High-energy Phosphate Levels Research Articles

Comparison of "Huaxi-1" or "histidine-tryptophan-ketoglutarate" cardioplegia in an animal model.

Using a pig model of cardiopulmonary bypass, we compared outcomes after cardioplegia either with our in-house "Huaxi-1" solution containing natural blood and crystalloid or with the entirely crystalloid, commercially available "histidine-tryptophan-ketoglutarate" solution. Cardiopulmonary bypass was established in 12 healthy male pigs, who were randomized to receive a single dose of either Huaxi-1 or entirely crystalloid. All animals were then subjected to whole-heart ischemia for 90 min, followed by 2 h of reperfusion, after which myocardial injury was assessed in terms of cardiac function, myocardial pathology and levels of biomarkers in plasma, while levels of high-energy phosphate in myocardium were assayed using liquid chromatography. Animals given Huaxi-1 cardioplegia required significantly less time to be weaned off bypass, they received significantly lower doses of norepinephrine, and they showed significantly higher levels (mean ± SD) of adenosine triphosphate (14 ± 4 vs. 8 ± 2 µg/mg, P = 0.005), adenosine diphosphate (16 ± 2 vs. 13 ± 2 µg/mg, P = 0.046), and total adenine nucleotide (37 ± 4 vs. 30 ± 3 µg/mg, P = 0.006) in myocardium after 2 h of reperfusion. They also showed less severe bleeding, edema and injury to mitochondria and myofibers in myocardium. The two groups did not differ significantly in doses of inotropic drugs received, cardiac output or levels of biomarkers in plasma. In this animal model of healthy hearts subjected to 90 min of ischemia, Huaxi-1 cardioplegia may be superior to entirely crystalloid cardioplegia for promoting energy generation and attenuating ischemia/reperfusion injury in myocardium.

Inflammation and mitochondria in the pathogenesis of chronic Chagas disease cardiomyopathy.

Chagas disease (CD), caused by the protozoan parasite Trypanosoma cruzi, is a neglected disease affecting around 6 million people. About 30% of CD patients develop chronic Chagas disease cardiomyopathy (CCC), an inflammatory cardiomyopathy that occurs decades after the initial infection, while most infected patients (60%) remain asymptomatic in the so-called indeterminate form (IF). Death results from heart failure or arrhythmia in a subset of CCC patients. Myocardial fibrosis, inflammation, and mitochondrial dysfunction are involved in the arrhythmia substrate and triggering events. Survival in CCC is worse than in other cardiomyopathies, which may be linked to a Th1-T cell rich myocarditis with abundant interferon (IFN)-γ and tumor necrosis factor (TNF)-α, selectively lower levels of mitochondrial energy metabolism enzymes in the heart, and reduced levels of high-energy phosphate, indicating poor adenosine triphosphate (ATP) production. IFN-γ and TNF-α signaling, which are constitutively upregulated in CD patients, negatively affect mitochondrial function in cardiomyocytes, recapitulating findings in CCC heart tissue. Genetic studies such as whole-exome sequencing (WES) in nuclear families with multiple CCC/IF cases has disclosed rare heterozygous pathogenic variants in mitochondrial and inflammatory genes segregating in CCC cases. In this minireview, we summarized studies showing how IFN-γ and TNF-α affect cell energy generation, mitochondrial health, and redox homeostasis in cardiomyocytes, in addition to human CD and mitochondria. We hypothesize that cytokine-induced mitochondrial dysfunction in genetically predisposed patients may be the underlying cause of CCC severity and we believe this mechanism may have a bearing on other inflammatory cardiomyopathies.

In vivo magnetic resonance spectrometry imaging demonstrates comparable adaptation of brain energy metabolism to metabolic stress induced by 72 h of fasting in depressed patients and healthy volunteers

Major depressive disorder (MDD) is characterized by dysregulation of stress systems and by abnormalities in cerebral energy metabolism. Stress induction has been shown to impact neurometabolism in healthy individuals. Contrarily, neurometabolic changes in response to stress are insufficiently investigated in MDD patients. Metabolic stress was induced in MDD patients (MDD, N = 24) and in healthy individuals (CTRL, N = 22) by application of an established fasting protocol in which calorie intake was omitted for 72 h. Both study groups were comparable regarding age, gender distribution, and body mass index (BMI). Fasting-induced effects on brain high-energy phosphate levels and membrane phospholipid metabolism were assessed using phosphorus-31 magnetic resonance spectroscopy (31P-MRS). Two-way repeated measures ANOVAs did not reveal significant interaction effects (group x fasting) or group differences in adenosine triphosphate (ATP), phosphocreatine (PCr), inorganic phosphate (Pi), phosphomonoesters (PME), phosphodiesters (PDE), or pH levels between MDD and CTRL. Fasting, independent of group, significantly increased ATP and decreased Pi levels and an overall increase in PME/PDE ratio as marker for membrane turnover was observed. Overall these results indicate reactive changes in cerebral energetics and in membrane phospholipid metabolism in response to fasting. The observed effects did not significantly differ between CTRL and MDD, indicating that neurometabolic adaptation to metabolic stress is preserved in MDD patients.

Exercise intolerance and rapid skeletal muscle energetic decline in human age-associated frailty.

BACKGROUNDPhysical frailty in older individuals is characterized by subjective symptoms of fatigue and exercise intolerance (EI). Objective abnormalities in skeletal muscle (SM) mitochondrial high-energy phosphate (HEP) metabolism contribute to EI in inherited myopathies; however, their presence or link to EI in the frail older adult is unknown.METHODSHere, we studied 3 groups of ambulatory, community-dwelling adults with no history of significant coronary disease: frail older (FO) individuals (81 ± 2.7 years, mean ± SEM), nonfrail older (NFO) individuals (79 ± 2.0 years), and healthy middle-aged individuals, who served as controls (CONT, 51 ± 2.1 years). Lower extremity SM HEP levels and mitochondrial function were measured with 31P magnetic resonance (MR) techniques during graded multistage plantar flexion exercise (PFE). EI was quantified by a 6-minute walk (6MW) and peak oxygen consumption during cardiopulmonary testing (peak VO2).RESULTSDuring graded exercise, FO, NFO, and CONT individuals all fatigued at similar SM HEP levels, as measured by 31P-MR. However, FO individuals fatigued fastest, with several-fold higher rates of PFE-induced HEP decline that correlated closely with shorter exercise duration in the MR scanner and with 6MW distance and lower peak oxygen consumption on cardiopulmonary testing (P < 0.001 for all). SM mitochondrial oxidative capacity was lower in older individuals and correlated with rapid HEP decline but less closely with EI.CONCLUSIONSeveral-fold faster SM energetic decline during exercise occurs in FO individuals and correlates closely with multiple measures of EI. Rapid energetic decline represents an objective, functional measure of SM metabolic changes and a potential new target for mitigating frailty-associated physical limitations.FUNDINGThis work was supported by NIH R21 AG045634, R01 AG063661, R01 HL61912, the Johns Hopkins University Claude D. Pepper Older Americans Independence Center P30AG021334, and the Clarence Doodeman Endowment in Cardiology at Johns Hopkins.

Fingolimod (FTY720) Preserves High Energy Phosphates and Improves Cardiac Function in Heterotopic Heart Transplantation Model.

During heart transplantation, donor heart leads to reduced oxygen supply resulting in low level of high energy phosphate (HEP) reserves in cardiomyocyte. Lower HEP is one of the underlying reasons of cell death due to ischemia. In this study we investigated the role of Fingolimod (FTY720) in heart transplantation ischemia. Eight groups of Sprague-Dawley rats (n = 5 for each subgroup) were made, A1 and C1 were given FTY720 1 mg/kg while B1 and D1 were given normal saline. The hearts were implanted into another set of similar rats after preservation period of 1 h at 4–8 °C. Significantly higher Left ventricular systolic pressure (LVSP), dP/dT maximum (p < 0.05), dP/dT minimum (p < 0.05) were recorded in the FTY720 treated group after 24 h of reperfusion while after 1 h of reperfusion, there were no significant differences in LVSP, maximum and negative dP/dT, and Left ventricular end diastolic pressure (LVEDP) between the control and the FTY720-treated transplant groups. Coronary blood flow (CBF) was enhanced (p < 0.05) in the FTY720 treated group after 1 and 24 h. ATP p < 0.001, p < 0.05 at 1 and 24 h, ADP p < 0.001, p > 0.05 at 1 and 24 h, and phosphocreatine p < 0.05, p > 0.05 at 1 and 24 h were better preserved by FTY720 treatment as compared to control group. The study concluded that pretreatment of grafted hearts with FTY720 improved hemodynamics, CBF, high energy phosphate reserves, reduces the peroxynitrite level and poly (ADP ribose) polymerase (PARP) inhibition that prevents ischemia-reperfusion injury.

Down-Regulation of Mitochondrial Metabolism after Tendon Release Primes Lipid Accumulation in Rotator Cuff Muscle

Atrophy and fat accumulation are debilitating aspects of muscle diseases and are rarely prevented. Using a vertical approach combining anatomic techniques with omics methodology in a tenotomy-induced sheep model of rotator cuff disease, we tested whether mitochondrial dysfunction is implicated in muscle wasting and perturbed lipid metabolism, speculating that both can be prevented by the stimulation of β-oxidation with l-carnitine. The infraspinatus muscle lost 22% of its volume over the first 6 weeks after tenotomy before the area-percentage of lipid increased from 8% to 18% at week 16. Atrophy was associated with the down-regulation of mitochondrial transcripts and protein and a slow-to-fast shift in muscle composition. Correspondingly, amino acid levels were increased 2 weeks after tendon release, when the levels of high-energy phosphates and glycerophospholipids were lowered. l-Carnitine administration (0.9 g/kg per day) prevented atrophy over the first 2 weeks, and mitigated alterations of glutamate, glycerophospholipids, and carnitine levels in released muscle, but did not prevent the level decrease in high-energy phosphates or protein constituents of mitochondrial respiration, promoting the accumulation of longer lipids with an increasing saturation. We conclude that the early phase of infraspinatus muscle degeneration after tendon release involves the elimination of oxidative characteristics associated with an aberrant accumulation of lipid species but is largely unrelated to the prevention of atrophy with oral l-carnitine administration.

Differences in Metabolic Profiles of Human Hearts after 6 Hour Preservation with a Novel Hypothermic Machine Perfusion Preservation Device Compared to Cold Storage

While cardiac transplantation remains the most effective therapy for end-stage heart failure, it continues to be plagued by a severe donor shortage. Many potentially suitable organs are not transplanted due to concerns of inadequate preservation with hypothermic static storage. Experimental and clinical studies suggest that machine perfusion preservation improves myocardial preservation. The purpose of this study is to test the hypothesis that a novel hypothermic machine perfusion preservation device maintains allograft oxidative metabolism, limits lactate accumulation, and more effectively preserves high energy phosphate stores compared to cold storage in a human model. Human donor hearts rejected or not offered for transplantation are eligible for this study through an organ procurement organization approved research protocol. Offered organs will be assigned to 6 hours of preservation either by conventional hypothermic static storage or hypothermic machine perfusion with a novel device by Paragonix Technologies, Inc. Donor demographic and diagnostic data will be collected. Myocardial oxygen consumption, lactate accumulation, temperature regulation and coronary flow will be measured in perfused hearts. After completion of the storage interval, myocardial biopsies will be obtained for proton and phosphorus-31 magnetic resonance spectroscopy, gas chromatography/mass spectroscopy and histologic studies. TCA cycle intermediates, high energy phosphates (ATP, phosphocreatine), lactate/alanine ratios will be measured. Cell death will be quantified by TUNEL. Groups will be compared by the Student's t-test or ANOVA as appropriate. A p-value < 0.05 will be considered significant. We expect to show that the machine perfusion device maintains an oxidative metabolic profile and low lactate levels. We anticipate machine perfusion preservation better preserves citric acid cycle intermediates, reduces cell death and increases high energy phosphate levels compared to static storage.

In Vivo Mitochondrial Function in Idiopathic and Genetic Parkinson's Disease.

Parkinson’s disease (PD) is associated with brain mitochondrial dysfunction. High-energy phosphates (HEPs), which rely on mitochondrial functioning, may be considered potential biomarkers for PD. Phosphorus magnetic resonance spectroscopy (31P-MRS) is a suitable tool to explore in vivo cerebral energetics. We considered 10 31P-MRS studies in order to highlight the main findings about brain energetic compounds in patients affected by idiopathic PD and genetic PD. The studies investigated several brain areas such as frontal lobes, occipital lobes, temporoparietal cortex, visual cortex, midbrain, and basal ganglia. Resting-state studies reported contrasting results showing decreased as well as normal or increased HEPs levels in PD patients. Functional studies revealed abnormal PCr + βATP levels in PD subjects during the recovery phase and abnormal values at rest, during activation and recovery in one PD subject with PINK1 gene mutation suggesting that mitochondrial machinery is more impaired in PD patients with PINK1 gene mutation. PD is characterized by energetics impairment both in idiopathic PD as well as in genetic PD, suggesting that mitochondrial dysfunction underlies the disease. Studies are still sparse and sometimes contrasting, maybe due to different methodological approaches. Further studies are needed to better assess the role of mitochondria in the PD development.

An Assessment of Melatonin's Therapeutic Value in the Hypoxic-Ischemic Encephalopathy of the Newborn.

Hypoxic-ischemic encephalopathy (HIE) is one of the most frequent causes of brain injury in the newborn. From a pathophysiological standpoint, a complex process takes place at the cellular and tissue level during the development of newborn brain damage in the absence of oxygen. Initially, the lesion is triggered by a deficit in the supply of oxygen to cells and tissues, causing a primary energy insufficiency. Subsequently, high energy phosphate levels recover transiently (the latent phase) that is followed by a secondary phase, in which many of the pathophysiological mechanisms involved in the development of neonatal brain damage ensue (i.e., excitotoxicity, massive influx of Ca2+, oxidative and nitrosative stress, inflammation). This leads to cell death by necrosis or apoptosis. Eventually, a tertiary phase occurs, characterized by the persistence of brain damage for months and even years after the HI insult. Hypothermia is the only therapeutic strategy against HIE that has been incorporated into neonatal intensive care units with limited success. Thus, there is an urgent need for agents with the capacity to curtail acute and chronic damage in HIE. Melatonin, a molecule of unusual phylogenetic conservation present in all known aerobic organisms, has a potential role as a neuroprotective agent both acutely and chronically in HIE. Melatonin displays a remarkable antioxidant and anti-inflammatory activity and is capable to cross the blood-brain barrier readily. Moreover, in many animal models of brain degeneration, melatonin was effective to impair chronic mechanisms of neuronal death. In animal models, and in a limited number of clinical studies, melatonin increased the level of protection developed by hypothermia in newborn asphyxia. This review article summarizes briefly the available therapeutic strategies in HIE and assesses the role of melatonin as a potentially relevant therapeutic tool to cover the hypoxia-ischemia phase and the secondary and tertiary phases following a hypoxic-ischemic insult.

Inhibition of CD38 with the Thiazoloquin(az)olin(on)e 78c Protects the Heart against Postischemic Injury.

Inhibition of and genetic deletion of the NAD(P)+ hydrolase [NAD(P)ase] CD38 have been shown to protect against ischemia/reperfusion (I/R) injury in rat and mouse hearts. CD38 has been shown to enhance salvage of NADP(H), which in turn prevents impairment of endothelial nitric oxide synthase function, a hallmark of endothelial dysfunction. Despite growing evidence for a role of CD38 in postischemic injury, until recently there had been a lack of potent CD38 inhibitors. Recently, a new class of thiazoloquin(az)olin(on)e compounds were identified as highly potent and specific CD38 inhibitors. Herein, we investigate the ability of one of these compounds, 78c, to inhibit CD38 and protect the heart in an ex vivo model of myocardial I/R injury. The potency and mechanism of CD38 inhibition by 78c was assessed in vitro using recombinant CD38. The dose-dependent tissue uptake of 78c in isolated mouse hearts was determined, and high tissue permeability of 78c was observed when delivered in perfusate. Treatment of hearts with 78c was protective against both postischemic endothelial and cardiac myocyte injury, with preserved nitric oxide synthase-dependent vasodilatory and contractile function, respectively. Myocardial infarction was also significantly decreased in 78c-treated hearts, with preserved levels of high-energy phosphates. Protective effects peaked at 10 μM treatment, and similar protection without toxicity was seen at 5-fold higher doses. Overall, 78c was shown to be a potent and biologically active CD38 inhibitor with favorable tissue uptake and marked protective effects against I/R injury with enhanced preservation of contractile function, coronary flow, and decreased infarction.

Abstract 289: Relationship Between Myocardial Glycogen Concentrations and Amplitude Spectrum Area During Ventricular Fibrillation in a Rat Model of Cardiac Arrest and Resuscitation

Introduction: Myocardial high-energy phosphate (ATP) levels has been demonstrated correlating with amplitude spectrum area (AMSA) during ventricular fibrillation (VF) in previous experimental studies. In the present study, we investigated the relationship between AMSA and myocardial glycogen content (MGC),which can be used to reflect the status of myocardial energy metabolism indirectly during VF. Hypothesis: AMSA has a significantly correlation with MGC during VF in a rat model of cardiac arrest and resuscitation. Methods: Twenty male Sprague-Dawley rats weighing 350 to 450 g were utilized and randomized into two groups: VF and cardiopulmonary resuscitation (CPR) (VF/CPR group) or untreated VF (VF group). 5 mins of CPR was performed after 10 mins of untreated VF in VF/CPR animals. Amplitude spectrum area (AMSA) at VF 5, 10 and 15 mins were calculated from ECG signals. The rats’ hearts were quickly removed at the predetermined time of 15 min for determines the glycogen contents by the anthrone reagent method using a glycogen assay kit. Results: AMSA values significantly decreased during untreated VF in both VF and VF/CPR animals. However, much greater AMSA during CPR was achieved by the VF/CPR group in comparison with the VF group. There was a marked and negative relationship between AMSA at VF 15 min and MGC. (Figure). Conclusion: MGC was significantly and negatively correlated with AMSA during VF in this rat model of cardiac arrest and resuscitation. In clinical practice, we can use AMSA to reflect the state of myocardial energy metabolism indirectly. Figure The changes of AMSA and relationship between AMSA and glycogen content:(A) The change of AMSA between VF/CRP group and VF group;(B) The relationship between AMSA and glycogen content. AMSA, amplitude spectrum area; V, time of ventricular fibrillation; # p &lt;0.05 vs. V4.

Hypothermia Decreases O2 Cost for Ex Vivo Contraction in Mouse Skeletal Muscle.

Evidence suggests that the energy efficiency of key ATPases involved in skeletal muscle contractile activity is improved in a hypothermic condition. However, it is unclear how a decrease in temperature affects skeletal muscle O2 consumption (mVO2) induced by muscle contraction. Isolated mouse extensor digitorum longus (EDL) muscles were incubated in a temperature-controlled (37°C or 25°C) bath that included an O2 probe. EDL muscles from one limb were subjected to the measurement of resting mVO2, and the contralateral EDL muscles were used for the measurement of mVO2 with electrically stimulated contraction. For the resting protocol, muscles were suspended at resting tension for 15 min with continuous O2 recordings. For the contraction protocol, EDL muscles underwent 10 electrically stimulated isometric contractions with continuous O2 recordings for 15 min. The rate of O2 disappearance was quantified as micromoles of O2 per minute and normalized to the wet weight of the muscle. Resting mVO2 was greater at 37°C than at 25°C, consistent with the idea that lower temperature reduces basal metabolic rate. Electrically stimulated contraction robustly increased mVO2 at both 37°C and 25°C, which was sustained for ~3 min postcontraction. During that period, mVO2 was elevated approximately fivefold at both 37°C and 25°C. Greater contraction-induced mVO2 at 37°C compared with 25°C occurred despite lower force generated at 37°C than at 25°C. Together, O2 cost for muscle contraction (force-time integral per O2 consumed) was greater at 37°C than at 25°C. Levels of high-energy phosphates were consistent with greater energy demand at 37°C compared with 25°C. In conclusion, these results indicate that muscle contraction that occurs at subnormal temperature requires less O2 than at 37°C.

Myocardial fatty acid uptake through CD36 is indispensable for sufficient bioenergetic metabolism to prevent progression of pressure overload-induced heart failure

The energy metabolism of the failing heart is characterized by reduced fatty acid (FA) oxidation and an increase in glucose utilization. However, little is known about how energy metabolism-function relationship is relevant to pathophysiology of heart failure. Recent study showed that the genetic deletion of CD36 (CD36KO), which causes reduction in FA use with an increased reliance on glucose, accelerates the progression from compensated hypertrophy to heart failure. Here, we show the mechanisms by which CD36 deletion accelerates heart failure in response to pressure overload. CD36KO mice exhibited contractile dysfunction and death from heart failure with enhanced cardiac hypertrophy and interstitial fibrosis when they were subjected to transverse aortic constriction (TAC). The pool size in the TCA cycle and levels of high-energy phosphate were significantly reduced in CD36KO-TAC hearts despite an increase in glycolytic flux. De novo synthesis of non-essential amino acids was facilitated in CD36KO-TAC hearts, which could cause a further decline of the pool size. The ingestion of a diet enriched in medium-chain FA improved cardiac dysfunction in CD36KO-TAC hearts. These findings suggest that myocardial FA uptake through CD36 is indispensable for sufficient ATP production and for preventing an increased glycolytic flux-mediated structural remodeling during pressure overload-induced hypertrophy.

NF-κB-mediated metabolic remodelling in the inflamed heart in acute viral myocarditis

Acute viral myocarditis (VM), characterised by leukocyte infiltration and dysfunction of the heart, is an important cause of sudden cardiac death in young adults. Unfortunately, to date, the pathological mechanisms underlying cardiac failure in VM remain incompletely understood. In the current study, we investigated if acute VM leads to cardiac metabolic rewiring and if this process is driven by local inflammation. Transcriptomic analysis of cardiac biopsies from myocarditis patients and a mouse model of VM revealed prominent reductions in the expression of a multitude of genes involved in mitochondrial oxidative energy metabolism. In mice, this coincided with reductions in high-energy phosphate and NAD levels, as determined by Imaging Mass Spectrometry, as well as marked decreases in the activity, protein abundance and mRNA levels of various enzymes and key regulators of cardiac oxidative metabolism. Indicative of fulminant cardiac inflammation, NF-κB signalling and inflammatory cytokine expression were potently induced in the heart during human and mouse VM. In cultured cardiomyocytes, cytokine-mediated NF-κB activation impaired cardiomyocyte oxidative gene expression, likely by interfering with the PGC-1 (peroxisome proliferator-activated receptor (PPAR)-γ co-activator) signalling network, the key regulatory pathway controlling cardiomyocyte oxidative metabolism. In conclusion, we provide evidence that acute VM is associated with extensive cardiac metabolic remodelling and our data support a mechanism whereby cytokines secreted primarily from infiltrating leukocytes activate NF-κB signalling in cardiomyocytes thereby inhibiting the transcriptional activity of the PGC-1 network and consequently modulating myocardial energy metabolism.

Is visual activation associated with changes in cerebral high-energy phosphate levels?

Phosphorus magnetic resonance spectroscopy (31P MRS) has been employed before to assess phosphocreatine (PCr) and other high-energy phosphates in the visual cortex during visual stimulation with inconsistent results. We performed functional 31P MRS imaging in the visual cortex and control regions during a visual stimulation paradigm at an unprecedented sensitivity, exploiting a dedicated RF coil design at a 7 T MR system. Visual stimulation in a 3 min 24 s on–off paradigm in eight young healthy adults generated a clear BOLD effect with traditional 1H functional MRI in the visual cortex (average z score 9.9 ± 0.2). However, no significant event-related changes in any of the 31P metabolite concentrations, linewidths (7.9 ± 1.8 vs 7.8 ± 1.9 Hz) or tissue pH (7.07 ± 0.13 vs 7.06 ± 0.07) were detectable. Overall, our study of 31P MRSI in 15 cm3 voxels had a detection threshold for changes in PCr, Pi and γ-ATP between stimulation and rest of 5, 17 and 10%, respectively. In individual subjects, the mean coefficients of variance for PCr and Pi levels of control voxels were 6 ± 3 and 19 ± 8% (three time point average of 3 min 24 s). Altogether this indicates that energy supply for neuronal activation at this temporal resolution does not drain global PCr resources.

Interaction of Calmodulin with the cSH2 Domain of the p85 Regulatory Subunit.

Calmodulin (CaM) is a calcium sensor protein that directly interacts with the dual-specificity (lipid and protein) kinase PI3Kα through the SH2 domains of the p85 regulatory subunit. In adenocarcinomas, the CaM interaction removes the autoinhibition of the p110 catalytic subunit of PI3Kα, leading to activation of PI3Kα and promoting cell proliferation, survival, and migration. Here we demonstrate that the cSH2 domain of p85α engages its two CaM-binding motifs in the interaction with the N- and C-lobes of CaM as well as the flexible central linker, and our nuclear magnetic resonance experiments provide structural details. We show that in response to binding CaM, cSH2 exposes its tryptophan residue at the N-terminal region to the solvent. Because of the flexible nature of both CaM and cSH2, multiple binding modes of the interactions are possible. Binding of CaM to the cSH2 domain can help release the inhibition imposed on the p110 subunit, similar to the binding of the phosphorylated motif of RTK, or phosphorylated CaM (pCaM), to the SH2 domains. Amino acid sequence analysis shows that CaM-binding motifs are common in SH2 domains of non-RTKs. We speculate that CaM can also activate these kinases through similar mechanisms.

Naringenin reduces oxidative stress and improves mitochondrial dysfunction via activation of the Nrf2/ARE signaling pathway in neurons.

Oxidative stress and mitochondrial dysfunction are considered to be major contributing factors in the development and progression of many neurodegenerative diseases. Naringenin (NAR) is an abundant flavanone in the Citrus genus and has been found to exert antioxidant, anticarcinogenic and antimutagenic effects. However, the potential underlying mechanism of its antioxidant effects remains unclear. In the present study, the authors investigated the antioxidant effect of NAR on neurons invitro. Neurons isolated from the brains of Sprague-Dawley rats were randomly divided into a control group, model group, NAR-L group, NAR-M group and NAR-H group. The model group received hypoxia and re-oxygenation treatment, and the NAR-L, NAR-M and NAR-H groups received 20, 40 and 80µM NAR, respectively. The levels of reactive oxygen species (ROS) in each group were detected by chloromethyl-2',7'dichlorodihydro fluorescein diacetate staining, and differences in mitochondrial dysfunction were analyzed through measurement of mitochondrial membrane potential (∆ψm), adenine nucleotide translocase transport activity and adenine nucleotide levels. MTT and flow cytometry assays were also used to analyze cell proliferation and apoptosis, and the effects of NAR on the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway were investigated using small interfering RNA methods. The authors detected an increased accumulation of ROS in the model group, and high-dose NAR could significantly reduce the levels of ROS. Furthermore, NAR could improve mitochondrial dysfunction, as indicated by increased levels of high-energy phosphates, enhanced mitochondrial ANT transport activity and increased mitochondrial membrane potential. Moreover, NAR increased cell viability and decreased the rate of cell apoptosis. NAR also increased the expression of Nrf2 and its downstream target genes. These findings demonstrated that NAR could reduce oxidative stress and improve mitochondrial dysfunction via activation of the Nrf2/ARE signaling pathway in neurons.

Evaluation of Energy Balance on Human Telomerase Reverse Transcriptase (hTERT) Alternative Splicing by Semi-quantitative RT-PCR in Human Umbilical Vein Endothelial Cells.

Background:Decreased high-energy phosphate level is involved in endothelial cell injury and dysfunction. Reduced telomerase activity in endothelial cells in parallel with reduced energy levels might be due to altered direction of alternative splicing machine as a complication of depleted energy during the process of atherosclerosis.Materials and Methods:Isolated human umbilical vein endothelial cells (HUVECs) were treated for 24 hours by oligomycine (OM) and 2-deoxy glucose (2-DG). After 24 hours, the effect of energy depletion on telomerase splicing pattern was evaluated using RT-PCR. Indeed, in both treated and untargeted cells, nitric oxide (NO) and von Willebrand factor (vWF) were measured.Results:ATP was depleted in treated cells by 43.9% compared with control group. We observed a slight decrease in NO levels (P = 0.09) and vWF (P = 0.395) in the setting of 49.36% ATP depletion. In both groups, no telomerase gene expression was seen. Telomerase and housekeeping gene expression were found in positive control group (colon cancer tissue) and sample tissue.Conclusions:The absence of telomerase gene expression in HUVECs might be due to the mortality of these cells or the low level of telomerase gene expression in these cells under normal circumstances.

Tissue-dependent cerebral energy metabolism in adolescents with bipolar disorder

ObjectivesTo investigate tissue-dependent cerebral energy metabolism by measuring high energy phosphate levels in unmedicated adolescents diagnosed with bipolar I disorder. MethodsPhosphorus-31 magnetic resonance spectroscopic imaging data were acquired over the entire brain of 24 adolescents with bipolar I disorder and 19 demographically matched healthy comparison adolescents. Estimates of phosphocreatine (PCr) and adenosine triphosphate (ATP, determined from the γ-resonance) in homogeneous gray and white matter in the right and left hemispheres of the cerebrum of each subject were obtained by extrapolation of linear regression analyses of metabolite concentrations vs. voxel gray matter fractions. ResultsMultivariate analyses of variance showed a significant effect of group on high energy phosphate concentrations in the right cerebrum (p=0.0002) but not in the left (p=0.17). Post-hoc testing in the right cerebrum revealed significantly reduced concentrations of PCr in gray matter and ATP in white matter in both manic (p=0.002 and 0.0001, respectively) and euthymic (p=0.004 and 0.002, respectively) bipolar I disorder subjects relative to healthy comparisons. LimitationsThe small sample sizes yield relatively low statistical power between manic and euthymic groups; cross-sectional observations limit the ability to determine if these findings are truly independent of mood state. ConclusionsOur results suggest bioenergetic impairment – consistent with downregulation of creatine kinase – is an early pathophysiological feature of bipolar I disorder.

Hypothermic continuous machine perfusion enables preservation of energy charge and functional recovery of heart grafts in an ex vivo model of donation following circulatory death.

Cardiac transplantation using hearts from donors after circulatory death (DCD) is critically limited by the unavoidable warm ischaemia and its related unpredictable graft function. Inasmuch as hypothermic machine perfusion (MP) has been shown to improve heart preservation, we hypothesized that MP could enable the use of DCD hearts for transplantation. We recovered 16 pig hearts following anoxia-induced cardiac arrest and cardioplegia. Grafts were randomly assigned to two different groups of 4-h preservation using either static cold storage (CS) or MP (Modified LifePort© System, Organ Recovery Systems©, Itasca, Il). After preservation, the grafts were reperfused ex vivo using the Langendorff method for 60 min. Energetic charge was quantified at baseline, post-preservation and post-reperfusion by measuring lactate and high-energy phosphate levels. Left ventricular contractility parameters were assessed both in vivo prior to ischaemia and ex vivo during reperfusion. Following preservation, the hearts that were preserved using CS exhibited higher lactate levels (57.1 ± 23.7 vs 21.4 ± 12.2 µmol/g; P < 0.001), increased adenosine monophosphate/adenosine triphosphate ratio (0.53 ± 0.25 vs 0.11 ± 0.11; P < 0.001) and lower phosphocreatine/creatine ratio (9.7 ± 5.3 vs 25.2 ± 11; P < 0.001) in comparison with the MP hearts. Coronary flow was similar in both groups during reperfusion (107 ± 9 vs 125 ± 9 ml/100 g/min heart; P = ns). Contractility decreased in the CS group, yet remained well preserved in the MP group. MP preservation of DCD hearts results in improved preservation of the energy and improved functional recovery of heart grafts compared with CS.