Project 11: Redox regulation of human heart muscle function

PI Göttingen: W.-H. Zimmermann; PI London: M. Mayr; PhD student: Pierre-Luc Satin

Scientific background and preliminary results

Increasing knowledge and the availability of imaging tools to resolve signaling and function in cellular compartments have unearthed cell type specific, compartmentalized ROS signals. Their relevance for individual cell function and cross talk between the different cell types in the heart are largely unknown. In the past funding periods we have gained experience in genetically targeting the cytosol and mitochondria in human fibroblasts and cardiomyocytes with Grx1-roGFP2 ROS (GSH redox potential - EGSH) indicators (kindly provided by T. Dick; Heidelberg). After initial experiments with lentiviral transduction (by Dr. E. Heta, first IRTG cohort), we have subsequently invested significant efforts into the development of human embryonic stem cell models with the stable genomic expression of Grx1-roGFP2 for imaging of EGSH in the cytosol and mitochondria. In this model, we could confirm the finding of distinct redox potentials in human cardiomyocytes (EGSH in mV: -320±1 vs. -282±1 in thy cytosolic vs. the mitochondrial compartment). The biological and pathophysiological relevance of these findings as well as compartment specific alterations during disease states are large unknown.

Hypothesis of the PhD Project

We aim to entrust a student from the third IRTG cohort with investigating the overarching hypothesis that distinct entities of heart failure will alter compartmentalized ROS signaling in human cardiomyocyte and fibroblasts. We will make use of the established Grx1-roGFP2 (mito and cyto) reporter human embryonic stem cell lines (developed by I. Eckhardt, student form the second IRTG cohort) and a novel engineered human myocardium (EHM)-based model of heart failure with reduced (HFrEF; Tiburcy et al. 2017) and preserved (HFpEF; unpublished) ejection fraction. The following specific hypotheses will be tested:

Cardiomyocytes and fibroblasts exhibit in its specific cytosolic and mitochondrial compartments distinct EGSH in EHM before and after induction of a HFrEF phenotype.

Cardiomyocytes and fibroblasts exhibit in its specific cytosolic and mitochondrial compartments distinct EGSH in EHM before and after induction of a HFpEF phenotype.

The induction of HFrEF and HFpEF phenotypes in EHM will result in distinct proteome and phosphoproteome profiles.

Induced HFrEF and HFpEF will have an impact on the ETC proteome assembly and induce distinct metabolic signatures.

Work programme

The recruited student will make use of the now available unique human embryonic stem cell ROS reporter models. The generation of EHM will be according to protocols developed and refined recently in the Zimmermann lab, which enable fully controlled engineering and culture of human EHM (Tiburcy et al. 2017, Circulation); the controlled culture conditions with no serum are key for the phenocopying of HFrEF and HFpEF. In collaboration with the Mayr lab at King’s we will study the induced HFrEF and HFpEF proteome signatures in EHM as well as in EHM-derived cardiomyocytes and fibroblasts; comparative analyses to human heart muscle biopsies from patients with HFrEF and HFpEF will be performed (collaboration with Prof. Hasenfuß and the DZHK). We will make use of the cell type specific color-indicator EHM model (Tiburcy et al. 2017) and FACS to separate the cardiomyocyte and fibroblast pools for subsequent mass spec analyses in the Mayr lab. In addition, to the investigation of cell specific proteome and phospho-proteome differences at large, we will isolate mitochondria from the distinct cardiomyocyte and fibroblast populations to study the distinct mitochondrial proteome signatures with a particular focus on the components of the electron transport chain (ECT). The latter will involve blue native gel electrophoreses studies of ETC composition in collaboration with the Rehling lab at the UMG. Proteome alterations will be associated with anticipated changes in metabolic function, which will be investigated using the Seahorse metabolic flux technology in the Zimmermann lab.

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