Citations

Modulation of mitochondrial respiratory chain, dehydrogenase, and nucleotide-metabolizing enzyme activities is fundamental to cellular protection. Here, we demonstrate that the potassium channel opener diazoxide, within its cardioprotective concentration range, modulated the activity of flavin adenine dinucleotide-dependent succinate dehydrogenase with an IC50of 32 μM and reduced the rate of succinate-supported generation of reactive oxygen species (ROS) in heart mitochondria. 5-Hydroxydecanoic fatty acid circumvented diazoxide-inhibited succinate dehydrogenase-driven electron flow, indicating a metabolism-dependent supply of redox equivalents to the respiratory chain. In perfused rat hearts, diazoxide diminished the generation of malondialdehyde, a marker of oxidative stress, which, however, increased on diazoxide washout. This effect of diazoxide mimicked ischemic preconditioning and was associated with reduced oxidative damage on ischemia-reperfusion. Diazoxide reduced cellular and mitochondrial ATPase activities, along with nucleotide degradation, contributing to preservation of myocardial ATP levels during ischemia. Thus, by targeting nucleotide-requiring enzymes, particularly mitochondrial succinate dehydrogenase and cellular ATPases, diazoxide reduces ROS generation and nucleotide degradation, resulting in preservation of myocardial energetics under stress.

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-Piand creatine phosphate-to-Piratios. Postischemic AK1 knockout hearts maintained ∼40% of β-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.

K+ channel openers have been recently recognized for their ability to protect mitochondria from anoxic injury. Yet the mechanism responsible for mitochondrial preservation under oxidative stress is not fully understood. Here, mitochondria were isolated from rat hearts and subjected to 20-min anoxia, followed by reoxygenation. At reoxygenation, increased generation of reactive oxygen species (ROS) was associated with reduced ADP-stimulated oxygen consumption, blunted ATP production, and disrupted mitochondrial structural integrity coupled with cytochrome c release. The prototype K+ channel opener diazoxide markedly reduced mitochondrial ROS production at reoxygenation with a half-maximal effect of 29 μM. Diazoxide also preserved oxidative phosphorylation and mitochondrial membrane integrity, as indicated by electron microscopy and reduced cytochrome c release. The protective effect of diazoxide was reproduced by the structurally distinct K+ channel opener nicorandil and antagonized by 5-hydroxydecanoic acid, a short-chain fatty acid derivative and presumed blocker of mitochondrial ATP-sensitive K+ channels. Opener-mediated mitochondrial protection was simulated by the free radical scavenger system composed of superoxide dismutase and catalase. However, the effect of openers on ROS production was maintained in nominally K+-free medium in the presence or absence of the K+ ionophore valinomycin and was mimicked by malonate, a modulator of the mitochondrial redox state. This suggests the existence of a K+ conductance-independent pathway for mitochondrial protection targeted by K+ channel openers. Thus the cardioprotecive mechanism of K+ channel openers includes direct attenuation of mitochondrial oxidant stress at reoxygenation.

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.

AbstractTo meet high cellular demands, the energy metabolism of cardiac muscles is organized by precise and coordinated functioning of intracellular energetic units (ICEUs). ICEUs represent structural and functional modules integrating multiple fluxes at sites of ATP generation in mitochondria and ATP utilization by myofibrillar, sarcoplasmic reticulum and sarcolemma ion‐pump ATPases. The role of ICEUs is to enhance the efficiency of vectorial intracellular energy transfer and fine tuning of oxidative ATP synthesis maintaining stable metabolite levels to adjust to intracellular energy needs through the dynamic system of compartmentalized phosphoryl transfer networks. One of the key elements in regulation of energy flux distribution and feedback communication is the selective permeability of mitochondrial outer membrane (MOM) which represents a bottleneck in adenine nucleotide and other energy metabolite transfer and microcompartmentalization. Based on the experimental and theoretical (mathematical modelling) arguments, we describe regulation of mitochondrial ATP synthesis within ICEUs allowing heart workload to be linearly correlated with oxygen consumption ensuring conditions of metabolic stability, signal communication and synchronization. Particular attention was paid to the structure–function relationship in the development of ICEU, and the role of mitochondria interaction with cytoskeletal proteins, like tubulin, in the regulation of MOM permeability in response to energy metabolic signals providing regulation of mitochondrial respiration. Emphasis was given to the importance of creatine metabolism for the cardiac energy homoeostasis.

Abstract —Communication between the cytoplasm and nucleoplasm of cardiac cells occurs by molecular transport through nuclear pores. In lower eukaryotes, nuclear transport requires the maintenance of cellular energetics and ion homeostasis. Although heart muscle is particularly sensitive to metabolic stress, the regulation of nuclear transport through nuclear pores in cardiomyocytes has not yet been characterized. With the use of laser confocal and atomic force microscopy, we observed nuclear transport in cardiomyocytes and the structure of individual nuclear pores under different cellular conditions. In response to the depletion of Ca 2+ stores or ATP/GTP pools, the cardiac nuclear pore complex adopted 2 distinct conformations that led to different patterns of nuclear import regulation. Depletion of Ca 2+ indiscriminately prevented the nuclear import of macromolecules through closure of the nuclear pore opening. Depletion of ATP/GTP only blocked facilitated transport through a simultaneous closure of the pore and relaxation of the entire complex, which allowed other molecules to pass into the nucleus through peripheral routes. The current study of the structural plasticity of the cardiac nuclear pore complex, which was observed in response to changes in cellular conditions, identifies a gating mechanism for molecular translocation across the nuclear envelope of cardiac cells. The cardiac nuclear pore complex serves as a conduit that differentially regulates nuclear transport of macromolecules and provides a mechanism for the control of nucleocytoplasmic communication in cardiac cells, in particular under stress conditions associated with disturbances in cellular bioenergetics and Ca 2+ homeostasis.

Abstract —Although the downregulation of creatine kinase activity has been associated with heart failure, creatine kinase–deficient transgenic hearts have a preserved contractile function. This suggests the existence of alternative phosphotransfer pathways in the myocardium, the identity of which is still unknown. In this study, we examined the contribution of adenylate kinase–catalyzed phosphotransfer to myocardial energetics. In the isolated mitochondria/actomyosin system, which possesses endogenous adenylate kinase activity in both compartments, substrates for adenylate kinase promoted the rate and amplitude of actomyosin contraction that was further enhanced by purified adenylate kinase. Inhibition of adenylate kinase activity diminished both actomyosin contraction and mitochondrial respiration, which indicated reduced energy flow between mitochondria and myofibrils. In intact myocardium, the net adenylate kinase–catalyzed phosphotransfer rate was 10% of the total ATP turnover rate as measured by 18 O-phosphoryl labeling in conjunction with gas chromatography and mass spectrometry. In pacing-induced failing heart, adenylate kinase–catalyzed phosphotransfer increased by 134% and contributed 21% to the total ATP turnover. Concomitantly, the contribution by creatine kinase dropped from 89% in normal hearts to 40% in failing hearts. These phosphotransfer changes were associated with reduced levels of metabolically active ATP but maintained overall ATP turnover rate. Thus, this study provides evidence that adenylate kinase facilitates the transfer of high-energy phosphoryls and signal communication between mitochondria and actomyosin in cardiac muscle, with an increased contribution to cellular phosphotransfer in heart failure. This phosphotransfer function renders adenylate kinase an important component for optimal myocardial bioenergetics and a compensatory mechanism in response to impaired intracellular energy flux in the failing heart.

Next-generation screening of disease-related metabolomic phenotypes requires monitoring of both metabolite levels and turnover rates. Stable isotope18O-assisted31P nuclear magnetic resonance (NMR) and mass spectrometry uniquely allows simultaneous measurement of phosphometabolite levels and turnover rates in tissue and blood samples. The18O labeling procedure is based on the incorporation of one18O into Pifrom [18O]H2O with each act of ATP hydrolysis and the distribution of18O-labeled phosphoryls among phosphate-carrying molecules. This enables simultaneous recording of ATP synthesis and utilization, phosphotransfer fluxes through adenylate kinase, creatine kinase, and glycolytic pathways, as well as mitochondrial substrate shuttle, urea and Krebs cycle activity, glycogen turnover, and intracellular energetic communication. Application of expanded18O-labeling procedures has revealed significant differences in the dynamics of G-6-P[18O] (glycolysis), G-3-P[18O] (substrate shuttle), and G-1-P[18O] (glycogenolysis) between human and rat atrial myocardium. In human atria, the turnover of G-3-P[18O], which defects are associated with the sudden death syndrome, was significantly higher indicating a greater importance of substrate shuttling to mitochondria. Phosphometabolomic profiling of transgenic hearts deficient in adenylate kinase (AK1−/−), which altered levels and mutations are associated to human diseases, revealed a stress-induced shift in metabolomic profile with increased CrP[18O] and decreased G-1-P[18O] metabolic dynamics. The metabolomic profile of creatine kinase M-CK/ScCKmit−/−-deficient hearts is characterized by a higher G-6-[18O]P turnover rate, G-6-P levels, glycolytic capacity, γ/β-phosphoryl of GTP[18O] turnover, as well as β-[18O]ATP and β-[18O]ADP turnover, indicating altered glycolytic, guanine nucleotide, and adenylate kinase metabolic flux. Thus,18O-assisted gas chromatography-mass spectrometry and31P NMR provide a suitable platform for dynamic phosphometabolomic profiling of the cellular energetic system enabling prediction and diagnosis of metabolic diseases states.