Inhibition of desmin expression blocks myoblast fusion and interferes with the myogenic regulators MyoD and myogenin.

The muscle-specific intermediate filament protein, desmin, is one of the earliest myogenic markers whose functional role during myogenic commitment and differentiation is unknown. Sequence comparison of the presently isolated and fully characterized mouse desmin cDNA clones revealed a single domain of polypeptide similarity between desmin and the basic and helix-loop-helix region of members of the myoD family myogenic regulators. This further substantiated the need to search for the function of desmin. Constructs designed to express anti-sense desmin RNA were used to obtain stably transfected C2C12 myoblast cell lines. Several lines were obtained where expression of the anti-sense desmin RNA inhibited the expression of desmin RNA and protein down to basal levels. As a consequence, the differentiation of these myoblasts was blocked; complete inhibition of myoblast fusion and myotube formation was observed. Rescue of the normal phenotype was achieved either by spontaneous revertants, or by overexpression of the desmin sense RNA in the defective cell lines. In several of the cell lines obtained, inhibition of desmin expression was followed by differential inhibition of the myogenic regulators myoD and/or myogenin, depending on the stage and extent of desmin inhibition in these cells. These data suggested that myogenesis is modulated by at least more than one pathway and desmin, which so far was believed to be merely an architectural protein, seems to play a key role in this process.

A single MEF2 site governs desmin transcription in both heart and skeletal muscle during mouse embryogenesis.

Desmin, the muscle-specific intermediate filament protein is one of the earliest known myogenic makers both in heart and in somites. We have previously shown that high levels of desmin expression in the skeletal cell line C2C12 are due to a distal enhancer, which contains a muscle-specific factor-(2MEF2) binding site, adjacent to an E box, the binding site of the myogenic Helix-Loop-Helix (mHLH) regulators. We have further shown that MEF2C, a myocyte restricted member of the MEF2 family and all four mHLH factors can bind to their corresponding sites and through a cooperation with a second proximal E box can transactivate the desmin promoter. To study the significance of these regulatory elements in vivo, we have generated transgenic mice with desmin-lacZ reporters, intact or mutated at the MEF2 and E box of the enhancer. We show that the cis-acting DNA sequences within the 1-kb 5' flanking region of the mouse desmin gene are sufficient to direct appropriate temporal transcription both in heart and in skeletal muscle during mouse embryogenesis. Mutation at the MEF2 site completely suppressed transcription of the linked lacZ transgene in both developing heart and somites of the embryos. Mutation of the E box only suppressed activation in skeletal muscle precursors (somites and limb buds) but not in cardiac muscle. These data demonstrate that the MEF2 site is indispensable for the desmin enhancer function both in heart and in skeletal muscle. In addition, MEF2 cooperation with the mHLH regulators is absolutely necessary for proper transcriptional activity during embryonic skeletal muscle development.