Cardiac Myocyte-specific Knock-out of Calcium-independent Phospholipase A2γ (iPLA2γ) Decreases Oxidized Fatty Acids during Ischemia/Reperfusion and Reduces Infarct Size

Abstract

Calcium-independent phospholipase A 2 ␥ (iPLA 2 ␥) is a mitochondrial enzyme that produces lipid second messengers that facilitate opening of the mitochondrial permeability transition pore (mPTP) and contribute to the production of oxidized fatty acids in myocardium. To specifically identify the roles of iPLA 2 ␥ in cardiac myocytes, we generated cardiac myocyte-specific iPLA 2 ␥ knock-out (CMiPLA 2 ␥KO) mice by removing the exon encoding the active site serine (Ser-477). Hearts of CMiPLA 2 ␥KO mice exhibited normal hemodynamic function, glycerophospholipid molecular species composition, and normal rates of mitochondrial respiration and ATP production. In contrast, CMiPLA 2 ␥KO mice demonstrated attenuated Ca 2؉ -induced mPTP opening that could be rapidly restored by the addition of palmitate and substantially reduced production of oxidized polyunsaturated fatty acids (PUFAs). Furthermore, myocardial ischemia/reperfusion (I/R) in CMiPLA 2 ␥KO mice (30 min of ischemia followed by 30 min of reperfusion in vivo) dramatically decreased oxidized fatty acid production in the ischemic border zones. Moreover, CMiPLA 2 ␥KO mice subjected to 30 min of ischemia followed by 24 h of reperfusion in vivo developed substantially less cardiac necrosis in the area-atrisk in comparison with their WT littermates. Furthermore, we found that membrane depolarization in murine heart mitochondria was sensitized to Ca 2؉ by the presence of oxidized PUFAs. Because mitochondrial membrane depolarization and calcium are known to activate iPLA 2 ␥, these results are consistent with salvage of myocardium after I/R by iPLA 2 ␥ loss of function through decreasing mPTP opening, diminishing production of proinflammatory oxidized fatty acids, and attenuating the deleterious effects of abrupt increases in calcium ion on membrane potential during reperfusion.The salvage of jeopardized regions of myocardium during ischemia/reperfusion (I/R) 3 has been a long-standing goal of heart research. Because mortality and morbidity are related to infarct size, a variety of hemodynamic, metabolic, and pharmacological approaches have been used to reduce the severity of myocardial infarction during ischemia (1-3). Recent studies have accumulated evidence that the irreversible opening of the mitochondrial permeability transition pore (mPTP) upon oxidative stress is a principal mechanism of apoptotic/necrotic cardiac cell death accounting for the majority of I/R injury (4 -6). Although therapies for acute ischemia (e.g. reperfusion) have been extensively studied, at present there is no therapy for attenuating mPTP opening during reperfusion of ischemic zones in myocardium.Although the precise chemical composition of the mPTP is incompletely understood (6), a variety of initiators and modulators of mPTP opening has been identified (7,8). For example, during reperfusion, the reoxygenation of ischemic tissue results in mitochondrial Ca 2ϩ overload and renormalization of intracellular and matrix pH, which are accompanied by the prodigious generation of reactive oxygen s...

Heart failure–induced activation of phospholipase iPLA2γ generates hydroxyeicosatetraenoic acids opening the mitochondrial permeability transition pore

“…Consistent with this notion, our recent findings have demonstrated that murine myocardial mitochondrial iPLA 2 ␥ is activated by calcium and that iPLA 2 ␥ loss of function in cardiac myocytes markedly attenuates Ca 2ϩ -induced mPTP opening, generation of proinflammatory oxidized lipid metabolites (e.g. eicosanoids, docosanoids, and oxidized linoleic acid metabolites), and membrane potential dissipation resulting in reduction of infarct size during cardiac I/R in mice (13,27). ** ** * Figure 5.…”

“…Through sequential chromatographies in conjunction with activity-based protein profiling and high mass accuracy proteomics (45, 46), we quite unexpectedly identified the predominant phospholipase activity associated with nonfailing human heart mitochondria as cPLA 2 . In stark contrast, the predominant mitochondrial phospholipase activity in failing hearts was catalyzed by iPLA 2 ␥ generating toxic HETEs which, in combination with a prominent decrease in protective EETs, likely results in alterations in surface charge and potential in the mitochondrial membrane (27,47). Moreover, previous studies by our group reported the regiospecific PLA 1 activity of iPLA 2 ␥ with phospholipids containing polyunsaturated fatty acids at the sn-2 position.…”

“…This results in palmitate release in the plane of the inner membrane. Palmitate has previously been shown to be a potent activator of mPTP opening that, when generated in the plane of the inner membrane, could rapidly diffuse laterally to amplify mPTP opening (11,27,48). Thus, a single enzyme, iPLA 2 ␥, has dual regulating pathways for opening of the mPTP, each of which are dependent upon the generation of lipid-signaling molecules (e.g.…”

“…Previously, we identified the prominent production of eicosanoids (including HETEs, EETs, and prostaglandins) in calcium-activated murine heart tissue and mitochondria that were markedly reduced by genetic ablation of iPLA 2 ␥ (13,27). Accordingly, we hypothesized that abrupt activation of mitochondrial phospholipases by calcium uptake into the mitochondria in conjunction with the loss of membrane potential would result in the pathological activation of iPLA 2 ␥ leading to the generation of a complex array of eicosanoid metabolites and fatty acids that contribute to the opening of the mPTP and progression of human heart failure.…”

“HETE”ing up mitochondria in human heart failure

“…As a result, the prolonged opening of mPTP has been implicated in mitochondrial swelling, cytochrome c release, and necrotic cell death; however, the precise mechanism mediating mPTP opening is incompletely understood (1,2). A series of investigations by Gross and colleagues have also pointed to a role for the mitochondrial phospholipase isoform iPLA 2 ␥ in these pathological processes (3)(4)(5)(6). This enzyme cleaves one of the carbon chains from phospholipids to release the fatty acid arachidonic acid (AA), which can be oxidized into signaling-active eicosanoids.…”