The goal of this investigation was to determine the effects of EPA+DHA supplementation on cardiac mitochondrial membrane composition, mitochondrial respiration, and MPTP opening in normal hearts and following myocardial infarction. We found that EPA+DHA altered fatty acid composition of mitochondrial phospholipids, specifically decreasing arachidonic acid and increasing DHA. EPA+DHA delayed Ca2+-induced MPTP opening in both SSM and IFM in sham rats. This effect was enhanced by the addition of cyclosporin A, and was not accompanied by changes in mitochondrial respiration, coupling, or cyclophilin D protein expression. On the other hand, supplementation with EPA+DHA did not improve LV function nor delay MPTP opening in failure myocardium. Nevertheless our findings suggest the novel concept that dietary intake of ω-3 polyunsaturated fatty acids can alter MPTP opening in normal hearts.
A delay in MPTP opening with EPA+DHA has not been previously reported, and could be clinically useful if it translates into less myocardial injury in response to acute and chronic cardiac stress (e.g. ischemia/reperfusion or hypertension). The EPA+DHA-induced delay in MPTP opening was enhanced by cyclosporin A, an inhibitor of the isomerase activity of cyclophilin D, which plays a regulatory role in MPTP opening. Cyclophilin D protein expression did not change with diet or surgery in this study (Supplementary Figure 1
), suggesting that changes in cyclophilin D do not mediate the effects of EPA+DHA. More definitive studies with EPA+DHA supplementation in cycolphilin D knockout mice are warranted [42
]. VDAC is another protein that has been implicated in the regulation of MPTP opening, however the infarct-induced reduction in VDAC1 and 2 that we observed were not related to MPTP opening. At present the molecular components and regulation of the MPTP are unclear[17
], which makes it difficult to draw conclusions regarding how EPA+DHA affect MPTP opening at a molecular level. One limitation of the method used to measure MPTP opening in the present study is that the assay conditions minimized the potential contribution of oxidative stress. Future studies should assess whether diet and heart failure affect MPTP opening at different mitochondrial redox states and in response to acute oxidative stress, e.g., that induced by the metabolism of exogenous peroxides. In addition, the lack of beneficial effect of EPA+DHA on LV remodeling and function in infarcted animals may be due to the delayed initiation of treatment 7 days after coronary ligation. Since MPTP opening is associated with apoptosis and myocardial damage during ischemia and chronic stress [17
], EPA+DHA treatment prior to infarction may lead to less MPTP opening and thus prevent myocardial damage and initiation of LV remodeling in the week following infarction.
Previous studies show a decrease in arachidonic acid in total myocardial phospholipids with EPA+DHA[6
], and in the present study we extend this observation to show a similar decrease in arachidonic acid in total mitochondrial phospholipids and in cardiolipin. A number of studies found that arachidonic acid plays an important role in Ca2+
mediated MPTP opening and apoptosis. Arachidonic release by Ca2+
-independent phospholipase A2γ
mediates MPTP opening in renal cortex mitochondria[46
]. The release of cytochrome c from the mitochondria, which leads to downstream apoptosis, may also be regulated by arachidonic acid release from the membrane in mouse embryonic fibroblasts[47
] and in rat liver mitochondria[20
]. The role of arachidonic acid may differ in cardiac mitochondria compared to mitochondria from other tissues, nonetheless there is clear evidence that arachidonic acid can contribute to MPTP opening[49
]. In the present study there may have been a decrease in arachidonic acid release from the membrane with EPA+DHA treatment, which would delay or inhibit MPTP opening. In addition, alterations in cardiolipin composition (i.e. decreased arachidonic acid and increased DHA incorporation) may also play a role in MPTP opening. Further studies are needed to fully assess the role of mitochondrial arachidonic acid on MPTP opening.
Supplementation with EPA+DHA was ineffective in delaying Ca2+
-induced MPTP opening in mitochondria from infarcted hearts despite increasing DHA and decreasing in arachidonic acid in cardiac phospholipids. This suggests that changes in phospholipid composition are not responsible for the delay in MPTP opening observed in sham rats. Myocardial infarction resulted in myocardial pathology, as evidenced by LV dilation and contractile dysfunction, decreased activity of mitochondrial marker enzymes in LV tissue, and a lower IFM yield. Consistent with previous studies there was no impairment in mitochondrial respiration in either SSM or IFM[7
]. At present it is unclear why the beneficial effects of EPA+DHA supplementation on MPTP opening are absent in mitochondria from infarcted rats.
Our recent studies in rats with pressure overload found that supplementation with EPA+DHA attenuated pathological LV hypertrophy and development of heart failure[6
], however no such protective effect was observed in the present investigation in rats with infarct-induced heart failure. The reasons for the lack of benefit in the present investigation may be due to the timing and duration of treatment with EPA+DHA. In our previous study with abdominal aortic banding, EPA+DHA treatment was initiated either one week prior to aortic banding [6
] or immediately after banding [29
], and resulted in significant cardioprotection. Since the purpose of the present study was to determine if EPA+DHA exerts a beneficial effect in a model of irreversible myocardial injury, we initiated treatment 7 days after coronary ligation to insure that the myocardial infarct was established. Our previous results from the aortic constriction model suggests that if supplementation with EPA+DHA had been initiated prior to coronary ligation a protective effect may have been observed. Another possibility is that more prolonged treatment is needed to fully elicit and maintain the effects of EPA+DHA. Previous work in patients undergoing elective cardiac surgery established that supplementation at a dose equivalent to ours show changes in membrane DHA and arachidonic acid by 7 to 10 days of treatment, and a plateau by ~30 days. Additional studies are needed to assess the effects of earlier and more prolonged treatment with EPA+DHA.
In the present investigation myocardial infarction resulted in heart failure as evidenced by LV dilation and contractile dysfunction, decreased activity of mitochondrial marker enzymes in LV tissue, and a decrease in the yield of IFM. Consistent with previous studies there was no impairment in mitochondrial respiration in either SSM or IFM[7
]. On the other hand, infarct-induced alterations in cardiac mitochondria in this model prevented the beneficial effects of EPA+DHA supplementation on MPTP opening. Future studies should clarify the relationship among heart failure, mitochondrial dyfunction and EPA+DHA supplementation by assessing the effects of more advanced heart failure, and initiating treatment after mitochondrial dysfunction is established.
More severe and prolonged heart failure may derive benefit with EPA+DHA supplementation, as suggested by the results of the recent GISSI-HF trial[5
]. For example, EPA+DHA could improve long term survival in infarct-induced heart failure. Fiaccavento et al. showed that high intake of α-linolenic acid, a ω-3 polyunsaturated fatty acid found in vegetable oils, prolonged survival and prevented myocardial pathology in cardiomyopathic Syrian hamsters compared to those fed standard chow[50
]. It is important to note that the dose of EPA+DHA used in this study corresponds to an equivalent dose of in humans of approximately 5 g/day (assuming an energy intake of 2000 kcal/day), which is in the range of safe and effective doses recommended for the treatment of hypertriglyceridemia ( 3 to 4 g/day). In the GISSI-HF trial, a much lower dose of 0.85 g/day was used. Additional studies are needed to more fully assess the effects of various ω-3 polyunsaturated fatty acids at higher doses on outcomes in heart failure.
In summary, the results of the present investigation demonstrate that MPTP opening is delayed by dietary EPA+DHA supplementation in sham rat hearts. This effect was associated with alterations in membrane phospholipids, specifically a decrease in arachidonic acid and elevated DHA. In contrast, LV function and MPTP opening were not affected by EPA+DHA supplementation in infarcted myocardium. Nevertheless, inhibition of MPTP opening in cardiac mitochondria should be cardioprotective, and thus the novel effect observed here may contribute to the lower incidence of heart failure observed in people consuming high amount of EPA+DHA in population based studies[3