MOLECULAR SYSTEM BIOENERGETICS – MOLSYBEN

1-Scientific background and objectives : Besides a prominent role in energy metabolism, mitochondria are central in several major cell functions including control of cell death. Studies on isolated mitochondria are insufficient to understand their regulation in living cells. The intracellular medium in muscles is highly organized and macromolecules and organelles, including mitochondria, are involved in multiple structural and functional interactions. The basic modern paradigm of cellular metabolism is the compartmentation of enzymes and both compartmentation, microcompartmentation and channelling of metabolites, including such important regulators of the cell life as calcium and ATP. One of the systems of high degree of organisation and coordination is the system of energy transfer networks in muscle and brain cells. The important task of highest complexity is to describe these processes quantitatively with the aim of better understanding of their mechanisms and regulation. This is the purpose of a new area of research which involves mathematical modeling in combination with experimental studies, entitled molecular system bioenergetics. The main objectives of the project are to quantify and to model the heterogeneous compartmentation of adenine nucleotides and the complex intracellular structural and functional communications of mitochondria with other cellular structures in normal cells. This project will be run by biologists, biophysicists and specialists of computer simulation. 2-Description of the project, methodology : On the basis of the results of the experimental investigations planned in this project, the complete reaction-diffusion mathematical model of mitochondrial reactions and energy transfer processes between different cellular compartments in muscle cells will be developed. Mathematical model of mitochondrial reactions will include models of respiratory chain, mitochondrial metabolite carriers, ATP synthase, Krebs cycle, mitochondrial calcium cycle and reactions of beta oxidation of fatty acids. Compartmentized energy transfer model will include coupled creatine and adenylate kinase reactions and diffusion processes. In the experiments, the isolated rat cardiomyocytes, contracting and non-contracting HL-1 cardiac cell line, isolated synaptosomes and mitochondria will be studied. Oxygraphy, HPLC and spectrophotometry will be used to study the kinetics of respiration regulation in all types of cells. Confocal microscopy and bioluminescence technique, fluorescence correlation spectroscopy and fluorescence resonance energy transfer (FRET) will be used to measure the parameters of the intracellular diffusion of adenine nucleotides between different cellular compartments with the quantitative analysis by mathematical models. The possible role of mitochondria movement will be investigated. The local concentrations of ATP will be measured in contracting and non-contracting cardiac cells with the use of recombinant forms of the ATP receptor luciferase in combination with green and yellow fluorescent proteins localised in different cell compartments: in mitochondrial matrix; at the outer side of mitochondrial inner membrane in the microcompartment between adenine nucleotide translocator (ANT) and mitochondrial creatine kinase (MtCK); in myofibrils in close connection to myosin molecules in A-band; at the membrane of sarcoplasmic reticulum, at the sarcolemma close to K ATP channel. The direct functional coupling and channelling of substrates between ANT and MtCK, and between K ATP channel and MM creatine kinase at the sarcolemma will be studied by using fluorescent analogues of adenine nucleotides and FRET. The experimental data will be used to verify the model predictions. 3-Expected results : Mathematical model the heterogeneous compartmentation of adenine nucleotides and the complex intracellular structural and functional communications of mitochondria with other cellular structures in normal cells will be created for wide practical use. Local concentrations of adenine nucleotides will be measured in different parts of the cells. Functional coupling mechanisms in different multienzyme complexes will be characterized. These quantitative methods will be used to investigate the pathological states of cells such as ischemia, and the cardioprotective methods, such as preconditioning phenomenon.