MAB3876 Sigma-AldrichAnti-Myogenin Antibody

Skeletal muscle atrophy is a critical component of the ageing process. Age-related muscle wasting is due to disrupted muscle protein turnover, a process mediated in part by the ubiquitin proteasome pathway (UPP). Additionally, older subjects have been observed to have an attenuated anabolic response, at both the molecular and physiological levels, following a single-bout of resistance exercise (RE). We investigated the expression levels of the UPP-related genes and proteins involved in muscle protein degradation in 10 older (60-75 years) vs. 10 younger (18-30 years) healthy male subjects at basal as well as 2 h after a single-bout of RE. MURF1, atrogin-1 and FBXO40, their substrate targets PKM2, myogenin, MYOD, MHC and EIF3F as well as MURF1 and atrogin-1 transcriptional regulators FOXO1 and FOXO3 gene and/or protein expression levels were measured via real time PCR and western blotting, respectively. At basal, no age-related difference was observed in the gene/protein levels of atrogin-1, MURF1, myogenin, MYOD and FOXO1/3. However, a decrease in FBXO40 mRNA and protein levels was observed in older subjects, while PKM2 protein was increased. In response to RE, MURF1, atrogin-1 and FBXO40 mRNA were upregulated in both the younger and older subjects, with changes observed in protein levels. In conclusion, UPP-related gene/protein expression is comparably regulated in healthy young and old male subjects at basal and following RE. These findings suggest that UPP signaling plays a limited role in the process of age-related muscle wasting. Future studies are required to investigate additional proteolytic mechanisms in conjunction with skeletal muscle protein breakdown (MPB) measurements following RE in older vs. younger subjects.

Mitogen-activated protein kinases (MAPKs) are highly conserved protein kinase modules, and they control fundamental cellular processes. While the activation of MAPKs has been well studied, little is known on the mechanisms driving their inactivation. Here we uncover a role for ubiquitination in the inactivation of a MAPK module. Extracellular-signal-regulated kinase 5 (ERK5) is a unique, conserved member of the MAPK family and is activated in response to various stimuli through a three-tier cascade constituting MEK5 and MEKK2/3. We reveal an unexpected role for Inhibitors of Apoptosis Proteins (IAPs) in the inactivation of ERK5 pathway in a bimodal manner involving direct interaction and ubiquitination. XIAP directly interacts with MEKK2/3 and competes with PB1 domain-mediated binding to MEK5. XIAP and cIAP1 conjugate predominantly K63-linked ubiquitin chains to MEKK2 and MEKK3 which directly impede MEK5-ERK5 interaction in a trimeric complex leading to ERK5 inactivation. Consistently, loss of XIAP or cIAP1 by various strategies leads to hyperactivation of ERK5 in normal and tumorigenic cells. Loss of XIAP promotes differentiation of human primary skeletal myoblasts to myocytes in a MEKK2/3-ERK5-dependent manner. Our results reveal a novel, obligatory role for IAPs and ubiquitination in the physical and functional disassembly of ERK5-MAPK module and human muscle cell differentiation.

The purpose of this study was to determine whether low-intensity pulsed ultrasound (LIPUS) could enhance the regeneration of myofibers and shorten the healing time in injured muscle. NIH C2C12 cells, a well-known myoblastic cell line, are subclones derived from the mouse myoblast cell line established from normal adult C3H mouse leg muscle. The cells differentiate rapidly and produce extensive contracting myotubes expressing characteristic muscle proteins. We exposed C2C12 cells to LIPUS therapy using the EXOGEN 2000+ system ultrasound apparatus (Exogen Inc., Piscataway, NJ, USA) with a total treatment of 20 min every 24 h. At intervals of 2, 4, 6 and 8 days, cell growth was measured by the increase in cell number and western blot analysis of myogenin and actin. Forty mice (C57BL10J+/+) were divided into five groups of eight animals each and used in the published laceration injury model. The gastrocnemius muscle of the left leg was lacerated in all the animals. The control group (sham ultrasound) did not undergo LIPUS therapy. The ultrasound 7-, 14-, 21- and 28-day groups (only changing the number of days during which the ultrasound was applied to the injured muscle) were treated with LIPUS (20 min/day) for 7, 14, 21 and 28 consecutive days, respectively. All animals were sacrificed at 4 weeks after the injury. Evaluation methods included muscle regeneration and muscle contractile properties. LIPUS therapy produced a significantly higher proliferative rate and cell number at days 6 and 8 (p < 0.05). Densitometric evaluation revealed an increase in myogenin and actin proteins in cells treated with LIPUS in the 4-, 6- and 8-day groups. The regeneration of myofibers, fast-twitch and tetanus of LIPUS-treated muscles (21 and 28 days) was significantly greater relative to control muscles. There was no major strength difference between the normal non-injured muscle and the group treated with LIPUS for 28 days. In conclusion, this was the first experimental study to show that LIPUS therapy is able to enhance the regeneration of myofibers with better physiologic performance in injured mice muscles after laceration, especially prior to postoperative week 4. Findings of this study demonstrate a scientific basis for future clinical trials and establish an indication for LIPUS in enhancing muscle healing after laceration injury.

We established cardiac pluripotent stem-like cells from the left atrium (LA-PCs) of adult rat hearts. These cells could differentiate not only into beating myocytes but also into cells of other lineages, including adipocytes and endothelial cells in the methylcellulose-based medium containing interleukin-3 (IL-3), interleukin-6 (IL-6), and stem cell factor (SCF). In particular, IL-3 and SCF contributed to the differentiation into cardiac troponin I-positive cells. Notably, small population of LA-PCs coexpressed GATA4 and myogenin, which are markers specific to cardiomyocytes and skeletal myocytes, respectively, and could differentiate into both cardiac and skeletal myocytes. Therefore, we investigated the involvement of these two tissue-specific transcription factors in the cardiac transcriptional activity. Coexpression of GATA4 and myogenin synergistically activated GATA4-specific promoter of the atrial natriuretic peptide gene. This combinatorial function was shown to be dependant on the GATA site, but independent of the E-box. The results of chromatin immunoprecipitation and electrophoretic mobility shift assays suggested that myogenin bound to GATA4 on the GATA elements and the C-terminal Zn-finger domain of GATA4 and the N-terminal region of myogenin were required for this synergistic activation of transcription. Taken together, these two transcription factors could be involved in the myogenesis of LA-PCs.