Figure 1.
Murine cardiac tissue expresses NOD1. Specific stimulation of NOD1 induces NF-κB pathway activation.
(A) Histograms show NOD1 mRNA levels in different mouse tissues. (B) NOD1 protein levels in mouse hearts were analyzed by western-blot. Animals received i.p. 150 µg of iEDAP (iE)/day, a selective agonist of NOD1, or vehicle (Veh.). After 2 weeks of treatment an up-regulation of P-RIP2/RIP2, P-IKK/IKK, P-IκBα/IκBα protein ratio (B), higher p65 binding to κB motifs determined by ELISA (C), and NOS2 and COX2 (D) protein levels were observed in iE treated hearts. NOS2 and COX2 protein values were normalized with GAPDH. Representative blots are shown in the left panels and right panels illustrate the histograms representing the mean (band ratio)±SEM values vs. Veh. (100%); n = 4–6 animals. *p<0.05, ***p<0.001 vs. vehicle.
Figure 2.
Specific activation of NOD1 promotes cardiac dysfunction.
(A) Ejection fraction obtained in mice treated for 2 weeks (daily) with vehicle or the indicated concentrations of iE. (B) Ejection fraction obtained in mice treated during 3, 7 or 14 days with vehicle or 150 µg of iE. (C,D) Representative echocardiographic images (M mode) of mouse hearts treated for two weeks with vehicle (C) or 150 µg of iE (D). End Systolic Diameter (ESD), End Diastolic Diameter (EDD), and left ventricle Posterior Walls (PW) are indicated. *p<0.05, ***p<0.001 vs. vehicle.
Table 1.
Cardiac parameters collected after M-mode ultrasound evaluation of mice.
Figure 3.
NOD1 activation increases cardiac fibrosis and TGF-β pathway in hearts isolated from iE treated mice.
(A) Histological analysis of the hearts from mice treated with vehicle (Veh.), with 150 µg of iEDAP (iE) or with 150 µg of iE-Lys (NOD1 inactive analogue of iEDAP). Representative whole-heart cross-sections are shown (magnification x20). Myocardial fibrosis was evaluated after staining with Masson’s trichrome. (B) Histograms show the quantitative analysis of subendocardial fibrosis is shown as collagen area vs. total tissue area (100%, mean ± SEM; n = 4 animals per condition). (C) Histograms show type I and III collagen mRNA levels in cardiac tissue from iE and Veh. treated mice. (D) TGF-β signaling was activated in hearts from iE-treated mice. Left panel, representative blots of TRβ1, TRβ2, P-Smad, Smad, PAI-1 and FGF-2 of vehicle and iE treated mouse hearts (150 µg for 2 weeks). Right panel, shows the corresponding histograms representing the mean values vs. vehicle (100%, n = 4–6 animals). GAPDH was used to normalize all the target protein levels. *p<0.05, ***p<0.001 vs. vehicle.
Figure 4.
Selective stimulation of NOD1 induces cardiac apoptosis.
Animals received i.p. 150 µg/day of iEDAP (iE), 150 µg/day of iE-Lys or vehicle for 2 weeks. (A) TUNEL staining of cells undergoing apoptosis in cardiac tissue sections from vehicle, iE or iE-Lys-treated mice. Light transmission of the preparations indicated that apoptotic cells were predominantly cardiomyocytes. Representative images of TUNEL positive (green) and DAPI (blue) staining (magnification x 40). The percentages of positive TUNEL cells are indicated in the images. (B) Caspase 3, Bax and X-IAP levels determined by Western blot from hearts of vehicle and iE mice. Left panel shows the histograms representing the mean±SEM vs. vehicle (100%). *p<0.05, ***p<0.001 vs. vehicle; n = 4–6 animals.
Figure 5.
The NOD1 agonist iE induces NF-κB activation and apoptosis in cardiomyocytes and H9c2 cells. Effect of NOD1 ablation with siRNAs.
(A) Native murine cardiomyocytes were incubated for 15 to 60 min, or 48 h with 20 µg/ml of iE. iE treatment induces an up-regulation of P-RIP2/RIP2, P-IKK/IKK, P-IκBα/IκBα (15–60 min) and COX2 (48 h) protein levels. (B) iE induces a decrease in cardiomyocyte viability in a dose-response form. Cardiomyocytes incubated for 2 h with 10–30 µg/ml of iE; staurosporine (Stau, 100 ng/ml) were used as a positive control to induce apoptosis. (C). iE treatment induces an up-regulation of caspase 3 and Bax protein levels and down-regulates the BcL-xL and X-IAP protein levels. (D) iE or Stau administration for 24 h in H9c2 promoted a decrease in viability as determined by MTT activity. NOD1 suppression by siRNA prevented the effect of iE on cell viability. (E) Representative blot of NOD1, caspase 3 and GAPDH obtained in vehicle and iE treated cells, NOD1 siRNAs (siNOD1) and NOD1 siRNA+iE in H9c2 cells. All targets protein levels were normalized by GAPDH. Data are expressed in histograms representing the mean±SEM vs. vehicle (100%); n = 3–5 samples per condition.*p<0.05, **p<0.01 and ***p<0.001 vs. vehicle.
Figure 6.
Selective activation of NOD1 stimulates NF-κB and TGF-β pathways in cardiac fibroblasts.
(A) Confocal microscopy images of cardiac fibroblasts isolated from mouse hearts stained for NOD1. (B) Isolated murine cardiac fibroblasts were incubated for 15 to 60 min, 24 h or 48 h with 40 µg/ml iE. iE-treatment induces an up-regulation of P-RIP2/RIP2, P-IKK/IKK, P-IκBα/IκBα (15–60 min), NOS2 (24 h) and COX2 (48 h) protein levels. (C) Treatment of fibroblasts with iE for 72 h promoted an increase of TRβ2, P-Smad/Smad, PAI-1 and FGF-2 protein levels. GAPDH was used to normalize all target protein levels. Data are illustrated in histograms as mean±SEM vs. vehicle (100%; n = 3–5 samples).*p<0.05, **p<0.01 and ***p<0.001 vs. vehicle.
Figure 7.
Effect of NOD1 siRNAs on TGF-β pathway. Proposed role for NOD1 activation in cardiac cells.
(A) Representative blot of NOD1, TRβ2, P-Smad/Smad, PAI-1, and GAPDH levels in vehicle, iE, NOD1 siRNAs (siNOD1) and NOD1 siRNA + iE (siNOD1+iE) NIH-3T3 cells. Scrambled siRNA sequences did not modify the basal levels after iE-treatment. (B)The selective agonist iEDAP induces NOD1 activation. Both in cardiomyocytes and cardiac fibroblasts, NOD1 promotes the activation of the NF-κB pathway through IKK/IκBα signaling. NF-κB activation induces a proinflammatory response, including the expression of NOS2 and COX2. Moreover, TGF-β is activated in cardiac fibroblasts resulting in PAI-1 transcriptional activation by TRβ and Smad factors. Finally, the enhancement and activation of Bax and caspases and the downregulation of X-IAP promoted by NOD1 agonist in cardiomyocytes, contributes to elicit an apoptotic response associated with NOD1 activation. Altogether, cardiomyocyte apoptosis and the pro-fibrotic profile observed in cardiac fibroblasts might contribute to the onset of cardiomyopathy linked to proinflammatory disease.