In this study, we diverged from the usual focusing on the atherosclerotic changes induced by an HCD in the arterial system, including the coronary arteries, and instead investigated its possible effects on the myocardium. In the great majority of these factors, a common pattern was seen: with continued HCD, TLRs and other noxious factors increased steadily, until the 90th day. Interestingly, this increase usually persisted throughout the 30 additional days after resumption of normal feeding (G120) but only started decreasing after statin treatment, with Ros, as a more potent statin, having a stronger effect than Flu.
4.1. TLR Overexpression
The two more abundant TLRs in the myocardium, TLR2 and TLR4, and the less prevalent TLR8 showed a similar course of increase with HCD, as already described. TLR3 showed a slightly different course, increasing later (at 60 days), possibly because it is situated in the endosomal compartment and not the membrane, thus being less promptly altered with HCD [4
]. This was also shown by our group in the rabbit aorta [5
]. TLR3 is an essential component of the innate stress response in virus-induced cardiac injury, with TLR3-/- mice showing less marked inflammatory changes [9
]. It has an “unexpected” protective role in the arterial wall, potentially through a repair mechanism [10
]. In the myocardium, Fattahi et al. [11
] have found that it is involved in cardiac dysfunction developing during polymicrobial sepsis. Gao et al. [12
] also found that it contributes to persistent autophagy and heart failure in mice after an experimental myocardial infarction, while it does not promote inflammation.
TLR8 is often lumped together with TLR7. In humans, Jurk et al. [13
] postulate that it can activate gene transcription of NFkB. Salagianni et al. [14
] have found that TLR7 has a protective antiatherosclerotic role by decreasing monocyte/macrophage proinflammatory activity. Triantafillou et al. [15
] have shown that in the human myocardium, inflammatory responses are triggered by Coxsackie B viruses through TLR8 and to a lesser extent through TLR7. It recognizes viral or bacterial single-stranded RNA and activates innate immune systems and early inflammatory responses.
It has been well described that TLRs are involved in ischemia-induced inflammation [3
]. It is unknown through which exact mechanisms high cholesterol levels can induce inflammation and TLR increase and activation of the cardiac immune response. However, TLRs are regulated by hypercholesteremia, hyperlipidemia and hyperglycemia [17
]. Ruysschaert and Lanez [18
] have shown that cholesterol and lipid microdomains influence TLR activity. Methe et al. [19
] have found that statins decrease TLR4 expression and downstream signaling in human CD14+ monocytes.
Our finding that chronic HCD induces myocardial TLR overexpression has an important clinical corollary: since high-fat feeding also has been shown to induce severe coronary atherosclerosis, a clinical situation can emerge in which a myocardial infarct due to exacerbated coronary atherosclerosis can be precipitated in a high-TLR-expressing myocardium. This may have important unfavorable clinical effects: (a) TLRs exacerbate I/R injury [3
]. Actually, TLR4 deficiency has been found to reduce myocardial infarction size [20
]. The TLR4 specific antagonist eritoran reduced infarct size [21
]. (b) TLRs contribute to postinfarct remodeling. Shishido et al. [22
] found that TLR2-deficient mice developed less cardiac remodeling after myocardial infarction. Timmers et al. [23
] found the same for TLR4-deficient mice. Frantz et al. [24
] also showed that TLR4 is increased in the total failing myocardium.
As shown in a previous study from our group, statins can diminish TLR mRNA expression in the aorta [5
]. Fluvastatin, simvastatin and atorvastatin have all shown anti-TLR4 activity [25
]. We showed that Ros is more potent than Flu in this aspect. It also caused a greater reduction in plasma cholesterol [5
]; thus, the question of whether cholesterol levels directly influence TLR expression remains. As regards the interaction of TLRs and apoptosis, Aliprantis et al. [26
] have shown that bacterial lipoprotein (BLP) stimulates TLR2, which signals for apoptosis through MYD88 via Fas-associated death domain protein and caspase 8. They also found that BLP activates caspase 1 through TLR2. Ruckdeschel et al. [27
] found that Yersinia infection can initiate apoptosis through TLR4 signaling.
4.2. Other Harmful Factors
The increase in IL6 in TNFa and HCD groups, indicating inflammation in the myocardium, is in accordance with the results of Ternacle et al. [28
], which will be discussed later. Bartekova et al. [29
] remark that circulating IL1b is elevated in dilated cardiomyopathy and associated with an adverse prognosis.
Interestingly, an opposite course with HCD was seen between the MMP2 and MMP9 collagen-degrading proteins and the profibrotic TIMP1, suggestive of increasing myocardial fibrosis. MMP2 and MMP9 increased very early (30 days) with HCD and declined thereafter, although remaining at much higher levels than control. However, they also started increasing again after statin treatment, with Ros showing a stronger effect than the generally weaker Flu. Fujimoto et al. [30
] found increased vascular MMP deposition after a 4-month HCD following balloon de-endothelization of the abdominal aorta; this regressed after atherogenic diet withdrawal and Flu treatment. Galis et al. [31
] found that MMP2 together with TIMP1 and TIMP2 were expressed by VSMCs in all layers of atherosclerotic arteries, while MMP1, MMP3 and MMP9 were localized to macrophages, VSMCs and endothelium in the fibrous cap and shoulder of the lesion. Ikeda et al. [32
] also mention the increased expression of several MMPs, including MMP2 and MMP9, in the shoulder areas of plaques. In human atherosclerotic lesions, Orbe et al. [33
] found an increase not only in MMPs but also in TIMP1 in calcification areas. Thus, the further increase in both MMPs with statin treatment may represent an adaptive phenomenon: MMPs may increase as an antagonistic response to TIMP1 increase. In fact, TIMP1 increased progressively in our HCD group, decreasing with resumption of a normal diet, but to a greater degree with statins, especially Ros. This factor is associated with the buildup of the fibrous plaque of coronary lesions with an increase in the TIMP1/MMPs ratio [34
]. Actually, in our study, the TIMP1/MMP2 and TIMP1/MMP9 ratios decreased, implying an antifibrotic action with statin treatment. Both TIMP1 and MMPs are increased in the myocardium in dilated cardiomyopathy [35
]; however, more information is needed. MMP2 structure and function are correlated with increased remodeling after an acute myocardial infarction [36
]. MMP9 is also correlated with remodeling and mortality after an infarct [37
]. MMP2 degrades troponin I in IRI ischemia–reperfusion injury [38
], while in aortic stenosis, TIMP1 and TIMP2 are related to fibrosis [39
]. TIMP1 also promotes myocardial fibrosis in pressure overload [40
]. In 669 patients in the Framingham Heart Study, MMP9 was positively correlated to left ventricular mass and thickness and negatively to fractional shortening [41
]. Moreover, serum MMP9 and TMP1 are also significant risk factors in population studies [42
As regards apoptosis, the transcriptional factor p53 showed similar behavior to the already mentioned markers. It stimulates the proapoptotic protein bax [44
]. Caspase 3, a main effector of apoptotic cell death, also showed a significant increase up to 120 days with HCD. Again resumption of a normal diet was not enough to cause a reversal of this trend, which was only seen with statin treatment, again with Ros being more potent than Flu. Dixon et al. [45
] underline the role of caspase 1 in nonalcoholic steatohepatitis induced by a high-fat diet.
We report, for the first time, an increase in BNP in the myocardium with an HCD together with the other fibrotic, inflammatory and apoptotic factors. BNP is produced in increased quantities by the ventricular cardiomyocytes when endoventricular pressure is increased; it is also increased in ischemia, left ventricular hypertrophy and remodeling [46
]. BNP is also produced by fibroblasts [47
]. Interestingly, it also induces the expression of MMP2 and TIMP2, according to Tsuruda et al. [48
]. It was interesting that all biomarkers showed a strong correlation in their increase. This is not surprising, since strong interactions exist among them in their rise and fall.
In accordance with our findings, Ternacle et al. [2
] found that 4- and 20-week high-fat (13.5% fat) diets compromised myocardial function as expressed by radial strain rate, although the LVEF did not change, suggesting that novel, more advanced echocardiographic indices are needed to detect subtle myocardial function alterations. They also noticed an increase in myocardial fibrosis, inflammation, tissue oxidation and apoptosis. These authors ascribed these changes to obesity. Their animals (mice) increased in weight by around 40%, while ours only increased by 16%. With these numbers, we cannot ascribe our results to obesity, but rather to a direct action of the HCD.
Carbone et al. [49
] fed mice for 4 weeks with a Western diet, i.e., a diet high in saturated fat and sugar. They found a decrease in LVEF and an impairment of diastolic function. Switching to standard diet, it was found that inhibition of the proinflammatory IL18 in mice fed a Western-type diet attenuated cardiac dysfunction despite a body weight gain of 38%. This finding also suggests that weight gain is not the determining factor in cardiac dysfunction. Resuming a normal diet for 4 weeks partially reversed this dysfunction. Carbone et al. [50
], in another study, and Drosatos and Schulze [51
] found that obesity-related cardiomyopathy and diabetic cardiomyopathy are caused by excess cardiac lipid accumulation. They describe that lipotoxicity can signal apoptotic pathways, and the treatment of neonatal rat ventricular myocytes with palmitic acid alters mitochondrial physiology, leading to apoptosis. It is interesting that both lipotoxicity and the more commonly studied myocardial glucotoxicity [52
] do not only affect the myocardium. In fact, a lipotoxic model of pancreatic B cell failure has been produced, which involves histone modifications, linked to epigenetics [53
]. Both hyperglycemic [54
] and lipotoxic [55
] cellular epigenetic memory can be established. The fact that normal diet resumption alone was not adequate for reversing molecular changes but statins were additionally needed may also suggest intensive treatment in hypercholesterolemic patients, especially if they are hypertensive and hyperglycemic, to avoid not only direct but also epigenetic changes. Thus, our study offers further evidence that an HCD not only affects the arterial system but also the myocardium. From the above, it can be seen that apart from our rabbit model, mice showed the same behavior [28
Importantly, the role of statin administration in heart failure is intensively discussed. Lee et al. [56
] have shown that pravastatin (a relatively weak statin) administration can cause regression of LV mass in hypercholesterolemic patients.
In a meta-analysis of 4500 patients from six studies, Deo et al. [57
] showed that statins improve long-term survival in nonischemic cardiomyopathy. Gastelurrutia et al. [58
], in a prospective study of 960 patients with both ischemic and nonischemic heart failure etiology, showed an improvement in mortality regardless of etiology. Two very experienced authors have raised the question of whether we need a large-scale outcome trial of statins in chronic heart failure [59
]. Statins can induce important side effects, particularly in skeletal muscle, especially in aged rats [60
]. However, the effects of statins on the cardiomyocytes have not been specifically reported.