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Cardiac actin is the major actin gene product in skeletal muscle cell differentiation in vitro.

We examined the expression of alpha-skeletal, alpha-cardiac, and beta- and gamma-cytoskeletal actin genes in a mouse skeletal muscle cell line (C2C12) during differentiation in vitro. Using isotype-specific cDNA probes, we showed that the alpha-skeletal actin mRNA pool reached only 15% of the level reached in adult skeletal muscle and required several days to attain this peak, which was then stably maintained. However, these cells accumulated a pool of alpha-cardiac actin six times higher than the alpha-skeletal actin mRNA peak within 24 h of the initiation of differentiation. After cells had been cultured for an additional 3 days, this pool declined to 10% of its peak level. In contrast, over 95% of the actin mRNA in adult skeletal muscle coded for alpha-actin. This suggests that C2C12 cells express a pattern of sarcomeric actin genes typical of either muscle development or regeneration and distinct from that seen in mature, adult tissue. Concurrently in the course of differentiation the beta- and gamma-cytoskeletal actin mRNA pools decreased to less than 10% of their levels in proliferating cells. The decreases in beta- and gamma-cytoskeletal actin mRNAs are apparently not coordinately regulated.

Differential patterns of transcript accumulation during human myogenesis

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 alpha-skeletal, alpha-cardiac, beta- and gamma-actin, total myosin heavy chain, and alpha- and beta-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 alpha-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 alpha-actin. Whereas beta- and gamma-actin displayed a sharp decrease after initiation of fusion and thereafter did not change, alpha- and beta-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.

Evolution of the human sarcomeric-actin genes: Evidence for units of selection within the 3′ untranslated regions of the mRNAs

SummaryThe complete 3′ untranslated region (3′UTR) sequence of the human skeletal-actin gene has been compared with the corresponding regions of the rat and chicken skeletal-actin genes. This comparison reveals that the skeletal-actin 3′UTR is composed of conserved and nonconserved segments. By using genomic Southern transfer blots and thermal stability (Tm) measurements, we found that the cardiac-actin gene 3′UTR also consists of conserved and nonconserved segments. Comparison of human andXenopus laevis cardiac-actin mRNA sequences confirms the presence of a region of high similarity in the 3′UTR. We conclude that subsegments of the 3′UTRs of both skeletal- and cardiac-actin genes of birds and mammals are under considerable selective pressure. This suggests that these conserved sequences may have functional roles in actin-gene expression or regulation, and that these roles might be different for each actin isoform.

Expression of human cardiac actin in mouse L cells: A sarcomeric actin associates with a nonmuscle cytoskeleton

A cloned human cardiac actin gene, introduced into mouse Ltk- cells, is expressed in several thymidine kinase (tk)-positive cotransfectants. The clones not only produce authentic polyadenylated human cardiac actin mRNA but also synthesize human cardiac actin protein. The cardiac actin protein, normally found only in myofibrils, is stably accumulated at a high level, about one-third that of the endogenous mouse beta-actin. Furthermore, this sarcomeric protein partitions between the Triton X-100 insoluble and soluble phases to the same extent as the endogenous beta-actin. This suggests that a sarcomeric actin can participate in the formation of Triton X-100-insoluble cytoskeletal structures.