RationaleHigh-fat diet with obesity-associated co-morbidities triggers cardiac remodeling and renders the heart more vulnerable to ischemia/reperfusion injury. However, the effect of high-fat diet without obesity and associated co-morbidities is presently unknown.ObjectivesTo characterize a non-obese mouse model of high-fat diet, assess the vulnerability of hearts to reperfusion injury and to investigate cardiac cellular remodeling in relation to the mechanism(s) underlying reperfusion injury.Methods and ResultsFeeding C57BL/6J male mice high-fat diet for 20 weeks did not induce obesity, diabetes, cardiac hypertrophy, cardiac dysfunction, atherosclerosis or cardiac apoptosis. However, isolated perfused hearts from mice fed high-fat diet were more vulnerable to reperfusion injury than those from mice fed normal diet. In isolated cardiomyocytes, high-fat diet was associated with higher diastolic intracellular Ca2+ concentration and greater damage to isolated cardiomyocytes following simulated ischemia/reperfusion. High-fat diet was also associated with changes in mitochondrial morphology and expression of some related proteins but not mitochondrial respiration or reactive oxygen species turnover rates. Proteomics, western blot and high-performance liquid chromatography techniques revealed that high-fat diet led to less cardiac oxidative stress, higher catalase expression and significant changes in expression of putative components of the mitochondrial permeability transition pore (mPTP). Inhibition of the mPTP conferred relatively more cardio-protection in the high-fat fed mice compared to normal diet.ConclusionsThis study shows for the first time that high-fat diet, independent of obesity-induced co-morbidities, triggers changes in cardiac oxidative state, calcium handling and mitochondria which are likely to be responsible for increased vulnerability to cardiac insults.
Background: Rupture of advanced atherosclerotic plaques accounts for most life-threatening myocardial infarctions. Classical (M1) and alternative (M2) macrophage activation could promote atherosclerotic plaque progression and rupture by increasing production of proteases, including matrix metalloproteinases (MMPs). Lymphocyte-derived cytokines may be essential for generating M1 and M2 phenotypes in plaques, although this has not been rigorously tested until now.Methods and results: We validated the expression of M1 markers (iNOS and COX-2) and M2 markers (arginase-1, Ym-1, and CD206) and then measured MMP mRNA levels in mouse macrophages during classical and alternative activation in vitro. We then compared mRNA expression of these genes ex vivo in foam cells from subcutaneous granulomas in fat-fed immune-competent ApoE knockout (KO) and immune-compromised ApoE/Rag-1 double-KO mice, which lack all T and B cells. Furthermore, we performed immunohistochemistry in subcutaneous granulomas and in aortic root and brachiocephalic artery atherosclerotic plaques to measure the extent of M1/M2 marker and MMP protein expression in vivo. Classical activation of mouse macrophages with bacterial lipopolysaccharide in vitro increased MMPs-13, -14, and -25 but decreased MMP-19 and TIMP-2 mRNA expressions. Alternative activation with IL-4 increased MMP-19 expression. Foam cells in subcutaneous granulomas expressed all M1/M2 markers and MMPs at ex vivo mRNA and in vivo protein levels, irrespective of Rag-1 genotype. There were also similar percentages of foam cell macrophages (FCMs) carrying M1/M2 markers and MMPs in atherosclerotic plaques from ApoE KO and ApoE/Rag-1 double-KO mice.Conclusion: Classical and alternative activation leads to distinct MMP expression patterns in mouse macrophages in vitro. M1 and M2 polarization in vivo occurs in the absence of T and B lymphocytes in either granuloma or plaque FCMs.
Atherosclerosis has been studied in animals for almost a century, yet the events leading up to the rupture of an atherosclerotic plaque (the underlying cause of the majority of fatal thrombosis formation) have only been studied in the past decade, due in part to the development of a mouse model of spontaneous plaque rupture. Apolipoprotein E knockout mice, when fed a high-fat diet, consistently develop lesions in the brachiocephalic artery that rupture at a known time point. It is therefore now possible to observe the development of lesions to elucidate the mechanisms behind the rupture of plaques. Critics argue that the model does not replicate the appearance of human atherosclerotic plaque ruptures. The purpose of this review is to highlight the reasons why we should be looking to the apolipoprotein E knockout mouse to further our understanding of plaque rupture.
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