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Transcriptomic analyses of postmortem brains have begun to elucidate molecular abnormalities in autism spectrum disorder (ASD). However, a crucial pathway involved in synaptic development, RNA editing, has not yet been studied on a genome-wide scale. Here we profiled global patterns of adenosine-to-inosine (A-to-I) editing in a large cohort of postmortem brains of people with ASD. We observed a global bias for hypoediting in ASD brains, which was shared across brain regions and involved many synaptic genes. We show that the Fragile X proteins FMRP and FXR1P interact with RNA-editing enzymes (ADAR proteins) and modulate A-to-I editing. Furthermore, we observed convergent patterns of RNA-editing alterations in ASD and Fragile X syndrome, establishing this as a molecular link between these related diseases. Our findings, which are corroborated across multiple data sets, including dup15q (genomic duplication of 15q11.2-13.1) cases associated with intellectual disability, highlight RNA-editing dysregulation in ASD and reveal new mechanisms underlying this disorder.

BackgroundRNA editing generates modifications to the RNA sequences, thereby increasing protein diversity and shaping various layers of gene regulation. Recent studies have revealed global shifts in editing levels across many cancer types, as well as a few specific mechanisms implicating individual sites in tumorigenesis or metastasis. However, most tumor-associated sites, predominantly in noncoding regions, have unknown functional relevance.ResultsHere, we carry out integrative analysis of RNA editing profiles between epithelial and mesenchymal tumors, since epithelial-mesenchymal transition is a key paradigm for metastasis. We identify distinct editing patterns between epithelial and mesenchymal tumors in seven cancer types using TCGA data, an observation further supported by single-cell RNA sequencing data and ADAR perturbation experiments in cell culture. Through computational analyses and experimental validations, we show that differential editing sites between epithelial and mesenchymal phenotypes function by regulating mRNA abundance of their respective genes. Our analysis of RNA-binding proteins reveals ILF3 as a potential regulator of this process, supported by experimental validations. Consistent with the known roles of ILF3 in immune response, epithelial-mesenchymal differential editing sites are enriched in genes involved in immune and viral processes. The strongest target of editing-dependent ILF3 regulation is the transcript encoding PKR, a crucial player in immune and viral response.ConclusionsOur study reports widespread differences in RNA editing between epithelial and mesenchymal tumors and a novel mechanism of editing-dependent regulation of mRNA abundance. It reveals the broad impact of RNA editing in cancer and its relevance to cancer-related immune pathways.

In the mammalian heart, the left ventricle (LV) rapidly becomes more dominant in size and function over the right ventricle (RV) after birth. The molecular regulators responsible for this chamber-specific differential growth are largely unknown. We found that cardiomyocytes in the neonatal mouse RV had lower proliferation, more apoptosis, and a smaller average size compared with the LV. This chamber-specific growth pattern was associated with a selective activation of p38 mitogen-activated protein kinase (MAPK) activity in the RV and simultaneous inactivation in the LV. Cardiomyocyte-specific deletion of both the Mapk14 and Mapk11 genes in mice resulted in loss of p38 MAPK expression and activity in the neonatal heart. Inactivation of p38 activity led to a marked increase in cardiomyocyte proliferation and hypertrophy but diminished cardiomyocyte apoptosis, specifically in the RV. Consequently, the p38-inactivated hearts showed RV-specific enlargement postnatally, progressing to pulmonary hypertension and right heart failure at the adult stage. Chamber-specific p38 activity was associated with differential expression of dual-specific phosphatases (DUSPs) in neonatal hearts, including DUSP26. Unbiased transcriptome analysis revealed that IRE1α/XBP1-mediated gene regulation contributed to p38 MAPK-dependent regulation of neonatal cardiomyocyte proliferation and binucleation. These findings establish an obligatory role of DUSP/p38/IRE1α signaling in cardiomyocytes for chamber-specific growth in the postnatal heart.

Recent studies revealed global shifts in RNA editing, the modification of RNA sequences, across many cancers. Besides a few sites implicated in tumorigenesis or metastasis, most tumor-associated sites, predominantly in noncoding regions, have unknown function. Here, we characterize editing profiles between epithelial (E) and mesenchymal (M) phenotypes in seven cancer types, as epithelial-mesenchymal transition (EMT) is a key paradigm for metastasis. We observe distinct editing patterns between E and M tumors and EMT induction upon loss of ADAR enzymes in cultured cells. E-M differential sites are highly enriched in genes involved in immune and viral processes, some of which regulate mRNA abundance of their respective genes. We identify a novel mechanism in which ILF3 preferentially stabilizes edited transcripts. Among editing-dependent ILF3 targets is the transcript encoding PKR, a crucial player in immune response. Our study demonstrates the broad impact of RNA editing in cancer and relevance of editing to cancer-related immune pathways.

Alcohol (ethanol, EtOH) consumption during pregnancy can result in fetal alcohol spectrum disorders (FASDs), which are characterized by prenatal and postnatal growth restriction and craniofacial dysmorphology. Recently, cell-derived extracellular vesicles, including exosomes and microvesicles containing several species of RNAs (exRNAs), have emerged as a mechanism of cell-to-cell communication. However, EtOH’s effects on the biogenesis and function of non-coding exRNAs during fetal development have not been explored. Therefore, we studied the effects of maternal EtOH exposure on the composition of exosomal RNAs in the amniotic fluid (AF) using rat fetal alcohol exposure (FAE) model. Through RNA-Seq analysis we identified and verified AF exosomal miRNAs with differential expression levels specifically associated with maternal EtOH exposure. Uptake of purified FAE AF exosomes by rBMSCs resulted in significant alteration of molecular markers associated with osteogenic differentiation of rBMSCs. We also determined putative functional roles for AF exosomal miRNAs (miR-199a-3p, miR-214-3p and let-7g) that are dysregulated by FAE in osteogenic differentiation of rBMSCs. Our results demonstrate that FAE alters AF exosomal miRNAs and that exosomal transfer of dysregulated miRNAs has significant molecular effects on stem cell regulation and differentiation. Our results further suggest the usefulness of assessing molecular alterations in AF exRNAs to study the mechanisms of FAE teratogenesis that should be further investigated by using an in vivo model.

Autism spectrum disorder (ASD) is a genetically complex, clinically heterogeneous neurodevelopmental disease. Recently, our understanding of the molecular abnormalities in ASD has been expanded through transcriptomic analyses of postmortem brains. However, a crucial molecular pathway involved in synaptic development, RNA editing, has not yet been studied on a genome-wide scale. Here, we profiled the global patterns of adenosine-to-inosine (A-to-I) editing in a large cohort of post-mortem ASD brains. Strikingly, we observed a global bias of hypo-editing in ASD brains, common to different brain regions and involving many genes with known neurobiological functions. Through genome-wide protein-RNA binding analyses and detailed molecular assays, we show that the Fragile X proteins, FMRP and FXR1P, interact with ADAR proteins and modulate A-to-I editing. Furthermore, we observed convergent patterns of RNA editing alterations in ASD and Fragile X syndrome, thus establishing RNA editing as a molecular link underlying these two highly related diseases. Our findings support a role for RNA editing dysregulation in ASD and highlight novel mechanisms for RNA editing regulation.

Background: RBFox1 is known to be an RNA splicing regulator with enriched expression in cardiac muscle. Loss of RBFox1 expression is a molecular hallmark associated with heart failure in mouse and human. Genetic manipulation of RBFox1 reveals a major function of RBFox1 in the pathogenesis of cardiac hypertrophy, fibrosis and dysfunction under pathological stresses. However, much of our current knowledge about RBFox1 focuses on the nuclear RBFox1 with a major impact on global alternative splicing changes. Yet, RBFox1 gene also generates a cytosolic isoform-RBFox1c through alternative splicing, and the specific function of RBFox1c has not been characterized. Goal and Methods: This study investigated the functional impact and the underlying mechanism of the RBFox1c in cardiac pathological remodeling and targeted gene regulation. Results: RBFox1c expression is significantly repressed in stressed cardiomyocytes in vivo and in vitro. RNA-Seq combined with IPA analyses from cardiomyocytes revealed RBFox1c but not the nucleus RBFox1 specifically suppressed pro-inflammatory genes. In the cardiac specific RBFox1 knockout mice, enhanced cardiac fibrosis is observed following I/R injury associated with elevated expression of pro-inflammatory genes. In contrast, cardiac specific expression of RBFox1c reduced cardiac fibrosis and inflammatory gene expression following pressure-overload and myocardial infarct injury associated with improved ejection fraction. Motif enrichment analysis identified significant enrichment of the RBFox1 binding motif in the 3’UTR of the RBFox1c regulated genes. We performed CLIP analysis followed by RT-PCR and observed RBFox1c interacted with inflammatory gene 3’UTR. Lastly, we explored the interactome of RBFox1c and found RBFox1c specifically interacted with a component of RNA decay machinery-Upf1. Indeed, while expression of RBFox1c repressed inflammatory gene expression, inactivation of Upf1 abolished this effect in cardiomyocytes. Conclusion: RBFox1 regulates cardiac transcriptome reprogramming at two post-transcriptional steps. The RBFox1 nuclei isoform regulates RNA splicing reprogramming, and the RBFox1c represses proinflammatory genes via recruitment of Upf1 mediated RNA degradation.

Human speech is one of the few examples of vocal learning among mammals yet ~half of avian species exhibit this ability. Its neurogenetic basis is largely unknown beyond a shared requirement for FoxP2 in both humans and zebra finches. We manipulated FoxP2 isoforms in Area X, a song-specific region of the avian striatopallidum analogous to human anterior striatum, during a critical period for song development. We delineate, for the first time, unique contributions of each isoform to vocal learning. Weighted gene coexpression network analysis of RNA-seq data revealed gene modules correlated to singing, learning, or vocal variability. Coexpression related to singing was found in juvenile and adult Area X whereas coexpression correlated to learning was unique to juveniles. The confluence of learning and singing coexpression in juvenile Area X may underscore molecular processes that drive vocal learning in young zebra finches and, by analogy, humans., eLife digest Songbirds, much like in humans, have a critical period in youth when they are best at learning vocal communication skills. In birds, this is when they learn a song they will use later in life as a courtship song. In humans, this is when language skills are most easily learned. After this critical period ends, it is much harder for people to learn languages, and for certain bird species to learn their song. When birds sing every morning, the activity of a gene called FoxP2 drops, which causes a coordinated change in the activity of thousands of other genes. It is suspected that FoxP2 – and the changes it causes – could be a part of the molecular basis for vocal learning. FoxP2 is also known to play a role in speech in humans, and both birds and humans have a long and a short version of this gene. Previous research has shown that when the long version of the gene was altered so its activity would no longer decrease when birds were singing, the birds failed to learn their song. Moreover, humans with a mutation in the long version have problems with their speech. However, until now, it was not known if modifications to the short version had the same effect. Burkett et al. investigated whether there was a noticeable pattern in the effects of FoxP2 before and after the critical period in a songbird. The analysis found that during the critical period, a set of genes changed together as young birds learned to sing. This particular pattern disappeared as the birds aged and the critical period ended. Burkett et al. confirmed that when birds had the long version of FoxP2 altered, they were less able to learn. However, changing the short version of FoxP2 had little effect on learning but led to changes in the birds’ song. The genetic pathways identified in the experiments are known to be present in many different species, including humans. Related pathways have also been found to play a role in non-vocal learning in organisms as distantly related as rats and snails. This suggests that they could be acting as a blueprint for learning new skills. Few treatments for language impairments have been developed so far due to poor understanding of the molecular basis for vocal communication. The findings of this study could help to create new treatments for speech problems in people, such as children with autism or people with mutated versions of FoxP2.

Ventricular chamber growth and development during perinatal circulatory transition is critical for functional adaptation of the heart. However, the chamber-specific programs of neonatal heart growth are poorly understood. We used integrated systems genomic and functional biology analyses of the perinatal chamber specific transcriptome and we identified Wnt11 as a prominent regulator of chamber-specific proliferation. Importantly, downregulation of Wnt11 expression was associated with cyanotic congenital heart defect (CHD) phenotypes and correlated with O2 saturation levels in hypoxemic infants with Tetralogy of Fallot (TOF). Perinatal hypoxia treatment in mice suppressed Wnt11 expression and induced myocyte proliferation more robustly in the right ventricle, modulating Rb1 protein activity. Wnt11 inactivation was sufficient to induce myocyte proliferation in perinatal mouse hearts and reduced Rb1 protein and phosphorylation in neonatal cardiomyocytes. Finally, downregulated Wnt11 in hypoxemic TOF infantile hearts was associated with Rb1 suppression and induction of proliferation markers. This study revealed a previously uncharacterized function of Wnt11-mediated signaling as an important player in programming the chamber-specific growth of the neonatal heart. This function influences the chamber-specific development and pathogenesis in response to hypoxia and cyanotic CHDs. Defining the underlying regulatory mechanism may yield chamber-specific therapies for infants born with CHDs.

To determine the role for mutations of MECP2 in Rett Syndrome, we generated isogenic lines of human iPSCs (hiPSCs), neural progenitor cells (NPCs), and neurons from patient fibroblasts with and without MECP2 expression in an attempt to recapitulate disease phenotypes in vitro. Molecular profiling uncovered neuronal specific gene expression changes including induction of a Senescence Associated Secretory Phenotype (SASP) program. Patient derived Neurons made without MECP2 show signs of stress, including induction of p53, and senescence. The induction of p53 appeared to affect dendritic branching in Rett neurons, as p53 inhibition restored dendritic complexity. These disease-in-a-dish data suggest that loss of MECP2 can lead to dendritic defects due to an increase in aspects of neuronal aging.

Mutations that lead to splicing defects can have severe consequences on gene function and cause disease. Here, we explore how human genetic variation affects exon recognition by developing a multiplexed functional assay of splicing using Sort-seq (MFASS). We assayed 27,733 variants in the Exome Aggregation Consortium (ExAC) within or adjacent to 2,198 human exons in the MFASS minigene reporter and found that 3.8% (1,050) of variants, most of which are extremely rare, led to large-effect splice-disrupting variants (SDVs). Importantly, we find that 83% of SDVs are located outside of canonical splice sites, are distributed evenly across distinct exonic and intronic regions, and are difficult to predict a priori. Our results indicate extant, rare genetic variants can have large functional effects on splicing at appreciable rates, even outside the context of disease, and MFASS enables their empirical assessment at scale.

Previous studies had shown that the integration of genome wide expression profiles, in metabolic tissues, with genetic and phenotypic variance, provided valuable insight into the underlying molecular mechanisms. We used RNA-Seq to characterize hypothalamic transcriptome in 99 inbred strains of mice from the Hybrid Mouse Diversity Panel (HMDP), a reference resource population for cardiovascular and metabolic traits. We report numerous novel transcripts supported by proteomic analyses, as well as novel non coding RNAs. High resolution genetic mapping of transcript levels in HMDP, reveals both local and trans expression Quantitative Trait Loci (eQTLs) demonstrating 2 trans eQTL 'hotspots' associated with expression of hundreds of genes. We also report thousands of alternative splicing events regulated by genetic variants. Finally, comparison with about 150 metabolic and cardiovascular traits revealed many highly significant associations. Our data provide a rich resource for understanding the many physiologic functions mediated by the hypothalamus and their genetic regulation. DOI: http://dx.doi.org/10.7554/eLife.15614.001, eLife digest Metabolism is a term that describes all the chemical reactions that are involved in keeping a living organism alive. Diseases related to metabolism – such as obesity, heart disease and diabetes – are a major health problem in the Western world. The causes of these diseases are complex and include both environmental factors, such as diet and exercise, and genetics. Indeed, many genetic variants that contribute to obesity have been uncovered in both humans and mice. However, it is only dimly understood how these genetic variants affect the underlying networks of interacting genes that cause metabolic disorders. Measuring gene activity or expression, and tracing how genetic instructions are carried from DNA into RNA and proteins, can reliably identify groups of genes that correlate with metabolic traits in specific organs. This strategy was successfully used in previous studies to reveal new information about abnormalities linked to obesity in specific tissues such as the liver and fat tissues. It was also shown that this approach might suggest new molecules that could be targeted to treat metabolic disorders. A brain region called the hypothalamus is key to the control of metabolism, including feeding behavior and obesity. Hasin-Brumshtein et al. set out to explore gene expression in the hypothalamus of 99 different strains of mice, in the hope that the data will help identify new connections between gene expression and metabolism. This approach showed that thousands of new and known genes are expressed in the mouse hypothalamus, some of which coded for proteins, and some of which did not. Hasin-Brumshtein et al. uncovered two genetic variants that controlled the expression of hundreds of other genes. Further analysis then revealed thousands of genetic variants that regulated the expression of, and type of RNA (so-called "spliceforms") produced from neighboring genes. Also, the expression of many individual genes showed significant similarities with about 150 metabolic measurements that had been evaluated previously in the mice. This new dataset is a unique resource that can be coupled with different approaches to test existing ideas and develop new ones about the role of particular genes or genetic mechanisms in obesity. Future studies will likely focus on new genes that show strong associations with attributes that are relevant to metabolic disorders, such as insulin levels, weight and fat mass. DOI: http://dx.doi.org/10.7554/eLife.15614.002

Background: Left ventricle (LV) and right ventricle (RV) in mouse heart undergo dramatically different chamber-specific remodeling after birth, leading to rapid increase in LV vs. RV chamber size. However, the underlying regulatory mechanism mediating chamber specific remodeling process remains enigmatic. Results and Methods: In neonatal mouse heart, p38 MAP kinase activity is dynamically activated in a chamber specific manner. p38 activity is specifically elevated in RV comparing to LV at E18.5, postnatal day 3 (P3) and P7 stages whereas p38 activity is lower in both ventricles at P0 and P1. In mouse heart with cardiomyocyte specific-knockout of p38α and β (p38ab-cdKO), total p38 activity was diminished in both chambers. The p38ab-cdKO mice had significant neonatal lethality associated with RV specific chamber enlargement and significant increase in both RV wall thickness (RVW) and inner diameter of RV (RVID) as early as P3. Interestingly, p38 inactivation suppressed myocyte apoptotic activity specifically in RV while increased RV myocyte proliferation and hypertrophy during neonatal period. Unexpectedly, RNA-seq results implicated Xbp1 mediated transcriptional regulation significantly contributing to p38 dependent transcriptome reprogramming in RV. Indeed, IRE1α expression in neonatal cardiomyocyte is sufficient to induce proliferation in vitro. Furthermore, knockdown of Xbp1 blunted p38 inhibition-induced myocyte proliferation, suggesting that IRE1a/Xbp1 mediate p38 signaling in neonatal myocyte proliferation. Conclusion: Chamber-specific remodeling in neonatal heart involves temporally regulated and RV specific p38 MAP kinase activity. RV specific myocyte proliferation and hypertrophy concurrent with RV specific programmed myocyte death is orchestrated by two innate stress-response pathways, p38 and Xbp1.

Background— Although metabolic reprogramming is critical in the pathogenesis of heart failure, studies to date have focused principally on fatty acid and glucose metabolism. Contribution of amino acid metabolic regulation in the disease remains understudied. Methods and Results— Transcriptomic and metabolomic analyses were performed in mouse failing heart induced by pressure overload. Suppression of branched-chain amino acid (BCAA) catabolic gene expression along with concomitant tissue accumulation of branched-chain α-keto acids was identified as a significant signature of metabolic reprogramming in mouse failing hearts and validated to be shared in human cardiomyopathy hearts. Molecular and genetic evidence identified the transcription factor Krüppel-like factor 15 as a key upstream regulator of the BCAA catabolic regulation in the heart. Studies using a genetic mouse model revealed that BCAA catabolic defect promoted heart failure associated with induced oxidative stress and metabolic disturbance in response to mechanical overload. Mechanistically, elevated branched-chain α-keto acids directly suppressed respiration and induced superoxide production in isolated mitochondria. Finally, pharmacological enhancement of branched-chain α-keto acid dehydrogenase activity significantly blunted cardiac dysfunction after pressure overload. Conclusions— BCAA catabolic defect is a metabolic hallmark of failing heart resulting from Krüppel-like factor 15–mediated transcriptional reprogramming. BCAA catabolic defect imposes a previously unappreciated significant contribution to heart failure.

Background: Fetal to neonatal transition of heart is an elaborate process, during which, neonatal cardiomyocytes undergo functional maturation and terminal exit from the cell cycle. However, transcriptome programming in neonatal cardiac chambers during perinatal stages is understudied. In particular, the changes in long non-coding RNAs (lncRNAs) in neonatal heart have not been explored. Objective: To achieve transcriptome-wide analysis of lncRNAs in neonatal left ventricle (LV) and right ventricle (RV) during maturation stages using deep RNA-Sequencing Methods: Deep RNA-sequencing was performed on male newborn mouse (C57 BL) LV and RV at 3 time points of perinatal circulatory transition: P0, P3 and P7. Reads were mapped to mouse genome (mm10). The lncRNAs annotated in NONCODE database were identified. Differentially expressed lncRNAs were defined as those with coefficient of variation ≥0.2, at a false discovery rate ≤0.05, and expressed at ≥3 RPKM in at least one sample. Correlated lncRNAs/ gene pairs were identified using Pearson’s (r2≥0.8, P≤0.05). A subset of LncRNAs/gene expression was validated using qRT-PCR. Results: Out of the 70, 983 observed unique lncRNAs, approximately 7000 were identified exhibiting significant variation during maturation windows with highly spatial-temporal dependent expression patterns, including approximately 5000 known and 2000 novel lncRNAs. Notably, 20% of these lncRNAs were located within 50 KB of a protein coding gene. Out of a total of 2400 lncRNAs/gene pairs, 10 % exhibited significantly concordant (lncRNA/gene) expression patterns. These correlated genes were significantly enriched in metabolism, cell cycle and contractility functional ontology. Interestingly, some of these lncRNAs exhibited concordance with their neighboring gene in human tissues with congenital heart defects, suggesting conserved, potentially significant, regulatory function. Conclusions: Transcriptome programming during neonatal heart maturation involves global changes in lncRNAs. Their expression concordance with neighboring protein coding genes implicates potential important regulatory role of lncRNAs in neonatal heart chamber specification and congenital diseases.

Although major genetic networks controlling early liver specification and morphogenesis are known, the mechanisms responsible for postnatal hepatic maturation are poorly understood. Here we employ global analyses of the mouse liver transcriptome to demonstrate that postnatal remodelling of the liver is accompanied by large-scale transcriptional and post-transcriptional transitions that are cell-type-specific and temporally coordinated. Combining detailed expression analyses with gain- and loss-of-function studies, we identify epithelial splicing regulatory protein 2 (ESRP2) as a conserved regulatory factor that controls the neonatal-to-adult switch of ∼20% of splice isoforms in mouse and human hepatocytes. The normal shift in splicing coincides tightly with dramatic postnatal induction of ESRP2 in hepatocytes. We further demonstrate that forced expression of ESRP2 in immature mouse and human hepatocytes is sufficient to drive a reciprocal shift in splicing and causes various physiological abnormalities. These findings define a direct role for ESRP2 in the generation of conserved repertoires of adult splice isoforms that facilitate terminal differentiation and maturation of hepatocytes., Alternative RNA splicing is important during organismal development. Here, the authors perform RNA-Seq on mouse and human liver samples to provide a comprehensive view of splicing events during liver development and growth, and identify Espr2 as a main regulator of these splicing processes.

Correct splice site recognition is critical in pre-mRNA splicing. We find that almost all of a diverse panel of exonic splicing silencer (ESS) elements alter splice site choice when placed between competing sites, consistently inhibiting use of intron-proximal 5' and 3' splice sites. Supporting a general role for ESSs in splice site definition, we found that ESSs are both abundant and highly conserved between alternative splice site pairs and that mutation of ESSs located between natural alternative splice site pairs consistently shifted splicing toward the intron-proximal site. Some exonic splicing enhancers (ESEs) promoted use of intron-proximal 5' splice sites, and tethering of hnRNP A1 and SF2/ASF proteins between competing splice sites mimicked the effects of ESS and ESE elements, respectively. Further, we observed that specific subsets of ESSs had distinct effects on a multifunctional intron retention reporter and that one of these subsets is likely preferred for regulation of endogenous intron retention events. Together, our findings provide a comprehensive picture of the functions of ESSs in the control of diverse types of splicing decisions.

Background: There are numerous anecdotal reports of ventricular arrhythmias during spaceflight; however, it is not known whether spaceflight or microgravity systematically increases the risk of cardiac dysrhythmias. Microvolt T wave alternans (MTWA) testing compares favorably with other noninvasive risk stratifiers and invasive electrophysiological testing in patients as a predictor of sudden cardiac death, ventricular tachycardia, and ventricular fibrillation. We hypothesized that simulated microgravity leads to an increase in MTWA. Methods: Twenty-four healthy male subjects underwent 9 to 16 days of head-down tilt bed rest (HDTB). MTWA was measured before and after the bed rest period during bicycle exercise stress. For the purposes of this study, we defined MTWA outcome to be positive if sustained MTWA was present with an onset heart rate ≤125 bpm. During various phases of HDTB, the following were also performed: daily 24-hour urine collections, serum electrolytes and catecholamines, and cardiovascular system identification (measure of autonomic function). Results: Before HDTB, 17% of the subjects were MTWA positive [95%CI: (0.6%, 37%)]; after HDTB, 42% of the subjects were MTWA positive [95%CI: (23%, 63%)] (P = 0.03). The subjects who were MTWA positive after HDTB compared with MTWA negative subjects had an increased versus decreased sympathetic responsiveness (P = 0.03) and serum norepinephrine levels (P = 0.05), and a trend toward higher potassium excretion (P = 0.06) after bed rest compared to baseline. Conclusions: HDTB leads to an increase in MTWA, providing the first evidence that simulated microgravity has a measurable effect on electrical repolarization processes. Possible contributing factors include loss in potassium and changes in sympathetic function.

Orthostatic intolerance (OI) is a major problem after spaceflight. Its etiology remains uncertain, but reports have pointed toward an individual susceptibility to OI. We hypothesized that individual predisposition plays an important role in post-bed rest OI. Twenty-four healthy male subjects were equilibrated on a constant diet, after which they underwent tilt-stand test (pre-TST). They then completed 14-16 days of head-down-tilt bed rest, and 14 of the subjects underwent repeat tilt-stand test (post-TST). During various phases, the following were performed: 24-h urine collections and hormonal measurements, plethysmography, and cardiovascular system identification (a noninvasive method to assess autonomic function and separately quantify parasympathetic and sympathetic responsiveness). Development of presyncope or syncope defined OI. During pre-TST, 11 subjects were intolerant and 13 were tolerant. At baseline, intolerant subjects had lower serum aldosterone ( P < 0.01), higher excretion of potassium ( P = 0.01), lower leg venous compliance ( P = 0.03), higher supine parasympathetic responsiveness ( P = 0.02), and lower standing sympathetic responsiveness ( P = 0.048). Of the 14 subjects who completed post-TST, 9 were intolerant and 5 were tolerant. Intolerant subjects had lower baseline serum cortisol ( P = 0.03) and a higher sodium level ( P = 0.02) compared with tolerant subjects. Thus several physiological characteristics were associated with increased susceptibility to OI. We propose a new model for OI, whereby individuals with greater leg venous compliance recruit compensatory mechanisms (activation of the renin-angiotensin-aldosterone system and sympathetic nervous system, and withdrawal of the parasympathetic nervous system) in the face of daily postural challenges, which places them at an advantage to face orthostatic stress. With head-down-tilt bed rest, the stimulus to recruit compensatory mechanisms disappears, and differences between the two subgroups attenuate.

Tumor metastasis remains the major cause of cancer-related death, but its molecular basis is still not well understood. Here we uncovered a splicing-mediated pathway that is essential for breast cancer metastasis. We show that the RNA-binding protein heterogeneous nuclear ribonucleoprotein M (hnRNPM) promotes breast cancer metastasis by activating the switch of alternative splicing that occurs during epithelial–mesenchymal transition (EMT). Genome-wide deep sequencing analysis suggests that hnRNPM potentiates TGFβ signaling and identifies CD44 as a key downstream target of hnRNPM. hnRNPM ablation prevents TGFβ-induced EMT and inhibits breast cancer metastasis in mice, whereas enforced expression of the specific CD44 standard (CD44s) splice isoform overrides the loss of hnRNPM and permits EMT and metastasis. Mechanistically, we demonstrate that the ubiquitously expressed hnRNPM acts in a mesenchymal-specific manner to precisely control CD44 splice isoform switching during EMT. This restricted cell-type activity of hnRNPM is achieved by competition with ESRP1, an epithelial splicing regulator that binds to the same cis-regulatory RNA elements as hnRNPM and is repressed during EMT. Importantly, hnRNPM is associated with aggressive breast cancer and correlates with increased CD44s in patient specimens. These findings demonstrate a novel molecular mechanism through which tumor metastasis is endowed by the hnRNPM-mediated splicing program.

Enhanced external counterpulsation (EECP) is a noninvasive, counterpulsative method to provide temporary aid to the failing heart by sequentially inflating cuffs on the lower extremity out-of-phase with the left ventricle. Optimization of the method necessitates consideration of the hemodynamics created by EECP and the mode of action providing patient benefit. A computational model based on the governing one-dimensional equations is developed that simulates cardiovascular hemodynamics during EECP. The model includes a 30-element arterial system including the left ventricle, bifurcations, and peripheral arterial vessels. Effects of vessel collapse as external pressure is applied, arterial refilling on pressure release, changes in aortic pressure, and shear stress generated in the arteries are each investigated. Device parameters are systematically varied to determine their effect on system performance. Results show the potential for significant collapse and shear augmentation throughout the arteries of the lower extremity. Performance is strongly influenced by the mean level of external pressurization and the timing of cuff inflation, but less so by the relative timing and pressure differences between cuff segments.

Background Exposure to microgravity induces cardiovascular deconditioning, manifested by orthostatic intolerance (OI). We assessed the renal, cardioendocrine, and cardiovascular responses of women and men to simulated microgravity to examine the impact of gender on OI. Methods Fifteen healthy female and 14 healthy male subjects were given a constant diet for 3 to 5 days, after which they underwent a tilt-stand test (pre-TST) and began 14 to 16 days of head-down tilt bed rest (HDTB), followed by a repeat tilt-stand test (post-TST). Female subjects began HDTB so that the post-TST was at the same time in their menstrual cycle as their pre-TST. Twenty-four-hour urine collections (daily), hormonal measurements, plethysmography, and cardiovascular system identification were performed. Results The times to presyncope were significantly different for men and women before ( p = .005) and after HDTB ( p = .001), with all of the women but only 50% of the men experiencing presyncope during the pre-TST ( p = .002) and all of the women but only 64% of the men experiencing presyncope during the post-TST. At baseline, the following differences between women and men were observed: women had higher serum aldosterone levels ( p = .02), higher parasympathetic responsiveness ( p = .01), lower sympathetic responsiveness ( p = .05), and lower venous compliance ( p = .05). Several parameters changed with HDTB in both men and women. In a double-blinded randomized trial, midodrine (5 mg orally) or placebo given to female subjects 1 hour before post-TST was ineffective in preventing OI. Conclusion In conclusion, the frequency of OI is higher in women than in men and is not modified by midodrine at the dose used. This increased susceptibility is likely secondary to intrinsic basal differences in the activity of volume-mediated parasympathetic and adrenergic systems and in venous tone. Thus, approaches to reduce OI in women are likely to differ from those effective in men.

A quantitative evaluation of parasympathetic and sympathetic function is often desirable in clinical practice. We previously proposed a noninvasive method to selectively quantify cardiac sympathetic and parasympathetic control. This method involves system identification analysis of the coupling mechanism between the instantaneous lung volume and heart rate signals. In the present study, we validated this method in conjunction with a newly developed Weighted Principal Component Regression-based system identification technique. We utilized data collected from 14 subjects in the control and autonomic blockade conditions in the standing posture. The results showed that a statistically significant reduction in parasympathetic function and sympathetic function was identified when atropine or propranolol was administered, respectively, and with autonomic double blockade, consistent with the wellaccepted physiologic knowledge.

Background Microgravity and simulated microgravity (SM) lead to important changes in orthostatic tolerance (OT), the autonomic nervous system (ANS), and the volume-regulating systems. After one is exposed to microgravity or SM, a period of readaptation to gravity is known to take place, but it is not certain if orthostatic function returns to baseline within the initial recovery and what mechanisms are involved. We hypothesized that after a period of recovery, OT, ANS, and volume-regulating systems would return to pre-SM levels. Methods To test this hypothesis, 24 healthy men were placed on a constant diet for 3 to 5 days, after which a tilt-stand test (pre-TST) was performed. The TST was repeated after 14 to 16 days of head-down tilt bed rest (HDTB) (post-TST) and a 3-day period of recovery (rec-TST), at which times measurements of renal, cardioendocrine, and cardiovascular systems were conducted. Results Presyncope occurred in 46% of subjects pre-TST, in 72% post-TST, and in 23% during rec-TST. OT was significantly better during the recovery period than at baseline ( p = .03). There was a significant decrease in urinary sodium and potassium excretion, along with a decrease in plasma renin activity and serum and urine aldosterone compared with baseline. Serum norepinephrine and sympathetic responsiveness remained below baseline values. Conclusion In summary, OT improved compared with baseline after a period of readaptation. Retention of electrolytes (sodium, potassium) could be involved. These findings indicate that recovery after SM is not simply a gradual return to baseline values but is instead a dynamic process reflecting interaction of multiple regulatory systems.

A quantitative evaluation of autonomic cardiovascular control is important in understanding basic pathophysiological mechanisms or for patient monitoring, treatment design and follow-up. Noninvasive techniques for this purpose have been the focus of many research endeavors. We previously proposed a method to extract pure parasympathetic and pure sympathetic indices based on the impulse response between instantaneous lung volume and heart rate. Identification of this impulse response involves a dual-input, single-output system in which one input interacts with the output in closed-loop. To identify this relatively complicated system, we propose here a new system identification technique based on a weighted-principal component regression method. Asymptotically, this technique implements model selection in the frequency domain. Therefore, in contrast to the conventional methods, it allows the data to play a significant role in determining candidate models. Moreover, the estimated model parameters reflect a trade-off between bias and variance to reach a relatively small mean squared prediction error. We employ experimental data to demonstrate that this technique is superior to a more traditional technique in terms of measuring cardiac autonomic indices.

Orthostatic intolerance (OI) is a major problem following spaceflight, and, during flight, astronauts also experience sleep restriction. We hypothesized that sleep restriction will compound the risk and severity of OI following simulated microgravity and exaggerate the renal, cardioendocrine, and cardiovascular adaptive responses to it. Nineteen healthy men were equilibrated on a constant diet, after which they underwent a tilt-stand test. They then completed 14–16 days of simulated microgravity [head-down tilt bed rest (HDTB)], followed by repeat tilt-stand test. During HDTB, 11 subjects were assigned to an 8-h sleep protocol (non-sleep restricted), and 8 were assigned to a sleep-restricted protocol with 6 h of sleep per night. During various phases, the following were performed: 24-h urine collections, hormonal measurements, and cardiovascular system identification. Development of presyncope or syncope defined OI. There was a significant decrease in time free of OI ( P = 0.02) and an increase in OI occurrence ( P = 0.06) after HDTB among all subjects. However, the increase in OI occurrence did not differ significantly between the two groups ( P = 0.60). The two groups also experienced similar physiological changes with HDTB (initial increase in sodium excretion; increased excretion of potassium at the end of HDTB; increase in plasma renin activity secretion without a change in serum or urine aldosterone). No significant change in autonomic function or catecholamines was noted. Simulated microgravity leads to increased OI, and sleep restriction does not additively worsen OI in simulated microgravity. Furthermore, conditions of sleep restriction and nonsleep restriction are similar with respect to renal, cardioendocrine, and cardiovascular responses to simulated microgravity.

Microgravity-induced orthostatic intolerance (OI) continues to be a primary concern for the human space program. To test the hypothesis that exposure to simulated microgravity significantly alters autonomic nervous control and, thus, contributes to increased incidence of OI, we employed the cardiovascular system identification (CSI) technique to evaluate quantitatively parasympathetic and sympathetic regulation of heart rate (HR). The CSI method analyzes second-to-second fluctuations in noninvasively measured HR, arterial blood pressure, and instantaneous lung volume. The coupling mechanisms between these signals are characterized by using a closed-loop model. Parameters reflecting parasympathetic and sympathetic responsiveness with regard to HR regulation can be extracted from the identified coupling mechanisms. We analyzed data collected from 29 human subjects before and after 16 days of head-down-tilt bed rest (simulated microgravity). Statistical analyses showed that parasympathetic and sympathetic responsiveness was impaired by bed rest. A lower sympathetic responsiveness and a higher parasympathetic responsiveness measured before bed rest identified individuals at greater risk of OI before and after bed rest. We propose an algorithm to predict OI after bed rest from measures obtained before bed rest.

Orthostatic intolerance following prolonged exposure to microgravity continues to be a primary concern of the human space program. Reduced autonomic tone has been demonstrated to contribute to this phenomenon, and the heart rate baroreflex, in particular, has been repeatedly shown to be impaired. However, only the works of Yelle et al. (1996, 2002) have attempted to address the role of the total peripheral resistance (TPR) baroreflex, a potentially more significant contributor to blood pressure regulation. We applied a previously developed method for estimating the static gains of both the arterial and cardiopulmonary TPR baroreflexes to data obtained before and after 16 day bed rest. Reductions in the estimated static gains of the arterial (statistically significant) and cardiopulmonary TPR baroreflex were found after bed rest. This study supports the works of Yelle et al., which imply that the TPR baroreflex is reduced after spaceflight.

Cardiovascular health is currently assessed through a variety of hemodynamic parameters, many of which can only be determined by invasive measurement often requiring hospitalization. A noninvasive method of evaluating several of these parameters such as systemic vascular resistance (SVR), maximum left ventricular elasticity E_LV end diastolic volume VED and cardiac output, is presented. The method has three elements: (1) a distributed model of the human cardiovascular system (Ozawa [et_al.], Ann. Biomed. Eng. 29:284–297, 2001) to generate a solution library that spans the anticipated range of parameter values, (2) a method for establishing the multidimensional relationship between features computed from the arterial blood pressure and/or flow traces (e.g., mean arterial pressure, pulse amplitude, mean flow velocity) and the critical hemodynamic parameters, and (3) a parameter estimation method that yields the best fit between measured and computed data. Sensitivity analyses were used to determine the critical parameters, and the influence of fixed model parameters. Using computer-generated brachial pressure and velocity profiles (which can be measured noninvasively), the error associated with this method was found to be less than 3% for SVR, and less than 10% for ELV and VED Simulations were also performed to test the ability of the approach to predict changes in SVR and ELV from an initial base line state. © 2002 Biomedical Engineering Society.

The transcription factor etsrp/Er71/Etv2 is a master control gene for vasculogenesis in all species studied to date. It is also required for hematopoiesis in zebrafish and mice. Several novel genes expressed in vasculature have been identified through transcriptional profiling of zebrafish embryos overexpressing etsrp by microarrays. Here we re-examined this transcriptional profile by Illumina RNA-sequencing technology, revealing a substantially increased number of candidate genes regulated by etsrp. Expression studies of 50 selected candidate genes from this dataset resulted in the identification of 39 new genes that are expressed in vascular cells. Regulation of these genes by etsrp was confirmed by their ectopic induction in etsrp overexpressing and decreased expression in etsrp deficient embryos. Our studies demonstrate the effectiveness of the RNA-sequencing technology to identify biologically relevant genes in zebrfish and produced a comprehensive profile of genes previously unexplored in vascular endothelial cell biology.

Microgravity-induced orthostatic intolerance continues to be a primary concern of the human space program. Reduced autonomic tone has been shown to contribute to this phenomenon. However, the techniques that have been utilized for quantitating autonomic tone are limited. In this paper, we present an improved, noninvasive method for quantitating distinctly parasympathetic and /spl beta/-sympathetic tone and demonstrate its validity with respect to postural changes. We then apply the method to data collected from 20 human subjects before and after exposure to simulated microgravity (16-day head down bedrest). Statistical analyses show that parasympathetic and /spl beta/-sympathetic tone are both impaired by bedrest and reduced 0-sympathetic tone before bedrest can predispose subjects to presyncope following bedrest.