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.     

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.     

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.     

Adenylate kinase I does not affect cellular growth characteristics under normal and metabolic stress conditions

Adenylate kinase (AK)-catalyzed phosphotransfer is essential in the maintenance of cellular energetic economy in cells of fully differentiated tissues with highly variable energy demand, such as muscle and brain. To investigate if AK isoenzymes have a comparable function in the energy-demand management of proliferating cells, AK1 and AK1beta were expressed in mouse neuroblastoma N2a cells and in human colon carcinoma SW480 cells. Glucose deprivation, galactose feeding, and metabolic inhibitor tests revealed a differential energy dependency for these two cell lines. N2a cells showed a faster proliferation rate and strongest coupling to mitochondrial activity, SW480 proliferation was more dependent on glycolysis. Despite these differences, ectopic expression of AK1 or AK1beta did not affect their growth characteristics under normal conditions. Also, no differential effects were seen under metabolic stress upon treatment with mitochondrial and glycolytic inhibitors in in vitro culture or in solid tumors grown in vivo. Although many intimate connections have been revealed between cell death and metabolism, our results suggest that AK1- or AK1beta-mediated high-energy phosphoryl transfer is not a modulating factor in the survival of tumor cells during episodes of metabolic crisis.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

1H, 13C, 15N backbone and side-chain resonance assignments of the human adenylate kinase 1 in apo form

AK1 (Adenylate Kinase 1) plays crucial roles in processes such as cellular phosphotransfer networks, neuronal maturation and regeneration, gating of ABC transporter CFTR, tumor cell metabolism and myocardial energetic homeostasis. Here we report ¹H, ¹⁵N and ¹³C backbone and side-chain resonance assignments of the human AK1 protein in apo form. This work lays the essential basis for the further structure determination of hAK1.     

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.     

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.     

ATP-sensitive potassium channels: metabolic sensing and cardioprotection.

The cardiovascular system operates under a wide scale of demands, ranging from conditions of rest to extreme stress. How the heart muscle matches rates of ATP production with utilization is an area of active investigation. ATP-sensitive potassium (K(ATP)) channels serve a critical role in the orchestration of myocardial energetic well-being. K(ATP) channel heteromultimers consist of inwardly-rectifying K(+) channel 6.2 and ATP-binding cassette sulfonylurea receptor 2A that translates local ATP/ADP levels, set by ATPases and phosphotransfer reactions, to the channel pore function. In cells in which the mobility of metabolites between intracellular microdomains is limited, coupling of phosphotransfer pathways with K(ATP) channels permits a high-fidelity transduction of nucleotide fluxes into changes in membrane excitability, matching energy demands with metabolic resources. This K(ATP) channel-dependent optimization of cardiac action potential duration preserves cellular energy balance at varying workloads. Mutations of K(ATP) channels result in disruption of the nucleotide signaling network and generate a stress-vulnerable phenotype with excessive susceptibility to injury, development of cardiomyopathy, and arrhythmia. Solving the mechanisms underlying the integration of K(ATP) channels into the cellular energy network will advance the understanding of endogenous cardioprotection and the development of strategies for the management of cardiovascular injury and disease progression.     

Increase of Adenylate Kinase Isozyme 1 Protein During Neuronal Differentiation in Mouse Embryonal Carcinoma P19 Cells and in Rat Brain Primary Cultured Cells

Abstract: Adenylate kinase (AK), which catalyzes the equilibrium reaction among AMP, ADP, and ATP, is considered to participate in the homeostasis of energy metabolism in cells. Among three vertebrate isozymes, AK isozyme 1 (AK1) is present prominently in the cytosol of skeletal muscle and brain. When mouse embryonal carcinoma P19 cells were differentiated by retinoic acid into neural cells, the amount of AK1 protein and enzyme activity increased about fivefold concomitantly with neurofilament (NF). Double-immunofluorescence staining showed that both AK1 and NF were located in neuronal processes as well as the perinuclear regions in neuron-like cells, but not in glia-like cells. The amount of brain-type creatine kinase increased only twofold during P19 differentiation. The AK isozyme 2, which was not detected in adult mouse brain, was found in P19 cells and did not increase during the differentiation. Mitochondrial AK isozyme 3, which uses GTP instead of ATP as a phosphate donor, was increased significantly. Immunohistochemical analysis with the primary cultured cells from rat cerebral cortex showed similar cellular localization of AK1 to those observed with differentiated P19 cells. These results suggest an important role of this enzyme in neuronal functions and in neuronal differentiation.     

Knockout of Kir6.2 negates ischemic preconditioning-induced protection of myocardial energetics

Although ischemic preconditioning induces bioenergetic tolerance and thereby remodels energy metabolism that is crucial for postischemic recovery of the heart, the molecular components associated with preservation of cellular energy production, transfer, and utilization are not fully understood. Here myocardial bioenergetic dynamics were assessed by (18)O-assisted (31)P-NMR spectroscopy in control or preconditioned hearts from wild-type (WT) or Kir6.2-knockout (Kir6.2-KO) mice that lack metabolism-sensing sarcolemmal ATP-sensitive K(+) (K(ATP)) channels. In WT vs. Kir6.2-KO hearts, preconditioning induced a significantly higher total ATP turnover (232 +/- 20 vs. 155 +/- 15 nmol x mg protein(-1) x min(-1)), ATP synthesis rate (58 +/- 3 vs. 46 +/- 3% (18)O labeling of gamma-ATP), and ATP consumption rate (51 +/- 4 vs. 31 +/- 4% (18)O labeling of P(i)) after ischemia-reperfusion. Moreover, preconditioning preserved cardiac creatine kinase-catalyzed phosphotransfer in WT (234 +/- 26 nmol x mg protein(-1) x min(-1)) but not Kir6.2-KO (133 +/- 18 nmol x mg protein(-1) x min(-1)) hearts. In contrast with WT hearts, preconditioning failed to preserve contractile recovery in Kir6.2-KO hearts, as tight coupling between postischemic performance and high-energy phosphoryl transfer was compromised in the K(ATP)-channel-deficient myocardium. Thus intact K(ATP) channels are integral in ischemic preconditioning-induced protection of cellular energetic dynamics and associated cardiac performance.     

Muscle mechanical work requirements during normal walking: the energetic cost of raising the body's center-of-mass is significant

Inverted pendulum models of walking predict that little muscle work is required for the exchange of body potential and kinetic energy in single-limb support. External power during walking (product of the measured ground reaction force and body center-of-mass (COM) velocity) is often analyzed to deduce net work output or mechanical energetic cost by muscles. Based on external power analyses and inverted pendulum theory, it has been suggested that a primary mechanical energetic cost may be associated with the mechanical work required to redirect the COM motion at the step-to-step transition. However, these models do not capture the multi-muscle, multi-segmental properties of walking, co-excitation of muscles to coordinate segmental energetic flow, and simultaneous production of positive and negative muscle work. In this study, a muscle-actuated forward dynamic simulation of walking was used to assess whether: (1). potential and kinetic energy of the body are exchanged with little muscle work; (2). external mechanical power can estimate the mechanical energetic cost for muscles; and (3.) the net work output and the mechanical energetic cost for muscles occurs mostly in double support. We found that the net work output by muscles cannot be estimated from external power and was the highest when the COM moved upward in early single-limb support even though kinetic and potential energy were exchanged, and muscle mechanical (and most likely metabolic) energetic cost is dominated not only by the need to redirect the COM in double support but also by the need to raise the COM in single support.     

31P-NMR observation of free ADP during fatiguing, repetitive contractions of murine skeletal muscle lacking AK1

Metabolic control within skeletal muscle is designed to limit ADP accumulation even during conditions where ATP demand is out of balance with ATP synthesis. This is accomplished by the reactions of adenylate kinase (AK; ADP+ADP AMP+ATP) and AMP deaminase (AMP+H₂O NH₃+IMP), which limit ADP accumulation under these conditions. The purpose of this study was to determine whether AK deficiency (AK^–/–) would result in sufficient ADP accumulation to be visible using ³¹P-NMRS during the high energy demands of frequent in situ tetanic contractions. To do this we examined the high-energy phosphates of the gastrocnemius muscle in the knockout mouse with AK1^–/– and wild-type (WT) control muscle over the course of 64 rapid (2/s) isometric tetanic contractions. Near-complete depletion of phosphocreatine was apparent after 16 contractions in both groups. By 40 contractions, ADP was clearly visible in AK1^–/– muscle. This transient concentration of the NMR visible free ADP was estimated to be 1.7 mM, and represents the first time free ADP has been directly measured in contracting skeletal muscle. Such an increase in free ADP is severalfold greater than previously thought to occur. This large accumulation of free ADP also represents a significant reduction in energy available from ATP, and has implications on cellular processes that depend on a high yield of energy from ATP such as calcium sequestration. Remarkably, the AK1^–/– and WT muscles exhibited similar fatigue profiles. Our findings suggest that skeletal muscle is surprisingly tolerant to a large increase in ADP and by extension, a decline in energy from ATP. muscle energetics; muscle relaxation; magnetic resonance spectroscopy