The tissue distribution and function of hemoglobin or myoglobin are well known; however, a newly found cytoglobin (CYGB), which also belongs to the globin family, remains to be characterized. To assess its expression in human malignancies, we sought to screen a number of cell lines originated from many tissues using northern blotting and real time PCR techniques. Unexpectedly, we found that several, but not all, melanoma cell lines expressed CYGB mRNA and protein at much higher levels than cells of other origins. Melanocytes, the primary origin of melanoma, also expressed CYGB at a high level. To verify these observations, immunostaining and immunoblotting using anti-CYGB antibody were also performed. Bisulfite-modified genomic sequencing revealed that several melanoma cell lines that abrogated CYGB expression were found to be epigenetically regulated by hypermethylation in the promoter region of CYGB gene. The RNA interference-mediated knockdown of the CYGB transcript in CYGB expression-positive melanoma cell lines resulted in increased proliferation in vitro and in vivo. Flow cytometric analysis using 2′-, 7′-dichlorofluorescein diacetate (DCFH-DA), an indicator of reactive oxygen species (ROS), revealed that the cellular ROS level may be involved in the proliferative effect of CYGB. Thus, CYGB appears to play a tumor suppressive role as a ROS regulator, and its epigenetic silencing, as observed in CYGB expression-negative melanoma cell lines, might function as an alternative pathway in the melanocyte-to-melanoma transition.

The pathogenesis of sarcopenia includes the dysfunction of calcium homeostasis associated with the sarcoplasmic reticulum; however, the localization in sarcoplasmic reticulum-related factors and differences by myofiber type remain unclear. Here, we investigated the effects of aging on sarcoplasmic reticulum-related factors in the soleus (slow-twitch) and gastrocnemius (fast-twitch) muscles of 3- and 24-month-old male C57BL/6J mice. There were no notable differences in the skeletal muscle weight of these 3- and 24-month-old mice. The expression of Atp2a1, Atp2a2, Sln, and Pln increased with age in the gastrocnemius muscles, but not in the soleus muscles. Subsequently, immunohistochemical analysis revealed ectopic sarcoplasmic reticulum calcium ion ATPase (SERCA) 1 and SERCA2a immunoreactivity only in the gastrocnemius muscles of old mice. Histochemical and transmission electron microscope analysis identified tubular aggregate (TA), an aggregation of the sarcoplasmic reticulum, in the gastrocnemius muscles of old mice. Dihydropyridine receptor α1, ryanodine receptor 1, junctophilin (JPH) 1, and JPH2, which contribute to sarcoplasmic reticulum function, were also localized in or around the TA. Furthermore, JPH1 and JPH2 co-localized with matrix metalloproteinase (MMP) 2 around the TA. These results suggest that sarcoplasmic reticulum-related factors are localized in or around TAs that occur in fast-twitch muscle with aging, but some of them might be degraded by MMP2.

The circadian system has been regarded as a limit cycle oscillator constructed by the integrated interaction of clock genes and proteins. Here, we investigated a mammalian circadian oscillation geometrically before and after a perturbation. We detected the singular point and transition from a type 1 to type 0 phase response curve (PRC) and determined the embedding dimension to show how many variables are needed to describe the limit cycle oscillation and relaxation process after a perturbation. As a perturbation, forskolin (FK) was administered to Rat-1 cells expressing the Per2::luc gene. By broadly and finely changing the phase and strength of the perturbation, we detected the transition of the PRC from type 1 to type 0 and a possible singular transition point, the property of which agreed quite well with our numerical simulation of the noisy Goodwin model, a simple yet canonical model for the transcription-translation feedback loop of the core clock genes. Furthermore, we estimated the embedding dimension of the limit cycle before and after the perturbation. The trajectory of the limit cycle was embedded in two dimensions but with the perturbation of the state point moved out of the trajectory, the relaxation process was generally embedded in higher dimensions. The average number of embedding dimensions at each dose of FK increased as the FK dose increased but most of the relaxation process was generally embedded within four dimensions. These findings support the existence of a circadian limit cycle oscillator in mammalian cells and suggest that a small number of variables determine the relaxation process after a perturbation.

Obesity and aging are known to affect the skeletal muscles. Obesity in old age may result in a poor basement membrane (BM) construction response, which serves to protect the skeletal muscle, thus making the skeletal muscle more vulnerable. In this study, older and young male C57BL/6J mice were divided into two groups, each fed a high-fat or regular diet for eight weeks. A high-fat diet decreased the relative gastrocnemius muscle weight in both age groups, and obesity and aging individually result in a decline in muscle function. Immunoreactivity of collagen IV, the main component of BM, BM width, and BM-synthetic factor expression in young mice on a high-fat diet were higher than that in young mice on a regular diet, whereas such changes were minimal in obese older mice. Furthermore, the number of central nuclei fibers in obese older mice was higher than in old mice fed a regular diet and young mice fed a high-fat diet. These results suggest that obesity at a young age promotes skeletal muscle BM formation in response to weight gain. In contrast, this response is less pronounced in old age, suggesting that obesity in old age may lead to muscle fragility.

Collagen I is the most abundant type of intramuscular collagen. Lysyl oxidase promotes collagen cross-link formation, which helps stabilize the extracellular matrix. Furthermore, matrix metalloproteinases, responsible for collagen degradation, maintain typical muscle structure and function through remodeling. Although it is well known that aging leads to delayed recovery of muscle fibers, the impact of aging on the remodeling of intramuscular collagen is not well understood. In this study, we investigated the impact of aging on collagen remodeling during muscle injury recovery using young and old mouse models. Muscle injury was induced in the right tibialis anterior (TA) muscle of male C57BL/6J mice [aged 21 weeks (young) and 92 weeks (old)] using intramuscular cardiotoxin injection, with the left TA serving as a sham with saline injection. Following a one-week recovery period, aging was found to delay the recovery of the fiber cross-sectional area. The intensity and area of immunoreactivity for collagen I were significantly increased in old mice compared to young mice post-injury. Additionally, Lox expression and the number of LOX (+) cells in the extracellular matrix significantly increased in old mice compared to young mice post-injury. Furthermore, Mmp9 and MMP9 expression levels after muscle injury were higher in old mice than in young mice. These results suggest that muscle injury in old mice can lead to increased collagen I accumulation, enhanced collagen cross-link formation, and elevated MMP9 expression compared to young mice.