This Special Issue is a significant step in developing a new direction of metabolic research— Molecular System Bioenergetics, which itself is a part of Systems Biology. As a new paradigm of biological sciences, Systems Biology aims at understanding of biological functions by studies and description of new, system level properties, resulting from interactions between components of biological systems at any level of organization, from molecular to population. Metabolism is the way of life of cells by exchanging mass and energy with the surrounding medium, and understanding its mechanisms requires knowledge of the complex interactions between cellular systems and components. While studies of metabolism have a long history, new concepts of Systems Biology provide useful tools for metabolic research. According to Schrödinger, living cells need to be open systems with energy and mass exchange with the surrounding medium, with the aim of maintaining their high structural and functional organization and thus their internal entropy low, achieving this by means of increasing the entropy of the medium by catabolic reactions. Thus, Schrödinger wrote: “The essential thing in metabolism is that the organism succeeds in freeing itself from all entropy it cannot help producing while alive”. Thus, free energy conversion in the cells is an important, central part of metabolism, and understanding the complex mechanisms of its regulation is the aim of Molecular System Bioenergetics. In this Special Issue, several important problems in this field were analyzed. Full article

In this article we continue our exploration of the historical roots of systems biology by considering the work of William Harvey. Central arguments in his work on the movement of the heart and the circulation of the blood can be shown to presage the concepts and methods of integrative systems biology. These include: (a) the analysis of the level of biological organization at which a function (e.g. cardiac rhythm) can be said to occur; (b) the use of quantitative mathematical modelling to generate testable hypotheses and deduce a fundamental physiological principle (the circulation of the blood) and (c) the iterative submission of his predictions to an experimental test. This article is the result of a tri-lingual study: as Harvey’s masterpiece was published in Latin in 1628, we have checked the original edition and compared it with and between the English and French translations, some of which are given as notes to inform the reader of differences in interpretation. Full article

The mitochondrial response to changes of cytosolic calcium concentration has a strong impact on neuronal cell metabolism and viability. We observed that Ca²⁺ additions to isolated rat brain mitochondria induced in potassium ion containing media a mitochondrial membrane potential depolarization and an accompanying increase ofmitochondrial respiration. These Ca²⁺ effects can be blocked by iberiotoxin and charybdotoxin, well known inhibitors of large conductance potassium channel (BK_Ca channel). Furthermore, NS1619 – a BK_Ca channel opener – induced potassium ion–specific effects on brain mitochondria similar to those induced by Ca²⁺. These findings suggest the presence of a calcium-activated, large conductance potassium channel (sensitive to charybdotoxin and NS1619), which was confirmed by reconstitution of the mitochondrial inner membrane into planar lipid bilayers. The conductance of the reconstituted channel was 265 pS under gradient (50/450 mM KCl) conditions. Its reversal potential was equal to 50 mV, which proved that the examined channel was cation-selective. We also observed immunoreactivity of anti-b₄ subunit (of the BK_Ca channel) antibodies with ~26 kDa proteins of rat brain mitochondria. Immunohistochemical analysis confirmed the predominant occurrence of b₄ subunit in neuronal mitochondria. We hypothesize that the mitochondrial BK_Ca channel represents a calcium sensor, which can contribute to neuronal signal transduction and survival. Full article

A key property of complex biological systems is the presence of interaction networks formed by its different components, primarily proteins. These are crucial for all levels of cellular function, including architecture, metabolism and signalling, as well as the availability of cellular energy. Very stable, but also rather transient and dynamic protein-protein interactions generate new system properties at the level of multiprotein complexes, cellular compartments or the entire cell. Thus, interactomics is expected to largely contribute to emerging fields like systems biology or systems bioenergetics. The more recent technological development of high-throughput methods for interactomics research will dramatically increase our knowledge of protein interaction networks. The two most frequently used methods are yeast two-hybrid (Y2H) screening, a well established genetic in vivo approach, and affinity purification of complexes followed by mass spectrometry analysis, an emerging biochemical in vitro technique. So far, a majority of published interactions have been detected using an Y2H screen. However, with the massive application of this method, also some limitations have become apparent. This review provides an overview on available yeast two-hybrid methods, in particular focusing on more recent approaches. These allow detection of protein interactions in their native environment, as e.g. in the cytosol or bound to a membrane, by using cytosolic signalling cascades or split protein constructs. Strengths and weaknesses of these genetic methods are discussed and some guidelines for verification of detected protein-protein interactions are provided. Full article

Mitochondrial dysfunction is a hallmark of almost all diseases. Acquired or inherited mutations of the mitochondrial genome DNA may give rise to mitochondrial diseases. Another class of disorders, in which mitochondrial impairments are initiated by extramitochondrial factors, includes neurodegenerative diseases and syndromes resulting from typical pathological processes, such as hypoxia/ischemia, inflammation, intoxications, and carcinogenesis. Both classes of diseases lead to cellular energetic depression (CED), which is characterized by decreased cytosolic phosphorylation potential that suppresses the cell’s ability to do work and control the intracellular Ca²⁺ homeostasis and its redox state. If progressing, CED leads to cell death, whose type is linked to the functional status of the mitochondria. In the case of limited deterioration, when some amounts of ATP can still be generated due to oxidative phosphorylation (OXPHOS), mitochondria launch the apoptotic cell death program by release of cytochrome c. Following pronounced CED, cytoplasmic ATP levels fall below the thresholds required for processing the ATP-dependent apoptotic cascade and the cell dies from necrosis. Both types of death can be grouped together as a mitochondrial cell death (MCD). However, there exist multiple adaptive reactions aimed at protecting cells against CED. In this context, a metabolic shift characterized by suppression of OXPHOS combined with activation of aerobic glycolysis as the main pathway for ATP synthesis (Warburg effect) is of central importance. Whereas this type of adaptation is sufficiently effective to avoid CED and to control the cellular redox state, thereby ensuring the cell survival, it also favors the avoidance of apoptotic cell death. This scenario may underlie uncontrolled cellular proliferation and growth, eventually resulting in carcinogenesis. Full article

Beyond their fundamental role in energy metabolism, mitochondria perform a great variety of other important cellular functions. However, the interplayamong these various roles of mitochondria is still poorly understood, and the underlying mechanisms can be related to system level properties. Importantly, mitochondria localized in different regions of a cell may display different morphology, dissimilar biochemical properties, or may differently interact with other intracellular structures. Recent advances in live imaging techniques have also revealed a functional heterogeneity of mitochondria with respect to mitochondrial redox state, membrane potential, respiratory activity, uncoupling proteins, mitochondrial ROS and calcium. An important and still unresolved question is how the heterogeneity of mitochondrial function and the regional specializations of mitochondria are mechanistically realized in the cell and to what extent this could be dependent on environmental aspects. Distinct mitochondrial subsets may also exhibit different responses to substrates and inhibitors and may vary in their sensitivity to pathology, resistance to apoptosis, oxidative stress, thus also demonstrating heterogeneous behavior. All these observations strongly suggest that the intracellular position, organization and the specific surroundings of mitochondria within the cell define their functional features, while also implying that different mitochondrial subpopulations, clusters or even single mitochondrion may execute diverse processes in a cell. The heterogeneity of mitochondrial function demonstrates an additional level of mitochondrial complexity and is a new, challenging area in mitochondrial research that potentially leads to the integration of mitochondrial bioenergetics and cell physiology with various physiological and pathophysiological implications. Full article

Adenylate kinase and downstream AMP signaling is an integrated metabolic monitoring system which reads the cellular energy state in order to tune and report signals to metabolic sensors. A network of adenylate kinase isoforms (AK1-AK7) are distributed throughout intracellular compartments, interstitial space and body fluids to regulate energetic and metabolic signaling circuits, securing efficient cell energy economy, signal communication and stress response. The dynamics of adenylate kinase-catalyzed phosphotransfer regulates multiple intracellular and extracellular energy-dependent and nucleotide signaling processes, including excitation-contraction coupling, hormone secretion, cell and ciliary motility, nuclear transport, energetics of cell cycle, DNA synthesis and repair, and developmental programming. Metabolomic analyses indicate that cellular, interstitial and blood AMP levels are potential metabolic signals associated with vital functions including body energy sensing, sleep, hibernation and food intake. Either low or excess AMP signaling has been linked to human disease such as diabetes, obesity and hypertrophic cardiomyopathy. Recent studies indicate that derangements in adenylate kinase-mediated energetic signaling due to mutations in AK1, AK2 or AK7 isoforms are associated with hemolytic anemia, reticular dysgenesis and ciliary dyskinesia. Moreover, hormonal, food and antidiabetic drug actions are frequently coupled to alterations of cellular AMP levels and associated signaling. Thus, by monitoring energy state and generating and distributing AMP metabolic signals adenylate kinase represents a unique hub within the cellular homeostatic network. Full article

Isotope labeling is one of the few methods of revealing the in vivo bidirectionality and compartmentalization of metabolic fluxes within metabolic networks. We argue that a shift from steady state to dynamic isotopomer analysis is required to deal with these cellular complexities and provide a review of dynamic studies of compartmentalized energy fluxes in eukaryotic cells including cardiac muscle, plants, and astrocytes. Knowledge of complex metabolic behaviour on a molecular level is prerequisite for the intelligent design of genetically modified organisms able to realize their potential of revolutionizing food, energy, and pharmaceutical production. We describe techniques to explore the bidirectionality and compartmentalization of metabolic fluxes using information contained in the isotopic transient, and discuss the integration of kinetic models with MFA. The flux parameters of an example metabolic network were optimized to examine the compartmentalization of metabolites and and the bidirectionality of fluxes in the TCA cycle of Saccharomyces uvarum for steady-state respiratory growth. Full article

The pattern of flux and concentration control coefficients in an integrated mitochondrial energetics model is examined by applying a generalized matrix method of control analysis to calculate control coefficients, as well as response coefficients The computational model of Cortassa et al. encompasses oxidative phosphorylation, the TCA cycle, and Ca²⁺ dynamics. Control of ATP synthesis, TCA cycle, and ANT fluxes were found to be distributed among various mitochondrial processes. Control is shared by processes associated with ATP/ADP production and transport, as well as by Ca²⁺ dynamics. The calculation also analyzed the control of the concentrations of key regulatory ions and metabolites (Ca²⁺, NADH, ADP). The approach we have used demonstrates how properties of integrated systems may be understood through applications of computational modeling and control analysis. Full article

We live in times of paradigmatic changes for the biological sciences. Reductionism, that for the last six decades has been the philosophical basis of biochemistry and molecular biology, is being displaced by Systems Biology, which favors the study of integrated systems. Historically, Systems Biology - defined as the higher level analysis of complex biological systems - was pioneered by Claude Bernard in physiology, Norbert Wiener with the development of cybernetics, and Erwin Schrödinger in his thermodynamic approach to the living. Systems Biology applies methods inspired by cybernetics, network analysis, and non-equilibrium dynamics of open systems. These developments follow very precisely the dialectical principles of development from thesis to antithesis to synthesis discovered by Hegel. Systems Biology opens new perspectives for studies of the integrated processes of energy metabolism in different cells. These integrated systems acquire new, system-level properties due to interaction of cellular components, such as metabolic compartmentation, channeling and functional coupling mechanisms, which are central for regulation of the energy fluxes. State of the art of these studies in the new area of Molecular System Bioenergetics is analyzed. Full article

Reduced glycolytic and mitochondrial respiration rates are common features of apoptosis that may reflect key events contributing to cell death. However, it is unclear to what extent the rate changes can be explained by direct alterations in the kinetics of the participating reactions, as changes in the concentrations of intermediates also affect reaction rates. Direct kinetic changes can be identified, ranked, and compared to the indirect effects mediated by the intermediates using top-down control analysis. Flux changes that are explained primarily by direct effects are likely to be prime targets of the pathways that signal death, and thus important contributors to apoptosis. Control analysis concepts relevant to identifying such effects are reviewed. Metabolic flux measurements are essential for this approach, but can be technically difficult, particularly when using adherent cells such as neurons. A simple method is described that renders such measurements feasible. Full article

A shift in glucose metabolism from oxidative phosphorylation to glycolysis is one of the biochemical hallmarks of tumor cells. Mitochondrial defects have been proposed to play an important role in the initiation and/or progression of various types of cancer. In the past decade, a wide spectrum of mutations and depletion of mtDNA have been identified in human cancers. Moreover, it has been demonstrated that activation of oncogenes or mutation of tumor suppressor genes, such as p53, can lead to the upregulation of glycolytic enzymes or inhibition of the biogenesis or assembly of respiratory enzyme complexes such as cytochrome c oxidase. These findings may explain, at least in part, the well documented phenomena of elevated glucose uptake and mitochondrial defects in cancers. In this article, we review the somatic mtDNA alterations with clinicopathological correlations in human cancers, and their potential roles in tumorigenesis, cancer progression, and metastasis. The signaling pathways involved in the shift from aerobic metabolism to glycolysis in human cancers are also discussed. Full article

Mitochondria play a key role in the energy metabolism in skeletal muscle. A new concept has emerged suggesting that impaired mitochondrial oxidative capacity in skeletal muscle may be the underlying defect that causes insulin resistance. According to current knowledge, the causes and the underlying molecular mechanisms at the origin of decreased mitochondrial oxidative capacity in skeletal muscle still remain to be elucidated. The present review focuses on recent data investigating these issues in the area of metabolic disorders and describes the potential causes, mechanisms and consequences of mitochondrial dysfunction in the skeletal muscle. Full article