Impaired Energetics Research Articles

Myocardial steatosis and impaired energetics are independent predictors of regional contractile function in patients with severe aortic stenosis

Background In aortic stenosis (AS), there is impairment of myocardial energetics and a substrate switch with a preference to glucose metabolism and downregulation of fatty acid utilization. Whether or not this results in cardiac steatosis is unknown. We hypothesi zed that cardiac steatosis is present in severe AS and this can be assessed by 1 H magnetic resonance spectroscopy (MRS). We aimed to investigate myocardial lipid content in patients with severe AS and to examine the associations amongst steatosis, energetics and myocardial function. Methods Thirty patients with severe AS (20 symptomatic, 10 asymptomatic) and 20 normal volunteers were enrolled. Inclusion criteria were peak aortic valve gradient >50 mmHg, no other significant valvular pathology, normal left ventricular systolic function, no obstructive coronary artery disease and blood pressure <160/90 mmHg. Myocardial lipid content and phosphocreatine/adenosine triphosphate (PCr/ATP) ratios were quantified using 1 Ha nd 31 P MRS, respectively. Global left ventricular systolic function and mass were assessed using cine cardiac magnetic

Coronary microvascular function, myocardial metabolism, and energetics in hypertrophic cardiomyopathy: insights from positron emission tomography

Hypertrophic cardiomyopathy (HCM) is a major cause of sudden cardiac death in adolescence, and may lead to heart failure at any age. However, significant heterogeneity in the clinical course and phenotypic expression exists. Next to left ventricular hypertrophy, an impaired myocardial blood flow (MBF) during stress and inefficient cardiac metabolism are other characteristics of HCM. Studies using positron emission tomography (PET) have led to an enhanced understanding of the role that myocardial ischaemia and impaired energetics play in the clinical course of HCM. The blunted vasodilator reserve in the absence of an epicardial coronary stenosis is the result of microvascular dysfunction. Microvascular dysfunction, in turn, represents a predisposing factor for myocardial ischaemia, which may lead to cardiac dysfunction and fibrosis. Correspondingly, the severity of microvascular dysfunction has been shown to serve as a major predictor of mortality. Myocardial energetics in HCM has been studied with similar interest as mounting evidence suggests that mechano-energetic uncoupling may play a central role in its pathogenesis. Although prognostic data related to an impaired energetic state in HCM are lacking, it may hold prognostic relevance. Consequently, enhancing perfusion and restoring energetics have gained considerable attention as potential strategies to alter the natural course of HCM. In this regard, myocardial perfusion and metabolic imaging serves as a valuable tool to monitor the effects of therapeutic interventions on the pathophysiology of HCM.

OP11: Metabolic and vascular determinants of increased left ventricular torsion in patients with type 2 diabetes mellitus

Left ventricular torsion is increased in uncomplicated type 2 diabetes (T2DM) which may be an early manifestation of cardiomyopathy. Our aim was to evaluate the relationship of torsion to energetic impairment and microvascular damage in healthy patients with T2DM. 36 patients with T2DM without coronary artery disease as measured by a normal dobutamine stress echocardiography and 20 healthy control (HC) subjects were recruited. Magnetic resonance spectroscopy, standard, speckle tracking and contrast echocardiography, cardiovascular autonomic function (CAN) testing and skin endothelial poly (ADP-ribose) polymerase assessments were performed. Left ventricular (LV) torsion was increased in subjects with T2DM (2.6 ± 0.8°/cm) compared to HC (2.0 ± 0.5°/cm, p=0.002). Cardiac energetics were reduced in patients with T2DM compared to HC (1.1 ± 0.4 vs. 1.8 ± 0.5, p<0.0001), but were not associated with increased torsion. Endothelial PARP was increased by 19% (p=0.0001) in subjects with T2DM and correlated with LV apical rotation ((R Square 0.424 p=0.017) after adjustment for weight and BMI. Torsion correlated significantly with CAN (F(2, 31) = 3.5, p=0.043, eta square 0.2). LV torsion is increased in healthy T2DM. Microvascular damage and autonomic dysfunction but not reduced cardiac energetics may play an important pathophysiological role in its development.

Pathophysiology of acute kidney injury.

Acute kidney injury (AKI) is the leading cause of nephrology consultation and is associated with high mortality rates. The primary causes of AKI include ischemia, hypoxia, or nephrotoxicity. An underlying feature is a rapid decline in glomerular filtration rate (GFR) usually associated with decreases in renal blood flow. Inflammation represents an important additional component of AKI leading to the extension phase of injury, which may be associated with insensitivity to vasodilator therapy. It is suggested that targeting the extension phase represents an area potential of treatment with the greatest possible impact. The underlying basis of renal injury appears to be impaired energetics of the highly metabolically active nephron segments (i.e., proximal tubules and thick ascending limb) in the renal outer medulla, which can trigger conversion from transient hypoxia to intrinsic renal failure. Injury to kidney cells can be lethal or sublethal. Sublethal injury represents an important component in AKI, as it may profoundly influence GFR and renal blood flow. The nature of the recovery response is mediated by the degree to which sublethal cells can restore normal function and promote regeneration. The successful recovery from AKI depends on the degree to which these repair processes ensue and these may be compromised in elderly or chronic kidney disease (CKD) patients. Recent data suggest that AKI represents a potential link to CKD in surviving patients. Finally, earlier diagnosis of AKI represents an important area in treating patients with AKI that has spawned increased awareness of the potential that biomarkers of AKI may play in the future.

Probucol modulates oxidative stress and excitotoxicity in Huntington's disease models in vitro

Huntington's disease (HD) is an autosomal dominant inherited neurodegenerative disease characterized by symptoms attributable to the death of striatal and cortical neurons. The molecular mechanisms mediating neuronal death in HD seem to be related to oxidative stress, excitotoxicity and misbalance in energetic metabolism. In this study we evaluated the potential relationship between energetic impairment, excitotoxicity and oxidative stress in rat striatal slices exposed to quinolinic acid (QA; as an excitotoxic model), 3-nitropropionic acid (3-NP; as an inhibitor of mitochondrial succinate dehydrogenase), as well as a combined model produced by the co-administration of these two toxins at subtoxic concentrations. We took advantage of the direct antioxidant/scavenger properties of Probucol in order to investigate the role of reactive oxygen species (ROS) in mediating the toxicity of both compounds alone or in association. Experiments with MK-801 (a NMDA type glutamate receptor antagonist) and succinate (an energy precursor agent) were also performed in an attempt to better comprehend the mechanisms of damage and neuroprotection. QA (1 mM), 3-NP (1 mM) and QA plus 3-NP (0.1 mM of both) significantly induced mitochondrial dysfunction and produced an increase in ROS generation, as well as a significant increase in lipid peroxidation in striatal slices. Probucol (10 and 30 μM) prevented ROS formation and lipid peroxidation in all used models, but did not protect against the mitochondrial dysfunction induced by 3-NP (only by QA or QA plus 3-NP). Sodium succinate (1 mM) protected the striatal slices only against 3-NP-induced mitochondrial dysfunction. On the other hand, MK-801 protected against mitochondrial dysfunction in all used models. Our data suggest that the two studied toxic models (QA and 3-NP) or the combined model (QA plus 3-NP) can generate complex patterns of damage, which involve metabolic compromise, ROS formation, and oxidative stress. Moreover, a partial inhibition of SDH by subtoxic 3-NP and moderate excitotoxicty by subtoxic QA are potentiated when both agents are associated. The toxic action of QA plus 3-NP seems to be involved with Ca2+ metabolism and ROS formation, and can be prevented or attenuated by antioxidant/scavenger compounds and NMDAr antagonists.

Intracellular Sodium Overload Contributes to Deterioration of Function in Dystrophic Heart

[Na+]i is found to be elevated in several models of heart failure. This is believed to favor Ca2+ extrusion from mitochondria via the mNCX and therefore reduce mitochondrial Ca2+ accumulation. As several dehydrogenases of the mitochondrial Krebs cycle are stimulated by Ca2+, the defects in cellular sodium homeostasis may induce mitochondrial oxidative stress. Previously we have estimated that resting [Na+]i is significantly elevated in ventricular myocytes isolated from dystrophic (mdx) mice compared to those from wild-type (WT) mice (24.2 ± 3.1 mM, n=13 vs. 14.0 ± 1.7 mM, n=9). Moreover, the mitochondrial matrix was significantly oxidized at resting conditions (35±3.1 %, n=27 vs. 53±5.2 %, of NADH reduction n=10, in mdx and WT respectively). Here maximal oxidation of NADH by FCCP/oligomycin was taken as 0% reduced NADH and maximal reduction by rotenone and β-hydroxy-butyrate was defined as 100% reduced NADH. We suggested that beat-to-beat mechanical activity in dystrophic heart might lead to greater [Na+]i accumulation and oxidation of the mitochondrial matrix. Here we investigated whether elevated [Na+]i affects mitochondrial energetics during excitation-contraction coupling in dystrophic myocytes. We measured NADH autofluorescence in WT and mdx cells under control conditions and during 4Hz stimulation in the presence of isoproterenol. An abrupt increase in workload resulted in significant oxidation of NADH both in mdx and WT cardiomyocytes. However, the extent of oxidation was significantly greater in dystrophic cells (50±9 %, n=10 vs 15±6 %, n=11 decrease in NADH, respectively). When workload was discontinued, the mitochondrial matrix quickly returned back to its base line levels in WT but not in mdx cells. These results support the notion that the elevated [Na+]i contributes to the impaired energetics in dystrophic cardiomyocytes and therefore contributes to the development of dystrophic cardiomyopathy.

Uncoupling of ATP-depletion and cell death in human dopaminergic neurons

The mitochondrial inhibitor 1-methyl-4-phenylpyridinium (MPP+) is the toxicologically relevant metabolite of 1-methyl-4-phenyltetrahydropyridine (MPTP), which causes relatively selective degeneration of dopaminergic neurons in the substantia nigra. Dopaminergic LUHMES cells were used to investigate whether ATP-depletion can be uncoupled from cell death as a downstream event in these fully post-mitotic human neurons. Biochemical assays indicated that in the homogeneously differentiated cell cultures, MPP+ was taken up by the dopamine transporter (DAT). MPP+ then triggered oxidative stress and caspase activation, as well as ATP-depletion followed by cell death. Enhanced survival of the neurons in the presence of agents interfering with mitochondrial pathology, such as the fission inhibitor Mdivi-1 or a Bax channel blocker suggested a pivotal role of mitochondria in this model. However, these compounds did not prevent cellular ATP-depletion. To further investigate whether cells could be rescued despite respiratory chain inhibition by MPP+, we have chosen a diverse set of pharmacological inhibitors well-known to interfere with MPP+ toxicity. The antioxidant ascorbate, the iron chelator desferoxamine, the stress kinase inhibitor CEP1347, and different caspase inhibitors reduced cell death, but allowed ATP-depletion in protected cells. None of these compounds interfered with MPP+ accumulation in the cells. These findings suggest that ATP-depletion, as the initial mitochondrial effect of MPP+, requires further downstream processes to result in neuronal death. These processes may form self-enhancing signaling loops, that aggravate an initial energetic impairment and eventually determine cell fate.

Abstract 17133: Cardiac Steatosis Detected in Hypertrophic Cardiomyopathy, Dilated Cardiomyopathy and Aortic Stenosis

BACKGROUND The failing heart is characterised by impaired myocardial energetics and a compensatory shift from fatty acid to glucose metabolism. Cardiac steatosis has been implicated in ventricular contractile dysfunction, hypertrophy, inflammation and arrhythmias.Therefore we assessed cardiac steatosis using 1H magnetic resonance spectroscopy in hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM, EF35 mmHg, LVEF&gt;45%). METHODS 75 prospectively enrolled subjects (15 DCM, LVEF34±15%; 17 HCM, LV mass 165±42g; 14 AS, gradient 44±2 mmHg and 29 normal volunteers) were scanned at 3T.1H magnetic resonance spectroscopy (MRS) was used to quantify myocardial lipid/water content(%) and 31P MRS to measure the phosphocreatine/adenosine triphosphate (PCr/ATP) ratio in the same region of the mid-ventricular septum. LVEF was assessed using cine imaging and tagging was used to assess global circumferential strain (% deformation normal -19±2; reduced contraction between -16 and 0%). RESULTS All subjects were well matched between cohorts for BMI, fasting glucose and total cholesterol (26±4 kg/m2, 5.0±0.6mmol/l, 5.1±1.2 mmol/l, p&lt;0.05). Cardiac lipid content was elevated in all patientcohorts compared with normal volunteers (HCM 0.7±0.4; DCM 0.7±0.3; AS 0.7±0.6 vs normals 0.33±0.2% lipid/water, p&lt;0.01 for all). PCr/ATP ratios were also impaired in all patientgroups (HCM 1.6±0.2; DCM 1.7±0.2; AS 1.7±0.2 vs normals 2.1±0.2, p&lt;0.01 for all).Peak systolic circumferential strain was impaired in HCM (-16±2%, p&lt;0.01) and DCM (-14±3%, p&lt;0.01) compared to normals (-19±2%) but unchanged in AS (-18±2%). CONCLUSION Cardiac steatosis occurs in HCM and DCM in the presence of impaired energetics and abnormal contractile function andin patients with AS in the presence of preserved global contractile function. This suggests a potential role for cardiac steatosis as a novel biomarker and mechanism inboth early and advanced non-ischemic cardiac disease.

Cytotoxicity of monocrotaline in isolated rat hepatocytes: Effects of dithiothreitol and fructose

Monocrotaline (MCT) is a pyrrolizidine alkaloid present in plants of the Crotalaria species that causes cytotoxicity and genotoxicity, including hepatotoxicity in animals and humans. It is metabolized by cytochrome P-450 in the liver to the alkylating agent dehydromonocrotaline (DHM). In previous studies using isolated rat liver mitochondria, we observed that DHM, but not MCT, inhibited the activity of respiratory chain complex I and stimulated the mitochondrial permeability transition with the consequent release of cytochrome c. In this study, we evaluated the effects of MCT and DHM on isolated rat hepatocytes. DHM, but not MCT, caused inhibition of the NADH-linked mitochondrial respiration. When hepatocytes of rats pre-treated with dexamethasone were incubated with MCT (5 mM), they showed ALT leakage, impaired ATP production and decreased levels of intracellular reduced glutathione and protein thiols. In addition, MCT caused cellular death by apoptosis. The addition of fructose or dithiotreitol to the isolated rat hepatocyte suspension containing MCT prevented the ATP depletion and/or glutathione or thiol oxidation and decreased the ALT leakage and apoptosis. These results suggest that the toxic effect of MCT on hepatocytes may be caused by metabolite-induced mitochondrial energetic impairment, together with a decrease of cellular glutathione and protein thiols.

Redox-Active Quinones and Ascorbate: An Innovative Cancer Therapy That Exploits the Vulnerability of Cancer Cells to Oxidative Stress

Cancer cells are particularly vulnerable to treatments impairing redox homeostasis. Reactive oxygen species (ROS) can indeed play an important role in the initiation and progression of cancer, and advanced stage tumors frequently exhibit high basal levels of ROS that stimulate cell proliferation and promote genetic instability. In addition, an inverse correlation between histological grade and antioxidant enzyme activities is frequently observed in human tumors, further supporting the existence of a redox dysregulation in cancer cells. This biochemical property can be exploited by using redox-modulating compounds, which represent an interesting approach to induce cancer cell death. Thus, we have developed a new strategy based on the use of pharmacologic concentrations of ascorbate and redox-active quinones. Ascorbate-driven quinone redox cycling leads to ROS formation and provoke an oxidative stress that preferentially kill cancer cells and spare healthy tissues. Cancer cell death occurs through necrosis and the underlying mechanism implies an energetic impairment (ATP depletion) that is likely due to glycolysis inhibition. Additional mechanisms that participate to cell death include calcium equilibrium impairment and oxidative cleavage of protein chaperone Hsp90. Given the low systemic toxicity of ascorbate and the impairment of crucial survival pathways when associated with redox-active quinones, these combinations could represent an original approach that could be combined to standard cancer therapy.

Metabolic Modulator Perhexiline Corrects Energy Deficiency and Improves Exercise Capacity in Symptomatic Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy patients exhibit myocardial energetic impairment, but a causative role for this energy deficiency in the pathophysiology of hypertrophic cardiomyopathy remains unproven. We hypothesized that the metabolic modulator perhexiline would ameliorate myocardial energy deficiency and thereby improve diastolic function and exercise capacity. Forty-six consecutive patients with symptomatic exercise limitation (peak Vo(2) <75% of predicted) caused by nonobstructive hypertrophic cardiomyopathy (mean age, 55±0.26 years) were randomized to perhexiline 100 mg (n=24) or placebo (n=22). Myocardial ratio of phosphocreatine to adenosine triphosphate, an established marker of cardiac energetic status, as measured by (31)P magnetic resonance spectroscopy, left ventricular diastolic filling (heart rate normalized time to peak filling) at rest and during exercise using radionuclide ventriculography, peak Vo(2), symptoms, quality of life, and serum metabolites were assessed at baseline and study end (4.6±1.8 months). Perhexiline improved myocardial ratios of phosphocreatine to adenosine triphosphate (from 1.27±0.02 to 1.73±0.02 versus 1.29±0.01 to 1.23±0.01; P=0.003) and normalized the abnormal prolongation of heart rate normalized time to peak filling between rest and exercise (0.11±0.008 to -0.01±0.005 versus 0.15±0.007 to 0.11±0.008 second; P=0.03). These changes were accompanied by an improvement in primary end point (peak Vo(2)) (22.2±0.2 to 24.3±0.2 versus 23.6±0.3 to 22.3±0.2 mL · kg(-1) · min(-1); P=0.003) and New York Heart Association class (P<0.001) (all P values ANCOVA, perhexiline versus placebo). In symptomatic hypertrophic cardiomyopathy, perhexiline, a modulator of substrate metabolism, ameliorates cardiac energetic impairment, corrects diastolic dysfunction, and increases exercise capacity. This study supports the hypothesis that energy deficiency contributes to the pathophysiology and provides a rationale for further consideration of metabolic therapies in hypertrophic cardiomyopathy.

P22 Derangements in energy, amino-acid and gut microbial metabolism in hepatic encephalopathy: a metabolomic approach

IntroductionHepatic encephalopathy (HE) pathogenesis is related to gut microbial products, which lead to deranged cerebral bioenergetics. Biofluid MR spectroscopy (MRS) can be used to determine changes in bioenergetics, gut microbial...

Age related acute effects of microcystin-LR on Daphnia magna biotransformation and oxidative stress

As a result of eutrophication among other factors, bloom forming species of cyanobacteria frequently thrive and dominate communities in freshwater ecosystems. Cyanobacteria are considered as a nuisance and hazardous due to the production of toxins. The most potent and studied cyanobacterial toxins are the microcystins, especially the variant microcystin-LR (MC-LR). Daphnia magna plays a central role in freshwater pelagic food webs and its populations are negatively affected by cyanobacteria due to nutritional inadequacy, feeding interference and toxic effects on growth and reproduction. Biotransformation and antioxidant enzymes, amongst others enable D. magna capacity to cope with MC-LR up to a certain concentration. Aim of our study was to investigate the age related acute effects of MC-LR on D. magna biotransformation and antioxidant enzymes (glutathione S-transferase, GST and catalase, CAT), subsequent lipid peroxidation and furthermore to lactate and the enzyme lactate dehydrogenase (LDH). D. magna neonates (<3 days old) and young adults (7 days old), were exposed to increasing concentrations of MC-LR (up to 100 μg/L) for 24 and 48 h. D. magna sGST activities at both ages, but more pronounced in adults, increased at low MC-LR concentrations followed by decreased activities at the higher ones suggesting diminished ability to biotransform the cyanotoxin. Oxidative protection was more efficient in neonates, where CAT was elevated strongly and contributed to buffer oxidative stress, evidenced by the constant TBARS concentrations. LDH was negatively affected by MC-LR exposure leading to constant and low concentrations of lactate. As this enzyme is involved in the production of rapidly required energy, the findings suggest an energetic impairment due to MC-LR.

Insulin differentially influences brain glucose and lactate in traumatic brain injured patients

Hypo- and hyperglycemia must be avoided to prevent additional brain damage following traumatic brain injury (TBI). However, the optimal blood glucose range requiring insulin remains unknown. Cerebral microdialysis is helpful in unmasking signs of metabolic impairment, thereby identifying deleterious blood glucose levels. A retrospective analysis of prospectively collected cerebral microdialysis samples obtained from 20 non-diabetic patients with severe TBI treated at the trauma surgical intensive care unit at the University Hospital Zürich, Switzerland. The impact of different arterial blood glucose values and concomitant insulin administration on cerebral interstitial glucose and lactate levels was investigated. In addition, energetic impairment was determined by calculating lactate-to-glucose ratios. Insulin administration was associated with significantly reduced cerebral glucose concentrations and significantly increased lactate-to-glucose ratios with arterial blood glucose levels <5 mM. At arterial blood glucose levels >7 mM, insulin administration was associated with significantly increased interstitial glucose values, significantly decreased lactate concentrations, and markedly diminished lactate-to-glucose ratios. Insulin exerts differential effects that depend strongly on the underlying arterial blood glucose concentrations. To avoid energetic impairment, insulin should not be administered at arterial blood glucose levels <5 mM. However, at arterial blood glucose levels >7-8 mM, insulin administration appears to be encouraged to increase extracellular glucose concentrations and decrease energetic impairment reflected by reduced interstitial brain lactate and decreased lactate-to-glucose ratios. Nevertheless, frequent analysis is required to minimize the risk of inducing impaired brain metabolism.

Modulation of myocardial metabolism: an emerging therapeutic principle

We focus on the molecular and cellular basis of the improvement in myocardial energetics, which might represent an attractive therapeutic option in some forms of acute and chronic heart disease. Myocardial dysfunction, whether related to left ventricular hypertrophy, heart failure or myocardial ischaemia, is frequently associated with impairment of myocardial energy balance. It is now apparent that this energetic impairment plays a pivotal role, not only in the evolution and outcomes of these disorders but also frequently in their pathogenesis. Despite the fact that energetic impairment may arise for many complex reasons, and the difficulty both in assessing the impairment in vivo and in determining its precise mechanism(s), a number of drugs have become available for treatment of ischaemia and heart failure, as well as potentially for limitation of pathological left ventricular hypertrophy, which act primarily by altering myocardial metabolism so as to improve energetic status. Recent studies with perhexiline and trimetazidine, agents which induce a 'metabolic shift' from long-chain fatty acid to glucose utilization, have demonstrated the utility of this therapeutic principle. There is ongoing need for more complete mechanistic understanding of the 'metabolic agents', as well as for the large-scale clinical trials of their impact on health outcomes.

061 Metabolic alteration in hypertrophic cardiomyopathy (METAL-HCM Study): randomised double blinded placebo controlled trial of perhexiline therapy in patient with hypertrophic cardiomyopathy

Background Patients with hypertrophic cardiomyopathy (HCM) exhibit myocardial energetic impairment, but a causative role for this energy deficiency in the pathogenesis of HCM remains unproven. We hypothesised that the metabolic modulator, perhexiline would ameliorate myocardial energy deficiency and thereby improve diastolic function and exercise capacity. Methods Forty-six consecutive patients with symptomatic exercise limitation (peak oxygen consumption – peak VO2 Results Perhexiline improved myocardial PCr/ATP ratios (from 1.27±0.02 to 1.73±0.02; p=0.003) and normalised the abnormal prolongation of nTTPF between rest and exercise (δ nTTPF: +0.11±0.008 s vs −0.01±0.005; p=0.03). These changes were accompanied by improvement in peak VO2 (22.2±0.2 to 24.29±0.2 ml/kg/min; p=0.003) and symptoms (NYHA class by 0.8 units p Conclusion In symptomatic HCM, perhexiline, a modulator of substrate metabolism, ameliorates cardiac energetic impairment, corrects diastolic dysfunction and increases exercise capacity. This study supports the hypothesis that energy deficiency contributes to the pathogenesis and provides a rationale for further consideration of metabolic therapies in HCM.

The pathophysiology of diastolic heart failure

Whilst resting disturbances of both diastolic and long-axis systolic function are observed in patients with heart failure who have normal left ventricular ejection fraction, recent evidence suggests that dynamic disturbances in cardiac function occur during exercise. A paradoxical slowing of left ventricular active relaxation during exercise limits cardiac filling and therefore stroke volume and appears to be due to the combination of cardiac energetic impairment and disturbed ventricular-vascular coupling.

Carcinoma cells activate AMP-activated protein kinase-dependent autophagy as survival response to kaempferol-mediated energetic impairment

Kaempferol, a dietary cancer chemopreventive polyphenol, has been reported to trigger apoptosis in several tumor histotypes, but the mechanism underlying this phenomenon is not fully understood. Here, we demonstrate that, in HeLa cells, kaempferol induces energetic failure due to inhibition of both glucose uptake and Complex I of the mitochondrial respiratory chain. As adaptive response, cells activate autophagy, the occurrence of which was established cytofluorometrically, upon acridine orange staining, and immunochemically, by following the increase of the autophagolysosome-associated form of the microtubule-associated protein light chain 3 (LC3-II). Autophagy is an early and reversible process occurring as survival mechanisms against apoptosis. Indeed, chemical inhibition of autophagy, by incubations with monensin, wortmannin, 3-methyladenine, or by silencing Atg5, significantly increases the extent of apoptosis, which takes place via the mitochondrial pathway, and shortens the time at which the apoptotic markers are detectable. We also demonstrate that autophagy depends on the early activation of the AMP-activated protein kinase (AMPK)/mTOR-mediated pathway. The over-expression of dominant negative AMPK results in a decrease of autophagic cells, a decrement of LC3-II levels, and a significant increase of apoptosis. Experiments performed with another carcinoma cell line yielded the same results, suggesting for kaempferol a unique mechanism of action.

Altered Mitochondrial Energetics and Increased ROS Generation Act Synergistically to Dampen β-Adrenergic Stimulated Contractility in the Diabetic Heart

Background: Excitation-contraction coupling and β-adrenergic activation are altered in diabetic hearts, contributing to contractile dysfunction. We hypothesized that mitochondrial dysfunction in diabetic hearts contributes to altered β-adrenergic responses via increased oxidative stress and respiratory uncoupling. Methods: Basal and isoproterenol (ISO)-induced changes in sarcomere shortening and Ca2+ transients were assessed in cardiomyocytes from wild-type (WT) and db/db mice under euglycemia (5.5mM) or hyperglycemia (30mM). Reactive oxygen species (ROS; H2O2 and O2.-), NADH, and ΔΨm were monitored using two photon laser scanning fluorescence microscopy. Results: Basal fractional shortening (FS) and Ca2+ transients were not significantly different between WT and db/db myocytes, regardless of glucose concentration. Following ISO (10nM), FS increased by ≅150% and Ca2+ transients by ≅30%, in both WT and db/db myocytes under euglycemia. Under hyperglycemia, the WT ISO response was intact, but the increase in FS and Ca2+ transients was blunted in db/db cells (68±2%, and 12±3%, respectively, both p<0.01 vs euglycemia). Under euglycemia, db/db cardiomyocytes had H2O2 signals 31% higher than WT (p<0.001), but under hyperglycemia, they possessed higher H2O2 (+12%; p<0.01) and lower O2.- levels (−22%; p<0.05) vs WT. Isolated WT and db/db mitochondria showed impaired respiration for substrates of Complex I (16±5 and 23±8 nmol O2min−1mg−1, respectively), but normal activity for substrates of Complexes II or IV. Impaired energetics correlated with high levels of ROS generation from Complex I or II observed under similar conditions. State 3 mitochondrial respiration with succinate and total respiratory capacity were significantly lower in db/db cells compared to WT. Conclusions: The findings suggest that the reduced effectiveness of ISO in diabetic hearts under hyperglycemia is mediated by impaired mitochondrial energetics coupled to increased oxidative stress, leading to a deleterious synergistic effect on β-adrenergic response.

The isatin-Schiff base copper(II) complex Cu(isaepy) 2 acts as delocalized lipophilic cation, yields widespread mitochondrial oxidative damage and induces AMP-activated protein kinase-dependent apoptosis

We previously demonstrated that Bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl)pyridine-N,N']copper(II) [Cu(isaepy)(2)] was an efficient inducer of the apoptotic mitochondrial pathway. Here, we deeply dissect the mechanisms underlying the ability of Cu(isaepy)(2) to cause mitochondriotoxicity. In particular, we demonstrate that Cu(isaepy)(2) increases NADH-dependent oxygen consumption of isolated mitochondria and that this phenomenon is associated with oxy-radical production and insensitive to adenosine diphosphate. These data indicate that Cu(isaepy)(2) behaves as an uncoupler and this property is also confirmed in cell systems. Particularly, SH-SY5Y cells show: (i) an early loss of mitochondrial transmembrane potential; (ii) a decrease in the expression levels of respiratory complex components and (iii) a significant adenosine triphosphate (ATP) decrement. The causative energetic impairment mediated by Cu(isaepy)(2) in apoptosis is confirmed by experiments carried out with rho(0) cells, or by glucose supplementation, where cell death is significantly inhibited. Moreover, gastric and cervix carcinoma AGS and HeLa cells, which rely most of their ATP production on oxidative phosphorylation, show a marked sensitivity toward Cu(isaepy)(2). Adenosine monophosphate-activated protein kinase (AMPK), which is activated by events increasing the adenosine monophosphate:ATP ratio, is deeply involved in the apoptotic process because the overexpression of its dominant/negative form completely abolishes cell death. Upon glucose supplementation, AMPK is not activated, confirming its role as fuel-sensing enzyme that positively responds to Cu(isaepy)(2)-mediated energetic impairment by committing cells to apoptosis. Overall, data obtained indicate that Cu(isaepy)(2) behaves as delocalized lipophilic cation and induces mitochondrial-sited reactive oxygen species production. This event results in mitochondrial dysfunction and ATP decrease, which in turn triggers AMPK-dependent apoptosis.