Proteomic analysis in valvular cardiomyopathy: aortic regurgitation vs. aortic stenosis

Left ventricular (LV) reverse remodeling after aortic valve (AV) surgery is less predictable in chronic aortic regurgitation (AR) than in aortic stenosis (AS). We aimed to disclose specific LV myocardial protein signatures possibly contributing to differential disease progression. Global protein profiling of LV myocardial samples excised from the subaortic interventricular septum in patients with isolated AR or AS undergoing AV surgery was performed using liquid chromatography–electrospray ionization–tandem mass spectrometry. Based on label-free quantitation protein intensities, a logistic regression model was calculated and adjusted for age, sex and protein concentration. Web-based functional enrichment analyses of phenotype-associated proteins were performed utilizing g:Profiler and STRING. Data are available via ProteomeXchange with identifier PXD039662. Lysates from 38 patients, including 25 AR and 13 AS samples, were analyzed. AR patients presented with significantly larger LV diameters and volumes (end-diastolic diameter: 61 (12) vs. 48 (13) mm, p < 0.001; end-diastolic volume: 180.0 (74.6) vs. 92.3 (78.4), p = 0.001). A total of 171 proteins were associated with patient phenotype: 117 were positively associated with AR and the enrichment of intracellular compartment proteins (i.e., assigned to carbohydrate and nucleotide metabolism, protein biosynthesis and the proteasome) was detected. Additionally, 54 were positively associated with AS and the enrichment of extracellular compartment proteins (i.e., assigned to the immune and hematopoietic system) was observed. In summary, functional enrichment analysis revealed specific AR- and AS-associated signatures of LV myocardial proteins

Transkriptomanalysen myokardialer Erkrankungen - Charakterisierung von krankheits und Therapie-assoziierten Genexpressionsänderungen in Patienten mit dilatativer Kardiomyopathie

Myokardiale Erkrankungen gehören zu den häufigsten Todesursachen. Das Verständnis der molekularen, patho-physiologischen Ereignisse ist somit entscheidend für die Suche nach geeigneten Therapien. Um einen Einblick in zelluläre Ereignisse der kardialen Erkrankungen zu erhalten, sollten in der hier vorgelegten Arbeit Genexpressionsanalysen unter Verwendung von DNA-Mikroarrays durchgeführt werden. Den Schwerpunkt dieser Arbeit bildete die Charakterisierung der Immunadsorptionstherapie (IA/IgG-Therapie), die eine Therapieoption der dilatativen Kardiomyopathie (DCM) darstellt. Anhand von Genexpressionsanalysen immunadsorbierter Patienten sollte der Therapieeffekt analysiert werden. Dafür wurde zunächst die Etablierung eines Protokolls zur Isolation von Protein und RNA aus humanen endomyokardialen Biopsien von DCM-Patienten nötig. Um die mit der Therapie verbundenen Genexpressionsänderungen einordnen zu können, sollte zunächst das Genexpressionsmuster von 47 DCM-Patienten mit Patienten ohne Einschränkung der Pumpfunktion (Kontrollen) verglichen und mittels Real-time PCR validiert werden. Die Betrachtung dieser DCM-Patienten und Patienten, die eine normale Pumpfunktion aufwiesen, ergab 649 Gene mit krankheitsbedingt veränderter Expression. Zu diesen gehören neben bekannten Herzinsuffizienz-Markern (BNP, ANP, MYH6) Gene, die Proteine des Protein-Ubiquitinylierungssystem kodieren oder an der Ausprägung von Hypoxie und Fibrose (Connective tissue growth factor) beteiligt sind und auf eine Dysregulation des Proteinabbaus, erhöhten oxidativen Stress und Matrix-Remodeling hinweisen. Darüber hinaus ist aufgrund der geringeren Expression von Genen, die der oxidativen Phosphorylierung und Glykolyse zuzuordnen sind, von einer Energielimitation in DCM-Patienten auszugehen. Unter Berücksichtigung von verschiedenen klinischen Parametern bestand weiterhin die Aufgabe, den Einfluss dieser Parameter auf die Genexpression zu klären. Während wenig Korrelationen zu Body-Mass-Index (BMI), Alter und Krankheitszeitraum auftrat, wurde eine Häufung von Genen festgestellt, deren Expression zur LVEF und LVIDd korreliert. Gene, die Regulatoren mit myokardialer Funktion kodieren (ADRA1A, ADRB2, PLN, RYR2), zeigten gleichzeitig Korrelationen zur LVEF und dem LVIDd (p<0,05). Darüber hinaus sollte das Genexpressionsprofil von Patienten, die von der IA/IgG-Therapie profitieren (Responder) mit den Patienten ohne Therapieerfolg (Nonresponder) vor und 6 Monate nach der IA/IgG-Therapie verglichen werden. In der Subgruppe der Responder wurde für 171 Gene eine signifikant unterschiedliche Expression bestimmt, während die Zahl der durch die Therapie in ihrer Expressionshöhe betroffenen Gene in Nonrespondern mit 72 wesentlich geringer ausfiel. Gene, die sowohl in Respondern nach Therapie als auch krankheitsbedingt verändert waren, konnten kaum beobachtet werden, so dass andere Mechanismen für den Therapieeffekt verantwortlich sein müssen. Neben einer geringeren Expression des ACE2 wurde auch eine Abnahme Fibrose-assoziierter Gene wie CTGF, Fibronectin und Collagen 1A2 in Respondern nach IA/IgG-Therapie beobachtet. Zudem war eine signifikante LVIDd-Abnahme in Respondern zu verzeichnen, die in Nonrespondern nicht zu erkennen war. In Nonrespondern wurde nach IA/IgG-Therapie dagegen die verminderte Expression einiger Komplementfaktoren beobachtet. Zudem bestand die Aufgabe in der Suche nach Genexpressionsänderungen immunadsorbierter DCM-Patienten, die mit der Änderung klinischer Parameter korrelieren. Weiterhin sollte im Rahmen dieser Arbeit eine beschreibende Signatur definiert werden, die eine Vorhersage des Therapieerfolges für den individuellen DCM-Patienten vor Durchführung der IA/IgG-Therapie ermöglicht. Unter Verwendung des Resamplings (Crossvalidierung) wurde mit Hilfe einer Support Vector Machine eine Signatur von 25 Genen definiert, die eine Klassifizierung der Subgruppen mit einer Fehlerrate von 3,7% erlaubt. Die in Abhängigkeit verschiedener, myokardialer Parameter gezeigten Genexpressionsunterschiede in DCM-Patienten spiegeln die Dynamik der Erkrankung wider. Der Einfluss der IA/IgG-Therapie auf die Genexpression von Patienten, die an der DCM erkrankt sind, betrifft eine Vielzahl von Genen verschiedener Kategorien. Korrelationsanalysen zeigen, dass ein Zusammenhang zwischen Genexpressionsänderung und den Änderungen der Parameter LVEF, LVIDd und Inflammation bestehen. Mit der Definition von Patienten, die von der IA/IgG-Therapie profitieren, wurden auch Unterschiede bezüglich klinischer Parameter (LVIDd, Zeitraum der Erkrankung) deutlich, die zum Verständnis der hier dargestellten Genexpressionsunterschiede beitragen. Die generierten Daten bieten neben dem besseren Verständnis der im Myokard ablaufenden Prozesse eine Reihe von Ansatzpunkten für weitere Untersuchungen, wie zum Beispiel zur Rolle des IGF-1-Signalweges oder des Protein-Ubiquitinylierungssystems bei der Ausprägung der DCM, die auch zu neuen Therapieansätzen beitragen können.Myocardial diseases represent prevalent causes of death. The understanding of molecular patho-physiological events is mandatory for the search of appropriate therapeutic approaches. In this present work gene expression analyses using DNA microarrays were performed to gain insights in cellular events of cardiac diseases. Focus of this PhD thesis was the analysis of gene expression changes after immunoadsorption therapy (IA/IgG) using endomyocardial biopsies of patients suffering from dilated cardiomyopathy (DCM). Therefore, a protocol for RNA and protein isolation was established. To rank the results of therapy associated gene expression, gene expression patterns of 47 DCM patients were compared to patients with normal (8) left ventricular function and results were validated by real time PCR. Investigation of myocardial mRNA levels in DCM patients and individuals with normal heart-function revealed differential expression of 649 disease associated genes. Beside known markers for heart failure (BNP, ANP, MYH6), differential expression included genes, which encode proteins of the protein ubiquitination system or hypoxia and fibrosis (CTGF). These genes may indicate dysregulation of protein degradation, elevated level of oxidative stress and matrix remodeling. Moreover, lower expression of genes, which were assigned to oxidative phosphorylation and glycolysis suspects an energy limitation in DCM patients. With respect to different clinical parameters as left ventricular ejection fraction (LVEF), left ventricular internal diameter at diastole (LVIDd) or inflammation influence of these parameters on gene expression should be clarified. Whereas a low number of correlations were found for body mass index (BMI), age and disease duration, an accumulation of correlating gene expression were established for left ventricular ejection fraction (LVEF) and left ventricular internal diameter at diastole (LVIDd). Expression of genes encoding regulators with myocardial function (ADRA1A, ADRB2, PLNB, RYR2) showed correlations to LVEF as well as LVIDd (p<0.05). Moreover, gene expression profiles of patients with positive outcome after IA/IgG therapy (responder) and patients without any improvement in heart-function (non-responder) were investigated. Comparison of responders before and 6 months after IA/IgG revealed 171 significant differentially expressed genes. Affected genes in the subgroup of non-responders after therapy revealed a lower number of differentially expressed genes (72). Genes which were affected in responders by therapy as well associated to disease could hardly be observed. The observed therapeutic effects on gene expression level probably arise from secondary mechanisms. Beside a lower expression of ACE2 in responders after IA/IgG also a decrease in fibrosis associated genes as CTGF (Connective tissue growth factor), Fibronectin and Collagen 1A2 was observed. Moreover, in responders a significant decrease in LVIDd was monitored which was not detected in non-responders. In contrast, in non-responders after IA/IgG therapy a reduced gene expression for a couple of complement factors was noticed. Furthermore, the analysis of gene expression changes of immunoadsorbed DCM patients which correlate to the change of parameters was another purpose of this present work. Moreover, a predictive molecular signature should be identified to allow differentiation of patients before therapy in reference of their outcome and minimization of ineffective therapy treatment. With application of re-sampling (cross validation) and support vector machine (SVM) a signature of 25 genes was identified, which allowed classification of subgroups with an error rate of 3.7%. The signature presents the first step for prediction of DCM patients. Further development shall enable estimation of therapy outcome of DCM patients without using DNA arrays. In this work pronounced differential gene expression in DCM patients reflected the dynamic of the disease in dependency of different myocardial parameters. A multiplicity of genes of different categories is influenced by IA/IgG therapy in DCM patients. Correlation analyses displayed the connection (interrelationship) between gene expression changes and changes of parameters as LVEF, LVIDd, and inflammation. Differences concerning clinical parameters (LVIDd, disease duration) were observed by definition of patients which benefit from IA/IgG therapy and contribute to a better understanding of demonstrated differences in gene expression. The data generated in context of this work provides, besides a better understanding of processes in the myocardium, a lot of new starting points for further analyses e.g. the relevance of IGF-1 signaling or the role of the protein ubiquitin system in development of DCM, which may also contribute to new therapy approaches

Improved reconstitution of Trizol derived protein extracts provides high quality samples for comprehensive proteomic characterization of cell cultures

Background: The study of RNA, DNA, and protein from the same sample is a great advantage but can be challenging. Using Trizol, one can simultaneously extract RNA, DNA, and protein, leading to efficient sample use and more comprehensive analysis. Although it is used routinely for RNA extraction, the frequency of use of Trizol extracts for proteomics applications is low. The aim of our study was to evaluate the results of a simple modification to the Trizol protocol in terms of extraction and protein recovery efficacy and compatibility of the extracts with proteomics technologies in comparison to our standard extraction protocol including freeze/thaw cycles in urea/ thiourea.Method: We used the human airway epithelial cell line S9 and extracted proteins either with a modified Trizol protocol or by freeze/thaw cycles in 8M urea/ 2M thiourea. Extracted proteins were quantified and subjected to 1D- and 2D-gel electrophoresis, Western Blotting and LC-coupled tandem mass spectrometry analysis. Results: Compared to urea/ thiourea extraction, the Trizol-extracted proteins exhibited a similar protein composition and identification rate in LC-coupled tandem mass spectrometry experiments. 1D- and 2D-PAGE of Trizol-extracted proteins revealed excellent protein resolution with better coverage of proteins in the low MW range than urea/ thiourea extraction. Conclusion: The modified Trizol-protocol enabled excellent protein extraction from cell culture samples and high compatibility with proteomics technologies, especially with LC-tandem mass spectrometry.

Overexpression of Renin-B Induces Warburg-like Effects That Are Associated with Increased AKT/mTOR Signaling

The classical secretory renin-a is known to be involved in angiotensin generation, thereby regulating not only blood pressure, but also promoting oxidative stress as well as apoptotic and necrotic cell death. In contrast, another cytosolic renin isoform named renin-b has been described, exerting protective effects under ischemia-related conditions in H9c2 cardiomyoblasts. Using microarray-based transcriptome analyses, we aimed to identify the signaling pathways involved in mediating cardioprotection in H9c2 cells overexpressing renin-b. By transcriptome profiling, we identified increased gene expression of several genes encoding glycolytic enzymes and glucose transporters, while the transcript levels of TCA-cycle enzymes were decreased. Complementing data from metabolic analyses revealed enhanced glucose consumption and lactate accumulation due to renin-b overexpression. Renin-b overexpression further stimulated AKT/mTOR signaling, where numerous genes involved in this pathway showed altered transcript levels. For AKT, we also detected enhanced phosphorylation levels by means of Western blotting, suggesting an activation of this kinase. Moreover, analysis of the ROS levels identified an increase in ROS accumulation in renin-b-overexpressing cells. Altogether, our data demonstrate that renin-b overexpression induces the metabolic remodeling of H9c2 cells similar to that seen under oxygen deprivation. This metabolic phenotype exerting so-called aerobic glycolysis is also known as the Warburg effect

APOE ε4 in Depression-Associated Memory Impairment—Evidence from Genetic and MicroRNA Analyses

(1) Background: The aim of this study was to replicate a reported interaction between APOE ε4 status and depression on memory function in two independent, nondemented samples from the general population and to examine the potential role of circulating plasma miRNAs. (2) Methods: The impact of the APOE ε4 allele on verbal memory and the interaction with depression is investigated in two large general-population cohorts from the Study of Health in Pomerania (SHIP, total n = 6286). Additionally, biological insights are gained by examining the potential role of circulating plasma miRNAs as potential epigenetic regulators. Analyses are performed using linear regression models adjusted for relevant biological and environmental covariates. (3) Results: Current depression as well as carrying the APOE ε4 allele were associated with impaired memory performance, with increasing effect for subjects with both risk factors. In a subcohort with available miRNA data subjects with current depressive symptoms and carrying APOE e4 revealed reduced levels of hsa-miR-107, a prominent risk marker for early Alzheimer’s Disease. (4) Conclusions: Our results confirm the effect of depressive symptoms and APOE ε4 status on memory performance. Additionally, miRNA analysis identified hsa-miR-107 as a possible biological link between APOE ε4, depressive symptoms, and cognitive impairment

Deciphering the Effect of Different Genetic Variants on Hippocampal Subfield Volumes in the General Population

The aim of this study was to disentangle the effects of various genetic factors on hippocampal subfield volumes using three different approaches: a biologically driven candidate gene approach, a hypothesis-free GWAS approach, and a polygenic approach, where AD risk alleles are combined with a polygenic risk score (PRS). The impact of these genetic factors was investigated in a large dementia-free general population cohort from the Study of Health in Pomerania (SHIP, n = 1806). Analyses were performed using linear regression models adjusted for biological and environmental risk factors. Hippocampus subfield volume alterations were found for APOE ε4, BDNF Val, and 5-HTTLPR L allele carriers. In addition, we were able to replicate GWAS findings, especially for rs17178139 (MSRB3), rs1861979 (DPP4), rs7873551 (ASTN2), and rs572246240 (MAST4). Interaction analyses between the significant SNPs as well as the PRS for AD revealed no significant results. Our results confirm that hippocampal volume reductions are influenced by genetic variation, and that different variants reveal different association patterns that can be linked to biological processes in neurodegeneration. Thus, this study underlines the importance of specific genetic analyses in the quest for acquiring deeper insights into the biology of hippocampal volume loss, memory impairment, depression, and neurodegenerative diseases

Brain Derived Neurotrophic Factor Contributes to the Cardiogenic Potential of Adult Resident Progenitor Cells in Failing Murine Heart

<div><p>Aims</p><p>Resident cardiac progenitor cells show homing properties when injected into the injured but not to the healthy myocardium. The molecular background behind this difference in behavior needs to be studied to elucidate how adult progenitor cells can restore cardiac function of the damaged myocardium. Since the brain derived neurotrophic factor (BDNF) moderates cardioprotection in injured hearts, we focused on delineating its regulatory role in the damaged myocardium.</p><p>Methods and Results</p><p>Comparative gene expression profiling of freshly isolated undifferentiated Sca-1 progenitor cells derived either from heart failure transgenic αMHC-CyclinT1/Gαq overexpressing mice or wildtype littermates revealed transcriptional variations. Bdnf expression was up regulated 5-fold during heart failure which was verified by qRT-PCR and confirmed at protein level. The migratory capacity of Sca-1 cells from transgenic hearts was improved by 15% in the presence of 25ng/ml BDNF. Furthermore, BDNF-mediated effects on Sca-1 cells were studied via pulsed Stable Isotope Labeling of Amino acids in Cell Culture (pSILAC) proteomics approach. After BDNF treatment significant differences between newly synthesized proteins in Sca-1 cells from control and transgenic hearts were observed for CDK1, SRRT, HDGF, and MAP2K3 which are known to regulate cell cycle, survival and differentiation. Moreover BDNF repressed the proliferation of Sca-1 cells from transgenic hearts.</p><p>Conclusion</p><p>Comparative profiling of resident Sca-1 cells revealed elevated BDNF levels in the failing heart. Exogenous BDNF (i) stimulated migration, which might improve the homing ability of Sca-1 cells derived from the failing heart and (ii) repressed the cell cycle progression suggesting its potency to ameliorate heart failure.</p></div

Gene expression alterations in Sca-1 cells in response to heart failure.

<p>(A) Relative fold change in expression of genes associated with heart failure in Cyc compared to Wt cells. (B) Top canonical pathways overrepresented by differentially expressed genes in Cyc vs Wt cells.—log <i>p-</i>value displays the significance of association dependent of the number of genes in the class calculated by Fisher’s exact test in IPA. Numbers of up-regulated/down-regulated genes are indicated above each bar. (C) Biological functions predicted to be differentially affected based on the differentially expressed genes in Cyc vs Wt cells based on activation z-score calculated in IPA. Black dots denote functional activation (z-score ≥ 1.5) and grey dots functional inhibition (z-score ≤ −1.5). (D) Validation of microarray results by quantitative RT-PCR (n = 3; <i>p</i><0.05; ANOVA).</p

pSILAC analysis of BDNF induced changes in protein synthesis.

<p>(A) Schematic representation of pulsed SILAC analysis. Sca-1 cells were cultured for 8days in light medium (red) followed by exchange to medium with heavy lysine and arginine (blue) containing 25ng/ml BDNF or 400nM K252a or a combination of both and allowed to grow for 24 hours. Cell lysates were digested and then subjected to mass spectrometric analysis. Newly synthesized proteins were determined based on H/L ratios. (B) Comparison of BDNF induced changes in the protein synthesis of Cyc and Wt cells (n = 3). Majority of proteins showed moderate change in protein synthesis depicted as grey spots; those identified with statistical significance (<i>p</i> < 0.05) are highlighted in black while only differentially regulated proteins in Cyc and Wt cells (<i>p</i> < 0.05, log2 ratio ≥ ±0.3) are highlighted in red and white spots respectively. (C) Venn diagram illustrates the overlap of displays protein numbers with altered synthesis rate as a result of BDNF treatment (BDNF vs Co, <i>p</i> < 0.05) in both groups of cells.</p

BDNF enhances migration of progenitor cells.

<p>Migration of Sca-1 cells (4 x 10⁴) was evaluated using a modified Boyden chamber assay. Freshly isolated cells were exposed to varying concentrations of BDNF (10, 25, 50ng/ml) and allowed to migrate for 2 hours (n = 3; mean ± SD; ANOVA; ***<i>p</i> < 0.0001, **<i>p</i> < 0.001, *<i>p</i> < 0.05 vs Control; §§<i>p</i> < 0.001, §<i>p</i> < 0.05 vs BDNF).</p