Valdur Saks

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.

Water content and its intracellular distribution in intact and saline perfused rat hearts revisited.

Precise estimation of cellular water content is a necessary basis for quantitative studies of metabolic control in the heart; however, marked discrepancies in water spaces of heart tissue are found in the literature. Reasons for this wide diversity are analyzed, and the conclusion is that the most probable value of total intracellular water content is 615 ml H(2)O/kg of wet mass (wm) and intracellular content of dry substance is 189 g/kg wm in intact in vivo rat heart. An extracellular water of 174 ml per kg wm and 22 g of dry mass per kg wm in vascular and interstitium spaces account for the rest of the tissue mass. These values can be directly related to normoosmotic saline perfused hydrated hearts, characterized by water accumulation in the extracellular spaces. Due to essentially intact heart cells, the experimentally determined dry mass, water and metabolite contents of these hydrated hearts can be extrapolated to the original morphological configuration of an intact heart muscle before the onset of edema. Such an extrapolated heart is defined as a standardized perfused heart (SPH). SPH is the heart in its original morphological configuration, characterized by cell density and cellular water contents of the intact heart, but with perfusate in the extracellular spaces. The total cellular water is distributed in the cell compartments of SPH and intact hearts according to volumes of particular compartments and density of their dry mass. The volumes of bulk water phases in different organelles, accessible to diffusion of low molecular metabolites, were obtained after corrections for the fraction of bound water of 0.3 g per g of compartmental dry mass content. The diffusible water spaces are proposed to be 321, 55, 153, 21 and 8 ml/kg wm for myofibrils, sarcoplasm, mitochondria, sarcoplasmic reticulum and nuclei, respectively. The SPH model allows direct comparison of metabolic data for intact and perfused hearts. We used this model to analyze the penetration of extracellular marker into cells of intact and hydrated perfused rat hearts.

Macrocompartmentation of total creatine in cardiomyocytes revisited

Distribution of total creatine (free creatine + phosphocreatine) between two subcellular macrocompartments – mitochondrial matrix space and cytoplasm – in heart and skeletal muscle cells was reinvestigated by using a permeabilized cell technique. Isolated cardiomyocytes were treated with saponin (50 μg/ml for 30 min or 600 μg/ml for 1 min) to open the outer cellular membrane and release the metabolites from cytoplasm (cytoplasmic fraction, CF). All mitochondrial population in permeabilized cells remained intact: the outer membrane was impermeable for exogenous cytochrome c, the acceptor control index of respiration exceeded 10, the mitochondrial creatine kinase reaction was fully coupled to the adenine nucleotide translocator. Metabolites were released from mitochondrial fraction (MF) by 2–5% Triton X100. Total cellular pool of free creatine + phosphocreatine (69.6 ± 2.1 nmoles per mg of protein) was found exclusively in CF and was practically absent in MF. When fibers were prepared from perfused rat hearts, cellular distribution of creatine was not dependent on functional state of the heart and only slightly modified by ischemia. It is concluded that there is no stable pool of creatine or phosphocreatine in the mitochondrial matrix in the intact muscle cells, and the total creatine pool is localized in only one macrocompartment – cytoplasm.

The phosphocreatine–creatine kinase system helps to shape muscle cells and keep them healthy and alive

An article by OConnor et al. (2008) in this issue of The Journal of Physiology significantly expands our knowledge of the role of the phosphocreatine (PCr)–creatine kinase (CK) system in muscle cells. The role of this system in muscle, brain and other tissue with high energy requirements and fluxes has been intensively studied and debated during the last four decades. The main results and current state of art of these studies were recently summarized in a number of chapters of three almost simultaneously published books (Vial, 2006; Wallimann et al. 2007; Dzeja et al. 2007; Saks et al. 2007; Wyss & Salomon, 2007). These studies helped to develop the new paradigm of bioenergetic studies – molecular system bioenergetics, a part of systems biology (Saks et al. 2006, 2007). n nThe important role of the PCr–CK system is based on the metabolic compartmentation of adenine nucleotides and modular organization of energy metabolism – both are system level properties not predictable from properties of isolated components of the cell (Weiss et al. 2006; Saks et al. 2007). Abundant experimental evidence shows the existence of distinct cellular ATP microdomains (compartments) localized in mitochondria and at the sites of ATP utilization. These are connected by phosphotransfer networks, notably via the PCr–creatine kinase pathway (reviewed by Wallimann et al. 2007; Dzeja et al. 2007; Saks et al. 2007; Wyss & Salomon, 2007). These networks function to bypass and overcome local restrictions on ATP or ADP diffusion and thus perform the important tasks of both energy supply and metabolic feedback regulation of respiration (Saks et al. 2006). This explains many classical observations that while all kinds of cellular work is based on the use of ATP, the total content of ATP in cells has no diagnostic value – contractile function of muscle cells and action potential duration decrease in pathological conditions (hypoxia, ischaemia) at an almost constant total level of ATP but follow changes in PCr; and ATP content itself can be changed by 70% by deoxyglucose treatment of perfused aerobic hearts without changes in contractile function if the PCr–CK system is intact and functional (data reviewed by Saks et al. 2007). n nThis general phenomenon – dissociation of total content of ATP from cell function – is again clearly demonstrated by OConnor et al. (2008). The authors show that it is the PCr–CK system which sustains localized ATP-dependent reactions during actin polymerization in myoblast fusion. Myoblasts treated with exogenous creatine showed enhanced intracellular PCr stores without any effect on ATP levels. This increase in PCr induced myoblast fusion and myotube formation during the initial 24 h of myogenesis. Interestingly, during this time CK-BB became localized and after 36 and 48 h was found to be close to the ends of myotubes. Actin polymerization is critical for myoblast fusion and occurs with involvement of ATP during both the addition of actin monomers to the growing ends of filaments and the dissociation of monomeres at the tail (OConnor et al. 2008). It is this localized ATP which is rapidly regenerated by CK-BB at the expense of PCr, and it seems that the formation of these ATP microdomains is a dynamic process during actin cytoskeleton remodelling. OConnor et al. (2008) showed also that local injection of creatine into injured skeletal muscle increased growth of regenerating myofibres from satellite cells via differentiation and fusion of myoblasts. All these results add new insight into the functioning of the PCr–CK system in muscle cells, showing its new role in energy supply for cytoskeletal remodelling. These results may help to better explain the therapeutic effects of creatine supplementation (Wallimann et al. 2007; Brosnan & Brosnan, 2007; Wyss & Salomon, 2007). n nThe vital importance of ATP in functional microdomains (compartments) connected to the PCr–CK system was also recently well documented in clinical studies by Neubauers group in Oxford on a large number of patients with heart disease – dilated cardiomyopathy – involving a follow-up study using nuclear magnetic resonance spectroscopy to record the 31P-NMR spectra in the heart (Neubauer, 2007). While again almost no differences were seen in ATP content in the healthy control and cardiomyopathic hearts, the mortality rate of patient during the follow-up period correlated with a decrease in the PCr/ATP ratio below 1.6 (Neubauer, 2007). Low PCr/ATP ratios mean decreased regeneration of ATP by the PCr–CK system in microdomains (compartments) which are critically important for function of the heart. These microdomains are localized in myofibrils, near the sarcolemma and the membrane of sarcoplasmic reticulum (Wallimann et al. 2007; Saks et al. 2006, 2007). n nThere is now a general consensus among the researchers in muscle and brain energy metabolism that the next challenge and most urgent need is to develop better bioprobes to image metabolic microdomains of ATP, and functional proteomics to identify physical interactions between key proteins responsible for their formation (Weiss et al. 2006; Neubauer, 2007; Wallimann et al. 2007; Saks et al. 2006, 2007). The article by OConnor et al. (2008) published in this issue of The Journal of Physiology emphasizes further the importance of this new perspective for research in cellular physiology and bioenergetics.

Comparative investigation of bioenergetic properties of human colorectal and breast cancer

Today, there is only very limited information of the regulation of energy metabolism in neuroblastoma (NB) cells, although this is a prerequisite condition for development of more efficient treatment protocols. It is believed that NBs have increased rates of aerobic glycolysis and display a Warburg phenotype. The aim of the present studywas to determine if there are specific alterations of aerobic energy metabolism in NBs, or if there is an overall down regulation of oxidative phosphorylation (OXPHOS) complexes. To clarify the mechanisms of regulation of mitochondrial respiration in NBs, we determined the apparent Km value for exogenously-added ADP. We found that the Km for ADP in non-differentiated and differentiated (normal) Neuro-2a (N2A) cells have very similar values, 20.3±1.4 μM and 19.4±3.2 μM, respectively, and the maximal (in the presence of 2 mM ADP) rate of O2 consumption by differentiated N2A cells exceeds considerably (N2 times) that measured for non-differentiated cells. Our findings suggest that NB cells have, in comparison with normal differentiated neural cells, a decreased activity of OXPHOS. Metabolic control analysis (MCA) performed onN2A cells suggest that in NBs the key sites of the regulation of OXPHOS are Complex-I (Flux control coefficient, FCC=1.11), Complex-II (FCC=0.99) and IV (FCC=0.92), since FCC(s) for other mitochondrial complexes were found to be substantially lower and they have approximately equal values. In the mitochondria of differentiated N2A cells the key sites of respiratory regulation were found to be Complex-II (FCC=1.34) and IV (FCC=0.99).Moreover, our data suggest that in differentiatedNB cells the Complex-II activitymay exceed considerably that inmalignant cells. It ismost interesting that inN2Acells (like for example in breast cancer cells in situ) the sum of FCC(s) for ADP activated respiration exceeds significantly 1 normally observed in oxidative tissues and isolated mitochondria and is close to 4. This indicates the altered structure of the mitochondrial respiratory chain in N2A cells. Indeed, our results suggest that the mitochondrial respiratory chain and OXPHOS system contain large respiratory supercomplexes with direct substrate transfer inside these complexes.