Christophe Ribuot

Intermittent hypoxia and sleep-disordered breathing: current concepts and perspectives

There are three major types of sleep-disordered breathing (SDB) with respect to prevalence and health consequences, i.e. obstructive sleep apnoea syndrome (OSAS), Cheyne–Stokes respiration and central sleep apnoea (CSR-CSA) in chronic heart failure, and obesity hypoventilation syndrome (OHS). In all three conditions, hypoxia appears to affect body functioning in different ways. Most of the molecular and cellular mechanisms that occur in response to SDB-related hypoxia remain unknown. In OSAS, an inflammatory cascade mainly dependent upon intermittent hypoxia has been described. There is a strong interaction between haemodynamic and inflammatory changes in promoting vascular remodelling. Moreover, during OSAS, most organ, tissue or functional impairment is related to the severity of nocturnal hypoxia. CSR-CSA occurring during heart failure is primarily a consequence of cardiac impairment. CSR-CSA has deleterious consequences for cardiac prognosis and mortality since it favours sympathetic activation, ventricular ectopy and atrial fibrillation. Although correction of CSR-CSA seems to be critical, there is a need to establish therapy guidelines in large randomised controlled trials. Finally, OHS is a growing health concern, owing to the worldwide obesity epidemic and OHS morbidities. The pathophysiology of OHS remains largely unknown. However, resistance to leptin, obesity and severe nocturnal hypoxia lead to insulin resistance and endothelial dysfunction. In addition, several adipokines may be triggered by hypoxia and explain, at least in part, OHS morbidity and mortality. Overall, chronic intermittent hypoxia appears to have specific genomic effects that differ notably from continuous hypoxia. Further research is required to fully elucidate the molecular and cellular mechanisms.

Major role for hypoxia inducible factor-1 and the endothelin system in promoting myocardial infarction and hypertension in an animal model of obstructive sleep apnea.

OBJECTIVES Our aim was to investigate the involvement of the endothelin (ET) system in the cardiovascular consequences of intermittent hypoxia (IH). BACKGROUND Obstructive sleep apnea (OSA) syndrome is an important risk factor for cardiovascular morbidity. Chronic IH, a major component of OSA, is thought to be responsible for most of the cardiovascular complications occurring during OSA, but the underlying mechanisms remain to be determined. METHODS Chronic IH was applied in rats genetically prone to develop hypertension (spontaneous hypertensive rats [SHR]) and their normotensive controls. The cardiovascular effects were assessed in vivo and in Langendorff perfused hearts. Hypoxia inducible factor (HIF)-1 activity and targeting of the myocardial ET-1 gene and activation of the ET system were investigated using tissue chromatin immunoprecipitation, enzyme-linked immunoadsorbent assay, immunostaining, and Western blotting. RESULTS Chronic IH enhanced hypertension development and infarct size in SHR compared with that seen in control rats. This was accompanied by an increase in myocardial big ET-1, ET-1, and ET-A receptor expression and by an enhanced coronary vascular reactivity to ET-1 in SHR only. Myocardial HIF-1 activity was increased, and HIF-1 was shown to be linked to the promoter of the myocardial ET-1 gene after chronic IH only. Moreover, administration of bosentan, a mixed ET receptor antagonist, during chronic IH prevented both the increase in blood pressure and in infarct size. CONCLUSIONS In SHR, activation of the ET system, mediated by HIF-1 activity, is responsible for the enhanced susceptibility to chronic IH and for its associated cardiovascular consequences leading to hypertension and ischemic injury. Furthermore, the beneficial effects of bosentan suggest exploring ET antagonists as possible therapeutic tools in OSA.

Oxidative stress mediates cardiac infarction aggravation induced by intermittent hypoxia

We have previously shown that chronic intermittent hypoxia (IH), a component of the obstructive sleep apnea syndrome, increases heart sensitivity to infarction. We investigate here the deleterious mechanisms potentially involved in the IH‐induced infarction aggravation, investigating the role of oxidative stress. Male Wistar rats were subjected to chronic IH or normoxia (N). IH consisted of repetitive 1‐min cycles (30 s with inspired O2 fraction 5% followed by 30 s normoxia) and was applied for 8 h during daytime, for 14 days. After the 14‐day exposure, mean arterial blood pressure (MABP) was higher in the hypoxic compared with the normoxic group. Infarct size, measured on isolated hearts after ischemia‐reperfusion, was significantly increased in IH compared with normoxic group (36.0 ± 2.8% vs. 21.8 ± 3.1% for tempol corresponding control groups and 40.3 ± 3.5% vs. 29.4 ± 3.7% for melatonin corresponding control groups). Tempol or melatonin administration during the 14‐day IH exposure prevented both IH‐induced increase in MABP and infarction aggravation (24.8 ± 2.8% vs. 25.9 ± 4.0% for tempol‐treated groups and 32.3 ± 3.2% vs. 34.5 ± 4.2% for melatonin‐treated groups). Myocardial oxidative stress was induced by IH, as measured by dihydroethidium (DHE) level and p47‐phox expression (the cytosolic protein required for the activation of the NADPH oxidase). This effect was abolished by tempol and melatonin treatments, which were able to normalize DHE level and NADPH expression. In conclusion, oxidative stress appears to mediate the deleterious cardiovascular effects of IH and, in particular, the increased myocardial susceptibility to infarction.

Erythropoietin and myocardial protection: what's new?

Erythropoietin (EPO), the principal hematopoietic cytokine produced by the kidney and the liver in fetuses, regulates mammalian erythropoiesis and exhibits diverse cellular effects in non‐hematopoietic tissues. The introduction of recombinant human EPO (rhEPO) has marked a significant advance in the management of anemia associated with chronic renal failure. At the same time, experimental studies have unveiled its potential cardioprotective actions. As with other preconditioning agents, administration of exogenous rhEPO can confer myocardial protection against ischemia–reperfusion injury, in terms of reduction in cellular apoptosis and necrosis as well as improvement in myocardial functional recovery. The purpose of this study is to review current information regarding the various protocols used to investigate the effects of rhEPO administration as well as its cardioprotective properties. We also address the potential mechanisms underlying the protective effects of EPO. A better understanding of these mechanisms is essential for the development of clinical applications and the design of novel therapeutical strategies.

Improvement of the comprehension of written information given to healthy volunteers in biomedical research: a single-blind randomized controlled study.

Writing an informed consent form (ICF) for biomedical research is a difficult task. We conducted a multicenter single‐blind randomized controlled trial to identify whether a working group or the systematic improvement in lexico‐syntactic readability or an association of the two could increase the comprehension of the written information given to healthy volunteers enrolled in biomedical research. Participants were randomized to read one of four versions of the ICF: unchanged ICF (A), ICF with systematic lexico‐syntactic readability improvement (B), ICF modified by a working group (C), and ICF modified by the working group followed by systematic lexico‐syntactic improvement (D). The primary end‐point was the objective comprehension score at day 0 for each study group. The scores of objective comprehension at day 0 were statistically different between the four study groups (anovaP = 0.020). The pairwise analysis showed an improvement in the working group vs. the unchanged group (P = 0.003), and a tendency to improvement in the group who read the ICF modified using lexico‐syntactic readability and in the group who read the ICF modified using the two methods (P = 0.020 and 0.027 respectively). We conducted a two‐way anova to identify some characteristics of the population which could explain this score. There was a significant interaction between the type of informed consent document (ICD) and the gender. Improving the ICD in phase I biomedical research leads to better comprehension, whether the method used is systematic lexico‐syntactic improvement or a review by a working group. The improvement is specifically observed in men compared with women. Conversely, while both methods diverge in their effect on lexico‐syntactic readability, their association is not mandatory. We suggest that in all phase I clinical trials, the ICF be improved by either method.

Early pharmacological preconditioning by erythropoietin mediated by inducible NOS and mitochondrial ATP-dependent potassium channels in the rat heart.

Administration of recombinant human erythropoietin (rhEPO) is known to induce protection against cardiac ischaemia injury improving functional recovery and reducing apoptosis. But the underlying mechanisms are not elucidated. We determined the role of nitric oxide synthases (NOS) as well as ATP‐dependent (KATP) and calcium‐activated (KCa) potassium channels in the early cardioprotection induced by rhEPO. Wistar male rats were divided into two experimental groups treated by rhEPO (5000 IU/kg, i.p.) or saline (control group). One hour later, rats were anaesthetized, hearts isolated, retrogradely perfused and submitted to a 30‐min no‐flow global ischaemia followed by 120 min of reperfusion sequence. Cardiac functional recovery (left ventricular developed pressure, LVDP) was significantly higher in the group treated by rhEPO (LVDP at 30 min reperfusion: 71.7 ± 2.3 mmHg) compared with the control group (57.4 ± 5.8 mmHg). We observed the same significant effect on its derivative (dP/dt). The rhEPO‐induced improvement in ventricular function was abolished by perfusion prior to ischaemia with either N‐nitro‐l‐arginine methyl ester (l‐NAME, a nonspecific NOS inhibitor) or N‐(3‐(aminomethyl)benzyl)acetamidine (1400W, a specific inducible NOS inhibitor) or 5‐hydroxydecanoic acid (5HD, a mitochondrial KATP channel blocker) but not with paxilline (a KCa channel inhibitor). Thus, in vivo rhEPO administration provides early preconditioning against ischaemic injury in the isolated perfused rat heart that is dependent on iNOS and mitochondrial KATP channels.

Fructose-Fed Rat Hearts are Protected Against Ischemia-Reperfusion Injury

High fructose-fed (HFF) rat model is known to develop the insulin-resistant syndrome with a very similar metabolic profile to the human X syndrome. Such metabolic modifications have been associated with a high incidence of cardiovascular disease. The role of free radical attack in diabetes mellitus and its cardiovascular complications have been abundantly documented. The present study examined the susceptibility to myocardial ischemic injury and the involvement of free radical attack and/or protection in the metabolic disorders of high FF rats. Rats were divided into two experimental groups that received diet for 4 weeks: a control group (C, n = 28) receiving a standard diet and a HFF group (FF, n = 28), in which 58% of the total carbohydrate was fructose. The euglycemic clamp technique was performed to assess insulin resistance. For the ischemia-reperfusion procedure, rat hearts were isolated and perfused at constant pressure before they were subjected to a 30-min occlusion of the left coronary artery followed by 120 mins of reperfusion. Hemodynamic parameters were measured throughout the protocol. Infarct-to-risk ratio (I/R) was assessed at the end of the protocol by 2,3,4-triphenyltetrazolium chloride staining and planimetric analysis. Lipid peroxidation, antioxidant enzyme activity, level of vitamin E, and trace element status were measured in blood samples from both groups. Rats of the FF group developed an insulin resistance indicated by the glucose infusion rate, which was decreased by 47%. Infarct size was significantly reduced in rats from the FF group (19.9% ± 6.6%) compared to rats from the control group (34.6% ± 4.9%), and cardiac functional recovery at reperfusion was improved in the FF group. Lipid peroxidation and oxidative stress were higher in the FF group, as indicated by higher malonedialdehyde level, whereas plasma vitamin E/triacylglycerol ratio was also enhanced in this group. This study indicates that fructose feeding affords protection against in vitro ischemia-reperfusion injury, potentially implicating vitamin E.

Erythropoietin improved initial resuscitation and increased survival after cardiac arrest in rats.

INTRODUCTION Recent data have demonstrated potent cardioprotective and neuroprotective effects of the application of growth hormones like erythropoietin (EPO) after focal cardiac or cerebral ischemia. In order to assess possible benefits regarding survival and resuscitation conditions, EPO was tested against placebo in a model of cardiac arrest in the rat. METHODS Thirty-four male Wistar rats were randomized into two groups (EPO versus control; n=17 per group). Under anesthesia, cardiac arrest was induced by asphyxia after neuromuscular blockade. After 6 min of global ischemia, animals were resuscitated by external chest compression combined with epinephrine administration. An intravenous bolus of recombinant human EPO (rhEPO, 3000 UIkg(-1) body weight, i.v.) or saline (in control group) was performed 15 min before cardiac arrest, by a blinded investigator. Restoration of spontaneous circulation (ROSC), survival at 1, 24, 48 and 72 h and hemodynamic changes after cardiac arrest were studied. RESULTS Survival to 72 h was significantly improved in the EPO group (n=15/17) compared to the control group (n=7/17). All the EPO-treated rats were successfully resuscitated whereas only 13 of 17 control animals resuscitated. EPO-treated animals required a significantly smaller dose of epinephrine before resuscitation, compared to control rats. Time course of systolic arterial blood pressure after resuscitation revealed no significant differences between both groups. CONCLUSION EPO, when administrated before cardiac arrest, improved initial resuscitation and increased the duration of post-resuscitation survival.

Atorvastatin protects against deleterious cardiovascular consequences induced by chronic intermittent hypoxia

Chronic intermittent hypoxia (IH), a major component of obstructive sleep apnea (OSA), contributes to the high risk of cardiovascular morbidity. We have previously demonstrated that IH-induced oxidative stress is involved in the hypertension and in the hypersensitivity to myocardial infarction. However, the mechanisms underlying these cardiovascular alterations are still unclear, as well as the role of potential protective treatment. Atorvastatin has pleiotropic actions, including increasing nitric oxide (NO) bioavailability and reducing inflammation and oxidative damage. The aim of this study was to evaluate the beneficial effect of a two time course of this treatment against the deleterious cardiovascular consequences of IH. Rats were divided into two groups subjected to chronic IH or normoxic (N) exposure. IH consisted of repetitive one-minute cycles (with only 30 s of a 5% inspired O2 fraction) and was applied for eight hours during daytime, for 14 (simultaneous protocol) or 28 d (delayed protocol). Atorvastatin (10 mg/kg/ d) or its vehicle was administered during the 14 d simultaneous protocol or the last 14 d of the delayed protocol. For both protocols, systolic arterial pressure was significantly increased by 14 d IH exposure. Atorvastatin prevented this deleterious effect in the simultaneous protocol. Carotid artery compliance and endothelial function were significantly altered after 28 d but not after 14 d of IH exposure. Delayed atorvastatin administration preserved these vascular parameters. IH also increased hypersensitivity to myocardial infarction after 14 d exposure, and atorvastatin abolished this deleterious effect. IH also enhanced cardiac NADPH expression and decreased aortic superoxide dismutase activity after 14 d exposure. Atorvastatin significantly restored these activities. In conclusion, whereas IH rapidly increased blood pressure, myocardial infarction hypersensitivity and oxidative stress, compliance, endothelial function and the structural wall of the carotid artery were only altered after a longer IH exposure. Atorvastatin prevented all these deleterious cardiovascular effects, leading to a potentially novel pharmacological therapeutic strategy for OSA syndrome.

Cathodal iontophoresis of treprostinil and iloprost induces a sustained increase in cutaneous flux in rats.

The treatment of scleroderma‐related digital ulcers is still a therapeutic challenge. The most effective drugs are prostacyclin analogues. However, their usage is limited to an intravenous route of administration and by their frequent side effects. The objective of this study was to test whether treprostinil, iloprost and epoprostenol can induce sustained vasodilatation in rats when delivered locally using cutaneous iontophoresis.