Marie Demion

Disruption of calcium transfer from ER to mitochondria links alterations of mitochondria-associated ER membrane integrity to hepatic insulin resistance

Aims/hypothesisMitochondria-associated endoplasmic reticulum membranes (MAMs) are regions of the endoplasmic reticulum (ER) tethered to mitochondria and controlling calcium (Ca2+) transfer between both organelles through the complex formed between the voltage-dependent anion channel, glucose-regulated protein 75 and inositol 1,4,5-triphosphate receptor (IP3R). We recently identified cyclophilin D (CYPD) as a new partner of this complex and demonstrated a new role for MAMs in the control of insulin’s action in the liver. Here, we report on the mechanisms by which disruption of MAM integrity induces hepatic insulin resistance in CypD (also known as Ppif)-knockout (KO) mice.MethodsWe used either in vitro pharmacological and genetic inhibition of CYPD in HuH7 cells or in vivo loss of CYPD in mice to investigate ER–mitochondria interactions, inter-organelle Ca2+ exchange, organelle homeostasis and insulin action.ResultsPharmacological and genetic inhibition of CYPD concomitantly reduced ER–mitochondria interactions, inhibited inter-organelle Ca2+ exchange, induced ER stress and altered insulin signalling in HuH7 cells. In addition, histamine-stimulated Ca2+ transfer from ER to mitochondria was blunted in isolated hepatocytes of CypD-KO mice and this was associated with an increase in ER calcium store. Interestingly, disruption of inter-organelle Ca2+ transfer was associated with ER stress, mitochondrial dysfunction, lipid accumulation, activation of c-Jun N-terminal kinase (JNK) and protein kinase C (PKC)ε and insulin resistance in liver of CypD-KO mice. Finally, CYPD-related alterations of insulin signalling were mediated by activation of PKCε rather than JNK in HuH7 cells.Conclusions/interpretationDisruption of IP3R-mediated Ca2+ signalling in the liver of CypD-KO mice leads to hepatic insulin resistance through disruption of organelle interaction and function, increase in lipid accumulation and activation of PKCε. Modulation of ER–mitochondria Ca2+ exchange may thus provide an exciting new avenue for treating hepatic insulin resistance.

Non-enzymatic cyclic oxygenated metabolites of omega-3 polyunsaturated fatty acid: Bioactive drugs?

Non-enzymatic oxygenated metabolites derived from polyunsaturated fatty acids (PUFA) are formed inxa0vivo through free radical reaction under oxidative stress conditions. It has been over twenty-five years since the discovery of cyclic oxygenated metabolites derived from arachidonic acid (20:4 n-6), the isoprostanes, and since then they have become biomarkers of choice for assessing inxa0vivo OS in humans and animals. Chemical synthesis of n-3 PUFA isoprostanoids such as F3-Isoprostanes from eicosapentaenoic acid (20:5 n-3), and F4-Neuroprostanes from docosahexaenoic acid (22:6 n-6) unravelled novel and unexpected biological properties of such omega-3 non-enzymatic cyclic metabolites as highlighted in this review.

Trpm4 Gene Invalidation Leads to Cardiac Hypertrophy and Electrophysiological Alterations

Rationale TRPM4 is a non-selective Ca2+-activated cation channel expressed in the heart, particularly in the atria or conduction tissue. Mutations in the Trpm4 gene were recently associated with several human conduction disorders such as Brugada syndrome. TRPM4 channel has also been implicated at the ventricular level, in inotropism or in arrhythmia genesis due to stresses such as ß-adrenergic stimulation, ischemia-reperfusion, and hypoxia re-oxygenation. However, the physiological role of the TRPM4 channel in the healthy heart remains unclear. Objectives We aimed to investigate the role of the TRPM4 channel on whole cardiac function with a Trpm4 gene knock-out mouse (Trpm4 -/-) model. Methods and Results Morpho-functional analysis revealed left ventricular (LV) eccentric hypertrophy in Trpm4 -/- mice, with an increase in both wall thickness and chamber size in the adult mouse (aged 32 weeks) when compared to Trpm4+/+ littermate controls. Immunofluorescence on frozen heart cryosections and qPCR analysis showed no fibrosis or cellular hypertrophy. Instead, cardiomyocytes in Trpm4-/- mice were smaller than Trpm4+/+with a higher density. Immunofluorescent labeling for phospho-histone H3, a mitosis marker, showed that the number of mitotic myocytes was increased 3-fold in the Trpm4-/-neonatal stage, suggesting hyperplasia. Adult Trpm4 -/- mice presented multilevel conduction blocks, as attested by PR and QRS lengthening in surface ECGs and confirmed by intracardiac exploration. Trpm4-/-mice also exhibited Luciani-Wenckebach atrioventricular blocks, which were reduced following atropine infusion, suggesting paroxysmal parasympathetic overdrive. In addition, Trpm4 -/- mice exhibited shorter action potentials in atrial cells. This shortening was unrelated to modifications of the voltage-gated Ca2+ or K+ currents involved in the repolarizing phase. Conclusions TRPM4 has pleiotropic roles in the heart, including the regulation of conduction and cellular electrical activity which impact heart development.

Non-enzymatic oxidized metabolite of DHA, 4(RS)-4-F4t-neuroprostane protects the heart against reperfusion injury.

Abstract Acute myocardial infarction leads to an increase in oxidative stress and lipid peroxidation. 4(RS)‐4‐F4t‐Neuroprostane (4‐F4t‐NeuroP) is a mediator produced by non‐enzymatic free radical peroxidation of the cardioprotective polyunsaturated fatty acid, docosahexaenoic acid (DHA). In this study, we investigated whether intra‐cardiac delivery of 4‐F4t‐NeuroP (0.03 mg/kg) prior to occlusion (ischemia) prevents and protects rat myocardium from reperfusion damages. Using a rat model of ischemic‐reperfusion (I/R), we showed that intra‐cardiac infusion of 4‐F4t‐NeuroP significantly decreased infarct size following reperfusion (−27%) and also reduced ventricular arrhythmia score considerably during reperfusion (−41%). Most notably, 4‐F4t‐NeuroP decreased ventricular tachycardia and post‐reperfusion lengthening of QT interval. The evaluation of the mitochondrial homeostasis indicates a limitation of mitochondrial swelling in response to Ca2+ by decreasing the mitochondrial permeability transition pore opening and increasing mitochondria membrane potential. On the other hand, mitochondrial respiration measured by oxygraphy, and mitochondrial ROS production measured with MitoSox red® were unchanged. We found decreased cytochrome c release and caspase 3 activity, indicating that 4‐F4t‐NeuroP prevented reperfusion damages and reduced apoptosis. In conclusion, 4‐F4t‐NeuroP derived from DHA was able to protect I/R cardiac injuries by regulating the mitochondrial homeostasis. HighlightsThe lipid mediator 4(RS)‐4‐F4t‐neuroprostane derived from non‐enzymatic peroxidation of DHA contributes to cardioprotective properties of this PUFA following an ischemia/reperfusion event.4(RS)‐4‐F4tneuroprostane displays a strong anti‐apoptotic property involving the normalization of calcium homeostasis by the stabilization of ryanodine receptor complex and to a decrease of mPTP opening leading to the reduction of pro‐apoptotic factors.This study suggests that some well‐known effects of n‐3 fatty acids are mediated by their non‐enzymatic cyclic oxygenated metabolites.This discovery opens new perspectives for non‐enzymatic oxidized products of n‐3 polyunsaturated fatty acids as potent preventive therapeutic way in acute myocardial infarction.

The TRPM4 channel is functionally important for the beneficial cardiac remodeling induced by endurance training

Cardiac hypertrophy (CH) is an adaptive process that exists in two distinct forms and allows the heart to adequately respond to an organism’s needs. The first form of CH is physiological, adaptive and reversible. The second is pathological, irreversible and associated with fibrosis and cardiomyocyte death. CH involves multiple molecular mechanisms that are still not completely defined but it is now accepted that physiological CH is associated more with the PI3-K/Akt pathway while the main signaling cascade activated in pathological CH involves the Calcineurin-NFAT pathway. It was recently demonstrated that the TRPM4 channel may act as a negative regulator of pathological CH by regulating calcium entry and thus the Cn-NFAT pathway. In this study, we examined if the TRPM4 channel is involved in the physiological CH process. We evaluated the effects of 4 weeks endurance training on the hearts of Trpm4+/+ and Trpm4−/− mice. We identified an elevated functional expression of the TRPM4 channel in cardiomyocytes after endurance training suggesting a potential role for the channel in physiological CH. We then observed that Trpm4+/+ mice displayed left ventricular hypertrophy after endurance training associated with enhanced cardiac function. By contrast, Trpm4−/− mice did not develop these adaptions. While Trpm4−/− mice did not develop gross cardiac hypertrophy, the cardiomyocyte surface area was larger and associated with an increase of Tunel positive cells. Endurance training in Trpm4+/+ mice did not increase DNA fragmentation in the heart. Endurance training in Trpm4+/+ mice was associated with activation of the classical physiological CH Akt pathway while Trpm4−/− favored the Calcineurin pathway. Calcium studies demonstrated that TRPM4 channel negatively regulates calcium entry providing support for activation of the Cn-NFAT pathway in Trpm4−/− mice. In conclusion, we provide evidence for the functional expression of TRPM4 channel in response to endurance training. This expression may help to maintain the balance between physiological and pathological hypertrophy.

p11 modulates calcium handling through 5-HT4R pathway in rat ventricular cardiomyocytes

BACKGROUNDnThe role of the serotonin receptor 4 (5-HT4R) pathway in cardiac excitation-contraction coupling (ECC) remains unclear. In the brain, induction of the calcium (Ca(2+))-binding protein p11 enhances 5-HT4R translocation and signaling and could therefore be considered as a modulator of the 5-HT4R pathway in the myocardium. p11 expression is increased by brain-derived neurotrophic factor (BDNF) or antidepressant drugs (imipramine). Thus, we investigated whether p11 regulates the 5-HT4R pathway in the heart in physiological conditions or under pharmacological induction and the effects on calcium handling.nnnMETHODS AND RESULTSnp11 expression was induced in vivo in healthy Wistar rats by imipramine (10 mg/kg/21 days) and in vitro in left ventricular cardiomyocytes exposed to BDNF (50 ng/ml/8h). Cell shortening and real-time Ca(2+) measurements were processed on field-stimulated intact cardiomyocytes with the selective 5-HT4R agonist, prucalopride (1 μM). Both imipramine and BDNF-induced cardiomyocyte p11 expression unmasked a strong response to prucalopride characterized by an increase of both cell shortening and Ca(2+) transient amplitude compared to basal prucalopride associated with a high propensity to trigger diastolic Ca(2+) events. Healthy rats treated with BDNF (180 ng/day/14 days) exhibited a sustained elevated heart rate following a single injection of prucalopride (0.1 mg/kg) which was not observed prior to treatment.nnnCONCLUSIONSnWe have identified a novel role for p11 in 5-HT4R signaling in healthy rat ventricular cardiomyocytes. Increased p11 expression by BDNF and imipramine unraveled a 5-HT4R-mediated modulation of cardiac Ca(2+) handling and ECC associated with deleterious Ca(2+) flux disturbances. Such mechanism could partly explain some cardiac adverse effects induced by antidepressant treatments.

No Additive Effects of Polyphenol Supplementation and Exercise Training on White Adiposity Determinants of High-Fat Diet-Induced Obese Insulin-Resistant Rats

One of the major insulin resistance instigators is excessive adiposity and visceral fat depots. Individually, exercise training and polyphenol intake are known to exert health benefits as improving insulin sensitivity. However, their combined curative effects on established obesity and insulin resistance need further investigation particularly on white adipose tissue alterations. Therefore, we compared the effects on different white adipose tissue depot alterations of a combination of exercise and grape polyphenol supplementation in obese insulin-resistant rats fed a high-fat diet to the effects of a high-fat diet alone or a nutritional supplementation of grape polyphenols (50u2009mg/kg/day) or exercise training (1u2009hr/day to 5u2009days/wk consisting of treadmill running at 32u2009m/min for a 10% slope), for a total duration of 8 weeks. Separately, polyphenol supplementation and exercise decreased the quantity of all adipose tissue depots and mesenteric inflammation. Exercise reduced adipocytes size in all fat stores. Interestingly, combining exercise to polyphenol intake presents no more cumulative benefit on adipose tissue alterations than exercise alone. Insulin sensitivity was improved at systemic, epididymal, and inguinal adipose tissues levels in trained rats thus indicating that despite their effects on adipocyte morphological/metabolic changes, polyphenols at nutritional doses remain less effective than exercise in fighting insulin resistance.

Biological activities of non-enzymatic oxygenated metabolites of polyunsaturated fatty acids (NEO-PUFAs) derived from EPA and DHA: New anti-arrhythmic compounds?

ω3 Polyunsaturated fatty acids (ω3 PUFAs) have several biological properties including anti-arrhythmic effects. However, there are some evidences that it is not solely ω3 PUFAs per se that are biologically active but the non-enzymatic oxygenated metabolites of polyunsaturated fatty acids (NEO-PUFAs) like isoprostanes and neuroprostanes. Recent question arises how these molecules take part in physiological homeostasis, show biological bioactivities and anti-inflammatory properties. Furthermore, they are involved in the circulations of childbirth, by inducing the closure of the ductus arteriosus. In addition, oxidative stress which can be beneficial for the heart in given environmental conditions such as the presence of ω3 PUFAs on the site of the stress and the signaling pathways involved are also explained in this review.

0509 : Beta 2 adrenegic receptor expression and activation of endogenous progenitor cells

Background Endogenous progenitor cells may participate in cardiac repair after a myocardial infarction. The beta adrenergic pathway has been proposed to induce proliferation and migration of progenitor cells. However the mechanisms have not yet been clarified. Methods and results The mechanism underlying beta adrenergic signaling on endogenous c-kit+/CD45 – cardiac cells was investigated by inducing myocardial infarction in adult mice. Hearts were dissociated and flow cytometry analysis demonstrated that one week after ligation, the percentage of c-kit+/ CD45 – cells expressing beta 1 or beta 2 adrenergic receptor was significantly increased (88.1±3% and 106.8±36.5% increase compared to sham respectively). Flow cytometry studies on cultured cardiac c-kit+/CD45 – cells confirmed increased beta 1 and 2 adrenergic receptor expression in response to stress conditions, specifically hypoxia (5%) or serum starvation. Interestingly, stress conditions altered localization of the beta 2 adrenergic receptor by increasing membrane expression. The beta 2 adrenergic receptor signaling pathway was stimulated in adult sham mice with the agonist fenoterol (0.25xa0mg/kg/day) administered in drinking water. Seven days after treatment the mice and non-treated controls were sacrificed and progenitor cells were measured by flow cytometry in the heart and blood. Fenoterol increased the proliferation and percentage of c-kit+/CD45 – cells in the heart (123.3±86.2% and 70.9±44.6% increase compared to control respectively). Fenoterol treatment also elevated levels of circulating endothelial progenitor cells (158.5±87.9% compared to control) and c-kit/CD45 – cells (70.6±33.5% increase) in the peripheral blood. Conclusion Beta adrenergic receptor expression is increased after coronary ligation in vivo and in stress conditions in vitro . A beta 2 adrenergic receptor agonist may be used to improve endogenous cardiac repair through the activation of progenitor cells. The author hereby declares no conflict of interest