Inhaltspezifische Aktionen

B07

Selective biomarkers for right heart hypertrophy and failure and their functional analysis in a human stem cell model

In pulmonary hypertension (PH) the right ventricle (RV) is exposed to pressure overload, contributing to RV hypertrophy.RV hypertrophy can lead to RV failure that is associated with a poor clinical prognosis. To date, there is lack of evidence for specific biomarkers indicating RV hypertrophy, including cases with maintained RV function (adaptation) or RV decompensation (maladaptation). We recently established a project-specific biobank comprising a total of >800 well-characterized patients with PH (chronic thromboembolic pulmonary hypertension, idiopathic pulmonary arterial hypertension, left ventricular (LV) adaptive hypertrophy, and LV heart failure) as well as healthy controls. In the last funding period, we identified Cartilage Intermediate Layer Protein 1 (CILP1) and further biomarkers (SPARC Like-1 (SPARCL1), osteopontin, galectin-3, fibroblast growth factor-23 (FGF23), Suppression Of Tumorigenicity 2 (ST2), growth differentiation factor15 (GDF15)) as novel and promising biomarkers indicating adaptive RV hypertrophy or maladaptive RV failure. Importantly, these biomarkers were linked with assessing RV-pulmonary artery (PA) coupling versus uncoupling. Cardiac MRI analysis further validated cut-off values for their ability to predict adaptive or maladaptive RV function in PH and their diagnostic and predictive value. Human induced pluripotent stem cells (iPSCs) provide a unique tool for investigating several cardiac conditions in vitro. We developed human patient-specific iPSC-derived ventricular and atrial cardiomyocytes (iPSC-CM) in 2D cultures and 3D engineered human myocardium to investigate heart failure (HF) pathologies, including the distinct contribution of mutations in cardiomyopathy- and arrhythmia-associated genes (e.g., Takotsubo Syndrome, RBM20-dependent dilated cardiomyopathy, Nav1.8-dependent arrhythmogenic HF triggers, anthracycline-induced cardiomyopathy). Combining expertise in iPSC-technology and cardiac patient-specific disease modeling with the established biobank of patients with PH, we will be able to functionally assess already identified biomolecules associated with RV pathological remodeling and establish PH stem cell models for predisposing genetics, further biomarker identification, and new therapeutic RV treatment strategies in PH on a patient-specific level. Accordingly, in the third funding period, we aim 1) to further validate putative biomolecules, determined a) from the ventricular zebrafish fibrosis model (project B01 - Reischauer: Matrilin 4 (MATN4), Glutathione S-Transferase Mu 1 (GSTM1), Prostaglandin-Endoperoxide Synthase 2 (PTGS2)), b) from the RNA sequencing analysis in donors with adaptive and maladaptive RV function (project A01 – Savai-Pullamsetti; A05 – Savai-Pullamsetti/Bauer: Epidermal Growth Factor (EGF9), Cartilage Acidic Protein 1 (CRTAC1), Nidogen 1 (NID1), and C1q And TNF Related 1 (C1QTNF1)), or c) from human RV biopsies (project CP01 – Wiedenroth/Dorfmüller/Yogeswaran) as specific indicators of RV failure in PH, that will be measured in peripheral blood samples; 2) to generate clinical mechanistic knowledge of the identified biomarkers by association with clinical characteristics and specific functional parameters of the RV, including 3D-RV echo, PV-loop analysis, and cMRI parameters (project B08 – Tello/Ghofrani). With this work a basis will be provided for translation into clinical use by the development of a diagnostic model, including multi-biomarker assessment as well as individual clinical, functional and imaging parameters to discriminate adaptive from maladaptive RV function, independent from LV; 3) to use patient-specific PH stem cell models for the identification and analysis of RV phenotypic (maladaptive RV function in PH) and genotypic correlations (CRISPR/Cas9 genome editing of single nucleotides) by focusing on previously described specific mutations associated with RV function in e.g., Bone Morphogenetic Protein Receptor Type 2 (BMPR2), supervillain (SVIL), obscurin (OBSCN), palladin (PALLD), or others. These same patient-specific stem cell models will also be used for the demonstration of individual right and left ventricular cardiomyocyte, and cardiac fibroblast contribution to RV dysfunction by mix and match of healthy and diseased cell types in 2D and 3D, and the identification of new biomolecules secreted from PH-patients’ iPSC-cardiomyocytes/ cardiac fibroblasts (after stress induction) that could serve as biomarkers of individual differences in disease pathogenesis in LV and RV dysfunction of PH. The selection of the patients used for iPSC generation will be based (a) on severity of the disease based on clinical PH parameters at constant afterload and (b) genetic variants in BMPR2, SVIL, OBSCN, PALLD (and other RV functionassociated genes) in RV myocardium of patients with most severe RV dysfunction (most preferred, same as used for iPSCs generation will be analyzed by single nucleus sequencing); 4) to generate cellular, molecular, and functional knowledge about specific biomolecules (candidate biomolecules derived from our project or CP01, A01, A05, B01; e.g. CILP1, SPARCL1)) involved in the development of RV dysfunction in PH by using iPSC-derived (right) ventricular cardiomyocytes, iPSC cardiac fibroblasts, iPSC endothelial cells in 2D and 3D (knockout/overexpression of genes of interest (CILP1 and SPARCL1) by CRISPR/Cas9) as well as human cardiac tissue (RV trabeculae for acute stress induction by isometrically twitching mimicking excessive afterload). In a back-to-bench approach, we will specify these biomarkers as cause or result of RV function by using iPSC-derivatives and cardiac tissue slices from RV and LV of PH patients for long-term treatment.