Failing atrial myocardium: energetic deficits accompany structural remodeling and electrical instability

The failing ventricular myocardium is characterized by reduction of high-energy phosphates and reduced activity of the phosphotransfer enzymes creatine kinase (CK) and adenylate kinase (AK), which are responsible for transfer of high-energy phosphoryls from sites of production to sites of utilization, thereby compromising excitation-contraction coupling. In humans with chronic atrial fibrillation (AF) unassociated with congestive heart failure (CHF), impairment of atrial myofibrillar energetics linked to oxidative modification of myofibrillar CK has been observed. However, the bioenergetic status of the failing atrial myocardium and its potential contribution to atrial electrical instability in CHF have not been determined. Dogs with (n = 6) and without (n = 6) rapid pacing-induced CHF underwent echocardiography (conscious) and electrophysiological (under anesthesia) studies. CHF dogs had more pronounced mitral regurgitation, higher atrial pressure, larger atrial area, and increased atrial fibrosis. An enhanced propensity to sustain AF was observed in CHF, despite significant increases in atrial effective refractory period and wavelength. Profound deficits in atrial bioenergetics were present with reduced activities of the phosphotransfer enzymes CK and AK, depletion of high-energy phosphates (ATP and creatine phosphate), and reduction of cellular energetic potential (ATP-to-ADP and creatine phosphate-to-Cr ratios). AF duration correlated with left atrial area (r = 0.73, P = 0.01) and inversely with atrial ATP concentration (r = -0.75, P = 0.005), CK activity (r = -0.57, P = 0.054), and AK activity (r = -0.64, P = 0.02). Atrial levels of malondialdehyde, a marker of oxidative stress, were significantly increased in CHF. Myocardial bioenergetic deficits are a conserved feature of dysfunctional atrial and ventricular myocardium in CHF and may constitute a component of the substrate for AF in CHF.     

Defective metabolic signaling in adenylate kinase AK1 gene knock-out hearts compromises post-ischemic coronary reflow

Matching blood flow to myocardial energy demand is vital for heart performance and recovery following ischemia. The molecular mechanisms responsible for transduction of myocardial energetic signals into reactive vasodilatation are, however, elusive. Adenylate kinase, associated with AMP signaling, is a sensitive reporter of the cellular energy state, yet the contribution of this phosphotransfer system in coupling myocardial metabolism with coronary flow has not been explored. Here, knock out of the major adenylate kinase isoform, AK1, disrupted the synchrony between inorganic phosphate P(i) turnover at ATP-consuming sites and gamma-ATP exchange at ATP synthesis sites, as revealed by (18)O-assisted (31)P NMR. This reduced energetic signal communication in the post-ischemic heart. AK1 gene deletion blunted vascular adenylate kinase phosphotransfer, compromised the contractility-coronary flow relationship, and precipitated inadequate coronary reflow following ischemia-reperfusion. Deficit in adenylate kinase activity abrogated AMP signal generation and reduced the vascular adenylate kinase/creatine kinase activity ratio essential for the response of metabolic sensors. The sarcolemma-associated splice variant AK1beta facilitated adenosine production, a function lost in the absence of adenylate kinase activity. Adenosine treatment bypassed AK1 deficiency and restored post-ischemic flow to wild-type levels, achieving phenotype rescue. AK1 phosphotransfer thus transduces stress signals into adequate vascular response, providing linkage between cell bioenergetics and coronary flow.     

Mapping hypoxia-induced bioenergetic rearrangements and metabolic signaling by 18O-assisted 31P NMR and 1H NMR spectroscopy

Brief hypoxia or ischemia perturbs energy metabolism inducing paradoxically a stress-tolerant state, yet metabolic signals that trigger cytoprotection remain poorly understood. To evaluate bioenergetic rearrangements, control and hypoxic hearts were analyzed with 18O-assisted 31P NMR and 1H NMR spectroscopy. The 18O-induced isotope shift in the 31P NMR spectrum of CrP, betaADP and betaATP was used to quantify phosphotransfer fluxes through creatine kinase and adenylate kinase. This analysis was supplemented with determination of energetically relevant metabolites in the phosphomonoester (PME) region of 31P NMR spectra, and in both aromatic and aliphatic regions of 1H NMR spectra. In control conditions, creatine kinase was the major phosphotransfer pathway processing high-energy phosphoryls between sites of ATP consumption and ATP production. In hypoxia, creatine kinase flux was dramatically reduced with a compensatory increase in adenylate kinase flux, which supported heart energetics by regenerating and transferring beta- and gamma-phosphoryls of ATP. Activation of adenylate kinase led to a build-up of AMP, IMP and adenosine, molecules involved in cardioprotective signaling. 31P and 1H NMR spectral analysis further revealed NADH and H+ scavenging by alpha-glycerophosphate dehydrogenase (alphaGPDH) and lactate dehydrogenase contributing to maintained glycolysis under hypoxia. Hypoxia-induced accumulation of alpha-glycerophosphate and nucleoside 5'-monophosphates, through alphaGPDH and adenylate kinase reactions, respectively, was mapped within the increased PME signal in the 31P NMR spectrum. Thus, 18O-assisted 31P NMR combined with 1H NMR provide a powerful approach in capturing rearrangements in cardiac bioenergetics, and associated metabolic signaling that underlie the cardiac adaptive response to stress.     

Adenylate kinase AK1 knockout heart: energetics and functional performance under ischemia-reperfusion

Deletion of the major adenylate kinase AK1 isoform, which catalyzes adenine nucleotide exchange, disrupts cellular energetic economy and compromises metabolic signal transduction. However, the consequences of deleting the AK1 gene on cardiac energetic dynamics and performance in the setting of ischemia-reperfusion have not been determined. Here, at the onset of ischemia, AK1 knockout mice hearts displayed accelerated loss of contractile force compared with wild-type controls, indicating reduced tolerance to ischemic stress. On reperfusion, AK1 knockout hearts demonstrated reduced nucleotide salvage, resulting in lower ATP, GTP, ADP, and GDP levels and an altered metabolic steady state associated with diminished ATP-to-P(i) and creatine phosphate-to-P(i) ratios. Postischemic AK1 knockout hearts maintained approximately 40% of beta-phosphoryl turnover, suggesting increased phosphotransfer flux through remaining adenylate kinase isoforms. This was associated with sustained creatine kinase flux and elevated cellular glucose-6-phosphate levels as the cellular energetic system adapted to deletion of AK1. Such metabolic rearrangements, along with sustained ATP-to-ADP ratio and total ATP turnover rate, maintained postischemic contractile recovery of AK1 knockout hearts at wild-type levels. Thus deletion of the AK1 gene reveals that adenylate kinase phosphotransfer supports myocardial function on initiation of ischemic stress and safeguards intracellular nucleotide pools in postischemic recovery.     

Cellular energetics in the preconditioned state: protective role for phosphotransfer reactions captured by 18O-assisted 31P NMR

Cell survival is critically dependent on the preservation of cellular bioenergetics. However, the metabolic mechanisms that confer resistance to injury are poorly understood. Phosphotransfer reactions integrate ATP-consuming with ATP-producing processes and could thereby contribute to the generation of a protective phenotype. Here, we used ischemic preconditioning to induce a stress-tolerant state and (18)O-assisted (31)P nuclear magnetic resonance spectroscopy to capture intracellular phosphotransfer dynamics. Preconditioning of isolated perfused hearts triggered a redistribution in phosphotransfer flux with significant increase in creatine kinase and glycolytic rates. High energy phosphoryl fluxes through creatine kinase, adenylate kinase, and glycolysis in preconditioned hearts correlated tightly with post-ischemic functional recovery. This was associated with enhanced metabolite exchange between subcellular compartments, manifested by augmented transfer of inorganic phosphate from cellular ATPases to mitochondrial ATP synthase. Preconditioning-induced energetic remodeling protected cellular ATP synthesis and ATP consumption, improving contractile performance following ischemia-reperfusion insult. Thus, the plasticity of phosphotransfer networks contributes to the effective functioning of the cellular energetic system, providing a mechanism for increased tolerance toward injury.     

Nucleotide-gated KATP channels integrated with creatine and adenylate kinases: Amplification,tuning and sensing of energetic signals in the compartmentalized cellular environment

Transmission of energetic signals to membrane sensors, such as the ATP-sensitive K+ (KATP) channel, is vital for cellular adaptation to stress. Yet, cell compartmentation implies diffusional hindrances that hamper direct reception of cytosolic energetic signals. With high intracellular ATP levels, KATP channels may sense not bulk cytosolic, but rather local submembrane nucleotide concentrations set by membrane ATPases and phosphotransfer enzymes. Here, we analyzed the role of adenylate kinase and creatine kinase phosphotransfer reactions in energetic signal transmission over the strong diffusional barrier in the submembrane compartment, and translation of such signals into a nucleotide response detectable by KATP channels. Facilitated diffusion provided by creatine kinase and adenylate kinase phosphotransfer dissipated nucleotide gradients imposed by membrane ATPases, and shunted diffusional restrictions. Energetic signals, simulated as deviation of bulk ATP from its basal level, were amplified into an augmented nucleotide response in the submembrane space due to failure under stress of creatine kinase to facilitate nucleotide diffusion. Tuning of creatine kinase-dependent amplification of the nucleotide response was provided by adenylate kinase capable of adjusting the ATP/ADP ratio in the submembrane compartment securing adequate KATP channel response in accord with cellular metabolic demand. Thus, complementation between creatine kinase and adenylate kinase systems, here predicted by modeling and further supported experimentally, provides a mechanistic basis for metabolic sensor function governed by alterations in intracellular phosphotransfer fluxes.     

Phosphotransfer dynamics in skeletal muscle from creatine kinase gene-deleted mice

To assess the significance of energy supply routes in cellular energetic homeostasis, net phosphoryl fluxes catalyzed by creatine kinase (CK), adenylate kinase (AK) and glycolytic enzymes were quantified using 18O-phosphoryl labeling. Diaphragm muscle from double M-CK/ScCKmit knockout mice exhibited virtually no CK-catalyzed phosphotransfer. Deletion of the cytosolic M-CK reduced CK-catalyzed phosphotransfer by 20%, while the absence of the mitochondrial ScCKmit isoform did not affect creatine phosphate metabolic flux. Contribution of the AK-catalyzed phosphotransfer to total cellular ATP turnover was 15.0, 17.2, 20.2 and 28.0% in wild type, ScCKmit, M-CK and M-CK/ScCKmit deficient muscles, respectively. Glycolytic phosphotransfer, assessed by G-6-P 18O-phosphoryl labeling, was elevated by 32 and 65% in M-CK and M-CK/ScCKmit deficient muscles, respectively. Inhibition of glyceraldehyde 3-phosphate dehydrogenase (GAPDH)/phosphoglycerate kinase (PGK) in CK deficient muscles abolished inorganic phosphate compartmentation and redirected high-energy phosphoryl flux through the AK network. Under such conditions, AK phosphotransfer rate was equal to 86% of the total cellular ATP turnover concomitant with almost normal muscle performance. This indicates that near-equilibrium glycolytic phosphotransfer reactions catalyzed by the GAPDH/PGK support a significant portion of the high-energy phosphoryl transfer in CK deficient muscles. However, CK deficient muscles displayed aberrant ATPase-ATPsynthase communication along with lower energetic efficiency (P/O ratio), and were more sensitive to metabolic stress induced by chemical hypoxia. Thus, redistribution of phosphotransfer through glycolytic and AK networks contributes to energetic homeostasis in muscles under genetic and metabolic stress complementing loss of CK function.     

Developmental enhancement of adenylate kinase-AMPK metabolic signaling axis supports stem cell cardiac differentiation

Background

Energetic and metabolic circuits that orchestrate cell differentiation are largely unknown. Adenylate kinase (AK) and associated AMP-activated protein kinase (AMPK) constitute a major metabolic signaling axis, yet the role of this system in guiding differentiation and lineage specification remains undefined.

Methods and Results

Cardiac stem cell differentiation is the earliest event in organogenesis, and a suitable model of developmental bioenergetics. Molecular profiling of embryonic stem cells during cardiogenesis revealed here a distinct expression pattern of adenylate kinase and AMPK genes that encode the AK-AMP-AMPK metabolic surveillance axis. Cardiac differentiation upregulated cytosolic AK1 isoform, doubled AMP-generating adenylate kinase activity, and increased AMP/ATP ratio. At cell cycle initiation, AK1 translocated into the nucleus and associated with centromeres during energy-consuming metaphase. Concomitantly, the cardiac AMP-signal receptor AMPKα2 was upregulated and redistributed to the nuclear compartment as signaling-competent phosphorylated p-AMPKα(Thr172). The cardiogenic growth factor TGF-β promoted AK1 expression, while knockdown of AK1, AK2 and AK5 activities with siRNA or suppression by hyperglycemia disrupted cardiogenesis compromising mitochondrial and myofibrillar network formation and contractile performance. Induction of creatine kinase, the alternate phosphotransfer pathway, compensated for adenylate kinase-dependent energetic deficits.

Conclusions

Developmental deployment and upregulation of the adenylate kinase/AMPK tandem provides a nucleocytosolic energetic and metabolic signaling vector integral to execution of stem cell cardiac differentiation. Targeted redistribution of the adenylate kinase-AMPK circuit associated with cell cycle and asymmetric cell division uncovers a regulator for cardiogenesis and heart tissue regeneration.     

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.     

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.     

Decline of Phosphotransfer and Substrate Supply Metabolic Circuits Hinders ATP Cycling in Aging Myocardium

Integration of mitochondria with cytosolic ATP-consuming/ATP-sensing and substrate supply processes is critical for muscle bioenergetics and electrical activity. Whether age-dependent muscle weakness and increased electrical instability depends on perturbations in cellular energetic circuits is unknown. To define energetic remodeling of aged atrial myocardium we tracked dynamics of ATP synthesis-utilization, substrate supply, and phosphotransfer circuits through adenylate kinase (AK), creatine kinase (CK), and glycolytic/glycogenolytic pathways using ¹⁸O stable isotope-based phosphometabolomic technology. Samples of intact atrial myocardium from adult and aged rats were subjected to ¹⁸O-labeling procedure at resting basal state, and analyzed using the ¹⁸O-assisted HPLC-GC/MS technique. Characteristics for aging atria were lower inorganic phosphate Pi[¹⁸O], γ-ATP[¹⁸O], β-ADP[¹⁸O], and creatine phosphate CrP[¹⁸O] ¹⁸O-labeling rates indicating diminished ATP utilization-synthesis and AK and CK phosphotransfer fluxes. Shift in dynamics of glycolytic phosphotransfer was reflected in the diminished G6P[¹⁸O] turnover with relatively constant glycogenolytic flux or G1P[¹⁸O] ¹⁸O-labeling. Labeling of G3P[¹⁸O], an indicator of G3P-shuttle activity and substrate supply to mitochondria, was depressed in aged myocardium. Aged atrial myocardium displayed reduced incorporation of ¹⁸O into second (¹⁸O₂), third (¹⁸O₃), and fourth (¹⁸O₄) positions of Pi[¹⁸O] and a lower Pi[¹⁸O]/γ-ATP[¹⁸ O]-labeling ratio, indicating delayed energetic communication and ATP cycling between mitochondria and cellular ATPases. Adrenergic stress alleviated diminished CK flux, AK catalyzed β-ATP turnover and energetic communication in aging atria. Thus, ¹⁸O-assisted phosphometabolomics uncovered simultaneous phosphotransfer through AK, CK, and glycolytic pathways and G3P substrate shuttle deficits hindering energetic communication and ATP cycling, which may underlie energetic vulnerability of aging atrial myocardium.     

Relating structure to mechanism in creatine kinase

Found in all vertebrates, creatine kinase catalyzes the reversible reaction of creatine and ATP forming phosphocreatine and ADP. Phosphocreatine may be viewed as a reservoir of "high-energy phosphate" which is able to supply ATP, the primary energy source in bioenergetics, on demand. Consequently, creatine kinase plays a significant role in energy homeostasis of cells with intermittently high energy requirements. The enzyme is of clinical importance and its levels are routinely used as an indicator of myocardial and skeletal muscle disorders and for the diagnosis of acute myocardial infarction. First identified in 1928, the enzyme has undergone intensive investigation for over 75 years. There are four major isozymes, two cytosolic and two mitochondrial, which form dimers and octamers, respectively. Depending on the pH, the enzyme operates by a random or an ordered bimolecular mechanism, with the equilibrium lying towards phosphocreatine production. Evidence suggests that conversion of creatine to phosphocreatine occurs via the in-line transfer of a phosphoryl group from ATP. A recent X-ray structure of creatine kinase bound to a transition state analog complex confirmed many of the predictions based on kinetic, spectroscopic, and mutagenesis studies. This review summarizes and correlates the more significant mechanistic and structural studies on creatine kinase.     

Compromised energetics in the adenylate kinase AK1 gene knockout heart under metabolic stress

Rapid exchange of high energy carrying molecules between intracellular compartments is essential in sustaining cellular energetic homeostasis. Adenylate kinase (AK)-catalyzed transfer of adenine nucleotide beta- and gamma-phosphoryls has been implicated in intracellular energy communication and nucleotide metabolism. To demonstrate the significance of this reaction in cardiac energetics, phosphotransfer dynamics were determined by [(18)O]phosphoryl oxygen analysis using( 31)P NMR and mass spectrometry. In hearts with a null mutation of the AK1 gene, which encodes the major AK isoform, total AK activity and beta-phosphoryl transfer was reduced by 94% and 36%, respectively. This was associated with up-regulation of phosphoryl flux through remaining minor AK isoforms and the glycolytic phosphotransfer enzyme, 3-phosphoglycerate kinase. In the absence of metabolic stress, deletion of AK1 did not translate into gross abnormalities in nucleotide levels, gamma-ATP turnover rate or creatine kinase-catalyzed phosphotransfer. However, under hypoxia AK1-deficient hearts, compared with the wild type, had a blunted AK-catalyzed phosphotransfer response, lowered intracellular ATP levels, increased P(i)/ATP ratio, and suppressed generation of adenosine. Thus, although lack of AK1 phosphotransfer can be compensated in the absence of metabolic challenge, under hypoxia AK1-knockout hearts display compromised energetics and impaired cardioprotective signaling. This study, therefore, provides first direct evidence that AK1 is essential in maintaining myocardial energetic homeostasis, in particular under metabolic stress.     

Protection of tubular epithelial cells during renal injury via post-transcriptional control of BMP7

The role of mitochondria in alterations that take place in the muscle cell during healthy aging is a matter of debate during recent years. Most of the studies in bioenergetics have a focus on the model of isolated mitochondria, while changes in the crosstalk between working myofibrils and mitochondria in senescent cardiomyocytes have been less studied. The aim of our research was to investigate the modifications in the highly regulated ATP production and energy transfer systems in heart cells in old rat cardiomyocytes. The results of our work demonstrated alterations in the diffusion restrictions of energy metabolites, manifested by changes in the apparent Michaelis–Menten constant of mitochondria to exogenous ADP. The creatine kinase (CK) phosphotransfer pathway efficiency declines significantly in senescence. The ability of creatine to stimulate OXPHOS as well as to increase the affinity of mitochondria for ADP is falling and the most critical decline is already in the 1-year group (middle-age model in rats). Also, a moderate decrease in the adenylate kinase phosphotransfer system was detected. The importance of glycolysis increases in senescence, while the hexokinase activity does not change during healthy aging. The main result of our study is that the decline in the heart muscle performance is not caused by the changes in the respiratory chain complexes activity but mainly by the decrease in the energy transfer efficiency, especially by the CK pathway.     

Impaired intracellular energetic communication in muscles from creatine kinase and adenylate kinase (M-CK/AK1) double knock-out mice

Previously we demonstrated that efficient coupling between cellular sites of ATP production and ATP utilization, required for optimal muscle performance, is mainly mediated by the combined activities of creatine kinase (CK)- and adenylate kinase (AK)-catalyzed phosphotransfer reactions. Herein, we show that simultaneous disruption of the genes for the cytosolic M-CK- and AK1 isoenzymes compromises intracellular energetic communication and severely reduces the cellular capability to maintain total ATP turnover under muscle functional load. M-CK/AK1 (MAK=/=) mutant skeletal muscle displayed aberrant ATP/ADP, ADP/AMP and ATP/GTP ratios, reduced intracellular phosphotransfer communication, and increased ATP supply capacity as assessed by 18O labeling of [Pi] and [ATP]. An analysis of actomyosin complexes in vitro demonstrated that one of the consequences of M-CK and AK1 deficiency is hampered phosphoryl delivery to the actomyosin ATPase, resulting in a loss of contractile performance. These results suggest that MAK=/= muscles are energetically less efficient than wild-type muscles, but an apparent compensatory redistribution of high-energy phosphoryl flux through glycolytic and guanylate phosphotransfer pathways limited the overall energetic deficit. Thus, this study suggests a coordinated network of complementary enzymatic pathways that serve in the maintenance of energetic homeostasis and physiological efficiency.     

Functional changes in the mitochondrial site of adenylate kinase and creatine kinase systems of energy transport induced by myocardial ischemia and adriablastin

Under effects of myocardial ischemia (30 min), the activities of the intermembrane enzymes of rabbit heart mitochondria, i.e., adenylate kinase and creatine kinase, are inhibited by 20% and 23%, respectively. Consequently, the creatine- and AMP-activated respiration of mitochondria diminishes by 52% and 39%, respectively. An inhibitory analysis of ADP-, AMP- and creatine-activated mitochondrial respiration performed in the presence of atractyloside has demonstrated that ischemia (30 min), adriblastin (0.688 mM) and succinate (10 mM) cause alterations in the functional coupling of adenylate kinase and creatine kinase with the adenine nucleotide translocator. These alterations lead to the diminution of the rate and efficiency of energy transfer from mitochondria to hexokinase, as an arbitrary site of energy consumption. An addition of cytochrome c to ischemic heart mitochondria results in an increase in the rate of ATP synthesis; however, the efficiency of this process is lowered. The toxic effect of the anticancer drug--adriblastin on heart mitochondria respiration is enhanced in the presence of creatine in the bathing solution.     

Coupling of cell energetics with membrane metabolic sensing. Integrative signaling through creatine kinase phosphotransfer disrupted by M-CK gene knock-out

Transduction of metabolic signals is essential in preserving cellular homeostasis. Yet, principles governing integration and synchronization of membrane metabolic sensors with cell metabolism remain elusive. Here, analysis of cellular nucleotide fluxes and nucleotide-dependent gating of the ATP-sensitive K+ (K(ATP)) channel, a prototypic metabolic sensor, revealed a diffusional barrier within the submembrane space, preventing direct reception of cytosolic signals. Creatine kinase phosphotransfer, captured by 18O-assisted 31P NMR, coordinated tightly with ATP turnover, reflecting the cellular energetic status. The dynamics of high energy phosphoryl transfer through the creatine kinase relay permitted a high fidelity transmission of energetic signals into the submembrane compartment synchronizing K(ATP) channel activity with cell metabolism. Knock-out of the creatine kinase M-CK gene disrupted signal delivery to K(ATP) channels and generated a cellular phenotype with increased electrical vulnerability. Thus, in the compartmentalized cell environment, phosphotransfer systems shunt diffusional barriers and secure regimented signal transduction integrating metabolic sensors with the cellular energetic network.     

Oxidative Myocytes of Heart and Skeletal Muscle Express Abundant Sarcomeric Mitochondrial Creatine Kinase

Sarcomeric mitochondrial creatine kinase catalyzes the reversible transfer of a high energy phosphate between ATP and creatine. To study cellular distribution of the kinase, we performed immunocytochemical studies using a peptide antiserum specific for the kinase protein. Our results demonstrated that the sarcomeric mitochondrial creatine kinase gene is abundantly expressed in heart and skeletal muscle, with no protein detected in other tissues examined, including brain, lung, liver, spleen, kidney, bladder, testis, stomach, intestine, and colon. RNA blot study showed that there is no detectable expression of the kinase mRNA in the thymus gland. In heart and skeletal muscle, the kinase protein is expressed in atrial and ventricular cardiomyocytes and a subpopulation of skeletal myofibres. In skeletal muscle, fast myosin heavy chain co-localization studies demonstrated that the sarcomeric mitochondrial creatine kinase is highly expressed in type 1, slow-oxidative and type 2A, fast-oxidative-glycolytic myofibres. We conclude that the kinase gene is abundantly expressed in oxidative myocytes of heart and skeletal muscle and may contribute to oxidative capacity of these cells.