Table 1.
Hemodynamic and functional characteristics.
Conductance and echocardiographic results presented as median (range) as the data is not normally distributed. Pes: end-systolic pressure, Ped: end-diastolic pressure. Sham (n = 3), PAB (n = 5), and PAB+ERB (n = 5). The groups were compared using Kruskal-Wallis multiple comparisons. Post hoc test showed:
Fig 1.
Circulating ET-1, and macitentan levels and ventricular morphology.
A) Plasma concentrations of endothelin-1 (ET-1) were unchanged in Sham (n = 3) compared to PAB (n = 11), but increased significantly with addition of macitentan (PAB+ERB n = 7) **p≤0.001. B) Bar graph showing a significant increase in myocyte size in the PAB group vs. sham for the RV (*p≤0.0001), measured in transverse cut myocytes (n = 4 Sham group, n = 7 for the PAB group, and n = 13 for the PAB+ERB group). Addition of endothelin receptor blocker (PAB+ERB) resulted in a significantly decreased cardiomyocyte size vs. PAB in the RV, ** p≤0.0001. For the LV there were no change in myocyte size. C) Hematoxylin-eosin staining, magnification × 40 the black lines is a micron bar indicating 20μm. RV: right ventricle, LV: left ventricle, PAB: pulmonary artery banding, and ERB: endothelin receptor blocker.
Fig 2.
A) Picrosirius red staining showed a significant increase of collagen in both the RV & LV in the groups with only pulmonary arterial banding (PAB). Addition of endothelin receptor blocker (PAB+ERB) significantly lowered the amount of collagen in both RV and LV. Magnification ×10. B) Quantitative collagen volume fractions in the RV. PAB increased collagen compared with sham (*p≤0.0001), macitentan decreased collagen compared with PAB (**p≤0.0001) C) Quantitative collagen volume fractions in the LV. PAB increased collagen compared with sham (§p≤0.0001), macitentan decreased collagen compared with PAB (‡ p≤0.0001). Note the scale difference in the collagen content graphs between the RV and LV. Volume fractions in the various groups were analyzed using Adobe Photoshop (n = 4 for the sham group, n = 7 for the PAB group, and n = 11 for the PAB+ERB group). RV: right ventricle, LV: left ventricle, SP: septum, PAB: pulmonary artery banding, and ERB: endothelin receptor blocker.
Fig 3.
Western blot results of proteins involved in the signaling pathway of fibrosis.
Western blot analyses with quantification of Connective Tissue Growth Factor (CTGF), platelet derived growth factor (PDGF), and matrix metalloproteinases 2 and 9 (MMP 2 and 9) proteins level at 6-weeks after PAB. A) Western blot examples of the respective proteins. B) CTGF increased significantly with PAB compared to sham in the RV (*p≤0.01), in the LV CTGF also increased with PAB compared to sham (§ p<0.05). Addition of macitentan did not significantly decrease CTGF. C) the protein level of endothelin-1 (ET-1) decreased significantly with addition of macitentan compared with the PAB group (*p≤0.05). D) PDGF increased in the RV with PAB compared with sham (*p≤0.05). E) MMP-2 in the RV showed a trending increase with PAB and decreased with addition of macitentan compared with PAB (*p≤0.05) F) MMP-9 in the RV increased with PAB compared with sham (*p<0.05), addition of macitentan decreased MMP-9 (**p≤0.05). Glyceraldehyde-3–phosphate dehydrogenase (GAPDH) was used as the internal control. Sham (n = 4), PAB-group (n = 5), and PAB+ERB (n = 5). RV: right ventricle, LV: left ventricle, PAB: pulmonary artery banding, and ERB: endothelin receptor blocker.
Fig 4.
Real-time PCR results of mRNA expression in the myocardium.
Real-time PCR data show levels of gene expression in response to endothelin receptor blocker treatment at 6-weeks after PAB. A & B) Genes related to profibrotic mediators: transforming growth factor–β1 (TGF-β1) and connective tissue growth factor (CTGF). C & D) Matrix metalloproteinase (MMP) -2 and MMP-9 gene expression. In the LV MMP-2 showed significant up-regulation in response to pulmonary arterial banding (sham vs. PAB §p≤0.05) and decrease with addition of macitentan ‡ p≤0.05). E & F) Endothelin receptor type A (ENDRA) and endothelin receptor type B (ENDRB); ENDRA was significantly up-regulated in the LV as a response to PAB, §p≤0.01. G & H) α- and β-MHC: myocyte heavy chain. Sham (n = 4), PAB-group (n = 5), and PAB+ERB (n = 5). RV: right ventricle, LV: left ventricle, PAB: pulmonary artery banding, and ERB: endothelin receptor blocker.
Fig 5.
Zymographic image of activated MMP-2 and MMP-9.
Representative MMP-9 and MMP2 activity in RV and LV of sham using zymography. Results corresponds with western blot results, showing activated MMP-9 and MMP-2 up regulated in PAB compare with sham; and down regulated after ERB treatment compare with PAB in the RV, but less in the LV.
Fig 6.
Immunofluoresence staining showing the immunolabeling of ET-1 Receptor of myocytes.
Using myocardial samples from sham, PAB, and PAB + ERB treatment from both the RV and LV. Images a-f represents ET-1 receptor (in green), images g-l (in red) represents α-actinin (myocyte marker). Merged images shown in m-r, nucleis are colored blue. The staining shows the expression pattern of ET receptor on cardiomyocytes membrane (arrow), and interstitial area (*). Original magnification, ×400. In sham myocardium, the ET-1 receptor was positively labeled in the membrane of cardiac myocytes and interstitial cells (a, b). After PAB, ET-1 receptor strongly labeled myocytes membrane as well as interstitial cells (c, d) and decreased after ERB treatment (e, f) in both RV and LV.
Fig 7.
Immunofluoresence staining of ET-1 receptor expressed in cardiac fibroblasts (a-d).
(a) represent ET-1 receptor (in green) and (b) Vimentin, a fibroblast marker (in red) from myocardial samples of the RV from a PAB heart. The orange colour in images c and d indicates overlapping of ET-1 receptor and Vimentin staining. (d) shows the merged image (a and b) including nucleis colored blue. Original magnification, ×400.
Fig 8.
TUNEL results of myocardial apoptosis.
A) Apoptosis measured by TUNEL assay in sham operated controls (n = 3), in response to pulmonary arterial banding (PAB, n = 3) and endothelin receptor blocker (ERB, n = 4) treatment at 6-weeks after PAB. TUNEL analysis of myocardium from sham RV (A, B), LV(C, D); PAB RV (E, F), LV(G, H); PAB with ERB treatment RV (I, J), LV (K, L) rats heart. Arrows show apoptotic nuclei indicated by green (A, C, E, G, I, K). Blue spots represent nuclear stained by DAPI (4',6-diamidino-2-phenylindole dihydrochloride) and merged images are shown in B, D, F, H, J and L (magnification ×400). B) Bar graph with quantification of apoptosis in the right and left ventricles. In the right ventricle apoptosis increased with PAB compared to sham (*p≤0.0001) and diminished with macitentan (compared with PAB **p≤0.05). For the LV, apoptosis increased with PAB compared with sham (*p≤0.01), but there were no significant changes with macitentan. RV: right ventricle, LV: left ventricle, PAB: pulmonary artery banding, and ERB: endothelin receptor blocker.
Fig 9.
Western blot results of Caspase 3 and 8, peptides involved in apoptosis.
Illustrated in the graphs are the quantification of Caspase 3 and 8. A) Shows caspase 3 increase with PAB compared with sham (*p≤0.05) and diminish with macitentan compared with PAB (**p≤0.05). B) Shows caspase 8 increase with PAB compared to sham (*p≤0.01) however, macitentan did not significantly diminish Caspase 8. C) Western blot examples are shown below the graphs of the respective proteins. Glyceraldehyde-3–phosphate dehydrogenase (GAPDH) was used as the internal control. Sham (n = 4), PAB-group (n = 5), and PAB+ERB (n = 5). RV: right ventricle, LV: left ventricle, PAB: pulmonary artery banding, and ERB: endothelin receptor blocker.
Fig 10.
Immunostaining of CD31 represented cardiac vessel volume in sham (A), PAB (B), and PAB+ERB (C) The black lines indicate 100μm. D) Quantification of capillary volume (expressed as percentage of tissue volume) shows the PAB group compared to sham, and the PAB+ERB group compared to sham or PAB, did not change.
Sham (n = 4), PAB-group (n = 4), and PAB+ERB (n = 4).