Dysfonction myocardique septique (conséquences des altérations de l'homéostasie calcique et mitochondriales)

La dysfonction myocardique observée au cours du choc septique est souvent attribuée aux effets délétères de nombreux médiateurs circulants issus de la réponse inflammatoire systémique (cytokines pro-inflammatoires, NO, sphingosine, etc). L'activation des voies biochimiques de l'apoptose joue également un rôle important dans l'apparition des anomalies contractiles. A l'échelon cellulaire, ces anomalies sont associées à une baisse du transitoire calcique et du potentiel de membrane mitochondrial. En revanche, à l'échelon subcellulaire, les mécanismes de la dysfonction myocardique ne sont pas encore élucidés. Notre travail a consisté, dans un premier temps, à étudier l'implication du pore de transition de perméabilité (PTP) mitochondriale dans la dysfonction cardiaque. Pour ce faire, nous avons développé une approche multiparamétrique in vitro d'étude de la fonction mitochondriale en vérifiant que les effets de la sphingosine connus sur cellules étaient similaires sur mitochondries isolées. Ensuite, l'utilisation de dérivés non-immunosuppresseurs de la cyclosporine ou la surexpression de Bcl-2 a permis de prévenir la dysfonction cardiaque consécutive à la péritonite chez la souris, prouvant ainsi l'implication du PTP dans la dysfonction myocardique septique. Dans un second travail, nous avons cherché par quels mécanismes l'activation des caspases participe à la baisse du transitoire calcique du cardiomyocyte septique. Nos résultats ont mis en évidence une augmentation de la fuite calcique et un déficit de recapture du calcium dépendant de la pompe Ca2+/ATPase (SERCA2) sur des fractions vésiculaires enrichies en réticulum sarcoplasmique (RS) isolées de coeurs de rats traités par l'endotoxine. Ces anomalies sont prévenues par le zVAD.fmk, un inhibiteur de caspases à large spectre. D'un point de vue moléculaire, nous avons pu montré que le clivage de la phosphatase de type PP2A par la caspase-3 provoque une augmentation de l'activité phosphatasique qui est alors responsable de ces anomalies du RS. Ainsi, les connaissances fondamentales acquises au cours de ce travail seront directement valorisables par la mise au point de nouvelles stratégies thérapeutiques dans la prise en charge de la dysfonction myocardiqueLILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF

Macrophage migration inhibitory factor inhibition is deleterious for high-fat diet-induced cardiac dysfunction.

AimsDevelopment of metabolic syndrome is associated with impaired cardiac performance, mitochondrial dysfunction and pro-inflammatory cytokine increase, such as the macrophage migration inhibitory factor MIF. Depending on conditions, MIF may exert both beneficial and deleterious effects on the myocardium. Therefore, we tested whether pharmacological inhibition of MIF prevented or worsened metabolic syndrome-induced myocardial dysfunction.Methods and resultsC57BL/6J mice were fed for ten weeks with 60% fat-enriched diet (HFD) or normal diet (ND). MIF inhibition was obtained by injecting mice twice a week with ISO-1, for three consecutive weeks. Then, triglycerides, cholesterol, fat mass, glucose intolerance, insulin resistance, ex vivo cardiac contractility, animal energetic substrate utilization assessed by indirect calorimetry and mitochondrial respiration and biogenesis were evaluated. HFD led to fat mass increase, dyslipidemia, glucose intolerance and insulin resistance. ISO-1 did not alter these parameters. However, MIF inhibition was responsible for HFD-induced cardiac dysfunction worsening. Mouse capacity to increase oxygen consumption in response to exercise was reduced in HFD compared to ND, and further diminished in ISO-1-treated HFD group. Mitochondrial respiration was reduced in HFD mice, treated or not with ISO-1. Compared to ND, mitochondrial biogenesis signaling was upregulated in the HFD as demonstrated by mitochondrial DNA amount and PGC-1α expression. However, this increase in biogenesis was blocked by ISO-1 treatment.ConclusionMIF inhibition achieved by ISO-1 was responsible for a reduction in HFD-induced mitochondrial biogenesis signaling that could explain majored cardiac dysfunction observed in HFD mice treated with MIF inhibitor

LRRK2 impairs PINK1/Parkin-dependent mitophagy via its kinase activity: pathologic insights into Parkinson’s disease

International audienceMutations of LRRK2, encoding leucine-rich repeat kinase 2 (LRRK2), are the leading cause of autosomal dominant Parkinson's disease (PD). The most frequent of these mutations, G2019S substitution, increases kinase activity, but it remains unclear how it causes PD. Recent studies suggest that LRRK2 modulates mitochondrial homeostasis. Mitochondrial dysfunction plays a key role in the pathogenesis of autosomal recessive PD forms linked to PARK2 and PINK1, encoding the cytosolic E3 ubiquitin-protein ligase Parkin and the mitochondrial kinase PINK1, which jointly regulate mitophagy. We explored the role of LRRK2 and its kinase activity in PINK1/Parkin-dependent mitophagy. LRRK2 increased mitochondrial aggregation and attenuated mitochondrial clearance in cells coexpressing Parkin and exposed to the protonophore carbonylcyanide m-chlorophenylhydrazone. Förster resonance energy transfer imaging microscopy showed that LRRK2 impaired the interactions between Parkin and Drp1 and their mitochondrial targets early in mitophagy. The inhibition of LRRK2 kinase activity by a 'kinase-dead' LRRK2 mutation or with a pharmacological inhibitor (LRRK2-IN-1) restored these interactions. The monitoring of mitophagy in human primary fibroblasts with the novel dual-fluorescence mtRosella reporter and a new hypothermic shock paradigm revealed similar defects in PD patients with the G2019S LRRK2 substitution or PARK2 mutations relative to healthy subjects. This defect was restored by LRRK2-IN-1 treatment in LRRK2 patients only. Our results suggest that PD forms due to LRRK2 and PARK2 mutations involve pathogenic mechanisms converging on PINK1/Parkin-dependent mitophagy

ISO-1 reduced aerobic capacity. (A) Basal <i>V</i>O₂ and (B) <i>V</i>CO₂ measured by indirect calorimetry.

<p>(C) Maximal <i>V</i>O₂ (<i>V</i>O₂max) obtained from mice subjected to treadmill exercise. (D) Difference between <i>V</i>O₂ and <i>V</i>O₂max (Δ<i>V</i>O₂) in ND, HFD and ISO-1-treated HFD mice. n = 6–10, *p<0.05 vs. ND, ^†p<0.05 vs. HFD.</p

ISO-1 treatment did not modify metabolic syndrome-associated parameters.

<p>(A) Abdominal fat mass, (B) subcutaneous fat mass, (C) abdominal to subcutaneous fat mass ratio, (D) plasma true triglyceride levels, (E) plasma cholesterol concentration and (F) fasting blood glucose. Data are means ± SEM, n = 5; *p<0.05 vs. ND. (G) Oral glucose tolerance test and (H) insulin tolerance test performed on ND, HFD and HFD+ISO-1 mice. n = 5–10 in each group. *p<0.05 vs. ND.</p

HFD induced systemic and cardiac MIF expression.

<p>(A) Tautomerase activity measured on plasma sample prepared from ND and HFD mice. Consumption of L-dopachrome-methyl ester was followed by measuring optical density at 475 nm. (B) Cardiac <i>Mif</i> transcript expression detected by RT-qPCR normalized to <i>β-actin</i>. (C) MIF protein cardiac expression observed by western-blot. Left panel is a representative western-blot, right panel is a semi-quantitation calculated after densitometric analysis. Data are means ± SEM. n = 6–10; *p<0.05 vs. ND.</p

ISO-1 amplified HFD-induced cardiac dysfunction.

<p>(A) Coronary perfusion pressure (CPP) measured in presence of rising concentration of sodium nitroprusside (SNP) on isolated heart preparations obtained from ND, ISO-1-injected ND mice (ND+ISO-1), HFD and ISO-1-treated HFD mice (HFD+ISO-1). (B) Left ventricular developed pressure (LVDP), (C) +d<i>P</i>/d<i>t</i> max and (D) −d<i>P</i>/d<i>t</i> min measured on ND, ND+ISO-1, HFD, HFD+ISO-1 hearts, in presence of the β-adrenergic receptor agonist isoproterenol. Data are means ± SEM of 5 experiments. *p<0.05 vs. ND; ^†p<0.05 vs. HFD.</p