Mitochondrial protein import in sepsis: Pathomechanistic and therapeutic implications

• Applicant: Privatdozent Dr. Tim Rahmel
• Subject Area: Anaesthesiology
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 464384825

Sepsis is a metabolic disorder with life-threatening organ dysfunction due to an infection-related dysregulated immune response. Mitochondrial dysfunction is assumed to play an important pathophysiological role and accompanied by a mitochondrial ROS formation during the inflammatory reaction. In particular, excessive ROS production and insufficient ROS neutralization may damage mitochondrial structures resulting in mitochondrial and cellular dysfunction. Mitochondrial biogenesis is of central importance for the repair of ROS-induced damage. However, it is unclear why functional recovery may fail despite induction of biogenesis with increased expression of mitochondrial transcription factor A (TFAM), a key protein in mitochondrial biogenesis. Our preliminary work shows decreased intramitochondrial TFAM concentrations despite its increased cytoplasmic expression accompanied by mitochondrial dysfunction. We, therefore, hypothesize that intracellular maldistribution of TFAM is caused by a disturbed protein import from the cytoplasm into the mitochondria, a pathomechanism not yet investigated in sepsis.The proposed funding will be used for the detection and characterization of a ROS-induced mitochondrial protein import disorder as a putative effector of mitochondrial dysfunction in sepsis and assessment of its pharmacological influenceability. We hypothesize that ROS production evokes a disturbance of protein translocation from the cytoplasm across the inner mitochondrial membrane via a reduction of the mitochondrial membrane potential and results in mitochondrial dysfunction. This will be tested by 1) determining the protein expression of import-relevant mitochondrial structures in cells from sepsis patients and in an in vitro LPS model and 2) quantifying the amount of imported proteins. In parallel, we measure ROS production and mitochondrial membrane potential by fluorescence assays. The effects of protein import disruption on mitochondrial function will be assessed by measuring cellular ATP concentration and respiratory chain variables (e.g. extracellular flux analyzer, to be funded). To identify particularly affected proteins, changes in the mitochondrial proteome will also be described under manifestation of the suspected import disorder. Finally, the hypothesis that antioxidants (ascorbic acid, MitoQ) attenuate the import disorder will be tested and different dosages and application times will be assessed. The aim here is not only to identify a ROS-induced import disorder as a factor of mitochondrial dysfunction in sepsis but also to explore the use of antioxidants as a potential therapeutic option.

• DFG Programme: Research Grants
• Co-Investigators: Professor Dr. Michael Adamzik; Privatdozent Dr. Björn Koos; Professor Dr. Jürgen Peters