Prolonged severe ischemia leads to cardiac cell death. Necrosis previously was regarded as the only mode of cell death, whereas, now there is accumulating evidence that in addition to overt necrosis, a subset of cells also die by apoptosis, (the programmed cell death). The relative contributions of necrosis and apoptosis to cell death in ischemia and reperfusion are still open to debate, although necrosis appears to dominate during ischemia, and apoptosis may dominate during reperfusion. There is now evidence that apoptosis occurs during sustained ischemia and when reperfusion follows shorter periods of ischemia [14, 15].
The progressive loss of cardiomyocytes in a heart that is already compromised leads to further deterioration of cardiac function, conduction disturbances due to degeneration of SA, AV and inter-nodal pathway, cardiac remodeling and cardiomyopathy [3, 4]. The fact that apoptosis plays a role in the tissue damage seen after myocardial infarction has pathological and therapeutic implications. If indeed cardiomyocyte apoptosis plays an important role in initiation and progression of cardiac diseases, drugs that effectively and specifically inhibit apoptosis might be useful therapeutic agents for attenuating myocardial injury due to I-R .
In reperfused ischemic hearts increase in oxidative stress, and decrease in antioxidant defense has been reported to lead to cardiac dysfunction partly due to apoptosis [4, 17]. However, whether or not plant derived agents, known to possess antioxidant activity may reduce myocardial apoptosis induced by I-R, and thus improve ventricular function and thus attenuate MI has not been directly investigated.
In the present study, TUNEL positivity and the immunohistochemical localization of Bax, an inducer of apoptosis and Bcl-2 proteins, inhibitors of apoptosis were studied to delineate the involvement of apoptosis in I-R induced injury. In order to correlate whether the inhibition of apoptosis has any direct effect on cardiac function, hemodynamic function was also monitored and recorded at preset time points throughout the experimental period. Cardioprotective activity of these herbs was confirmed by assessing the severity of pathological changes.
Results of the present study demonstrated that in the control IR group myocardial I-R injury triggered apoptotic cell death. Increase in TUNEL staining observed in the control IR group suggests a role of apoptosis in contribution of myocardial injury following I-R. A slight reduction in Bcl-2 expression and significant increase in Bax expression as compared with sham group was observed in the control IR group. This observation receives support from earlier studies [18, 19]. In addition, consistent with increase in apoptotic cell death, post ischemic reperfusion injury also resulted in significant depression of left ventricular dynamics, peripheral hemodynamics (MAP) and HR.
Cl treatment significantly reduced the percentage of TUNEL positive cells Vs the control IR group, demonstrating its significant anti-apoptotic activity. Treatment with Cl was associated with greater Bcl-2 and attenuated Bax expression as compared to the control IR group. Curcumin, the active ingredient of the rhizome of the turmeric plant (Curcuma longa), a commonly used spice, has been reported to prevent cancer in animal tumor models possibly by its apoptosis-inducing and antiproliferative influences [20–22]. However, in the present study, in contrast to earlier reports, marked anti-apoptotic activity of Cl was observed.
Previously we reported that cardioprotective effect of Cl results from the suppression of oxidative stress and correlates with the improved ventricular function . However, till date there are no in vivo studies, which have explored the relationship between the putative anti-apoptotic effects of Cl on the functional recovery of ischemia reperfused myocardium. The present study demonstrates that Cl treatment resulted in preserved left ventricular function as reflected by a significant increase in the indices of contractility (+) LVdP/dt, relaxation (-) LVdP/dt and decrease in preload (LVEDP). It is speculated that, Cl treatment may have indirectly restored blood flow in the ischemic regions towards normal as assessed by its efficacy in improving cardiac performance, especially correcting the ischemia and reperfusion-induced increase in LVEDP. However, Cl did not significantly affect MAP and HR. The anti-apoptotic effect of Cl, improved ventricular functions with improved histologic features suggests that treatment with this agent may exert cardioprotective effects following coronary ligation and reperfusion.
Os treatment did not demonstrate any significant anti-apoptotic activity as determined by TUNEL staining and immunohistochemical results. No significant change in the expression of Bax and Bcl-2 proteins was observed with Os treatment as compared to control IR in the present study. It markedly increased MAP, HR and significantly reduced the surrogate preload marker LVEDP as compared to control IR. However, it failed to significantly improve the left ventricular contractility and relaxation and failed to significantly modulate the histopathologic alterations compared to control IR group. Results of the present study demonstrate that Os does not possess significant cardioprotective effects.
The relationship between the possible anti-apoptotic effects of Cl on the functional recovery of ischemic reperfusion injury of the heart has been well elucidated by the dosing protocol of the present study. The exact mechanism by which Cl may reduce myocardial ischemia and reperfusion induced myocardial apoptosis is far from clear presently. Recently it has been reported that Curcumin, an active principle of Curcuma longa reduces cardiomyocytic apoptosis. Curcumin an inhibitor of NF-kappaB, ameliorated the surge of pro-inflammatory cytokines during cardiopulmonary bypass (CBP) and decreased the occurrence of cardiomyocytic apoptosis after global cardiac ischemia/reperfusion injury. The authors proposed that by inhibiting NF-kappaB activation, the up-regulation of cardiac proinflammatory genes can be ameliorated, and the activation of matrix metalloproteinase can be decreased during CPB, thereby lessening severity of cardiac mechanical dysfunction after global cardiac ischemia/reperfusion injury [24, 25]. However, in the present study, we have used the aqueous extract of Cl and have proposed a different mechanism for its antiapoptotic effect and correlated it with the ventricular function. Based on the present findings it can be speculated that Cl may attenuate apoptosis via a number of mechanisms: Upregulation of Bcl-2 may result in formation of heterodimers with Bax, resulting in no/fewer free Bax protein available for homodimerization. If Bax homodimers predominate cell death will occur, but when Bcl-2 and Bax heterodimererization prevails cells can survive. Substantial evidence indicates that the mitochondria play a critical regulatory role in the signal transduction pathway leading to apoptosis [4, 26]. Loss of contractile cells in the heart poses an additional workload on the remaining viable myocytes that may be unbearable, resulting in pathologic stimuli and death signals. In the present study, In contrast to Os, Cl treatment may have salvaged these myocytes and prevented cell loss induced by apoptosis. Histopathologic evaluation further confirms the cardioprotective potential of such a treatment.
In order to elucidate, the additional mechanisms by which Cl may reduce myocardial apoptosis and the potential clinical implications of such actions, we need to further investigate the relationship of the detrimental effects of key oxidants and apoptotic signals with reperfusion injury. This will lead to a better understanding of basic physiological and pathological mechanisms relevant to myocardial ischemia and reperfusion injury and give new insight to novel therapeutic targets and strategies for its treatment. However, the present study provides a lead for further exploring other mechanisms contributing to the cardioprotective effect of Cl. Whether the conclusions drawn on the basis of the current data can be extrapolated to clinical setting, remains to be defined by well-controlled studies in-patients. Nonetheless, the results of the present study are rather encouraging, because they could unravel a new therapeutic approach for the prevention and/or treatment of ischemic heart disease.