Abstract
Background and aim. Adult cardiac muscle cells predominantly use mitochondrial oxidative phosphorylation for production of ATP and utilize fatty acids as their main source of reducing power. Mitochondria in cardiac cells are positioned within intermyofibrillar space at the level of A-band in a crystal-l ike pattern and together with actomyosin system, sarcoplasmatic reticulum, cytoskeletal proteins and ion pumps are integrated into structuralfunctional complex, the Intracellular Energetic Unit ( ICEU), which regulates energy production and transport through phosphotransfer network. In perinatal cardiomyocytes regular arrangement of mitochondria is missing, ICEU has not been established and the bioenergetic metabolism and energy transfer regulation are different.
The aim of our study was to investigate the timeline of formation of regular mitochondrial arrangement, functional interactions with cytoskeletal proteins and regulation of oxidative phosphorylation in mitochondria, characteristic of adult cardiomyocyte. These data could give valuable information about the causes of pathological processes related the deficiencies in the structure-function relationship of cardiac cell bioenergetics.
Methods. Cardiomyocytes were isolated after perfusion of the rat heart with collagenase. Isolated cells were kept on ice during further experiments. Skinned cardiac fibers were isolated according to the method developed by V. Saks and A. Kuznetsov. Bundles of fibers were isolated from endocardial surface of left ventricle. By using sharp-ended needles muscle fibers were separated, leaving only small areas of contact. Fibers were permeabilized with 50 μg/ml saponin for 30 min and then washed to remove metabolites. The images of the cells and fibers were acquired with Zeiss LSM 510 and Olympus FluoView FV10i-W confocal microscopes, using antibodies from Abcam. The VDAC antibody was a generous gift from Dr. C. Brenner. The rates of oxygen uptake in different conditions were determined with high-resolution respirometer Oxygraph-2K.
Results. Results of this study show that during first postnatal weeks, significant changes in regulation of energy transfer in cardiac cells are taking place. Remodeling of subcellular organization of mitochondria occurs in liaison with shift in cellular metabolism. While localization of mitochondria becomes progressively more organized within cellular interior Michaelis-Menten constant Km(ADP) for mitochondrial respiration increases from 75,0・}4,5 μM (3-day old rat cardiomyocytes) to 317・}29,5 μM (84-day old rat cardiomyocytes). This reflects the increased diffusion restrictions for ADP and is a manifestation of higher regulation of energy transfer. Also, activation of mitochondrial creatine kinase – phosphotransfer system was concurrently seen with emergence of tubulin 4 and 2 colocalization and formation of regular localization pattern reminiscent to that of mitochondria. Similar to timeline of creatine/phosphotranfer activation the maturation of cellular cytoarchitecture of tubul in 4 and 2 tubul in isoforms residing in vicinity of the mitochondria, suggests that both 4 and 2 tubulin could participate in regulation of VDAC permeability for adenine nucleotides. Additionally progressive covering of 4 and 2 tubulin by mitochondria indicates that these tubulin isoforms could act as directional cues for targeting mitochondria to their specific location.
Conclusions. The results of the study indicate that dynamic changes in the structure of cardiomyocytes during postnatal development are fol lowed by the functional changes. Existence of functional interactions between mitochondria and the components of cytoskeleton in cardiac cells are necessary prerequisites for highly organized metabolic networks, like in adult cardiomyocytes.