Expression of myogenin, MyoD and MHC isoforms in regenerating skeletal muscle.

骨格筋再生過程におけるミオシン重鎖(MHC)アイソフォーム発現とmyogenin，MyoDタンパクの発現様式との関連性を検討するために，塩酸ブピバカインを用いてマウスヒラメ筋損傷モデルを作成し，損傷筋の再生過程を組織形態学的に確認すると同時に，再生各段階におけるMHCアイソフォームと，myogeninおよびMyoDタンパク発現を経時的に検索した．本研究における筋損傷は塩酸ブピバカインをマウス(C57BL/10SnSlc)のヒラメ筋に注入することで作成した．組織学的には，塩酸ブピバカイン投与後3日目で筋線維はほとんど消失し，処置後6日目で中心核を有する再生筋線維がかなり出現し，処置後28日目では対照群のものと同程度まで回復した．生化学的分析では，対照群ヒラメ筋はMHCⅠ(34．3±1．7%)とMHCⅡa(65．7±1．7%)で構成されていた．実験群ヒラメ筋ではMHCⅠは処置後14日目まで減少し，その後増加傾向を示し，処置後90日目では36．3±2．9%となった．また，正常ヒラメ筋では検出されない速筋型MHC(MHC Ⅱd，MHC Ⅱb)が処置後3日目から28日目まで検出された．Western blotを用いた分析では，myogeninタンパク正常ヒラメ筋（遅筋）で検出された一方，前脛骨筋（速筋）においては検出できなかった．実験群ヒラメ筋では，myogeninは対照群と比較して処置後３日目より増加し（3．1±0．5），処置後６日目でピークに達した（5．8±0．8）．それからmyogeninタンパクは徐々に減少していったが，処置後90日目においてもなお対照群ヒラメ筋の1．8倍の発現を維持し続けた．一方，MyoDタンパクは正常前脛骨筋において正常ヒラメ筋の3．3倍の発現が認められた．MyoDは処置後３日目で対照群ヒラメ筋と比較して5．4倍になりピークに達した．その後は徐々に減少し始めた．しかし処置後90日目においても2．2倍の発現があった．これらのことから筋の再生過程においては速筋タイプの筋細胞が出現するmyogeninとMyoDは衛星細胞の分化と筋の再生に密接に関係していることが示唆された．To investigate the precise mechanism of skeletal muscle cell regeneration, the changing pattern ofmyosin heavy chain(MHC)isoforms during the regenerating process was observed with relation to theactivation of myogenin and MyoD. In addition, histopathological observation of the damaged muscles wasperformed throughout the experiment.In this study, muscle damage was induced by intramuscular injection of bupivacaine hydrochloride in thesoleus muscle of mice (C57BL/10SnSc). In the light microscopic observation, muscle cells had almost disappeared at 3 days after bupivacainetreatment with severe inflammatory cell infiltration. At 6 days after treatment, a considerable number ofregenerating muscle cells containing centrally located nuclei appeared in the damaged soleus muscle. At28 days, these regenerating muscle cells showed almost the same appearance as the control muscle cellscontaining subsarcolemmal nuclei, although a small number of muscle cells with central nuclei were stillrecognized.In the biochemical analysis, control soleus muscles contained only MHC I (34.3±1.7 %)and MHC IIa(65.7±1.7 %). In the damaged muscles, MHC I was decreased toward 14 days after treatment, and thengradually increased. At 90 days, the contents of MHC I was finally recovered to 36.3±2.9 %.0 In addition,MHC IId and MHC IIb appeared in the damaged muscle from 3 to 28 days after treatment. However, theyhad disappeared at 90 days.Using western blot analysis, myogenin protein was recognized in the control soleus muscles (slow typemuscle), while the myogenin could not be found in the first type muscle of the anterior tibial muscle. Themyogenin contents increased to about three fold (3.1±0.5)at 3 days after treatment compared withthose of control muscles and reached the maximum level (5.8±0.8)at 6 days after treatment. Then, myogenin contents gradually decreased,although they still remained high (1.8 times)at the end of experiment (90 days after treatment). Incontrast to the myogenin protein, a high level (3.3 times)of MyoD protein was detected in the anteriortibial muscle compared with that of control soleus muscles. In the damaged soleus muscles, MyoDcontents reached a maximum level (5.4 times)at 3 days after treatment compared with that of controlsoleus muscles, and then gradually decreased toward the end of experiment. However, MyoD protein stillremained 2.2 times compared with that of control soleus muscles. These findings described above indicate that, 1)a property of fast type muscle cells appeared in theregenerating muscle cells during the regenerating process, and 2)myogenin and MyoD are closelyrelated to the differentiation of the satellite cells and regeneration of the skeletal muscle cells

The role of astrocytes during repair of cerebral infarction in mdx mice

様々な大きさのジストロフィンアイソフォーム(427kDa, 260kDa, 140kDa, 116kDa, 71-75kDa)が広く体内に存在していることはよく知られている.中枢神経系においては71-75kDaのDp71が著明に多く,毛細血管の内皮の基底膜に接しているアストロサイトの細胞質に局在することが報告されている.しかしながらDp71の機能についてはよくわかっていないことが多い.そこで今回,脳組織におけるDp71の役割を調べるために,コントロールマウス(wild-typeマウス)およびデュシャンヌ型筋ジストロフィーモデル動物であるmdxマウスを用いて実験的脳梗塞を作成し,その治癒過程を形態学的に観察した.また,GFAPおよびDp71に関して生化学的に分析をおこなった.HE染色およびGFAP免疫組織学的染色の結果から,形態学的にはmdxマウスとコントロールマウスの脳に違いは認められなかった.しかしながら,mdxマウスの脳において,Dp71の発現量がコントロールマウスよりも少ないことがわかった.またmdxマウスにおいて,脳梗塞の修復過程におけるアストロサイトの反応がコントロールマウスよりも弱いことがわかった.これらの結果から,mdxマウスの脳において,アストロサイトの機能,アストロサイトの血管新生に関わる機能の障害されていることが示唆された.It is now well known that dystrophin isoforms (427kDa, 260kDa, 140kDa, 116kDa, 71-75kDa) are widely distributed throughout our body. In the central nervous system a considerable amount of Dp71 (71-75kDa) is found in the perivascular cytoplasm of the astrocytes. However, the function of this dystrophin is still unknown. To investigate the role of Dp71 in the brain tissue, cerebral infarction was induced in the control (wide-type) mouse and mdx mouse which is known as an animal model of human muscle dystrophy (Duchenne type), and morphological changes of the infarcted area were observed during repair of the infarction. In addition, biochemical analysis of GFAP and Dp71 was carried out in the brain of the control and mdx mouse. In our present study, there were no differences in brain morphology between mdx and control mouse as revealed in H-E stain and GFAP immunohistochemistry. However, the Dp71 were smaller in quantity in the brain of the mdx mouse than that of the control mouse. The reaction of astrocytes during repair of serebral infarction was distinctly delayed in the mdx mouse compared with that of the control mouse. These findings suggest that the astrocytes in the brain of the mdx mouse are functionally impaired including perivascular cytoplasmic processes with relation to neo-vascularization

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure < 100 mmHg (n = 1127), estimated glomerular filtration rate < 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Efficient preservation of sprouting vegetables under simulated microgravity conditions.

The effectiveness of a simulated microgravity environment as a novel method for preserving the freshness of vegetables was investigated. Three types of vegetables were selected: vegetable soybean, mung bean sprouts, and white radish sprouts. These selected vegetables were fixed on a three-dimensional rotary gravity controller, rotated slowly. The selected vegetables were stored at 25°C and 66% of relative humidity for 9, 6, or 5 d while undergoing this process. The simulated microgravity was controlled utilizing a gravity controller around 0 m s-2. The mung bean sprouts stored for 6 d under simulated microgravity conditions maintained higher thickness levels than the vegetable samples stored under normal gravity conditions (9.8 m s-2) for the same duration. The mass of all three items decreased with time without regard to the gravity environment, though the samples stored within the simulated microgravity environment displayed significant mass retention on and after 3 d for mung bean sprout samples and 1 d for white radish sprout samples. In contrast, the mass retention effect was not observed in the vegetable soybean samples. Hence, it was confirmed that the mass retention effect of microgravity was limited to sprout vegetables. As a result of analysis harnessing a mathematical model, assuming that the majority of the mass loss is due to moisture loss, a significant difference in mass reduction coefficient occurs among mung bean sprouts and white radish sprouts due to the microgravity environment, and the mass retention effect of simulated microgravity is quantitatively evaluated utilizing mathematical models. Simulated microgravity, which varies significantly from conventional refrigeration, ethylene control, and modified atmosphere, was demonstrated effective as a novel method for preserving and maintaining the freshness of sprout vegetables. This founding will support long-term space flight missions by prolonging shelf life of sprout vegetables

Hypergravity enhances RBM4 expression in human bone marrow-derived mesenchymal stem cells and accelerates their differentiation into neurons

Introduction: The regulation of stem cell differentiation is important in determining the quality of transplanted cells in regenerative medicine. Physical stimuli are involved in regulating stem cell differentiation, and in particular, research on the regulation of differentiation using gravity is an attractive choice. We have shown that microgravity is useful for maintaining undifferentiated mesenchymal stem cells (MSCs). However, the effects of hypergravity on the differentiation of MSCs, especially on neural differentiation related to neural regeneration, have not been elucidated. Methods: We induced neural differentiation of human bone marrow-derived MSCs (hbMSCs) for 10 days under normal gravity (1G) or hypergravity (3G) conditions using a gravity controller, Gravite®. HbMSCs were collected, and cell number and viability were measured 3 and 10 days after induction. RNA was also extracted from the collected hbMSCs, and the expression of neuron-associated genes and regulator markers of neural differentiation was analyzed using real-time polymerase chain reaction (PCR). Additionally, we evaluated the NF-M-positive cell rate 10 days after induction using immunofluorescent staining. Results: Neural gene expression and the NF-M-positive cell rate were increased in hbMSCs under the 3G condition 10 days after induction. mRNA expression of RNA binding motif protein 4 (RBM4) and pyruvate kinase M 1 (PKM1) in the 3G condition was also higher than that in the 1G group. Conclusions: Hypergravity can enhance RBM4 and PKM1, promoting the neural differentiation of hbMSCs

Impact of the Microgravity Environment in a Three–dimensional Clinostat on Osteoblast- and Osteoclast-like Cells

Mechanical unloading conditions result in decreases in bone mineral density and quantity, which may be partly attributed to an imbalance in bone formation and resorption. To investigate the effect of mechanical unloading on osteoblast and osteoclast differentiation, and the expression of RANKL and OPG genes in osteoblasts, we used a three-dimensional (3D) clinostat system simulating microgravity to culture MC3T3-E1 and RAW264.7 cells. Long-term exposure (7 days) of MC3T3-E1 cells to microgravity in the 3D clinostat inhibited the expression of Runx2, Osterix, type I collagen αI chain, RANKL and OPG genes. Similarly, 3D clinostat exposure inhibited the enhancement of β3-integrin gene expression, which normally induced by sRANKL stimulation in RAW264.7 cells. These results, taken together, demonstrate that long-term 3D clinostat exposure inhibits the differentiation of MC3T3-E1 cells together with suppression of RANKL and OPG gene expression, as well as the RANKL-dependent cellular fusion of RAW264.7 cells, suggesting that long-term mechanical unloading suppresses bone formation and resorption