Unchanged mitochondrial organization and compartmentation of high-energy phosphates in creatine-deficient GAMT-/- mouse hearts.

Disruption of the creatine kinase (CK) system in hearts of CK-deficient mice leads to changes in the ultrastructure and regulation of mitochondrial respiration. We expected to see similar changes in creatine-deficient mice, which lack the enzyme guanidinoacetate methyltransferase (GAMT) to produce creatine. The aim of this study was to characterize the changes in cardiomyocyte mitochondrial organization, regulation of respiration, and intracellular compartmentation associated with GAMT deficiency. Three-dimensional mitochondrial organization was assessed by confocal microscopy. On populations of permeabilized cardiomyocytes, we recorded ADP and ATP kinetics of respiration, competition between mitochondria and pyruvate kinase for ADP produced by ATPases, ADP kinetics of endogenous pyruvate kinase, and ATP kinetics of ATPases. These data were analyzed by mathematical models to estimate intracellular compartmentation. Quantitative analysis of morphological and kinetic data as well as derived model fits showed no difference between GAMT-deficient and wild-type mice. We conclude that inactivation of the CK system by GAMT deficiency does not alter mitochondrial organization and intracellular compartmentation in relaxed cardiomyocytes. Thus, our results suggest that the healthy heart is able to preserve cardiac function at a basal level in the absence of CK-facilitated energy transfer without compromising intracellular organization and the regulation of mitochondrial energy homeostasis. This raises questions on the importance of the CK system as a spatial energy buffer in unstressed cardiomyocytes.

Intracellular diffusion restrictions in isolated cardiomyocytes from rainbow trout.

BACKGROUND: Restriction of intracellular diffusion of adenine nucleotides has been studied intensively on adult rat cardiomyocytes. However, their cause and role in vivo is still uncertain. Intracellular membrane structures have been suggested to play a role. We therefore chose to study cardiomyocytes from rainbow trout (Oncorhynchus mykiss), which are thinner and have fewer intracellular membrane structures than adult rat cardiomyocytes. Previous studies suggest that trout permeabilized cardiac fibers also have diffusion restrictions. However, results from fibers may be affected by incomplete separation of the cells. This is avoided when studying permeabilized, isolated cardiomyocytes. The aim of this study was to verify the existence of diffusion restrictions in trout cardiomyocytes by comparing ADP-kinetics of mitochondrial respiration in permeabilized fibers, permeabilized cardiomyocytes and isolated mitochondria from rainbow trout heart. Experiments were performed at 10, 15 and 20 degrees C in the absence and presence of creatine. RESULTS: Trout cardiomyocytes hypercontracted in the solutions used for mammalian cardiomyocytes. We developed a new solution in which they retained their shape and showed stable steady state respiration rates throughout an experiment. The apparent ADP-affinity of permeabilized cardiomyocytes was different from that of fibers. It was higher, independent of temperature and not increased by creatine. However, it was still about ten times lower than in isolated mitochondria. CONCLUSIONS: The differences between fibers and cardiomyocytes suggest that results from trout heart fibers were affected by incomplete separation of the cells. However, the lower ADP-affinity of cardiomyocytes compared to isolated mitochondria indicate that intracellular diffusion restrictions are still present in trout cardiomyocytes despite their lower density of intracellular membrane structures. The lack of a creatine effect indicates that trout heart lacks mitochondrial creatine kinase tightly coupled to respiration. This argues against diffusion restriction by the outer mitochondrial membrane. These results from rainbow trout cardiomyocytes resemble those from other low-performance hearts such as neonatal rat and rabbit hearts. Thus, it seems that metabolic regulation is related to cardiac performance, and it is likely that rainbow trout can be used as a model animal for further studies of the localization and role of diffusion restrictions in low-performance hearts.

Tight coupling of Na+/K+-ATPase with glycolysis demonstrated in permeabilized rat cardiomyocytes.

The effective integrated organization of processes in cardiac cells is achieved, in part, by the functional compartmentation of energy transfer processes. Earlier, using permeabilized cardiomyocytes, we demonstrated the existence of tight coupling between some of cardiomyocyte ATPases and glycolysis in rat. In this work, we studied contribution of two membrane ATPases and whether they are coupled to glycolysis--sarcoplasmic reticulum Ca2+ ATPase (SERCA) and plasmalemma Na+/K+-ATPase (NKA). While SERCA activity was minor in this preparation in the absence of calcium, major role of NKA was revealed accounting to ∼30% of the total ATPase activity which demonstrates that permeabilized cell preparation can be used to study this pump. To elucidate the contribution of NKA in the pool of ATPases, a series of kinetic measurements was performed in cells where NKA had been inhibited by 2 mM ouabain. In these cells, we recorded: ADP- and ATP-kinetics of respiration, competition for ADP between mitochondria and pyruvate kinase (PK), ADP-kinetics of endogenous PK, and ATP-kinetics of total ATPases. The experimental data was analyzed using a series of mathematical models with varying compartmentation levels. The results show that NKA is tightly coupled to glycolysis with undetectable flux of ATP between mitochondria and NKA. Such tight coupling of NKA to PK is in line with its increased importance in the pathological states of the heart when the substrate preference shifts to glucose.

ADP protects cardiac mitochondria under severe oxidative stress.

ADP is not only a key substrate for ATP generation, but also a potent inhibitor of mitochondrial permeability transition pore (mPTP). In this study, we assessed how oxidative stress affects the potency of ADP as an mPTP inhibitor and whether its reduction of reactive oxygen species (ROS) production might be involved. We determined quantitatively the effects of ADP on mitochondrial Ca(2+) retention capacity (CRC) until the induction of mPTP in normal and stressed isolated cardiac mitochondria. We used two models of chronic oxidative stress (old and diabetic mice) and two models of acute oxidative stress (ischemia reperfusion (IR) and tert-butyl hydroperoxide (t-BH)). In control mitochondria, the CRC was 344 ± 32 nmol/mg protein. 500 µmol/L ADP increased CRC to 774 ± 65 nmol/mg protein. This effect of ADP seemed to relate to its concentration as 50 µmol/L had a significantly smaller effect. Also, oligomycin, which inhibits the conversion of ADP to ATP by F0F1ATPase, significantly increased the effect of 50 µmol/L ADP. Chronic oxidative stress did not affect CRC or the effect of 500 µmol/L ADP. After IR or t-BH exposure, CRC was drastically reduced to 1 ± 0.2 and 32 ± 4 nmol/mg protein, respectively. Surprisingly, ADP increased the CRC to 447 ± 105 and 514 ± 103 nmol/mg protein in IR and t-BH, respectively. Thus, it increased CRC by the same amount as in control. In control mitochondria, ADP decreased both substrate and Ca(2+)-induced increase of ROS. However, in t-BH mitochondria the effect of ADP on ROS was relatively small. We conclude that ADP potently restores CRC capacity in severely stressed mitochondria. This effect is most likely not related to a reduction in ROS production. As the effect of ADP relates to its concentration, increased ADP as occurs in the pathophysiological situation may protect mitochondrial integrity and function.

Cox17, a copper chaperone for cytochrome c oxidase: expression, purification, and formation of mixed disulphide adducts with thiol reagents.

Copper chaperone for cytochrome c oxidase (Cox17) is a 7 kDa copper-binding protein, which facilitates incorporation of copper ions into Cu(A) site of cytochrome c oxidase. Cox17 contains six conserved Cys residues and occurs in three different oxidative states, which display different metal-binding properties and stability. In the present study, we have elaborated technologies for production of partially oxidized human recombinant Cox17 in a bacterial expression system and purification of fully oxidized Cox17. For this purpose we used Escherichia coli Origami strain, which is deficient in thioredoxin and thioredoxin reductase systems and allows formation of disulfide bonds in cytoplasmic proteins. Fully oxidized Cox17 was purified by a simplified two-step procedure including gel filtration and cation exchange chromatography. By using mass spectrometry we demonstrated that application of 2-mercaptoethanol (2-ME) during purification leads to formation of its mixed disulfide adducts with Cox17. Moreover, partially reduced Cox17 can form mixed disulfide adducts also with the cellular reducing agent glutathione, which abolishes copper-binding ability of partially reduced Cox17.