Role of Toll-like receptors and interferon regulatory factors in different experimental heart failure models of diverse etiology: IRF7 as novel cardiovascular stress-inducible factor

Abstract

Heart failure (HF) is a leading cause of morbidity and mortality in the western world. Although optimal medical care and treatment is widely available, the prognosis of patients with HF is still poor. Toll-like receptors (TLRs) are important compartments of the innate immunity. Current studies have identified TLRs as critical mediators in cardiovascular diseases. In the present study, we investigated the involvement of TLRs and interferon (IFN) regulatory factors (IRFs) in different experimental HF models including viral myocarditis, myocardial ischemia, diabetes mellitus, and cardiac hypertrophy. In addition, we investigated for the first time comprehensive TLR and IRF gene and protein expression under basal conditions in murine and human cardiac tissue. We found that Tlr4, Tlr9 and Irf7 displayed highest gene expression under basal conditions, indicating their significant role in first-line defense in the murine and human heart. Moreover, induction of TLRs and IRFs clearly differs between the various experimental HF models of diverse etiology and the concomitant inflammatory status. In the HF model of acute viral-induced myocarditis, TLR and IRF activation displayed the uppermost gene expression in comparison to the remaining experimental HF models, indicating the highest amount of myocardial inflammation in myocarditis. In detail, Irf7 displayed by far the highest gene expression during acute viral infection. Interestingly, post myocardial infarction TLR and IRF gene expression was almost exclusively increased in the infarct zone after myocardial ischemia (Tlr2, Tlr3, Tlr6, Tlr7, Tlr9, Irf3, Irf7). With one exception, Irf3 showed a decreased gene expression in the remote zone post infarction. Finally, we identified Irf7 as novel cardiovascular stress-inducible factor in the pathologically stressed heart. These findings on TLR and IRF function in the inflamed heart highlight the complexity of inflammatory immune response and raise more interesting questions for future investigation.

Fig 1. Gene and protein expression of plasma membrane and intracellular localized TLRs and of the IFN regulatory factors IRF3 and IRF7 in murine and human cardiac tissue under basal conditions.

Cardiac tissue of healthy C57BL/6J wildtype mice was used for TaqMan based gene expression analysis of various TLRs and their IFN regulatory factors Irf3 and Irf7 under basal conditions. Gene expressions of Tlr4, Tlr9, Irf3 and Irf7 were highly expressed when compared to the remaining TLRs under basal conditions in murine cardiac tissue. The highest gene expression under basal condition was detected for Irf7. In addition, we detected similar human protein expression patterns when compared with murine gene expression in cardiac tissue under basal conditions. Data are presented in box plots as absolute mRNA expression normalized to the house keeping gene Cdkn1b and human protein abundance are presented as rhombus sign on the right-hand of each mRNA expression.

Fig 2. Gene expression levels of the plasma membrane localized TLRs Tlr1, Tlr2, Tlr4, Tlr5, and Tlr6 in different heart failure models of diverse etiology.

Cardiac tissue of healthy and diseased C57BL/6J mice was used for TaqMan based gene expression analysis of the plasma membrane localized TLRs Tlr1, Tlr2, Tlr4, Tlr5, and Tlr6. Expression levels of cardiac tissue from control mice are shown as white boxes, from diseased animals as red, blue, green or yellow boxes corresponding to the analyzed heart failure model. In viral-induced myocarditis (shown in red), gene expression of Tlr1, Tlr2, Tlr4, Tlr5, and Tlr6 was highly increased 7 days after infection compared to healthy controls. However, Tlr2 and Tlr6 displayed the highest increase during acute myocarditis. The initially increased gene expression levels returned almost with exception to basal levels 28 days after infection. In the model of myocardial infarction (shown in blue), gene expression of Tlr1, Tlr2, Tlr4, Tlr5, and Tlr6 was highly increased 5 days post infarction in the infarction zone when compared to the remote zone. In the remote zone, no increased TLR gene expression was observed. In STZ-induced diabetic cardiomyopathy (shown green), a decreased gene expression of Tlr5 in comparison to their healthy controls was detected; the remaining plasma membrane localized TLRs displayed no changes in gene expression levels. In the heart failure model caused by chronic AngII-infusion for 21 days (shown in yellow), all plasma membrane localized TLRs displayed a significant decrease when compared to their controls. Data are presented in box plots as relative mRNA expression in fold change to the corresponding untreated control using the formula 2^−ΔΔCt. * = significantly different compared to corresponding control; ^# = significantly different compared to VM (acute—7 days) or RZ (remote zone).

Fig 3. Gene expression levels of the intracellular localized TLRs Tlr3, Tlr7, Tlr8, and Tlr9 in different HF models of diverse etiology.

Cardiac tissue of healthy and diseased C57BL/6J mice was used for TaqMan based gene expression analysis of the plasma membrane localized TLRs Tlr3, Tlr7, Tlr8, and Tlr9. Expression levels of cardiac tissue from control mice are shown as white boxes, from diseased animals as red, blue, green, or yellow boxes corresponding to the analyzed heart failure model. In viral-induced myocarditis (shown in red), gene expression of Tlr3, Tlr7, Tlr8, and Tlr9 was highly increased 7 days after infection compared to healthy controls. However, Tlr3, Tlr7 and Tlr9 displayed the highest increase during acute myocarditis and the initially increased gene expression levels returned almost with exception to basal levels 28 days after infection. Moreover, in the model of myocardial infarction (shown in blue), gene expression of Tlr3, Tlr7, Tlr8, and Tlr9 was highly increased 5 days post infarction in the infarct zone when compared to the remote zone. In addition, the TLRs Tlr7, Tlr8, and Tlr9 showed the highest increase in gene expression levels after myocardial ischemia. In the remote zone, no increased TLR gene expression was observed. In STZ-induced diabetic cardiomyopathy (shown in green), a decreased gene expression of Tlr8 in comparison to their healthy controls was detected; the remaining TLRs displayed no changes in gene expression levels. In the heart failure model caused by chronic AngII-infusion for 21 days (shown in yellow) only the intracellular localized Tlr3 displayed a significant decrease when compared to their controls. Data are presented as relative mRNA expression in fold change to the corresponding untreated control using the formula 2^−ΔΔCt. * = significantly different compared to corresponding control; ^# = significantly different compared to VM (acute—7 days) or RZ (remote zone).

Fig 4. Gene expression levels of the IFN regulatory factors Irf3 and Irf7 in different HF models of diverse etiology.

Cardiac tissue of healthy and diseased C57BL/6J mice was used for TaqMan based gene expression analysis of the IFN regulatory factors Irf3 and Irf7. Expression levels of cardiac tissue from control mice are shown as white boxes, from diseased animals as red, blue, green or yellow boxes corresponding to the analyzed heart failure model. In viral-induced myocarditis (shown in red), gene expression of Irf7 showed by far the highest increase 7 days after infection compared to healthy controls. However, Irf3 displayed no changes in gene expression during viral myocarditis. Moreover, in the model of myocardial infarction (shown in blue), gene expression of Irf3 and Irf7 was highly increased 5 days post infarction in the scar tissue when compared to the non-infarcted LV or sham, whereas Irf7 showed a clearly higher gene expression when compared to the expression of Irf3. In STZ-induced diabetic cardiomyopathy (shown in green), an opposite effect on gene expression of Irf3 and Irf7 was observed. Whereas, Irf3 showed a decreased gene expression, Irf7 displayed an increased gene expression under diabetic conditions. However, in the heart failure model caused by chronic AngII-infusion for 21 days (shown in yellow), no differences in gene expression of Irf3 and Irf7 were detected. Data are presented in box plots as relative mRNA expression in fold change to the corresponding untreated control using the formula 2^−ΔΔCt. * = significantly different compared to corresponding control; ^# = significantly different compared to VM (acute—7 days) or RZ (remote zone).