The main results of the study could be summarized as follows: The LMW cod muscle aqueous extract decreased the mortality rate in mice following DOX-induced acute CHF. The extract is characterized by pronounced free-radical scavenging effects in vitro.
To the best of our knowledge, this is the first study to demonstrate the beneficial effect of an aqueous fish extract on survival rate in an
in vivo animal model of acute heart failure. Cardioprotection has been in the research focus for many years. Different pharmacological and non-pharmacological strategies have been proposed to decrease myocardial damage e.g. during ischemia-reperfusion injury and cardiotoxicity. However, only few experimental interventions have been translated into a clinical practice and with a limited success. Several studies have shown beneficial cardiovascular effects of fish and fish derived substances. By far the best known are the long chain n-3 polyunsaturated fatty acids[
12,
13]. These fatty acids may enter the cellular membrane and alter membrane functions resulting in, e.g., antiarrhythmic effects[
12]. Recent evidence indicates that dietary supplementation with fish oil preserves normal vasomotion of atherosclerotic coronary arteries and reduces damage to the myocardium after ischemia and reperfusion in animal models[
23,
24]. Much less is known whether aqueous components of fish, e.g. proteins, peptides, amino acids and other organic acids may protect the heart. On a wet weight basis, the aqueous phase usually contribute to 95–99.5% of the total edible parts of seafoods. Indeed, the results of the present study provide the evidence that also aqueous fish-derived compounds may play an important role in cardioprotection.
The mouse model of DOX induced acute heart failure is suitable for screening studies and is well characterized in the literature[
25]. We have verified the presence of acute CHF in the animals treated with DOX demonstrating that already one day after the exposure to the high-dose DOX, the indices of LV function were severely impaired with development of early pathologic LV remodeling (Figure
2). It is therefore not surprising that the mortality rate after high-dose DOX reached ~90% in the untreated animals. Although several organ systems sustain damage in this model, the central role in the progression of the multiorgan failure is the failing heart. Acute myocardial injury is a result of pathophysiological abnormalities that are caused by direct toxic intracellular actions of DOX and include inhibition of nucleic acid and protein synthesis, release of vasoactive amines, alteration in adrenergic function, mitochondrial abnormalities, lysosomal changes, modification of sarcolemma Ca
2+ transport, attenuation of adenyl-cyclase, Na
+-K
+-ATPase, and Ca
2++-ATPase activities, imbalance in myocardial electrolytes. However, most of the studies support the view that oxidative stress holds the central role in the development of these derangements (see ref. [
8] for review). Our study does not provide the exact explanation for the possible mechanisms behind the improved survival rate. Given the fact that cardiotoxicity with acute heart failure is the primary cause of death in this model, we speculate that the treatment was primarily cardioprotective. At the cellular level, this cardioprotection may have been mediated by antioxidative effects and/or by other mechanisms. Others have shown that increasing antioxidative capacity of the heart muscle suppresses cardiotoxicity of DOX[
26]. The aqueous LMW-cod extract used in this study was reported to possess strong antioxidative effects[
27]. However, it has not been previously evaluated whether this extract exerts antioxidative properties under physiological conditions. The results shown in Figure
3,
4 and Table
3 provide evidence for such an effect. The data from human monocytes in Figure
4 indicate that the capacity of the cod extract to prevent ROS-formation is ~50% at a 100-fold dilution! To evaluate the contribution of some individual compounds of the cod extract to its total antioxidative capacity (ORAC value) it was compared to some previously reported ORAC values for solutions of pure ascorbic acid and uric acid at the equimolar levels to those measured in the cod extract[
28]. These substances had ORAC values three orders of magnitude lower than the cod extract, indicating that other compounds in the extract appear to be involved in its radical scavenging effect and/or that antioxidative substances require other LMW-compounds present at the same time for regeneration purposes. Which are the most likely candidates in the extract that have provided organ protection and survival benefit in this study? The LMW-compounds of fish muscle that has been ascribed antioxidative properties include ascorbic acid, uric acid, glutathione, various polyamines, histidine containing dipeptides (anserine, carnosine) and free amino acids (taurine, histidine). Based on the quantitative analyses (Table
3,
4,
5) we speculate that taurine and anserine might have been responsible for the most part of the protection. Taurine is generally found in high levels in seafood[
29]. It is involved in radical scavenging, membrane regulation, osmoregulation and regulation of calcium homeostasis[
30]. In animal models and human trials in the settings of CHF, taurine was found to have beneficial effects on cardiac function and morphology[
31,
32]. Similarly, anserine has shown antioxidative effects at physiological levels in different in vitro systems[
33]. We did not find any differences between the groups in regard to myocardial contents of ATP, ADP or AMP. Unexpectedly the DOX treated groups did not show lower ATP levels. One possible explanation for this result may be the selection bias since this analysis was performed only on biopsies from the surviving mice.
Table 4
Composition of total amino acids in Cod LMW-PJ (<500 Da).
Taurine | 0.5 ± 0 |
Cysteine | <0.1 |
Methionine | <0.1 |
Aspartic acid | <0.1 |
Threonine | <0.1 |
Serine | <0.1 |
Glutamic acid | <0.1 |
Proline | <0.1 |
Glycine | 0.5 ± 0 |
Alanine | 0.3 ± 0 |
β-Alanine | 0.2 ± 0 |
Valine | <0.1 |
Isoleucine | <0.1 |
Leucine | <0.1 |
Tyrosine | <0.1 |
Phenyl alanine | <0.1 |
Histidine | <0.1 |
Ornithine | <0.1 |
Lysine | <0.1 |
Arginine | <0.1 |
Hydroxyproline | <0.1 |
Sum | 1.5 |
Table 5
Composition of free amino acids and certain dipeptides in Cod LMW-aqueous extract (<500 Da).
Phosphoserine | <0.02 |
Taurine | 0.55 ± 0 |
Phosphoethanolamine | <0.02 |
Urea | 0.07 ± 0.01 |
Aspartic acid | <0.02 |
Threonine | 0.04 ± 0 |
Serine | 0.03 ± 0 |
Aspargine | <0.02 |
Glutamic acid | 0.04 ± 0 |
Sarcosine | <0.02 |
α-Aminoadipitic acid | <0.02 |
Proline | 0.035 ± 0.005 |
Glycine | 0.325 ± 0.005 |
Alanine | 0.295 ± 0.005 |
Citrulline | <0.02 |
α-Amino-n-butyric acid | <0.02 |
Valine | 0.04 ± 0 |
Cysteine | <0.02 |
Methonine | 0.02 ± 0 |
Cystathionine | <0.02 |
Isoleucine | <0.02 |
Leucine | 0.04 ± 0 |
Tyrosine | <0.02 |
β-Alanine | 0.175 ± 0.015 |
Phenyl alanine | <0.02 |
β-Aminoisobutyric acid | <0.02 |
L-Homocystine | <0.02 |
γ-Amino-n-butyric acid | <0.02 |
Ethanolamine | <0.02 |
Ammonia | 0.08 ± 0 |
γ-Hydroxilysine | <0.02 |
Ornithine | <0.02 |
Lysine | <0.02 |
1-Methylhistidine | 0.095 ± 0.005 |
Histidine | <0.02 |
3-Metylhistidine | <0.02 |
Anserine | 0.09 ± 0 |
Carnosine | <0.02 |
Arginine | <0.02 |
Hydroxiproline | <0.02 |
Glutamine | <0.02 |
There are some limitations that deserve to be mentioned. We have not compared the efficacy of the cod muscle extract to other known cardioprotective agents such as dexrazoxane. We have not demonstrated specific cardioprotective effects of the extract in terms of cardiac function (echocardiography) or tissue structure (pathohistology). Furthermore, our study does not provide the answer to whether the survival benefit is mediated by few compounds in the extract (such as taurine and anserine) or by multiple compounds optimally mixed in this preparation.
In conclusion, the aqueous LMW cod muscles extract decreases mortality in the mouse model of DOX induced acute CHF. This effect may be mediated by cardioprotection through antioxidative mechanisms.