Permeabilized rat cardiomyocyte response demonstrates intracellular origin of diffusion obstacles.

Intracellular diffusion restrictions for ADP and other molecules have been predicted earlier based on experiments on permeabilized fibers or cardiomyocytes. However, it is possible that the effective diffusion distance is larger than the cell dimensions due to clumping of cells and incomplete separation of cells in fiber preparations. The aim of this work was to check whether diffusion restrictions exist inside rat cardiomyocytes or are caused by large effective diffusion distance. For that, we determined the response of oxidative phosphorylation (OxPhos) to exogenous ADP and ATP stimulation in permeabilized rat cardiomyocytes using fluorescence microscopy. The state of OxPhos was monitored via NADH and flavoprotein autofluorescence. By varying the ADP or ATP concentration in flow chamber, we determined that OxPhos has a low affinity in cardiomyocytes. The experiments were repeated in a fluorometer on cardiomyocyte suspensions leading to similar autofluorescence changes induced by ADP as recorded under the microscope. ATP stimulated OxPhos more in a fluorometer than under the microscope, which was attributed to accumulation of ADP in fluorometer chamber. By calculating the flow profile around the cell in the microscope chamber and comparing model solutions to measured data, we demonstrate that intracellular structures impose significant diffusion obstacles in rat cardiomyocytes.

Structure-function relationships in feedback regulation of energy fluxes in vivo in health and disease: mitochondrial interactosome.

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.

Distinct organization of energy metabolism in HL-1 cardiac cell line and cardiomyocytes.

Expression and function of creatine kinase (CK), adenylate kinase (AK) and hexokinase (HK) isoforms in relation to their roles in regulation of oxidative phosphorylation (OXPHOS) and intracellular energy transfer were assessed in beating (B) and non-beating (NB) cardiac HL-l cell lines and adult rat cardiomyocytes or myocardium. In both types of HL-1 cells, the AK2, CKB, HK1 and HK2 genes were expressed at higher levels than the CKM, CKMT2 and AK1 genes. Contrary to the saponin-permeabilized cardiomyocytes the OXPHOS was coupled to mitochondrial AK and HK but not to mitochondrial CK, and neither direct transfer of adenine nucleotides between CaMgATPases and mitochondria nor functional coupling between CK-MM and CaMgATPases was observed in permeabilized HL-1 cells. The HL-1 cells also exhibited deficient complex I of the respiratory chain. In conclusion, contrary to cardiomyocytes where mitochondria and CaMgATPases are organized into tight complexes which ensure effective energy transfer and feedback signaling between these structures via specialized pathways mediated by CK and AK isoforms and direct adenine nucleotide channeling, these complexes do not exist in HL-1 cells due to less organized energy metabolism.

Mitochondrial dynamics in heart cells: very low amplitude high frequency fluctuations in adult cardiomyocytes and flow motion in non beating Hl-1 cells.

The arrangement and movement of mitochondria were quantitatively studied in adult rat cardiomyocytes and in cultured continuously dividing non beating (NB) HL-1 cells with differentiated cardiac phenotype. Mitochondria were stained with MitoTracker Green and studied by fluorescent confocal microscopy. High speed scanning (one image every 400 ms) revealed very rapid fluctuation of positions of fluorescence centers of mitochondria in adult cardiomyocytes. These fluctuations followed the pattern of random walk movement within the limits of the internal space of mitochondria, probably due to transitions between condensed and orthodox configurational states of matrix and inner membrane. Mitochondrial fusion or fission was seen only in NB HL-1 cells but not in adult cardiomyocytes. In NB HL-1 cells, mitochondria were arranged as a dense tubular network, in permanent fusion, fission and high velocity displacements of approximately 90 nm/s. The differences observed in mitochondrial dynamics are related to specific structural organization and mitochondria-cytoskeleton interactions in these cells.

Metabolic compartmentation - a system level property of muscle cells: real problems of diffusion in living cells.

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.

Different kinetics of the regulation of respiration in permeabilized cardiomyocytes and in HL-1 cardiac cells. Importance of cell structure/organization for respiration regulation.

The aim of this study was to investigate the mechanism of cellular regulation of mitochondrial respiration in permeabilized cardiac cells with clearly different structural organization: (i) in isolated rat cardiomyocytes with very regular mitochondrial arrangement, (ii) in HL-1 cells from mouse heart, and (iii) in non-beating (NB HL-1 cells) without sarcomeres with irregular and dynamic filamentous mitochondrial network. We found striking differences in the kinetics of respiration regulation by exogenous ADP between these cells: the apparent Km for exogenous ADP was by more than order of magnitude (14 times) lower in the permeabilized non-beating NB HL-1 cells without sarcomeres (25+/-4 microM) and seven times lower in normally cultured HL-1 cells (47+/-15 microM) than in permeabilized primary cardiomyocytes (360+/-51 microM). In the latter cells, treatment with trypsin resulted in dramatic changes in intracellular structure that were associated with 3-fold decrease in apparent Km for ADP in regulation of respiration. In contrast to permeabilized cardiomyocytes, in NB HL-1 cells creatine kinase activity was low and the endogenous ADP fluxes from MgATPases recorded spectrophotometrically by the coupled enzyme assay were not reduced after activation of mitochondrial oxidative phosphorylation by the addition of mitochondrial substrates, showing the absence of ADP channelling in the NB HL-1 cells. While in the permeabilized cardiomyocytes creatine strongly activated mitochondrial respiration even in the presence of powerful competing pyruvate kinase-phosphoenolpyruvate system, in the NB HL-1 cells the stimulatory effect of creatine was not significant. The results of this study show that in normal adult cardiomyocytes and HL-1 cells intracellular local restrictions of diffusion of adenine nucleotides and metabolic feedback regulation of respiration via phosphotransfer networks are different, most probably related to differences in structural organization of these cells.

Structure-function relationships in the regulation of energy transfer between mitochondria and ATPases in cardiac cells.

The present study discusses the role of structural organization of cardiac cells in determining the mechanisms of regulation of oxidative phosphorylation and interaction between mitochondria and ATPases. In permeabilized adult cardiomyocytes, the apparent K(m) (Michaelis-Menten constant) for ADP in the regulation of respiration is far higher than in mitochondria isolated from the myocardium. Respiration of mitochondria in permeabilized cardiomyocytes is effectively activated by endogenous ADP produced by ATPases from exogenous ATP, and the activation of respiration is associated with a decrease in the apparent K(m) for ATP in the regulation of ATPase activity compared with this parameter in the absence of oxidative phosphorylation. It has also been shown that a large fraction of the endogenous ADP stimulating respiration remains inaccessible for the exogenous ADP trapping system, consisting of pyruvate kinase and phosphoenolpyruvate, unless the mitochondrial structures are modified by controlled proteolysis. These data point to the endogenous cycling of adenine nucleotides between mitochondria and ATPases. Accordingly, the current hypothesis is that in cardiac cells, mitochondria and ATPases are compartmentalized into functional complexes (ie, intracellular energetic units [ICEUs]), which appear to represent a basic pattern of organization of energy metabolism in these cells. Within the ICEUs, the mitochondria and ATPases interact via different routes: creatine kinase-mediated phosphoryltransfer; adenylate kinase-mediated phosphoryltransfer; and direct ATP and ADP channelling. The function of ICEUs changes not only after selective proteolysis, but also during contraction of cardiomyocytes caused by an increase in cytosolic Ca(2+) concentration up to micromolar levels. In these conditions, the apparent K(m) for exogenous ADP and ATP in the regulation of respiration markedly decreases, and more ADP becomes available for the exogenous pyruvate kinase-phosphoenolpyruvate system, which indicates altered barrier functions of the ICEUs. Thus, structural changes transmitted from the contractile apparatus to mitochondria clearly participate in the regulation of mitochondrial function due to alterations in localized restriction of the diffusion of adenine nucleotides. The importance of strict structural organization in cardiac cells emerged drastically from experiments in which the regulation of mitochondrial respiration was assessed in a novel cardiac cell line, that is, beating and nonbeating HL-1 cells. In these cells, the mitochondrial arrangement is irregular and dynamic, whereas the sarcomeric structures are either absent (in nonbeating HL-1 cells) or only rarely present (in beating HL-1 cells). In parallel, the apparent K(m) for exogenous ADP in the regulation of respiration was much lower than that in permeabilized primary cardiomyocytes, and trypsin treatment exerted no impact on the low K(m) value for ADP, in contrast to adult cardiomyocytes where it caused a marked decrease in this parameter. The HL-1 cells were also characterized by the absence of direct exchange of adenine nucleotides. The results further support the concept that the ICEUs in adult cardiomyocytes are products of complex structural organization developed to create the most optimal conditions for effective energy transfer and feedback between mitochondria and ATPases.

Comparative analysis of the bioenergetics of adult cardiomyocytes and nonbeating HL-1 cells: respiratory chain activities, glycolytic enzyme profiles, and metabolic fluxes.

Comparative analysis of the bioenergetic parameters of adult rat cardiomyocytes (CM) and HL-1 cells with very different structure but similar cardiac phenotype was carried out with the aim of revealing the importance of the cell structure for regulation of its energy fluxes. Confocal microscopic analysis showed very different mitochondrial arrangement in these cells. The cytochrome content per milligram of cell protein was decreased in HL-1 cells by a factor of 7 compared with CM. In parallel, the respiratory chain complex activities were decreased by 4-8 times in the HL-1 cells. On the contrary, the activities of glycolytic enzymes, hexokinase (HK), and pyruvate kinase (PK) were increased in HL-1 cells, and these cells effectively transformed glucose into lactate. At the same time, the creatine kinase (CK) activity was significantly decreased in HL-1 cells. In conclusion, the results of this study comply with the assumption that in contrast to CM in which oxidative phosphorylation is a predominant provider of ATP and the CK system is a main carrier of energy from mitochondria to ATPases, in HL-1 cells the energy metabolism is based mostly on the glycolytic reactions coupled to oxidative phosphorylation through HK.

Regulation of respiration in brain mitochondria and synaptosomes: restrictions of ADP diffusion in situ, roles of tubulin, and mitochondrial creatine kinase.

The role of ubiquitous mitochondrial creatine kinase (uMtCK) reaction in regulation of mitochondrial respiration was studied in purified preparations of rat brain synaptosomes and mitochondria. In permeabilized synaptosomes, apparent Km for exogenous ADP, Km (ADP), in regulation of respiration in situ was rather high (110 +/- 11 microM) in comparison with isolated brain mitochondria (9 +/- 1 microM). This apparent Km for ADP observed in isolated mitochondria in vitro dramatically increased to 169 +/- 52 microM after their incubation with 1 muM of dimeric tubulin showing that in rat brain, particularly in synaptosomes, mitochondrial outer membrane permeability for ADP, and ATP may be restricted by tubulin binding to voltage dependent anion channel (VDAC). On the other hand, in synaptosomes apparent Km (ADP) decreased to 25 +/- 1 microM in the presence of 20 mM creatine. To fully understand this effect of creatine on kinetics of respiration regulation, complete kinetic analysis of uMtCK reaction in isolated brain mitochondria was carried out. This showed that oxidative phosphorylation specifically altered only the dissociation constants for MgATP, by decreasing that from ternary complex MtCK.Cr.MgATP (K (a)) from 0.13 +/- 0.02 to 0.018 +/- 0.007 mM and that from binary complex MtCK.MgATP (K (ia)) from 1.1 +/- 0.29 mM to 0.17 +/- 0.07 mM. Apparent decrease of dissociation constants for MgATP reflects effective cycling of ATP and ADP between uMtCK and adenine nucleotide translocase (ANT). These results emphasize important role and various pathophysiological implications of the phosphocreatine-creatine kinase system in energy transfer in brain cells, including synaptosomes.

Mitochondrial regular arrangement in muscle cells: a "crystal-like" pattern.

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 +/- 0.43 and 1.43 +/- 0.43 microm 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 +/- 0.11 and 0.61 +/- 0.07 microm in soleus and white gastrocnemius, respectively. The distance between mitochondrial centers in different I bands is approximately 3.7 and approximately 3.3 microm 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 +/- 0.22 microm in soleus and 1.09 +/- 0.41 microm 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.