Myocardial Ischemia-Reperfusion/Injury (Pharmacotherapy of Ischemic Heart Disease) Part 2

Effects of vitamin E versus nitroglycerine on I/R injury

Our results demonstrated that a short course of vitamin E treatment induced preconditioning in the hearts. The vitamin E therapy, enhanced contractile, and vascular recovery, and attenuated oxidative stress in cardiac tissue, as demonstrated by the decrease of MDA in cardiac tissue. Moreover, this therapy protected the hearts against MPT opening as indicated by significant increase of NAD+ in cardiac tissue. Histological examination showed less mitochondrial injury induced by reperfusion in the hearts of this group, with preservation of the myocytes structure. Moreover, the present study clearly demonstrated that preischemic treatment with GTN; a NO donor did not provide significant protection of hearts against I/R-induced contractile dysfunction and tissue injury. Peroxynitrite (ONOO-), the reacting product of NO and O2-, is apotent cytotoxic agent. It is highly reactive with a wide variety of molecules, including deoxyribose, cellular lipids, and protein sulfhydryls, and results in oxidative tissue damage (Farshid et al., 2002). In this study, GTN treatment did not attenuate the reperfusion-mediated increase of MDA in cardiac tissues of GTN-treated rats. This finding suggested that ONOO- might be formed excessively in post-ischemic myocardial tissue. In the current study, supplementation of NO donor could have raised cardiac tissue NO concentration, with the coincident increase in free radical generation at reperfusion established the conditions of ONOO- formation. Therefore, we suggest that exogenous NO failed to provide cardioprotection, due to concomitant increase of ONOO-. In this study, the non significant increase of NAD+ content in isolated hearts from GTN-supplemented rats indicated failure of GTN to attenuate MPT opening. The opening of that channel lead to mitochondrial swelling, release of apoptotic molecules and eventually cell death. This suggestion was further confirmed by histological examination of isolated hearts from GTN-supplemented rats. Previous literature (Dhalla et al., 2000) reported a depletion of endogenous antioxidants in the ischemic hearts upon reperfusion. Various studies have reported the beneficial effects of antioxidants as these agents render resistance to the hearts against I/R injury. However, other investigators have failed to observe such results. The slow incorporation of vitamin E into tissues, due to its marked lipophilicity, is probably responsible for its failure as a cardioprotective compound as shown during the acute administration of a-tocopherol after I/R injury induced in the pig (Altavilla et al., 2000). Several studies reinforce the importance of localization and timing in cardioprotection. Delivery of the antioxidants to the right compartment in the right time period is very difficult to achieve in controlled animal studies, and even more difficult in patients. As a result of the controversy in the animal studies, and the failed clinical trials, it is often concluded that inhibition of ROS will not influence infarct size. A more realistic assessment is that to have a significant benefit in reducing infarct size requires the correct delivery of mitochondrial targeted antioxidants perhaps in conjunction with other therapies. So in this study, we tried to give the animals vitamin E in a large dose, with a sufficient time to provide its effects. The demonstration that prior exposure to a low concentration of H2O2 protects against MPT opening may be of pathophysiological importance for cardioprotection (Costa et al., 2006). By the same reasoning, in preconditioning, we speculate the need for an antioxidant that attenuates the large burst of oxidative stress at the start of reperfusion without completely neutralizing free radicals.

We suggest that vitamin E as a physiological antioxidant acted to scavenge free radicals without completely neutralizing them, thereby affording a significant preconditioning effect. Reduction of free radical formation inhibits MPT opening, thereby affording preconditioning. This is clearly demonstrated in the current study, since there was a significant increase of NAD+ content of reperfused hearts in the vitamin E-treated groups. Histological examination confirmed this result, as it revealed marked protection of the normal structure of myocytes and mitochondria. Addition of GTN treatment to vitamin E attenuated its cardioprotective effect. In summary, the findings of the present study provide evidence that a short course of vitamin E treatment protected the heart against reperfusion-injury compared to a NO donor. The MPT is an important target of this protection.

Effects of vitamin E treatment on age-associated changes in cardiac responses to the injury of post I/R

Aging is one of the most important risk factors for the development of cardiovascular disease. Aging is characterized by loss of myocytes, remodeling and impaired contractile function in the heart. The rate of programmed cell death in the left ventricle increases with age (Kwak et al., 2006). Meanwhile the aged heart faces a high risk of free radical injury owing to slow generation of antioxidant enzymes by its cells, and a general decline in this system may be another reason for the development of myocardial dysfunction (Asha Devi et al., 2003 a ).

It is well established that nitric oxide (NO), is constitutively generated within the heart, not only by the endothelium but also by the myocytes (Balligand et al., 1995). Numerous previous animal studies have reported that there is a loss of NO biological activity and /or biosynthesis during aging (Matz et al., 2000).

Oxygen free radicals increase in concentration upon reperfusion of ischemic cardiac tissue (Xia et al., 1996). It is well established that these reactive species can interact with and damage various cellular components (Berlett & Stadtman, 1997). Free radical production in the heart has also been reported to increase with age (Sohal et al., 1990). Thus, although restoration of blood flow is the sole method for salvaging ischemic tissue, oxidative damage may occur during reoxygenation and contribute to ischemia-reperfusion injury.

Regular exercise is a key component of cardiovascular risk prevention strategies. Physical activity is known to cause generation of free radicals. Exercise training in old animals failed to enhance antioxidant activity (Hatao et al., 2006). However swim training especially at low-intensity, was found to be beneficial as a major protective adaptation against oxidative stress particularly in the older myocardium (Kiran et al., 2004). The tocopherols (major vitamers of vitamin E) are believed to play a role in the prevention of human aging-related changes, and are of particular interest, mainly because of their antioxidative properties (Asha Devi et al., 2003 b).

From the aforementioned data, it seems possible that free radical – mediated injury is a major issue in the increase of aging related reperfusion-induced myocardial dysfunction. Exercise training program could not always protect the aging heart satisfactorily. We tried to test the hypothesis that using an antioxidant as vitamin E could lead to better protection of the aging hearts as compared with exercise training.

Therefore, a study was conducted to compare the effects of vitamin E treatment versus swim- exercise training on age- associated changes in cardiac responses to the injury of post I/R.

Methods

This work was undertaken on female Wistar rats aged 16-18 months. Rats were allocated into 3 groups: a) Control group of aged rats, b) Exercise -trained aged rats subjected to daily physical exercise by swimming in the water tank, for 2 hours daily, 6 days aweek for 2 weeks, and c) Vitamin E-treated aged rats subjected to daily injection of vitamin E (300 mg/kg) intraperitoneally, 6 days aweek for 2 weeks.

The swim-training program adopted was according to Refaat et al., (1989), in a swimming tank, filled with water and maintained at thermoneutral temperature of 30°c. A motor fan in the tank stirs strong water currents. This ensures uniformity of temperature and forces the rats to swim actively all the time.

Experimental procedures

They were done as previously described (Ayobe &Tarazi, 1983). Peak developed tension per left ventricular weight as well as coronary flow rate per left ventricular weight were calculated relative to left ventricular weight (PT/LV, g /100mg)& (CF/LV, ml /100mg/min).

Nitrate concentration in coronary effluents, as a stable product of nitric oxide (NO), was measured by an endpoint one-step enzymatic assay using nitrate reductase, as described by Bories and Bories, (1995), and modified in tissues by Kassim, (1997).

Effects of exercise on cardiac weights and post Ischemia-reperfusion (I/R) responses

In this study, exercise was found to exert a trophic effect on cardiac chambers manifested by significant increase of the absolute & relative weights of atria, right ventricle, and left ventricle. Swimming is well recognized for its effectiveness in inducing myocardial hypertrophy (Evangelista et al., 2003). Exercise can protect cardiac function of the aging heart by protection against; loss of cardiac myocytes, reduction in number of myonuclei, reactive hypertrophy of remaining myocytes, and increased connective tissue in left ventricle (LV) of the aging rat heart. This protection is achieved through attenuation of age- induced elevation in Bax/Bcl-2 ratio, thereby inhibits apoptosis (Asha Devi et al., 2003a), and exerts its trophic effect.

Although exercise-trained rats had significant increase in LV and LV/BW that indicates LV hypertrophy, they showed Peak developed tension similar to control which could be attributed to the increase of free radical in aged rats induced by exercise. Oxygen free radicals are known to have deleterious effects on contractile force of heart (Gao et al., 1996).

Fig. 6. Mean values± SEM of: absolute weights (mg) of atria (AT), right ventricle (RV), left ventricle (LV), and their relative weights (mg/g) (AT/BW), (RV/BW), (LV/BW) in control rats, exercise-trained rats _, and vit. E-treated rats. a. Significance calculated by least significant difference (LSD) at P < 0.05 from control group. b. Significance calculated by LSD at P < 0.05 between vit E-treated, and exercise-trained groups

The results of ischemia-reperfusion of isolated hearts from both control and exercise-trained aged rats have shown the lack of tolerance to ischemia-reperfusion injury as regard inotropic activities as well as myocardial flow rate. This could be explained in view of the increase in free radicals generation induced by exercise, even if small, was added to the increased free radicals generation induced by aging exceeding the antioxidant defense capacity of the heart leading to loss of beneficial cardio-protective effect of exercise on ischemia-reperfusion injury (Hatao et al., 2006).

Fig. 7. Mean absolute and percent values ± SEM of: heart beating rate(HR), Peak developed tension(PT), Peak developed tension per left ventricular weight(PT/LV), Time to peak tension (TPT), Half relaxation time(% RT), and Coronary flow rate per left ventricular weight (CF/LV), at 30 minutes reperfusion of hearts isolated from control rats, exercise-trained rats, and vit. E- treated rats. a. Significance calculated by least significant difference (LSD) at P < 0.05 from control group. b. Significance calculated by LSD at P < 0.05 between vit E-treated, and exercise-trained groups

The increased free radicals generation such as superoxide anion could react with NO to form peroxynitrate, decreasing NO biological activity. Peroxynitrate molecule by itself might play a significant role in oxidative tissue damage. This is supported in this study by the absence of increase of nitrates in the coronary effluent from exercise- trained aged rats at 30 min reperfusion.

Fig. 8. Mean values ±SEM of nitrates in coronary effluents (nmol/100mg LV/min) at baseline values, and 30 minutes of reperfusion in control rats, exercise-trained rats, and vit. E-treated rats. a. Significance calculated by least significant difference (LSD) at P < 0.05 from control group. b. Significance calculated by LSD at P < 0.05 between vit E-treated, and exercise-trained groups

Also, the heart beating rate of the exercise-trained rats could not be preserved after 30 min of reperfusion. This could be attributed to the increased free oxygen radicals in these rats, since oxyradicals have been implicated as a possible cause of reperfusion arrhythmias (Tanguy et al., 1998). However, the rate of relaxation of hearts excised from exercise-trained rats showed better tolerance to I/R after initial intolerance, and such improvement is in accordance to a recent report by Libonati et al., (2005), who found that sprint training in rats improves post-ischemic left ventricular diastolic stiffness due to up-regulation of myocardial glycolysis, with ATP production that protects the heart from ischemia-reperfusion as it plays an important role in actin-myosin rigor bond dissociation, and regulating myocardial diastolic function.

Effects of vitamin E on cardiac weights and Post Ischemia-Reperfusion (I/R) responses:

Vitamin E has been reported to stimulate upregulation at the expression level of B cl-2 gene, B cl-2 molecule is involved in the inhibition of apoptosis (Azzi et al., 2004).

Therefore, vitamin E by preventing apoptosis of myocardial cells could be expected to induce hypertrophy of the heart (trophic effect). However in case of vitamin E-treated rats, results have shown that the effect of vitamin E on the rate of growth of right and left ventricles was not similar. While the rate of growth of right ventricle was increased by the anti-apoptosis effects of vitamin E. The increase in left ventricle & left ventricle/ body weight was insignificant. This could be attributed to the favorable effects of vitamin E on reduction of blood pressure and blood viscosity (Costa et al., 2005). These two factors have been claimed to induce growth of left ventricle during aging (Ghali et al., 1997). Thereby, by eliminating these two factors, the rate of growth of left ventricle was decreased under the influence of vitamin E supplementation counteracting its trophic effect in aged rats.

In contrast to aged control rats and exercise-trained aged rats, the isolated hearts from vitamin E-treated rats showed better tolerance as regard inotropic activity, especially force generation measured as peak developed tension as well as coronary flow rate. In accordance, Venditti et al., (1999) showed a protective action of vitamin E treatment against lipid peroxidation and cardiac dysfunction associated with ischemia- reperfusion in rats. Vitamin E decreases free radical deleterious effect on cell membrane, proteins, DNA, and decreases both ROS mediated direct ischemia-reperfusion mediated injury, as well as peroxynitrite-mediated injury and thereby preserving NO through preventing its inactivation by its transformation to peroxynitrite (Kamat, 2006). In support, this study showed significant increase of nitrates in coronary effluent from vitamin E- treated aged rats measured at 30 min of reperfusion.

In view of the aforementioned data, vitamin E proved to be a cardio-protective agent as it attenuated the injury of post ischemia-reperfusion on the aged myocardium. This beneficial effect of vitamin E could be partially attributable to preservation of NO biological activity by preventing its transformation to the toxic compound peroxynitrite.

On the other hand, the swim-training program adopted in this study, as regard tolerance to ischemia-reperfusion apart from improvement of half relaxation time, the swim-training program did not show other promising cardio-protective effects against cardiac changes associated with the aging process.

Therefore, vitamin E supplementation could be recommended to aged people especially patients suffering from ischemic heart disease. Vitamin E could be used either alone or in combination with exercise training taking into consideration the duration and intensity of exercise program.