Exploring the impact of the PNPLA3 I148M variant on primary human hepatic stellate cells using 3D extracellular matrix models.

BACKGROUND & AIMS: The PNPLA3 rs738409 C>G (encoding for I148M) variant is a risk locus for the fibrogenic progression of chronic liver diseases, a process driven by hepatic stellate cells (HSCs). We investigated how the PNPLA3 I148M variant affects HSC biology using transcriptomic data and validated findings in 3D-culture models. METHODS: RNA sequencing was performed on 2D-cultured primary human HSCs and liver biopsies of individuals with obesity, genotyped for the PNPLA3 I148M variant. Data were validated in wild-type (WT) or PNPLA3 I148M variant-carrying HSCs cultured on 3D extracellular matrix (ECM) scaffolds from human healthy and cirrhotic livers, with/without TGFB1 or cytosporone B (Csn-B) treatment. RESULTS: Transcriptomic analyses of liver biopsies and HSCs highlighted shared PNPLA3 I148M-driven dysregulated pathways related to mitochondrial function, antioxidant response, ECM remodelling and TGFB1 signalling. Analogous pathways were dysregulated in WT/PNPLA3-I148M HSCs cultured in 3D liver scaffolds. Mitochondrial dysfunction in PNPLA3-I148M cells was linked to respiratory chain complex IV insufficiency. Antioxidant capacity was lower in PNPLA3-I148M HSCs, while reactive oxygen species secretion was increased in PNPLA3-I148M HSCs and higher in bioengineered cirrhotic vs. healthy scaffolds. TGFB1 signalling followed the same trend. In PNPLA3-I148M cells, expression and activation of the endogenous TGFB1 inhibitor NR4A1 were decreased: treatment with the Csn-B agonist increased total NR4A1 in HSCs cultured in healthy but not in cirrhotic 3D scaffolds. NR4A1 regulation by TGFB1/Csn-B was linked to Akt signalling in PNPLA3-WT HSCs and to Erk signalling in PNPLA3-I148M HSCs. CONCLUSION: HSCs carrying the PNPLA3 I148M variant have impaired mitochondrial function, antioxidant responses, and increased TGFB1 signalling, which dampens antifibrotic NR4A1 activity. These features are exacerbated by cirrhotic ECM, highlighting the dual impact of the PNPLA3 I148M variant and the fibrotic microenvironment in progressive chronic liver diseases. IMPACT AND IMPLICATIONS: Hepatic stellate cells (HSCs) play a key role in the fibrogenic process associated with chronic liver disease. The PNPLA3 genetic mutation has been linked with increased risk of fibrogenesis, but its role in HSCs requires further investigation. Here, by using comparative transcriptomics and a novel 3D in vitro model, we demonstrate the impact of the PNPLA3 genetic mutation on primary human HSCs' behaviour, and we show that it affects the cell's mitochondrial function and antioxidant response, as well as the antifibrotic gene NR4A1. Our publicly available transcriptomic data, 3D platform and our findings on NR4A1 could facilitate the discovery of targets to develop more effective treatments for chronic liver diseases.

Ester Prodrugs of Malonate with Enhanced Intracellular Delivery Protect Against Cardiac Ischemia-Reperfusion Injury In Vivo.

PURPOSE: Mitochondrial reactive oxygen species (ROS) production upon reperfusion of ischemic tissue initiates the ischemia/reperfusion (I/R) injury associated with heart attack. During ischemia, succinate accumulates and its oxidation upon reperfusion by succinate dehydrogenase (SDH) drives ROS production. Inhibition of succinate accumulation and/or oxidation by dimethyl malonate (DMM), a cell permeable prodrug of the SDH inhibitor malonate, can decrease I/R injury. However, DMM is hydrolysed slowly, requiring administration to the heart prior to ischemia, precluding its administration to patients at the point of reperfusion, for example at the same time as unblocking a coronary artery following a heart attack. To accelerate malonate delivery, here we developed more rapidly hydrolysable malonate esters. METHODS: We synthesised a series of malonate esters and assessed their uptake and hydrolysis by isolated mitochondria, C2C12 cells and in mice in vivo. In addition, we assessed protection against cardiac I/R injury by the esters using an in vivo mouse model of acute myocardial infarction. RESULTS: We found that the diacetoxymethyl malonate diester (MAM) most rapidly delivered large amounts of malonate to cells in vivo. Furthermore, MAM could inhibit mitochondrial ROS production from succinate oxidation and was protective against I/R injury in vivo when added at reperfusion. CONCLUSIONS: The rapidly hydrolysed malonate prodrug MAM can protect against cardiac I/R injury in a clinically relevant mouse model.

Histological sub-classification of cirrhosis using collagen proportionate area in patients with chronic hepatitis C.

Collagen proportionate area (CPA, %) is used to quantify liver fibrosis. Here, we assessed CPA performance to sub-classify cirrhosis. CPA was measured in explanted livers from consecutively transplanted patients for hepatitis C virus-related cirrhosis. Model for end-stage liver disease (MELD), Child-Pugh score and decompensating events (ascites, variceal bleeding, non-obstructive jaundice and encephalopathy) were recorded at the time of liver transplant. Of the 154 patients, 24%, 12%, 35%, 24% and 5% had zero, one, two, three and four previous decompensating events. Patients with decompensation had significantly higher CPA than those without (25.1 ± 8.4 vs 15.8 ± 5.5, P < .001). Decompensation was independently associated with CPA, bilirubin and albumin or with CPA and MELD score. CPA did not differ between patients with one, two, three or four decompensating events (22.2 ± 6.3 vs 26.6 ± 8.9 vs 24.5 ± 7.7 vs 24.4 ± 10.9, P = .242). Overall, CPA correlates with the clinical severity of cirrhosis until the advent of decompensation but not with subsequent decompensating events.

Respiratory chain signalling is essential for adaptive remodelling following cardiac ischaemia.

Cardiac ischaemia-reperfusion (I/R) injury has been attributed to stress signals arising from an impaired mitochondrial electron transport chain (ETC), which include redox imbalance, metabolic stalling and excessive production of reactive oxygen species (ROS). The alternative oxidase (AOX) is a respiratory enzyme, absent in mammals, that accepts electrons from a reduced quinone pool to reduce oxygen to water, thereby restoring electron flux when impaired and, in the process, blunting ROS production. Hence, AOX represents a natural rescue mechanism from respiratory stress. This study aimed to determine how respiratory restoration through xenotopically expressed AOX affects the re-perfused post-ischaemic mouse heart. As expected, AOX supports ETC function and attenuates the ROS load in post-anoxic heart mitochondria. However, post-ischaemic cardiac remodelling over 3 and 9 weeks was not improved. AOX blunted transcript levels of factors known to be up-regulated upon I/R such as the atrial natriuretic peptide (Anp) whilst expression of pro-fibrotic and pro-apoptotic transcripts were increased. Ex vivo analysis revealed contractile failure at nine but not 3 weeks after ischaemia whilst label-free quantitative proteomics identified an increase in proteins promoting adverse extracellular matrix remodelling. Together, this indicates an essential role for ETC-derived signals during cardiac adaptive remodelling and identified ROS as a possible effector.

Control of mitochondrial superoxide production by reverse electron transport at complex I.

The generation of mitochondrial superoxide (O2̇̄) by reverse electron transport (RET) at complex I causes oxidative damage in pathologies such as ischemia reperfusion injury, but also provides the precursor to H2O2 production in physiological mitochondrial redox signaling. Here, we quantified the factors that determine mitochondrial O2̇̄ production by RET in isolated heart mitochondria. Measuring mitochondrial H2O2 production at a range of proton-motive force (Δp) values and for several coenzyme Q (CoQ) and NADH pool redox states obtained with the uncoupler p-trifluoromethoxyphenylhydrazone, we show that O2̇̄ production by RET responds to changes in O2 concentration, the magnitude of Δp, and the redox states of the CoQ and NADH pools. Moreover, we determined how expressing the alternative oxidase from the tunicate Ciona intestinalis to oxidize the CoQ pool affected RET-mediated O2̇̄ production at complex I, underscoring the importance of the CoQ pool for mitochondrial O2̇̄ production by RET. An analysis of O2̇̄ production at complex I as a function of the thermodynamic forces driving RET at complex I revealed that many molecules that affect mitochondrial reactive oxygen species production do so by altering the overall thermodynamic driving forces of RET, rather than by directly acting on complex I. These findings clarify the factors controlling RET-mediated mitochondrial O2̇̄ production in both pathological and physiological conditions. We conclude that O2̇̄ production by RET is highly responsive to small changes in Δp and the CoQ redox state, indicating that complex I RET represents a major mode of mitochondrial redox signaling.

Mitochondria-derived ROS activate AMP-activated protein kinase (AMPK) indirectly.

Mitochondrial reactive oxygen species (ROS) production is a tightly regulated redox signal that transmits information from the organelle to the cell. Other mitochondrial signals, such as ATP, are sensed by enzymes, including the key metabolic sensor and regulator, AMP-activated protein kinase (AMPK). AMPK responds to the cellular ATP/AMP and ATP/ADP ratios by matching mitochondrial ATP production to demand. Previous reports proposed that AMPK activity also responds to ROS, by ROS acting on redox-sensitive cysteine residues (Cys-299/Cys-304) on the AMPK α subunit. This suggests an appealing model in which mitochondria fine-tune AMPK activity by both adenine nucleotide-dependent mechanisms and by redox signals. Here we assessed whether physiological levels of ROS directly alter AMPK activity. To this end we added exogenous hydrogen peroxide (H2O2) to cells and utilized the mitochondria-targeted redox cycler MitoParaquat to generate ROS within mitochondria without disrupting oxidative phosphorylation. Mitochondrial and cytosolic thiol oxidation was assessed by measuring peroxiredoxin dimerization and by redox-sensitive fluorescent proteins. Replacing the putative redox-active cysteine residues on AMPK α1 with alanines did not alter the response of AMPK to H2O2 In parallel with measurements of AMPK activity, we measured the cell ATP/ADP ratio. This allowed us to separate the effects on AMPK activity due to ROS production from those caused by changes in this ratio. We conclude that AMPK activity in response to redox changes is not due to direct action on AMPK itself, but is a secondary consequence of redox effects on other processes, such as mitochondrial ATP production.

CD36 palmitoylation disrupts free fatty acid metabolism and promotes tissue inflammation in non-alcoholic steatohepatitis.

BACKGROUND AND AIMS: Fatty acid translocase CD36 (CD36) is a membrane protein with multiple immuno-metabolic functions. Palmitoylation has been suggested to regulate the distribution and functions of CD36, but little is known about its significance in non-alcoholic steatohepatitis (NASH). METHODS: Human liver tissue samples were obtained from patients undergoing liver biopsy for diagnostic purposes. CD36 knockout mice were injected with lentiviral vectors expressing wild-type CD36 or CD36 with mutated palmitoylation sites. Liver histology, immunofluorescence, mRNA expression profile, subcellular distributions and functions of CD36 protein were assessed. RESULTS: The localization of CD36 on the plasma membrane of hepatocytes was markedly increased in patients with NASH compared to patients with normal liver and those with simple steatosis. Increased CD36 palmitoylation and increased localization of CD36 on the plasma membrane of hepatocytes were also observed in livers of mice with NASH. Furthermore, inhibition of CD36 palmitoylation protected mice from developing NASH. The absence of palmitoylation decreased CD36 protein hydrophobicity reducing its localization on the plasma membrane as well as in lipid raft of hepatocytes. Consequently, a lack of palmitoylation decreased fatty acid uptake and CD36/Fyn/Lyn complex in HepG2 cells. Inhibition of CD36 palmitoylation not only ameliorated intracellular lipid accumulation via activation of the AMPK pathway, but also inhibited the inflammatory response through the inhibition of the JNK signaling pathway. CONCLUSIONS: Our findings demonstrate the key role of palmitoylation in regulating CD36 distributions and its functions in NASH. Inhibition of CD36 palmitoylation may represent an effective therapeutic strategy in patients with NASH. LAY SUMMARY: Fatty acid translocase CD36 (CD36) is a multifunctional membrane protein which contributes to the development of liver steatosis. In the present study, we demonstrated that the localization of CD36 on the plasma membrane of hepatocytes is increased in patients with non-alcoholic steatohepatitis. Blocking the palmitoylation of CD36 reduces CD36 distribution in hepatocyte plasma membranes and protects mice from non-alcoholic steatohepatitis. The inhibition of CD36 palmitoylation not only improved fatty acid metabolic disorders but also reduced the inflammatory response in vitro and in vivo. The present study suggests that CD36 palmitoylation is important for non-alcoholic steatohepatitis development and inhibition of CD36 palmitoylation could be used to cure non-alcoholic steatohepatitis.

Dexmedetomidine protects the heart against ischemia-reperfusion injury by an endothelial eNOS/NO dependent mechanism.

The alpha2-adrenergic receptor agonist Dexmedetomidine (Dex) is a sedative medication used by anesthesiologists. Dex protects the heart against ischemia-reperfusion (IR) and can also act as a preconditioning mimetic. The mechanisms involved in Dex-dependent cardiac preconditioning, and whether this action occurs directly or indirectly on cardiomyocytes, still remain unclear. The endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) signaling pathway and endothelial cells are known to play key roles in cardioprotection against IR injury. Therefore, the aims of this work were to evaluate whether the eNOS/NO pathway mediates the pharmacological cardiac effect of Dex, and whether endothelial cells are required in this cardioprotective action. Isolated adult rat hearts were treated with Dex (10nM) for 25min and the dimerization of eNOS and production of NO were measured. Hearts were then subjected to global IR (30/120min) and the role of the eNOS/NO pathway was evaluated. Dex promoted the activation of eNOS and production of NO. Dex reduced the infarct size and improved the left ventricle function recovery, but this effect was reversed when Dex was co-administered with inhibitors of the eNOS/NO/PKG pathway. In addition, Dex was unable to reduce cell death in isolated adult rat cardiomyocytes subjected to simulated IR. Cardiomyocyte death was attenuated by co-culturing them with endothelial cells pre-treated with Dex. In summary, our results show that Dex triggers cardiac protection by activating the eNOS/NO signaling pathway. This pharmacological effect of Dex requires its interaction with the endothelium.

Equilibrium contrast-enhanced CT imaging to evaluate hepatic fibrosis: initial validation by comparison with histopathologic sampling.

PURPOSE: To prospectively evaluate hepatic extracellular volume (ECV) fraction measurement at equilibrium computed tomographic (CT) imaging compared with both fibrosis quantified with histologic analysis and the enhanced liver fibrosis panel (ELF) in a cohort of patients with chronic hepatitis. MATERIALS AND METHODS: This prospective study was approved by the regional ethics committee. All patients gave fully informed written consent. Forty patients with a clinical indication for liver biopsy were prospectively recruited for liver ECV quantitation at equilibrium CT imaging. Biopsy samples underwent digital image analysis and assessment of collagen content expressed as the collagen-proportionate area (CPA). Spearman correlation was used to evaluate association between ECV, ELF, and CPA. Multiple regression analysis was used to test prediction of CPA by a model that combined ECV and ELF. ECV, ELF score, and CPA were compared with Ishak stage by using the Kruskal-Wallis test. RESULTS: Complete ECV, ELF, and CPA were available in 33 patients. Liver ECV, CPA, and ELF had a median of 0.26 (interquartile range [IQR], 0.24-0.29), 5.0 (IQR, 3.0-15.0), and 9.71 (IQR, 8.14-10.92), respectively. Hepatic ECV demonstrated good association with CPA (r = 0.64; P < .001) and ELF score (r = 0.38; P < .027), with no significant difference in strength of correlation (P = .177). The regression model that combined ELF and ECV achieved good prediction of CPA (R(2) = 0.67; P < .001). Significant variation in ECV and ELF was seen between fibrosis stage groups. CONCLUSION: Hepatic ECV measured with equilibrium CT imaging is associated with biopsy-derived CPA and serum ELF-validated markers of liver fibrosis. This suggests that equilibrium CT imaging can quantify diffuse fibrosis in chronic liver disease.

Hypoxia signaling controls postnatal changes in cardiac mitochondrial morphology and function.

Fetal cardiomyocyte adaptation to low levels of oxygen in utero is incompletely understood, and is of interest as hypoxia tolerance is lost after birth, leading to vulnerability of adult cardiomyocytes. It is known that cardiac mitochondrial morphology, number and function change significantly following birth, although the underlying molecular mechanisms and physiological stimuli are undefined. Here we show that the decrease in cardiomyocyte HIF-signaling in cardiomyocytes immediately after birth acts as a physiological switch driving mitochondrial fusion and increased postnatal mitochondrial biogenesis. We also investigated mechanisms of ATP generation in embryonic cardiac mitochondria. We found that embryonic cardiac cardiomyocytes rely on both glycolysis and the tricarboxylic acid cycle to generate ATP, and that the balance between these two metabolic pathways in the heart is controlled around birth by the reduction in HIF signaling. We therefore propose that the increase in ambient oxygen encountered by the neonate at birth acts as a key physiological stimulus to cardiac mitochondrial adaptation.

A novel dihydropyridine with 3-aryl meta-hydroxyl substitution blocks L-type calcium channels in rat cardiomyocytes.

RATIONALE: Dihydropyridines are widely used for the treatment of several cardiac diseases due to their blocking activity on L-type Ca(2+) channels and their renowned antioxidant properties. METHODS: We synthesized six novel dihydropyridine molecules and performed docking studies on the binding site of the L-type Ca(2+) channel. We used biochemical techniques on isolated adult rat cardiomyocytes to assess the efficacy of these molecules on their Ca(2+) channel-blocking activity and antioxidant properties. The Ca(2+) channel-blocking activity was evaluated by confocal microscopy on fluo-3AM loaded cardiomyocytes, as well as using patch clamp experiments. Antioxidant properties were evaluated by flow cytometry using the ROS sensitive dye 1,2,3 DHR. RESULTS: Our docking studies show that a novel compound with 3-OH substitution inserts into the active binding site of the L-type Ca(2+) channel previously described for nitrendipine. In biochemical assays, the novel meta-OH group in the aryl in C4 showed a high blocking effect on L-type Ca(2+) channel as opposed to para-substituted compounds. In the tests we performed, none of the molecules showed antioxidant properties. CONCLUSIONS: Only substitutions in C2, C3 and C5 of the aryl ring render dihydropyridine compounds with the capacity of blocking LTCC. Based on our docking studies, we postulate that the antioxidant activity requires a larger group than the meta-OH substitution in C2, C3 or C5 of the dihydropyridine ring.

The use of guideline images to improve histological estimation of hepatic steatosis.

BACKGROUND & AIMS: Guideline images of specific fat proportionate area (FPA) percentages have recently been published to aid the histological assessment of liver steatosis as subjective estimates of FPA are usually overestimated. To assess, (i) the effect of guideline images on accuracy and concordance of estimated FPA (eFPA), (ii) experience of steatosis grading systems on eFPA, (iii) the effect of magnification on assessment of FPA (iv) and produce a range of guideline images at x4 objective magnification (OM). METHODS: Two circulations of sample images (C1 and C2) were circulated to UK liver external quality assessment histopathology scheme members who were asked to independently evaluate steatosis. Each circulation consisted of 15 images taken at both x20 and x4OM representing the full range of steatosis. C1 was distributed first, then C2 with guideline images of FPA 6 weeks later. RESULTS: Participants overestimated FPA in C1. In C2, there was significant improvement in accuracy (P < 0.001) of eFPA for sample images with mFPA >5%. Concordance of x4OM eFPA was substantial in both circulations (C1 K = 0.878, C2 K = 0.724). CONCLUSION: The tendency to overestimate eFPA has been corroborated and can be largely corrected with the use of guideline images (without needing digital image analysis). There is a need to redefine steatosis grades that are clinically significant and validated using an accurate quantification of steatosis.

Morphological and molecular preservation through universal preparation of fresh-frozen tissue samples for multimodal imaging workflows.

The landscape of tissue-based imaging modalities is constantly and rapidly evolving. While formalin-fixed, paraffin-embedded material is still useful for histological imaging, the fixation process irreversibly changes the molecular composition of the sample. Therefore, many imaging approaches require fresh-frozen material to get meaningful results. This is particularly true for molecular imaging techniques such as mass spectrometry imaging, which are widely used to probe the spatial arrangement of the tissue metabolome. As high-quality fresh-frozen tissues are limited in their availability, any sample preparation workflow they are subjected to needs to ensure morphological and molecular preservation of the tissues and be compatible with as many of the established and emerging imaging techniques as possible to obtain the maximum possible insights from the tissues. Here we describe a universal sample preparation workflow, from the initial step of freezing the tissues to the cold embedding in a new hydroxypropyl methylcellulose/polyvinylpyrrolidone-enriched hydrogel and the generation of thin tissue sections for analysis. Moreover, we highlight the optimized storage conditions that limit molecular and morphological degradation of the sections. The protocol is compatible with human and plant tissues and can be easily adapted for the preparation of alternative sample formats (e.g., three-dimensional cell cultures). The integrated workflow is universally compatible with histological tissue analysis, mass spectrometry imaging and imaging mass cytometry, as well as spatial proteomic, genomic and transcriptomic tissue analysis. The protocol can be completed within 4 h and requires minimal prior experience in the preparation of tissue samples for multimodal imaging experiments.

Nitric oxide regulates cardiac intracellular Na⁺ and Ca²âº by modulating Na/K ATPase via PKCε and phospholemman-dependent mechanism.

In the heart, Na/K-ATPase regulates intracellular Na(+) and Ca(2+) (via NCX), thereby preventing Na(+) and Ca(2+) overload and arrhythmias. Here, we test the hypothesis that nitric oxide (NO) regulates cardiac intracellular Na(+) and Ca(2+) and investigate mechanisms and physiological consequences involved. Effects of both exogenous NO (via NO-donors) and endogenously synthesized NO (via field-stimulation of ventricular myocytes) were assessed in this study. Field stimulation of rat ventricular myocytes significantly increased endogenous NO (18 ± 2 µM), PKCε activation (82 ± 12%), phospholemman phosphorylation (at Ser-63 and Ser-68) and Na/K-ATPase activity (measured by DAF-FM dye, western-blotting and biochemical assay, respectively; p<0.05, n=6) and all were abolished by Ca(2+)-chelation (EGTA 10mM) or NOS inhibition l-NAME (1mM). Exogenously added NO (spermine-NONO-ate) stimulated Na/K-ATPase (EC50=3.8 µM; n=6/grp), via decrease in Km, in PLM(WT) but not PLM(KO) or PLM(3SA) myocytes (where phospholemman cannot be phosphorylated) as measured by whole-cell perforated-patch clamp. Field-stimulation with l-NAME or PKC-inhibitor (2 µM Bis) resulted in elevated intracellular Na(+) (22 ± 1.5 and 24 ± 2 respectively, vs. 14 ± 0.6mM in controls) in SBFI-AM-loaded rat myocytes. Arrhythmia incidence was significantly increased in rat hearts paced in the presence of l-NAME (and this was reversed by l-arginine), as well as in PLM(3SA) mouse hearts but not PLM(WT) and PLM(KO). We provide physiological and biochemical evidence for a novel regulatory pathway whereby NO activates Na/K-ATPase via phospholemman phosphorylation and thereby limits Na(+) and Ca(2+) overload and arrhythmias. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes".

Hepatic steatosis estimated microscopically versus digital image analysis.

BACKGROUND & AIMS: Evaluate in liver biopsies: (i) interobserver agreement of estimates of fat proportionate area (eFPA) and steatosis grading, (ii) the relationship between steatosis grades and measured fat proportionate area (mFPA, digital image analysis), (iii) the accuracy of eFPA, (iv) to present images to aid standardization and accuracy of eFPA. METHODS: Twenty-one liver biopsies were selected from the Royal Free Hospital (RFH) histopathology archive to represent the full range of histopathological steatosis severity. As many non-overlapping fields of parenchyma as possible were photographed at ×20 objective magnification from the biopsies (n = 651). A total of 15 sample images were selected to represent the range of steatosis seen. Twelve hepatopathologists from 11 sites worldwide independently evaluated the sample images for steatosis grade [normal (none)/mild/moderate/severe], and eFPA (% area of liver parenchyma occupied by fat). RESULTS: The hepatopathologists had good linear correlation between eFPA and mFPA for sample images (r = 0.924, P < .001) and excellent concordance (kappa = 0.91, P < 0.001). Interobserver concordance of steatosis grade showed 'substantial agreement' (kappa = 0.64). There was significant difference between eFPA and mFPA in the sample images for mild, moderate and severe steatosis (P = 0.024, P < 0.001, P < 0.001 respectively): the observers consistently over-estimated the eFPA. CONCLUSION: Hepatopathologists showed 'excellent' interobserver agreement in eFPA and 'substantial' agreement in assigning steatosis grade (precision was high). However, compared with mFPA, eFPA was inaccurate. eFPA systematically exceeds mFPA; generally the overestimation increases with severity of steatosis. Considering that non-invasive technologies for estimating liver fat utilize histopathology as reference, such assessments would benefit from quantitative validation of visually estimated microscopic liver fat percentages.

Chronic metformin associated cardioprotection against infarction: not just a glucose lowering phenomenon.

PURPOSE: Clinical and experimental investigations demonstrated that metformin, a widely used anti-diabetic drug, exhibits cardioprotective properties against myocardial infarction. Interestingly, metformin was previously shown to increase the expression of PGC-1α a key controller of energy metabolism in skeletal muscle, which is down-regulated in diabetic conditions. We hypothesized that chronic treatment with metformin could protect the aged, diabetic heart against ischemia-reperfusion injury (IRI) by up-regulating PGC-1α and improving the impaired functionality of diabetic mitochondria. METHODS: Following 4 weeks of metformin (300 mg/kg) administered in the drinking water, 12 month-old diabetic Goto Kakizaki and non-diabetic Wistar rat hearts were assigned for infarct measurement following 35 min ischemia and 60 min reperfusion or for electron microscopy (EM) and Western blotting (WB) investigations. RESULTS: Metformin elicited a cardioprotective effect in both non-diabetic and diabetic hearts. In contrast with the diabetic non-treated hearts, the diabetic hearts treated with metformin showed more organized and elongated mitochondria and demonstrated a significant increase in phosphorylated AMPK and in PGC-1α expression. CONCLUSIONS: In summary these results show for the first time that chronic metformin treatment augments myocardial resistance to ischemia-reperfusion injury, by an alternative mechanism in addition to the lowering of blood glucose. This consisted of a positive effect on mitochondrial structure possibly via a pathway involving AMPK activation and PGC-1α. Thus, metformin prescribed chronically to patients may lead to a basal state of cardioprotection thereby potentially limiting the occurrence of myocardial damage by cardiovascular events.

Simultaneously discovering the fate and biochemical effects of pharmaceuticals through untargeted metabolomics.

Untargeted metabolomics is an established approach in toxicology for characterising endogenous metabolic responses to xenobiotic exposure. Detecting the xenobiotic and its biotransformation products as part of the metabolomics analysis provides an opportunity to simultaneously gain deep insights into its fate and metabolism, and to associate the internal relative dose directly with endogenous metabolic responses. This integration of untargeted exposure and response measurements into a single assay has yet to be fully demonstrated. Here we assemble a workflow to discover and analyse pharmaceutical-related measurements from routine untargeted UHPLC-MS metabolomics datasets, derived from in vivo (rat plasma and cardiac tissue, and human plasma) and in vitro (human cardiomyocytes) studies that were principally designed to investigate endogenous metabolic responses to drug exposure. Our findings clearly demonstrate how untargeted metabolomics can discover extensive biotransformation maps, temporally-changing relative systemic exposure, and direct associations of endogenous biochemical responses to the internal dose.

Mitochondrial metabolism and bioenergetic function in an anoxic isolated adult mouse cardiomyocyte model of in vivo cardiac ischemia-reperfusion injury.

Cell models of cardiac ischemia-reperfusion (IR) injury are essential to facilitate understanding, but current monolayer cell models poorly replicate the in vivo IR injury that occurs within a three-dimensional tissue. Here we show that this is for two reasons: the residual oxygen present in many cellular hypoxia models sustains mitochondrial oxidative phosphorylation; and the loss of lactate from cells into the incubation medium during ischemia enables cells to sustain glycolysis. To overcome these limitations, we incubated isolated adult mouse cardiomyocytes anoxically while inhibiting lactate efflux. These interventions recapitulated key markers of in vivo ischemia, notably the accumulation of succinate and the loss of adenine nucleotides. Upon reoxygenation after anoxia the succinate that had accumulated during anoxia was rapidly oxidized in association with extensive mitochondrial superoxide/hydrogen peroxide production and cell injury, mimicking reperfusion injury. This cell model will enable key aspects of cardiac IR injury to be assessed in vitro.

Role of cardiac mitofusins in cardiac conduction following simulated ischemia-reperfusion.

Mitochondrial dysfunction induced by acute cardiac ischemia-reperfusion (IR), may increase susceptibility to arrhythmias by perturbing energetics, oxidative stress production and calcium homeostasis. Although changes in mitochondrial morphology are known to impact on mitochondrial function, their role in cardiac arrhythmogenesis is not known. To assess action potential duration (APD) in cardiomyocytes from the Mitofusins-1/2 (Mfn1/Mfn2)-double-knockout (Mfn-DKO) compared to wild-type (WT) mice, optical-electrophysiology was conducted. To measure conduction velocity (CV) in atrial and ventricular tissue from the Mfn-DKO and WT mice, at both baseline and following simulated acute IR, multi-electrode array (MEA) was employed. Intracellular localization of connexin-43 (Cx43) at baseline was evaluated by immunohistochemistry, while Cx-43 phosphorylation was assessed by Western-blotting. Mfn-DKO cardiomyocytes demonstrated an increased APD. At baseline, CV was significantly lower in the left ventricle of the Mfn-DKO mice. CV decreased with simulated-ischemia and returned to baseline levels during simulated-reperfusion in WT but not in atria of Mfn-DKO mice. Mfn-DKO hearts displayed increased Cx43 lateralization, although phosphorylation of Cx43 at Ser-368 did not differ. In summary, Mfn-DKO mice have increased APD and reduced CV at baseline and impaired alterations in CV following cardiac IR. These findings were associated with increased Cx43 lateralization, suggesting that the mitofusins may impact on post-MI cardiac-arrhythmogenesis.