Hepatic fibrosis progresses with right heart failure, and becomes cardiac cirrhosis in a severe case. Although its causal factor still remains unclear. Here we evaluated the progression of hepatic fibrosis using a pulmonary artery banding (PAB)-induced right heart failure model and investigated whether cardiac output (CO) is responsible for the progression of hepatic fibrosis.

Endothelial progenitor cell (EPC) transplantation is beneficial for ischemic diseases such as critical limb ischemia and ischemic heart disease. The scarcity of functional EPCs in adults is a limiting factor for EPC transplantation therapy. The quality and quantity culture (QQc) system is an effective ex vivo method for enhancing the number and angiogenic potential of EPCs. Further, microgravity environments have been shown to enhance the functional potential of stem cells. We therefore hypothesized that cells cultured with QQc under microgravity may have enhanced functionality. We cultured human peripheral blood mononuclear cells using QQc under normal (E), microgravity (MG), or microgravity followed by normal (ME) conditions and found that ME resulted in the most significant increase in CD34+ and double positive Dil-Ac-LDL-FITC-Ulex-Lectin cells, both EPC markers. Furthermore, angiogenic potential was determined by an EPC-colony forming assay. While numbers of primitive EPC-colony forming units (pEPC-CFU) did not change, numbers of definitive EPC-CFU colonies increased most under ME conditions. Gene-expression profiling also identified increases in angiogenic factors, including vascular endothelial growth factor, under MG and ME conditions. Thus, QQc along with ME conditions could be an efficient system for significantly enhancing the number and angiogenic potential of EPCs.

Reorientation of cucumber seedlings induces re-localization of CsPIN1 auxin efflux carriers in endodermal cells of the transition zone between hypocotyl and roots. This study examined whether the re-localization of CsPIN1 was due to the graviresponse. Immunohistochemical analysis indicated that, when cucumber seedlings were grown entirely under microgravity conditions in space, CsPIN1 in endodermal cells was mainly localized to the cell side parallel to the minor axis of the elliptic cross-section of the transition zone. However, when cucumber seeds were germinated in microgravity for 24 h and then exposed to 1g centrifugation in a direction crosswise to the seedling axis for 2 h in space, CsPIN1 was re-localized to the bottom of endodermal cells of the transition zone. These results reveal that the localization of CsPIN1 in endodermal cells changes in response to gravity. Furthermore, our results suggest that the endodermal cell layer becomes a canal by which auxin is laterally transported from the upper to the lower flank in response to gravity. The graviresponse-regulated re-localization of CsPIN1 could be responsible for the decrease in auxin level, and thus for the suppression of peg formation, on the upper side of the transition zone in horizontally placed seedlings of cucumber.

The effects of heat stress on the morphological properties and intracellular signaling of innervated and denervated soleus muscles were investigated. Heat stress was applied to rats by immersing their hindlimbs in a warm water bath (42°C, 30 min/day, every other day following unilateral denervation) under anesthesia. During 14 days of experimental period, heat stress for a total of seven times promoted growth-related hypertrophy in sham-operated muscles and attenuated atrophy in denervated muscles. In denervated muscles, the transcription of ubiquitin ligase, atrogin-1/muscle atrophy F-box (Atrogin-1), and muscle RING-finger protein-1 (MuRF-1), genes was upregulated and ubiquitination of proteins was also increased. Intermittent heat stress inhibited the upregulation of Atrogin-1, but not MuRF-1 transcription. And the denervation-caused reduction in phosphorylated protein kinase B (Akt), 70-kDa heat-shock protein (HSP70), and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), which are negative regulators of Atrogin-1 and MuRF-1 transcription, was mitigated. In sham-operated muscles, repeated application of heat stress did not affect Atrogin-1 and MuRF-1 transcription, but increased the level of phosphorylated Akt and HSP70, but not PGC-1α Furthermore, the phosphorylation of Akt and ribosomal protein S6, which is known to stimulate protein synthesis, was increased immediately after a single heat stress particularly in the sham-operated muscles. The effect of a heat stress was suppressed in denervated muscles. These results indicated that the beneficial effects of heat stress on the morphological properties of muscles were brought regardless of innervation. However, the responses of intracellular signaling to heat stress were distinct between the innervated and denervated muscles.

Duchenne muscular dystrophy (DMD) and the less severe Becker muscular dystrophy (BMD) are due to mutations in the DMD gene. Previous reports show that in-frame deletion of exons 45-55 produces an internally shorted, but functional, dystrophin protein resulting in a very mild BMD phenotype. In order to elucidate the molecular mechanism leading to this phenotype, we generated exon 45-55 deleted dystrophin transgenic/mdx (Tg/mdx) mice. Muscular function of Tg/mdx mice was restored close to that of wild type (WT) mice but the localization of the neuronal type of nitric oxide synthase was changed from the sarcolemma to the cytosol. This led to hyper-nitrosylation of the ryanodine receptor 1 causing increased Ca2+ release from the sarcoplasmic reticulum. On the other hand, Ca2+ reuptake by the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) was restored to the level of WT mice, suggesting that the Ca2+ dysregulation had been compensated by SERCA activation. In line with this, expression of sarcolipin (SLN), a SERCA-inhibitory peptide, was upregulated in mdx mice, but strongly reduced in Tg/mdx mice. Furthermore, knockdown of SLN ameliorated the cytosolic Ca2+ homeostasis and the dystrophic phenotype in mdx mice. These findings suggest that SLN may be a novel target for DMD therapy.

Myocardial fibrosis is often associated with cardiac hypertrophy; indeed, fibrosis is one of the most critical factors affecting prognosis. We aimed to identify the molecules involved in promoting fibrosis under hypertrophic stimuli. We previously established a rat model of cardiac hypertrophy by pulmonary artery banding, in which approximately half of the animals developed fibrosis in the right ventricle. Here, we first comprehensively analyzed mRNA expression in the right ventricle with or without fibrosis in pulmonary artery banding model rats by DNA microarray analysis (GSE141650 at NCBI GEO). The expression levels of 19 genes were up-regulated more than 1.5-fold in fibrotic hearts compared with non-fibrotic hearts. Among them, fibrosis growth factor (FGF) 23 showed one of the biggest increases in expression. Real-time PCR analysis also revealed that, among the FGF receptor (FGFR) family, FGFR1 was highly expressed in fibrotic hearts. We then found that FGF23 was expressed predominantly in cardiomyocytes, while FGFR1 was predominantly expressed in fibroblasts in the rat ventricle. Next, we added FGF23 and transforming growth factor (TGF)-β1 (10-50 ng/mL of each) to isolated fibroblasts from normal adult rat ventricles and cultured them for three days. While FGF23 itself did not directly affect the expression levels of any fibrosis-related mRNAs, FGF23 enhanced the effect of TGF-β1 on increasing the expression levels of α-smooth muscle actin (α-SMA) mRNA. This increase in xx-SMA mRNA levels due to the combination of TGF-β1 and FGF23 was attenuated by the inhibition of FGFR1 or the knockdown of FGFR1 in fibroblasts. Thus, FGF23 synergistically promoted the activation of fibroblasts with TGF-β1, transforming fibroblasts into myofibroblasts via FGFR1. Thus, we identified FGF23 as a paracrine factor secreted from cardiomyocytes to promote cardiac fibrosis under conditions in which TGF-β1 is activated. FGF23 could be a possible target to prevent fibrosis following myocardial hypertrophy.

The ductus arteriosus, an essential embryonic blood vessel between the pulmonary artery and the descending aorta, constricts after birth or hatching and eventually closes to terminate embryonic circulation. Chicken embryos have two long ductus arteriosi, which anatomically differ from mammal ductus arteriosus. Each long ductus arteriosus is divided into two parts: the pulmonary artery-sided and descending aorta-sided ductus arteriosi. Although the pulmonary artery-sided and descending aorta-sided ductus arteriosi have distinct functional characteristics, such as oxygen responsiveness, the difference in their transcriptional profiles has not been investigated. We performed a DNA microarray analysis (GSE 120116 at NCBI GEO) with pooled tissues from the chicken pulmonary artery-sided ductus arteriosus, descending aorta-sided ductus arteriosus, and aorta at the internal pipping stage. Although several known ductus arteriosus-dominant genes such as tfap2b were highly expressed in the pulmonary artery-sided ductus arteriosus, we newly found genes that were dominantly expressed in the chicken pulmonary artery-sided ductus arteriosus. Interestingly, cluster analysis showed that the expression pattern of the pulmonary artery-sided ductus arteriosus was closer to that of the descending aorta-sided ductus arteriosus than that of the aorta, whereas the morphology of the descending aorta-sided ductus arteriosus was closer to that of the aorta than that of the pulmonary artery-sided ductus arteriosus. Subsequent pathway analysis with DAVID bioinformatics resources revealed that the pulmonary artery-sided ductus arteriosus showed enhanced expression of the genes involved in melanogenesis and tyrosine metabolism compared with the descending aorta-sided ductus arteriosus, suggesting that tyrosinase and the related genes play an important role in the proper differentiation of neural crest-derived cells during vascular remodeling in the ductus arteriosus. In conclusion, the transcription profiles of the chicken ductus arteriosus provide new insights for investigating the mechanism of ductus arteriosus closure.

The effects of long-term exposure to extreme space conditions on astronauts were investigated by analyzing hair samples from ten astronauts who had spent six months on the International Space Station (ISS). Two samples were collected before, during and after their stays in the ISS; hereafter, referred to as Preflight, Inflight and Postflight, respectively. The ratios of mitochondrial (mt) to nuclear (n) DNA and mtRNA to nRNA were analyzed via quantitative PCR. The combined data of Preflight, Inflight and Postflight show a significant reduction in the mtDNA/nDNA in Inflight, and significant reductions in the mtRNA/nRNA ratios in both the Inflight and Postflight samples. The mtRNA/mtDNA ratios were relatively constant, except in the Postflight samples. Using the same samples, the expression of redox and signal transduction related genes, MnSOD, CuZnSOD, Nrf2, Keap1, GPx4 and Catalase was also examined. The results of the combined data from Preflight, Inflight and Postflight show a significant decrease in the expression of all of the redox-related genes in the samples collected Postflight, with the exception of Catalase, which show no change. This decreased expression may contribute to increased oxidative stress Inflight resulting in the mitochondrial damage that is apparent Postflight.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors are oral antidiabetic drugs that promote urinary glucose excretion. Conversely, they cause behavioural changes, such as hyperphagia, that result in a positive energy balance. The relationship between energy homeostasis and SGLT2 inhibitors-induced behavioural changes remains unclear. Here we show that ipragliflozin, a SGLT2 inhibitor, time-dependently affects behaviour and enhances energy expenditure in normal and type 2 diabetic Goto-Kakizaki (GK) rats, using continuous glucose telemetry. Alongside increased urinary glucose excretion, ipragliflozin increased total food and water intakes in normal and GK rats. In normal rats, ipragliflozin treatment acutely disturbed the circadian rhythms of food and water intakes, activity, and body temperature. Subsequently, these rhythms gradually returned towards a normal state. However, activity and body temperature remained suppressed. In GK rats, ipragliflozin did not affect circadian rhythms. Blood glucose values assessed by glucose telemetry were significantly reduced in both ipragliflozin-treated groups. Despite these behavioural and glycaemic changes, ipragliflozin significantly increased oxygen consumption during dark and light periods in both groups. Ipragliflozin reduced body weight in normal rats only. Thus, ipragliflozin decreases blood glucose beyond compensatory hyperphagia in normal and GK rats, resulting in enhanced basal energy expenditure, despite acutely altering circadian rhythms in normoglycaemic individuals.

Ductus arteriosus (DA) closure follows constriction and remodeling of the entire vessel wall. Patent ductus arteriosus occurs when the DA does not close after birth, and this condition is currently treated using cyclooxygenase inhibitors. However, the efficacy of cyclooxygenase inhibitors is often limited. Our previous study demonstrated that low-dose thromboxane A2 receptor (TP) stimulation constricted the DA with minimal adverse effects in rat neonates. However, its effect on DA remodeling remains unknown. In this study, we focused on the impact of the exogenous TP stimulation on the DA remodeling, especially intimal thickening. Using DA explants from rat fetuses at embryonic day 19 as a ex vivo model and primary cultured rat DA smooth muscle cells from embryonic day 21 as a in vitro model, we evaluated the effect of TP stimulation on the DA remodeling. The selective TP agonists U46619 and I-BOP promoted neointima formation in the ex vivo DA explants, and TP stimulation increased DA SMC migration in a dose-dependent manner. Both effects were inhibited by the selective TP antagonist SQ29548 or the siRNA against TP. TP stimulation also increased DA SMC proliferation in the presence of 10% fetal bovine serum. LC/MS/MS analysis revealed that TP stimulation increased secretion of several extracellular matrix proteins that may contribute to an increase in neointima formation. In conclusion, we uncovered that exogenous administration of TP agonist promotes neointima formation through the induction of migration and proliferation of DA SMC, which could contribute to DA closure and also to its vasoconstrictive action.

Endothelial cells (ECs) lining the blood vessels serve a variety of functions and play a central role in the homeostasis of the circulatory system. Since the ductus arteriosus (DA) has different arterial characteristics from its connecting vessels, we hypothesized that ECs of the DA exhibited a unique gene profile involved in the regulation of DA-specific morphology and function. Using a fluorescence-activated cell sorter, we isolated ECs from pooled tissues from the DA or the descending aorta of Wistar rat fetuses at full-term of gestation (F group) or neonates 30 minutes after birth (N group). Using anti-CD31 and anti-CD45 antibodies as cell surface markers for ECs and hematopoietic derived cells, respectively, cDNAs from the CD31-positive and CD45-negative cells were hybridized to the Affymetrix GeneChip® Rat Gene 1.0 ST Array. Among 26,469 gene-level probe sets, 82 genes in the F group and 81 genes in the N group were expressed at higher levels in DA ECs than in aortic ECs (p<0.05, fold change>2.0). In addition to well-known endothelium-enriched genes such as Tgfb2 and Vegfa, novel DA endothelium-dominant genes including Slc38a1, Capn6, and Lrat were discovered. Enrichment analysis using GeneGo MetaCore software showed that DA endothelium-related biological processes were involved in morphogenesis and development. We identified many overlapping genes in each process including neural crest-related genes (Hoxa1, Hoxa4, and Hand2, etc) and the second heart field-related genes (Tbx1, Isl1, and Fgf10, etc). Moreover, we found that regulation of epithelial-to-mesenchymal transition, cell adhesion, and retinol metabolism are the active pathways involved in the network via potential interactions with many of the identified genes to form DA-specific endothelia. In conclusion, the present study uncovered several significant differences of the transcriptional profile between the DA and aortic ECs. Newly identified DA endothelium-dominant genes may play an important role in DA-specific functional and morphologic characteristics.

Sarcomeric contraction in cardiomyocytes serves as the basis for the heart's pump functions in mammals. Although it plays a critical role in the circulatory system, myocardial sarcomere length (SL) change has not been directly measured in vivo under physiological conditions because of technical difficulties. In this study, we developed a high speed (100-frames per second), high resolution (20-nm) imaging system for myocardial sarcomeres in living mice. Using this system, we conducted three-dimensional analysis of sarcomere dynamics in left ventricular myocytes during the cardiac cycle, simultaneously with electrocardiogram and left ventricular pressure measurements. We found that (a) the working range of SL was on the shorter end of the resting distribution, and (b) the left ventricular-developed pressure was positively correlated with the SL change between diastole and systole. The present findings provide the first direct evidence for the tight coupling of sarcomere dynamics and ventricular pump functions in the physiology of the heart.

Skeletal muscle wasting is a major obstacle for long-term space exploration. Similar to astronauts, the nematode Caenorhabditis elegans displays negative muscular and physical effects when in microgravity in space. It remains unclear what signaling molecules and behavior(s) cause these negative alterations. Here we studied key signaling molecules involved in alterations of C. elegans physique in response to fluid dynamics in ground-based experiments. Placing worms in space on a 1G accelerator increased a myosin heavy chain, myo-3, and a transforming growth factor-β (TGF-β), dbl-1, gene expression. These changes also occurred when the fluid dynamic parameters viscosity/drag resistance or depth of liquid culture were increased on the ground. In addition, body length increased in wild type and body wall cuticle collagen mutants, rol-6 and dpy-5, grown in liquid culture. In contrast, body length did not increase in TGF-β, dbl-1, or downstream signaling pathway, sma-4/Smad, mutants. Similarly, a D1-like dopamine receptor, DOP-4, and a mechanosensory channel, UNC-8, were required for increased dbl-1 expression and altered physique in liquid culture. As C. elegans contraction rates are much higher when swimming in liquid than when crawling on an agar surface, we also examined the relationship between body length enhancement and rate of contraction. Mutants with significantly reduced contraction rates were typically smaller. However, in dop-4, dbl-1, and sma-4 mutants, contraction rates still increased in liquid. These results suggest that neuromuscular signaling via TGF-β/DBL-1 acts to alter body physique in response to environmental conditions including fluid dynamics.

In cucumber seedlings, gravitropism interferes with hydrotropism, which results in the nearly complete inhibition of hydrotropism under stationary conditions. However, hydrotropic responses are induced when the gravitropic response in the root is nullified by clinorotation. Columella cells in the root cap sense gravity, which induces the gravitropic response. In this study, we found that removing the root tip induced hydrotropism in cucumber roots under stationary conditions. The application of auxin transport inhibitors to cucumber seedlings under stationary conditions suppressed the hydrotropic response induced by the removal of the root tip. To investigate the expression of genes related to hydrotropism in de-tipped cucumber roots, we conducted transcriptome analysis of gene expression by RNA-Seq using seedlings exhibiting hydrotropic and gravitropic responses. Of the 21 and 45 genes asymmetrically expressed during hydrotropic and gravitropic responses, respectively, five genes were identical. Gene ontology (GO) analysis indicated that the category auxin-inducible genes was significantly enriched among genes that were more highly expressed in the concave side of the root than the convex side during hydrotropic or gravitropic responses. Reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) analysis revealed that root hydrotropism induced under stationary conditions (by removing the root tip) was accompanied by the asymmetric expression of several auxin-inducible genes. However, intact roots did not exhibit the asymmetric expression patterns of auxin-inducible genes under stationary conditions, even in the presence of a moisture gradient. These results suggest that the root tip inhibits hydrotropism by suppressing the induction of asymmetric auxin distribution. Auxin transport and distribution not mediated by the root tip might play a role in hydrotropism in cucumber roots.

Phospholamban (PLN) is a key regulator of sarcolemma calcium uptake in cardiomyocyte, its inhibitory activity to SERCA is regulated by phosphorylation. PLN hypophosphorylation is a common molecular feature in failing heart. The current study provided evidence at molecular, cellular and whole heart levels to implicate a sarcolemma membrane targeted protein phosphatase, PP2Ce, as a specific and potent PLN phosphatase. PP2Ce expression was elevated in failing human heart and induced acutely at protein level by β -adrenergic stimulation or oxidative stress in cardiomyocytes. PP2Ce expression in mouse heart blunted β-adrenergic response and exacerbated ischemia/reperfusion injury. Therefore, PP2Ce is a new regulator for cardiac function and pathogenesis.

Interstitial myocardial fibrosis is one of the factors responsible for dysfunction of the heart. However, how interstitial fibrosis affects cardiac function and excitation-contraction coupling (E-C coupling) has not yet been clarified. We developed an animal model of right ventricular (RV) hypertrophy with fibrosis by pulmonary artery (PA) banding in rats. Two, four, and six weeks after the PA-banding operation, the tension and intracellular Ca2+ concentration of RV papillary muscles were simultaneously measured (n = 33). The PA-banding rats were clearly divided into two groups by the presence or absence of apparent interstitial fibrosis in the papillary muscles: F+ or F- group, respectively. The papillary muscle diameter and size of myocytes were almost identical between F+ and F-, although the RV free wall weight was heavier in F+ than in F-. F+ papillary muscles exhibited higher stiffness, lower active tension, and lower Ca2+ responsiveness compared with Sham and F- papillary muscles. In addition, we found that the time to peak Ca2+ had the highest correlation coefficient to percent of fibrosis among other parameters, such as RV weight and active tension of papillary muscles. The phosphorylation level of troponin I in F+ was significantly higher than that in Sham and F-, which supports the idea of lower Ca2+ responsiveness in F+. We also found that connexin 43 in F+ was sparse and disorganized in the intercalated disk area where interstitial fibrosis strongly developed. In the present study, the RV papillary muscles obtained from the PA-banding rats enabled us to directly investigate the relationship between fibrosis and cardiac dysfunction, the impairment of E-C coupling in particular. Our results suggest that interstitial fibrosis worsens cardiac function due to 1) the decrease in Ca2+ responsiveness and 2) the asynchronous activation of each cardiac myocyte in the fibrotic preparation due to sparse cell-to-cell communication.

Mammalian embryos differentiate into the inner cell mass (ICM) and trophectoderm at the 8-16 cell stage. The ICM forms a single cluster that develops into a single fetus. However, the factors that determine differentiation and single cluster formation are unknown. Here we investigated whether embryos could develop normally without gravity. As the embryos cannot be handled by an untrained astronaut, a new device was developed for this purpose. Using this device, two-cell frozen mouse embryos launched to the International Space Station were thawed and cultured by the astronauts under microgravity for 4 days. The embryos cultured under microgravity conditions developed into blastocysts with normal cell numbers, ICM, trophectoderm, and gene expression profiles similar to those cultured under artificial-1 g control on the International Space Station and ground-1 g control, which clearly demonstrated that gravity had no significant effect on the blastocyst formation and initial differentiation of mammalian embryos.

Although muscle atrophy is a serious problem during spaceflight, little is known about the sequence of molecular events leading to atrophy in response to microgravity. We carried out a spaceflight experiment using Caenorhabditis elegans onboard the Japanese Experiment Module of the International Space Station. Worms were synchronously cultured in liquid media with bacterial food for 4 days under microgravity or on a 1-G centrifuge. Worms were visually observed for health and movement and then frozen. Upon return, we analyzed global gene and protein expression using DNA microarrays and mass spectrometry. Body length and fat accumulation were also analyzed. We found that in worms grown from the L1 larval stage to adulthood under microgravity, both gene and protein expression levels for muscular thick filaments, cytoskeletal elements, and mitochondrial metabolic enzymes decreased relative to parallel cultures on the 1-G centrifuge (95% confidence interval (P⩽0.05)). In addition, altered movement and decreased body length and fat accumulation were observed in the microgravity-cultured worms relative to the 1-G cultured worms. These results suggest protein expression changes that may account for the progressive muscular atrophy observed in astronauts.

Multipotent Isl1(+) heart progenitors give rise to three major cardiovascular cell types: cardiac, smooth muscle, and endothelial cells, and play a pivotal role in lineage diversification during cardiogenesis. A critical question is pinpointing when this cardiac-vascular lineage decision is made, and how this plasticity serves to coordinate cardiac chamber and vessel growth. The posterior domain of the Isl1-positive second heart field contributes to the SLN-positive atrial myocardium and myocardial sleeves in the cardiac inflow tract, where myocardial and vascular smooth muscle layers form anatomical and functional continuity. Herein, using a new atrial specific SLN-Cre knockin mouse line, we report that bipotent Isl1(+)/SLN(+) transient cell population contributes to cardiac as well as smooth muscle cells at the heart-vessel junction in cardiac inflow tract. The Isl1(+)/SLN(+) cells are capable of giving rise to cardiac and smooth muscle cells until late gestational stages. These data suggest that the cardiac and smooth muscle cells in the cardiac inflow tract share a common developmental origin. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".

Adaptation to the space environment can sometimes pose physiological problems to International Space Station (ISS) astronauts after their return to earth. Therefore, it is important to develop healthcare technologies for astronauts. In this study, we examined the feasibility of using hair follicles, a readily obtained sample, to assess gene expression changes in response to spaceflight adaptation. In order to investigate the gene expression changes in human hair follicles during spaceflight, hair follicles of 10 astronauts were analyzed by microarray and real time qPCR analyses. We found that spaceflight alters human hair follicle gene expression. The degree of changes in gene expression was found to vary among individuals. In some astronauts, genes related to hair growth such as FGF18, ANGPTL7 and COMP were upregulated during flight, suggesting that spaceflight inhibits cell proliferation in hair follicles.