|dc.description.abstract||Ischemia reperfusion (IR) injury in cardiac myocytes is well known to provoke membrane damage that is mediated by oxygen free radicals and that is associated with iron accumulation, energy dysregulation and opening of the mitochondrial permeability transition pore (MPTP). Nonetheless, the exact mechanism for how the oxygen free radicals develop in the mitochondria due to IR injury is currently ambiguous and need to be unraveled. In the present work, therefore, we explicitly focus upon understanding the role of frataxin in regulating mitochondrial damage which is associated with IR injury. Frataxin, which is a highly conserved small acidic mitochondrial matrix protein, has been recognized to regulate the mitochondrial iron metabolism and modulate the function of the electron transport chain (ETC) and production of reactive oxygen species (ROS). Loss of frataxin, in Friedreich’s ataxia, is coupled with impaired mitochondrial iron homeostasis, increased ROS production and respiratory chain dysfunction which prompt tissue damages. In the first project, we propose that enhanced expression of frataxin will salvage cardiomyocytes against IR injury by promoting and preserving the myocardial bioenergetics and that will improve the anti-oxidants effect in response to IR injury. We reported for the first time that frataxin expression is increased during reoxygenation stage of IR injury in cardiomyocytes and that led to mitigation the mitochondrial iron accumulation, prevented the MPTP opening and ROS formation, which in turn preserved the integrity of the mitochondria and increased the cardiomyocyte viability. Furthermore, cells that over-express frataxin had improvement in the mitochondrial respiratory chain complexes I and IV and in the cellular oxygen consumption compared to frataxin knockdown cells. Unexpectedly, we observed that enhanced frataxin expression revealed elevated levels of glutathione (GSH) and superoxide dismutase (SOD) and that protect the cardiomyocytes against IR injury. Together, our data present novel findings that over-expression of frataxin is cardioprotective against reperfusion injury-mediated cellular damage through its anti-oxidant effect, improving the mitochondrial bioenergetics and by inhibiting MPTP opening.
It is evident that cardiomyocytes express different types of P2 purinergic receptors. P2 receptors, which can be divided into P2X (ligand gated ion channels) and P2Y (G-protein coupled receptors) receptors, are known to be activated by extracellular nucleotides. Uridine triphosphates (UTP) is an extracellular nucleotide that is known to activate specific P2Y G-protein coupled receptor, including P2Y2 and P2Y4, in most cells to mediate numerous biological functions. It has been shown that UTP induced cardioprotection against IR injury and that protection was reported to be induced via P2Y2 and/or P2Y4 receptors. However, the exact mechanism how UTP is cardioprotective against hypoxic and/or IR injury conditions is still to be elucidated. In the second project, therefore, we examined the expression of P2Y2 receptors in cardiomyocytes and we tested the hypothesis that activation of P2Y2 receptors in cardiomyocytes by UTP is cardioprotective through the activation of mitogen activated protein kinases (MAPKs) signaling pathway. In the current study, we found that P2Y2 receptors were highly expressed in H9C2 cardiomyocytes as validated by gene analysis studies. Although P2Y2 receptors are highly expressed in these cells, extracellular nucleotides (UTP and ATP) pre-treatments could not induce any changes in the intracellular calcium mobilization, suggesting that these receptors are not Gq-coupled receptors that mediate the activation of PLC (phospholipase C). Unexpectedly, we found that UTP significantly induced the phosphorylation of ERK1/2 MAPK in a time- and dose-dependent manner. Additionally, UTP triggered minimal but insignificant activation of p-p38 and p-JNK and had no effect on p-Akt. Surprisingly, ARC-118925XX (P2Y2 selective antagonist), MRS-2578 (P2Y6 selective antagonist) and pertussis toxin (PTX, Gi-protein inhibitor) did not inhibit the induction of p-ERK1/2 by UTP, suggesting that these receptors are not P2Y2, P2Y4 or P2Y6 receptors and possibly these are new P2Y-like receptors because UTP induced ERK1/2 phosphorylation was abolished by suramin, non-selective antagonist of all P2Y receptors. In addition, we provide evidence that activation of these receptors by UTP increased the cell viability of cardiomyocytes in response to serum starvation. Therefore, activation of these receptors could be a potential cardioprotective mechanism, especially under stress conditions.||en_US