In the present study we investigated the response of Atlantic salmon to TTA during the seawater phase. The results from our study show that feeding TTA had profound effects on the cardiac gene expression at sampling point 17.weeks, 9 weeks after TTA feeding ended. The level of TTA applied in the study was, with 0.25%, lower than previous studies that have been conducted in Atlantic salmon (compare
[1, 6, 37]). The mortality rates previously observed in Atlantic salmon in response to high TTA levels was not observed in this study.
Gene set over-representation of the transcription profile at 17.weeks shows an increased capacity of fat catabolism, glycolysis and activity of the TCA cycle as well as cardiac contractility and cardiac hypertrophy. Overall, the results suggest a scenario where cardiac ventricles of TTA pre-fed fish are able to generate more energy via a TCA-cycle that is fueled by metabolites from fat catabolism and glycolysis. TTA functions as a ligand for all three PPAR subtypes
[3, 4], which have crucial functions in the transcriptional regulation of cardiac metabolism. In mice the transcriptional effects of TTA in the heart have been shown to be mediated almost exclusively via PPARα. Gain-of-function and loss-of-function mutations have shown that PPARα is a crucial transcription factor in the cardiac metabolism, regulating mainly cardiac fatty acid uptake and oxidation
[10, 11]. Furthermore, activation of PPARαhas been demonstrated to shift cardiac energy utilization away from glucose and towards fatty acid oxidation, actually mimicking the cardiac phenotype observed in diabetic hearts
. Interestingly the cardiac phenotype of PPARβ differs from that of PPARα, indicating that both transcription factors regulate, at least partly, different subsets of genes in the heart. PPARβ loss-of-function hearts suffer from myocardial lipid accumulation and cardiomyopathy
. Gain-of-function mutations on the other hand clearly show that PPARβ positively regulates cardiac glucose utilization
, and also stimulates cardiac growth
. Thus, the significantly higher cardiac transcription of PPARα and the elevated mean transcription of PPARβ/ in concert with the activation of their down-stream pathways, fat catabolism and the glycolysis pathway suggest that cardiac effects of TTA in Atlantic salmon are mediated by both PPARα and PPARβ. Intriguingly, over-expression of a constitutively active form of PPARβin murine skeletal muscle has been reported to mimic training-based muscle adaptation
. Hence, it has been speculated, in accordance with the results from PPARβ over-expression in mice
, that PPARβcauses “physiological” cardiac hypertrophy
Between the 8.weeks and 17.weeks sampling points, the hearts grew by a considerable portion in absolute and relative terms. The gene expression profile in hearts of TTA fed fish at 17.weeks suggests that the cardiac growth of TTA fed fish is shifted towards “physiological” hypertrophy, which may translate to an increased cardiac output. This notion is supported by the expression profile found for the category “cardiac performance” at 17.weeks, unanimously pointing to an increased cardiac contractility and also showing up-regulation of crucial cardiac transcription factors. In particular the higher transcription of the cardiac transcription factors Nkx2.5 and Mef2C can be regarded as markers for cardiac hypertrophy/growth. It has been demonstrated in mice that over-expression of Mef2C is sufficient to induce cardiac hypertrophy
. Furthermore, both Mef2C and Nkx2.5 have been shown, in vitro, to be regulated by PPARα in cardiomyocytes
It should also be noted that although we did not find significant differences in relative heart weight in this study, in other studies we found that TTA significantly increases heart size in Atlantic salmon
[8, 9], and that the effect seems to be correlated to the dose of TTA (Rørvik, unpublished data). Thus, it is tempting to speculate that the increase in relative heart weight may be related to the cardiac transcriptional changes induced by TTA. A “cardiac exercise” stimulating effect is of high relevance for salmonid aquaculture. Atlantic salmon, having a circulatory system that is naturally adapted to long migration routes and high activity, show alteration in cardiac morphology and a reduced relative heart weight in captivity
. In addition, circulatory failure has been identified as an important cause of mortality in salmon farming
. Thus, using TTA may be one way to support the cardiac performance of fish in captivity.
The highest tissue concentrations of TTA in Atlantic salmon, as well as in mice, can be found in the heart
[1, 45]. In accordance, the heart was also the tissue where the strongest transcriptional response of PPARαwas detected. The main transcriptional effects were found nine weeks after the TTA feeding stopped and where our data suggested that the cardiac tissue levels of TTA were neglectable. However, we have no information about the course of gene expression between both sampling points, thus it might very well be that the effects of sampling at 17.weeks are the remains of earlier, stronger transcriptional effects. It is remarkable that a similar, delayed response in expression of lipid metabolism related genes to TTA has been observed in our previous Atlantic salmon studies
[8, 9], indicating a common underlying mechanism. It is possible that the delay in transcriptional response is caused by a common, yet unknown, mechanism.