No abstract

Maintenance of skeletal and cardiac muscle structure and function requires precise control of the synthesis, assembly, and turnover of contractile proteins of the sarcomere. Abnormalities in accumulation of sarcomere proteins are responsible for a variety of myopathies. However, the mechanisms that mediate turnover of these long-lived proteins remain poorly defined. We show that muscle RING finger 1 (MuRF1) and MuRF3 act as E3 ubiquitin ligases that cooperate with the E2 ubiquitin-conjugating enzymes UbcH5a, -b, and -c to mediate the degradation of β/slow myosin heavy chain (β/slow MHC) and MHCIIa via the ubiquitin proteasome system (UPS) in vivo and in vitro. Accordingly, mice deficient for MuRF1 and MuRF3 develop a skeletal muscle myopathy and hypertrophic cardiomyopathy characterized by subsarcolemmal MHC accumulation, myofiber fragmentation, and diminished muscle performance. These findings identify MuRF1 and MuRF3 as key E3 ubiquitin ligases for the UPS-dependent turnover of sarcomeric proteins and reveal a potential basis for myosin storage myopathies.

The RING-finger domain is a novel zinc-binding Cys-His protein motif found in a growing number of proteins involved in signal transduction, ubiquitination, gene transcription, differentiation, and morphogenesis. We describe a novel muscle-specific RING-finger protein (MURF) expressed specifically in cardiac and skeletal muscle cells throughout pre- and postnatal mouse development. MURF belongs to the RING-B-box-coiled-coil subclass of RING-finger proteins, characterized by an NH2-terminal RING-finger followed by a zinc-finger domain (B-box) and a leucine-rich coiled-coil domain. Expression of MURF is required for skeletal myoblast differentiation and myotube fusion. The leucine-rich coiled-coil domain of MURF mediates association with microtubules, whereas the RING-finger domain is required for microtubule stabilization and an additional region is required for homo-oligomerization. Expression of MURF establishes a cellular microtubule network that is resistant to microtubule depolymerization induced by alkaloids, cold and calcium. These results identify MURF as a myogenic regulator of the microtubule network of striated muscle cells and reveal a link between microtubule organization and myogenesis.

Changes in actin dynamics influence diverse cellular processes and couple the actin-based cytoskeleton to changes in gene transcription. Members of the Rho GTPase family regulate cytoskeletal organization by stimulating actin polymerization and stress fiber formation when activated by extracellular signaling. The transcriptional activity of serum response factor (SRF) is stimulated in response to changes in actin dynamics and Rho signaling, but the proteins that mediate this phenomenon have not been fully identified. We describe a novel, evolutionarily conserved actin-binding protein, called STARS (striated muscle activator of Rho signaling), that is expressed specifically in cardiac and skeletal muscle cells. STARS binds to the I-band of the sarcomere and to actin filaments in transfected cells, where it activates Rho-signaling events. STARS stimulates the transcriptional activity of SRF through a mechanism that requires actin binding and involves Rho GTPase activation. STARS provides a potential mechanism for specifically enhancing Rho-dependent transcription in muscle cells and for linking changes in actin dynamics to gene transcription.

Fibulin (fbln)-5 is an elastin-binding protein required for assembly and organization of elastic fibers. To examine the potential role of fbln-5 in vascular remodeling and neointima formation, we induced vascular injury by carotid artery ligation in fbln-5 ؊/؊ mice. Mutant mice displayed an exaggerated vascular remodeling response that was accompanied by severe neointima formation with thickened adventitia. These abnormalities were not observed in elastin ؉/؊ mice that exhibited a comparable reduction of vessel extensibility to fbln-5 ؊/؊ mice. Thus, the severe remodeling response could not be attributed to altered extensibility of the vessel wall alone. Vascular smooth muscle cells cultured from fbln-5 ؊/؊ mice displayed enhanced proliferative and migratory responses to mitogenic stimulation relative to wild-type cells, and these responses were inhibited by overexpression of fbln-5. These findings demonstrate the importance of the elastic laminae in vascular injury, and reveal an unexpected role of fbln-5 as an inhibitor of vascular smooth muscle cell proliferation and migration.elastic fibers ͉ extracellular matrix ͉ neointima ͉ elastin V ascular obstructive abnormalities, such as atherosclerosis and restenosis after percutaneous coronary intervention, are triggered by damage to the vessel wall, initiating a series of biological responses, including up-regulation of adhesion molecules, recruitment of inflammatory cells, secretion of cytokines, and activation of smooth muscle cells (SMCs). Activated SMCs secrete growth factors, extracellular matrix proteins, and matrix proteases, thereby altering the microenvironment of the injured vessel wall. Much attention has focused on the signaling pathways responsible for the proliferation and migration of SMCs associated with vascular obstruction, but relatively little is known of the potential contributions of extracellular matrix proteins to these processes (reviewed in ref. 1).Structural changes of the vessel wall induced by mechanical force or enzymatic digestion also influence the development of vascular obstructive disease (2, 3). The elastic lamina plays a critical role in maintaining the integrity of the vessel wall. Rupture of the external elastic lamina (EEL) is a more potent stimulus for neointima formation than injury involving the internal elastic lamina (IEL) alone after coronary artery stent-induced injury (4). In addition, exposure of the adventitia to the blood lumen by disruption of the EEL during percutaneous angioplasty increases the activation of adventitial myofibroblasts, leading to adventitial fibrosis that eventually constricts the vessel wall (5).The perception of the elastic lamina (or elastic fibers) as the sole structural component of the vessel wall was recently challenged by the view that elastic fibers actively modulate intercellular signaling. Elastin (eln), a major component of the elastic fibers in the arterial wall, inhibits proliferation of SMCs (6). Eln is secreted as a tropoelastin monomer and subsequently undergoes cross-linking t...