hypothesis that myoblasts isolated from adult tissue recapitulate a myogenic developmental program. However, we also detected a transient appearance of adult skeletal muscle-specific transcripts in high-confluence myoblast cultures. This indicates that the initial differentiation of these myoblasts may reflect a more complex process than simple recapitulation of development.

Identification of a program of contractile protein gene expression initiated upon skeletal muscle differentiation

“…Human myogenic cells when differentiated in vitro form myotubes that express an isoform phenotype differing from that of adult human muscle for 8 contractile protein gene families (Gunning et al, 1987;Wade et al, 1990). It would appear, then, that newly differentiated skeletal muscle cells, both in vivo and in vitro, exhibit a contractile protein isoform phenotype that is not like the isoform phenotype of the adult muscle fiber.…”

A combination of closely associated positive and negative cis-acting promoter elements regulates transcription of the skeletal alpha-actin gene.

“…from replicating myoblasts to terminally differentiated myotubes appears to regulate the coexpression of other musclespecific contractile gene products such as myosin heavy chains (42,49), cardiac troponin T (10), and cardiac cx-actin (26). Transfection studies of these contractile protein genes showed regulated expression in muscle cell culture, as well, by sequences often present within a few hundred base pairs of the transcription start site (6,40,44).…”

Muscle-specific expression of the cardiac alpha-actin gene requires MyoD1, CArG-box binding factor, and Sp1.

“…expressed in both skeletal muscle and heart during embryogenesis (Minty et al 1982), and is the dominant ~-actin gene expressed during differentiation of both established cell lines (Bains et al 1984) and primary myogenic cells (Minty et al 1986a;Gunning et al 1987). The HCA gene promoter is expressed exclusively in myogenic cells (Minty and Kedes 1986a, b) and contains numerous nuclear protein-binding domains upstream of the TATA box (Gustafson and Kedes 1989), several of which display cooperative transcriptional activating interactions .…”