Role In Muscle Development Research Articles

An alternatively-spliced mRNA in the carboxy terminus of the neurofibromatosis type 1 (NF1) gene is expressed in muscle.

The gene for neurofibromatosis type 1 (NF1) was identified by positional cloning and found to contain two alternatively spliced exons. The first described alternatively spliced exon (exon 23a) is located within the GAP-related domain of the gene and inserts an additional 63 nucleotides into the NF1 mRNA. The second alternatively spliced exon (exon 48a) is located near the extreme carboxy terminus of the gene and inserts an additional 54 nucleotides into the mRNA. This second isoform, termed 3'ALT, was originally detected while screening a fetal brain cDNA library. Examination of its expression by reverse-transcribed RNA PCR demonstrates high level of expression in cardiac muscle, skeletal muscle and smooth muscle. Trace levels of expression are detected in brain and nerve. The 3'ALT isoform is expressed in fetal cardiac muscle, adult left ventricle and cardiac Purkinje cells. Further confirmation of the existence of this isoform was obtained by blotting the PCR products with a radiolabeled oligonucleotide entirely derived from sequences contained within exon 48a and by direct sequencing of the PCR products. Additionally, this isoform is expressed in muscle tissues from other vertebrate species. The expression of this isoform in muscle suggests that the NF1 gene may play additional tissue-specific roles in muscle development and signal transduction.

Skeletal Muscle Regeneration and Plasticity of Grafts

The sequence of molecular and cellular events of muscle ontogeny leads to the proliferation, fusion, and differentiation of myoblasts to muscle cells. This sequence is closely paralleled in the grafting-ischemia model in which adult myoblast-satellite cells function as the muscle precursor cells. The study of skeletal muscle regeneration is a fertile and promising area of research in myogenesis. The early regenerative development and maturation of muscle is similar regardless of the perturbation that induced the degeneration-regeneration sequelae. In light of this, we maintain that the skeletal muscle graft model is useful to rigorously evaluate many regulatory aspects of skeletal muscle development and maturation in an adult animal host. One advantage of the graft model is that manipulation of the adult host, such as with exercise or hormone treatment, allows insight into their regulatory roles in muscle development and maturation. These approaches are often not possible for developing skeletal muscle in utero or in ovo. After skeletal muscle grafting, many structural and functional characteristics change with time until they reach a stable value. Successful regeneration requires revascularization, cellular infiltration, phagocytosis of necrotic muscle fibers, proliferation and fusion of muscle precursor cells, reinnervation, and recruitment and loading. The time taken to reach stable values varies among different structural and functional variables, and many reach stable values that are less than those of control skeletal muscle. There are differences in the degree of regenerative success because of the size of muscle mass grafted. In small and large grafts, regeneration is enhanced by facilitation of the reinnervation. Regeneration is evident without vascular repair in grafts of up to approximately 6 g, although in all but the 100 to 150-mg grafts in rats, a significant necrotic core is present. Regeneration is typically unsuccessful when muscle masses greater than 6 g are grafted without vascular repair. Large muscles can be grafted with vascular repair, and in this case, the cellular response is quite different, as the majority of fibers survive rather than degenerate and regenerate. Changing the components of physical activity during skeletal muscle regeneration can alter several attributes of the graft phenotype. The consensus of several experiments supports the interpretation that proper recruitment and force development by grafts are essential variables in the regulation of the development and maturation of muscle grafts. Morphological and physiological attributes of grafts adapt to changes in the habitual level of physical activity in a qualitatively similar fashion to control muscle.(ABSTRACT TRUNCATED AT 400 WORDS)

Cardiac fibroblasts: function, regulation of gene expression, and phenotypic modulation.

Cardiac fibroblasts constitute the majority of the non-myocyte cell population in the ventricular myocardium. These cells are located in the interstitium, in areas between and surrounding cardiac myocytes. Cardiac fibroblasts are responsible for the synthesis of extracellular matrix proteins such as fibrillar collagen types I and III, basement membrane type IV collagen, fibronectin, and laminin. In addition to its role in muscle development and myoblast differentiations, extracellular matrix consisting primarily of fibrillar collagen is an intricate and highly organized structure that serves to support cardiac myocytes and to maintain functional integrity of the myocardium. Balanced synthesis and degradation of this matrix is the key to normal development of cardiac muscle and perfect myocardial function. Collagen remodeling and accumulation has been demonstrated in several experimental models of cardiac hypertrophy. To gain insights into molecular and cellular mechanisms that affect cardiac fibroblast behavior, cardiac fibroblasts from rat and rabbit ventricular myocardium were cultured and the impact of neurotransmitters and growth factors such as norepinephrine and transforming growth factor--beta (TGF-beta 1), to which cardiac fibroblasts are exposed in vivo, was studied. Results of these studies, with regards to gene expression, proliferation and differentiation of cardiac fibroblasts in culture, and their biological implications are discussed.

Cdc2 and the regulation of mitosis: six interacting mcs genes

A cdc2-3w weel-50 double mutant of fission yeast displays a temperature-sensitive lethal phenotype that is associated with gross abnormalities of chromosome segregation and has been termed mitotic catastrophe. In order to identify new genetic elements that might interact with the cdc2 protein kinase in the regulation of mitosis, we have isolated revertants of the lethal double mutant. The suppressor mutations define six mcs genes (mcs: mitotic catastrophe suppressor) that are not allelic to any of the following mitotic control genes: cdc2, wee 1, cdc13, cdc25, suc1 or nim1. Each mcs mutation is recessive with respect to wild-type in its ability to suppress mitotic catastrophe. None confer a lethal phenotype as a single mutant but few of the mutants are expected to be nulls. A diverse range of genetic interactions between the mcs mutants and other mitotic regulators were uncovered, including the following examples. First, mcs2 cdc2w or mcs6 cdc2w double mutants display a cell cycle defect dependent on the specific wee allele of cdc2. Second, both mcs1 cdc25-22 or mcs4 cdc25-22 double mutants are nonconditionally lethal, even at a temperature normally permissive for cdc25-22. Finally, the characteristic suppression of the cdc25 phenotype by a loss-of-function wee1 mutation is reversed in a mcs3 mutant background. The mcs genes define new mitotic elements that might be activators or substrates of the cdc2 protein kinase.

Fetal rat myoblasts release both rat somatomedin-C (SM-C)/insulin-like growth factor I (IGF I) and multiplication-stimulating activity in vitro: partial characterization and biological activity of myoblast-derived SM-C/IGF I.

The relative release of rat somatomedin-C (SM-C)/insulin-like growth factor I (IGF I) and multiplication-stimulating activity (MSA) immunoreactivity and bioactivity from isolated fetal rat myoblasts was assessed by a partial characterization of the SM peptides present in concentrated myoblast-conditioned culture medium (MCM). The SM bioactivity of MCM, measured by [3H]thymidine or [35S]sulfate uptake by fetal rat cartilage explants, eluted with an apparent size of 50-80K on Sephadex G-200 at pH 7.5, and was associated with SM-C/IGF I immunoreactivity. Chromatography of MCM on Bio-Gel P-10 or Sephadex G-75 at acidic pH resulted in a peak of SM bioactivity associated with both SM-C/IGF I and MSA immunoreactivity in the 6-9K region. SM-binding activity, measured by competition with activated charcoal for [125I]SM-C or MSA, eluted in the void volume. When these fractions were incubated with [125I]SM-C and chromatographed on Sephadex G-200 at neutral pH, a heterogeneous pattern of binding proteins was seen, with a major component of 50-80K. After chromatofocusing of proteins in the 6-9K region from Bio-Gel P-10, three peaks of SM bioactivity were recovered, each associated with SM-C immunoreactivity, with pI values of 8.5, 7.1, and 6.5. Although both the basic and neutral peaks enhanced [3H]thymidine uptake by growth-restricted fetal rat myoblasts in vitro, only the bioactivity of the former could be blocked by incubation with a monoclonal antibody to human SM-C. Both human SM-C/IGF I and MSA purified from Buffalo rat liver cell-conditioned medium enhanced thymidine incorporation by growth-restricted fetal rat myoblasts. The results suggest that unlike reports of other fetal rat tissues, fetal rat myoblasts released approximately equal amounts of rat SM-C/IGF I and MSA during culture. The myoblast-derived SM-C/IGF I was biologically active on the cell type of origin and may play a paracrine role in muscle development.

Neuromuscular transmission in the athymic nude mouse

No major differences were observed in the mechanical properties of diaphragm, extensor digitorum longus and soleus muscles from athymic nude and control mice. Denervated soleus muscles from nudes and controls showed no significant differences in their sensitivities to the cholinoceptor agonists acetylcholine and carbachol, either in the absence or presence of the anticholinesterase, physostigmine, suggesting that postjunctional receptor function is essentially normal. Phrenic nerve-diaphragm preparations from nudes were less sensitive to the twitch-augmenting effects of neostigmine. No difference in the time course of endplate potentials (epps) between nudes and controls was seen either in the absence or presence of neostigmine. Hence the observed differences in twitch augmentation are unlikely to be due to differences in acetylcholinesterase activity in the two muscles. In normal mice miniature endplate potential (mepp) amplitude decreased and mepp frequency increased with age. These changes were associated with an increase in muscle fibre diameter and a concomitant decrease in membrane resistance. Such changes did not occur in nude mice; thus mepp amplitude remained, high as in young normal muscle. It is suggested that the thymus may play a role in muscle development and that the effects on neuromuscular transmission are secondary to changes in development. In cut diaphragm muscles transmitter reversal potentials in nudes and controls were not different. Although there was no difference in the amplitude of the first epp of a train, or in the immediately releasable acetylcholine store, the quantal content of the first epp, the probability of transmitter release, the total nerve terminal acetylcholine store and the transmitter mobilization rate were all reduced. It is considered probable that all the measurable differences in transmitter release can be explained in terms of the nude muscle fibre diameter being small and being associated with a small nerve terminal size.