Intermittent hypoxia increases lipid insulin resistance in healthy humans: A randomized crossover trial.

Sympathetic overactivity caused by chronic intermittent hypoxia is a hallmark of obstructive sleep apnea. A high sympathetic tone elicits increases in plasma free fatty acid and insulin. Our objective was to assess the impact of 14 nights of chronic intermittent hypoxia exposure on sympathetic activity, glucose control, lipid profile and subcutaneous fat tissue remodelling in non-obese healthy humans. In this prospective, double-blinded crossover study, 12 healthy subjects were randomized, among them only nine underwent the two phases of exposures of 14 nights chronic intermittent hypoxia versus air. Sympathetic activity was measured by peroneal microneurography (muscle sympathetic nerve activity) before and after each exposure. Fasting glucose, insulin, C-peptide and free fatty acid were assessed at rest and during a multisampling oral glucose tolerance test. We assessed histological remodelling, adrenergic receptors, lipolysis and lipogenesis genes expression and functional changes of the adipose tissue. Two weeks of exposure of chronic intermittent hypoxia versus ambient air significantly increased sympathetic activity (p = 0.04). Muscle sympathetic nerve activity increased from 24.5 [18.9; 26.8] before to 21.7 [13.8; 25.7] after ambient air exposure, and from 20.6 [17.4; 23.9] before to 28.0 [24.4; 31.5] bursts per min after exposure to chronic intermittent hypoxia. After chronic intermittent hypoxia, post-oral glucose tolerance test circulating free fatty acid area under the curve increased (p = 0.05) and free fatty acid sensitivity to insulin decreased (p = 0.028). In adipocyte tissue, intermittent hypoxia increased expression of lipolysis genes (adipocyte triglyceride lipase and hormone-sensitive lipase) and lipogenesis genes (fatty acid synthase; p < 0.05). In this unique experimental setting in healthy humans, chronic intermittent hypoxia induced high sympathetic tone, lipolysis and decreased free fatty acid sensitivity to insulin. This might participate in the trajectory to systemic insulin resistance and diabetes for patients with obstructive sleep apnea.

Activin-A limits Th17 pathogenicity and autoimmune neuroinflammation via CD39 and CD73 ectonucleotidases and Hif1-α-dependent pathways.

In multiple sclerosis (MS), Th17 cells are critical drivers of autoimmune central nervous system (CNS) inflammation and demyelination. Th17 cells exhibit functional heterogeneity fostering both pathogenic and nonpathogenic, tissue-protective functions. Still, the factors that control Th17 pathogenicity remain incompletely defined. Here, using experimental autoimmune encephalomyelitis, an established mouse MS model, we report that therapeutic administration of activin-A ameliorates disease severity and alleviates CNS immunopathology and demyelination, associated with decreased activation of Th17 cells. In fact, activin-A signaling through activin-like kinase-4 receptor represses pathogenic transcriptional programs in Th17-polarized cells, while it enhances antiinflammatory gene modules. Whole-genome profiling and in vivo functional studies revealed that activation of the ATP-depleting CD39 and CD73 ectonucleotidases is essential for activin-A-induced suppression of the pathogenic signature and the encephalitogenic functions of Th17 cells. Mechanistically, the aryl hydrocarbon receptor, along with STAT3 and c-Maf, are recruited to promoter elements on Entpd1 and Nt5e (encoding CD39 and CD73, respectively) and other antiinflammatory genes, and control their expression in Th17 cells in response to activin-A. Notably, we show that activin-A negatively regulates the metabolic sensor, hypoxia-inducible factor-1α, and key inflammatory proteins linked to pathogenic Th17 cell states. Of translational relevance, we demonstrate that activin-A is induced in the CNS of individuals with MS and restrains human Th17 cell responses. These findings uncover activin-A as a critical controller of Th17 cell pathogenicity that can be targeted for the suppression of autoimmune CNS inflammation.

Intermittent hypoxia-related alterations in vascular structure and function: a systematic review and meta-analysis of rodent data.

BACKGROUND: Obstructive sleep apnoea and the related intermittent hypoxia (IH) are widely recognised as risk factors for incident cardiovascular diseases. Numerous studies support the deleterious vascular impact of IH in rodents but an overall interpretation is challenging owing to heterogeneity in rodent species investigated and the severity and duration of IH exposure. To clarify this major issue, we conducted a systematic review and meta-analysis to quantify the impact of IH on systemic artery structure and function depending on the different IH exposure designs. METHODS: We searched PubMed, Embase and Web of Science, and included 125 articles in a meta-analysis, among them 112 using wild-type rodents and 13 using apolipoprotein E knockout (ApoE-/-) mice. We used the standardised mean difference (SMD) to compare results between studies. RESULTS: IH significantly increased mean arterial pressure (+13.90 (95% CI 11.88-15.92) mmHg), and systolic and diastolic blood pressure. Meta-regressions showed that mean arterial pressure change was associated with strain and year of publication. IH altered vasodilation in males but not in females and increased endothelin-1-induced but not phenylephrine-induced vasoconstriction. Intima-media thickness significantly increased upon IH exposure (SMD 1.10 (95% CI 0.58-1.62); absolute values +5.23 (2.81-7.84) µm). This increase was observed in mice but not in rats and was negatively associated with age. Finally, IH increased atherosclerotic plaque size in ApoE-/- mice (SMD 1.08 (95% CI 0.80-1.37)). CONCLUSIONS: Our meta-analysis established that IH, independently of other confounders, has a strong effect on vascular structure and physiology. Our findings support the interest of identifying and treating sleep apnoea in routine cardiology practice.

Impact of obstructive sleep apnoea and intermittent hypoxia on blood rheology: a translational study.

BACKGROUND: Haemorheological alterations are reported in obstructive sleep apnoea (OSA) and reversed with continuous positive airway pressure (CPAP), observations potentially explained by intermittent hypoxia (IH)-induced oxidative stress. Our objective was to investigate whether IH causes haemorheological alterations via oxidative stress. METHODS: Wistar rats were exposed to normoxia (n=7) or IH (n=8) for 14 days. 23 moderate-to-severe OSA patients were assessed at three time-points: baseline, after randomisation to either 2 weeks of nocturnal oxygen (n=13) or no treatment (n=10) and after 1 month of CPAP treatment (n=17). Furthermore, an OSA-free control group (n=13) was assessed at baseline and after time-matched follow-up. We measured haemorheological parameters (haematocrit, blood viscosity, plasma viscosity (rats only), erythrocyte aggregation and deformability (humans only)) and redox balance (superoxide dismutase (SOD), glutathione peroxidase, protein oxidation (advanced oxidation protein products (AOPPs)) and lipid peroxidation (malondialdehyde)). We also tested the haemorheological sensitivity of erythrocytes to reactive oxygen species (ROS) in our human participants using the oxidant t-butyl hydroperoxide (TBHP). RESULTS: In rats, IH increased blood viscosity by increasing haematocrit without altering the haemorheological properties of erythrocytes. IH also reduced SOD activity and increased AOPPs. In humans, baseline haemorheological properties were similar between patients and control participants, and properties were unaltered following oxygen and CPAP, except erythrocyte deformability was reduced following oxygen therapy. Redox balance was comparable between patients and control participants. At baseline, TBHP induced a greater reduction of erythrocyte deformability in patients while CPAP reduced TBHP-induced increase in aggregation strength. CONCLUSIONS: IH and OSA per se do not cause haemorheological alterations despite the presence of oxidative stress or higher sensitivity to ROS, respectively.

Cardiovascular and metabolic responses to passive hypoxic conditioning in overweight and mildly obese individuals.

Although severe intermittent hypoxia (IH) is well known to induce deleterious cardiometabolic consequences, moderate IH may induce positive effects in obese individuals. The present study aimed to evaluate the effect of two hypoxic conditioning programs on cardiovascular and metabolic health status of overweight or obese individuals. In this randomized single-blind controlled study, 35 subjects (54 ± 9.3 yr, 31.7 ± 3.5 kg/m2) were randomized into three 8-wk interventions (three 1-h sessions per week): sustained hypoxia (SH), arterial oxygen saturation ([Formula: see text]) = 75%; IH, 5 min [Formula: see text] = 75% - 3 min normoxia; normoxia. Ventilation, heart rate, blood pressure, and tissue oxygenation were measured during the first and last hypoxic conditioning sessions. Vascular function, blood glucose and insulin, lipid profile, nitric oxide metabolites, and oxidative stress were evaluated before and after the interventions. Both SH and IH increased ventilation in hypoxia (+1.8 ± 2.1 and +2.3 ± 3.6 L/min, respectively; P < 0.05) and reduced normoxic diastolic blood pressure (-12 ± 15 and -13 ± 10 mmHg, respectively; P < 0.05), whereas changes in normoxic systolic blood pressure were not significant (+3 ± 9 and -6 ± 13 mmHg, respectively; P > 0.05). IH only reduced heart rate variability (e.g., root-mean-square difference of successive normal R-R intervals in normoxia -21 ± 35%; P < 0.05). Both SH and IH induced no significant change in body mass index, vascular function, blood glucose, insulin and lipid profile, nitric oxide metabolites, or oxidative stress, except for an increase in superoxide dismutase activity following SH. This study indicates that passive hypoxic conditioning in obese individuals induces some positive cardiovascular and respiratory improvements despite no change in anthropometric data and even a reduction in heart rate variability during IH exposure.

Effects of acute nitric oxide precursor intake on peripheral and central fatigue during knee extensions in healthy men.

NEW FINDINGS: What is the central question of this study? What is the effect of acute NO precursor intake on vascular function, muscle and cerebral oxygenation and peripheral and central neuromuscular fatigue during knee-extension exercise? What is the main finding and its importance? Acute NO precursor ingestion increases the plasma concentrations of NO precursors (nitrate, arginine and citrulline) and enhances post-ischaemic vasodilatation, but has no significant effect on muscle and cerebral oxygenation, peripheral and central mechanisms of neuromuscular fatigue and, consequently, does not improve exercise performance. ABSTRACT: Nitric oxide (NO) plays an important role in matching blood flow to oxygen demand in the brain and contracting muscles during exercise. Previous studies have shown that increasing NO bioavailability can improve muscle function. The aim of this study was to assess the effect of acute NO precursor intake on muscle and cerebral oxygenation and on peripheral and central neuromuscular fatigue during exercise. In four experimental sessions, 15 healthy men performed a thigh ischaemia-reperfusion test followed by submaximal isometric knee extensions (5 s on-4 s off; 45% of maximal voluntary contraction) until task failure. In each session, subjects drank a nitrate-rich beetroot juice containing 520 mg nitrate (N), N and citrulline (6 g; N+C), N and arginine (6 g; N+A) or a placebo (PLA). Prefrontal cortex and quadriceps near-infrared spectroscopy parameters were monitored continuously. Transcranial magnetic stimulation and femoral nerve electrical stimulation were used to assess central and peripheral determinants of fatigue. The post-ischaemic increase in thigh blood total haemoglobin concentration was larger in N (10.1 ± 3.7 mmol) and N+C (10.9 ± 3.3 mmol) compared with PLA (8.2 ± 2.7 mmol; P < 0.05). Nitric oxide precursors had no significant effect on muscle and cerebral oxygenation or on peripheral and central mechanisms of neuromuscular fatigue during exercise. The total number of knee extensions did not differ between sessions (N, 71.9 ± 33.2; N+A, 73.3 ± 39.4; N+C, 74.6 ± 34.0; PLA, 71.8 ± 39.9; P > 0.05). In contrast to the post-ischaemic hyperaemic response, NO bioavailability in healthy subjects might not be the limiting factor for tissue perfusion and oxygenation during submaximal knee extensions to task failure.

Cysteinyl-leukotriene pathway as a new therapeutic target for the treatment of atherosclerosis related to obstructive sleep apnea syndrome.

AIMS: Obstructive sleep apnea (OSA) characterized by nocturnal intermittent hypoxia (IH) is associated with atherosclerosis and cysteinyl-leukotrienes (CysLT) pathway activation. We aimed to identify the determinants of CysLT pathway activation and the role of CysLT in OSA-related atherosclerosis. METHODS AND RESULTS: Determinants of the urinary excretion of LTE4 (U-LTE4) including history of cardiovascular events, polysomnographic and biological parameters were studied in a cohort of 170 OSA patients and 29 controls, and in a subgroup of OSA patients free of cardiovascular event (n = 136). Mechanisms linking IH, the CysLT pathway and atherogenesis were investigated in Apolipoprotein E deficient (ApoE-/-) mice exposed to 8-week IH. In the whole cohort, U-LTE4 was independently influenced by age, minimal oxygen saturation, and a history of cardiovascular events, and correlated significantly with intima-media thickness. In the subgroup of OSA patients free of cardiovascular event, increased U-LTE4 was increased compared to controls and independently related to hypoxia severity and traditional risk factors aggregated in the 10-year cardiovascular risk score of European Society of Cardiology. In IH mice, atherosclerosis lesion size and mRNA levels of 5-lipoxygenase, 5-lipoxygenase activating protein (FLAP) and CysLT1 receptor were significantly increased. This transcriptional activation was associated with the binding of HIF-1 to the FLAP promoter and was strongly associated with atherosclerosis lesion size. CysLT1 receptor antagonism (montelukast) significantly reduced atherosclerosis progression in IH mice. CONCLUSIONS: IH-related CysLT pathway activation contributes to OSA-induced atherogenesis. In the era of personalized medicine, U-LTE4 may be a useful biomarker to identify OSA patients for whom CysLT1 blockade could represent a new therapeutic avenue for reducing cardiovascular risk.

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

AIMS/HYPOTHESIS: Mitochondria-associated endoplasmic reticulum membranes (MAMs) are regions of the endoplasmic reticulum (ER) tethered to mitochondria and controlling calcium (Ca(2+)) 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. METHODS: We 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 Ca(2+) exchange, organelle homeostasis and insulin action. RESULTS: Pharmacological and genetic inhibition of CYPD concomitantly reduced ER-mitochondria interactions, inhibited inter-organelle Ca(2+) exchange, induced ER stress and altered insulin signalling in HuH7 cells. In addition, histamine-stimulated Ca(2+) 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 Ca(2+) 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/INTERPRETATION: Disruption of IP3R-mediated Ca(2+) 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 Ca(2+) exchange may thus provide an exciting new avenue for treating hepatic insulin resistance.

High-intensity training reduces intermittent hypoxia-induced ER stress and myocardial infarct size.

Chronic intermittent hypoxia (IH) is described as the major detrimental factor leading to cardiovascular morbimortality in obstructive sleep apnea (OSA) patients. OSA patients exhibit increased infarct size after a myocardial event, and previous animal studies have shown that chronic IH could be the main mechanism. Endoplasmic reticulum (ER) stress plays a major role in the pathophysiology of cardiovascular disease. High-intensity training (HIT) exerts beneficial effects on the cardiovascular system. Thus, we hypothesized that HIT could prevent IH-induced ER stress and the increase in infarct size. Male Wistar rats were exposed to 21 days of IH (21-5% fraction of inspired O2, 60-s cycle, 8 h/day) or normoxia. After 1 wk of IH alone, rats were submitted daily to both IH and HIT (2 × 24 min, 15-30m/min). Rat hearts were either rapidly frozen to evaluate ER stress by Western blot analysis or submitted to an ischemia-reperfusion protocol ex vivo (30 min of global ischemia/120 min of reperfusion). IH induced cardiac proapoptotic ER stress, characterized by increased expression of glucose-regulated protein kinase 78, phosphorylated protein kinase-like ER kinase, activating transcription factor 4, and C/EBP homologous protein. IH-induced myocardial apoptosis was confirmed by increased expression of cleaved caspase-3. These IH-associated proapoptotic alterations were associated with a significant increase in infarct size (35.4 ± 3.2% vs. 22.7 ± 1.7% of ventricles in IH + sedenary and normoxia + sedentary groups, respectively, P < 0.05). HIT prevented both the IH-induced proapoptotic ER stress and increased myocardial infarct size (28.8 ± 3.9% and 21.0 ± 5.1% in IH + HIT and normoxia + HIT groups, respectively, P = 0.28). In conclusion, these findings suggest that HIT could represent a preventive strategy to limit IH-induced myocardial ischemia-reperfusion damages in OSA patients.

Exercise does not activate the ß3 adrenergic receptor-eNOS pathway, but reduces inducible NOS expression to protect the heart of obese diabetic mice.

Obesity and diabetes are associated with higher cardiac vulnerability to ischemia-reperfusion (IR). The cardioprotective effect of regular exercise has been attributed to ß3-adrenergic receptor (ß3AR) stimulation and increased endothelial nitric oxide synthase (eNOS) activation. Here, we evaluated the role of the ß3AR-eNOS pathway and NOS isoforms in exercise-induced cardioprotection of C57Bl6 mice fed with high fat and sucrose diet (HFS) for 12 weeks and subjected or not to exercise training during the last 4 weeks (HFS-Ex). HFS animals were more sensitive to in vivo and ex vivo IR injuries than control (normal diet) and HFS-Ex mice. Cardioprotection in HFS-Ex mice was not associated with increased myocardial eNOS activation and NO metabolites storage, possibly due to the ß3AR-eNOS pathway functional loss in their heart. Indeed, a selective ß3AR agonist (BRL37344) increased eNOS activation and had a protective effect against IR in control, but not in HFS hearts. Moreover, iNOS expression, nitro-oxidative stress (protein s-nitrosylation and nitrotyrosination) and ROS production during early reperfusion were increased in HFS, but not in control mice. Exercise normalized iNOS level and reduced protein s-nitrosylation, nitrotyrosination and ROS production in HFS-Ex hearts during early reperfusion. The iNOS inhibitor 1400 W reduced in vivo infarct size in HFS mice to control levels, supporting the potential role of iNOS normalization in the cardioprotective effects of exercise training in HFS-Ex mice. Although the ß3AR-eNOS pathway is defective in the heart of HFS mice, regular exercise can protect their heart against IR by reducing iNOS expression and nitro-oxidative stress.

Depressing mitochondria-reticulum interactions protects cardiomyocytes from lethal hypoxia-reoxygenation injury.

BACKGROUND: Under physiological conditions, Ca(2+) transfer from the endoplasmic reticulum (ER) to mitochondria might occur at least in part at contact points between the 2 organelles and involves the VDAC1/Grp75/IP3R1 complex. Accumulation of Ca(2+) into the mitochondrial matrix may activate the mitochondrial chaperone cyclophilin D (CypD) and trigger permeability transition pore opening, whose role in ischemia/reperfusion injury is well recognized. We questioned here whether the transfer of Ca(2+) from ER to mitochondria might play a role in cardiomyocyte death after hypoxia-reoxygenation. METHODS AND RESULTS: We report that CypD interacts with the VDAC1/Grp75/IP3R1 complex in cardiomyocytes. Genetic or pharmacological inhibition of CypD in both H9c2 cardiomyoblasts and adult cardiomyocytes decreased the Ca(2+) transfer from ER to mitochondria through IP3R under normoxic conditions. During hypoxia-reoxygenation, the interaction between CypD and the IP3R1 Ca(2+) channeling complex increased concomitantly with mitochondrial Ca(2+) content. Inhibition of either CypD, IP3R1, or Grp75 decreased protein interaction within the complex, attenuated mitochondrial Ca(2+) overload, and protected cells from hypoxia-reoxygenation. Genetic or pharmacological inhibition of CypD provided a similar effect in adult mice cardiomyocytes. Disruption of ER-mitochondria interaction via the downregulation of Mfn2 similarly reduced the interaction between CypD and the IP3R1 complex and protected against hypoxia-reoxygenation injury. CONCLUSIONS: Our data (1) point to a new role of CypD at the ER-mitochondria interface and (2) suggest that decreasing ER-mitochondria interaction at reperfusion can protect cardiomyocytes against lethal reperfusion injury through the reduction of mitochondrial Ca(2+) overload via the CypD/VDAC1/Grp75/IP3R1 complex.

Cardioprotective effect of VEGF and venom VEGF-like protein in acute myocardial ischemia in mice: effect on mitochondrial function.

Coronary endothelial dysfunction is involved in cardiac ischemia-reperfusion (IR) injury. Vascular endothelial growth factor (VEGF) activates endothelial cells and exerts cardioprotective effects in isolated hearts. The recently discovered viper venom protein called increasing capillary permeability protein (ICPP) exerts VEGF-like effects in endothelial cells. We examined whether VEGF or ICPP can influence IR outcome in vivo in mice. Dosages of VEGF and ICPP were determined by preliminary blood pressure study. In IR, both the proteins administered intravenously at reperfusion reduced infarct size (IS) by 57% for VEGF and 52% for ICPP (P < 0.01). Pretreatment with a selective VEGFR2 receptor antagonist abolished the reduction in IS. VEGF and ICPP induced ERK phosphorylation in the myocardium. IR triggered mitochondrial pore opening and impaired mitochondrial respiratory function. These effects of IR were prevented by VEGF or ICPP, which increased mitochondrial calcium retention capacity by 37% compared with saline (P < 0.05) and improved mitochondrial respiratory function (by 71% and 65%, respectively for state 3, and 51% and 38% for state 4, P < 0.01 for VEGF). Thus, intravenous administration of VEGF or ICPP at reperfusion largely reduces IS in IR, through stimulation of VEGFR2 receptors. This effect is mediated, at least in part, by improvement of IR-induced mitochondrial dysfunction.

Interplay between hypoxia inducible Factor-1 and mitochondria in cardiac diseases.

Ischemic heart diseases and cardiomyopathies are characterized by hypoxia, energy starvation and mitochondrial dysfunction. HIF-1 acts as a cellular oxygen sensor, tuning the balance of metabolic and oxidative stress pathways to provide ATP and sustain cell survival. Acting on mitochondria, HIF-1 regulates different processes such as energy substrate utilization, oxidative phosphorylation and mitochondrial dynamics. In turn, mitochondrial homeostasis modifications impact HIF-1 activity. This underlies that HIF-1 and mitochondria are tightly interconnected to maintain cell homeostasis. Despite many evidences linking HIF-1 and mitochondria, the mechanistic insights are far from being understood, particularly in the context of cardiac diseases. Here, we explore the current understanding of how HIF-1, reactive oxygen species and cell metabolism are interconnected, with a specific focus on mitochondrial function and dynamics. We also discuss the divergent roles of HIF in acute and chronic cardiac diseases in order to highlight that HIF-1, mitochondria and oxidative stress interaction deserves to be deeply investigated. While the strategies aiming at stabilizing HIF-1 have provided beneficial effects in acute ischemic injury, some deleterious effects were observed during prolonged HIF-1 activation. Thus, deciphering the link between HIF-1 and mitochondria will help to optimize HIF-1 modulation and provide new therapeutic perspectives for the treatment of cardiovascular pathologies.

Loss of testosterone induces postprandial insulin resistance and increases the expression of the hepatic antioxidant flavin-containing monooxygenases in mice exposed to intermittent hypoxia.

AIM: We tested the hypothesis that low testosterone alters the effects of intermittent hypoxia (IH) on glucose homeostasis, hepatic oxidative stress, and transcriptomic profile in male mice. METHODS: We used sham-operated or orchiectomized (ORX) mice exposed to normoxia (Nx) or IH for 2 weeks. We performed fasting insulin and glucose tolerance tests and assessed fasting and postprandial insulin resistance with the HOMA-IR. The activity of hepatic prooxidant (NADPH oxidase-NOX), antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase-SOD, Cat, GPx), lipid peroxidation (MDA concentration), and the total concentration of glutathione (GSH) were measured under postprandial conditions. mRNA sequencing and pathway enrichment analyses were used to identify hepatic genes underlying the interactions between IH and testosterone. RESULTS: In Sham mice, IH improves fasting insulin sensitivity and glucose tolerance, while there are no effects of IH in ORX mice. In ORX mice, IH induces postprandial hyperinsulinemia, insulin resistance, and a prooxidant profile of enzyme activity (low SOD activity) without altering hepatic MDA and GSH content. ORX and IH altered the expression of genes involved in oxidoreductase activities, cytochromes-dependent pathways, and glutathione metabolism. Among the genes upregulated in ORX-IH mice, the flavin-containing monooxygenases (FMO) are particularly relevant since these are potent hepatic antioxidants that could help prevent overt oxidative stress in ORX-IH mice. CONCLUSION: Low levels of testosterone in male mice exposed to IH induce post-prandial hyperinsulinemia and insulin resistance and determine the mechanisms by which the liver handles IH-induced oxidative stress.

Chronic intermittent hypoxia triggers cardiac fibrosis: Role of epididymal white adipose tissue senescent remodeling?

AIM: Obstructive sleep apnea (OSA) is a growing health problem affecting nearly 1 billion people worldwide. The landmark feature of OSA is chronic intermittent hypoxia (CIH), accounting for multiple organ damage, including heart disease. CIH profoundly alters both visceral white adipose tissue (WAT) and heart structure and function, but little is known regarding inter-organ interaction in the context of CIH. We recently showed that visceral WAT senescence drives myocardial alterations in aged mice without CIH. Here, we aimed at investigating whether CIH induces a premature visceral WAT senescent phenotype, triggering subsequent cardiac remodeling. METHODS: In a first experiment, 10-week-old C57bl6J male mice (n = 10/group) were exposed to 14 days of CIH (8 h daily, 5%-21% cyclic inspired oxygen fraction, 60 s per cycle). In a second series, mice were submitted to either epididymal WAT surgical lipectomy or sham surgery before CIH exposure. Finally, we used p53 deficient mice or Wild-type (WT) littermates, also exposed to the same CIH protocol. Epididymal WAT was assessed for fibrosis, DNA damages, oxidative stress, markers of senescence (p16, p21, and p53), and inflammation by RT-qPCR and histology, and myocardium was assessed for fibrosis and cardiomyocyte hypertrophy. RESULTS: CIH-induced epididymal WAT remodeling characterized by increased fibrosis, oxidative stress, DNA damage response, inflammation, and increased expression of senescent markers. CIH-induced epididymal WAT remodeling was associated with subtle and early myocardial interstitial fibrosis. Both epididymal WAT surgical lipectomy and p53 deletion prevented CIH-induced myocardial fibrosis. CONCLUSION: Short-term exposure to CIH induces epididymal WAT senescent remodeling and cardiac interstitial fibrosis, the latter being prevented by lipectomy. This finding strongly suggests visceral WAT senescence as a new target to mitigate OSA-related cardiac disorders.

Endoplasmic reticulum stress contributes to heart protection induced by cyclophilin D inhibition.

Preventing cyclophilin D (cypD) translocation to the inner mitochondrial membrane can limit lethal reperfusion injury through the inhibition of the opening of the mitochondrial permeability transition pore. Inhibition or loss of function of cypD may also result into an endoplasmic reticulum (ER) stress that has been shown to alter cell survival. We therefore questioned whether ER stress might play a role in the protection induced by CypD deficiency or inhibition. CypD-KO and NIM811 (a CypD inhibitor)-treated mice were subjected to a prolonged ischemia-reperfusion (I/R). Area at risk and infarct size was measured using blue dye and triphenyltetrazolium chloride staining. ER stress markers were measured in the hearts during the reperfusion phase. As expected, cypD-KO mice exhibited a decreased infarct size when compared to wild-type mice (8 ± 1 vs. 20 ± 4% of left ventricular weight; p < 0.01). CypD-deficient mice displayed an increased expression of ER stress proteins such as eukaryotic initiation factor 2α (eIF2α) or glucose regulated protein 78 (Grp78 or Bip). The ER stress inhibitor TUDCA prevented the infarct size reduction afforded by the loss of cypD function (mean infarct size averaged 21 ± 4% of LV weight, p < 0.01 vs. cypD-KO). Similar results were obtained when NIM811, an analog of cyclosporine A, was used to pharmacologically (instead of genetically) inhibit cypD function. This study suggests that the ER stress induced by the inhibition of cypD function plays a key role in protecting the heart against lethal ischemia-reperfusion injury.

Cyclosporine A normalizes mitochondrial coupling, reactive oxygen species production, and inflammation and partially restores skeletal muscle maximal oxidative capacity in experimental aortic cross-clamping.

OBJECTIVE: By binding to cyclophilin D, cyclosporine A (CsA) inhibits mitochondrial permeability transition pore (mPTP) opening and prevents mitochondrial dysfunction and ultimately cell death after ischemia-reperfusion (IR) injury in cardiac muscle. This study tested whether CsA would decrease skeletal muscle oxidative stress and mitochondrial dysfunctions after aortic cross-clamping related IR. METHODS: Forty-five Wistar rats were investigated. The sham group (n = 8) had aortic exposure but no ischemia, the IR group (n = 10) had aortic cross-clamping for 3 hours followed by 2 hours of reperfusion, and the IR+CsA group (n = 9) had two intraperitoneal injections of 10 mg of CsA at 90 and 150 minutes of ischemia before reperfusion. Mitochondrial coupling (acceptor control ratio) and mitochondrial respiratory chain complexes' activities were measured. Reactive oxygen species (ROS) production, cyclophilin D expression, and muscle inflammation were determined using dihydroethidium staining, Western blot, and immunohistochemistry, respectively. An additional 18 sham rats were investigated to determine CsA blood levels and the effects of CsA on mitochondrial respiration and calcium retention capacity, a marker of mPTP opening, both in myocardium and gastrocnemius with and without CsA. RESULTS: Compared with sham, IR decreased mitochondrial coupling (1.38 ± 0.06 vs 1.98 ± 0.20; P = .0092), increased ROS production (3992 ± 706 arbitrary units [AU] vs 1812 ± 322 AU; P = .033), was associated with macrophage infiltration, and decreased maximal oxidative capacity (V(max): 4.08 ± 0.38 µmol O(2)/min/g vs 5.98 ± 0.56 µmol O(2)/min/g; P = .015). Despite IR, CsA treatment totally restored mitochondrial coupling (1.93 ± 0.12; P = .023 vs IR), normalized ROS (1569 ± 348 AU; P = .0098 vs IR), and decreased inflammation. The V(max) was slightly enhanced (5.02 ± 0.39 µmol O(2)/min/g; P = .33 vs IR; P = .35 vs sham). Compared with myocardium, gastrocnemius muscle was characterized by a decreased cyclophilin D content (-50%) associated with an earlier opening of mPTP (calcium retention capacity increased from 10.85 ± 1.35 µM/mg dry weight [DW] to 12.11 ± 2.77 µM/mg DW; P = .65; and from 11.07 ± 1.67 to 37.65 ± 11.41 µM/mg DW; P = .0098 in gastrocnemius and heart, respectively). CONCLUSIONS: Cyclosporine A normalized ROS production, decreased inflammation, and restored mitochondrial coupling during aortic cross-clamping. Incomplete Vmax protection might be due to low cyclophilin D expression in gastrocnemius, preventing CsA from blocking mPTP opening.

Chronic intermittent hypoxia due to obstructive sleep apnea slightly alters nutritional status: a pre-clinical study.

Obstructive sleep apnea syndrome (OSAS) is associated with chronic intermittent hypoxia (cIH) that causes disturbances in glucose and lipid metabolism. Animals exposed to cIH show lower body weight and food intake, but the protein-energy metabolism has never been investigated. Here, to address the gap, we studied the impact of cIH on nutritional status in rats. A total of 24 male Wistar rats were randomized into 3 groups (n = 8): a control group (Ctrl), a cIH group (cIH) exposed to cIH (30 s 21-30 s 5% fraction of inspired oxygen, 8 h per day, for 14 days), and a pair-fed group (PF) exposed to normoxia with food intake adjusted to the intake of the cIH group rats with anorexia. Body weight and food intake were measured throughout the study. After 14 days, the rats were euthanized, the organs were collected, weighed, and the liver, intestine mucosa, and muscles were snap-frozen to measure total protein content. Food intake was decreased in the cIH group. Body weight was significantly lower in the cIH group only (-11%, p < 0.05). Thymus and liver weight as well as EDL protein content tended to be lower in the cIH group than in the Ctrl and PF groups. Jejunum and ileum mucosa protein contents were lower in the cIH group compared to the PF group. cIH causes a slight impairment of nutritional status and immunity. This pre-clinical work argues for greater consideration of malnutrition in care for OSAS patients. Further studies are warranted to devise an adequate nutritional strategy.

Cardiac consequences of intermittent hypoxia: a matter of dose? A systematic review and meta-analysis in rodents.

AIM: Intermittent hypoxia (IH) is considered to be a major contributor to obstructive sleep apnoea-related cardiovascular consequences. The present meta-analysis aimed to assess the effects of IH on cardiac remodelling, function and infarct size after myocardial ischaemia across different rodent species and IH severities. METHODS AND RESULTS: Relevant articles from PubMed, Embase and Web of Science were screened. We performed a random effect meta-analysis to assess the effect of IH on myocardium in rodents by using standardised mean difference (SMD). Studies using rodents exposed to IH and outcomes related to cardiac remodelling, contractile function and response to myocardial ischaemia-reperfusion were included. 5217 articles were screened and 92 were included, demonstrating that IH exposure induced cardiac remodelling, characterised by cardiomyocyte hypertrophy (cross-sectional area: SMD=2.90, CI (0.82-4.98), I2=94.2%), left ventricular (LV) dilation (LV diameter: SMD=0.64, CI (0.18-1.10), I2=88.04%), interstitial fibrosis (SMD=5.37, CI (3.22-7.53), I2=94.8) and apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labelling: SMD=6.70, CI (2.96-10.44), I2=95.9). These structural changes were accompanied by a decrease in LV ejection fraction (SMD=-1.82, CI (-2.52--1.12), I2=94.22%). Importantly, most of the utilised IH protocols mimicked extremely severe hypoxic disease. Concerning infarct size, meta-regression analyses highlighted an ambivalent role of IH, depending on its severity. Indeed, IH exposure with inspiratory oxygen fraction (F IO2 ) <7% was associated with an increase in infarct size, whereas a reduced infarct size was reported for F IO2 levels above 10%. Heterogeneity between studies, small study effect and poor reporting of methods in included articles limited the robustness of the meta-analysis findings. CONCLUSION: This meta-analysis demonstrated that severe IH systematically induces cardiac remodelling and contractile dysfunction in rodents, which might trigger or aggravate chronic heart failure. Interestingly, this meta-analysis showed that, depending on stimulus severity, IH exhibits both protective and aggravating effects on infarct size after experimental ischaemia-reperfusion procedures.

Intermittent Hypoxia-Induced Cardiomyocyte Death Is Mediated by HIF-1 Dependent MAM Disruption.

RATIONALE: Intermittent hypoxia (IH) is one of the main features of sleep-disordered breathing (SDB). Recent findings indicate that hypoxia inducible factor-1 (HIF-1) promotes cardiomyocytes apoptosis during chronic IH, but the mechanisms involved remain to be elucidated. Here, we hypothesize that IH-induced ER stress is associated with mitochondria-associated ER membrane (MAM) alteration and mitochondrial dysfunction, through HIF-1 activation. METHODS: Right atrial appendage biopsies from patients with and without SDB were used to determine HIF-1α, Grp78 and CHOP expressions. Wild-type and HIF-1α+/- mice were exposed to normoxia (N) or IH (21-5% O2, 60 cycles/h, 8 h/day) for 21 days. Expressions of HIF-1α, Grp78 and CHOP, and apoptosis, were measured by Western blot and immunochemistry. In isolated cardiomyocytes, we examined structural integrity of MAM by proximity ligation assay and their function by measuring ER-to-mitochondria Ca2+ transfer by confocal microscopy. Finally, we measured mitochondrial respiration using oxygraphy and calcium retention capacity (CRC) by spectrofluorometry. MAM structure was also investigated in H9C2 cells incubated with 1 mM CoCl2, a potent HIF-1α inducer. RESULTS: In human atrial biopsies and mice, IH induced HIF-1 activation, ER stress and apoptosis. IH disrupted MAM, altered Ca2+ homeostasis, mitochondrial respiration and CRC. Importantly, IH had no effect in HIF-1α+/- mice. Similar to what observed under IH, HIF-1α overexpression was associated with MAM alteration in H9C2. CONCLUSION: IH-induced ER stress, MAM alterations and mitochondrial dysfunction were mediated by HIF-1; all these intermediate mechanisms ultimately inducing cardiomyocyte apoptosis. This suggests that HIF-1 modulation might limit the deleterious cardiac effects of SDB.