Délétion de Tspo chez le rat : caractérisation du phénotype des animaux et effet sur le transport de cholestérol & les fonctions des mitochondries cardiaques

Cholesterol is essential for eukaryotic cells and its homeostasis is tightly regulated. Hypercholesterolemia is a major risk factor for myocardial infarction, which corresponds to myocardial necrosis following an insufficient oxygen supply. The only therapeutic option is the restoration of blood circulation but this causes additional lesions, called reperfusion lesions. Mitochondria play a major role in these lesions which are associated with a massive accumulation of cholesterol in these organelles. In steroidogenic tissues, cholesterol is transported to the mitochondria by a specialized multiprotein complex, composed in particular of the translocator protein (TSPO) and steroid acute regulatory protein (STAR), whose role in the myocardium remains to be determined. In this context, TSPO ligands have shown cardioprotective effects, linked to the preservation of mitochondria. In this work, we developed a colony of Tspo-deleted rats and characterized their phenotype, and developed a hypoxia-reoxygenation model on cells of the AC16 line. We then identified, using deleted rats, a mechanism involving TSPO and STAR in the mitochondrial accumulation of cholesterol observed during reperfusion of an ischemic myocardium. This mechanism represents a potential target for developing cardioprotective strategies. On the other hand, the rats deleted for Tspo do not show an altered phenotype, whether at the cardiac level, steroidogenesis or heme biosynthesis. The TSPO remains to this day an enigma as to its function.Le cholestérol est fondamental pour les cellules eucaryotes et son homéostasie est étroitement régulée. L’hypercholestérolémie est un facteur de risque majeur de l’infarctus du myocarde,qui correspond à la nécrose du myocarde suite à un apport insuffisant en oxygène. La seule option thérapeutique est la restauration de la circulation sanguine mais elle engendre des lésions supplémentaires, dites lésions de reperfusion. Les mitochondries jouent un rôle majeur dans ces lésions qui sont associées à une accumulation massive de cholestérol dans ces organites. Dans les tissus stéroïdogènes, le cholestérol est transporté à la mitochondrie par un complexe multiprotéique spécialisé, composé notamment de la protéine translocatrice (TSPO) et de la protéine de régulation aiguë de la stéroïdogenèse (STAR), dont le rôle dans le myocarde reste à déterminer. Dans ce contexte,des ligands de TSPO ont montré des effets cardioprotecteurs, lié à la préservation des mitochondries. Dans ce travail, nous avons développé une colonie de rats délétés pour Tspo et caractérisé leur phénotype, et développé un modèle d’hypoxie-réoxygénation sur des cellules de la lignée AC16. Nous avons ensuite identifié, à l’aide des rats délétés, un mécanisme impliquant TSPO et STAR dans l’accumulation mitochondriale de cholestérol observé lors de la reperfusion d’un myocarde ischémié. Ce mécanisme représente une cible potentielle pour développer des stratégies cardioprotectrices. En revanche, les rats délétés pour Tspo ne présentent pas de phénotype altéré,que ce soit au niveau cardiaque, de la stéroïdogenèse et de la biosynthèse de l’hème. La TSPO reste à ce jour une énigme quant à sa fonction

Délétion de Tspo chez le rat : caractérisation du phénotype des animaux et effet sur le transport de cholestérol & les fonctions des mitochondries cardiaques

Cholesterol is essential for eukaryotic cells and its homeostasis is tightly regulated. Hypercholesterolemia is a major risk factor for myocardial infarction, which corresponds to myocardial necrosis following an insufficient oxygen supply. The only therapeutic option is the restoration of blood circulation but this causes additional lesions, called reperfusion lesions. Mitochondria play a major role in these lesions which are associated with a massive accumulation of cholesterol in these organelles. In steroidogenic tissues, cholesterol is transported to the mitochondria by a specialized multiprotein complex, composed in particular of the translocator protein (TSPO) and steroid acute regulatory protein (STAR), whose role in the myocardium remains to be determined. In this context, TSPO ligands have shown cardioprotective effects, linked to the preservation of mitochondria. In this work, we developed a colony of Tspo-deleted rats and characterized their phenotype, and developed a hypoxia-reoxygenation model on cells of the AC16 line. We then identified, using deleted rats, a mechanism involving TSPO and STAR in the mitochondrial accumulation of cholesterol observed during reperfusion of an ischemic myocardium. This mechanism represents a potential target for developing cardioprotective strategies. On the other hand, the rats deleted for Tspo do not show an altered phenotype, whether at the cardiac level, steroidogenesis or heme biosynthesis. The TSPO remains to this day an enigma as to its function.Le cholestérol est fondamental pour les cellules eucaryotes et son homéostasie est étroitement régulée. L’hypercholestérolémie est un facteur de risque majeur de l’infarctus du myocarde,qui correspond à la nécrose du myocarde suite à un apport insuffisant en oxygène. La seule option thérapeutique est la restauration de la circulation sanguine mais elle engendre des lésions supplémentaires, dites lésions de reperfusion. Les mitochondries jouent un rôle majeur dans ces lésions qui sont associées à une accumulation massive de cholestérol dans ces organites. Dans les tissus stéroïdogènes, le cholestérol est transporté à la mitochondrie par un complexe multiprotéique spécialisé, composé notamment de la protéine translocatrice (TSPO) et de la protéine de régulation aiguë de la stéroïdogenèse (STAR), dont le rôle dans le myocarde reste à déterminer. Dans ce contexte,des ligands de TSPO ont montré des effets cardioprotecteurs, lié à la préservation des mitochondries. Dans ce travail, nous avons développé une colonie de rats délétés pour Tspo et caractérisé leur phénotype, et développé un modèle d’hypoxie-réoxygénation sur des cellules de la lignée AC16. Nous avons ensuite identifié, à l’aide des rats délétés, un mécanisme impliquant TSPO et STAR dans l’accumulation mitochondriale de cholestérol observé lors de la reperfusion d’un myocarde ischémié. Ce mécanisme représente une cible potentielle pour développer des stratégies cardioprotectrices. En revanche, les rats délétés pour Tspo ne présentent pas de phénotype altéré,que ce soit au niveau cardiaque, de la stéroïdogenèse et de la biosynthèse de l’hème. La TSPO reste à ce jour une énigme quant à sa fonction

Ferutinine as a calcium ionophore for the investigation of mitochondrial permeability transition pore opening in murine isolated adult cardiomyocytes

International audienc

Opening of mitochondrial permeability transition pore in cardiomyocytes: is ferutinin a suitable tool for its assessment?

International audienceMitochondrial permeability transition pore (mPTP) opening is a critical event leading to cell injury during myocardial ischemia-reperfusion but having a reliable cellular model to study the effect of drugs targeting mPTP is an unmet need. This study evaluated whether the Ca2+ electrogenic ionophore ferutinin is a relevant tool to induce mPTP in cardiomyocytes. mPTP opening was monitored using the calcein/cobalt fluorescence technique in adult cardiomyocytes isolated from wild-type and cyclophylin D (CypD) knock-out mice. Concomitantly, the effect of ferutinin was assessed in isolated myocardial mitochondria. Our results confirmed the Ca2+ ionophoric effect of ferutinin in isolated mitochondria and cardiomyocytes. Ferutinin induced all the hallmarks of mPTP opening in cells (loss of calcein, of mitochondrial potential and cell death), but none of them could be inhibited by CypD deletion or cyclosporine A, indicating that mPTP opening was not the major contributor to the effect of ferutinin. This was confirmed in isolated mitochondria where ferutinin acts by different mechanisms dependent and independent of the mitochondrial membrane potential. At low ferutinin/mitochondria concentration ratio, ferutinin displays protonophoric-like properties, lowering the mitochondrial membrane potential and limiting oxidative phosphorylation without mitochondrial swelling. At high ferutinin/mitochondria ratio, ferutinin induced a sudden Ca2+ independent mitochondrial swelling, which is only partially inhibited by cyclosporine A.Together, these result show that ferutinin is not a suitable tool to investigate CypD-dependent mPTP opening in isolated cardiomyocytes because it possesses other mitochondrial properties such as swelling induction and mitochondrial uncoupling properties which impede its utilization

Effects of TSPO and STAR inhibitors on cell death in a cardiomyocyte model of hypoxia-reoxygenation

International audienceHypoxia-reoxygenation TSPO-STAR Introduction We previously demonstrated that reperfusion of an ischemic myocardium induces an increase in mitochondrial cholesterol (CL) content accompanied by a generation of oxysterols. The translocator protein (TSPO) and the steroid acute regulatory protein (STAR) are involved in CL transport at the mitochondrial membrane in steroidogenic tissues but in the heart their role remain uncertain. The TSPO ligand 4'-chlorodiazepam (4'CDZ) has been demonstrated to inhibit STAR and sterols mitochondrial accumulations and to reduce infarct size. These data suggest that targeting mitochondrial sterol accumulation could participate to the protective effects of TSPO ligands. Objective To analyze the mechanisms and the role of the mitochondrial CL transport in cell death with a cardiomyocyte model of hypoxiareoxygenation (HR). Method AC16 human cardiomyocytes were submitted to different durations of hypoxia (1% O2) followed by reoxygenation (21% O2) to achieve 50% mortality. Cells were then treated at reoxygenation with 4'CDZ and novel TSPO and STAR inhibitors, known to delay steroidogenesis in vitro by targeting CL specific binding sites of these proteins (CRAC and START, respectively). Cell mortality was assessed with MTT and crystal violet assay and CL was identified by means of fluorescent probes. Results HR induced 41±3% mortality and modified membrane CL pattern in the cells. In this model, 4'-CDZ (10 µM) did not display cardioprotective effect (44±3% mortality). Similar results were observed with the TSPO inhibitors (CRAC benzamide 100 µM, CRAC triol 100 µM) and the STAR inhibitor (START triol 100 µM) (46±1% 43±3% and 54±3% mortality, respectively). Conclusion This preliminary study suggests that TSPO ligands do not exert cardioprotection through a direct action on cardiomyocytes. This conclusion needs to be confirmed with the use of adult primary cardiomyocytes

Identification of a mechanism promoting mitochondrial sterol accumulation during myocardial ischemia–reperfusion: role of TSPO and STAR

International audienceHypercholesterolemia is a major risk factor for coronary artery diseases and cardiac ischemic events. Cholesterol per se could also have negative effects on the myocardium, independently from hypercholesterolemia. Previously, we reported that myocardial ischemia-reperfusion induces a deleterious build-up of mitochondrial cholesterol and oxysterols, which is potentiated by hypercholesterolemia and prevented by translocator protein (TSPO) ligands. Here, we studied the mechanism by which sterols accumulate in cardiac mitochondria and promote mitochondrial dysfunction. We performed myocardial ischemia-reperfusion in rats to evaluate mitochondrial function, TSPO and steroidogenic acute regulatory protein (STAR) levels and the related mitochondrial concentrations of sterols. Rats were treated with the cholesterol synthesis inhibitor pravastatin or the TSPO ligand 4’-chlorodiazepam. We used Tspo deleted rats, which were phenotypically characterized. Inhibition of cholesterol synthesis reduced mitochondrial sterol accumulation and protected mitochondria during myocardial ischemia-reperfusion. We found that cardiac mitochondrial sterol accumulation is the consequence of enhanced influx of cholesterol and not of the inhibition of its mitochondrial metabolism during ischemia-reperfusion. Mitochondrial cholesterol accumulation at reperfusion was related to an increase in mitochondrial STAR but not to changes in TSPO levels. 4’-Chlorodiazepam inhibited this mechanism and prevented mitochondrial sterol accumulation and mitochondrial ischemia-reperfusion injury, underlying the close cooperation between STAR and TSPO. Conversely, Tspo deletion, which did not alter cardiac phenotype, abolished the effects of 4’-chlorodiazepam. This study reveals a novel mitochondrial interaction between TSPO and STAR to promote cholesterol and deleterious sterol mitochondrial accumulation during myocardial ischemia-reperfusion. This interaction regulates mitochondrial homeostasis and plays a key role during mitochondrial injury

Dysregulated Phenylalanine Catabolism Plays a Key Role in the Trajectory of Cardiac Aging

International audienceBackground: Aging myocardium undergoes progressive cardiac hypertrophy and interstitial fibrosis with diastolic and systolic dysfunction. Recent metabolomics studies shed light on amino acids in aging. The present study aimed to dissect how aging leads to elevated plasma levels of the essential amino acid phenylalanine and how it may promote age-related cardiac dysfunction. Methods: We studied cardiac structure and function, together with phenylalanine catabolism in wild-type (WT) and p21 −/− mice (male; 2–24 months), with the latter known to be protected from cellular senescence. To explore phenylalanine’s effects on cellular senescence and ectopic phenylalanine catabolism, we treated cardiomyocytes (primary adult rat or human AC-16) with phenylalanine. To establish a role for phenylalanine in driving cardiac aging, WT male mice were treated twice a day with phenylalanine (200 mg/kg) for a month. We also treated aged WT mice with tetrahydrobiopterin (10 mg/kg), the essential cofactor for the phenylalanine-degrading enzyme PAH (phenylalanine hydroxylase), or restricted dietary phenylalanine intake. The impact of senescence on hepatic phenylalanine catabolism was explored in vitro in AML12 hepatocytes treated with Nutlin3a (a p53 activator), with or without p21-targeting small interfering RNA or tetrahydrobiopterin, with quantification of PAH and tyrosine levels. Results: Natural aging is associated with a progressive increase in plasma phenylalanine levels concomitant with cardiac dysfunction, whereas p21 deletion delayed these changes. Phenylalanine treatment induced premature cardiac deterioration in young WT mice, strikingly akin to that occurring with aging, while triggering cellular senescence, redox, and epigenetic changes. Pharmacological restoration of phenylalanine catabolism with tetrahydrobiopterin administration or dietary phenylalanine restriction abrogated the rise in plasma phenylalanine and reversed cardiac senescent alterations in aged WT mice. Observations from aged mice and human samples implicated age-related decline in hepatic phenylalanine catabolism as a key driver of elevated plasma phenylalanine levels and showed increased myocardial PAH-mediated phenylalanine catabolism, a novel signature of cardiac aging. Conclusions: Our findings establish a pathogenic role for increased phenylalanine levels in cardiac aging, linking plasma phenylalanine levels to cardiac senescence via dysregulated phenylalanine catabolism along a hepatic-cardiac axis. They highlight phenylalanine/PAH modulation as a potential therapeutic strategy for age-associated cardiac impairment

Dysregulated Phenylalanine Catabolism Plays a Key Role in the Trajectory of Cardiac Aging

International audienceBackground: Aging myocardium undergoes progressive cardiac hypertrophy and interstitial fibrosis with diastolic and systolic dysfunction. Recent metabolomics studies shed light on amino acids in aging. The present study aimed to dissect how aging leads to elevated plasma levels of the essential amino acid phenylalanine and how it may promote age-related cardiac dysfunction. Methods: We studied cardiac structure and function, together with phenylalanine catabolism in wild-type (WT) and p21 −/− mice (male; 2–24 months), with the latter known to be protected from cellular senescence. To explore phenylalanine’s effects on cellular senescence and ectopic phenylalanine catabolism, we treated cardiomyocytes (primary adult rat or human AC-16) with phenylalanine. To establish a role for phenylalanine in driving cardiac aging, WT male mice were treated twice a day with phenylalanine (200 mg/kg) for a month. We also treated aged WT mice with tetrahydrobiopterin (10 mg/kg), the essential cofactor for the phenylalanine-degrading enzyme PAH (phenylalanine hydroxylase), or restricted dietary phenylalanine intake. The impact of senescence on hepatic phenylalanine catabolism was explored in vitro in AML12 hepatocytes treated with Nutlin3a (a p53 activator), with or without p21-targeting small interfering RNA or tetrahydrobiopterin, with quantification of PAH and tyrosine levels. Results: Natural aging is associated with a progressive increase in plasma phenylalanine levels concomitant with cardiac dysfunction, whereas p21 deletion delayed these changes. Phenylalanine treatment induced premature cardiac deterioration in young WT mice, strikingly akin to that occurring with aging, while triggering cellular senescence, redox, and epigenetic changes. Pharmacological restoration of phenylalanine catabolism with tetrahydrobiopterin administration or dietary phenylalanine restriction abrogated the rise in plasma phenylalanine and reversed cardiac senescent alterations in aged WT mice. Observations from aged mice and human samples implicated age-related decline in hepatic phenylalanine catabolism as a key driver of elevated plasma phenylalanine levels and showed increased myocardial PAH-mediated phenylalanine catabolism, a novel signature of cardiac aging. Conclusions: Our findings establish a pathogenic role for increased phenylalanine levels in cardiac aging, linking plasma phenylalanine levels to cardiac senescence via dysregulated phenylalanine catabolism along a hepatic-cardiac axis. They highlight phenylalanine/PAH modulation as a potential therapeutic strategy for age-associated cardiac impairment