Project 7: Redox control of the mitochondrial calcium uniporter is important for an acute adaptation towards hypoxia

PI Göttingen: D.M. Katschinski, I. Bogeski; PI London: A.M. Shah, M. Mayr; PhD student: Ana M. Vergel Leon

Scientific background and preliminary results

Hypoxia triggers several mechanisms to adapt cells to a low oxygen environment including activating a highly conserved transcriptional programme. Major hypoxia-inducible genes depend on the hypoxia-inducible factor (HIF). HIF comprises two subunits, i.e. the oxygen dependently regulated α-subunit and the constitutively expressed β-subunit. In normoxia the α-subunit is rapidly marked by the oxygen-dependent prolyl-4-hydroxylase domain enzymes (PHD), which results in a subsequent proteasomal degradation via the von Hippel Lindau E3 ligase. Although stabilization of HIFα is instantaneous there is a critical pause of the cellular response towards hypoxia based on the time needed for initiating transcription and translation of the HIF target genes. Aside from classical target genes affecting angiogenesis, erythropoiesis, iron metabolism etc., HIF is likewise affecting mitochondrial function. Mitochondria are major consumers of oxygen and a potential source of reactive oxygen species (ROS). In response to hypoxia they exchange or modify distinct subunits of the respiratory chain and adjust their metabolism. HIF causes multiple changes in the composition of ETC complexes. These changes are required to keep mitochondria intact under low oxygen conditions and to prevent excessive ROS formation. Most of the HIF dependent changes in complex composition occur within complex I, a dominant acceptor of electrons, and complex IV, facilitating electron transport to molecular oxygen. Some of these alterations comprise the exchange of subunits within ETC complexes others modify complex structure, while subunit depletion also occurs. Aside from these HIF-dependent effects on mitochondria in hypoxia, there is strong evidence that these organelles respond towards a decrease in oxygen availability in a HIF-independent manner. In our previous work we could show a rapid reductive response of the mitochondrial glutathione redox potential (E_GSH) upon onset of hypoxia, which was quickly reversible upon re-oxygenation. Such a rapid response could cover the time gap between HIFα stabilization and activation of the full HIF response via transcription and translation. In these experiments we made use of our newly established E_GSHcardiomyocyte-specific biosensor mouse lines. In these mice the Grx1-roGFP2 biosensor is either targeted to the cytoplasm or the mitochondrial matrix of cardiomyocytes (Swain et al, Circ Res, 2016 Oct 14;119(9):1004-1016). RoGFP2 enables real-time visualization of the E_GSH via disulfide-bond formation between cysteine residues, which is reflected in a shift of fluorescence intensities at 405 nm and 488 nm. Distinct E_GSH were observed in the cytoplasm (−257.2±0.7 mV) and the mitochondrial matrix (−278.9±0.4 mV) of isolated cardiomyocytes. Interestingly the E_GSH in the mitochondrial matrix was highly depending on the oxygen concentration. In line with an impact of hypoxia in mitochondrial function, we next demonstrated a lower H₂O₂ release and complex III activity in hypoxic mitochondria. Aside from their respiratory function, mitochondria integrate Ca²⁺ and oxidative stress signals to control the cellular supply of ATP via mitochondrial dehydrogenase activity and the release of cell death factors. We have first evidence that the Ca²⁺ uptake and ATP release is enhanced in mitochondria upon hypoxia in line with an altered activity of the mitochondrial calcium uniporter (MCU) complex.

Hypotheses of the PhD project

In the proposed project, we will test the hypotheses that the mitochondrial Ca²⁺ uptake and ATP production are enhanced under hypoxic conditions via redox regulation of the MCU. Thus, alterations in the MCU redox state might be the basis for the rapid HIF-independent adaptation of the cellular response towards hypoxia.

Aims:

1. Characterize mitochondrial Ca²⁺ uptake and ATP production in normoxic and hypoxic mitochondria

Experimental Platforms: isolated mitochondria from cardiac myocytes and MEFs, intact cardiac myocytes and MEFs.

Genetic manipulations: transient and stable overexpression and transduction, transgenic mice (roGFP-GRX1 + mito Ca²⁺ sensor).

2. Biochemical characterization of the MCU redox state and composition

Experimental Platforms: isolated mitochondria from cardiac myocytes and MEFs + alternative oxidase (AOX) mice.

3. Evaluate the role of MCU redox regulation in disease models

Two mouse models (inspiratory hypoxia and LAD-ligation) in wild type, Grx1-roGFP2 mice plus AOX mice as control.

Work programme

Ca²⁺ uptake and ATP production in isolated mitochondria and mouse hearts in normoxia and hypoxia

Ca²⁺ uptake and ATP measurements will be performed with mitochondria isolated from mouse hearts. To gain insight, if any observed effects are depending on HIF we will likewise include a genetically modified cell model, i.e. mouse embryonic fibroblasts (MEF) isolated from HIF+/+ or HIF-/- embryos. Mitochondrial calcium dynamics will be recorded in freshly isolated mitochondria using the calcium-sensitive fluorescent dye Calcium-Green. To this end, the entire procedure including mitochondria isolation, decalcification, Ca²⁺ injection and fluorescence recording will be done in normoxia (20.9% O₂) or in hypoxia (0.1% O₂). At the end of the experiment, mitochondria will be lysed and ATP concentrations in the lysates will be determined. In addition, to measure mitochondrial calcium dynamics as well as ATP production in intact living cells under normoxic and hypoxic conditions, we will use genetically encoded mitochondrial calcium (D₃CPV, TN-XL) and ATP sensors (Ateam). These measurements will be initially performed in MEFs and at a later stage in cardiac myocytes.

MCU composition in 1D and 2D Blue native SDS page (BN-PAGE) gel electrophoresis

Preliminary experiments demonstrate that we are able to analyze the MCU complex via Blue native gel electrophoresis. Mitochondria isolated from mouse hearts and MEF cells reveal a typical band pattern with two major complexes at around 400 and 800 kDa in 1D gels. The band pattern changes upon incubation of cells in hypoxia. We will next set up 2D BN/SDS-PAGE. In the 2D BN the proteins and protein complex samples are denatured by SDS in the gel strip after the separation by BN-PAGE before they are applied to a second-dimension SDS-PAGE gel. We plan a thorough analysis of the composition of the 400 and 800 kDA complexes in normoxia and hypoxia including known MCU interaction partners like MCUb, Micu1, Micu2 and EMRE. Additionally we will analyze the redox state and cysteine oxidative modifications of the MCU and Micu.

Impact of changes in complex III activity for the MCU activity in hypoxia

In our preliminary experiments we have detected a specific decrease in complex III activity in hypoxia. We will apply AOX transgenic mice to further gain insight into the role of complex III for the adaptation of cardiac mitochondria to hypoxia (Szibor et al, Dis Model Mech. 2017 Feb 1;10(2):163-171). Plants and many lower organisms, but not mammals, express AOXs that branch the mitochondrial respiratory chain, transferring electrons directly from ubiquinol to oxygen without proton pumping and thus limiting excess production of ROS. First experiments with cardiac mitochondria isolated from the AOX mice revealed that the changes seen in the E_GSH in wild type mice are ameliorated in the AOX mice indicating that hypoxia affects the glutathione pool via complex III. Therefore, we will use this mouse model to analyse, if changes in Ca²⁺ uptake and MCU complex composition in normoxia versus hypoxia are likewise mediated via complex III.

Composition of the MCU complex in the hypoxic and ischemic hearts

To gain insight if the observed effects of hypoxia on MCU composition and thereby calcium uptake, play a role in the in vivo adaptation to hypoxia; we will use two different mouse disease models. (i) We will expose the intact mouse to inspiratory hypoxia (6% O₂) for 6 hrs. (ii) We will apply a LAD-ligation ischemia model to induce myocardial infarction. After exposure to hypoxia or LAD-ligation we will subsequently analyze Ca²⁺ content, MCU composition and MCU redox state in cardiac mitochondria.

Contact