Mitochondria-mediated mechanisms of ferroptosis in response to cardiac ischemia-reperfusion injury - ABSTRACT Coronary heart disease is the leading cause of mortality and morbidity worldwide that occurs due to the detrimental effects of myocardial infarction (MI)/ischemia-reperfusion injury (IRI). Mechanisms of MI/IRI are not completely clear, and hence, there are no effective therapeutic strategies; current therapeutic approaches to mitigate heart damage mostly focus on restoring coronary perfusion but not limiting reperfusion injury. Multiple forms of cell death occur at different stages of post-MI/IRI depending on the severity of the disease. This project will elucidate the regulatory mechanisms of a recently discovered iron- dependent programmed cell death, ferroptosis, in cardiac IRI. Ferroptosis arises via excessive oxygenation of phospholipids (PLs) accompanied by the insufficient capacity of a selenoprotein glutathione peroxidase 4 (GPX4) to eliminate oxidized PLs, particularly, phosphatidylethanolamine (PEox) at the expense of reduced glutathione (GSH). We have recently identified and quantified pro-ferroptotic PEox species in response to RSL3 (GPX4 inhibitor, ferroptosis inducer) in cardiomyocytes by a state-of-art technique. In addition, our preliminary studies revealed accumulation of ferroptotic PEox species in mitochondria isolated from hearts exposed to global IRI as well as from cardiomyocytes challenged with RSL3. We propose that mitochondria participate in MI/IRI-induced ferroptotic signaling through two major GSH- dependent mechanisms: i) IR-induced mitochondrial ROS generation by ETC and TCA cycle deplete GSH, and hence, inactivate the GSH/GPX4 system, and ii) glutamate deficiency due to inhibition of glutaminolysis in mitochondria inhibits GSH synthesis as a result of low glutamate and cysteine levels. Thus, this project will explore a novel paradigm by investigating the role of mitochondria in cardiac IRI ferroptosis. Our central hypothesis is that mitochondria are engaged in ferroptosis induced by cardiac IRI through diminishing GPX4 activity and inability to control the accumulation of pro-ferroptotic PEox species. Also, we propose that major cell death mechanisms have different impacts during post-MI/IRI depending on the severity and duration of post-MI/IRI. Hence, we will evaluate specific biomarkers to distinguish the contributions of apoptosis, ferroptosis, necroptosis, and pyroptosis to post-MI/IRI. Two approaches will be employed to mitigate cardiac IRI via i) specific suppression of mtROS production/GSH depletion, and ii) replenishment of the GSH pool by a potent reductant lipoic/dihydrolipoic acid (LA/DHLA). Specific Aims of the project will (i) determine comparative contributions of ferroptosis to cardiac dysfunction during IRI, (ii) explore molecular mechanisms of mitochondria-mediated ferroptotic signaling in cardiac cells, and (iii) examine the effectiveness of novel inhibitors of ferroptosis against cardiac IRI. The project will establish the contribution of ferroptosis, in comparison with other major cell death mechanisms, to MI/IRI-induced cardiac dysfunction, and discover new mitochondria-mediated mechanisms of ferroptotic signaling that will lead to innovative therapeutic strategies for the treatment of cardiac IRI.
