The aim of this work was to characterize quantitatively the arrangement of mitochondria in heart and skeletal muscles. We studied confocal images of mitochondria in nonfixed cardiomyocytes and fibers from soleus and white gastrocnemius muscles of adult rats. The arrangement of intermyofibrillar mitochondria was analyzed by estimating the densities of distribution of mitochondrial centers relative to each other (probability density function). In cardiomyocytes (1,820 mitochondrial centers marked), neighboring mitochondria are aligned along a rectangle, with distance between the centers equal to 1.97 Ϯ 0.43 and 1.43 Ϯ 0.43 m in the longitudinal and transverse directions, respectively. In soleus (1,659 mitochondrial centers marked) and white gastrocnemius (621 pairs of mitochondria marked), mitochondria are mainly organized in pairs at the I-band level. Because of this organization, there are two distances characterizing mitochondrial distribution in the longitudinal direction in these muscles. The distance between mitochondrial centers in the longitudinal direction within the same I band is 0.91 Ϯ 0.11 and 0.61 Ϯ 0.07 m in soleus and white gastrocnemius, respectively. The distance between mitochondrial centers in different I bands is ϳ3.7 and ϳ3.3 m in soleus and gastrocnemius, respectively. In the transverse direction, the mitochondria are packed considerably closer to each other in soleus than in white gastrocnemius, with the distance equal to 0.75 Ϯ 0.22 m in soleus and 1.09 Ϯ 0.41 m in gastrocnemius. Our results show that intermyofibrillar mitochondria are arranged in a highly ordered crystal-like pattern in a muscle-specific manner with relatively small deviation in the distances between neighboring mitochondria. This is consistent with the concept of the unitary nature of the organization of the muscle energy metabolism. confocal microscopy; quantitative analysis; cardiac and skeletal muscles; probability density function; unitary structure of cells RECENT STUDIES HAVE SHOWN the existence of multiple specific functional interactions among mitochondria, sarcoplasmic reticulum (SR), and myofibrils in permeabilized muscle fibers (5,14,30,34). Namely, endogenous ATP has been shown to be more efficient than exogenous ATP in maintaining calcium uptake into SR (14). In addition, kinetic studies have shown a direct supply of endogenous ADP from ATPases to mitochondria (30, 34). Such interaction can be explained by the existence of localized intracellular diffusion restrictions (28, 39). A mild treatment of the fibers with trypsin leads to the removal of these diffusion restrictions, and at the same time, distribution of mitochondria in the fiber is changed from regular arrangement in the control to random distribution after the treatment (28). Similarly, in ischemic hearts, various alterations in mitochondrial function such as the significant decrease in maximal respiration rate and half-saturation constant for ADP were observed in parallel with the changes in structural organization of the cardiac muscle cells (7,1...

The aim of this review is to analyze the results of experimental research of mechanisms of regulation of mitochondrial respiration in cardiac and skeletal muscle cells in vivo obtained by using the permeabilized cell technique. Such an analysis in the framework of Molecular Systems Bioenergetics shows that the mechanisms of regulation of energy fluxes depend on the structural organization of the cells and interaction of mitochondria with cytoskeletal elements. Two types of cells of cardiac phenotype with very different structures were analyzed: adult cardiomyocytes and continuously dividing cancerous HL-1 cells. In cardiomyocytes mitochondria are arranged very regularly, and show rapid configuration changes of inner membrane but no fusion or fission, diffusion of ADP and ATP is restricted mostly at the level of mitochondrial outer membrane due to an interaction of heterodimeric tubulin with voltage dependent anion channel, VDAC. VDAC with associated tubulin forms a supercomplex, Mitochondrial Interactosome, with mitochondrial creatine kinase, MtCK, which is structurally and functionally coupled to ATP synthasome. Due to selectively limited permeability of VDAC for adenine nucleotides, mitochondrial respiration rate depends almost linearly upon the changes of cytoplasmic ADP concentration in their physiological range. Functional coupling of MtCK with ATP synthasome amplifies this signal by recycling adenine nucleotides in mitochondria coupled to effective phosphocreatine synthesis. In cancerous HL-1 cells this complex is significantly modified: tubulin is replaced by hexokinase and MtCK is lacking, resulting in direct utilization of mitochondrial ATP for glycolytic lactate production and in this way contributing in the mechanism of the Warburg effect. Systemic analysis of changes in the integrated system of energy metabolism is also helpful for better understanding of pathogenesis of many other diseases.

Problems of quantitative investigation of intracellular diffusion and compartmentation of metabolites are analyzed. Principal controversies in recently published analyses of these problems for the living cells are discussed. It is shown that the formal theoretical analysis of diffusion of metabolites based on Fick's equation and using fixed diffusion coefficients for diluted homogenous aqueous solutions, but applied for biological systems in vivo without any comparison with experimental results, may lead to misleading conclusions, which are contradictory to most biological observations. However, if the same theoretical methods are used for analysis of actual experimental data, the apparent diffusion constants obtained are orders of magnitude lower than those in diluted aqueous solutions. Thus, it can be concluded that local restrictions of diffusion of metabolites in a cell are a system-level properties caused by complex structural organization of the cells, macromolecular crowding, cytoskeletal networks and organization of metabolic pathways into multienzyme complexes and metabolons. This results in microcompartmentation of metabolites, their channeling between enzymes and in modular organization of cellular metabolic networks. The perspectives of further studies of these complex intracellular interactions in the framework of Systems Biology are discussed. OPEN ACCESSInt. J. Mol. Sci. 2008, 9 752