Role of cytoskeleton in regulation of mitochondrial function

The control of mitochondrial oxidative phosphorylation is a cardinal issue in the field of muscle energetics, and the analysis of mitochondrial regulation is central to basic research of mitochondrial physiology and the diagnosis of many diseases. In the myocardium, a linear relationship exists between oxygen consumption and contractile performance, showing that this muscle should permanently adjust the balance between mitochondrial energy production and energy utilization. The precise nature of the signals involved in this process is as yet unresolved. Mitochondrial oxidative capacity and affinity to the main regulator ADP are key components of mitochondrial physiology. Studies of mitochondria in situ in permeabilized cells and muscle fibers have shown remarkably increased apparent Kms for ADP in the regulation of mitochondrial respiration as compared with isolated mitochondria. It has been suggested that this effect is related to the local restrictions on ADP in the cells, due to interaction between cytoskeletal proteins in particular intermediate filament (IF) proteins and VDAC of the mitochondrial outer membrane, indicating that IF proteins can actively participate in mitochondrial regulation. Although various cytoskeletal proteins have been demonstrated to associate and interact with mitochondria, their role in regulating mitochondrial bioenergetics is still unclear. The best candidates for key roles here are the cytoskeletal proteins plectin and desmin. The general focus of the project, therefore, will be to determine the physiological role of mitochondria-cytoskeleton interactions in controlling mitochondrial sensitivity to ADP. This will be achieved by analyzing the kinetics of ATP synthesis and the sensitivity of mitochondria to ADP in situ in permeabilized fibers isolated from various muscles of plectin and desmin knockout mice (including desmin/plectin isoform 1d-double knockout) and comparing these parameters to those of control wild-type animals. The project proposes to conduct in situ analysis of mitochondria, which is based on the selective permeabilization of the plasma membrane. As compared with isolated mitochondria, this approach has a number of advantages: 1) artefacts of mitochondrial isolation are avoided; 2) only very small tissue samples are required; 3) the total cellular population of mitochondria can be investigated; 4) most importantly, it allows mitochondria to be analyzed within an integrated cellular system, in their normal intracellular position and assembly, thus preserving essential interactions with the cytoskeleton. This work will be performed in collaboration with Dr. G. Wiche`s group, MFPL, Vienna. His collaboration and previously generated animal models will be essential for the project. By combining high- resolution respirometry and live imaging of mitochondria we also plan to identify the relation between mitochondrial intracellular organization and ADP sensitivity. Using complementary approaches in collaboration with national and international partners we are confident that the goals of the project can be reached in a highly efficient way.

The proposed project provides new data and a more complete understanding of the role of mitochondria-cytoskeleton interactions in controlling and in regulating mitochondrial respiration in the heart and oxidative skeletal muscles. This will help to reveal a missing molecular link between mechanisms regulating mitochondrial organization and the basic mechanisms of regulating mitochondrial respiratory function in normal cells and their alteration under conditions of energy metabolism impairment. Moreover, the new knowledge will substantially contribute to our understanding of more general mechanisms of mitochondrial interaction with/crosstalk to the rest of the cell (mitochondria-cell communication), as well as our knowledge of the impairment of such interaction in various pathologies. Taken together, the new scientific information obtained on this project will certainly find a use and will also promote important advances both in the basic research of mitochondrial physiology and in clinically oriented mitochondrial studies. Studies of mitochondrial interactions with other intracellular systems like the cytoskeleton will have various implications for many areas of cell biology and pathology. The proposed analysis of the alteration in mitochondrial function and energy transfer provides valuable tool for precise interpretation of the results obtained in clinical studies of various human metabolic diseases (for example molecular mechanisms of ischemia-reperfusion injury in various organs, mechanisms of human cardiac and muscle diseases). This will be useful for further understanding of mitochondrial biology as well as molecular mechanisms of mitochondria-mediated cell dysfunction to develop a number of new therapeutic approaches for the treatment of various human diseases by targeting mitochondria. Moreover, muscle energy metabolism is sensitive to nutrition and exercise training and fiber-type composition. Therefore, the proposed mitochondrial analysis will certainly find use also in sport medicine.