Project 1: Compartmentalized changes of H2O2 production and the glutathione redox potential during heart failure develo

PI Göttingen: D.M. Katschinski; PI London: C.X.C. Santos, A.M. Shah; PhD: M.S. Nanadikar - finished PhD

Scientific background and preliminary results

There is increased recognition that changes in the cellular redox potential are important for cellular feedback and signalling pathways. NOXs are major enzymes affecting redox sensitive signalling pathways in many cells and tissues. Among the seven NOX family members especially NOX2 and NOX4 are expressed in the heart. Both exhibit significant isoform-specific features. Despite these specific functions, both are known to be involved in the modulation of heart disease processes including the response to ischemia or mechanical load.

Cardiomyocytes feature a defined organization of their cytoplasm containing the sarcomeric-contractile apparatus and their confined subcellular organellar compartments. The intracellular environment is assumed to be mostly reductive with the exception of the oxidizing mitochondria and the endoplasmic reticulum. Excitation contraction coupling is thus under tight redox control. The possibility to quantify the redox state in cardiomyocytes at these subcellular levels is challenging but could provide new insights into the impact of the cellular redox potential for cardiac physiology. Up to now it is still unclear (i) if subcellular compartments create redox microdomains in cardiomyocytes and (ii) how they are spatially and temporally affected by changes in the cellular context. A better understanding of these open questions relies on the establishment of tools to specifically measure defined redox-changes in a quantitative manner. Recent advances in generating redox sensitive sensor probes like the redox sensitive (ro) green fluorescent protein(GFP)2 or the H2O2 sensitive HyPer in combination with established genetically modified transgenic mouse models offer new technical opportunities to answer the open questions. Coupling roGFP2 to oxidant sensing enzymes like the glutaredoxin (Grx) additionally allows to measure clearly defined redox processes as the sensor is coupled to the glutathione redox potential (EGSH).

We have recently developed αMHC-driven cardiomyocyte-specific Grx1-roGFP2 sensor mice, in which the redox biosensor is expressed as transgene in the cytosol (cyto-Grx1-roGFP2) or targeted to the mitochondrial matrix (mito-Grx1-roGFP2). Generation of these mice allows for the first time quantitative spatial and temporal measurements of redox changes in cardiomyocytes in the physiological context. To this end we established a setup, which allows performing ratiometric measurements of the biosensor within isolated cardiomyocytes but also Langendorff-perfused whole hearts. First experimental evidence revealed that the calculated glutathione redox potential (EGSH) for resting cardiomyocytes in the mitochondria is -278.51 ± 0.71 mV and in the cytoplasm is -254.62 ± 0.86 mV. Stimulation of the cardiomyocytes with Isoprenaline or Angiotensin II resulted in compartment-specific alterations of the redox potential.

Hypotheses of the PhD Project

Based on evidence gained with the newly established genetically encoded redox-biosensor mouse lines, we hypothesize that the glutathione redox-potential is highly compartimentalized in cardiomyocytes.

In the project the PhD student will test the hypotheses that

in the failing heart the glutathione redox potential is altered in the cytosol and the
mitochondrial compartment.
alterations in H2O2 production add to the changes in the glutathione redox
potential in the failing heart (Grx1-roGFP2/HyPer co-expression)
NOX2 and 4 contribute to the heart failure development in an isoform-specific
manner by altering the H2O2 production and glutathione redox potential in
cardiomyocytes.

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