Abstract
Epoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxygenase metabolites of arachidonic acid involved in regulating pathways promoting cellular protection. We have previously shown that EETs trigger a protective response limiting mitochondrial dysfunction and reducing cellular death. Considering it is unknown how EETs regulate cell death processes, the major focus of the current study was to investigate their role in the autophagic response of HL-1 cells and neonatal cardiomyocytes (NCMs) during starvation. We employed a dual-acting synthetic analog UA-8 (13-(3-propylureido)tridec-8-enoic acid), possessing both EET-mimetic and soluble epoxide hydrolase (sEH) inhibitory properties, or 14,15-EET as model EET molecules. We demonstrated that EETs significantly improved viability and recovery of starved cardiac cells, whereas they lowered cellular stress responses such as caspase-3 and proteasome activities. Furthermore, treatment with EETs resulted in preservation of mitochondrial functional activity in starved cells. The protective effects of EETs were abolished by autophagy-related gene 7 (Atg7) short hairpin RNA (shRNA) or pharmacological inhibition of autophagy. Mechanistic evidence demonstrated that sarcolemmal ATP-sensitive potassium channels (pmKATP) and enhanced activation of AMP-activated protein kinase (AMPK) played a crucial role in the EET-mediated effect. Our data suggest that the protective effects of EETs involve regulating the autophagic response, which results in a healthier pool of mitochondria in the starved cardiac cells, thereby representing a novel mechanism of promoting survival of cardiac cells. Thus, we provide new evidence highlighting a central role of the autophagic response in linking EETs with promoting cell survival during deep metabolic stress such as starvation.
Highlights
Free Arachidonic acid (AA) can be metabolized by cytochrome P450 epoxygenases to epoxyeicosatrienoic acids (EETs) that are further metabolized to dihydroxyeicosatrienoic acids (DHETs) (via soluble epoxide hydrolase) or incorporated into membranes.[4,5]
We found that UA-8 prevented the decrease in citrate synthase, succinate dehydrogenase and c oxidase (COX) cytochrome c oxidase (IV) enzymatic activities observed in control groups following 24 h of starvation; no significant protective effect was observed for SDH in HL-1 cells (Figures 5a–f)
We found that neonatal cardiomyocyte (NCM) starved for 24 h had an elevated level of mitochondrial marker proteins such as VIDAC, SDH and COX IV (Figures 5g–i)
Summary
Arachidonic acid (AA) is a polyunsaturated fatty acid normally found esterified to cell membranes that can be released in response to several stimuli including ischemia and stress.[1,2,3] Free AA can be metabolized by cytochrome P450 epoxygenases to epoxyeicosatrienoic acids (EETs) that are further metabolized to dihydroxyeicosatrienoic acids (DHETs) (via soluble epoxide hydrolase (sEH)) or incorporated into membranes.[4,5] EETs are lipid mediators that act as potent cellular signaling molecules regulating key cellular processes, such as limiting mitochondrial damage, inhibiting apoptosis and reducing inflammatory responses.[6,7,8,9] Despite extensive research efforts investigating the biological effects of EETs, their intrinsic mechanism(s) of action remains poorly understood.[10]. AMPK acts as an intracellular sensor of energy status that is activated by an increase in the intracellular AMP/ATP ratio, including response to metabolic stress observed in starvation.[24] Once activated, AMPK switches on catabolic pathways that generate ATP while switching off ATP-consuming processes, such as cell growth and proliferation, and activating autophagy.[25] Other critical molecules like pmKATP channels are involved in the cellular response by regulating ionic homeostasis under conditions of metabolic stress; these channels have demonstrated cardioprotective effects, their role in regulating cell death pathways is limited.[26] Excessive injury of cardiomyocytes in the heart results in collapse of cardiac function. We report a novel EET-mediated protective mechanism for cardiac cell survival during starvation
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