Functional improvement and maturation of rat and human engineered heart tissue by chronic electrical stimulation

Spontaneously beating engineered heart tissue (EHT) represents an advanced in vitro model for drug testing and disease modeling, but cardiomyocytes in EHTs are less mature and generate lower forces than in the adult heart. We devised a novel pacing system integrated in a setup for videooptical recording of EHT contractile function over time and investigated whether sustained electrical field stimulation improved EHT properties. EHTs were generated from neonatal rat heart cells (rEHT, n=96) or human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hEHT, n=19). Pacing with biphasic pulses was initiated on day 4 of culture. REHT continuously paced for 16-18 days at 0.5Hz developed 2.2× higher forces than nonstimulated rEHT. This was reflected by higher cardiomyocyte density in the center of EHTs, increased connexin-43 abundance as investigated by two-photon microscopy and remarkably improved sarcomere ultrastructure including regular M-bands. Further signs of tissue maturation include a rightward shift (to more physiological values) of the Ca(2+)-response curve, increased force response to isoprenaline and decreased spontaneous beating activity. Human EHTs stimulated at 2Hz in the first week and 1.5Hz thereafter developed 1.5× higher forces than nonstimulated hEHT on day 14, an ameliorated muscular network of longitudinally oriented cardiomyocytes and a higher cytoplasm-to-nucleus ratio. Taken together, continuous pacing improved structural and functional properties of rEHTs and hEHTs to an unprecedented level. Electrical stimulation appears to be an important step toward the generation of fully mature EHT

Pilotvorhaben Erdbecken-Heisswasserwaermespeicher in Rottweil Abschlussbericht

The concept for an underground hot water heat store was developed, and a 600 m"3 pilot store was constructed at Rottweil as part of the district heating system. Heat from cogeneration units is transferred to a reinforced-concrete tank lined with steel sheets and thermally insulated with rockwool. Leaktightness of the steel liner was found to be a problem, so that automatic operation was impossible and the floor had to be relined in summer 1997. After reconstruction of the heat supply system, the overall system including the heat store was scheduled for full-scale operation by the end of 1997. The PC-supported data acquisition system with modem for remote transmission of data functioned reliably. Owing to its modular structure, it is able to process and evaluate all relevant measured data. The experience gained and the cost calculations made provide a basis for the construction of further underground heat stores which may also be used for seasonal storage of heat. (orig.)Im Rahmen dieses Vorhabens wurde ein Konzept fuer einen Erdbecken-Heisswasser-Waermespeicher entwickelt und in Rottweil ein Pilotspeicher mit 600 m"3 Volumen errichtet. Dieser Waermespeicher ist Bestandteil einer Fernwaermeversorgung und wird mit Waerme aus Blockheizkraftwerken beladen. Der Behaelter aus Stahlbeton ist aussen an Decke und Wand mit Mineralwolle waermegedaemmt und innen vollstaendig mit einem Edelstahlblech ausgekleidet. Die wasserdichte Verbindung der Edelstahlbahnen stellte sich dabei als kritischer Faktor heraus, da durch Undichtigkeiten der Betrieb des Waermespeichers stark beeinflusst wird. So konnte trotz verlaengerter Projektlaufzeit kein automatischer Betrieb des Waermespeichers stattfinden. Die erneute Auskleidung des Bodenbereichs wurde im Sommer 1997 durchgefuehrt. Nach Umbau der Waermeerzeugungsanlagen soll das Gesamtsystem inkl. Waermespeicher Ende 1997 in Betrieb genommen werden. Die installierte PC gestuetzte Messdatenerfassung mit Modemanlage zur Datenfernuebertragung hat sich als ein zuverlaessiges System erwiesen. Ihr modularer Aufbau erlaubt es, alle relevanten Messgroessen aufzubereiten und auszuwerten. Die Online-Darstellung gibt dabei einen schnellen Ueberblick ueber die aktuellen Systemdaten. Die Messergebnisse waehrend des Probebetriebes bestaetigten die vorausberechneten Temperaturgradienten im Speicher und in der Behaelterwand. Die Undichtigkeiten der Auskleidung bedingten eine zeitliche Verzoegerung, so dass nur ein Fehlbetrieb einiger Komponenten nicht aber eine Betriebsoptimierung stattfinden konnte. Die verbunden mit dem Bau des Waermespeichers prognostizierte Steigerung der mittels Kraft-Waermekopplung erzeugten Waerme und die Verschiebung der Blockheizkraftwerk-Laufzeit von der Nacht auf den Tag konnte deshalb nicht nachgewiesen werden. Ausgehend von dem erarbeiteten Baukonzept wurden Kostenschaetzungen fuer Speichervolumina von 5000 m"3 und 10000 m"3 durchgefuehrt. Die mit den Einheitspreisen auf Basis des Ausschreibungsergebnisses in Rottweil ermittelten Baukosten liegen demnach zwischen 207 und 189 DM/m"3. Mit den gewonnenen Erfahrungen und den ermittelten Kosten ist somit die Grundlage fuer den Bau von weiteren Erdbecken-Heisswasserwaermespeichern geschaffen worden. Diese koennen dann auch als saisonale Waermespeicher errichtet werdenSIGLEAvailable from TIB Hannover: F98B1281+a / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Bildung, Wissenschaft, Forschung und Technologie, Bonn (Germany)DEGerman

DNA methylation in an engineered heart tissue model of cardiac hypertrophy: common signatures and effects of DNA methylation inhibitors

DNA methylation affects transcriptional regulation and constitutes a drug target in cancer biology. In cardiac hypertrophy, DNA methylation may control the fetal gene program. We therefore investigated DNA methylation signatures and their dynamics in an in vitro model of cardiac hypertrophy based on engineered heart tissue (EHT). We exposed EHTs from neonatal rat cardiomyocytes to a 12-fold increased afterload (AE) or to phenylephrine (PE 20 microM) and compared DNA methylation signatures to control EHT by pull-down assay and DNA methylation microarray. A 7-day intervention sufficed to induce contractile dysfunction and significantly decrease promoter methylation of hypertrophy-associated upregulated genes such as Nppa (encoding ANP) and Acta1 (alpha-skeletal actin) in both intervention groups. To evaluate whether pathological consequences of AE are affected by inhibiting de novo DNA methylation we applied AE in the absence and presence of DNA methyltransferase (DNMT) inhibitors: 5-aza-2'-deoxycytidine (aza, 100 microM, nucleosidic inhibitor), RG108 (60 microM, non-nucleosidic) or methylene disalicylic acid (MDSA, 25 microM, non-nucleosidic). Aza had no effect on EHT function, but RG108 and MDSA partially prevented the detrimental consequences of AE on force, contraction and relaxation velocity. RG108 reduced AE-induced Atp2a2 (SERCA2a) promoter methylation. The results provide evidence for dynamic DNA methylation in cardiac hypertrophy and warrant further investigation of the potential of DNA methylation in the treatment of cardiac hypertrophy

Ataxin-10 is part of a cachexokine cocktail triggering cardiac metabolic dysfunction in cancer cachexia.

Objectives Cancer cachexia affects the majority of tumor patients and significantly contributes to high mortality rates in these subjects. Despite its clinical importance, the identity of tumor-borne signals and their impact on specific peripheral organ systems, particularly the heart, remain mostly unknown. Methods and Results By combining differential colon cancer cell secretome profiling with large-scale cardiomyocyte phenotyping, we identified a signature panel of seven “cachexokines”, including Bridging integrator 1, Syntaxin 7, Multiple inositol-polyphosphate phosphatase 1, Glucosidase alpha acid, Chemokine ligand 2, Adamts like 4, and Ataxin-10, which were both sufficient and necessary to trigger cardiac atrophy and aberrant fatty acid metabolism in cardiomyocytes. As a prototypical example, engineered secretion of Ataxin-10 from non-cachexia-inducing cells was sufficient to induce cachexia phenotypes in cardiomyocytes, correlating with elevated Ataxin-10 serum levels in murine and human cancer cachexia models. Conclusions As Ataxin-10 serum levels were also found to be elevated in human cachectic cancer patients, the identification of Ataxin-10 as part of a cachexokine cocktail now provides a rational approach towards personalized predictive, diagnostic and therapeutic measures in cancer cachexia

Ataxin-10 is part of a cachexokine cocktail triggering cardiac metabolic dysfunction in cancer cachexia (vol 5, 67, 2015).

The authors regret that the original paper was published with an error in the supplementary data (Table 1). In order to identify tumor-secreted factors that contribute to cardiac atrophy under the condition of cancer cachexia, in the original study, we performed a differential secretome analysis comparing cell conditioned media from cachexia-inducing C26 colon carcinoma cells and non-cachexia-inducing MC38 colon carcinoma cells. Secreted proteins which were at least 2-fold more abundantly secreted from C26 cells were selected for further functional validation. Functional validation was performed by overexpressing candidate proteins in HEK293A cells and collecting the candidate-enriched cell conditioned media for assaying their atrophy-inducing potential on primary neonatal rat cardiomyocytes. In the context of subsequent studies focusing on different aspects of cancer-induced cachexia, we performed a differential transcriptomics analysis comparing RNAseq data from C26 and MC38 cells. While differential protein secretion does not necessarily need to be fully reflected by corresponding differences at the level of gene expression, we were still surprised about the lack of congruency between the corresponding regulation of gene expression and protein secretion. Surprisingly, for the overlap of genes and proteins being differentially regulated between C26 and MC38 cells (regardless of the direction of change), we found a significant negative correlation between differential gene expression and differential protein secretion (Corrigendum Figure 1 A). In order to elucidate the basis of this unexpected finding, we decided to repeat the differential secretome analysis in the same manner as it has been performed in the original study. Notably, the comparison between the secretome analyses (old vs new) revealed a remarkably strong negative correlation concerning the difference in protein secretion between C26 and MC38 cells (Corrigendum Figure 1B). Furthermore, when we then compared the new secretome analysis with the differences in the transcription between C26 and MC38 cells, there was a highly significant positive correlation (Corrigendum Figure 1C). The found consistency for the differences between the cell lines at distinct levels of regulation (transcription vs secretion) suggested that these datasets were correctly associated, in contrast to the previous comparison with the original differential secretome analysis. Taken together, these new analyses strongly indicate that in the original (old) secretome analysis, a swap in the sample allocation must have occurred, either during sample preparation, the subsequent proteomic or data analysis. Despite extensive evaluation of the respective experiment records, it was not possible to detect at which exact point in the course of the experimental work this mistake was made. Remarkably enough, the high-throughput functional validation of 109 candidates performed in the original study (original manuscript Figure 3C), using selected candidates now considered to be more abundantly secreted from non-cachexia-inducing MC38 instead of cachexia-inducing C26 cells, still revealed a set of candidates showing the expected atrophy effects upon treatment of primary cardiomyocytes with the respective candidate-enriched conditioned media. These effects were comparable to the effects of C26 conditioned medium on cardiomyocyte atrophy (original Figure 2A and B) and were therefore applied as primary selection criterion for putative cachexokines (mediators of cachexia). Additionally, further analysis selected a subset of 7 candidates which, similar to C26 conditioned medium, increased the fatty acid oxidation rate in neonatal rat cardiomyocytes treated with candidate-enriched medium (original Figure 4A). We speculate that the high-throughput functional analysis, albeit being based on an erroneous initial secretome analysis, contained a sufficient high number of protein candidates in order to contain protein factors which eventually turned out to be still relevant with respect to their capability to mediate a specific component of the cachexia phenotype (cardiomyocyte atrophy). This is exemplified by the main candidate Ataxin-10 (Atxn10), for which we could confirm elevated levels in different models of experimental cachexia, including C26 tumor-bearing mice but not in MC38 tumor-bearing mice (original Figure 5A–D). In a model pancreatic of cancer based on orthotopic cell implantation, circulating Atxn10 levels closely correlated with the degree of weight loss (Figure 5E and F). Finally, we found Atxn10 levels to be elevated in cancer patients with cachexia compared to weight-stable patients (Figure 5G). Therefore, we would like to emphasize that the majority of data provided in the publication is still correct. We apologize for any inconvenience that might have resulted from providing incorrect differential secretome data as supplemental material of the study and now provide the data of the new and correct differential secretome analysis (Corrigendum supplemental data)

10-year stroke prevention after successful carotid endarterectomy for asymptomatic stenosis (ACST-1); a multicentre randomised trial

BACKGROUND: If carotid artery narrowing remains asymptomatic (ie, has caused no recent stroke or other neurological symptoms), successful carotid endarterectomy (CEA) reduces stroke incidence for some years. We assessed the long-term effects of successful CEA. METHODS: Between 1993 and 2003, 3120 asymptomatic patients from 126 centres in 30 countries were allocated equally, by blinded minimised randomisation, to immediate CEA (median delay 1 month, IQR 0·3-2·5) or to indefinite deferral of any carotid procedure, and were followed up until death or for a median among survivors of 9 years (IQR 6-11). The primary outcomes were perioperative mortality and morbidity (death or stroke within 30 days) and non-perioperative stroke. Kaplan-Meier percentages and logrank p values are from intention-to-treat analyses. This study is registered, number ISRCTN26156392. FINDINGS: 1560 patients were allocated immediate CEA versus 1560 allocated deferral of any carotid procedure. The proportions operated on while still asymptomatic were 89·7% versus 4·8% at 1 year (and 92·1%vs 16·5% at 5 years). Perioperative risk of stroke or death within 30 days was 3·0% (95% CI 2·4-3·9; 26 non-disabling strokes plus 34 disabling or fatal perioperative events in 1979 CEAs). Excluding perioperative events and non-stroke mortality, stroke risks (immediate vs deferred CEA) were 4·1% versus 10·0% at 5 years (gain 5·9%, 95% CI 4·0-7·8) and 10·8% versus 16·9% at 10 years (gain 6·1%, 2·7-9·4); ratio of stroke incidence rates 0·54, 95% CI 0·43-0·68, p<0·0001. 62 versus 104 had a disabling or fatal stroke, and 37 versus 84 others had a non-disabling stroke. Combining perioperative events and strokes, net risks were 6·9% versus 10·9% at 5 years (gain 4·1%, 2·0-6·2) and 13·4% versus 17·9% at 10 years (gain 4·6%, 1·2-7·9). Medication was similar in both groups; throughout the study, most were on antithrombotic and antihypertensive therapy. Net benefits were significant both for those on lipid-lowering therapy and for those not, and both for men and for women up to 75 years of age at entry (although not for older patients). INTERPRETATION: Successful CEA for asymptomatic patients younger than 75 years of age reduces 10-year stroke risks. Half this reduction is in disabling or fatal strokes. Net benefit in future patients will depend on their risks from unoperated carotid lesions (which will be reduced by medication), on future surgical risks (which might differ from those in trials), and on whether life expectancy exceeds 10 years. FUNDING: UK Medical Research Council, BUPA Foundation, Stroke Association