Human SIRT1 controls various physiological responses including cell fate, stress, and aging, through deacetylation of its specific substrate protein. In processing DNA damage signaling, SIRT1 attenuates a cellular apoptotic response by deacetylation of p53 tumor suppressor. The present study shows that, upon exposure to radiation, SIRT1 could enhance DNA repair capacity and deacetylation of repair protein Ku70. Ectopically over-expressed SIRT1 resulted in the increase of repair of DNA strand breakages produced by radiation. On the other hand, repression of endogenous SIRT1 expression by SIRT1 siRNA led to the decrease of this repair activity, indicating that SIRT1 can regulate DNA repair capacity of cells with DNA strand breaks. In addition, we found that SIRT1 physically complexed with repair protein Ku70, leading to subsequent deacetylation. The dominant-negative SIRT1, a catalytically inactive form, did not induce deacetylation of Ku70 protein as well as increase of DNA repair capacity. These observations suggest that SIRT1 modulates DNA repair activity, which could be regulated by the acetylation status of repair protein Ku70 following DNA damage.

Muscle stem (satellite) cells are relatively resistant to cell-autonomous aging. Instead, their endogenous signaling profile and regenerative capacity is strongly influenced by the aged P-Smad3, differentiated niche, and by the aged circulation. With respect to muscle fibers, we previously established that a shift from active Notch to excessive transforming growth factor-beta (TGF-β) induces CDK inhibitors in satellite cells, thereby interfering with productive myogenic responses. In contrast, the systemic inhibitor of muscle repair, elevated in old sera, was suggested to be Wnt. Here, we examined the age-dependent myogenic activity of sera TGF-β1, and its potential cross-talk with systemic Wnt. We found that sera TGF-β1 becomes elevated within aged humans and mice, while systemic Wnt remained undetectable in these species. Wnt also failed to inhibit satellite cell myogenicity, while TGF-β1 suppressed regenerative potential in a biphasic fashion. Intriguingly, young levels of TGF-β1 were inhibitory and young sera suppressed myogenesis if TGF-β1 was activated. Our data suggest that platelet-derived sera TGF-β1 levels, or endocrine TGF-β1 levels, do not explain the age-dependent inhibition of muscle regeneration by this cytokine. In vivo, TGF-β neutralizing antibody, or a soluble decoy, failed to reduce systemic TGF-β1 and rescue myogenesis in old mice. However, muscle regeneration was improved by the systemic delivery of a TGF-β receptor kinase inhibitor, which attenuated TGF-β signaling in skeletal muscle. Summarily, these findings argue against the endocrine path of a TGF-β1-dependent block on muscle regeneration, identify physiological modalities of age-imposed changes in TGF-β1, and introduce new therapeutic strategies for the broad restoration of aged organ repair.

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Background/AimsChronic liver disease is a major widespread cause of death, and whole liver transplantation is the only definitive treatment for patients with end-stage liver diseases. However, many problems, including donor shortage, surgical complications and cost, hinder their usage. Recently, tissue-engineering technology provided a potential breakthrough for solving these problems. Three-dimensional (3D) printing technology has been used to mimic tissues and organs suitable for transplantation, but applications for the liver have been rare.MethodsA 3D bioprinting system was used to construct 3D printed hepatic structures using alginate. HepG2 cells were cultured on these 3D structures for 3 weeks and examined by fluorescence microscopy, histology and immunohistochemistry. The expression of liver-specific markers was quantified on days 1, 7, 14, and 21.ResultsThe cells grew well on the alginate scaffold, and liver-specific gene expression increased. The cells grew more extensively in 3D culture than two-dimensional culture and exhibited better structural aspects of the liver, indicating that the 3D bioprinting method recapitulates the liver architecture.ConclusionsThe 3D bioprinting of hepatic structures appears feasible. This technology may become a major tool and provide a bridge between basic science and the clinical challenges for regenerative medicine of the liver.

Cell membrane consists of various lipids such as phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidlethanolamine (PE). Among them, PS is a molecular marker of apoptosis, because it is located to the inner leaflet of plasma membrane generally but it is moved to the outer leaflet during programmed cell death. The process of apoptosis has been implicated in the fusion of muscle progenitor cells, myoblasts, into myotubes. However, it remained unclear whether PS regulates muscle cell differentiation directly. In this paper, localization of PS to the outer leaflet of plasma membrane in proliferating primary myoblasts and during fusion of these myoblasts into myotubes is validated using Annexin V. Moreover, we show the presence of PS clusters at the cell-cell contact points, suggesting the importance of membrane ruffling and PS exposure for the myogenic cell fusion. Confirming this conclusion, experimentally constructed PS, but not PC liposomes dramatically enhance the formation of myotubes from myoblasts, thus demonstrating a direct positive effect of PS on the muscle cell fusion. In contrast, myoblasts exposed to PC liposomes produce long myotubes with low numbers of myonuclei. Moreover, pharmacological masking of PS on the myoblast surface inhibits fusion of these cells into myotubes in a dose-dependent manner

Aim: To study the influence of acute experimental diabetes on the regenerative potential of muscle stem (satellite) cells in mice. Methods: Male C57BL/6 young mice were injected with a single dose of streptozotocin (STZ, 180 mg/kg, ip) to induce diabetes. The diabetic mice were treated with insulin (0.75 U/kg, ip), follistatin (12 μg/kg, im) or Alk5 inhibitor (5 μmol/L per kg, sc) once a day. On the first day when high glucose levels were found, cardiotoxin (CTX) was focally injected into tibialis anterior and gastronemius muscles of the mice. The muscles were harvested 3 d and 5 d after CTX injection, and myofibers and satellite cells were isolated. Quantitative ex-vivo and in-vivo assays of myogenic potential were used to evaluate the muscle regenerative responses. Results: The satellite cells from the diabetic mice 3 d after CTX injection fail to activate, and the repair of muscle deteriorates, resembling that observed in old control mice. Furthermore, the satellite cells have excessive levels of myostatin, TGF-β receptor 1, pSmad3 and the cell cycle inhibitor p15, while the level of TGF-β1 remain unchanged. Treatment of the diabetic mice with insulin rescued muscle regenerative responses, and restored the expression levels of myostatin, TGF-β receptor 1, pSmad3, and p15 to those similar of healthy controls. Treatment of the diabetic mice with the myostatin antagonist follistatin, or with the Alk5 inhibitor of TGF-β receptor 1 (which did not diminish the blood glucose levels) rescued muscle regenerative responses and attenuated the myostatin/ TGFβ receptor/pSmad3 signaling. Conclusion: The muscle regenerative responses are incapacitated and repair of the tissue fails within hours after the initiation of hyperglycemia in a mouse model of type 1 diabetes, but stem cell function is rescued by insulin, as well as follistatin or an Alk5 inhibitor that blocks TGF-β receptor signaling.