Our study demonstrates for the first time that the intraperitoneal administration of Br provides cardioprotection by limiting myocardial injury in a global I/R model. This was determined by an improved postischemic ventricular recovery and a reduction in the myocardial infarct size as well as a reduction in the degree of apoptosis following I/R injury when compared with those values of the nontreated control rats. The present study was designed to pharmacologically precondition the heart with Br before the induction of ischemia, to slow down the rate of development of ischemic injury, and to avoid postischemic reperfusion injury. There are several important findings of the present study that indicate that Br may function as a pharmacological preconditioning agent. Br was found to precondition the heart, as evidenced by its ability to lower the infarct size by reducing apoptosis and improved postischemic functional recovery. It was also found to be a potent coronary vasodilator, as documented by increased CF.
From our observations, we suggest that the Br-mediated cardioprotection might be mediated by the activation of the Akt-FOXO signaling cascade. In our present study, we have observed an increased phosphorylation of Akt on Br treatment, which might have resulted in decreased cardiomyocyte apoptosis and myocardial infarct size following I/R injury. Apoptosis occurs in a wide variety of cardiovascular disorders and is now known as one of the prime causes that attributes to the compromised cardiac function (9
). Reperfusion of the ischemic myocardium is associated with apoptotic cell death in concert with DNA fragmentation. It was recently documented that apoptosis is the dominant form of myocardial cell death in the infarct area immediately after infarction as well as during the later stages in the remote viable myocardium (3
). Serine or threonine phosphorylation of Akt plays a crucial role in the cell survival cascade (22
) since Akt activation phosphorylates multiple protein substrates, which regulate the gene transcription of which one is the set of FOXO proteins. An increased expression of Akt has also been associated with better myocardial contractility (6
). In addition, the phosphorylation of Akt was shown to play an important role in facilitating growth factor-mediated cell survival and in blocking apoptotic cell death (7
Recently, the Akt-FOXO signaling cascade and the regulation and translocation of FOXO have been suggested and frequently cited as important signaling mechanisms for the prosurvival pathway. The FOXO subfamily of forkhead transcription factors is a downstream target of Akt. This subfamily consists of three members: FOXO1 (FKHR), FOXO3A (FKHRL-1), and FOXO4 (AFX), which are all inactivated by Akt (2
). FOXO transcription factors have been implicated in regulating diverse cellular functions, including differentiation, metabolism, proliferation, and survival (1
). Active FOXO transcription factors have been associated with the activation of several proapoptotic genes, thereby activating apoptosis. However, the phosphorylation of FOXO by Akt has been shown to bring about the exclusion of FOXO from the nucleus and the inhibition of the forkhead transcriptional program.
Br treatment has demonstrated an increased phosphorylation of FOXO3A both in the nucleus and the cytoplasm, thereby inhibiting the proapoptotic signaling pathway. We assume after nuclear phosphorylation of FOXO3A by p-Akt that it is expelled from the nucleus and gets inactivated. It has been proposed that the phosphorylation of the forkhead domains in FOXO by Akt leads to the disruption of its DNA binding capacity (21
). Furthermore, this phosphorylated state of FOXO seems to expose a high-affinity binding site for 14-3-3 proteins (21
). This FOXO is then shuttled to the cytosol where it becomes a target for the ubiquitin-proteasome system (21
). The improvement in cardiac functions on Br treatment might be attributed to the decreased cardiomyocyte apoptosis by the activation of the prosurvial Akt and, thereby, the inhibition of the proapoptotic FOXO3A.
For the first time, the results of our study demonstrate that the beneficial effects of Br are due to its ability to phosphorylate Akt, which in turn leads to the phosphorylation of FOXO3A. This phosphorylation sequence can be linked to the reduction of apoptotic cell death. We demonstrate that under our experimental circumstances, Br significantly reduced the extent of myocardial infarction and improved postischemic cardiac function, providing evidence that Br could mediate cardioprotection at least in part against I/R-induced injury.
In conclusion, Br demonstrated a significant cardioprotective effect by inhibiting the degree of apoptosis and reducing infarct size, leading to improved cardiac function, which may be due to the phosphorylation of Akt and FOXO3A. Therefore, adjunctive therapies by natural products such as Br can target specific molecular pathways involved in cell survival, which may prove to be efficient in the treatment of human heart disease and ischemic pathology.