MITOCARD

The main objective of MITOCARD is to lead to a major progress in the understanding of cardiac physiology by integrating the mitochondrial properties of cell signaling in the comprehensive view of cardiac energetics and rhythm pathologies. It was recently demonstrated that in the heart, in striking contrast with skeletal muscle, a parallel activation by calcium of mitochondria and myofibrils occurs during contraction, which indicates that mitochondria actively participate to Ca2+ signaling in the cardiomyocyte. We hypothesize that the mitochondrial permeability transition pore (mPTP), by rhythmically depolarizing inner mitochondrial membrane, plays a crucial role in mitochondrial Ca2+ regulation and, as a result, of cardiomyocyte Ca2+ homeostasis. Moreover, mitochondrial ROS may play a key role in the regulation of the mPTP by “sensing” mitochondrial energetics balance. Consequently, a deeper understanding of mitochondrial “electrophysiology” is mandatory to decipher their exact role in heart excitation-contraction coupling processes. However, this is currently prevented by the absence of adequate methodological tools (lack of sensitivity or selectivity, time resolution, averaged responses of numerous biological entities); we propose to solve that issue by developing innovative analytical tools and biophysical approaches to monitor kinetically and quantitatively the Ca2+ handling by isolated mitochondria in the cardiomyocyte. MITOCARD is a multi-disciplinary project involving 4 partners of different scientific fields. Two partners (ISM, LAAS) will develop devices and methods to monitor in real-time key mitochondrial signaling parameters: Ca2+, membrane potential, quinone reduction status, O2 consumption, ROS production. We plan to monitor activities at different biological levels, from small populations of mitochondria, then on isolated cells, while targeting the single unit level so as to measure exact fluxes and kinetics of metabolites. To reach these goals, we will first develop chips integrating 4 different electrochemical microsensors. These will allow to measure simultaneously on the same mitochondria or cells their O2 consumption, quinone reduction status and ROS production (ISM, CRCTB, LAAS). Subsequently, highly innovative microwell arrays integrating ring nanoelectrodes will be developed to trap single mitochondria within micrometric chambers and measure locally by combined fluorescence microscopy and electrochemical techniques intra- (by fluorescence) and extra-mitochondrial (electrochemistry) metabolites. This approach should demonstrate the direct correlations between energetics, redox signaling and calcium handling via the PTP on a single biological entity (CRCTB, CARMEN). Mitochondria from two different origins, i.e. from the rat heart and skeletal muscle because of their mechanistic differences will be challenged with diverse activators and inhibitors of calcium transport, respiratory chain and ROS production. We will further switch from the level of isolated mitochondria to a single myocyte (CRCTB, ISM), so as to evaluate general integrated behaviors of mitochondrial populations interacting with cell structures (SC in particular for calcium). We aim also at working on a cell in physiological conditions, meaning a beating cell whose contraction is not pharmacologically inhibited. This approach will necessitate the adaptation of methodologies to measure sufficiently rapidly and locally the dynamics of calcium, quinone, ROS, and oxygen. All these unprecedented data will stimulate the development of a new numerical dynamic model of excitation-contraction coupling by including mitochondrial electrophysiology (CARMEN). The model may serve both to assess biological assumptions on the role of mitochondria in Ca2+ signaling and to integrate pathological data and provide clues for their global understanding