The major finding of this present study is that acute alcohol abuse increases the susceptibility of donor hearts to ischemia/reperfusion injury in a rat model of heart transplantation even though global heart contractile function recovers 6 h after ethanol administration.
In the present study, we focussed our attention on acute ethanol exposure in which the total consumption is compressed into a short period of time to mimic binge drinking. A bolus of ethanol at a standard dose of 3.45 g/kg body weight, intraperitoneally ensures a complete bioavailability and succeeded in producing rapid rises in circulating level of ethanol. The resulting plasma ethanol concentrations were approximately 375, 185 and 0 mg/100 ml at 1 h, 6 h and 24 h respectively and were similar to pathological levels occurring clinically 
. It has been shown that ethanol damage to heart becomes evident if alcohol consumption exceeds 90 to 100 g/day in humans 
, which can be transpired to a dosage of ~1.5 g/kg for an adult weighing 70 kg. In the present study we therefore used the single dose of 3.45 g/kg of ethanol which corresponds to a state of excessive ethanol consumption, given that rodents are less sensitive than human to intoxicating effects of ethanol.
The principal indicator of myopathic alteration following ethanol exposure is characterized by compromised myocardial contractility 
. This is supported by our results which revealed that after 1 h, ethanol elicited deterioration in the heart function, as evidenced by reduced mean arterial pressure, load-dependent (dP/dtmax
), and load-independent (dP/dtmax
-EDV) contractility indexes. Moreover, we observed decreased LV end-diastolic pressure at 1 h which recovered 6 h and 24 h after ethanol administration. This can be due to the peripheral vasodilatation and relative hypovolemia induced by ethanol, which decreases mean arterial pressure, presumably reduces the LV afterload, and subsequently preload with the consequence that the central venous pressure is lowered. It is conceivable that the systolic function was recovered at 6 h and 24 h when the ethanol levels were either low or absent but not at 1 h. However, the continuation of ethanol's deleterious effects in the absence of circulating plasma levels, suggests that interacting phenomena are occurring. Our data demonstrated that 6 h and 24 h after alcohol administration, myocardial contractility (shown by decreased dP/dtmax
) and relaxation (evidenced by reduced dP/dtmin
) were impaired in the recipients, i.e., these rats show increased susceptibility to myocardial ischemia/reperfusion injury after heart transplantation. Although results of animal experimental studies should not be directly extrapolated to human biology, these novel results have an important implication in cardiac transplantation: acute alcohol intoxication could significantly affect myocardial sensitivity to ischemia/reperfusion even if the donor heart seems to have normal contractile function. In a mouse model of coronary artery occlusion and reperfusion, aldehydes may increase the myocardial susceptibility to ischemia/reperfusion injury 
and moreover another model of myocardial infarction has shown that with aldehyde dehydrogenase-2 knockout condition, which is accompanied by elevated cardiac acetaldehyde levels, regional ischemia/reperfusion injury was accentuated 
. The EDPVR reflects the passive properties of the LV and used to obtain a measure of diastolic stiffness. Impaired myocardial relaxation in this study is shown by increased EDPVR for up to 24 h after ethanol, indicating that alcohol administration in rats also induces abnormal diastolic function. Moreover, alterations of active phase of relaxation index (as reflected by decreased dP/dtmin
) were found to recover slowly at 24 h after ethanol administration, when there is no measurable blood alcohol. Taken together, these results suggest that contrary to systolic dysfunction, which was recovered 6 h following ethanol, active relaxation and passive stiffness are slowly improved or sustained during 24 h. We can speculate that the diastolic dysfunction might be at least partially due to the cellular swelling. Due to the direct proportionality of the surface area and myocyte volume, the increase in myocyte profile surface area as evidence in the present work with an enlarged cardiomyocytes size 24 h following ethanol exposure indicates cellular swelling 
The donor's cardiac function has been shown to be an important prognostic factoring the clinical outcome of heart transplantation. In the present study we showed elevated circulating plasma cardiac troponin-T levels, the sensitive marker of myocardial injury in heart donors after alcohol intoxication even when hemodynamic measurement showed no evidence of impaired contractile function. A clinical study showed cardiac troponin-T >0.1 µg/l in the serum of heart donors to be predictive of early impaired graft function after transplantation 
. Previously with the same bolus of ethanol on rat significant elevation in cardiac troponin-T concentration was shown at 2.5 h and 6 h, but at 24 h, in contrast to our results, the levels were not significantly different from controls 
. It is possible that acute myocardial damage will arise as a consequence of ischemia and occurrence of cardiac arrhythmias. It has been argued that high blood acetaldehyde level might be responsible for the development of myocardial ischemia 
. In the present study, 24 h after ethanol administration, ischemic damage was assessed histologically by measuring cardiomyocytes swelling and in ECG by ischemic repolarization changes by ST-segment elevation. Alcohol in modest and higher doses has the potential to induce cardiac arrhythmias. The link between alcohol intake and acute disturbances of cardiac electrophysiology has been long suggested. Administration of alcohol in humans even in moderate doses was reported to induce acute prolongation of various electrocardiographic time intervals supporting the evidence of a potential arrhythmogenic effect of ethanol 
. We observed at 6 h and 24 h following ethanol injection, increased corrected QT-interval, a biomarker for ventricular tachyarrhythmia, which occurred while the heart rate was normal, suggesting that the ventricular repolarization time had lengthened. When the movement of ions across the channels is disturbed, ventricular repolarization is prolonged, resulting in prolongation of the QT-interval. Despite the apparent significance of ethanol interaction with the ion channels on cardiomyocytes membranes, the data published so far describing the effect of ethanol on ionic currents of cardiac cells are scarce. Some authors reported an alteration of calcium homeostasis, especially an up-regulation of cardiac L-type calcium channel in mice exposed to acute alcohol consumption 
. However, our data showed that following acute ethanol administration both mRNA and protein expression of L-type calcium channel, SERCA-2 and Na+
-ATPase remained unchanged.
Excessive consumption of alcohol over a short period of time (binge drinking) induces a systemic inflammatory reaction 
, which might lead to alcohol-induced myocardial inflammation. Over expression of TNF-α in the myocardium contributes to cardiac dysfunction caused by systemic and local insults. In the present study down-regulation of TNF- α and inducible NOS accounts for the absence of inflammation, which is also supported by our H&E staining showing no sign of myocardial inflammation. It has been shown in monocytes and macrophages that likely acute alcohol exposure directly increases transcription of heat shock protein 70, which in turn could repress TNF-α gene expression 
. Taken together, decreased LV contractility of the donor heart in the present study seems to be specific to ethanol-induced myocardial injury than inflammation. Moreover, impaired myocardial contractility after transplantation also bolsters this mechanism.
Accumulation of reactive oxygen species in response to ethanol exposure is believed to play an important role. Ethanol or acetaldehyde, the primary metabolic product of ethanol, is known to trigger both oxidative stress and apoptosis 
. This is also supported by our experimental findings that anti-oxidant enzyme, SOD-1 one of first line defense enzymes mRNA level was upregulated at 24 h and protein level after 6 h and 24 h following acute ethanol exposure. Additionally, both mRNA and protein levels for GPx-4 was increased 24 h after ethanol administration, depicting initiation of oxidative stress. In the present study, we evaluated two antioxidant enzymes, which do not exclude the involvement of other enzymes or non-enzymatic antioxidants following acute ethanol exposure. Moreover, it is known that both in vivo
and in vitro
, oxidative stress activates latent resident myocardial matrix MMPs 
. We showed in this study that MMP-9 mRNA-expression was upregulated at 6 h and 24 h and protein level was increased after 24 h following ethanol administration, which may indicate an increase in oxidative stress. In addition, we showed in this study that acute ethanol exposure resulted in formation of DNA-strand breaks as evidenced by our positive TUNEL staining. Recently, mitochondrial dysfunction also received some attention in the onset of alcoholic complications 
. Data from our current study revealed that 24 h following acute ethanol administration, there was increased both mRNA and protein expression of myocardial cytochrome-c oxidase, a terminal enzyme in the mitochondrial electron transport chain. This suggests a role of the mitochondrial death pathway in ethanol-induced apoptosis. A previous work showed in an in vitro
model that isolated cardiomyocytes treated with high-dose of ethanol induced oxidative stress and apoptosis 
. Consistent also with our data, Guo et al. showed that in mice acute ethanol exposure-induced myocardial dysfunction is associated with mitochondrial damage and apoptosis, supporting an essential role of acetaldehyde and mitochondrial dysfunction in ethanol-elicit alcoholic myopathic alteration 
. Moreover, ethanol exposure impairs glucose homeostasis. In humans, ethanol consumption is associated with increased circulating glucose concentration, glucose intolerance, and glucose homeostasis 
. It has been reported that short-term ethanol treatment in rats leads to glucose intolerance similar to that reported in humans 
. Recent data suggested that chronic alcohol consumption could not only result in glucose intolerance in hepatocytes and skeletal muscles but could also lead to alcoholic cardiomyopathy 
. In this present study we showed that acute alcohol administration resulted in elevated plasma glucose levels, which peaked at 1 h, time corresponding to decreased cardiac contractility and declined at 6 h and 24 h, times corresponding to normal systolic function of donor hearts.
The use of hearts from donors with a history of “alcohol abuse” remains uncertain 
. We sought to explore if acute alcohol abuse associated with a potential heart transplant donor can influence recipient graft function because the transplantation of these hearts may unmask abnormalities that may manifest after ischemia/reperfusion injury. The underlying pathophysiological mechanisms of ischemia/reperfusion injury include oxidative stress which could induce apoptosis and mitochondrial dysfunction, changes in calcium homeostatasis, inflammation, osmotic swelling, metabolic modulation, rapid restoration of physiologic pH 
. In a rat model of heart transplantation, donor rats received ethanol 6 h or 24 h before explantation, these time points were selected to be relevant to clinical cardiac transplantation and the measurements were done 1 h or 24 h after transplantation. Although the explanted heart function was recovered from ethanol-induced systolic dysfunction, we showed impaired contractile function 1 h and also unexpectedly 24 h after heart transplantation. In our laboratory, we previously showed in canine orthotopic heart transplantation 
and cardiopulmonary bypass models of global ischemia/reperfusion 
and in a rat model of transplantation-induced ischemia/reperfusion injury 
decreased LV contractility. However, we and others have already demonstrated that 24 h after transplantation systolic and diastolic function return to normal 
. In the present study persistence of graft dysfunction 24 h after transplantation needs to be considered during heart transplantation when hearts from donor with alcohol abuse are used. Systolic LV dysfunction is relatively common in even asymptomatic alcoholics, but whether diastolic function is also altered is much less well-studied 
. Care must be taken because in the present study our data additionally showed that acute alcohol abuse affects diastolic function of donor hearts and the myocardial alterations are at the cellular level. Moreover, in the recipients LV diastolic dysfunction was still present after transplantation. The possible explanation for normal contractile function after 6 h and 24 h following ethanol exposure and depressed systolic and diastolic function after heart transplantation could in part involve the alteration of SOD-1, GPx-4, MMP-9, cytochrome-c oxidase both at mRNA and protein levels due to ethanol exposure. Moreover the increased protein expression levels were still present 24 h after heart transplantation. The changes in calcium homeostasis and the inflammatory process do not seem to play an important role. One hour and also 24 h after heart transplantation, in which donors received ethanol 24 h prior to explantation, an increase in myocardial oxidative stress has been shown as evidenced by elevated myocardial TBARS levels. Thus, all these mentioned events may be further aggravated by ischemia/reperfusion injury with subsequent development of graft dysfunction.