Calciumtransienten in Peroxisomen
by Uta Bickmeyer
Date of Examination:2024-12-09
Date of issue:2024-11-12
Advisor:Prof. Dr. Sven Thoms
Referee:Prof. Dr. Sven Thoms
Referee:PD Dr. Sören Brandenburg
Referee:Prof. Dr. Dieter Kube
Persistent Address:
http://dx.doi.org/10.53846/goediss-10842
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Abstract
English
Introduction: Peroxisomes are small cell organelles that play an important role in metabolic functions such as alpha- and beta-oxidation of fatty acids and ROS-homeostasis. So far the data of calcium concentrations and function in Peroxisomes is inconsistent. The function of peroxisomes in the heart is still under ongoing investigation. Peroxisomes are described to potentially have cardioprotective functions as peroxisomal diseases e.g. the adult Refsum-Syndrom lead to cardiomyopathy, arrhythmias and eventually heart failure. They occur in high numbers in the myocard especially in the left ventricle where there is an especially high energy demand. As a result of functional translational readthrough the Malate-Dehydrogenase is localized in peroxisomes. So as oxidation of fatty acids occur in peroxisomes a malate shuttle equivalent to the one in mitochondria is assumed to also play a role in peroxisomal regeneration of reducing equivalents. The aspartate/glutamate carriers 1 and 2 (AGC1 and 2) as part of the malate shuttle have calcium-binding EF-hands localised in their N-terminal domains. The thesis focuses on measuring peroxisomal calcium transients in cardiomyocytes as well as investigating the topology of AGC1 and 2 in the peroxisomal membrane as potential targets of calcium signalling. Methods: Cloned FRET-sensors localised in peroxisomes and the peroxisomal membrane with extraluminal orientation were transfected in Hela and HEK293 cells. Live-cell imaging was performed and the cells were stimulated chemically with histamine or treated with ionomycin to evoke an intracellular rise of calcium ion concentrations. To measure calcium transients in cardiac peroxisomes iPSC cardiomyocytes and neonatal rat cardiomyocytes were used. These cells were stimulated with SERCA-Inhibitor thapsigargin as well as electrical stimulation. Furthermore a modified form of Fluorescence Protein Protection Assay was used to investigate the topology of peroxisomal AGC1 and 2. Here permeabilising the cells with digitonin and treating them with Proteinase K was combined with immunofluorescence. Results: It was shown that calcium ions can enter peroxisomes in non-electric cells as well as in cardiomyocytes. In both iPSC and neonatal rat cardiomyocytes peroxisomal calcium concentrations increased after chemical stimulation with thapsigargin. Neonatal rat cardiomyocytes showed rhythmic calcium transients under electrical stimulation on a beat to beat basis. With immunofluorescence peroxisomal localisation of AGC1 and partly AGC2 was shown. The Fluorescence Protein protection Assay provided hints for an intraluminal peroxisomal localisation of the calcium-binding EF-hands thus identifying a potential target for calcium signalling. Discussion: The advantage of FRET-sensors is the high spatial resolution especially for measuring small organelles like peroxisomes. A limiting factor might have been the lower time resolution due to conformational changes that the FRET-Sensor needs to undergo after calcium binding. Calcium can enter cardiac peroxisomes in case of neonatal rat cardiomyocytes even on a beat-to-beat basis which hints that peroxisomal functions might be regulated through calcium signals. With the likely peroxisomal localisation of AGC1 and 2 potential targets of calcium signalling could be found.
Keywords: Calcium imaging; Peroxisomes; Aspartate/Glutamate Carrier; cardiomyocytes; GECI
Schlagwörter: Peroxisomen; Calciummessungen; FRET-Sensor; Aspartat/Glutatamat Carrier; Kardiomyozyten
