A diet supplemented with ALA rich flaxseed prevents cardiomyocyte apoptosis by regulating Caveolin 3 expression

January 1, 2013 Human Health and Nutrition Data 0 Comments

A diet supplemented with ALA rich flaxseed prevents cardiomyocyte apoptosis by regulating Caveolin 3 expression

Year: 2013
Authors: Carotenuto, F. Minieri, M. Monego, G. Fiaccavento, R. Bertoni, A. Sinigaglia, F. Vecchini, A. Carosella, L.
Publication Name: Cardiac Res.
Publication Details: doi:10.1093/cvr/cvt211


n3 polyunsaturated fatty acids (PUFAs) induce beneficial effects on the heart, but the mechanisms through which these effects are operated are not completely clarified yet. Among others, cardiac diseases are often associated with increased levels of cytokines, such as tumour necrosis factor alpha (TNF), that cause degeneration and death of cardiomyocytes. The present study has been carried out to investigate (i) the potential anti-apoptotic effects induced by the n3 polyunsaturated a-linolenic acid (ALA) in experimental models of cardiac diseases characterized by high levels of TNF, and (ii) the potential role of caveolin 3 (Cav3) in the mechanisms involved in this process. An ALA rich flaxseed diet, administered from weaning to hereditary cardiomyopathic hamsters, prevented the onset of myocardial apoptosis associated with high plasma and tissue levels of TNF preserving caveolin3 expression. To confirm these findings, isolated neonatal mouse cardiomyocytes were exposed to TNF to induce apoptosis. ALA pre-treatment greatly enhanced Cav3 expression hampering the internalization of the caveolar TNF receptor and, thus, determining the abortion of the apoptotic vs. survival cascade. This study unveiled the Cav3 pivotal role in defending cardiomyocytes against the TNF pro-apoptotic action and theALA capacity to regulate this mechanism preventing cardiac degenerative diseases.(Authors abstract)
Information about ALA ability to regulate cardiomyocyte apoptosis is lacking, although a flaxseed enriched diet has shown to preserve skeletal and myocardial muscle structure and function in an animal model of hereditary cardiomyopathy, in which myocardial apoptosis largely contributes to myocardial derangement. In the heart, apoptosis contributes to the development of heart failure and ischaemic and non-ischaemic cardiomyopathies and is induced by tumour necrosis factor (TNF), a pro inflammatory cytokine produced within the context of myocardial ischaemia and heart failure.  In the myocardium, the sustained TNF stimulation leads to activation of multiple cell death pathways (intrinsic or extrinsic) that contribute to progressive cardiomyocyte apoptosis. Invaginations called caveolae may function to bring different proteins into proximity and promote interactions among different signalling receptors and between receptors and signalling proteins. n3 PUFAs can alter the composition of lipid caveolae and cell signal transduction pathways. The present study has been carried out to investigate (i) potential flaxseed ALA anti apoptotic effects in in vivo and in vitro models of cardiac diseases and (ii) the role of rafts caveolae and Cav3 in the mechanisms involved in the anti-apoptotic process. The in vivo model has been the hereditary cardiomyopathic hamster (CMPH), in which increased plasma and cardiac tissue TNF concentrations associated with widespread myocardial apoptosis. CMPH were fed with a diet supplemented with ALA rich flaxseed, assuming that the natural food would be more
palatable than the pure fatty acid. To verify the effects of ALA and to dissect the related cell mechanism, isolated neonatal mouse cardiomyocytes (nCMCs), in which apoptosis was induced by TNF, were challenged with pure fatty acid. The present investigation showed that an extensive regulation of the expression of Cav3 governs the internalization of TNFR1 and, thus, the capability of TNF to induce cardiomyocyte apoptosis. ALA enacts antiapoptotic effects both in vivo and in vitro preventing the reduction of Cav3 typically associated with TNF action. The administration of a flaxseed enriched diet determined the simultaneous increase in Cav3 expression and ALA plasma and myocardial levels. As a consequence, the cardiomyopathic heart structure and function remained within physiological limits and the animal longevity was dramatically elongated. The simultaneous presence of large amounts of ALA and TNF in CMPH plasma and myocardium implied that the antiapoptotic protection was caused by a direct effect on cardiac cells rather than by a reduced inflammatory potential. FS exerted beneficial effects only when administered from weaning, i.e. before the myocardial damage was established.
ALA pretreatment prevented TNFR1 internalization enhancing Cav3 expression and clustering into caveolae, and its interaction with the receptor, as demonstrated by the immunoprecipitation analysis. As result, the TNF pro-apoptotic effects were hampered, while the capability to activate the pro-survival Akt pathway from cell surface was amplified. The crucial role of the Cav3/caveolae system in the regulation of the TNF/TNFR1 anti- vs. pro-apoptotic effects has been confirmed challenging cardiomyocytes with MbCD, a lipid rafts disintegrator. Caveolae disruption by MbCD completely abolished the protective effect induced by ALA, further increasing apoptosis and heavily decreasing Akt phosphorylation in cardiomyocytes. Akt partitions into rafts and caveolae, whose integrity is required for facilitating the activation of the PI3K/Akt signalling pathway.  Very likely, ALA exerts a dual effect: (i) enhancement of Cav3 accumulation into caveolae facilitated by ALA incorporation into the membrane and (ii) modulation of Cav3 expression through a still unknown mechanism: in fact, although the Cav3 promoter does not display any CpG islands allowing a direct ALA demethylating epigenetic effect,  it cannot be excluded that protein expression could be indirectly modulated by the demethylation of other genes more susceptible to ALA action.
In conclusion, a cellular mechanism through which ALA can prevent the derangement of the myocardial structure and function induced by TNF has been defined. The core of the mechanism is the regulation of the Cav3 expression and location into caveolae that prevents TNFR1 internalization and the consequent cardiomyocyte apoptosis, while promoting the TNF induced survival pathway. Possible ALA induced membrane and epigenetic modifications could determine other beneficial effects cooperating to preserve cardiomyocyte integrity.  Cardiac beneficial effects could also be induced or enhanced by other flaxseed components. However, the massive presence of ALA in the FS myocardium as well as the results obtained in the in vitro experiments in the absence of other flaxseed components substantiated the prominent ALA role in protecting cardiomyocytes against apoptosis. (Editors comments)

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