Our results demonstrate by four independent assays (viability using MTT, nuclear fragmentation by Hoechst staining, increase in the early apoptosis marker annexin V, and activation of the late apoptotic protease, caspase-3) that EETs attenuate HR-induced cell death/apoptosis of cardiomyocytes cultured from the hearts of 2 rodent species, rats and mice. Importantly, incubation with EETs not only enhanced cell survival but also maintained contractile function of neonatal myocytes that was attenuated by HR. Currently there is no accepted mechanism for protection of cardiomyocytes by EETs. Our results strongly support the hypothesis that EETs maintain cellular structure and function by stimulation of the PI3K-Akt cell survival pathway. We identified 5 targets that mediate the action of EETs, viz. PI3K, Akt, BAD, XIAP and caspase 9 (see schematic ). Each one of these proteins was affected by EETs in an anti-apoptotic manner, defining a mechanism for the observed protection of cardiomyocytes after injury by HR. EETs may stimulate other pathways in the cell that also have a pro-survival role (eg. opening of KATP channels). Future studies are needed to address if activation of PI3K regulates channel function or if multiple pro-survival pathways must be functional at the same time to protect cardiomyocytes. Understanding clearly how EETs are protective will be a first step towards developing these fatty acids into therapeutic agents for cardiovascular disease.
Schematic representation of a pathway for protection of cardiomyocytes by EET via the PI3K/Akt pro-survival pathway
The PI3K/Akt pathway is one of the most potent intracellular mechanisms to promote cell survival. For example, in a study of four downstream effectors of growth factor receptors, PI3K, Ras, Raf and Src, PI3K was the only one to inhibit apoptosis after serum withdrawal (30
). Our results indicate that PI3K activity is enhanced by EET within 30 minutes of application, and also hours later during the first hour of reoxygenation. It is not clear if EETs directly stimulate PI3K or act indirectly. The best known activators of PI3K include G-protein coupled receptors (GPCR) (10
), receptor tyrosine kinases (23
), and glycoprotein 130 (12
). Heterotrimeric G-proteins and receptor tyrosine kinases mediate the action of EET. Evidence of cholera toxin sensitive, high affinity binding of EETs to mononuclear cells (61
) or activation of K+
channels via the Gsα subunit of heterotrimeric G proteins, raises the possibility that EETs may stimulate PI3K via GPCR. The sulfonamide derivative of 14,15-EET (14,15-EET-SI, 20 μM) induced association between the EGF receptor and Src-like kinases within 1 minute of application at a concentration of 20 μM in renal epithelial cells (7
). This high concentration of 14,15-EET-SI stimulated phosphorylation of the 85 kD regulatory subunit of PI3K (7
). Cross-talk with the epidermal growth factor receptor, EGFR, regulates the angiogenic action of EET in the chick chorioallantoic vessels (38
). In addition EETs can transcriptionally upregulate proteins such as tissue plasminogen activator (t-PA) via Gs (45
), implying secondary or late signaling may occur after synthesis of new proteins or factors that are induced by EET. These observations may explain why a single exposure to free fatty acids which are rapidly metabolized in cells may trigger events appearing many hour later.
One downstream target of PI3K is the kinase Akt (67
) which is reported to be activated during intracellular signal transduction of many receptors and survival factors. Members of this family include Akt1, Akt2 and Akt3 (42
). Akt, also known as protein kinase B (PKB) or RAC-PK, is a serine-threonine kinase. PI3K phosphorylates inositol lipids that recruit and modify several targets containing Pleckstrin homology (PH) domains including Akt and 3-phosphoinositide–dependent kinase 1 (PDK-1) (4
). Akt1 is activated by phosphorylation first on Thr308
by PDK-1 and subsequently on Ser473
). PDK-1 has a Pleckstrin homology domain and is therefore also recruited to the membrane after activation of PI3K. A number of downstream targets that regulate apoptosis are modulated by p-Akt including members of the Bcl-2/CED9 family Bax, Bak and BAD (14
), XIAP, caspase 9, GSK3β (glycogen synthase kinase 3β), transcription factors of the forkhead (FKH) and NFkB families and eNOS (18
). Bad and caspase 9 are inactivated after phosphorylation by Akt (1
) while XIAP is stabilized by it. Therefore, Akt promotes cell survival by ultimately modifying the core death machinery.
Akt primarily triggers phosphorylation of BAD at Ser-136 which is sufficient to promote survival. The phosphorylation of BAD leads to the prevention of cell death via a mechanism that involves the selective association of p-BAD with isoforms of the scaffold protein 14-3-3. This interaction induces release of anti-apoptotic binding partners of BAD such as Bcl-XL or Bcl-2 (14
). 14-3-3 isoforms associate with a number of cellular signaling molecules including KSR, cdc25, Raf-1 and PI3K (3
). Growth factor deprivation or apoptotic signals cause removal of the phosphates which allow BAD to bind to anti-apoptotic BCl-2 ultimately resulting in displacement of voltage-dependent anion channel 2 (VDAC2) of the mitochondria, increasing mitochondrial outer membrane permeabilization and inducing apoptosis. BAD is therefore a pro-apoptotic molecule when it is not phosphorylated.
XIAP is, as its name (X-linked inhibitor of apoptosis) suggests, an inhibitor of apoptosis which belongs to the IAP family of proteins with an evolutionarily conserved role in regulating programmed cell death (15
). It binds to and blocks caspases 9, 3 and 7 (16
). The protein contains BIR (baculovirus
inhibitor of apoptosis repeats) that limit substrate access to catalytic sites of activated caspases. In addition, this protein prevents dimerization of procaspase 9 (57
) and protects the cells from accidental activation of caspases. XIAP is an important anti-apoptotic protein since it attenuates the major inhibitor of the extrinsic pathway, caspase 9, as well as the common mediator of both the intrinsic as well as extrinsic pathways, caspase 3 (13
). Our results () demonstrate reduced levels of this protein after HR in both types of cultured myocytes. Pretreatment with EETs impressively reduces this fall in the level of XIAP induced by HR.
Though this is the first report describing anti-apoptotic actions of EETs on cardiomyocytes, enhanced survival of other cultured cells by these fatty acids and also by epoxygenase overexpression, have been reported. Examples of cell types so affected include LLCPKc14 (porcine kidney proximal tubule-like epithelial cell line (6
), primary human coronary and pulmonary microvascular endothelial cells (17
), bovine aortic endothelial cells (66
), and the human carcinoma cell line Tca-8113 (29
). The PI3K/Akt pathway has been implicated in all these studies, though much higher concentrations of 14-15-EET (10-20 μM) were used in the kidney cells (6
), where Akt kinase activity was increased 10 minutes after addition of 14,15-EET to the LLCPKc14s. Levels of pAkt were higher after at least 12 hours of treatment of the carcinoma cells with 8,9-, 11,12- or 14,15-EET (100 nM) followed by induction of apoptosis by tumor necrosis factor α for 12 hours, (29
). Interestingly expression of the 110 kD PI3K subunit was increased after overexpression of epoxygenase enzymes that catalyze formation of EET, in bovine aortic endothelial cells (66
Hence we demonstrate that EETs prevent apoptosis in rat neonatal cardiac myocytes and a mouse atrial cardiomyocyte cell line (HL-1), subjected to HR injury. We report for the first time that treatment with EET enhances PI3K activity. This treatment also attenuates both the increase in activity of caspase 9 and the fall in the levels of intracellular XIAP induced by HR. We have confirmed that EETs increase phosphorylation of Akt that is upstream of these effectors. Together these observations support mechanistic evidence for the protective effect of EETs in cardiomyocytes to prevent HR-induced cell death. These data make it essential to investigate if activation of potassium channels (especially KATP channels), the mechanism favored by other investigators for protection of myocardium by EETs, is independent of PI3K or occurs by cross-talk in parallel to this pro-survival pathway.