Dzeja, Petras P.

Energy metabolism and metabolic signaling circuits orchestrate cell commitment to self-renewal, lineage specification, differentiation and regeneration. When energy resources are plenty, cell can grow, proliferate and regenerate, however when energy is low - augmented adenylate kinase (AK)-mediated AMP signaling turns on AMPK which silences p53/p21/cyclin cell cycle metabolic checkpoint and halts cell division. Using mouse neonatal hearts, with high and low regenerative capacity, we have determined metabolomic profiles and dynamics of phosphotransfer circuits using 18O-phosphoryl labeling mass-spectrometric and 18O-assisted 31P NMR techniques. We demonstrate that loss of heart regenerative capacity after birth is associated with marked changes in heart AK-catalyzed phosphotransfer flux and AMP signaling along with changes in expression levels of p21, cyclin A and E and thymidine kinase. It appears, that in adult heart increased expression of AK isoforms (AK1, AK2 and AK1β) and augmented high energy phosphoryl and AMP signal dynamics is misread by AMPK-sensor as "low energy" state inducing blockade of cell cycle metabolic checkpoint and cardiomyocyte proliferation and renewal. Using AK-GFP constructs and immunocytochemistry we further demonstrate the distribution of AK1, AK1β, AK2 and AMPK between cytosol and nucleus and association with mitotic spindle and cytokinetic apparatus during cell division cycle. AK1 translocation to the nucleus, however, doesn’t occur in adult cardiomyocytes deficient in cytokinesis. Protein knockdown using siRNA indicates that AK2 is critical for cardiomyocyte mitochondrial biogenesis and network formation. Furthermore, we have discovered that deficiency of the AK2 isoform, which is localized in mitochondria intermembrane-intra-cristae space, arrests developmental programming and is embryonically lethal in mice. The uncovered shift in metabolic signaling mechanisms opens new avenues for targeted regulation of heart regenerative potential critical for repair of injured hearts.

Metabolic signaling mechanisms of tissue regeneration is still an enigma. Energy state and metabolite signals regulate cell commitment to self-renewal, lineage specification, differentiation and regeneration. In a favorable metabolic environment, cells can grow and proliferate, however when energy is low - metabolic monitoring system sends signals to cell cycle checkpoint to halt cell division and preserve fuel resources. We demonstrate that loss of heart regenerative capacity after birth in mice is associated with marked changes in metabolome, AK-catalyzed phosphotransfer flux (β-ATP[ 18 O]), ATP turnover (γ-ATP[ 18 O]) and AMP-AMPK signaling along with changes in expression levels of p21, cyclins A and E, pGSK3β and thymidine kinase. Marked reduction of thymidine phosphorylation capacity prevents DNA synthesis and cell proliferation. It emerges, that in adult heart augmented ATP turnover and AMP signal dynamics is misread by AMPK-sensor as "low energy" state inducing blockade of cell cycle metabolic checkpoint and cardiomyocyte proliferation and regeneration after injury. This occurs through augmented adenylate kinase (AK)-mediated AMP signaling which turns on AMPK consequently silencing p53/p21/cyclin cell cycle checkpoint. Changes in expression levels AK1, AK1β, AK2 and AK5 isoforms occur with arrest of heart regeneration. Protein knockdown using siRNA and CRISPR/Cas9 approach indicates that AK2 is critical for cardiomyocyte mitochondrial biogenesis and network formation. Furthermore, we have discovered that deficiency of the AK2 isoform, which is localized in mitochondria intermembrane-intra-cristae space, arrests developmental programming and is embryonically lethal in mice. The uncovered shift in metabolic signaling mechanisms opens new avenues for targeted regulation of heart regenerative potential critical for repair of injured hearts.

The mammalian heart loses its regenerative potential within a week after birth. Although under certain circumstances adult cardiomyocytes can re-enter the cell cycle, initiate DNA synthesis and progress towards mitosis, however, they do not complete a conventional mitosis cycle and proceed towards endomitosis resulting in polyploidy. This suggests that high-energy dependent steps in cell cycle and myofibrillar disassembly are malfunctioning in adult cardiomyocytes. It is known that nucleotide-based energetic signaling and metabolic environment are critical for initiation of tissue regeneration after injury. The regulatory mechanisms and energetic-metabolic signaling circuits that govern cardiomyocyte cell cycle withdrawal and binucleation are poorly understood. Using mouse neonatal (2 and 10 days) hearts, with high and low regenerative capacity, respectively, we have determined metabolomic profiles and dynamics of phosphotransfer circuits and AMP-signaling using 18O-phosphoryl labeling mass-spectrometric and 18O-assisted 31P NMR techniques. The results demonstrate that loss of heart regenerative capacity 10 days after birth is associated with marked changes in heart metabolome and adenylate kinase (AK)-catalyzed phosphotransfer flux. Among most altered metabolites were putrescine and myo-inositol, involved in tissue regeneration, Krebs cycle and amino acid metabolites, creatinine, glucose-6-phosphate and lactate indicating rearrangements in energy and substrate metabolic systems. AK flux was the only phosphotransfer flux significantly upregulated in hearts at 10 days of age along with increased in mitochondrial and glycolytic capacities and mitochondrial AK2 expression. Increased AK2 expression was associated with augmented AMP phosphorylation, measured by generation of β-ATP[18O], and reduction of AMPK activity. Thus, increased channeling of AMP into mitochondria and reduction of AK-AMP-AMPK signaling axis could contribute to the withdrawal from completion of cell cycle in terminally differentiated adult cardiomyocytes. The uncovered metabolic signaling mechanism opens new avenues for targeted regulation of heart regenerative potential critical for repair of injured hearts.

ATP‐sensitive K + (K ATP ) channels are nucleotide‐gated channels that couple the metabolic status of a cell with membrane excitability and regulate a number of cellular functions, including hormone secretion and cardioprotection. Although intracellular ATP is the endogenous inhibitor of K ATP channels and ADP serves as the channel activator, it is still a matter of debate whether changes in the intracellular concentrations of ATP, ADP, and/or in the ATP/ADP ratio could account for the transition from the ATP‐liganded to the ADP‐liganded channel state. Here, we overview evidence for the role of cellular phosphotransfer cascades in the regulation of K ATP channels. The microenvironment of the K ATP channel harbors several phosphotransfer enzymes, including adenylate, creatine, and pyruvate kinases, as well as other glycolytic enzymes that are able to transfer phosphoryls between ATP and ADP in the absence of major changes in cytosolic levels of adenine nucleotides. These phosphotransfer reactions are governed by the metabolic status of a cell, and their phosphotransfer rate closely correlates with K ATP channel activity. Adenylate kinase catalysis accelerates the transition from ATP to ADP, leading to K ATP channel opening, while phosphotransfers driven by creatine and pyruvate kinases promote ADP to ATP transition and channel closure. Thus, through delivery and removal of adenine nucleotides at the channel site, phosphotransfer reactions could regulate ATP/ADP balance in the immediate vicinity of the channel and thereby the probability of K ATP channel opening. In this way, phosphotransfer reactions could provide a transduction mechanism coupling cellular metabolic signals with K ATP channel‐associated functions.

Discovered in the cardiac sarcolemma, ATP-sensitive K+(KATP) channels have more recently also been identified within the inner mitochondrial membrane. Yet the consequences of mitochondrial KATPchannel activation on mitochondrial function remain partially documented. Therefore, we isolated mitochondria from rat hearts and used K+channel openers to examine the effect of mitochondrial KATPchannel opening on mitochondrial membrane potential, respiration, ATP generation, Ca2+transport, and matrix volume. From a mitochondrial membrane potential of −180 ± 15 mV, K+channel openers, pinacidil (100 μM), cromakalim (25 μM), and levcromakalim (20 μM), induced membrane depolarization by 10 ± 7, 25 ± 9, and 24 ± 10 mV, respectively. This effect was abolished by removal of extramitochondrial K+or application of a KATPchannel blocker. K+channel opener-induced membrane depolarization was associated with an increase in the rate of mitochondrial respiration and a decrease in the rate of mitochondrial ATP synthesis. Furthermore, treatment with a K+channel opener released Ca2+from mitochondria preloaded with Ca2+, an effect also dependent on extramitochondrial K+concentration and sensitive to KATPchannel blockade. In addition, K+channel openers, cromakalim and pinacidil, increased matrix volume and released mitochondrial proteins, cytochrome cand adenylate kinase. Thus, in isolated cardiac mitochondria, KATPchannel openers depolarized the membrane, accelerated respiration, slowed ATP production, released accumulated Ca2+, produced swelling, and stimulated efflux of intermembrane proteins. These observations provide direct evidence for a role of mitochondrial KATPchannels in regulating functions vital for the cardiac mitochondria.

Heart failure (HF) is associated with changes in cardiac substrate utilization and energy metabolism, including a decline in high‐energy phosphate content, mitochondrial dysfunction, and phosphotransfer enzyme deficiency. A shift toward glucose metabolism was noted in the end stage of HF in animals, although HF in humans may not be associated with a shift toward predominant glucose utilization. Deficiencies of micronutrients are well‐established causes of cardiomyopathy. Correction of these deficits can improve heart function. The genes governing the energy metabolism were predominantly underexpressed in nonischemic cardiomyopathy and hypertrophic cardiomyopathy but were overexpressed in ischemic cardiomyopathy. Cardiac resynchronization therapy (CRT) has been proven to increase cardiac efficiency without increasing myocardial oxygen consumption. Altered myocardial metabolism is normalized by CRT to improve ventricular function.

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 by18O-assisted31P-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+(KATP) channels. In WT vs. Kir6.2-KO hearts, preconditioning induced a significantly higher total ATP turnover (232 ± 20 vs. 155 ± 15 nmol · mg protein−1· min−1), ATP synthesis rate (58 ± 3 vs. 46 ± 3%18O labeling of γ-ATP), and ATP consumption rate (51 ± 4 vs. 31 ± 4%18O labeling of Pi) after ischemia-reperfusion. Moreover, preconditioning preserved cardiac creatine kinase-catalyzed phosphotransfer in WT (234 ± 26 nmol · mg protein−1· min−1) but not Kir6.2-KO (133 ± 18 nmol · mg protein−1· 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 KATP-channel-deficient myocardium. Thus intact KATPchannels are integral in ischemic preconditioning-induced protection of cellular energetic dynamics and associated cardiac performance.

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.