The total content of myosin heavy chains (MHC) and their isoform pattern were studied by biochemical methods in the slow-twitch (soleus) and fast-twitch (extensor digitorum longus) muscles of adult rat during atrophy after denervation and recovery after self-reinnervation. The pattern of fibre types, in terms of ultrastructure, was studied in parallel.After denervation, total MHC content decreased sooner in the slow-twitch muscle than in the fast-twitch. The ratio of MHC-1 and the MHC-2B isoforms to the MHC-2A isoform decreased in the slow and the fast denervated muscles, respectively. After reinnervation of the slow muscle, the normal pattern of MHC recovered within 10 days and the type 1 isoform increased above the normal. In the reinnervated fast muscle, the 2B/2A isoform ratio continued to decrease. Traces of the embryonic MHC isoform, identified by immunochemistry, were found in both denervated and reinnervated slow and fast muscles. A shift in fibre types was similar to that found in the MHC isoforms. Within 2 months of recovery a tendency to normalization was observed.The results show that (a) MHC-2B isoform and the morphological characteristics of the 2B-type muscle fibres are susceptible to lack of innervation, similar to those of type 1, (b) during muscle recovery induced by reinnervation the MHC isoforms and muscle fibres shift transiently to type 1 in the soleus and to type 2A in the extensor digitorum longus muscles, and (c) the embryonic isoform of MHC may appear in the adult skeletal muscles if innervation is disturbed.During the last decade, plasticity of the striated muscle has been intensively studied (results are summarized in [l -31) and the general rules of denervation atrophy and of reinnervation recovery are well known. However, it is not fully understood how in the mature striated muscle a disturbed innervation influences remodelling of the contractile apparatus. We would like to stress that in the slow leg muscle (in adult rats) atrophying after denervation, the myosin filaments disappear before the actin filaments [4] ; simultaneously the muscle content of myosin heavy chains (MHC) decreases considerably. After reinnervation, when the muscle begins to recover, its MHC content increases rapidly in parallel with recovery of the correct proportion and arrangement of thin and thick filaments [4, 51. Nevertheless, it is not known how the total content of MHC changes in the denervated and reinnervated fast muscle.Myosin polymorphism is characteristic of different types of skeletal muscle fibres. The isoform composition of myosin may become adapted to modifications of functional requirements (for review see [6, 71). In chronically denervated mixed muscles (2 -6 months after the operation) expression of the fast isoform of myosin prevails, while after a long-term reinnervation (6 months of recovery) myosin composition is simi- lar to that of the normal muscle [8, 91. On the other hand, it is not clear how the myosin isoforms change in the shortterm denervated muscle and whether any lack of inne...

We tested the hypothesis that the MEK/Erk/caldesmon phosphorylation cascade regulates PKCmediated podosome dynamics in A7r5 cells. We observed the phosphorylation of MEK, Erk and caldesmon, and their translocation to the podosomes upon phorbol dibutyrate (PDBu) stimulation, together with the nuclear translocation of phospho-MEK and phospho-Erk. After MEK inhibition by U0126, Erk translocated to the interconnected actin-rich columns but failed to translocate to the nucleus, suggesting that podosomes served as a site for Erk phosphorylation. The interconnected actin-rich columns in U0126-treated, PDBu-stimulated cells contained α-actinin, caldesmon, vinculin, and metalloproteinase-2. Caldesmon and vinculin became integrated with F-actin at the columns, in contrast to their typical location at the ring of podosomes. Live-imaging experiments suggested the growth of these columns from podosomes that were slow to disassemble. The observed modulation of podosome size and life time in A7r5 cells overexpressing wild-type and phosphorylation-deficient caldesmon-GFP mutants in comparison to untransfected cells suggests that caldesmon and caldesmon phosphorylation modulate podosome dynamics in A7r5 cells. These results suggest that Erk1/2 and caldesmon differentially modulate PKC-mediated formation and/or dynamics of podosomes in A7r5 vascular smooth muscle cells.

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SummaryThe actin-binding protein caldesmon (CaD) exists both in smooth muscle (the heavy isoform, h-CaD) and non-muscle cells (the light isoform, l-CaD). In smooth muscles h-CaD binds to myosin and actin simultaneously and modulates the actomyosin interaction. In non-muscle cells l-CaD binds to actin and stabilizes the actin stress fibers; it may also mediate the interaction between actin and non-muscle myosins. Both h-and l-CaD are phosphorylated in vivo upon stimulation. The major phosphorylation sites of h-CaD when activated by phorbol ester are the Erk-specific sites, modification of which is attenuated by the MEK inhibitor PD98059. The same sites in l-CaD are also phosphorylated when cells are stimulated to migrate, whereas in dividing cells l-CaD is phosphorylated more extensively, presumably by cdc2 kinase. Both Erk and cdc2 are members of the MAPK family. Thus it appears that CaD is a downstream effector of the Ras signaling pathways. Significantly, the phosphorylatable serine residues shared by both CaD isoforms are in the C-terminal region that also contains the actin-binding sites. Biochemical and structural studies indicated that phosphorylation of CaD at the Erk sites is accompanied by a conformational change that partially dissociates CaD from actin. Such a structural change in h-CaD exposes the myosin-binding sites on the actin surface and allows actomyosin interactions in smooth muscles. In the case of non-muscle cells, the change in l-CaD weakens the stability of the actin filament and facilitates its disassembly. Indeed, the level of l-CaD modification correlates very well in a reciprocal manner with the level of actin stress fibers. Since both cell migration and cell division require dynamic remodeling of actin cytoskeleton that leads to cell shape changes, phosphorylation of CaD may therefore serve as a plausible means to regulate these processes. Thus CaD not only links the smooth muscle contractility and non-muscle motility, but also provides a common mechanism for the regulation of cell migration and cell proliferation.Abbreviations: BPM -benzophenone maleimide; CaD -caldesmon; CaM -calmodulin; EM -electron microscopy; Erk -extracellular signal-regulated kinase; FRET -fluorescence resonance energy transfer; GFP -green fluorescent protein; h-CaD -smooth muscle caldesmon; IAEDANS -5-(iodoacetamidoethyl) aminonaphthalene-1-sulfonic acid; l-CaD -non-muscle caldesmon; MAPK -mitogen activated protein kinase; MLCK -myosin light chain kinase; MS -mass spectrometry; Pak -p21-activated protein kinase; PMA -phorbol 12-myristate 13-acetate; Raf -rat aorta fibroblast cells; Tm -tropomyosin Remodeling of actin cytoskeleton plays a central role in a variety of cellular processes that involve shape change and movement. Malfunction of these processes could lead to pathological consequences, but how actin-mediated motility is regulated is only beginning to be understood. With recent

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