1 Division of Health Promotion Science, College of Public Health and
2 Cardiovascular and Thoracic Surgery and The Sarver Heart Center, School of Medicine, University of Arizona, Tucson, AZ 85724, USA
Received 23 November 2001; in revised form 17 July 2002; accepted 30 July 2002
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ABSTRACT |
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INTRODUCTION |
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Recent epidemiological studies suggest that moderate ethanol consumption reduces the risk for developing coronary artery disease, angina pectoris, acute myocardial infarction and sudden death (Iso et al., 1995; Poikolainen et al., 1996
; McKee and Britton, 1998
; van Tol and Hendriks, 2001
). Regular moderate drinking may have cardioprotective effects. However, it is unknown if chronic ethanol consumption can attenuate myocardial ischaemiareperfusion injury in AIDS.
Neutrophils are a principal mediator of ischaemia reperfusion injury (Ritter and McDonagh, 1997; Cavanagh et al., 1998
; Gale et al., 2001
). Upon neutrophil activation, they express a higher affinity CD11/CD18 to mediate firm adhesion to vascular endothelial cells and eventually transmigrate to heart tissue. Accumulated neutrophils in hearts release proteolytic enzymes and cytotoxic H2O2, resulting in severe ischaemiareperfusion injury. We found that neutrophils were highly activated in murine AIDS and down-regulated during chronic ethanol consumption (unpublished results). Therefore, we hypothesized in the present work that murine AIDS mice are susceptible to severe neutrophil-mediated reperfusion injury, and that chronic ethanol consumption attenuates the severity of ischaemiareperfusion injury.
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MATERIALS AND METHODS |
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Murine heart model for myocardial ischaemia and reperfusion
We modified a murine model for myocardial ischaemia and reperfusion based on the protocol of Michael et al. (1995). Female C57BL/6 mice were anaesthetized with sodium pentobarbital (55 mg/kg, i.p.). A tracheotomy was performed to facilitate breathing. A section of polyethylene (PE) 90 tubing was inserted into the mouses trachea and connected via PE 160 to a respirator (Harvard Rodent Ventilator Model 683, Holliston, MA, USA). The respirators tidal volume was set at ~1.0 ml/min with 100% oxygen supplementation, and the rate was set at 120 strokes/min. Normal chest expansion was monitored for adjusting optimal tidal volume. The right carotid artery was then cannulated with PE-10 tubing to monitor arterial blood pressure and heart rate. The arterial cannula was filled with heparinized phosphate-buffered saline (2 U/ml) and connected to a blood pressure transducer and a blood pressure monitor (Gould Windograf, Valley View, OH, USA). After an equilibration period of 10 min, a thoracotomy was performed. With an electrocautery, an incision was made to the left of the sternum. The pericardial sac was then removed. Ligation of the left anterior descending branch (LAD) was performed using a 70 silk suture attached to a needle. A small piece of PE-50 was used to secure the ligature without damaging the artery. The animals were subjected to 30 min of LAD occlusion and 120 min of reflow. Blood samples were taken from the carotid catheter for monitoring blood gas. At the end of the 2-h period of reperfusion, the LAD was re-ligated with a 70 silk suture. Trypan blue (1.2 ml, 0.4%; Sigma Chemical Co., St Louis, MO, USA) was injected retrogradely into the carotid artery catheter to delineate the in vivo area at risk. At the end of the protocol, the heart was excised and four 1-mm transverse sections were made with one section at the site of the ligature. Each section was scanned with a high-resolution scanner (1200 dpi Hewlett-Packard-model 5370C, Palo Alto, CA, USA). Each slice was then counterstained with 1.0% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma) solution for 5 min at 37°C. The sections were placed in a 10% buffered formalin solution. The next day, each section was scanned again to account for formalin-induced shrinkage and to determine infarct size.
Measurement of the area at risk and infarct size
A scanned transverse section from 1 mm distal to ligature was analysed using Adobe software (Adobe Photoshop 5.5) and measured for the area at risk and the infarct size (Fig. 1). Using a toolbar, a 1 mm2 calibration square was produced and converted to pixels. The entire area of the myocardial section and area at risk (the area not stained by Trypan blue) were outlined and computed into mm2. The fractional area at risk in this section was calculated by dividing the area at risk by the total area. To measure the infarct size, the same section (stained with TTC) was used. The fractional infarct area in the fixed section was calculated by dividing the TTC-stained area by the total area of the fixed section. The fractional infarct area was divided by the fractional area at risk to determine the ratio of the infarct area/area at risk.
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RESULTS |
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DISCUSSION |
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Attenuation of ischaemiareperfusion injury with regular alcohol consumption could lead to improved myocardial recovery and survival after myocardial infarction. One potential mechanism by which regular drinking may improve survival after myocardial infarction is to reduce ischaemia reperfusion injury, analogous to experimental ischaemic preconditioning (Klatsky et al., 1990; Kawano et al., 1992
; Hu and Nattel, 1995
). Ischaemic preconditioning occurs when brief periods of ischaemia and reperfusion protect hearts against injury from subsequent prolonged ischaemia reperfusion. Recent evidence in experimental animals indicates that ischaemiareperfusion injury can be reduced by preconditioning the heart with brief episodes of ischaemia and reperfusion prior to prolonged ischaemia (Kawano et al., 1992
; Headrick, 1996
). A study demonstrated that hearts from rats fed ethanol for 8 weeks could be preconditioned with a single 5-min episode of ischaemia prior to prolonged ischaemiareperfusion, whereas hearts from control animals were not protected (McDonough, 1997
). Studies in guinea-pigs found that regular ethanol consumption mimicked ischaemic preconditioning and reduced ischaemiareperfusion injury (Miyamae et al., 1997
). Furthermore, ischaemic preconditioning, a cardioprotective effect of alcohol, is mediated by adenosine and
1-adrenergic signalling in many species, including guinea-pigs, humans and rats (Miyamae et al., 1998
).
The incidence and severity of myocardial infarction in AIDS is increasing (Rickerts et al., 2000). Severe ischaemic attacks may contribute to sudden death in AIDS patients. Our results strongly support the idea that retrovirus-infected hearts are more vulnerable to a heart attack. When cardiovascular ischaemic events occur in the late stage of AIDS, hearts have reduced protective ability against ischaemia. This may be due to underlying cardiovascular complications by direct and/or indirect retroviral infection. Several causative factors contributing to cardiovascular complications of AIDS may amplify ischaemiareperfusion injury, such as retroviral infection, multi-opportunistic infection, autoimmune reaction, neutrophil activation and cytokine dysregulation. Overall, ischaemic attack could exaggerate the cardiovascular complications and rigorously affect heart performance.
Retrovirus itself may directly attack the hearts. Researchers have found the presence of HIV-1 in the myocardium of AIDS patients (Calabrese et al., 1987; Lipshultz et al., 1990
). Barbaro et al. (1998)
found that cardiac myocytes were infected with HIV-1 in 58 patients and nearly two-thirds of those samples had myocarditis. Cardiac complications, such as myocarditis, dilated cardiomyopathy, endocarditis, pericardial effusion, arteriopathy and cardiac malignant neoplasms, have been found in AIDS patients (Currie et al., 1995
; Odeh et al., 1995
; Barbaro et al., 1998
; Shannon et al., 2000
). These findings suggest that myocarditis is related to a direct action of the retrovirus. Endothelial cells infected by retrovirus may also contribute to cardiovascular dysfunction in AIDS. Altered function of vascular endothelial cells is associated with hyperactivity of the microcirculation and with coronary vasospasm resembling the changes seen in cocaine abuse (Mohan et al., 1995
). Coronary artery spasm may lead to ischaemic attack and myocardial cellular necrosis, subsequently causing hypertrophy. This evidence suggests that the retrovirus itself causes the cardiovascular complications.
A number of opportunistic infections involving the hearts have been reported in AIDS (Kostianovsky et al., 1987; Wu et al., 1992
). Disseminated cytomegalovirus (CMV) infection occurs frequently in HIV-infected patients. CMV antigen and CMV-mediated early gene expression were found in myocytes from HIV-infected patients (Wu et al., 1992
). EpsteinBarr virus is another opportunistic pathogen involved in the aetiology of cardiac lymphomas (Gill et al., 1987
). Multiple infections may trigger cellular and humoral-mediated cardiac injury.
An autoimmune reaction in AIDS may also compromise heart function. Acierno et al. (1989) suggested that myocardial damage is related to uncontrolled hyper-gamma-globulinaemia. The murine model of AIDS characteristically develops hyper-gamma-globulinaemia. Many researchers (Hastillo et al., 1991; Lieberman et al., 1993
) proposed an autoimmune mechanism for HIV-related myocardial disease similar to those described with antimyosin antibodies. Viral genes may alter the cell surface of the muscle fibre. Some of these cell surface proteins become immunogenic and elicit a progressive autoimmune reaction. A series of experiments has revealed the presence of circulating cardiac autoantibodies to heavy chain myosin in AIDS patients having cardiovascular complications.
Neutrophil activation and cytokine dysregulation may contribute to the cardiovascular complications in AIDS. We found that neutrophils were significantly activated in murine AIDS (unpublished results). A number of cytokines, including TNF-, IL-1, IL-6, and platelet-activating factor (PAF) were increased in AIDS individuals (Akarid et al., 1995
; Westmoreland et al., 1996
; Liang et al., 1997
; Sei et al., 1997
). These cytokines affect heart performance to various degrees. IL-1 has a suppressive effect on adrenergic agonist-mediated increase in cyclic adenosine monophosphate (cAMP) in cardiomyocytes. IL-2 and IL-6 have reversible myocardial depressant effects in vivo (Finkel, 1992; Barry, 1994
). Long-term treatment of cardiomyocytes with IL-1 and TNF-
reduces contractility. The myocardial depressant effects of TNF-
infusion cause left ventricle dysfunction (Pagani et al., 1992
). TNF-
induces cell apoptosis, and PAF acts as a cofactor in accelerating apoptosis (Westmoreland et al., 1996
). Increased expression of iNOS is found in vitro in cardiac myocytes treated with TNF-
, IL-1 and interferon-
(Pinsky et al., 1995
). Therefore, increased cytokines may contribute to the initiation and perpetuation of activated blood cells, causing heart dysfunction. Upon ischaemiareperfusion, hypersensitive neutrophils may become more activated. iNOS may be over-expressed and more cytokines released in response to ischaemiareperfusion injury. Overall, activated neutrophils release reactive oxygen species and the proteolytic enzymes that directly destroy heart tissue. Over-produced cytokines and over-expressed iNOS amplify the local pathological inflammatory reaction and perturb myocardial function. Our data strongly support this notion, because survival in the 2-month AIDS group was poor and a larger infarct size was observed in the 1-month murine AIDS hearts.
In summary, the present study clearly demonstrates that 2-month exposure to murine AIDS increases the vulnerability of the heart to acute ischaemic attack. Even though early stage (1 month) murine AIDS hearts survived the ischaemia reperfusion protocol, infarction of the myocardium was severe. Chronic ethanol consumption improved survival but did not completely reverse the deleterious effects on hearts due to retroviral infection. These results support the cardioprotective effects of moderate ethanol consumption.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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