Enn Seppet

Functional complexes of mitochondria with Ca,MgATPases of myofibrils and sarcoplasmic reticulum in muscle cells

Regulation of mitochondrial respiration in situ in the muscle cells was studied by using fully permeabilized muscle fibers and cardiomyocytes. The results show that the kinetics of regulation of mitochondrial respiration in situ by exogenous ADP are very different from the kinetics of its regulation by endogenous ADP. In cardiac and m. soleus fibers apparent K(m) for exogenous ADP in regulation of respiration was equal to 300-400 microM. However, when ADP production was initiated by intracellular ATPase reactions, the ADP concentration in the medium leveled off at about 40 microM when about 70% of maximal rate of respiration was achieved. Respiration rate maintained by intracellular ATPases was suppressed about 20-30% during exogenous trapping of ADP with excess pyruvate kinase (PK, 20 IU/ml) and phosphoenolpyruvate (PEP, 5 mM). ADP flux via the external PK+PEP system was decreased by half by activation of mitochondrial oxidative phosphorylation. Creatine (20 mM) further activated the respiration in the presence of PK+PEP. It is concluded that in oxidative muscle cells mitochondria behave as if they were incorporated into functional complexes with adjacent ADP producing systems - with the MgATPases in myofibrils and Ca,MgATPases of sarcoplasmic reticulum.

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.

Functional coupling as a basic mechanism of feedback regulation of cardiac energy metabolism

In this review we analyze the concepts and the experimental data on the mechanisms of the regulation of energy metabolism in muscle cells. Muscular energetics is based on the force–length relationship, which in the whole heart is expressed as a Frank–Starling law, by which the alterations of left ventricle diastolic volume change linearly both the cardiac work and oxygen consumption. The second basic characteristics of the heart is the metabolic stability – almost constant levels of high energy phosphates, ATP and phosphocreatine, which are practically independent of the workload and the rate of oxygen consumption, in contrast to the fast-twitch skeletal muscle with no metabolic stability and rapid fatigue. Analysis of the literature shows that an increase in the rate of oxygen consumption by order of magnitude, due to Frank–Starling law, is observed without any significant changes in the intracellular calcium transients. Therefore, parallel activation of contraction and mitochondrial respiration by calcium ions may play only a minor role in regulation of respiration in the cells. The effective regulation of the respiration under the effect of Frank–Starling law and metabolic stability of the heart are explained by the mechanisms of functional coupling within supramolecular complexes in mitochondria, and at the subcellular level within the intracellular energetic units. Such a complex structural and functional organisation of heart energy metabolism can be described quantitatively by mathematical models.

Intracellular diffusion of adenosine phosphates is locally restricted in cardiac muscle

Recent studies have revealed the structural and functional interactions between mitochondria, myofibrils and sarcoplasmic reticulum in cardiac cells. Direct channeling of adenosine phosphates between organelles identified in the experiments indicates that diffusion of adenosine phosphates is limited in cardiac cells due to very specific intracellular structural organization. However, the mode of diffusion restrictions and nature of the intracellular structures in creating the diffusion barriers is still unclear, and, therefore, a subject of active research. The aim of this work is to analyze the possible role of two principally different modes of restriction distribution for adenosine phosphates (a) the uniform diffusion restriction and (b) the localized diffusion limitation in the vicinity of mitochondria, by fitting the experimental data with the mathematical model. The reaction-diffusion model of compartmentalized energy transfer was used to analyze the data obtained from the experiments with the skinned muscle fibers, which described the following processes: mitochondrial respiration rate dependency on exogenous ADP and ATP concentrations; inhibition of endogenous ADP-stimulated respiration by pyruvate kinase (PK) and phosphoenolpyruvate (PEP) system; kinetics of oxygen consumption stabilization after addition of 2 mM MgATP or MgADP; ATPase activity with inhibited mitochondrial respiration; and buildup of MgADP concentration in the medium after addition of MgATP. The analysis revealed that only the second mechanism considered – localization of diffusion restrictions – is able to account for the experimental data. In the case of uniform diffusion restrictions, the model solution was in agreement only with two measurements: the respiration rate as a function of ADP or ATP concentrations and inhibition of respiration by PK + PEP. It was concluded that intracellular diffusion restrictions for adenosine phosphates are not distributed uniformly, but rather are localized in certain compartments of the cardiac cells.

Impaired Regulation of Brain Mitochondria by Extramitochondrial Ca2+ in Transgenic Huntington Disease Rats

Huntington disease (HD) is characterized by polyglutamine expansions of huntingtin (htt), but the underlying pathomechanisms have remained unclear. We studied brain mitochondria of transgenic HD rats with 51 glutamine repeats (htt51Q), modeling the adult form of HD. \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Ca}_{\mathrm{free}}^{2+}\) \end{document} up to 2 μm activated state 3 respiration of wild type mitochondria with glutamate/malate or pyruvate/malate as substrates. \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Ca}_{\mathrm{free}}^{2+}\) \end{document} above 2 μm inhibited respiration via cyclosporin A-dependent permeability transition (PT). Ruthenium red, an inhibitor of the mitochondrial Ca2+ uniporter, did not affect the Ca2+-dependent activation of respiration but reduced Ca2+-induced inhibition. Thus, Ca2+ activation was mediated exclusively by extramitochondrial Ca2+, whereas inhibition was promoted also by intramitochondrial Ca2+. In contrast, htt51Q mitochondria showed a deficient state 3 respiration, a lower sensitivity to Ca2+ activation, and a higher susceptibility to Ca2+-dependent inhibition. Furthermore htt51Q mitochondria exhibited a diminished membrane potential stability in response to Ca2+, lower capacities and rates of Ca2+ accumulation, and a decreased Ca2+ threshold for PT in a substrate-independent but cyclosporin A-sensitive manner. Compared with wild type, Ca2+-induced inhibition of respiration of htt51Q mitochondria was less sensitive to ruthenium red, indicating the involvement of extramitochondrial Ca2+. In conclusion, we demonstrate a novel mechanism of mitochondrial regulation by extramitochondrial Ca2+. We suggest that specific regulatory Ca2+ binding sites on the mitochondrial surface, e.g. the glutamate/aspartate carrier (aralar), mediate this regulation. Interactions between htt51Q and distinct targets such as aralar and/or the PT pore may underlie mitochondrial dysregulation leading to energetic depression, cell death, and tissue atrophy in HD.

Mitochondria and Energetic Depression in Cell Pathophysiology

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.

Lack of dystrophin is associated with altered integration of the mitochondria and ATPases in slow-twitch muscle cells of MDX mice

The potential role of dystrophin-mediated control of systems integrating mitochondria with ATPases was assessed in muscle cells. Mitochondrial distribution and function in skinned cardiac and skeletal muscle fibers from dystrophin-deficient (MDX) and wild-type mice were compared. Laser confocal microscopy revealed disorganized mitochondrial arrays in m. gastrocnemius in MDX mice, whereas the other muscles appeared normal in this group. Irrespective of muscle type, the absence of dystrophin had no effect on the maximal capacity of oxidative phosphorylation, nor on coupling between oxidation and phosphorylation. However, in the myocardium and m. soleus, the coupling of mitochondrial creatine kinase to adenine nucleotide translocase was attenuated as evidenced by the decreased effect of creatine on the Km for ADP in the reactions of oxidative phosphorylation. In m. soleus, a low Km for ADP compared to the wild-type counterpart was found, which implies increased permeability for that nucleotide across the mitochondrial outer membrane. In normal cardiac fibers 35% of the ADP flux generated by ATPases was not accessible to the external pyruvate kinase-phosphoenolpyruvate system, which suggests the compartmentalized (direct) channeling of that fraction of ADP to mitochondria. Compared to control, the direct ADP transfer was increased in MDX ventricles. In conclusion, our data indicate that in slow-twitch muscle cells, the absence of dystrophin is associated with the rearrangement of the intracellular energy and feedback signal transfer systems between mitochondria and ATPases. As the mechanisms mediated by creatine kinases become ineffective, the role of diffusion of adenine nucleotides increases due to the higher permeability of the mitochondrial outer membrane for ADP and enhanced compartmentalization of ADP flux.

De novo SCN8A mutation identified by whole-exome sequencing in a boy with neonatal epileptic encephalopathy, multiple congenital anomalies, and movement disorders.

Epileptic encephalopathies represent a clinically and genetically heterogeneous group of disorders, majority of which are of unknown etiology. We used whole-exome sequencing of a parent-offspring trio to identify the cause of early infantile epileptic encephalopathy in a boy with neonatal seizures, movement disorders, and multiple congenital anomalies who died at the age of 17 months because of respiratory illness and identified a de novo heterozygous missense mutation (c.3979A>G; p.Ile1327Val) in SCN8A (voltage-gated sodium-channel type VIII alpha subunit) gene. The variant was confirmed in the proband with Sanger sequencing. Because the clinical phenotype associated with SCN8A mutations has previously been identified only in a few patients with or without epileptic seizures, these data together with our results suggest that mutations in SCN8A can lead to early infantile epileptic encephalopathy with a broad phenotypic spectrum. Additional investigations will be worthwhile to determine the prevalence and contribution of SCN8A mutations to epileptic encephalopathies.

Sarcoplasmic reticulum function in determining atrioventricular contractile differences in rat heart

The relationships between the contractile characteristics and the sarcoplasmic reticulum (SR) function of rat atrial and ventricular trabeculae were compared. The isometric developed tension (DT) and the rates of contraction (+dT/d t) and relaxation (-dT/d t) normalized to cross-sectional area were 3.7, 2.2, and 1.8 times lower, respectively, in intact atrial strips compared with ventricular strips, whereas +dT/d t and -dT/d t(normalized to DT) were 2.3 and 2.8 times higher, respectively, in atria. Atria exhibited a maximal potentiation of DT after shorter rest periods than ventricles and a lower reversal for prolonged rest periods. Caffeine-induced tension transients in saponin-permeabilized fibers suggested that the Ca2+concentration released in atrial myofibrils reached a lower maximum and decayed more slowly than in ventricular preparations. However, the tension-time integrals indicated an equivalent capacity of sequestrable Ca2+ in SR from both tissues. In atrial, as in ventricular myocardium, the SR Ca2+ uptake was more efficiently supported by ATP produced by the SR-bound MM form of creatine kinase (CK; MM-CK) than by externally added ATP, suggesting a tight functional coupling between the SR Ca2+adenosinetriphosphatase (ATPase) and MM-CK. The maximal rate of oxalate-supported Ca2+ uptake was two times higher in atrial than in ventricular tissue homogenates. The SR Ca2+-ATPase 2a mRNA content normalized to 18S RNA was 38% higher in atria than in ventricles, whereas the amount of mRNA encoding the α-myosin heavy chain, calsequestrin, and the ryanodine receptor was similar in both tissues. Thus a lower amount of readily releasable Ca2+ together with a faster uptake rate may partly account for the shorter time course and lower tension development in intact atrial myocardium compared with ventricular myocardium.The relationships between the contractile characteristics and the sarcoplasmic reticulum (SR) function of rat atrial and ventricular trabeculae were compared. The isometric developed tension (DT) and the rates of contraction (+ dT/dt) and relaxation (-dT/dt) normalized to cross-sectional area were 3.7, 2.2, and 1.8 times lower, respectively, in intact atrial strips compared with ventricular strips, whereas + dT/dt and -dT/dt (normalized to DT) were 2.3 and 2.8 times higher, respectively, in atria. Atria exhibited a maximal potentiation of DT after shorter rest periods than ventricles and a lower reversal for prolonged rest periods. Caffeine-induced tension transients in saponin-permeabilized fibers suggested that the Ca2+ concentration released in atrial myofibrils reached a lower maximum and decayed more slowly than in ventricular preparations. However, the tension-time integrals indicated an equivalent capacity of sequestrable Ca2+ in SR from both tissues. In atrial, as in ventricular myocardium, the SR Ca2+ uptake was more efficiently supported by ATP produced by the SR-bound MM form of creatine kinase (CK; MM-CK) than by externally added ATP, suggesting a tight functional coupling between the SR Ca2+ adenosinetriphosphatase (ATPase) and MM-CK. The maximal rate of oxalate-supported Ca2+ uptake was two times higher in atrial than in ventricular tissue homogenates. The SR Ca(2+)-ATPase 2a mRNA content normalized to 18S RNA was 38% higher in atria than in ventricles, whereas the amount of mRNA encoding the alpha-myosin heavy chain, calsequestrin, and the ryanodine receptor was similar in both tissues. Thus a lower amount of readily releasable Ca2+ together with a faster uptake rate may partly account for the shorter time course and lower tension development in intact atrial myocardium compared with ventricular myocardium.

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.