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Myosin plays a fundamental role in muscle contraction. Approximately 300 myosins form a bipolar thick filament, in which myosin is continuously replaced by protein turnover. However, it is unclear how rapidly this process occurs and whether the myosin exchange rate differs depending on the region of the thick filament. To answer this question, we first measured myosin release and insertion rates over a short period and monitored myotubes expressing a photoconvertible fluorescence protein-tagged myosin, which enabled us to monitor myosin release and insertion simultaneously. About 20% of myosins were replaced within 10 min, while 70% of myosins were exchanged over 10 h with symmetrical and biphasic alteration of myosin release and insertion rates. Next, a fluorescence pulse-chase assay was conducted to investigate whether myosin is incorporated into specific regions in the thick filament. Newly synthesized myosin was located at the tip of the thick filament rather than the center in the first 7 min of pulse-chase labeling and was observed in the remainder of the thick filament by 30 min. These results suggest that the myosin replacement rate differs depending on the regions of the thick filament. We concluded that myosin release and insertion occur concurrently and that myosin is more frequently exchanged at the tip of the thick filament.

Foreign body reaction (FBR) causes unexpected adverse effects due to implanted materials in humans and animals. Inflammation and subsequent fibrosis during FBR seems to be affected by recipient immunity, such as the balance of T helper (Th) response that has the potential to regulate FBR-related macrophage function. Here, the immunological effects of FBR on subcutaneously imbedded silicone tubes (ST) at 8 weeks were investigated histologically by comparing Th1-biased C57BL/6N, Th2-biased MRL/MpJ, and autoimmune disease-prone MRL/MpJ-Fas(lpr/lpr). Tissue surrounding ST (TSS) was analyzed at day (D) 7 and 14 (reaction phase) or D35 (stability phase) after surgery. In all strains, the TSS was composed of a thin layer (TL) containing fibrous tissues and loose connective tissues formed outside the TL. Few lymphocytes and mast cells, several neutrophils, and numerous macrophages infiltrated the TSS. Active vascularization was observed at D14 in all strains. For the examined indices, M1-type macrophage density in the TSS of C57BL/6N mice was significantly higher at D14 compared to other strains. No significant strain difference relating to M2-type macrophages was detected, suggesting the effects of Th1-biased immunity on FBR-related inflammation. Collagen fibers in the TSS increased in density and became stable with age in all strains. In particular, MRL/MpJ-Fas(lpr/lpr) showed progressive fibrotic features. Serum autoantibody levels in MRL/MpJ-Fas(lpr/lpr) mice were inversely correlated with M1-type macrophage density. These data from MRL/MpJ-Fas(lpr/lpr) mice suggested modifications of FBR-related inflammation and fibrosis by autoimmune abnormalities. The results provide crucial insights into the pathological modification of FBR by recipient immunity and emphasize its clinicopathological importance in humans and animals.

Resident myogenic stem cells (satellite cells) are attracting attention for their novel roles in myofiber type regulation. In the myogenic differentiation phase, satellite cells from soleus muscle (slow fiber-abundant) synthesize and secrete higher levels of semaphorin 3A (Sema3A, a multifunctional modulator) than those derived from extensor digitorum longus (EDL; fast fiber-abundant), suggesting the role of Sema3A in forming slow-twitch myofibers. However, the regulatory mechanisms underlying fast-twitch myotube commitment remain unclear. Herein, we focused on netrin family members (netrin-1, -3, and -4) that compete with Sema3A in neurogenesis and osteogenesis. We examined whether netrins affect fast-twitch myotube generation by evaluating their expression in primary satellite cell cultures. Initially, netrins are upregulated during myogenic differentiation. Next, we compared the expression levels of netrins and their cell membrane receptors between soleus- and EDL-derived satellite cells; only netrin-1 showed higher expression in EDL-derived satellite cells than in soleus-derived satellite cells. We also performed netrin-1 knockdown experiments and additional experiments with recombinant netrin-1 in differentiated satellite cell-derived myoblasts. Netrin-1 knockdown in myoblasts substantially reduced fast-type myosin heavy chain (MyHC) expression; exogenous netrin-1 upregulated fast-type MyHC in satellite cells. Thus, netrin-1 synthesized in EDL-derived satellite cells may promote myofiber type commitment of fast muscles.

Genistein is a soy isoflavone and shows various physiological activities, such as affinities for estrogen receptors (ERs) and inhibitory effects on the epidermal growth factor receptor (EGFR) pathway. A previous study reported that genistein downregulates milk production ability in mammary epithelial cells (MECs) while decreasing the phosphorylation of STAT5. The ER and EGFR pathways indirectly regulate STAT5. In this study, the repressing mechanism of genistein against the phosphorylation of STAT5 was investigated using a culture model of mouse MECs with milk production ability. The results revealed that genistein did not influence the behavior of ERa and ER beta, whereas genistein immediately repressed the phosphorylation of ERK1/2. However, the decrease in phosphorylated STAT5 occurred independent of the phosphorylation of EGFR. Genistein repressed new phosphorylation of STAT5 by prolactin without influencing the phosphorylation of JAK2. In conclusion, this study indicates that genistein directly inhibits the phosphorylation of STAT5 in lactating MECs.

Myosin, the most abundant myofibrillar protein in skeletal muscle, functions as a motor protein in muscle contraction. Myosin polymerizes into the thick filaments in the sarcomere where approximately 50% of embryonic myosin (Myh3) are replaced within 3 h (Ojima K, Ichimura E, Yasukawa Y, Wakamatsu J, Nishimura T, Am J Physiol Cell Physiol 309: C669-C679, 2015). The sarcomere structure including the thick filament is maintained by a balance between protein biosynthesis and degradation. However, the involvement of a protein degradation system in the myosin replacement process remains unclear. Here, we show that the muscle-specific ubiquitin ligase Ozz regulates replacement rate of Myh3. To examine the direct effect of Ozz on myosin replacement, eGFP-Myh3 replacement rate was measured in myotubes overexpressing Ozz by fluorescence recovery after photobleaching. Ozz overexpression significantly decreased the replacement rate of eGFP-Myh3 in the myofibrils, whereas it had no effect on other myosin isoforms. It is likely that ectopic Ozz promoted myosin degradation through increment of ubiquitinated myosin, and decreased myosin supply for replacement, thereby reducing myosin replacement rate. Intriguingly, treatment with a proteasome inhibitor MG132 also decreased myosin replacement rate, although MG132 enhanced the accumulation of ubiquitinated myosin in the cytosol where replaceable myosin is pooled, suggesting that ubiquitinated myosin is not replaced by myosin in the myofibril. Collectively, our findings showed that Myh3 replacement rate was reduced in the presence of overexpressed Ozz probably through enhanced ubiquitination and degradation of Myh3 by Ozz.