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

Sung Ho Moon,David J Mancuso,Harold F Sims,Xinping Liu,Annie L Nguyen,Kui Yang,Shaoping Guan,Beverly Gibson Dilthey,Christopher M Jenkins,Carla J Weinheimer,Attila Kovacs,Dana Abendschein,Richard W Gross

doi:10.1074/jbc.m116.740597

Abstract

Calcium-independent phospholipase A2γ (iPLA2γ) 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 iPLA2γ in cardiac myocytes, we generated cardiac myocyte-specific iPLA2γ knock-out (CMiPLA2γKO) mice by removing the exon encoding the active site serine (Ser-477). Hearts of CMiPLA2γKO mice exhibited normal hemodynamic function, glycerophospholipid molecular species composition, and normal rates of mitochondrial respiration and ATP production. In contrast, CMiPLA2γKO 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 CMiPLA2γKO 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, CMiPLA2γKO mice subjected to 30 min of ischemia followed by 24 h of reperfusion in vivo developed substantially less cardiac necrosis in the area-at-risk 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 iPLA2γ, these results are consistent with salvage of myocardium after I/R by iPLA2γ 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.

Highlights

The precise chemical composition of the mitochondrial permeability transition pore (mPTP) is incompletely understood [6], a variety of initiators and modulators of mPTP opening has been identified [7, 8]
Comparisons of WT Creϩ with CMiPLA2␥KO mice definitively demonstrate that the overwhelming majority of iPLA2␥ protein of murine myocardium is present in cardiac myocytes by tissue-specific knockout mediated by the specificity of cardiac myocyte-specific expression of Cre recombinase
Previous studies have emphasized the central roles of the mPTP in mediating cardiac damage during ischemia/reperfusion through opening of the channel precipitated by calcium overload, accumulation of inorganic phosphate, and induction of oxidative stress that is amplified by the production of saturated fatty acids and oxidized lipid metabolites [4, 37, 38]

Summary

The abbreviations used are

I/R, ischemia/reperfusion; CL, cardiolipin; CMiPLA2␥KO, cardiac myocyte-specific iPLA2␥ knock-out; EET, epoxyeicosatrienoic acid; FLP, flippase; HDoHE, hydroxydocosahexaenoic acid; HETE, hydroxyeicosatetraenoic acid; iPLA2␥, calcium-independent phospholipase A2␥; mPTP, mitochondrial permeability transition pore; oxoODE, oxo-octadecadienoic acid; PG, prostaglandin; I.S., internal standard; oxlam, oxidized linoleic acid metabolite; FCCP, trifluoromethoxy carbonylcyanide phenylhydrazone; MRM, multiple reaction monitoring; AMPP, N-(4-aminomethylphenyl) pyridinium; TTC, triphenyltetrazolium chloride; D, diacyl; ϩ. To begin the mechanistic dissection of the roles of iPLA2␥ in biological function in health and disease, we generated a germ line knock-out of iPLA2␥ in mice (iPLA2␥ KO) [22,23,24] These studies revealed that iPLA2␥ loss of function dramatically reduced the opening of the mitochondrial permeability transition pore (mPTP) in liver mitochondria and that calcium challenge of myocardial mitochondria obtained from the iPLA2␥ KO mouse markedly decreased the production of inflammatory eicosanoids in comparison with wild-type mice. Germ line iPLA2␥ KO mice displayed multiple defects in virtually every organ system studied, rendering definitive mechanistic interpretation of responses to in vivo cardiac ischemia difficult To traverse this difficulty, in this study we generated cardiac myocyte-specific iPLA2␥ knock-out mice (CMiPLA2␥KO) by inserting flox sites proximal and distal to the active site serine of iPLA2␥ (Ser-477 in exon 5) and subsequently excising the exon containing the active site by tamoxifen-activated cardiac myocyte-specific Cre recombinase. Through ablation of iPLA2␥-facilitated mPTP opening, generation of inflammatory lipid second messengers, and the release of toxic mitochondrial metabolites, a novel strategy to attenuate cardiac necrosis and inflammation during acute coronary syndromes has been identified

Results

E Kidney Heart Brain WT KO WT KO WT KO 38

WT KO 4

Discussion

Experimental Procedures