Marie Joyeux

Protective effects of melatonin against ischemia-reperfusion injury in the isolated rat heart.

There has been increased interest in melatonin recently, since it was shown to be a potent scavenger of toxic free radicals. Melatonin has been found to be effective in protecting against pathological states due to reactive oxygen species release. The present study was performed in order to determine whether melatonin or 5-methoxy-carbonylamino-N-acetyl-tryptamine (5-MCA-NAT), a structurally related indole compound, protect against ischemia-reperfusion injury in the isolated rat heart. Wistar rats were treated in vivo with either melatonin (1 or 10 mg/kg, i.p.) or 5-MCA-NAT (10 mg/kg, i.p.) or their vehicle, 30 min before their hearts were excised and perfused according to the Langendorff technique. Two different protocols were then applied. In the first one, a regional ischemia (5 min)-reperfusion (30 min) sequence was performed in order to record incidence and duration of reperfusion arrhythmias. In the second one, infarct size was assessed after a regional ischemia (30 min)-reperfusion (120 min) sequence. Results show a spectacular protection against ischemia-reperfusion injuries (on arrhythmias as well as on infarct size) in rats pre-treated with 10 mg/kg of melatonin or 5-MCA-NAT. In conclusion, both melatonin and its structural analog, 5-MCA-NAT, appear to confer protection against ischemia-reperfusion injury in the isolated rat heart. This observation suggests that melatonin could have a potential clinical application in the treatment of myocardial ischemia, even if the mechanisms underlying this protection remain to be determined.

Endocannabinoids are implicated in the infarct size-reducing effect conferred by heat stress preconditioning in isolated rat hearts

OBJECTIVE We have investigated the involvement of the endocannabinoid system in the delayed cardioprotection conferred by heat stress preconditioning in the isolated rat heart. METHODS Rats were divided into eight groups (n=7 in each group), subjected to either heat stress (42 degrees C for 15 min, HS groups) or sham anaesthesia (Sham groups). Twenty-four hours later, their hearts were isolated, retrogradely perfused, and subjected to a 30-min occlusion of the left coronary artery followed by 120 min of reperfusion. Some hearts were perfused with either SR 141716 (a cannabinoid CB(1) receptor antagonist, 1 microM), SR 144528 (a CB(2) receptor antagonist, 1 microM) or L-NAME (a NOS inhibitor, 3 microM) 5 min before ischaemia and during the ischaemic period. RESULTS The infarct size-reducing effect conferred by heat stress (35.7+/-1.8% in Sham to 14.1+/-0.6% in HS groups) was not altered by the perfusion of SR 141716 (11.2+/-1.5%) but was abolished by both SR 144528 (36.6+/-1.6%) and L-NAME (32.0+/-4.4%). In hearts from non-heat-stressed rats, perfusion with SR 141716 (32.8+/-1.6%), SR 144528 (33.4+/-2.2%) and L-NAME (31.6+/-2.9%) had no effect on infarct size. CONCLUSION These results suggest an involvement of endocannabinoids, acting through CB(2) receptors, and NO in the cardioprotection conferred by heat stress against myocardial ischaemia. The possible interaction between both mediators of the heat stress response remains to be determined.

Free‐radical production triggered by hyperthermia contributes to heat stress‐induced cardioprotection in isolated rat hearts

Heat stress (HS) is known to protect the myocardium against ischaemic damage. It has been reported that reactive oxygen species (ROS) are abundantly produced during this stress. Since mechanisms triggering the HS‐induced cardioprotection remain unknown, we investigated the role of ROS in the genesis of this protective phenomenon. Rats were divided into four groups (n=8 in each group), subjected to either hyperthermia (42°C internal temperature for 15 min) or sham anaesthesia and treated or not with N‐2‐mercaptopropionyl glycine (MPG), a synthetic antioxidant, 10 min before HS. Twenty‐four hours later, their hearts were isolated, retrogradely perfused, and subjected to a 30‐min occlusion of the left coronary artery followed by 120 min of reperfusion. Myocardial Hsp 27 and 70 expression was assessed by Western blot analysis (n=4). Cardiac activities of antioxidant enzymes (superoxide dismutase and glutathione peroxidase) were also examined (n=4). Infarct‐to‐risk zone ratio was significantly reduced in HS (17±1.3%) compared to Sham (34.3±1.7%) hearts. This effect was abolished by MPG pretreatment (40.6±1.9% in HS+MPG vs 39.8±2.5% in Sham+MPG hearts). This cardioprotection was associated with an enhanced Hsp 27 and 70 expression, which was not modified by MPG pretreatment. Antioxidant enzyme activities was not modified by heat stress or MPG pretreatment. Free radical production following hyperthermia appears to play a role in the heat stress induced cardioprotection, independently of Hsp levels. Antioxidant enzyme activities do not seem to be implicated in this cardioprotective mechanism.

iNOS is a mediator of the heat stress-induced preconditioning against myocardial infarction in vivo in the rat

OBJECTIVE The inducible isoform of nitric oxide synthase (iNOS) is known to be a trigger of the heat stress (HS)-induced cardioprotection. Since iNOS also appears to mediate various forms of myocardial preconditioning, the goal of this study was to investigate its role as a mediator of the HS response. METHODS AND RESULTS Male Wistar rats were divided in six groups, subjected or not to HS (42 degrees C internal temperature, for 15 min). Twenty-four hours later, they were treated or not with either L-NAME, a non-selective inhibitor of NO synthase isoforms, or 1400W, a selective iNOS inhibitor, 10 min before being subjected to a 30-min left coronary artery occlusion followed by a 120-min reperfusion, in vivo. The infarct size (tetrazolium staining) reducing effect conferred by heat stress (from 46.0+/-1.4% in sham to 26.8+/-3.8% in HS groups) was completely abolished by both L-NAME (53.9+/-3.1%) and 1400W (51.8+/-3.3%). Additional studies using Western blot analysis demonstrated a 3.8-fold increase in myocardial iNOS protein expression 24 h after HS. CONCLUSION These results suggest an involvement of iNOS as a mediator of the protection conferred by heat stress against myocardial ischaemia.

Resistance to myocardial infarction induced by heat stress and the effect of ATP-sensitive potassium channel blockade in the rat isolated heart

1 Heat stress (HS) is known to protect against myocardial ischaemia‐reperfusion injury by improving mechanical dysfunction and decreasing necrosis. However, the mechanisms responsible for this form of cardioprotection remain to be elucidated. ATP‐sensitive potassium (KATP) channels have been shown to be involved in the delayed phase of protection following ischaemic preconditioning, a phenomenon closely resembling the HS‐induced cardioprotection. The aim of this study was thus to investigate the role of KATP channels in HS‐induced protection of the isolated rat heart. 2 Twenty four hours after whole body heat stress (at 42°C for 15 min) or sham anaesthesia, isolated perfused hearts were subjected to a 15 min stabilization period followed by a 15 min infusion of either 10 μM glibenclamide (Glib), 100 μM sodium 5‐hydroxydecanoate (5HD) or vehicle (0.04% DMSO). Regional ischaemia (35 min) and reperfusion (120 min) were then performed. 3 Prior heat stress significantly reduced infarct‐to‐risk ratio (from 42.4±2.4% to 19.4±2.9, P<0.001). This resistance to myocardial infarction was abolished in both Glib‐treated (40.1±1.8% vs 42.3±1.8%) and 5HD‐treated (41.2±1.8% vs 41.8±1.2%) groups. 4 The results of this study suggest that KATP channel activation contributes to the cytoprotective response induced by heat stress.

Role of nitric oxide synthases in the infarct size-reducing effect conferred by heat stress in isolated rat hearts

Nitric oxide (NO) donors are known to induce both delayed cardioprotection and myocardial heat stress protein (HSP) expression. Moreover, heat stress (HS), which also protects myocardium against ischaemic damages, is associated with a NO release. Therefore, we have investigated the implication of NO in HS‐induced resistance to myocardial infarction, in the isolated rat heart model. Rats were divided in six groups (n=10 in each group), subjected or not to heat stress (42°C internal temperature, 15 min) and treated or not with nitro‐L‐arginine‐methylester (L‐NAME) a non‐selective inhibitor of NO synthase isoforms, or L‐N6‐(1‐imino‐ethyl)lysine (L‐NIL), a selective inhibitor of the inducible NO synthase. Twenty‐four hours after heat stress, their hearts were isolated, retrogradely perfused, and subjected to a 30‐min occlusion of the left coronary artery followed by 120 min of reperfusion. Infarct‐to‐risk ratio was significantly reduced in HS (18.7±1.6%) compared to Sham (33.0±1.7%) hearts. This effect was abolished in L‐NAME‐treated (41.7±3.1% in HS+L‐NAME vs 35.2±3.0% in Sham+L‐NAME) and L‐NIL‐treated (36.1±3.4% in HS+L‐NIL vs 42.1±4.6% in Sham+L‐NIL) groups. Immunohistochemical analysis of myocardial HSP 27 and 72 showed an HS‐induced increase of these proteins, which was not modified by L‐NAME pretreatment. We conclude that NO synthases, and in particular the inducible isoform, appear to play a role in the heat stress‐induced cardioprotection, independently of HSP 27 and 72 levels. Further investigations are required to elucidate the precise role of HSPs in this adaptive response.

Heat stress fails to protect myocardium of streptozotocin-induced diabetic rats against infarction

OBJECTIVE Protection conferred by heat stress (HS) against ischaemia-reperfusion injury, in term of mechanical function and myocardial necrosis, has been extensively studied. In contrast, the effects of disease states on this HS-induced cytoprotective response are less known. Therefore, we investigated the effects of prior heat stress on the infarct size in the isolated heart and on the myocardial heat stress protein (HSP) 72 synthesis, in a model of insulin-dependent diabetic rats. METHODS Three groups of animals were studied: D rats were rendered diabetic by 55 mg/kg streptozotocin i.v. injection. DI rats received the same treatment plus a daily injection of insulin started 2 weeks after and V rats received the vehicle of streptozotocin plus a daily injection of saline. Eight weeks later, D, DI and V rats were either heat-stressed (42 degrees C for 15 min) or sham-anaesthetised. Twenty-four hours later, their hearts were isolated, perfused using the Langendorff technique, and subjected to a 30 min occlusion of the left coronary artery followed by 120 min of reperfusion. Myocardial HSP72 content was measured 24 h after HS or sham treatment using an electrophoresis coupled with a Western blot analysis. RESULTS Infarct-to-risk ratio (I/R) was significantly reduced in hearts from heat-stressed (11.7 +/- 2.0%) compared to sham (30.0 +/- 3.2%) V rats. This cardioprotection was not observed in hearts from D (I/R: 31.4 +/- 3.3 vs. 34.3 +/- 3.5%) and DI (I/R: 28.7 +/- 1.6 vs. 30.3 +/- 1.6%) rats. Risk zones were similar between all experimental groups. The incidence of ventricular arrhythmias during ischaemia and reperfusion periods was not different between the six experimental groups. Western blot analysis of the myocardial HSP72 content showed a comparable heat stress-induced increase of this protein, in V, D and DI animals. CONCLUSION These results demonstrate that myocardial protective effect induced by heat stress could not extend to a pathological animal model like the diabetic rat and seems to be unrelated to the HSP72 level. Further investigations are required to elucidate the precise role of the heat stress proteins in this adaptive response.

SB 203580, a mitogen-activated protein kinase inhibitor, abolishes resistance to myocardial infarction induced by heat stress.

Heat stress (HS) is known to confer protection against ischemia-reperfusion injury, including mechanical dysfunction and myocardial necrosis. However, the mechanisms involved in this cardioprotection are yet to be elucidated. Mitogen-activated protein (MAP) kinase cascades have been demonstrated to be involved in cellular response to different stresses. In particular, p38 MAP kinase is known to be activated by HS. Therefore, we investigated the implication of this kinase in HS-induced resistance to myocardial infarction, in the isolated rat heart model, using SB 203580 (SB) to selectively inhibit p38 MAP kinase. Rats were treated with SB (2.83 mg/kg, i.p.) or vehicle (1% DMSO in saline, i.p.) before they were either heat stressed (42°C for 15 minutes) or sham anesthetized. Their hearts were isolated 24 hours later, retrogradely perfused, and subjected to a 35-minute occlusion of the left coronary artery followed by 120 minutes of reperfusion. The infarct-to-risk ratio was significantly reduced in HS (16.9 ± 2.0%) compared with sham (41.6 ± 2.5%) hearts. This reduction in infarct size was abolished in the SB 203580–treated group (37.8 ± 1.9% in HS + SB vs. 42.0 ± 1.9% in sham + SB). Risk zones were similar between experimental groups. Western blot analysis of the myocardial HSP72 showed an HS-induced increase of this protein, which was not modified by the p38 MAP kinase inhibitor, SB 203580. We conclude that activation of p38 MAP kinase appears to play a role in the functional cardioprotection associated with the heat stress response, which seems to be unrelated to the HSP72 level. Further investigations are required to elucidate the precise role of the p38 MAP kinase and heat stress proteins in this adaptative response.

Heat stress-induced protection of endothelial function against ischaemic injury is abolished by ATP-sensitive potassium channel blockade in the isolated rat heart

The protection conferred by heat stress (HS) against myocardial ischaemia‐reperfusion injury, in terms of mechanical function preservation and infarct size reduction, is well documented and mechanisms underlying these effects have been extensively explored. However, the effect of HS on coronary circulation is less known. The aim of this study was thus to investigate the role of ATP‐sensitive potassium (KATP) channels in the protection against ischaemic injury afforded by HS to the coronary endothelial function. Twenty‐four hours after whole body hyperthermia (42°C for 15 min, H groups) or sham anaesthesia (Sham groups), isolated perfused rat hearts were subjected to a 15 min stabilization period followed by a 30 min infusion of either 0.3 μM glibenclamide (Gli, a KATP channel blocker) or its vehicle (V). Hearts were then exposed to a low‐flow ischaemia (30 min)‐reperfusion (20 min) (I/R) or normally perfused (50 min), after which coronaries were precontracted with 0.1 μM U‐46619. Finally, the response to the endothelium‐dependent vasodilator, 5‐hydroxytryptamine (5‐HT, 10 μM) was compared to that of the endothelium‐independent vasodilator, sodium nitroprusside (SNP, 3 μM). In hearts from Sham‐V and Sham‐Gli groups, I/R selectively diminished 5‐HT‐induced vasodilatation without affecting the vasodilatation to SNP. In V‐treated groups, prior HS preserved the vasodilatation produced by 5‐HT. This HS‐induced protection was abolished by Gli treatment. In conclusion, these results suggest that KATP channel activation contributes to the preservation of coronary endothelial function conferred by heat stress against ischaemic insult.

Early effects of acute γ-radiation on vascular arterial tone

1 To determine the acute effects of irradiation on the functionality of vessel, rat aortic rings were mounted in an organ bath for isometric tension measurements and irradiated (60Co, 1 Gy min−1, 15 min). 2 Irradiation, which is without effect on non‐contracted or endothelium‐denuded vessels, led to an immediate and reversible increase in vascular tone on (−)‐phenylephrine (1 μM)‐precontracted aortic rings. The tension reached a plateau about 5 min after the beginning of irradiation. 3 The maximal radiation‐induced contraction occurred on aortic rings relaxed by acetylcholine (ACh) (1 μM). In this condition, the addition of catalase (1000 u ml−1), which reduces hydrogen peroxide, and DMSO (0.1% v/v), which scavenges hydroxyl radical, had no influence on tension level while superoxide dismutase (SOD) (100 u ml−1), a superoxide anion scavenger, reduced the observed contraction. A similar result was obtained in the presence of indomethacin (10 μM), a cyclo‐oxygenase blocker. 4 Pretreatment of rings with the nitric oxide synthase inhibitor, Nω‐nitro‐L‐arginine methyl ester (L‐NAME) (10–100 μM) inhibited the radiation‐induced contraction. 5 This effect was dose rate‐dependent and even occurred for a very low dose rate (0.06 Gy min−1). 6 The present results indicate that γ‐radiation induces an instantaneous vascular tone increase that is endothelium and dose rate‐dependent. This effect is (i) maximal when nitric oxide (NO) is produced, (ii) greatly reduced by SOD and (iii) inhibited by L‐NAME, suggesting a major involvement of complexes between NO and superoxide anion.