Dissecting novel regulatory mechanisms of S100A1 on cardiac function

6.1 Extracellular S100A1 inhibits apoptosis in ventricular cardiomyocytes: via activation of the extracellular signal-regulated protein kinase 1/2 (ERK1/2) Growing evidence indicates that members of the S100 protein family exert intracellular but also extracellular effects on their target cells (Donato, 2003). Moreover, it has been shown that S100A1 protein is released into the extracellular space in considerable amounts during ischemic myocardial injury (Kiewitz et al., 2000). This observation prompted us to explore the extracellular effect of S100A1 on neonatal ventricular cardiomyocytes (NVCMs). By directly coupling human recombinant S100A1 protein with rhodamine we were able to trace the uptake of extracellulary added S100A1 into the cytosolic compartment of cultured NVCMs (Chaper 2). Using confocal laser scanning microscopy, we have shown for the first time that S100A1 is internalized by NVCMs via a Ca2+-dependent pathway. The colocalization studies documented that the pathway of S100A1 internalization is clathrindependent. By using several inhibitors that are specific for different steps along the intracellular signal transduction pathway, we could identify that S100A1-mediated activation of the ERK1/2 signaling involves activation of PLC and PKC, both of which have been closely linked to the endosomal compartment. Taken together this part of our study demonstrated extracellular S100A1 to act as a novel anti-apoptotic factor that enhances survival of neonatal cardiomyocytes in vitro via activation of the PLC-PKC-MAP kinase kinase1-ERK1/2 pathway. So far, the receptor for S100A1 internalization is still unknown. Previous studies identified the cell surface receptor for advanced glycosylated end products (RAGE) as possible receptor for S100 proteins (Hofmann et al., 1999; Huttunen et al., 2000). This was shown for the isoforms S100B and S100A12 (Donato, 2003). Because our results suggest that RAGE is not involved in the endocytosis of S100A1 in NVCM, we predict that another cell surface receptor is responsible for S100A1 uptake in NVCMs (Figure 6.1) In future studies we will attempt to identify a possible receptor for S100A1 by employing GST-S100A1 fusion protein pull-down assays of plasma membranes. In addition, the cardioprotective effect of internalized S100A1 in vitro warrants further research into the release of S100A1 during heart failure. Moreover, our findings should initiate new perspectives on the pathophysiological relevance of the cardioprotective effect of S100A1 protein on cardiac cells in vivo. 6.2 Adenoviral-mediated S100A1 gene delivery rescues failing myocard: Since S100A1 protein has been shown to be downregulated in human and animal heart failure (HF) model (Remppis et al., 1996; Tsoporis et al., 2003), we undertook this study to address whether S100A1 gene addition might reserve ventricular contractile dysfunction in failing myocardium (Chapter 3). Using a postinfarct HF model in the rat, we provided evidence that adenoviral-mediated myocardial S100A1 gene delivery can restore S100A1 protein expression in failing myocardium. As a result of restored S100A1 levels, a normalisation of previously dysfunctional intracellular Ca2+- and Na+-handling occured. In additon S100A1 expression abolished aberrant fetal gene expression associated with HF, and most intringuely restored energy supply in failing myocardium. Overall, we have shown for the first time that restoring S100A1 protein expression can rescue defective contractile performance of failing myocardium in vitro and in vivo due to improved cytosolic and SR (sarcoplasmic reticulum) Ca2+-cycling. This effect is caused by both enhanced activity of SERCA (sarco(endo)plasmic reticulum Ca2+-ATPase) and modulation of RyR (ryanodine receptor). Our studies provide ample vidence that S100A1 is a key-regulator of cardiac function in vitro and in vivo. The effects of restored S100A1 protein level in failing myocardium argue for a novel clinical approach in the regimen of HF. Athough the study described first results on the application of adenoviral S100A1 expression in failing myocardium, there exist also limitations and open questions. For instance, we have used a first-generation adenoviral vector that limits study duration. Thus, we will analyze chronic effects of HF rescue by S100A1 gene delivery with improved vectors. However, in the future S100A1 HF gene therapy might proof to be a promising clinical treatment. 6.3 Distinct subcellular location of S100A1 differentially modulates Ca2+-cycling in ventricular rat cardiomyocytes: Because S100A1 is a Ca2+-sensor, we investigated the effect of an increasing S100A1 levels on the cycling of cytosolic Ca2+ in NVCMs by two different procedures: NVCMs were either transduced by an adenoviral S100A1-expression construct (AdS100A1) or incubated with recombinant human S100A1 protein (S100A1-treated) (Chapter 4). Ca2+-transient measurements revealed an increase on Ca2+-turnover for both procedures. By using different inhibitors of Ca2+-cycling, we have demonstrated that cells overexpressing S100A1 and cells treated with S100A1 use different mechanisms to increase the intracellular Ca2+-cycling. AdS100A1 cells arrived at an enhanced Ca2+-transient amplitude mainly through an increase in systolic [Ca2+]i. In contrast, a marked decrease in diastolic Ca2+-concentrations ([Ca2+]i) was the main cause for the enhanced Ca2+-transient amplitude in S100A1-treated NVCMs. The decreased diastolic [Ca2+]i in S100A1-treated cells was likely the result of increased sarcolemmal Ca2+-extrusion through the Na+/Ca2+-exchanger (NCX). Moreover, uptake of S100A1 into the endosomal compartment triggered endosome-associated PLC and PKC, which then activate NCX to increase sarcolemmal Ca2+-efflux. The enhanced systolic [Ca2+]i in AdS100A1 cells was brought about by an increased activity of RyR and SERCA. Consistently, immunofluorescence documented a colocalization of intracellular overexpressed S100A1 and these two Ca2+-regulatory proteins. In conclusion, we demonstrated that intracellular S100A1, depending on its subcellular location, modulates cardiac Ca2+-turnover via different Ca2+-regulatory proteins. After having shown that internalized S100A1 has a pro-survival effect on cultured cardiomyocytes and that adenoviral S100A1 gene delivery rescues failing myocardium in vitro and in vivo (Most et al., 2004), we are now facing the challenge of elucidating the effects of internalized S100A1 on cardiac function in vivo. 6.4 The Ca2+-dependent dependent interaction of S100A1 with the F1-ATPase leads to an increased ATP content in cardiomyoctes: Reports on the involvement of S100A1 in energy metabolism (Zimmer et al., 1995; Zimmer and Dubuisson, 1993), have prompted us to examine the effect of S100A1 overexpression on cardiac energy metabolism (Chapter 5). We found that AdS100A1 cells exhibited a significantly higher ATP content compared to control cells (Adcontrol). By using GST-S100A1 fusion protein pull-down assays we were able to identify several mitochondrial proteins, which are all involved in energy metabolism. Based on the relationship between S100A1 and ATP content, we primarily focussed on the interaciton of S100A1 with the F1-ATPase. Confocal and electron microscopy studies provided further evidence that S100A1 interacts with the mitochondrial F1-ATPase. Interestingly, several groups have reported the localization of S100A1 in mitochondria, but so far the functional consequence of this localization has not been addressed (Haimoto and Kato, 1988; Maco et al., 2001). In order to fill this gap, we dissected the S100A1-F1-ATPase complex using different biochemical assays. We found the interaction to be dependent on the presence of Ca2+ and on the pH. At physiological pH (7.4 ) and in the presence of Ca2+, S100A1 and F1-ATPase form a stable complex. For further structural analysis of the complex, we initialized a collaboration with the laboratory of Sir John Walkers in Cambridge. In a first step we have isolated an S100A1-F1-ATPase complex by gel filtration chromatography. To unravel the molecular interactions of S100A1 and F1-ATPase, we ultimately plan to crystallize the complex. This part of the projekt unveiled a novel mechanism of S100A1 on cardiac function. The high metabolic demand of the heart requires a close coordination of energy production (ATP) and workload (Cortassa et al., 2003). Approximately 2% of the cellular ATP is consumed with each heartbeat and almost all of this energy is replenished by mitochondrial oxidative phosphorylation under normoxic conditions (Das and Harris, 1991; Harris and Das, 1991). The fact that not only the F1-ATPase but also other mitochondrial proteins that participate oxidative phosphorylation associate with GST-S100A1 in pull-down experiments, convincingly argues for further investigations of these interactions (Figure 6.2). 6.5 Conclusions: The work presented in this thesis provides essential insights into the molecular mechanisms of S100A1 on cardiac function. First it reveals novel mechanisms of S100A1. By combining different localization techniques and biochemical assays, the internalization of S100A1 via a Ca2+-dependent, clathrinmediated process was discovered. Moreover, it is shown that internalized S100A1 exhibits anti-apoptotic effects on cardiomyocytes and increases Ca2+-turnover. Different molecular mechanisms were implicated in Ca2+-handling depending on the localization of the exogenous S100A1. In addition, the rescue of failing myocardium in response to S100A1 gene delivery holds promise for a treatment of heart failure by S100A1 gene therapy. Finally, the identification of new target proteins for S100A1 gives rise to new perspectives on the involvement of S100A1 in the energy metabolism

In Silico Approaches and the Role of Ontologies in Aging Research

The 2013 Rostock Symposium on Systems Biology and Bioinformatics in Aging Research was again dedicated to dissecting the aging process using in silico means. A particular focus was on ontologies, as these are a key technology to systematically integrate heterogeneous information about the aging process. Related topics were databases and data integration. Other talks tackled modeling issues and applications, the latter including talks focussed on marker development and cellular stress as well as on diseases, in particular on diseases of kidney and skin

Analysis of dynamic gene expression responses to altered gravity in the wildtype and auxin efflux carrier mutants of the model plant Arabidopsis thaliana

Plant roots are among most intensively studied biological systems in gravity research. Altered gravity induces asymmetric cell growth leading to root bending. Differential distribution of the phytohormone auxin underlies root's responses to gravity being coordinated by auxin efflux transporters from the PIN family. The objective of this study was to compare early transcriptomic changes in roots of Arabidopsis thaliana using experiments on board of parabolic flights, suborbital and orbital flights, and ground-based facilities for simulated microgravity conditions to correlate these changes to auxin distribution. By comparing immediate and initial responses of the gene expression to the different gravitational forces identified primary gravity regulated genes and resolved time-effects in gene expression leading to an understanding of the underlying physiological responses and adaptive processes. High-resolution imaging in combination with computational approaches further resolved phenotypic changes initiated by altered gravity at the cellular level. Our study provides important insights towards understanding signal transduction processes in altered gravity conditions by combining experimental platforms with the analysis of different genetic mutants in the model Arabidopsis

Requirements analysis and specification for a molecular tumor board platform based on cBioPortal

Clinicians in molecular tumor boards (MTB) are confronted with a growing amount of genetic high-throughput sequencing data. Today, at German university hospitals, these data are usually handled in complex spreadsheets from which clinicians have to obtain the necessary information. The aim of this work was to gather a comprehensive list of requirements to be met by cBioPortal to support processes in MTBs according to clinical needs. Therefore, oncology experts at nine German university hospitals were surveyed in two rounds of interviews. To generate an interview guideline a scoping review was conducted. For visual support in the second round, screenshot mockups illustrating the requirements from the first round were created. Requirements that cBioPortal already meets were skipped during the second round. In the end, 24 requirements with sometimes several conceivable options were identified and 54 screenshot mockups were created. Some of the identified requirements have already been suggested to the community by other users or are currently being implemented in cBioPortal. This shows, that the results are in line with the needs expressed by various disciplines. According to our findings, cBioPortal has the potential to significantly improve the processes and analyses of an MTB after the implementation of the identified requirements

MDM2 Inhibition Enhances Immune Checkpoint Inhibitor Efficacy by Increasing IL15 and MHC Class II Production

The treatment of patients with metastatic melanoma with immune checkpoint inhibitors (ICI) leads to impressive response rates but primary and secondary resistance to ICI reduces progression-free survival. Novel strategies that interfere with resistance mechanisms are key to further improve patient outcome during ICI therapy. P53 is often inactivated by mouse-double-minute-2 (MDM2), which may decrease immunogenicity of melanoma cells. We analyzed primary patient-derived melanoma cell lines, performed bulk sequencing analysis of patient-derived melanoma samples, and used melanoma mouse models to investigate the role of MDM2-inhibition for enhanced ICI therapy. We found increased expression of IL15 and MHC-II in murine melanoma cells upon p53 induction by MDM2-inhibition. MDM2-inhibitor induced MHC-II and IL15-production, which was p53 dependent as Tp53 knockdown blocked the effect. Lack of IL15-receptor in hematopoietic cells or IL15 neutralization reduced the MDM2-inhibition/p53-induction–mediated antitumor immunity. P53 induction by MDM2-inhibition caused anti-melanoma immune memory as T cells isolated from MDM2-inhibitor–treated melanoma-bearing mice exhibited anti-melanoma activity in secondary melanoma-bearing mice. In patient-derived melanoma cells p53 induction by MDM2-inhibition increased IL15 and MHC-II. IL15 and CIITA expressions were associated with a more favorable prognosis in patients bearing WT but not TP53-mutated melanom Implications: MDM2-inhibition represents a novel strategy to enhance IL15 and MHC-II–production, which disrupts the immunosuppressive tumor microenvironment. On the basis of our findings, a clinical trial combining MDM2-inhibition with anti–PD-1 immunotherapy for metastatic melanoma is planned

Erratum : Sorafenib promotes graft-versus-leukemia activity in mice and humans through IL-15 production in FLT3-ITD-mutant leukemia cells

This corrects the article DOI: 10.1038/nm.4484