|News and Events||Working Groups||Short-Term Scientific Missions||Management Committee||Members|
COST Action CA15203 (2016-2021): MitoEAGLE
Evolution-Age-Gender-Lifestyle-Environment: mitochondrial fitness mapping
MitoPedia topics: EAGLE
|Institution||University of Innsbruck|
|Address||Rennweg 10, 6020|
|O2k-Network Lab||AT Innsbruck Jansen-Duerr P|
|Gerna 2021 Int J Mol Sci||2021||Gerna D, Arc E, Holzknecht M, Roach T, Jansen-Duerr P, Weiss AKH, Kranner I (2021) AtFAHD1a: a new player influencing seed longevity and dormancy in arabidopsis?. Int J Mol Sci 22:2997.|
|Holzknecht 2017 MiPschool Obergurgl||2017|
MitoEAGLE Short-Term Scientific Mission
- Work Plan summary
- The applicant plans to visit the Max Plank Institute for Heart and Lung Research for around 4 weeks in order to learn new methods for the isolation and analysis of isolated cardiomyocytes from the mouse heart. These techniques will be applied at the Department for Biomedical Aging Research (IBA) in Innsbruck where the applicant is performing his PhD since spring 2019.
- The main focus of the current research at the IBA is cellular metabolism connected to mitochondrial dysfunction by investigating a mitochondrial protein, namely FAHD1, which was identified as an oxaloacetate decarboxylase. This enzyme seems to plays a role in the energetic homeostasis of the mitochondria by balancing the availability of TCA-metabolites. Beyond its regulatory features concerning the TCA cycle, the effect of FAHD1 elimination on whole organ morphology as well as cell and organelle structures in living organisms needs to be addressed. These questions can be answered using a FAHD1 knock-out (FAHD1-KO) mouse model, which will be used by the applicant to study the heart specific role of FAHD1. First echocardiographic screenings of FAHD1-KO and wild type (WT) littermates showed a reduced ejection fraction in FAHD1-KO mice which indicates a dilated cardiac hypertrophy (unpublished data). Comparison of isolated cardiomyocytes derived from the heart of WT and FAHD1-KO mice will provide a first insight into potential cellular phenotypes caused by apparent mitochondrial dysfunction due to FAHD1 deletion. Respirometric measurements (e.g. Seahorse) will be used to analyze the mitochondrial performance and overall oxygen metabolism of the isolated cardiomyocytes.
- Moreover, the applicant will also assist during the isolation of cardiac tissue from fetal and neonatal stages of WT mice to learn this technique at the Max-Plank Institute and to perform similar experiments once he is back in Innsbruck at the IBA. On one hand, these lysates can be used to study the expression pattern of FAHD1 during different stages of embryogenesis, on the other hand, lysates from WT and FAHD1-KO mice can be used to create a genetic profile of selected glycolytic and mitochondrial genes during early development.
- All together, the described experiments will complete the understanding of Fahd1 as an important enzyme for an efficient energy metabolism that could be involved in essential adaptations during fetal heart development. The proposed short term scientific mission will fit into the general objective of the MitoEAGLE action that aims to put mitochondrial dysfunction in the context of cellular function and disease.
- Work Plan summary
- Work plan and methods:
- Week 1 and 2:
- Isolation of cardiomyocytes from WT mice.
- Two different methods will be used: one based on a Langendorff-like glass cannula to perfuse the heart, the other is based on syringes to perfuse the ventricle manually.
- Cultivation of isolated cardiomyocytes.
- Once the cardiomyocytes are isolated they will be held in culture as primary cells for further analysis.
- Week 2 and 3
- Respirometry with isolated cardiomyocytes.
- After obtaining a stable cell culture, the Seahorse respirometer will be used to investigate the mitochondrial function of the isolated cardiomyocytes. Here, different aspects of mitochondrial metabolism can be addressed: Basal oxygen consumption rates, maximal oxygen flux and leak respiration can be measured. Additional information about glycolytic metabolism will be provided by measuring the extracellular acidification rate (ECAR).
- Week 3 and 4
- FAHD1 in the early development.
- The expression patterns of FAHD1 during embryonic development of the heart will be analyzed on protein levels via Western Blot (specific antibodies are available). More information about metabolic adaptations in the heart during embryogenesis will be gained by investigating mRNA levels (qPCR) of selected genes that are involved in the metabolic shift from glycolysis (early development) to oxidative phosphorylation (adult heart). Here, the detection of different gene expression levels between WT and FAHD1-KO mice could connect the regulatory effects of FAHD1 to pathways that control the metabolism in the mouse heart.