Sandrine Pons

Role of hypoxia inducible factor-1α in remote limb ischemic preconditioning

Remote ischemic preconditioning (RIPC) has emerged as a feasible and attractive therapeutic procedure for heart protection against ischemia/reperfusion (I/R) injury. However, its molecular mechanisms remain poorly understood. Hypoxia inducible factor-1α (HIF-1α) is a transcription factor that plays a key role in the cellular adaptation to hypoxia and ischemia. This studys aim was to test whether RIPC-induced cardioprotection requires HIF-1α upregulation to be effective. In the first study, wild-type mice and mice heterozygous for HIF1a (gene encoding the HIF-1α protein) were subjected to RIPC immediately before myocardial infarction (MI). RIPC resulted in a robust HIF-1α activation in the limb and acute cardioprotection in wild-type mice. RIPC-induced cardioprotection was preserved in heterozygous mice, despite the low HIF-1α expression in their limbs. In the second study, the role of HIF-1α in RIPC was evaluated using cadmium (Cd), a pharmacological HIF-1α inhibitor. Rats were subjected to MI (MI group) or to RIPC immediately prior to MI (R-MI group). Cd was injected 18 0min before RIPC (Cd-R-MI group). RIPC induced robust HIF-1α activation in rat limbs and significantly reduced infarct size (IS). Despite Cds inhibition of HIF-1α activation, RIPC-induced cardioprotection was preserved in the Cd-R-MI group. RIPC applied immediately prior to MI increased HIF-1α expression and attenuated IS in rats and wild-type mice. However, RIPC-induced cardioprotection was preserved in partially HIF1a-deficient mice and in rats pretreated with Cd. When considered together, these results suggest that HIF-1α upregulation is unnecessary in acute RIPC.

Regular treadmill exercise restores cardioprotective signaling pathways in obese mice independently from improvement in associated co-morbidities

Obesity is a major health issue that impedes the ability of preconditioning and postconditioning to protect the myocardium against infarction secondary to dysregulation of kinase signaling pathways. Moreover, exercise decreases cardiovascular mortality in obese patients but the mechanism remains to be established. Wild-type (WT) and obese (ob/ob) mice were assigned to sedentary conditions or regular treadmill exercise (1h/day, 5 days/7, 4 weeks, 4° slope, 10-30 cm/s) and underwent 30 min of coronary artery occlusion followed by 24h of reperfusion for infarct size measurement. In WT, exercise reduced infarct size by 60% and increased phosphorylation of kinases such as Akt, ERK 1/2, p70S6K, AMPK and GSK3β. Importantly, the level of corresponding phosphatases PTEN, MKP-3 and PP2C was decreased. Calcium concentration inducing the opening of mitochondrial permeability transition pore (mPTP) was increased by exercise. In ob/ob, regular exercise induced a robust cardioprotection by reducing infarct size (-67%), increasing kinase phosphorylation, decreasing phosphatase levels and improving the resistance to mPTP opening. However exercise did not modify hyperglycemia, hypercholesterolemia, hyperinsulinemia, fat mass and body weight in obese mice. In conclusion, regular exercise induces cardioprotection against myocardial infarction despite obesity and restores pro-survival signaling pathways with simultaneous increase in kinase phosphorylations, decreased levels of phosphatases and increased resistance of mPTP opening, independently from improvement in associated co-morbidities.

Mild hypothermia reduces per-ischemic reactive oxygen species production and preserves mitochondrial respiratory complexes

BACKGROUNDnMitochondrial dysfunction is critical following ischemic disorders. Our goal was to determine whether mild hypothermia could limit this dysfunction through per-ischemic inhibition of reactive oxygen species (ROS) generation.nnnMETHODSnFirst, ROS production was evaluated during simulated ischemia in an vitro model of isolated rat cardiomyocytes at hypothermic (32°C) vs. normothermic (38°C) temperatures. Second, we deciphered the direct effect of hypothermia on mitochondrial respiration and ROS production in oxygenated mitochondria isolated from rabbit hearts. Third, we investigated these parameters in cardiac mitochondria extracted after 30-min of coronary artery occlusion (CAO) under normothermic conditions (CAO-N) or with hypothermia induced by liquid ventilation (CAO-H; target temperature: 32°C).nnnRESULTSnIn isolated rat cardiomyocytes, per-ischemic ROS generation was dramatically decreased at 32 vs. 38°C (e.g., -55±8% after 140min of hypoxia). In oxygenated mitochondria isolated from intact rabbit hearts, hypothermia also improved respiratory control ratio (+22±3%) and reduced H2O2 production (-41±1%). Decreased oxidative stress was further observed in rabbit hearts submitted to hypothermic vs. normothermic ischemia (CAO-H vs. CAO-N), using thiobarbituric acid-reactive substances as a marker. This was accompanied by a preservation of the respiratory control ratio as well as the activity of complexes I, II and III in cardiac mitochondria.nnnCONCLUSIONnThe cardioprotective effect of mild hypothermia involves a direct effect on per-ischemic ROS generation and results in preservation of mitochondrial function. This might explain why the benefit afforded by hypothermia during regional myocardial ischemia depends on how fast it is instituted during the ischemic process.

The use of proteome similarity for the qualitative and quantitative profiling of reperfused myocardium.

An LC-MS-based approach is presented for the identification and quantification of proteins from unsequenced organisms. The method relies on the preservation of homology across species and the similarity in detection characteristics of proteomes in general. Species related proteomes share similarity that progresses from the amino acid frequency distribution to the complete amino sequence of matured proteins. Moreover, the comparative analysis between theoretical and experimental proteome distributions can be used as a measure for the correctness of detection and identification obtained through LC-MS-based schemes. Presented are means to the identification and quantification of rabbit myocardium proteins, immediately after inducing cardiac arrest, using a data-independent LC-MS acquisition strategy. The employed method of acquisition affords accurate mass information on both the precursor and associated product ions, whilst preserving and recording the intensities of the ions. The latter facilitates label-free quantification. The experimental ion density observations obtained for the rabbit sub proteome were found to share great similarity with five other mammalian samples, including human heart, human breast tissue, human plasma, rat liver and a mouse cell line. Redundant, species-homologues peptide identifications from other mammalian organisms were used for initial protein identification, which were complemented with peptide identifications of translated gene sequences. The feasibility and accuracy of label-free quantification of the identified peptides and proteins utilizing above mentioned strategy is demonstrated for selected cardiac rabbit proteins.

Chronic heart rate reduction with ivabradine improves systolic function of the reperfused heart through a dual mechanism involving a direct mechanical effect and a long-term increase in FKBP12/12.6 expression.

AIMSnTo investigate the adaptations of left ventricular function and calcium handling to chronic heart rate reduction with ivabradine in the reperfused heart.nnnMETHODS AND RESULTSnRabbits underwent 20 min coronary artery occlusion followed by 3 weeks of reperfusion. Throughout reperfusion, rabbits received ivabradine (10 mg/kg/day) or vehicle (control). Ivabradine reduced heart rate by about 20% and improved both ejection fraction (+35%) and systolic displacement (+26%) after 3 weeks of treatment. Interestingly, this was associated with a two-fold increase expression of FKBP12/12.6. There was no difference in the expressions of phospholamban, SERCA2a, calsequestrin, ryanodine, phospho-ryanodine, and Na(2+)/Ca(2+) exchanger in the two groups. Infarct scar and vascular density were similar in both groups. Administration of a single intravenous bolus of ivabradine (1 mg/kg) in control rabbits at 3 weeks of reperfusion also significantly improved acutely ejection fraction and systolic displacement.nnnCONCLUSIONnChronic heart rate reduction protects the myocardium against ventricular dysfunction induced by myocardial ischaemia followed by 3 weeks of reperfusion. Beyond pure heart rate reduction, ivabradine improves global and regional systolic function of the reperfused heart through a dual mechanism involving a direct mechanical effect and a long-term adaptation in calcium handling, as supported by the increase in FKBP12/12.6 expression.

Mitochondrial translocator protein (TSPO): From physiology to cardioprotection.

The mitochondrial translocator protein (TSPO) is a high affinity cholesterol binding protein which is primarily located in the outer mitochondrial membrane where it has been shown to interact with proteins implicated in mitochondrial permeability transition pore (mPTP) formation. TSPO is found in different species and is expressed at high levels in tissues that synthesize steroids but is also present in other peripheral tissues especially in the heart. TSPO has been involved in the import of cholesterol into mitochondria, a key step in steroidogenesis. This constitutes the main established function of the protein which was recently challenged by genetic studies. TSPO has also been associated directly or indirectly with a wide range of cellular functions such as apoptosis, cell proliferation, differentiation, regulation of mitochondrial function or porphyrin transport. In the heart the role of TSPO remains undefined but a growing body of evidence suggests that TSPO plays a critical role in regulating physiological cardiac function and that TSPO ligands may represent interesting drugs to protect the heart under pathological conditions. This article briefly reviews current knowledge regarding TSPO and discusses its role in the cardiovascular system under physiological and pathologic conditions. More particularly, it provides evidence that TSPO can represent an alternative strategy to develop new pharmacological agents to protect the myocardium against ischemia-reperfusion injury.

A model of hypoxia-reoxygenation on isolated adult mouse cardiomyocytes: characterization, comparison with ischemia-reperfusion, and application to the cardioprotective effect of regular treadmill exercise.

The use of in vitro experimental models of hypoxia-reoxygenation (H/R) that mimic in vivo ischemia-reperfusion represents a powerful tool to investigate cardioprotective strategies against myocardial infarction. Most in vitro studies are performed using neonatal cardiac cells or immortalized embryonic cardiac cell lines which may limit the extrapolation of the results. We developed an H/R model using adult cardiomyocytes freshly isolated from mice and compared its characteristics to the in vivo ischemia-reperfusion conditions. First, cell death was assessed at different values of pH medium during hypoxia (6.2 vs 7.4) to simulate extracellular pH during in vivo ischemia. Cardiomyocyte mortality was aggravated with hypoxia under acidic pH. We next evaluated the relationship between the duration of hypoxia and cell death. Hypoxia time-dependently reduced myocyte viability (−24%, −36%, −53%, and −74% with 1, 1.5, 2, and 3 hours of hypoxia followed by 17 hours of reoxygenation, respectively). We then focused on the duration of reoxygenation as cardioprotective strategies have been reported to have different effects with short and long durations of reperfusion. We observed that cardiomyocyte mortality was increased when the duration of reoxygenation was increased from 2 h to 17 hours. Finally, we used our characterized model to investigate the cardioprotective effect of regular treadmill exercise. Myocyte viability was significantly greater in exercised when compared to sedentary mice (44% and 26%, respectively). Similarly, mice submitted to in vivo ischemia-reperfusion elicited infarct sizes reaching 27%, 43%, and 55% with 20, 30, and 45 minutes of coronary artery occlusion. In addition, infarct size was significantly reduced by exercise. In conclusion, this H/R model of cardiomyocytes freshly isolated from adult mice shows similar characteristics to the in vivo ischemia-reperfusion conditions. The comparison of in vivo and in vitro settings represents a powerful approach to investigate cardioprotective strategies and to distinguish between direct and indirect cardiomyocyte-dependent mechanisms.

Cardioprotection against myocardial infarction with PTD-BIR3/RING, a XIAP mimicking protein

The purpose of the present study was to investigate the potential cardioprotective effects of an original approach based on the properties of the X chromosome-linked Inhibitor of Apoptosis (XIAP), the most effective endogenous inhibitor of apoptosis. For this purpose, the C-terminal part of XIAP (BIR3 and RING domains) was fused to the protein transduction domain (PTD) of the HIV1 transactivator of transcription, which confers to fused protein the ability to cross cell membranes. This protein, so-called PTD-BIR3/RING, was administered intravenously in C57BL/6J mice subjected to 30 min coronary artery occlusion and 24 h of reperfusion. Administration of PTD-BIR3/RING at 5 min before and 30 min after the onset of reperfusion reduced infarct size vs control (23+/-2% vs 41+/-4% and 27+/-4% vs 41+/-3%, respectively, p<0.05). Similar reduction in infarct size was observed when PTD-BIR3/RING was administered prior to ischemia (28+/-1% vs 44+/-3%). In addition to inhibition of caspase-3 and -9 activities, PTD-BIR3/RING induced an inhibition of caspase-8 and several other actors of the apoptotic pathways. In conclusion, this study demonstrates that the administration of PTD-BIR3/RING reduces myocardial infarct size even when injected during reperfusion through interruption of caspase activation by pharmacologically mimicking endogenous XIAP.

Regular treadmill exercise inhibits mitochondrial accumulation of cholesterol and oxysterols during myocardial ischemia-reperfusion in wild-type and ob/ob mice

Mitochondria play a central role in the irreversible damages induced to the heart by a prolonged period of ischemia followed by reperfusion. We previously demonstrated that (1) myocardial ischemia-reperfusion induces mitochondrial accumulation of cholesterol and oxysterols that are deleterious for the organelle; (2) inhibition of cholesterol and oxysterol accumulation prevents mitochondrial injury at reperfusion; (3) exercise is cardioprotective and remains efficient in the presence of co-morbidities such as obesity. The aim of this study was to investigate whether regular exercise limits mitochondrial cholesterol and oxysterol accumulation in wild-type and obese mice. Wild-type C57BL/6J and obese (ob/ob) mice were assigned to sedentary conditions or regular treadmill exercise and submitted to 30min of coronary artery occlusion followed by 15min of reperfusion. Regular exercise improved oxidative phosphorylation, restored the antioxidant capacity of the heart by increasing the expression of SOD1 and catalase and reduced the mitochondrial generation of oxysterols in wild-type as well as in ob/ob mice. In wild-type animals, exercise limited the production of oxysterols. In ob/ob mice, despite hypercholesterolemia, chronic exercise abolished the mitochondrial accumulation of cholesterol and concomitantly reduced the generation of 7α-hydroxycholesterol, 7-ketocholesterol and cholesterol-5α,6α-epoxide. In conclusion, regular exercise prevents the mitochondrial accumulation of cholesterol and oxysterols which occurs during early reperfusion of an ischemic myocardium in mice. This effect is observed in normo and hypercholesterolemic animals. It may be partly responsible for the antioxidant properties of regular exercise and contribute to its cardioprotective effect in obese conditions.

A TSPO ligand prevents mitochondrial sterol accumulation and dysfunction during myocardial ischemia-reperfusion in hypercholesterolemic rats

ABSTRACT A major cause of cell death during myocardial ischemia‐reperfusion is mitochondrial dysfunction. We previously showed that the reperfusion of an ischemic myocardium was associated with an accumulation of cholesterol into mitochondria and a concomitant strong generation of auto‐oxidized oxysterols. The inhibition of mitochondrial accumulation of cholesterol abolished the formation of oxysterols and prevented mitochondrial injury at reperfusion. The aim of this study was to investigate the impact of hypercholesterolemia on sterol and oxysterol accumulation in rat cardiac cytosols and mitochondria and to analyse the effect of the translocator protein ligand 4′‐chlorodiazepam on this accumulation and mitochondrial function. Hypercholesterolemic ZDF fa/fa rats or normocholesterolemic lean rats were submitted to 30 min of coronary artery occlusion followed by 15 min reperfusion where cardiac cytosols and mitochondria were isolated. Hypercholesterolemia increased the cellular cardiac concentrations of cholesterol, cholesterol precursors and oxysterols both in cytosol and mitochondria in non‐ischemic conditions. It also amplified the accumulation of all these compounds in cardiac cells and the alteration of mitochondrial function with ischemia‐reperfusion. Administration of 4′‐chlorodiazepam to ZDF fa/fa rats had no effect on the enhancement of sterols and oxysterols observed in the cytosols but inhibited cholesterol transfer to the mitochondria. It also alleviated the mitochondrial accumulation of all the investigated sterols and oxysterols. This was associated with a restoration of oxidative phosphorylation and a prevention of mitochondrial transition pore opening. The inhibition of cholesterol accumulation with TSPO ligands represents an interesting strategy to protect the mitochondria during ischemia‐reperfusion in hypercholesterolemic conditions.