I/R injury may be defined as the damage to myocardial tissue when blood supply is restored after a period of ischemia, resulting in oxidative damage, inflammation, intracellular calcium overload, apoptotic and necrotic myocytes death and cardiac dysfunction.[¹–⁴] Hhcy, a condition of elevated serum homocysteine concentration, is considered as an independent risk factor for various cardiovascular disorders such as atherosclerosis, endothelial dysfunction, hypertension, myocardial infarction and chronic heart failure.[^{5, 6}] Hhcy has been well reported to enhance the generation of reactive oxygen species (ROS), decrease endothelail nitric oxide synthase (eNOS) expression and consequently reduces the generation of NO to produce cardiac dysfunction.[⁷–⁹] Statins, the HMG-CoA reductase inhibitors, have been regarded as potent hypolipidemic agents that accounts for their role in reducing cardiovascular mortality and morbidity.[^{10, 11}] Simvastatin, a potent inhibitor of HMG-CoA reductase, has been well reported to be a potent cardioprotective agent due to its antioxidant properties.[¹²] Simvastatin has been noted to prevent aortic production of ROS. Moreover, simvastatin showed inhibitions of protein and lipid oxidation products such as thiobarbituric acid reactive oxygen species (TBARS) confirming its antioxidant potential.[¹³] In addition, experimental studies have shown that treatment with simvastatin resulted in reductions of malondialdehyde (MDA) levels and increases in the superoxide dysmutase (SOD) and NO levels accounting for its cardioprotective potential.[¹⁴] In addition, simvastatin has been reported to lessen myocardial contractile dysfunction and lethal ischemic injury in isolated Langendorff-perfused rat heart model.[^{15, 16}] Therefore, the present study was undertaken to investigate the cardioprotective effect of simvastatin against I/R-induced myocardial injury in hyperhomocysteinemic rat hearts.

Rats fed with L-methionine (1.7 g/kg/day, p.o.) for 4 weeks via oral gavage produced hyperhomocysteinemia (22.15±1.85 μM/L) when compared with normal rats (4.31±0.56 μM/L). In addition, L-methionine administration did not produce mortality in rats.

Cardiovascular diseases correspond to the leading cause of morbidity and mortality whose incidence is continuously increasing worldwide.[^{22, 23}] The myocardial, vascular or electrophysiological dysfunction induced by the restoration of blood flow to previously ischemic tissue refers to I/R injury, the manifestations of which include reperfusion arrhythmias, endothelial cell damage leading to microvascular dysfunction, myocardial stunning, myocyte death and infarction.[^{24, 25}] The increase in infarct size and the release of LDH and CK have been well reported to be an index of I/R-induced myocardial injury.[^{26, 27}] In the present study, 30 min of ischemia followed by 120 min of reperfusion produced myocardial injury, as assessed in terms of increased infarct size in the heart and elevated release of LDH and CK in the coronary effluent. The peak release of CK was observed after 5 min of reperfusion whereas the maximal release of LDH was noted immediately after reperfusion, which are in accordance with the earlier reports.[^{28, 29}] Moreover, increase in lipid peroxidation and superoxide anion generation have been reported to be the indicators of oxidative stress.[^{30, 31}] Lipid peroxidations, measured in terms of TBARS, and superoxide anion generation, assessed in terms of reduced NBT, were noted to be increased as a result of I/R. These indicators suggest the development of I/R-induced oxidative stress, which may be responsible for the noted I/R-induced myocardial injury.

Administration of L-methionine (1.7 g/kg/day orally) in rats for 4 weeks produced Hhcy.[²⁹] In the present study, a marked increase in infarct size and release of LDH and CK were noted in the hyperhomocysteinemic rat heart when compared with the normal rat heart. Hhcy has been noted to downregulate NO bioavailability by accumulating asymmetric dimethylarginine, which is an endogenous inhibitor of eNOS.[³²] In addition, Hhcy has been reported to produce high oxidative stress in the heart by activating NADPH oxidase-mediated ROS generation.[³³] Hhcy-induced oxidative stress may occur as a result of decreased expression and activity of key antioxidant enzymes, as well as increased enzymatic generation of superoxide anion.[³⁴] Thus, development of high degree of oxidative stress may be responsible for the observed marked increase in myocardial injury in the hyperhomocysteinemic rat heart. This contention is supported by the fact that a marked increase in lipid peroxidation and superoxide anion generation were noted in hyperhomocysteinemic rat hearts when compared with normal rat hearts.

Statins, commonly referred to as HMG-CoA reductase inhibitors, have been widely accepted to possess various pleiotropic effects in order to afford cardioprotection.[^{35, 36}] Simvastatin, a potent member of statins, has been well reported to inhibit HMG-CoA reductase and show cardioprotection.[³⁷] Numbers of studies have demonstrated simvastatin to reduce myocardial injury parameters in order to mimic cardioprotection. Treatment with simvastatin has been noted to improve endothelial function in mice.[³⁸] Moreover, simvastatin reduced the infarction volume and ameliorated the ischemic damage in rats that further confirmed its cardioprotective potential. Additionally, various studies have reported that Simvastatin lessened myocardial contractile dysfunction and lethal ischemic injury in isolated Langendorff-perfused rat heart model.[^{15, 16, 39, 40}] The present study investigated the cardioprotective potential of simvastatin against I/R injury in normal and hyperhomocysteinemic rat hearts when administered at the onset of reperfusion. The data demonstrates that administration of Simvastatin (10 μMol) at the onset of reperfusion results in significant attenuation of I/R-induced myocardial injury in normal and hyperhomocysteinemic rat hearts as assessed in terms of reductions in myocardial infarct size and decreased release of LDH and CK in coronary effluent, which is in accordance of our earlier reports.[³⁷]

Further, numerous studies have reported that simvastatin possesses cardioprotective effects due to its potent antioxidant properties.[¹²] In support, simvastatin has been noted to reduce the activity of NADPH-CoQ reductase, an enzyme required in generation of free radicals that evidenced its potent role as an antioxidant.[⁴¹] Moreover, treatment with simvastatin prevented the aortic productions of ROS along with inhibition of lipid oxidation products such as TBARS. In addition, experimental studies have shown that treatment with Simvastatin decreased MDA levels and increased the SOD activity.[^{14, 42}] Furthermore, another experimental study in rats showed that treatment with simvastatin decreased oxidative stress in diabetic-hypercholesterolemic rats that further confirmed its antioxidant potential.[⁴³] This contention is supported by the results obtained in the present study that treatment with simvastatin (10 μMol), markedly reduced the oxidative stress in normal and hyperhomocysteinemic rat hearts subjected to I/R, as assessed in terms of reductions in TBARS and superoxide anion generation.

On the basis of the above discussion, it may be concluded that I/R-injury prepares the myocardium susceptible to increased infarct size and enhanced oxidative stress. Simvastatin, due to its potent antioxidant effects, showed cardioprotection in normal and hyperhomocysteinemic rat hearts. Further studies are going on in our laboratory to reveal various mechanisms possessed by statins in attenuating myocardial injury in normal and hyperhomocysteinemic rat hearts.

Source of Support: Nil

Conflict of Interest: Nil.

We wish to express our gratitude to Respected Dr. Balvir Singh Tomar ji, Honorable Chairman; Dr. (Mrs) Shobha Tomar ji, Honorable Managing Director; Dr. K.C. Singhal ji, Honorable Vice Chancellor; Dr. K.P. Singh ji, Honorable Registrar and Prof. Govind Mohan ji, Director Pharmaceutical Sciences Division, of NIMS University Shobha Nagar, Jaipur - 303121, Rajasthan for their praiseworthy suggestions and constant support for excellant research area.