David Yaffe

Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle.

THE muscular dystrophies are a group of hereditary disorders manifested by a progressive wasting of the skeletal muscles. In spite of extensive studies, the nature of the primary lesion is unknown (for review see ref. 1). Because of the complex interaction between tissues, it is difficult to study this question in vivo. Therefore attempts have been made to investigate this question in cultures of dystrophic muscles of human or animal origin. Tissue explants as well as monolayer primary cell cultures contain, in addition to the myogenic cells, a heterogeneous cell population, the composition of which might differ in normal and dystrophic muscle cultures. It is difficult in such experiments to distinguish between properties intrinsic to the myogenic cells and effects exerted by other cell types. Indeed, previous experiments have yielded conflicting conclusions2–6. We therefore tested the possibility of obtaining cell cultures consisting of pure populations of myogenic cells obtained from dystrophic muscles. The present report describes the isolation of a cloned population of such cells, derived from adult dystrophic mouse muscle, that can proliferate and differentiate in cell culture.

Alterations of enzymatic activities during muscle differentiation in vitro

Abstract The activity of creatine phosphokinase, myokinase, and glycogen phosphorylase in muscle cells grown under controlled culture conditions was investigated. Cell fusion and formation of multinucleated muscle fibers were found to be closely associated with a severalfold increase in enzyme activity. The correlation is manifested both in primary cultures and during the differentiation of myogenic cell lines which were maintained for several months under conditions of continuous multiplication. Cytochemical staining for glycogen phosphorylase showed that the increased activity is localized within the multinucleated fibers. Experiments in which the duration of cell fusion was controlled by the concentration of Ca2+ in the nutritional medium suggested that the increase in enzyme activity is largely dependent on the continuation of the fusion process. Application of actinomycin D to cultures during the stage of rapid increase in enzymatic activity did not prevent activity from increasing for several hours, whereas application of inhibitors of protein synthesis did inhibit the increase in activity. In the presence of cyclohexamide, the process of cell fusion was almost totally inhibited while in the presence of actinomycin D fusion continued for several hours. These experiments suggest the possibility that the messenger RNA molecules which specify the synthesis of proteins essential for cell fusion and increased enzymatic activity are formed at a developmental stage preceding cell fusion.

The in vitro cultivation and differentiation capacities of myogenic cell lines.

Summary The ability of mononucleated muscle precursor cells to multiply continuously and differentiate in vitro was studied. Repeated selective serial passages of myoblasts obtained from newborn rat thigh muscle resulted in the establishment of 6 independently isolated myogenic cell lines. These lines manifested distinct differences in cell morphology and extent of differentiation. Clonal analysis has shown that the capacity to differentiate is retained by the cells of all lines, but that there are differences in the expression of this capacity.

Chapter 2 Cellular Aspects of Muscle Differentiation in Vitro

Publisher Summary This chapter reviews a series of experiments that utilize muscle cell cultures as a model for studying some of the inherent properties of differentiating cells. A main outcome of the experiments is the establishment of myogenic cell lines that are able to multiply for extended periods in culture and retain their capacity to differentiate. It is not clear why some of the attempts to establish myogenic lines resulted in the loss of the cells by degeneration and cessation of multiplication whereas others were successful. This experience is common for many kinds of mammalian or avian cells serially passaged in vitro . The establishment of myogenic cell lines is of special interest with relevance to this phenomenon as it shows that the changes, which take place in the growth characteristics during establishment of cell lines, may be distinct from the differentiation characteristics of these lines. The possibility of cloning and thus performing analyses and experiments on homogeneous populations of one kind of cell makes this system valuable and versatile for studying many aspects of cell differentiation. Differentiated primary cultures always contain a population of mononucleated cells that do not participate in fusion. The relative proportion between the amount of mononucleated and multinucleated cells is variable and is influenced by culture conditions. Also, under identical culture conditions, clones produced by different cell lines differ considerably in their mononucleated cell content.

Developmental changes preceding cell fusion during muscle differentiation in vitro.

Summary Myoblasts obtained from newborn rat skeletal muscle undergo changes in vitro before fusion into multinucleated fibers. This is reflected, under standard culture conditions, in a lag period of about 52 h between the plating of the cells and the onset of a period of rapid fusion. When myoblasts, maintained in culture for different periods are mixed together, fusion takes place only between cells which had completed these developmental changes. The time at which the cells complete their pre-fusionchanges can be controlled to a great extent by the composition of the nutritional medium. A change from an inhibitory to a permissive medium results in the onset of very intense cell fusion after a lag period of about 18 h.

The formation of hybrid multinucleated muscle fibers from myoblasts of different genetic origin

Abstract Radioautographic investigation of tissue cultures prepared from thymidine-H 3 -labeled and unlabeled cells showed that multinucleated muscle fibers were formed by the cytoplasmic fusion of mononucleated cells. When labeled thigh muscle cells of rat origin were mixed with unlabeled thigh muscle cells obtained from rabbit, calf, or chicken, hybrid fibers consisting of both types of nuclei were formed. The fusion was found to show cell type specificity, as indicated by the selective segregation and fusion of cells from thigh muscle origin, when mixed with cells of kidney or heart origin.

Dissecting muscle and neuronal disorders in a Drosophila model of muscular dystrophy.

Perturbation in the Dystroglycan (Dg)–Dystrophin (Dys) complex results in muscular dystrophies and brain abnormalities in human. Here we report that Drosophila is an excellent genetically tractable model to study muscular dystrophies and neuronal abnormalities caused by defects in this complex. Using a fluorescence polarization assay, we show a high conservation in Dg–Dys interaction between human and Drosophila. Genetic and RNAi‐induced perturbations of Dg and Dys in Drosophila cause cell polarity and muscular dystrophy phenotypes: decreased mobility, age‐dependent muscle degeneration and defective photoreceptor path‐finding. Dg and Dys are required in targeting glial cells and neurons for correct neuronal migration. Importantly, we now report that Dg interacts with insulin receptor and Nck/Dock SH2/SH3‐adaptor molecule in photoreceptor path‐finding. This is the first demonstration of a genetic interaction between Dg and InR.

Muscle-specific activation of a methylated chimeric actin gene

To understand how DNA methylation affects tissue-specific activation of genes, we have transfected in vitro methylated alpha-actin (skeletal) constructs into fibroblasts, which do not produce endogenous alpha-actin, and into a myogenic line, which is inducible for alpha-actin expression. Although methylation significantly inhibits the expression of these constructs in fibroblasts, it does not in myoblasts. The methylation pattern of the introduced methylated genes reveals specific demethylations in the transfected molecules in myoblasts but not in fibroblasts, and it precisely mimics the methylation pattern found in myoblasts in vivo.

The effect of actinomycin D on heart and thigh muscle cells grown in vitro

The in vitro morphogenetic effects of suppression of RNA synthesis by actinomycin D, on thigh and cardiac muscle cells, was studied. Nondifferentiated cells were found to be highly susceptible to the effects of the drug and were destroyed within 24 hours after actinomycin application. On the other hand, muscle cells that had already undergone differentiation were relatively resistant to the effects of actinomycin, although the drug was shown to have penetrated the cells and to have suppressed their RNA synthesis. The multinucleated muscle fibers increased rates of contraction following the application of the drug. Inhibitors of protein synthesis, such as puromycin and fluorophenylalanine, abolished this differential effect and damaged all cell types. It is therefore suggested that the synthesis of proteins essential for the maintenance of the functional properties of muscle cells continues under conditions of suppression of DNA-dependent RNA synthesis.

Dystrophin deficiency in Drosophila reduces lifespan and causes a dilated cardiomyopathy phenotype

A number of studies have been conducted recently on the model organism Drosophila to determine the function of genes involved in human disease, including those implicated in neurological disorders, cancer and metabolic and cardiovascular diseases. The simple structure and physiology of the Drosophila heart tube together with the available genetics provide a suitable in vivo assay system for studying cardiac gene functions. In our study, we focus on analysis of the role of dystrophin (Dys) in heart physiology. As in humans, the Drosophila dys gene encodes multiple isoforms, of which the large isoforms (DLPs) and a truncated form (Dp117) are expressed in the adult heart. Here, we show that the loss of dys function in the heart leads to an age‐dependent disruption of the myofibrillar organization within the myocardium as well as to alterations in cardiac performance. dys RNAi‐mediated knockdown in the mesoderm also shortens lifespan. Knockdown of all or deletion of the large isoforms increases the heart rate by shortening the diastolic intervals (relaxation phase) of the cardiac cycle. Morphologically, loss of the large DLPs isoforms causes a widening of the cardiac tube and a lower fractional shortening, a phenotype reminiscent of dilated cardiomyopathy. The dilated dys mutant phenotype was reversed by expressing a truncated mammalian form of dys (Dp116). Our results illustrate the utility of Drosophila as a model system to study dilated cardiomyopathy and other muscular‐dystrophy‐associated phenotypes.