The novel findings of this study are 1) DHA supplementation delayed MPTP opening in response to Ca2+
compared to animals fed the standard diet or supplemented with EPA, and 2) this effect is associated with a greater increase in total ω-3 PUFA in cardiac mitochondrial phospholipids with DHA supplementation compared to EPA, which corresponds with a greater reduction in the amount of ARA and an increase in L4
CL. These differences between DHA and EPA occurred despite equivalent triglyceride lowering effects in the present investigation and in clinical studies[12
], suggesting that the lipid lowering effects of DHA and EPA are independent of phospholipid remodeling, as previously proposed[16
]. Thus the results of the present study show a novel and potentially important difference between DHA and EPA supplementation: DHA causes more extensive alterations in mitochondrial phospholipid fatty acid composition and delays Ca2+
-induced MPTP opening, despite lipid lowering effects that are similar to EPA.
Opening of the MPTP contributes to cardiac pathology with acute stress associated with ischemia/reperfusion or with the chronic stress of heart failure, thus interventions that prevent MPTP opening may have profound clinical importance [17
]. Long term treatment with pharmacological doses of DHA may prevent MPTP and cardiomyocyte apoptosis, and improve clinical outcome in ischemia heart disease and heart failure. We previously showed that dietary supplementation with a mixture of DHA+EPA (70:30) prevented left ventricular dysfunction and lowered cardiomyocyte apoptosis in rats subjected to chronic arterial pressure overload, suggesting that MPTP opening was prevented[9
]. Additional studies are needed to assess the effect of treatment with DHA on MPTP, apoptosis and myocardial dysfunction and injury in hearts subjected to ischemia/reperfusion, and in models of heart failure.
The molecular mechanisms by which DHA supplementation delays Ca2+
-induced MPTP opening are not clear. The structure of the MPTP and its interaction with membrane phospholipids environment are currently poorly understood [2
]. ARA release fromrcell membranes has previously been implicated in MPTP opening[26
], suggesting the association between the decline in ARA in mitochondrial phospholipids and delay in Ca2+
-induced MPTP observed in the present investigation is causal. Inhibition of ARA release from membranes reduced myocardial infarct size following I/R, and this effect was lost by concurrent perfusion with free ARA[35
]. Similarly, inhibition of ARA release in isolated renal mitochondria prevented MPTP opening and addition of free ARA restored MPTP[18
]. Similar results are observed in liver mitochondria and cultured cells[26
]. Clearly additional studies are needed to fully assess the role of depletion of ARA in possibly mediating the effects of DHA on MPTP opening in cardiac mitochondria. It is important to note that although EPA significantly decreased membrane ARA, it did not affect measures of Ca2+
-induced MPTP opening. It is possible that a threshold content of ARA is necessary to elicit the differences observed with DHA feeding. Alternatively, the delay in calcium-induced MPTP might not be due to a decrease in membrane ARA, but rather attributable to an increase in membrane DHA, which is completely unaffected by EPA feeding.
Previous studies show that MPTP opening is delayed in cardiac mitochondria by the addition of CsA[13
]. In the present investigation we observed that CsA delayed Ca2+
-induced MPTP opening in rats fed either the standard diet or EPA, but not with DHA supplementation. This finding is in contrast to our recent observation that supplementation with DHA+EPA (70:30) delayed MPTP opening compared to a standard diet, with a further delay in pore opening in both groups with the addition of CsA. CsA inhibits MPTP by binding to cyclophilin-D[6
], however in the present study we found no effect of DHA supplementation on mitochondrial cyclophilin-D content, suggesting the loss of the CsA effect on MPTP was not due to changes in cyclophilin-D. Furthermore, DHA supplementation had no effect on the Ca2+
retention capacity of mitochondria in the presence of palmitoylcarnitine+malate as respiratory substrates. However, this substrate also decreased the capacity of mitochondria to retain Ca2+
when compared to other substrates in animals fed the CTRL diet. Previous studies suggest that lipid mediators can induce pathological processes in mitochondria [7
], and a high concentration of either palmitic or arachidonic acid can induce MPTP in isolated mitochondria [29
]. It is possible that MPTP is sensitized and Ca2+
retention capacity is decreased by the relatively high concentration of palmitoylcarnititne (40 μM) required to support respiration in our preparation.
In summary, dietary supplementation with DHA but not EPA, profoundly altered mitochondrial phospholipid fatty acid composition by increasing DHA and depleting ARA, and delaying Ca2+-induced MPTP opening. This suggest novel and potentially important differences between DHA and EPA supplementation: DHA causes more extensive alterations in mitochondrial phospholipid fatty acid composition and delays Ca2+-induced MPTP opening, despite lipid lowering effects that are similar to EPA. These results suggest a novel pharmacological effect of DHA, and suggest that clinical treatment with DHA may exert greater cardioprotective benefit than treatment with standard fish oil formulations that are high in EPA.