p94/calpain 3 is a skeletal muscle-specific Ca(2+)-regulated cysteine protease (calpain), and genetic loss of p94 protease activity causes muscular dystrophy (calpainopathy). In addition, a small in-frame deletion in the N2A region of connectin/titin that impairs p94-connectin interaction causes a severe muscular dystrophy (mdm) in mice. Since p94 via its interaction with the N2A and M-line regions of connectin becomes part of the connectin filament system that serves as a molecular scaffold for the myofibril, it has been proposed that structural and functional integrity of the p94-connectin complex is essential for health and maintenance of myocytes. In this study, we have surveyed the interactions made by p94 and connectin N2A inside COS7 cells. This revealed that p94 binds to connectin at multiple sites, including newly identified loci in the N2A and PEVK regions of connectin. Functionally, p94-N2A interactions suppress p94 autolysis and protected connectin from proteolysis. The connectin N2A region also contains a binding site for the muscle ankyrin repeat proteins (MARPs), a protein family involved in the cellular stress responses. MARP2/Ankrd2 competed with p94 for binding to connectin and was also proteolyzed by p94. Intriguingly, a connectin N2A fragment with the mdm deletion possessed enhanced resistance to proteases, including p94, and its interaction with MARPs was weakened. Our data support a model in which MARP2-p94 signaling converges within the N2A connectin segment and the mdm deletion disrupts their coordination. These results also implicate the dynamic nature of connectin molecule as a regulatory scaffold of p94 functions.

After skin wounding, the repair process is initiated by the release of growth factors, cytokines, and bioactive lipids from injured vessels and coagulated platelets. These signal molecules induce synthesis and deposition of a provisional extracellular matrix, as well as fibroblast invasion into and contraction of the wounded area. We previously showed that sphingosine-1-phosphate (S1P) triggers a signal transduction cascade mediating nuclear translocation of the LIM-only protein Fhl2 in response to activation of the RhoA GTPase (Muller, J.M., U. Isele, E. Metzger, A. Rempel, M. Moser, A. Pscherer, T. Breyer, C. Holubarsch, R. Buettner, and R. Schule. 2000. EMBO J. 19:359–369; Muller, J.M., E. Metzger, H. Greschik, A.K. Bosserhoff, L. Mercep, R. Buettner, and R. Schule. 2002. EMBO J. 21:736–748.). We demonstrate impaired cutaneous wound healing in Fhl2-deficient mice rescued by transgenic expression of Fhl2. Furthermore, collagen contraction and cell migration are severely impaired in Fhl2-deficient cells. Consequently, we show that the expression of α-smooth muscle actin, which is regulated by Fhl2, is reduced and delayed in wounds of Fhl2-deficient mice and that the expression of p130Cas, which is essential for cell migration, is reduced in Fhl2-deficient cells. In summary, our data demonstrate a function of Fhl2 as a lipid-triggered signaling molecule in mesenchymal cells regulating their migration and contraction during cutaneous wound healing.

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MuRF1 is a member of the RBCC (RING, B-box, coiled-coil) superfamily that has been proposed to act as an atrogin during muscle wasting. Here, we show that MuRF1 is preferentially expressed in type II muscle fibers. Five and 14 days after denervation, MuRF1 protein was further elevated but remained preferentially expressed in type-II muscle fibers. Consistent with a fiber-type dependent function of MuRF1, the tibialis anterior muscle (rich in type-II muscle fibers) was considerably more protected in MuRF1-KO mice from muscle wasting when compared to soleus muscle with mixed fiber-types. We also determined fiber type distributions in MuRF1/MuRF2 double deficient KO (dKO) mice, because MuRF2 is a close homolog of MuRF1. MuRF1/MuRF2 dKO mice showed a profound loss of type-II fibers in soleus muscle. As a potential mechanism we identified the interaction of MuRF1/MuRF2 with myozenin-1, a calcineurin/NFAT regulator and a factor required for maintenance of type-II muscle fibers. MuRF1/MuRF2 dKO mice had lost myozenin-1 expression in tibialis anterior muscle, implicating MuRF1/MuRF2 as regulators of the calcineurin/NFAT pathway. In summary, our data suggest that expression of MuRF1 is required for remodeling of type-II fibers under pathophysiological stress states, whereas MuRF1 and MuRF2 together are required for maintenance of type-II fibers, possibly via the regulation of myozenin-1.

The muscle-specific ubiquitin ligase MuRF1 regulates muscle catabolism during chronic wasting states, although its roles in general metabolism are less-studied. Here, we metabolically profiled MuRF1-deficient knockout mice. We also included knockout mice for MuRF2 as its closely related gene homolog. MuRF1 and MuRF2-KO (knockout) mice have elevated serum glucose, elevated triglycerides, and reduced glucose tolerance. In addition, MuRF2-KO mice have a reduced tolerance to a fat-rich diet. Western blot and enzymatic studies on MuRF1-KO skeletal muscle showed perturbed FoxO-Akt signaling, elevated Akt-Ser-473 activation, and downregulated oxidative mitochondrial metabolism, indicating potential mechanisms for MuRF1,2-dependent glucose and fat metabolism regulation. Consistent with this, the adenoviral re-expression of MuRF1 in KO mice normalized Akt-Ser-473, serum glucose, and triglycerides. Finally, we tested the MuRF1/2 inhibitors MyoMed-205 and MyoMed-946 in a mouse model for type 2 diabetes mellitus (T2DM). After 28 days of treatment, T2DM mice developed progressive muscle weakness detected by wire hang tests, but this was attenuated by the MyoMed-205 treatment. While MyoMed-205 and MyoMed-946 had no significant effects on serum glucose, they did normalize the lymphocyte–granulocyte counts in diabetic sera as indicators of the immune response. Thus, small molecules directed to MuRF1 may be useful in attenuating skeletal muscle strength loss in T2DM conditions.

Since the first description of Bolton et al., critical illness polyneuropathy (CIP) and critical illness myopathy (CIM) are increasingly observed as a complication in intensive care patients. CIP and CIM commonly occur in patients with an ICU length of stay exceeding one week. Typically, these patients show weakness of the limbs and difficulties in weaning from the respirator. Neurological and electrophysiological examinations as well as muscle biopsies if myopathy is of concern may help to characterize and identify polyneuropathy and myopathy.

Background: Inflammatory cytokines like tumor necrosis factor alpha (TNF-α) are known to impair skeletal muscle (SM) function. Furthermore, TNF-α induces the expression of atrogin-like muscle specific ubiquitin E3-ligases, presumed to mediate muscle atrophy. The relative contributions of respective ubiquitin ligases, like MuRF1 for the TNF-α induced reduction in muscle function are not known. Methods: TNF-α or saline was injected either into C57Bl6 or MuRF1 −/− mice. After 16 –24h the expression of MuRF1 in the SM was quantified by qRT-PCR and western blot. Muscle function was measured in an organ bath. To obtain a broader overview on potential alterations, 2D-gel electrophoresis was performed. Results: WT animals injected with TNF-α had higher MuRF1 mRNA (saline: 56.6±12.1 vs. TNF-α: 133.6±30.3 arb. Units; p<0.05) and protein expression (saline: 0.38±0.11 vs. TNF-α: 1.07±0.25 arb. Units; p<0.05) as compared to saline injected littermates. However, TNF- α was unable to induce MurRF1 expression in MuRF1 −/− mice. Furthermore, TNF- α reduced force development at 150Hz by 25% in C57Bl6 animals (saline: 2412±120 vs. TNF-α: 1799±114 g/cm2; p<0.05), but not in MuRF1 −/− mice (saline: 2424±198 vs. TNF-α: 2431±180 g/cm2; p=NS). The proteome analysis revealed a significant down-regulation of fast skeletal muscle troponin T (TNNT3) in WT animals treated with TNF- α as compared to MuRF1 −/− mice receiving TNF-α . In addition, TNF-α injection into C57Bl6 animals resulted in a down-regulation of eEF1γ ( WT: 0.60±0.02 vs. WT+TNF-α: 0.39±0.05 arb. Units; p<0.05). This reduction was not seen in MuRF1 −/− mice receiving TNF- α (KO: 0.59±0.03 vs. KO+TNF-α: 0.68±0.01 arb. Units; p<0.05) Conclusion: The results of this study demonstrate for the first time, that the TNF- α induced reduction in SM force development depends on the induction of the atrophy related E3-ubiquitin ligase MuRF1. A link for the reduction in muscle force may be the TNF-α-MuRF1-mediated down-regulation of TNNT3 and the elongation factor eEF1γ.