Structural and functional changes in heart mitochondria from sucrose-fed hypertriglyceridemic rats

In the heart of sugar-induced hypertriglyceridemic (HTG) rats, cardiac performance is impaired with glucose as fuel, but not with fatty acids. Accordingly, the glycolytic flux and the transfer of energy diminish in the HTG heart, in comparison to control heart. To further explore the biochemical nature of such alteration in the HTG heart, the components of the non-glycolytic energy systems involved were evaluated. Total creatine kinase (CK) activity in the myocardial tissue was depressed by 30% in the HTG heart whereas the activity of the mitochondrial CK (mitCK) isoenzyme fraction that is functionally associated with oxidative phosphorylation decreased in isolated HTG heart mitochondria by 45%. Adenylate kinase (AK) was 20% lower in the HTG heart. In contrast, respiratory rates with 2-oxoglutarate (2-OG) and pyruvate/malate (pyr) were significantly higher in HTG heart mitochondria than in control mitochondria. 2-OG dehydrogenase activity was also higher in HTG mitochondria. Respiration with succinate was similar in both groups. Content of cytochromes b, c + c₁ and a + a₃, and cytochrome c oxidase activity, were also similar in the two kinds of mitochondria. A larger content of saturated and monounsaturated fatty acids was found in the HTG mitochondrial membranes with no changes in phospholipids composition or cholesterol content. Mitochondrial membranes from HTG hearts were more rigid, which correlated with the generation of higher membrane potentials. As the mitochondrial function was preserved or even enhanced in the HTG heart, these results indicated that deficiency in energy transfer was associated with impairment in mitCK and AK. This situation brought about uncoupling between the site of ATP production and the site of ATP consumption (contractile machinery), in spite of compensatory increase in mitochondrial oxidative capacity and membrane potential generation.     

Cooperation and Competition between Adenylate Kinase,Nucleoside Diphosphokinase,Electron Transport,and ATP Synthase in Plant Mitochondria Studied by 31P-Nuclear Magnetic Resonance

Nucleotide metabolism in potato (Solanum tuberosum) mitochondria was studied using 31P-nuclear magnetic resonance spectroscopy and the O2 electrode. Immediately following the addition of ADP, ATP synthesis exceeded the rate of oxidative phosphorylation, fueled by succinate oxidation, due to mitochondrial adenylate kinase (AK) activity two to four times the maximum activity of ATP synthase. Only when the AK reaction approached equilibrium was oxidative phosphorylation the primary mechanism for net ATP synthesis. A pool of sequestered ATP in mitochondria enabled AK and ATP synthase to convert AMP to ATP in the presence of exogenous inorganic phosphate. During this conversion, AK activity can indirectly influence rates of oxidation of both succinate and NADH via changes in mitochondrial ATP. Mitochondrial nucleoside diphosphokinase, in cooperation with ATP synthase, was found to facilitate phosphorylation of nucleoside diphosphates other than ADP at rates similar to the maximum rate of oxidative phosphorylation. These results demonstrate that plant mitochondria contain all of the machinery necessary to rapidly regenerate nucleoside triphosphates from AMP and nucleoside diphosphates made during cellular biosynthesis and that AK activity can affect both the amount of ADP available to ATP synthase and the level of ATP regulating electron transport.     

Oxidative phosphorylation and its coupling to mitochondrial creatine and adenylate kinases in human gastric mucosa

Energy metabolism in gastrobiopsy specimens of the antral and corpus mucosa, treated with saponin to permeabilize the cells, was studied in patients with gastric diseases. The results show twice lower oxidative capacity in the antral mucosa than in the corpus mucosa and the relative deficiency of antral mitochondria in complex I. The mucosal cells expressed mitochondrial and cytosolic isoforms of creatine kinase and adenylate kinase (AK). Creatine (20 mM) and AMP (2 mM) markedly stimulated mitochondrial respiration in the presence of submaximal ADP or ATP concentrations, and creatine reduced apparent Km for ADP in stimulation of respiration, which indicates the functional coupling of mitochondrial kinases to oxidative phosphorylation. Addition of exogenous cytochrome c increased ADP-dependent respiration, and the large-scale cytochrome c effect (>or=20%) was associated with suppressed stimulation of respiration by creatine and AMP in the mucosal preparations. These results point to the impaired mitochondrial outer membrane, probably attributed to the pathogenic effects of Helicobacter pylori. Compared with the corpus mucosa, the antral mucosa exhibited greater sensitivity to such type of injury as the prevalence of the large-scale cytochrome c effect was twice higher among the latter specimens. Active chronic gastritis was associated with decreased respiratory capacity of the corpus mucosa but with its increase in the antral mucosa. In conclusion, human gastric mucosal cells express the mitochondrial and cytosolic isoforms of CK and AK participating in intracellular energy transfer systems. Gastric mucosa disease is associated with the altered functions of these systems and oxidative phosphorylation.     

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.     

Analyzing the functional properties of the creatine kinase system with multiscale 'sloppy' modeling

In this study the function of the two isoforms of creatine kinase (CK; EC 2.7.3.2) in myocardium is investigated. The 'phosphocreatine shuttle' hypothesis states that mitochondrial and cytosolic CK plays a pivotal role in the transport of high-energy phosphate (HEP) groups from mitochondria to myofibrils in contracting muscle. Temporal buffering of changes in ATP and ADP is another potential role of CK. With a mathematical model, we analyzed energy transport and damping of high peaks of ATP hydrolysis during the cardiac cycle. The analysis was based on multiscale data measured at the level of isolated enzymes, isolated mitochondria and on dynamic response times of oxidative phosphorylation measured at the whole heart level. Using 'sloppy modeling' ensemble simulations, we derived confidence intervals for predictions of the contributions by phosphocreatine (PCr) and ATP to the transfer of HEP from mitochondria to sites of ATP hydrolysis. Our calculations indicate that only 15±8% (mean±SD) of transcytosolic energy transport is carried by PCr, contradicting the PCr shuttle hypothesis. We also predicted temporal buffering capabilities of the CK isoforms protecting against high peaks of ATP hydrolysis (3750 μM*s(-1)) in myofibrils. CK inhibition by 98% in silico leads to an increase in amplitude of mitochondrial ATP synthesis pulsation from 215±23 to 566±31 μM*s(-1), while amplitudes of oscillations in cytosolic ADP concentration double from 77±11 to 146±1 μM. Our findings indicate that CK acts as a large bandwidth high-capacity temporal energy buffer maintaining cellular ATP homeostasis and reducing oscillations in mitochondrial metabolism. However, the contribution of CK to the transport of high-energy phosphate groups appears limited. Mitochondrial CK activity lowers cytosolic inorganic phosphate levels while cytosolic CK has the opposite effect.     

Nitric oxide inhibits cardiac energy production via inhibition of mitochondrial creatine kinase

Nitric oxide biosynthesis in cardiac muscle leads to a decreased oxygen consumption and lower ATP synthesis. It is suggested that this effect of nitric oxide is mainly due to the inhibition of the mitochondrial respiratory chain enzyme, cytochrome c oxidase. However, this work demonstrates that nitric oxide is able to inhibit soluble mitochondrial creatine kinase (CK), mitochondrial CK bound in purified mitochondria, CK in situ in skinned fibres as well as the functional activity of mitochondrial CK in situ in skinned fibres. Since mitochondrial isoenzyme is functionally coupled to oxidative phosphorylation, its inhibition also leads to decreased sensitivity of mitochondrial respiration to ADP and thus decreases ATP synthesis and oxygen consumption under physiological ADP concentrations.     

Compartmentation of energy metabolism in atrial myocardium of patients undergoing cardiac surgery

The parameters of oxidative phosphorylation and its interaction with creatine kinase (CK)- and adenylate kinase (AK)-phosphotransfer networks in situ were studied in skinned atrial fibers from 59 patients undergoing coronary artery bypass surgery, valve replacement/correction and atrial septal defect correction. In atria, the mitochondrial CK and AK are effectively coupled to oxidative phosphorylation, the MM-CK is coupled to ATPases and there exists a direct transfer of adenine nucleotides between mitochondria and ATPases. Elimination of cytoplasmic ADP with exogenous pyruvate kinase was not associated with a blockade of the stimulatory effects of creatine and AMP on respiration, neither could it abolish the coupling of MM-CK to ATPases and direct transfer of adenine nucleotides. Thus, atrial energy metabolism is compartmentalized so that mitochondria form functional complexes with adjacent ATPases. These complexes isolate a part of cellular adenine nucleotides from their cytoplasmic pool for participating in energy transfer via CK- and AK-networks, and/or direct exchange. Compared to atria in sinus rhythm, the fibrillating atria were larger and exhibited increased succinate-dependent respiration relative to glutamate-dependent respiration and augmented proton leak. Thus, alterations in mitochondrial oxidative phosphorylation may contribute to pathogenesis of atrial fibrillation. (Mol Cell Biochem 270: 49–61, 2005)     

Mitochondrial energy metabolism and redox responses to hypertriglyceridemia

In this work we review recent findings that explain how mitochondrial bioenergetic functions and redox state respond to a hyperlipidemic in vivo environment and may contribute to the maintenance of a normal metabolic phenotype. The experimental model utilized to evidence these adaptive mechanisms is especially useful for these studies since it exhibits genetic hypertriglyceridemia and avoids complications introduced by high fat diets. Liver from hypertrigliceridemic (HTG) mice have a greater content of glycerolipids together with increased mitochondrial free fatty acid oxidation. HTG liver mitochondria have a higher resting respiration rate but normal oxidative phosphorylation efficiency. This is achieved by higher activity of the mitochondrial potassium channel sensitive to ATP (mitoK_ATP). The mild uncoupling mediated by mitoK_ATP accelerates respiration rates and reduces reactive oxygen species generation. Although this response is not sufficient to inhibit lipid induced extra-mitochondrial oxidative stress in whole liver cells it avoids amplification of this redox imbalance. Furthermore, higher mitoK_ATP activity increases liver, brain and whole body metabolic rates. These mitochondrial adaptations may explain why these HTG mice do not develop insulin resistance and obesity even under a severe hyperlipidemic state. On the contrary, when long term high fat diets are employed, insulin resistance, fatty liver and obesity develop and mitochondrial adaptations are inefficient to counteract energy and redox imbalances.     

Simple oxygraphic analysis for the presence of adenylate kinase 1 and 2 in normal and tumor cells

The adenylate kinase (AK) isoforms network plays an important role in the intracellular energy transfer processes, the maintenance of energy homeostasis, and it is a major player in AMP metabolic signaling circuits in some highly-differentiated cells. For this purpose, a rapid and sensitive method was developed that enables to estimate directly and semi-quantitatively the distribution between cytosolic AK1 and mitochondrial AK2 localized in the intermembrane space, both in isolated cells and tissue samples (biopsy material). Experiments were performed on isolated rat mitochondria or permeabilized material, including undifferentiated and differentiated neuroblastoma Neuro-2a cells, HL-1 cells, isolated rat heart cardiomyocytes as well as on human breast cancer postoperative samples. In these samples, the presence of AK1 and AK2 could be detected by high-resolution respirometry due to the functional coupling of these enzymes with ATP synthesis. By eliminating extra-mitochondrial ADP with an excess of pyruvate kinase and its substrate phosphoenolpyruvate, the coupling of the AK reaction with mitochondrial ATP synthesis could be quantified for total AK and mitochondrial AK2 as a specific AK index. In contrast to the creatine kinase pathway, the AK phosphotransfer pathway is up-regulated in murine neuroblastoma and HL-1 sarcoma cells and in these malignant cells expression of AK2 is higher than AK1. Differentiated Neuro-2a neuroblastoma cells exhibited considerably higher OXPHOS capacity than undifferentiated cells, and this was associated with a remarkable decrease in their AK activity. The respirometric method also revealed a considerable difference in mitochondrial affinity for AMP between non-transformed cells and tumor cells.     

CHANGES DURING DEVELOPMENT OF MOUSE BRAIN IN THE ACTIVITIES AND SUBCELLULAR DISTRIBUTIONS OF CREATINE AND ADENYLATE KINASES

Abstract— The activities and electrophoretic patterns of creatine (CK) and adenylate (AK) phosphokinases were determined in the mitochondrial and high speed supernatant fractions of developing and adult mouse brain. The CK activity of the cytosol increases three-fold between 9 and 19 days of age and four-fold by 29 days. Mitochondrial CK activity increases from birth to reach a maximum four-fold that at birth by 19 days of age. The developmental changes in soluble AK activity parallels that of CK. Although the increase in CK activity of the mitochondria exceeds that of the mitochondrial proteins during the period of rapid brain development, mitochondrial AK specific activity changes little. No major changes were found during development in the electrophoretic patterns of proteins with CK or AK activity in the supernatant and mitochondrial fractions. The changes in mitochondrial CK and AK activity parallel the change from a predominantly glycolytic to an aerobic mode of brain metabolism and support the concept that these enzymes play an important role in mitochondrial energy metabolism. Soluble CK and AK activity increase concomitant with neuronal maturation and the associated greatly increased need for rapidly available high energy phosphate.     

Down-regulated energy metabolism genes associated with mitochondria oxidative phosphorylation and fatty acid metabolism in viral cardiomyopathy mouse heart

The majority of experimental and clinical studies indicates that the hypertrophied and failing myocardium are characterized by changes in energy and substrate metabolism that attributed to failing heart changes at the genomic level, in fact, heart failure is caused by various diseases, their energy metabolism and substrate are in different genetic variations, then the potential significance of the molecular mechanisms for the aetiology of heart failure is necessary to be evaluated. Persistent viral infection (especially coxsackievirus group B3) of the myocardium in viral myocarditis and viral dilated cardiomyopathy has never been neglected by experts. This study aimed to explore the role and regulatory mechanism of the altered gene expression for energy metabolism involved in mitochondrial oxidative phosphorylation, fatty acid metabolism in viral dilated cardiomyopathy. cDNA Microarray technology was used to evaluate the expression of >35,852 genes in a mice model of viral dilated cardiomyopathy. In total 1385 highly different genes expression, we analyzed 33 altered genes expression for energy metabolism involved in mitochondrial oxidative phosphorylation, fatty acid metabolism and further selected real-time-PCR for quantity one of regulatory mechanisms for energy including fatty acid metabolism—the UCP2 and assayed cytochrome C oxidase activity by Spectrophotometer to explore mitochondrial oxidative phosphorylation function. We found obviously different expression of 33 energy metabolism genes associated with mitochondria oxidative phosphorylation, fatty acid metabolism in cardiomyopathy mouse heart, the regulatory gene for energy metabolism: UCP2 was down-regulated and cytochrome C oxidase activity was decreased. Genes involved in both fatty acid metabolism and mitochondrial oxidative phosphorylation were down-regulated, mitochondrial uncoupling proteins (UCP2) expression did not increase but decrease which might be a kind of adaptive protection response to regulate energy metabolism for ATP produce.     

Computational model of excitable cell indicates ATP free energy dynamics in response to calcium oscillations are undampened by cytosolic ATP buffers

Mitochondria in excitable cells are recurrently exposed to pulsatile calcium gradients that activate cell function. Rapid calcium uptake by the mitochondria has previously been shown to cause uncoupling of oxidative phosphorylation. To test (i) if periodic nerve firing may cause oscillation of the cytosolic thermodynamic potential of ATP hydrolysis and (ii) if cytosolic adenylate (AK) and creatine kinase (CK) ATP buffering reactions dampen such oscillations, a lumped kinetic model of an excitable cell capturing major aspects of the physiology has been developed. Activation of ATP metabolism by low-frequency calcium pulses caused large oscillation of the cytosolic, but not mitochondrial ATP/ADP, ratio. This outcome was independent of net ATP synthesis or hydrolysis during mitochondrial calcium uptake. The AK/CK ATP buffering reactions dampened the amplitude and rate of cytosolic ATP/ADP changes on a timescale of seconds, but not milliseconds. These model predictions suggest that alternative sources of capacitance in neurons and striated muscles should be considered to protect ATP-free energy-driven cell functions.     

Reduced creatine kinase activity in transgenic amyotrophic lateral sclerosis mice

Creatine (Cr), the substrate of the creatine kinase (CK) isoenzymes, was shown to be neuroprotective in several models of neurodegeneration, including amyotrophic lateral sclerosis (ALS). In order to investigate the mechanism of this beneficial effect, we determined CK activities and mitochondrial respiration rates in tissues from G93A transgenic mice, which overexpress a mutant form of human superoxide dismutase associated with familial ALS (FALS). While respiration rates of mitochondria from G93A transgenic or wild-type control mice isolated from spinal cord showed no difference, a significant and dramatic loss of CK activity could be detected in these tissues. In homogenates from spinal cord of G93A transgenic mice, CK activity decreased to 49% and in mitochondrial fractions to 67% compared to CK activities in wild-type control mice. Feeding the G93A transgenic mice with 2% Cr, the same tissues showed no statistically significant increase of CK activity compared to regular fed G93A transgenic mice. Experiments with isolated mitochondria, however, showed that Cr and adenosine triphosphate (ATP) protected mitochondrial CK activity against peroxynitrite-induced inactivation, which may play a role in tissue damage in neurodegeneration. Our data provide evidence for oxidative damage to the CK system in ALS, which may contribute to impaired energy metabolism and neurodegeneration.     

31P NMR detection of subcellular creatine kinase fluxes in the perfused rat heart: contractility modifies energy transfer pathways

The subcellular fluxes of exchange of ATP and phosphocreatine (PCr) between mitochondria, cytosol, and ATPases were assessed by (31)P NMR spectroscopy to investigate the pathways of energy transfer in a steady state beating heart. Using a combined analysis of four protocols of inversion magnetization transfer associated with biochemical data, three different creatine kinase (CK) activities were resolved in the rat heart perfused in isovolumic control conditions: (i) a cytosolic CK functioning at equilibrium (forward, F(f) = PCr --> ATP, and reverse flux, F(r) = ATP --> PCr = 3.3 mm.s(-1)), (ii) a CK localized in the vicinity of ATPases (MM-CK bound isoform) favoring ATP synthesis (F(f) = 1.7 x F(r)), and (iii) a mitochondrial CK displaced toward PCr synthesis (F(f) = 0.3 and F(r) = 2.6 mm.s(-1)). This study thus provides the first experimental evidence that the energy is carried from mitochondria to ATPases by PCr (i.e. CK shuttle) in the whole heart. In contrast, a single CK functioning at equilibrium was sufficient to describe the data when ATP synthesis was partly inhibited by cyanide (0.15 mm). In this case, ATP was directly transferred from mitochondria to cytosol suggesting that cardiac activity modified energy transfer pathways. Bioenergetic implications of the localization and activity of enzymes within myocardial cells are discussed.     

Adenylate kinase: Kinetic behavior in intact cells indicates it is integral to multiple cellular processes

Monitoring the kinetic behavior of adenylate kinase (AK) and creatine kinase (CK) in intact cells by ¹⁸O-phosphoryl oxygen exchange analysis has provided new perspectives from which to more fully define the involvement of these phosphotransferases in cellular bioenergetics. A primary function attributable to both AK and CK is their apparent capability to couple ATP utilization with its generation by glycolytic and/or oxidative processes depending on cell metabolic status. This is evidenced by the observation that the sum of the net AK- plus CK-catalyzed phosphoryl transfer is equivalent to about 95% of the total ATP metabolic flux in non-contracting rat diaphragm; under basal conditions almost every newly generated ATP molecule appears to be processed by one or the other of these phosphotransferases prior to its utilization. Although CK accounts for the transfer of a majority of the ATP molecules generated/consumed in the basal state there is a progressive, apparently compensatory, shift in phosphotransfer catalysis from the CK to the AK system with increasing muscle contraction or graded chemical inhibition of CK activity. AK and CK appear therefore to provide similar and interrelated functions. Evidence that high energy phosphoryl transfer in some cell types or metabolic states can also be provided by specific nucleoside mono- and diphosphate kinases and by the phosphotransfer capability inherent to the glycolytic system has been obtained. Measurements by ¹⁸O-exchange analyses of net AK- and CK-catalyzed phosphoryl transfer in conjunction with 31P NMR analyses of total unidirectional phosphoryl flux show that each new energy-bearing molecule CK or AK generates subsequently undergoes about 50 or more unidirectional CK-or AK-catalyzed phosphotransfers en route to an ATP consumption site in intact muscle. This evidence of multiple enzyme catalyzed exchanges coincides with the mechanism of vectorial ligand conduction suggested for accomplishing intracellular high energy phosphoryl transfer by the AK and CK systems. AK-catalyzed phosphotransfer also appears to be integral to the transduction of metabolic signals influencing the operation of ion channels regulated by adenine nucleotides such as ATP-inhibitable K⁺ channels in insulin secreting cells; transition from the ATP to ADP liganded states closely coincides with the rate AK-catalyzes phosphotransfer transforming ATP (+AMP) to (2) ADP.     

Biochemical factors limiting myocardial energy in a chicken genotype selected for rapid growth

Broiler chickens (Gallus gallus) genetically selected for rapid growth are inherently predisposed to heart failure. In order to understand the biochemical mechanisms associated with the deterioration of heart function and development of congestive heart failure (CHF) in fast-growing chickens, this study examined several factors critical for myocardial energy metabolism. Measured variables included cardiac energy substrates [creatine phosphate (CrP), adenosine triphosphate (ATP), l-carnitine], activity of selected cytosolic enzymes [creatine kinase (CK; EC 2.7.3.2), lactate dehydrogenase (LDH; EC 1.1.1.27)] and mitochondrial enzymes [pyruvate dehydrogenase (PDH; EC 1.2.4.1), alpha-ketoglutarate dehydrogenase (alpha-KGDH; EC 1.2.4.2)]. The CK activities were higher in fast-growing and CHF broilers as compared to slow-growing broilers (p<0.05). Cardiac LDH and alpha-KGDH activities were not changed (p>0.05), whereas PDH activity was highest (p<0.05) in broilers with CHF. Deterioration of heart function is correlated with lowered cardiac ATP, CrP, and l-carnitine levels (all p<0.05). Depletion of high energy phosphate substrates, ATP and CrP, is evident in fast-growing chickens and those that developed CHF. Increased activity of CK suggests that cardiac energy management in fast-growing broilers and those with CHF largely depends on contribution of this pathway to regeneration of ATP from CrP. In this scenario, inadequate level of CrP is a direct cause of ATP insufficiency, whereas low cardiac l-carnitine, because of its role in fatty acid transport, is most likely an important factor contributing to shortage of key substrate required for synthesis of cardiac ATP. The insufficiencies in cardiac energy substrate synthesis provide metabolic basis of myocardial dysfunction in chickens predisposed to heart failure.     

Oxidative phosphorylation in a hybrid system containing bovine heart membranes and pea mitochondrial F1-ATPase

Purified pea mitochondrial F1-ATPase reconstituted oxidative phosphorylation in both partially and completely F1-depleted bovine heart mitochondrial membranes. The isolated plant enzyme exhibited high rates of ATP synthesis when combined with bovine heart membranes, suggesting great evolutionary conservation of the ATP synthase complex in mitochondria.     

Determination of molar content of creatine kinase in heart mitochondria by SH-reagents

The maximal content of mitochondrial isoenzyme of creatine kinase (CK) in rat heart mitochondria does not exceed 12.5 moles per mole of ATP-ADP translocase. This value was obtained by titration of mitochondrial CK activity in aged mitochondria by 5,5'-dithiobis-(2-nitrobenzoate) (DTNB) and 2,4-dinitrofluorobenzene (DNFB) and by a more complex and accurate method. The essential thiol groups of membrane-bound mitochondrial CK (and its enzymic activity) can be specifically protected by phosphocreatine (12 mM) + ADP (1-5 mM) against inactivation by DTNB. Mitochondria with protected SH-groups of CK and with groups inactivated by DTNB were repeatedly incubated with DTNB under identical conditions and the number of additionally reacted sulfhydryl groups and the changes in CK activity were measured. The differences in the number of additionally reacted SH-groups correlated with the changes in the CK activity, which made it possible to calculate the molar ratios of mitochondrial CK to cytochrome c oxidase and ATP-ADP translocase (2.16 +/- (0.4): 1:2, respectively).     

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.     

Structural and functional adaptations of striated muscles to CK deficiency

In adult mammalian muscle cells, energy consuming processes are mainly localized to the sarcolemma, sarcoplasmic reticulum (SR) and myofibrillar compartments, while energy production occurs within mitochondria or glycolytic complexes. Due to the restricted diffusion of adenine nucleotides near the active sites of ATPases involved in contractile activity and calcium homeostasis, there are multiple local systems that can locally rephosphorylate ADP and provide ATP. The creatine kinase (CK) system, with specific isoenzymes localized within each compartment, efficiently controls local adenylate pools and links energy production and utilization. However, mice lacking one or both of the MM-CK and mi-CK isoforms (CK-/-) are viable and develop almost normal cardiac and skeletal muscle function under the conditions of moderate workload, suggesting adaptations or other mechanisms that may ensure efficient energy transfer. While fixed CK is essentially important, other systems could also be involved as well, such as bound glycolytic enzymes or adenylate kinase. We have shown that, additionally, a direct functional interplay exists between mitochondria and sarcoplasmic reticulum, or between mitochondria and myofilaments in muscle cells, that catalyzes direct energy and signal transfer between organelles. In cardiac cells of CK-/- mice, marked cytoarchitectural modifications were observed, and direct adenine nucleotide channeling between mitochondria and organelles was very effective to rescue SR and myofilament functions. In fast skeletal muscles, increased oxidative capacity also indicates compensatory mechanisms. In mutant mice, mitochondrial capacity increases and a direct energy channeling occurs between mitochondria on one hand and ATP consuming sites on the other. However, these systems appear to be insufficient to fully compensate for the lack of CK at high workload. It can be concluded that local rephosphorylation of ADP is a crucial regulatory point in highly differentiated and organized muscle cells to ensure contractile diversity and efficiency and that the CK system is important to control energy fluxes and energy homeostasis.