Metabolic control of human cardiomyocyte function and maturation

Cardiovascular diseases remain a leading cause of morbidity and mortality in the world, despite advances in drug and therapeutic developments. The complex nature of cardiac diseases such as myocardial infarction and heart failure require innovative approaches to elucidate disease mechanisms, identify molecular targets and develop novel therapies. The advent of human pluripotent stem cell (hPSC) technologies allowed for robust and reliable generations of contracting human cardiomyocytes (CMs) in vitro. hPSC-CMs hold great promise for a broad range of research and clinical applications including studying myocardial physiology, modeling cardiac diseases, and transplanting healthy cells to repair the damaged heart. However, one major limitation of hPSC-CMs differentiated in vitro is that they are relatively immature and resemble embryonic CMs. These cells lack well defined cellular edges and mature sarcomeres, which makes it difficult to quantitatively assess contractile functions using traditional edge detection technologies. In addition, hPSC-CMs cultured in traditional glucose rich media lack metabolic and functional maturity, utilizing mainly glycolysis for energy production, similar to the embryonic heart. To address these limitations, we first devised a novel technology to simultaneously quantify hPSC-CMs’ contractile kinetics, force generation and electrical activities at the single cell resolution. This methodology allowed us to examine the impact of energy substrates and metabolic pathway utilization on CM physiology and function. We identified that Hypoxia Inducible Factor 1 alpha (HIF1α) and its transcriptional target Lactate Dehydrogenase A (LDHA) are aberrantly upregulated in hPSC-CMs cultured in traditional glucose rich media. By using small molecules and siRNA, we demonstrated that inhibition of HIF1α/LDHA shifts hPSC-CMs’ metabolism from glycolysis to oxidative phosphorylation, which resulted in improved CM structural and functional maturation. Furthermore, we investigated the energy substrate dependency of hPSC-CMs in response to in vitro hypoxic and ischemia-reperfusion injuries. We observed that hPSC-CMs cultured in glucose rich media lack physiological responses to hypoxic insults. On the other hand, in vitro coverslip ischemia-reperfusion resulted in CM death and apoptosis, independent of glucose cultures. These findings highlighted the importance of bioenergetics in modeling cardiac diseases in vitro and provided us with the basis for a potential drug screening platform using hPSC-CMs.2020-07-02T00:00:00

Engineering culture environment of human pluripotent stem cells to direct their commitment and maturation towards functional cardiomyocytes: An “-Omics” driven approach

The immature phenotype of human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) constrains their potential in cell therapy and drug discovery applications. In this study, we aim to overcome this hurdle by devising a novel strategy for generation and maturation of functional hPSC-CM. We relied on the aggregation of hPSC-derived cardiac progenitors to establish a scalable differentiation protocol capable of generating highly pure CM aggregate cultures. Whole-transcriptome analysis and 13C-metabolic flux analysis demonstrate at both molecular and fluxome levels that a 3D culture environment enhances metabolic maturation of hPSC-CMs. When compared to 2D, 3D cultures of hPSC-CMs displayed down-regulation of genes involved in glycolysis and lipid biosynthesis and increased expression of genes involved in OXPHOS. Accordingly, 3D hPSC-CMs had lower fluxes through glycolysis and fatty acid synthesis and increased TCA-cycle activity. We then assessed if alteration of culture medium composition to mimic in vivo substrate usage during cardiac development improves further hPSC-CM maturation in vitro. Our results showed that shifting hPSC-CMs from glucose-containing to galactose- and fatty acid-containing medium promotes their fast maturation into adult-like CMs with higher oxidative metabolism, transcriptional signatures closer to those of adult ventricular tissue, higher myofibril density and alignment, improved calcium handling, enhanced contractility, and more physiological action potential kinetics. Integrated “-Omics” analyses showed that addition of galactose to culture medium improves total oxidative capacity of the cells and ameliorates fatty acid oxidation avoiding the lipotoxicity that results from cell exposure to high fatty acid levels. This study provides an important link between substrate utilization and functional maturation of hPSC-CMs facilitating the application of these cells in preclinical research and regenerative medicine. Funding: This work was supported by FCT-funded projects CardioRegen (HMSP-ICT/0039/2013), CARDIOSTEM (MITPTB/ECE/0013/2013) and Netdiamond (SAICTPAC/0047/2015). iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344) is also acknowledged

Improving functional maturation of human pluripotent stem cells derived cardiomyocytes through metabolic understanding

In vitro differentiation of human pluripotent stem cells into cardiomyocytes (hPSC-CMs) is a crucial process to enable their application in cell therapy and drug discovery. Nevertheless, despite the remarkable efforts over the last decade towards the optimization of cardiac differentiation protocols, generated hPSC-CMs are still immature, closely reminiscent of fetal cardiomyocytes in what regards structure, metabolism and function. In this study, we aim to overcome this hurdle by devising a novel metabolic-based strategy to improve hPSC-CMs generation and functionality. Noteworthy, we integrated structural and functional analyses of hPSC-CM with powerful “omics” technologies (proteomics, transcriptomics, metabolomics and fluxomics) as complementary analytical tools to support process optimization and product characterization. We relied on the aggregation of hPSC-derived cardiac progenitors to establish a scalable differentiation protocol capable of generating highly pure CM aggregate cultures. Whole-transcriptome analysis and 13C-metabolic flux analysis demonstrated that a three-dimensional (3D) and agitated-based culture environment enhances metabolic maturation of hPSC-CMs. When compared to static monolayer, 3D cultures of hPSC-CMs displayed down-regulation of genes involved in glycolysis and lipid biosynthesis and increased expression of genes involved in OXPHOS. Accordingly, 3D hPSC-CMs had lower fluxes through glycolysis and fatty acid synthesis and increased TCA-cycle activity. We then assessed if alteration of culture medium composition to mimic in vivo substrate usage during cardiac development improved further hPSC-CM maturation in vitro. Our results showed that shifting hPSC-CMs from glucose-containing to galactose- and fatty acid-containing medium promotes their fast maturation into adult-like CMs with higher oxidative metabolism, transcriptional signatures closer to those of adult ventricular tissue, higher myofibril density and alignment, improved calcium handling, enhanced contractility, and more physiological action potential kinetics. “-Omics” analyses showed that addition of galactose to culture medium improves total oxidative capacity of the cells and ameliorates fatty acid oxidation avoiding the lipotoxicity that results from cell exposure to high fatty acid levels. This study demonstrated that metabolic shifts during differentiation/maturation of hPSC-CM are a cause, rather than a consequence, of the phenotypic and functional alterations observed. The metabolic-based strategy established herein holds technical and economic advantages over the existing protocols due to its scalability, simplicity and ease of application. Improved maturation and functionality of in vitro generated hPSC-CM will excel their application in cell therapy, drug discovery and cardiac disease modeling. Funding: This work was supported by FCT-funded projects CardioRegen (HMSP-ICT/0039/2013), NETDIAMOND (SAICTPAC/0047/2015) and MetaCardio (Ref.032566). iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344) is also acknowledged

Analysis of lncRNA-Associated ceRNA Network Reveals Potential lncRNA Biomarkers in Human Colon Adenocarcinoma

Background/Aims: Long non-coding RNAs (lncRNAs) acting as competing endogenous RNAs (ceRNAs) play significant roles in the development of tumors, but the functions of specific lncRNAs and lncRNA-related ceRNA networks have not been fully elucidated for colon adenocarcinoma (COAD). In this study, we aimed to clarify the lncRNA-microRNA (miRNA)-mRNA ceRNA network and potential lncRNA biomarkers in COAD. Methods: We extracted data from The Cancer Genome Atlas (TCGA) and identified COAD-specific mRNAs, miRNAs, and lncRNAs. The biological processes in Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were analyzed for COAD-specific mRNAs. We then constructed a ceRNA network of COAD-specific mRNAs, miRNAs and lncRNAs and analyzed the correlation between expression patterns and clinical features of the lncRNAs involved. After identifying potential mRNA targets of 4 lncRNAs related to overall survival (OS), we conducted stepwise analysis of these targets through GO and KEGG. Using tissue samples from our own patients, we also verified certain analytical results using quantitative real-time PCR (qRT-PCR). Results: Data from 521 samples (480 tumor tissue and 41 adjacent non-tumor tissue samples) were extracted from TCGA. A total of 258 specific lncRNAs, 206 specific miRNAs, and 1467 specific mRNAs were identified (absolute log2 [fold change] > 2, false discovery rate < 0.01). Analysis of KEGG revealed that specific mRNAs were enriched in cancer-related pathways. The ceRNA network was constructed with 64 lncRNAs, 18 miRNAs, and 42 mRNAs. Among these lncRNAs involved in the network, 3 lncRNAs (LINC00355, HULC, and IGF2-AS) were confirmed to be associated with certain clinical features and 4 lncRNAs (HOTAIR, LINC00355, KCNQ1OT1, and TSSC1-IT1) were found to be negatively linked to OS (log-rank p < 0.05). KEGG showed that the potential mRNA targets of these 4 lncRNAs may be concentrated in the MAPK pathway. Certain results were validated by qRT-PCR. Conclusion: This study providing novel insights into the lncRNA-miRNA-mRNA ceRNA network and reveals potential lncRNA biomarkers in COAD

Laminar flame characteristics of natural gas and dissociated methanol mixtures diluted by nitrogen

The effect of dissociated methanol (H2:CO=2:1 by volume) on laminar burning velocity of natural gas (methane as the main component) was studied by using a constant volume bomb (CVB). Nitrogen, as diluent gas, was added into the natural gas (CH4) - dissociated methanol (DM) mixtures to investigate the dilution effect. Experiments were conducted at initial temperature of 343 K and initial pressure of 0.3 MPa with equivalence ratios from 0.8 to 1.4. Laminar burning velocities were calculated through Schlieren photographs, correlation of in-cylinder pressure data and Chemkin-Pro. Results show an increase in laminar burning velocity with initial temperature and proportion of dissociated methanol but a decrease with initial pressure and proportion of nitrogen. The laminar burning velocities were 25.1 cm/s, 38.7 cm/s and 83.2 cm/s respectively at stoichiometric ratio when the proportions of the dissociated methanol were 0%, 40% and 80%. Adding more dissociated methanol tends to shift the peak burning velocity towards the richer side while adding nitrogen has the opposite effect. More dissociated methanol will lead to earlier cellularity

Detection of Essential Tremor at the S -Band

Essential tremor (ET) is a neurological disorder characterized by rhythmic, involuntary shaking of a part or parts of the body. The most common tremor is seen in the hands/arms and fingers. This paper presents an evaluation of ETs monitoring based on finger-to-nose test measurement as captured by small wireless devices working in shortwave or S-band frequency range. The acquired signals in terms of amplitude and phase information are used to detect a tremor in the hands. Linearly transforming raw phase data acquired in the S-band were carried out for calibrating the phase information and to improve accuracy. The data samples are used for classification using support vector machine algorithm. This model is used to differentiate the tremor and nontremor data efficiently based on secondary features that characterize ET. The accuracy of our measurements maintains linearity, and more than 90% accuracy rate is achieved between the feature set and data samples

Novel MicroRNA Regulators of Atrial Natriuretic Peptide Production

Atrial natriuretic peptide (ANP) has a central role in regulating blood pressure in humans. Recently, microRNA 425 (miR-425) was found to regulate ANP production by binding to the mRNA of NPPA, the gene encoding ANP. mRNAs typically contain multiple predicted microRNA (miRNA)-binding sites, and binding of different miRNAs may independently or coordinately regulate the expression of any given mRNA. We used a multifaceted screening strategy that integrates bioinformatics, next-generation sequencing data, human genetic association data, and cellular models to identify additional functional NPPA-targeting miRNAs. Two novel miRNAs, miR-155 and miR-105, were found to modulate ANP production in human cardiomyocytes and target genetic variants whose minor alleles are associated with higher human plasma ANP levels. Both miR-15 and miR-105 repressed NPPA mRNA in an allele-specific manner, with the minor allele of each respective variant conferrin resistance to the miRNA either by disruption of miRNA base pairing or by creation of wobble base pairing. Moreover, miR-15 enhanced the repressive effects of miR-425 on ANP production in human cardiomyocytes. Our study combines computational genomic, and cellular tools to identify novel miRNA regulators of ANP production that could be targeted to raise ANP levels which may have applications for the treatment of hypertension or heart failure

Upper ocean biogeochemistry of the oligotrophic North Pacific Subtropical Gyre : from nutrient sources to carbon export

Subtropical gyres cover 26–29% of the world’s surface ocean and are conventionally regarded as ocean deserts due to their permanent stratification, depleted surface nutrients, and low biological productivity. Despite tremendous advances over the past three decades, particularly through the Hawaii Ocean Time-series and the Bermuda Atlantic Time-series Study, which have revolutionized our understanding of the biogeochemistry in oligotrophic marine ecosystems, the gyres remain understudied. We review current understanding of upper ocean biogeochemistry in the North Pacific Subtropical Gyre, considering other subtropical gyres for comparison. We focus our synthesis on spatial variability, which shows larger than expected dynamic ranges of properties such as nutrient concentrations, rates of N2 fixation, and biological production. This review provides new insights into how nutrient sources drive community structure and export in upper subtropical gyres. We examine the euphotic zone in subtropical gyres as a two-layered vertically structured system: a nutrient-depleted layer above the top of the nutricline in the well-lit upper ocean and a nutrient-replete layer below in the dimly lit waters. These layers vary in nutrient supply and stoichiometries and physical forcing, promoting differences in community structure and food webs, with direct impacts on the magnitude and composition of export production. We evaluate long-term variations in key biogeochemical parameters in both of these euphotic zone layers. Finally, we identify major knowledge gaps and research challenges in these vast and unique systems that offer opportunities for future studies

Einfluss der Verarbeitungstechnologie und Werkstoffzusammensetzung auf die Struktur-Eigenschafts-Beziehungen von thermoplastischen Nanoverbundwerkstoffen

Die Einarbeitung von nanoskaligen Füllstoffen zur Steigerung von polymeren Eigenschaftsprofilen ist sehr viel versprechend und stößt daher heutzutage sowohl in der Forschung als auch in der Industrie auf großes Interesse. Bedingt durch ausgeprägte Oberflächen und hohe Anziehungskräfte, liegen Nanopartikel allerdings nicht singulär sondern als Partikelanhäufungen, so genannten Agglomeraten oder Aggregaten, vor. Zur Erzielung der gewünschten Materialverbesserungen gilt es, diese aufzuspalten und homogen in der polymeren Matrix zu verteilen. Bei thermoplastischen Kunststoffen ist die gleichläufige Doppelschneckenextrusion eines der gängigsten Verfahren zur Einarbeitung von Additiven und Füllstoffen. Aus diesem Grund war es Ziel dieser Arbeit, mittels dieses Verfahrens verbesserte Verbundwerkstoffe mit Polyamid 66- und Polyetheretherketon-Matrix, durch Einarbeitung von nanoskaligem Titandioxid (15 und 300 nm), zu generieren. In einem ersten Schritt wurden die verfahrenstechnischen Parameter, wie Drehzahl und Durchsatz, sowie die Prozessführung und damit deren Einfluss auf die Materialeigenschaften beleuchtet. Der spezifische Energieeintrag ist ausschlaggebend zur Deagglomeration der Nanopartikel. Dieser zeigte leichte Abhängigkeiten von der Drehzahl und dem Durchsatz und verursachte bei der Einarbeitung der Partikel keine wesentlichen Unterschiede in der Aufspaltung der Partikel sowie gar keine in den resultierenden mechanischen Eigenschaften. Die Prozessführung wurde unterteilt in Mehrfach- und Einfachextrusion. Die Herstellung eines hochgefüllten Masterbatches, dessen mehrfaches Extrudieren und anschließendes Verdünnen, führte zu einer sehr guten Deagglomeration und stark verbesserten Materialeigenschaften. Mittels Simulation des Extrusionsprozesses konnte festgestellt werden, dass das Vorhandensein von ungeschmolzenem Granulat in der Verfahrenszone zu einer Schmelze/Nanopartikel/ Feststoffreibung führt, die die Ursache für eine sehr gute Aufspaltung der Partikel zu sein scheint. Durch Modifikation des Extrusionsprozesses erreichte die Einfachextrusion annähernd den Grad an Deagglomeration bei Mehrfachextrusion, wobei die Materialien bei letzterem Verfahren die besten Eigenschaftsprofile aufwiesen. In einem zweiten Schritt wurde ein Vergleich der Einflüsse von unterschiedlichen Partikelgrößen und –gehalten auf die polymeren Matrizes vollzogen. Die 15 nm Partikel zeigten signifikant bessere mechanische Ergebnisse auf als die 300 nm Partikel, und die Wirkungsweise des 15 nm Partikels auf Polyetheretherketon war stärker als auf Polyamid 66. Es konnten Steigerungen in Steifigkeit, Festigkeit und Zähigkeit erzielt werden. Rasterelektronenmikroskopische Aufnahmen bestätigten diese Ergebnisse. Eine Berechnung der Plan-Selbstkosten von einem Kilogramm PEEK-Nanoverbundwerkstoff im Vergleich zu einem Kilogramm unverstärktem PEEK verdeutlichte, dass ein Material kreiert wurde, welches deutlich verbesserte Eigenschaften bei gleichem Preis aufweist. Zusammenfassend konnte in dieser Arbeit ein tieferes Verständnis des Extrusionsvorganges zur Herstellung von kostengünstigen und verbesserten Thermoplasten durch das Einbringen von Nanopartikeln gewonnen werden