The Effect of Sleep Deprivation on Cardiac Function and Tolerance to Ischemia-Reperfusion Injury in Male Rats

Abstract Background: Sleep deprivation (SD) is strongly associated with elevated risk for cardiovascular disease. Objective: To determine the effect of SD on basal hemodynamic functions and tolerance to myocardial ischemia-reperfusion (IR) injury in male rats. Method: SD was induced by using the flowerpot method for 4 days. Isolated hearts were perfused with Langendorff setup, and the following parameters were measured at baseline and after IR: left ventricular developed pressure (LVDP); heart rate (HR); and the maximum rate of increase and decrease of left ventricular pressure (±dp/dt). Heart NOx level, infarct size and coronary flow CK-MB and LDH were measured after IR. Systolic blood pressure (SBP) was measured at start and end of study. Results: In the SD group, the baseline levels of LVDP (19%), +dp/dt (18%), and -dp/dt (21%) were significantly (p < 0.05) lower, and HR (32%) was significantly higher compared to the controls. After ischemia, hearts from SD group displayed a significant increase in HR together with a low hemodynamic function recovery compared to the controls. In the SD group, NOx level in heart, coronary flow CK-MB and LDH and infarct size significantly increased after IR; also SD rats had higher SBP after 4 days. Conclusion: Hearts from SD rats had lower basal cardiac function and less tolerance to IR injury, which may be linked to an increase in NO production following IR.

Improving Reconstituted HDL Composition for Efficient Post-Ischemic Reduction of Ischemia Reperfusion Injury.

BACKGROUND: New evidence shows that high density lipoproteins (HDL) have protective effects beyond their role in reverse cholesterol transport. Reconstituted HDL (rHDL) offer an attractive means of clinically exploiting these novel effects including cardioprotection against ischemia reperfusion injury (IRI). However, basic rHDL composition is limited to apolipoprotein AI (apoAI) and phospholipids; addition of bioactive compound may enhance its beneficial effects. OBJECTIVE: The aim of this study was to investigate the role of rHDL in post-ischemic model, and to analyze the potential impact of sphingosine-1-phosphate (S1P) in rHDL formulations. METHODS AND RESULTS: The impact of HDL on IRI was investigated using complementary in vivo, ex vivo and in vitro IRI models. Acute post-ischemic treatment with native HDL significantly reduced infarct size and cell death in the ex vivo, isolated heart (Langendorff) model and the in vivo model (-48%, p<0.01). Treatment with rHDL of basic formulation (apoAI + phospholipids) had a non-significant impact on cell death in vitro and on the infarct size ex vivo and in vivo. In contrast, rHDL containing S1P had a highly significant, protective influence ex vivo, and in vivo (-50%, p<0.01). This impact was comparable with the effects observed with native HDL. Pro-survival signaling proteins, Akt, STAT3 and ERK1/2 were similarly activated by HDL and rHDL containing S1P both in vitro (isolated cardiomyocytes) and in vivo. CONCLUSION: HDL afford protection against IRI in a clinically relevant model (post-ischemia). rHDL is significantly protective if supplemented with S1P. The protective impact of HDL appears to target directly the cardiomyocyte.

Cannabidiol protects against hepatic ischemia/reperfusion injury by attenuating inflammatory signaling and response, oxidative/nitrative stress, and cell death.

Ischemia/reperfusion (I/R) is a pivotal mechanism of liver damage after liver transplantation or hepatic surgery. We have investigated the effects of cannabidiol (CBD), the nonpsychotropic constituent of marijuana, in a mouse model of hepatic I/R injury. I/R triggered time-dependent increases/changes in markers of liver injury (serum transaminases), hepatic oxidative/nitrative stress (4-hydroxy-2-nonenal, nitrotyrosine content/staining, and gp91phox and inducible nitric oxide synthase mRNA), mitochondrial dysfunction (decreased complex I activity), inflammation (tumor necrosis factor α (TNF-α), cyclooxygenase 2, macrophage inflammatory protein-1α/2, intercellular adhesion molecule 1 mRNA levels; tissue neutrophil infiltration; nuclear factor κB (NF-κB) activation), stress signaling (p38MAPK and JNK), and cell death (DNA fragmentation, PARP activity, and TUNEL). CBD significantly reduced the extent of liver inflammation, oxidative/nitrative stress, and cell death and also attenuated the bacterial endotoxin-triggered NF-κB activation and TNF-α production in isolated Kupffer cells, likewise the adhesion molecule expression in primary human liver sinusoidal endothelial cells stimulated with TNF-α and attachment of human neutrophils to the activated endothelium. These protective effects were preserved in CB(2) knockout mice and were not prevented by CB(1/2) antagonists in vitro. Thus, CBD may represent a novel, protective strategy against I/R injury by attenuating key inflammatory pathways and oxidative/nitrative tissue injury, independent of classical CB(1/2) receptors.

Myocardial tolerance to ischemia-reperfusion injury, training intensity and cessation.

Training has been shown to induce cardioprotection. The mechanisms involved remain still poorly understood. Aims of the study were to examine the relevance of training intensity on myocardial protection against ischemia/reperfusion (I/R) injury, and to which extent the beneficial effects persist after training cessation in rats. Sprague-Dawley rats trained at either low (60% [Formula: see text]) or high (80% [Formula: see text]) intensity for 10 weeks. An additional group of highly trained rats was detrained for 4 weeks. Untrained rats served as controls. At the end of treatment, rats of all groups were split into two subgroups. In the former, rats underwent left anterior descending artery (LAD) ligature for 30 min, followed by 90-min reperfusion, with subsequent measurement of the infarct size. In the latter, biopsies were taken to measure heat-shock proteins (HSP) 70/72, vascular endothelial growth factor (VEGF) protein levels, and superoxide dismutase (SOD) activity. Training reduced infarct size proportionally to training intensity. With detraining, infarct size increased compared to highly trained rats, maintaining some cardioprotection with respect to controls. Cardioprotection was proportional to training intensity and related to HSP70/72 upregulation and Mn-SOD activity. The relationship with Mn-SOD was lost with detraining. VEGF protein expression was not affected by either training or detraining. Stress proteins and antioxidant defenses might be involved in the beneficial effects of long-term training as a function of training intensity, while HSP70 may be one of the factors accounting for the partial persistence of myocardial protection against I/R injury in detrained rats.

Role of Mitogen-Activated Protein Kinases in Myocardial Ischemia-Reperfusion Injury during Heart Transplantation.

In solid organ transplantation, ischemia/reperfusion (IR) injury during organ procurement, storage and reperfusion is an unavoidable detrimental event for the graft, as it amplifies graft inflammation and rejection. Intracellular mitogen-activated protein kinase (MAPK) signaling pathways regulate inflammation and cell survival during IR injury. The four best-characterized MAPK subfamilies are the c-Jun NH2-terminal kinase (JNK), extracellular signal- regulated kinase-1/2 (ERK1/2), p38 MAPK, and big MAPK-1 (BMK1/ERK5). Here, we review the role of MAPK activation during myocardial IR injury as it occurs during heart transplantation. Most of our current knowledge regarding MAPK activation and cardioprotection comes from studies of preconditioning and postconditioning in nontransplanted hearts. JNK and p38 MAPK activation contributes to myocardial IR injury after prolonged hypothermic storage. p38 MAPK inhibition improves cardiac function after cold storage, rewarming and reperfusion. Small-molecule p38 MAPK inhibitors have been tested clinically in patients with chronic inflammatory diseases, but not in transplanted patients, so far. Organ transplantation offers the opportunity of starting a preconditioning treatment before organ procurement or during cold storage, thus modulating early events in IR injury. Future studies will need to evaluate combined strategies including p38 MAPK and/or JNK inhibition, ERK1/2 activation, pre- or postconditioning protocols, new storage solutions, and gentle reperfusion.

Contribution of CD4+ T cells to the early mechanisms of ischemia- reperfusion injury in a mouse model of acute renal failure

Renal ischemia-reperfusion (IR) injury is the major cause of acute renal failure in native and transplanted kidneys. Mononuclear leukocytes have been reported in renal tissue as part of the innate and adaptive responses triggered by IR. We investigated the participation of CD4+ T lymphocytes in the pathogenesis of renal IR injury. Male mice (C57BL/6, 8 to 12 weeks old) were submitted to 45 min of ischemia by renal pedicle clamping followed by reperfusion. We evaluated the role of CD4+ T cells using a monoclonal depleting antibody against CD4 (GK1.5, 50 µ, ip), and class II-major histocompatibility complex molecule knockout mice. Both CD4-depleted groups showed a marked improvement in renal function compared to the ischemic group, despite the fact that GK1.5 mAb treatment promoted a profound CD4 depletion (to less than 5% compared to normal controls) only within the first 24 h after IR. CD4-depleted groups presented a significant improvement in 5-day survival (84 vs 80 vs 39%; antibody treated, knockout mice and non-depleted groups, respectively) and also a significant reduction in the tubular necrosis area with an early tubular regeneration pattern. The peak of CD4-positive cell infiltration occurred on day 2, coinciding with the high expression of ßC mRNA and increased urea levels. CD4 depletion did not alter the CD11b infiltrate or the IFN-g and granzyme-B mRNA expression in renal tissue. These data indicate that a CD4+ subset of T lymphocytes may be implicated as key mediators of very early inflammatory responses after renal IR injury and that targeting CD4+ T lymphocytes may yield novel therapies.

Endothelium-dependent and -independent hepatic artery vasodilatation is not impaired in a canine model of liver ischemia-reperfusion injury

We investigated whether hepatic artery endothelium may be the earliest site of injury consequent to liver ischemia and reperfusion. Twenty-four heartworm-free mongrel dogs of either sex exposed to liver ischemia/reperfusion in vivo were randomized into four experimental groups (N = 6): a) control, sham-operated dogs, b) dogs subjected to 60 min of ischemia, c) dogs subjected to 30 min of ischemia and 60 min of reperfusion, and d) animals subjected to 45 min of ischemia and 120 min of reperfusion. The nitric oxide endothelium-dependent relaxation of hepatic artery rings contracted with prostaglandin F2a and exposed to increasing concentrations of acetylcholine, calcium ionophore A23187, sodium fluoride, phospholipase-C, poly-L-arginine, isoproterenol, and sodium nitroprusside was evaluated in organ-chamber experiments. Lipid peroxidation was estimated by malondialdehyde activity in liver tissue samples and by blood lactic dehydrogenase (LDH), serum aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT) activities. No changes were observed in hepatic artery relaxation for any agonist tested. The group subjected to 45 min of ischemia and 120 min of reperfusion presented marked increases of serum aminotransferases (ALT = 2989 ± 1056 U/L and AST = 1268 ± 371 U/L; P < 0.01), LDH = 2887 ± 1213 IU/L; P < 0.01) and malondialdehyde in liver samples (0.360 ± 0.020 nmol/mgPT; P < 0.05). Under the experimental conditions utilized, no abnormal changes in hepatic arterial vasoreactivity were observed: endothelium-dependent and independent hepatic artery vasodilation were not impaired in this canine model of ischemia/reperfusion injury. In contrast to other vital organs and in the ischemia/reperfusion injury environment, dysfunction of the main artery endothelium is not the first site of reperfusion injury.

Effects of sirolimus alone or in combination with cyclosporine A on renal ischemia/reperfusion injury

Calcineurin inhibitors exacerbate ischemic injury in transplanted kidneys, but it is not known if sirolimus protects or exacerbates the transplanted kidney from ischemic injury. We determined the effects of sirolimus alone or in combination with cyclosporin A (CsA) on oxygenated and hypoxic/reoxygenated rat proximal tubules in the following in vitro groups containing 6-9 rats per group: sirolimus (10, 50, 100, 250, 500, and 1000 ηg/mL); CsA (100 µg/mL); sirolimus (50 and 250 ηg/mL) + CsA (100 µg/mL); control; vehicle (20% ethanol). For in vivo studies, 3-week-old Wistar rats (150-250 g) were submitted to left nephrectomy and 30-min renal artery clamping. Renal function and histological evaluation were performed 24 h and 7 days after ischemia (I) in five groups: sham, I, I + SRL (3 mg·kg-1·day-1, po), I + CsA (3 mg·kg-1·day-1, sc), I + SRL + CsA. Sirolimus did not injure oxygenated or hypoxic/reoxygenated proximal tubules and did not potentiate the tubular toxic effects of CsA. Neither drug affected the glomerular filtration rate (GFR) at 24 h. GFR was reduced in CsA-treated rats on day 7 (0.5 ± 0.1 mL/min) but not in rats receiving sirolimus + CsA (0.8 ± 0.1 mL/min) despite the reduction in renal blood flow (3.9 ± 0.5 mL/min). Acute tubular necrosis regeneration was similar for all groups. Sirolimus alone was not toxic and did not enhance hypoxia/reoxygenation injury or CsA toxicity to proximal tubules. Despite its hemodynamic effects, sirolimus protected post-ischemic kidneys against CsA toxicity.

CPU0213, a novel endothelin type A and type B receptor antagonist, protects against myocardial ischemia/reperfusion injury in rats

The efficacy of endothelin receptor antagonists in protecting against myocardial ischemia/reperfusion (I/R) injury is controversial, and the mechanisms remain unclear. The aim of this study was to investigate the effects of CPU0123, a novel endothelin type A and type B receptor antagonist, on myocardial I/R injury and to explore the mechanisms involved. Male Sprague-Dawley rats weighing 200-250 g were randomized to three groups (6-7 per group): group 1, Sham; group 2, I/R + vehicle. Rats were subjected to in vivo myocardial I/R injury by ligation of the left anterior descending coronary artery and 0.5% sodium carboxymethyl cellulose (1 mL/kg) was injected intraperitoneally immediately prior to coronary occlusion. Group 3, I/R + CPU0213. Rats were subjected to identical surgical procedures and CPU0213 (30 mg/kg) was injected intraperitoneally immediately prior to coronary occlusion. Infarct size, cardiac function and biochemical changes were measured. CPU0213 pretreatment reduced infarct size as a percentage of the ischemic area by 44.5% (I/R + vehicle: 61.3 ± 3.2 vs I/R + CPU0213: 34.0 ± 5.5%, P < 0.05) and improved ejection fraction by 17.2% (I/R + vehicle: 58.4 ± 2.8 vs I/R + CPU0213: 68.5 ± 2.2%, P < 0.05) compared to vehicle-treated animals. This protection was associated with inhibition of myocardial inflammation and oxidative stress. Moreover, reduction in Akt (protein kinase B) and endothelial nitric oxide synthase (eNOS) phosphorylation induced by myocardial I/R injury was limited by CPU0213 (P < 0.05). These data suggest that CPU0123, a non-selective antagonist, has protective effects against myocardial I/R injury in rats, which may be related to the Akt/eNOS pathway.

Hydrogen sulfide postconditioning protects isolated rat hearts against ischemia and reperfusion injury mediated by the JAK2/STAT3 survival pathway

The JAK2/STAT3 signal pathway is an important component of survivor activating factor enhancement (SAFE) pathway. The objective of the present study was to determine whether the JAK2/STAT3 signaling pathway participates in hydrogen sulfide (H2S) postconditioning, protecting isolated rat hearts from ischemic-reperfusion injury. Male Sprague-Dawley rats (230-270 g) were divided into 6 groups (N = 14 per group): time-matched perfusion (Sham) group, ischemia/reperfusion (I/R) group, NaHS postconditioning group, NaHS with AG-490 group, AG-490 (5 µM) group, and dimethyl sulfoxide (DMSO; <0.2%) group. Langendorff-perfused rat hearts, with the exception of the Sham group, were subjected to 30 min of ischemia followed by 90 min of reperfusion after 20 min of equilibrium. Heart rate, left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), and the maximum rate of increase or decrease of left ventricular pressure (± dp/dt max) were recorded. Infarct size was determined using triphenyltetrazolium chloride (TTC) staining. Myocardial TUNEL staining was used as the in situ cell death detection method and the percentage of TUNEL-positive nuclei to all nuclei counted was used as the apoptotic index. The expression of STAT3, bcl-2 and bax was determined by Western blotting. After reperfusion, compared to the I/R group, H2S significantly improved functional recovery and decreased infarct size (23.3 ± 3.8 vs 41.2 ± 4.7%, P < 0.05) and apoptotic index (22.1 ± 3.6 vs 43.0 ± 4.8%, P < 0.05). However, H2S-mediated protection was abolished by AG-490, the JAK2 inhibitor. In conclusion, H2S postconditioning effectively protects isolated I/R rat hearts via activation of the JAK2/STAT3 signaling pathway.

Quercetin postconditioning attenuates myocardial ischemia/reperfusion injury in rats through the PI3K/Akt pathway

Quercetin (Que), a plant-derived flavonoid, has multiple benefical actions on the cardiovascular system. The current study investigated whether Que postconditioning has any protective effects on myocardial ischemia/reperfusion (I/R) injury in vivo and its potential cardioprotective mechanisms. Male Sprague-Dawley rats were randomly allocated to 5 groups (20 animals/group): sham, I/R, Que postconditioning, Que+LY294002 [a phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway inhibitor], and LY294002+I/R. I/R was produced by 30-min coronary occlusion followed by 2-h reperfusion. At the end of reperfusion, myocardial infarct size and biochemical changes were compared. Apoptosis was evaluated by both TUNEL staining and measurement of activated caspase-3 immunoreactivity. The phosphorylation of Akt and protein expression of Bcl-2 and Bax were determined by Western blotting. Que postconditioning significantly reduced infarct size and serum levels of creatine kinase and lactate dehydrogenase compared with the I/R group (all P<0.05). Apoptotic cardiomyocytes and caspase-3 immunoreactivity were also suppressed in the Que postconditioning group compared with the I/R group (both P<0.05). Akt phosphorylation and Bcl-2 expression increased after Que postconditioning, but Bax expression decreased. These effects were inhibited by LY294002. The data indicate that Que postconditioning can induce cardioprotection by activating the PI3K/Akt signaling pathway and modulating the expression of Bcl-2 and Bax proteins.