Original articleLong-chain polyunsaturated fatty acids protect the heart against ischemia/reperfusion-induced injury via a MAPK dependent pathway
Introduction
Coronary heart disease (CHD) remains one of the leading causes of death in all Western industrialized countries. Although epidemiology and human intervention trials consistently show an inverse relationship between dietary omega-3 fatty acid consumption and mortality from heart disease [1], [2], [3], the mechanism of action of these fatty acids remains to be elucidated. Long-chain polyunsaturated fatty acids (PUFAs) play an important role in various biological processes in cardiac muscle cells. PUFAs can either act as second messengers or as reversible modulators to amplify, attenuate or deviate a signal at a precise intracellular location [4].
Previous studies have shown that a variety of extracellular stimuli, including ischemia–reperfusion, profoundly affect the activation of the mitogen-activated protein kinases (MAPKs), which includes extracellular signal-regulated protein kinase (ERK), p38, and c-Jun NH2-terminal protein kinase (JNK) [5], [6], [7], [8]. All three MAPKs have been shown to play pivotal roles in transmission of signals from cell surface receptors to the nucleus and are involved in cell growth, differentiation and apoptosis [9], [10], [11]. In response to MAPK activation, a family of dual-specificity phosphatases becomes transcriptionally induced, leading to dephosphorylation and inactivation of specific MAPKs within 30–60 min [12]. Currently ≈9 dual-specificity phosphatase family members have been described, each of which has a slightly different substrate specificity, tissue distribution, subcellular localization, or inducible expression profile [12]. MKP-1 (mitogen-activated protein kinase phosphatase-1) is an important member of this family, which regulates inactivation of p38, JNK and ERK [13], [14], [15].
Very little or any information is available regarding the effects of long-chain PUFAs on the MAPK family and phosphatases in the heart. Chen and co-workers [16] have recently shown that EPA inhibits hypoxia/reoxygenation-induced injury by attenuating upregulation of MMP-1 in adult rat cardiac myocytes, while Mackay and Mochly-Rosen [17] demonstrated that ARA protects neonatal rat cardiac myocytes from ischemic injury through activation of PKCε. A potential target of long-chain PUFAs may also be the serine/threonine kinase PKB/Akt. There is increasing evidence that the PKB/Akt pathway participates in tissue salvage during ischemia/reperfusion-induced injury [18], [19].
However, as far as we know, no evidence exists for an interaction between EPA or ARA and the activation/inhibition of the MAPKs and the pro-survival kinase, PKB/Akt in neonatal rat cardiomyocytes during simulated ischemia (SI) and reperfusion. Therefore, in order to assess the mechanisms of protection of long-chain polyunsaturated fatty acids (PUFAs) in injured/apoptotic heart cells, we treated neonatal cardiomyocytes with EPA and ARA prior to and after simulated ischemia and determined their effects on cell viability, apoptosis and the activation patterns of the MAPKs and PKB/Akt. To further evaluate the significance of findings obtained in a cell model of simulated ischemia, the effect of the PUFA’s on functional recovery of isolated globally ischemic perfused rat hearts was also studied.
Section snippets
Antibodies and chemicals
Antibodies were purchased from Cell Signaling Technology, collagenase from Worthington Biochemical Corporation, and DMEM from Gibco Laboratories. All other chemicals were obtained from Sigma Chemical Co (St. Louis, MO).
Cell culture preparation
Neonatal cardiac myocytes were isolated and cultured by a modification of the method of Pinson [20] as previously described [21]. At least 30 neonatal rat hearts were used to prepare cardiomyocytes for one specific independent experiment. Confluent, spontaneously contracting
Myocyte loss during SI/R
To establish whether any of the cellular manipulations resulted in a significant detachment of myocytes during the incubation periods, the number of cells in the supernatant (media) was counted after each insult. The number of detached cells was less that 0.01% of the number of cells plated (data not shown). This was further corroborated by the fact that no protein loss (assessed by the Bradford technique [27]) was detected during SI and reperfusion. Therefore, changes in viability or apoptotic
Model of ischemia–reperfusion
The model used in this study combines the following two properties of ischemia: firstly, inhibition of mitochondrial respiration (cyanide prevents the oxidation of cytochrome a3, thereby obstructing the electron transport chain and oxidative phosphorylation, thereby mimicking cellular hypoxia); secondly, inhibition of glycolysis (2-deoxy-d-glucose in the buffer inhibits glycolysis and further decreases the energy supply).
We have shown that EPA and ARA, when present as free fatty acids rather
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