Figure 1.
Masson’s trichrome staining of rat heart sections shows scar formation 4weeks after MI and CAR treatment.
A-E, the representative views of the sham surgery control, AMI, CAR-L, CAR-M and CAR-H groups, respectively (Scale bar: 100 µm). F. The collagen volume fraction in the border zone of the infarcted myocardial region was significantly lower in the CAR-M and CAR-H groups. #p < 0.01, # # p < 0.001 vs. AMI group, *p < 0.05, **p < 0.01 vs. CAR-L group, N = 4–5.
Figure 2.
ECM-related Col1a1, Col3a1, and α-SMA expression and miR-29b expression in the border zone of the infarcted region.
A. Col1a1, Col3a1, and α-SMA mRNA expression by quantitative real-time PCR assay. #p < 0.01 vs. sham surgery control group; *p < 0.05, **p < 0.01 vs. AMI group, N = 6–8. B. Col1a1, Col3a1, and α-SMA protein expression by Western-blot assay. C. Mature miR-29b expression by quantitative real-time PCR assay. *p < 0.05, **p < 0.01 vs. AMI group, N = 6–8. D. miR-29b-1 and miR-29b-2 precursor expression by quantitative real-time PCR assay. ※p < 0.001 vs. miR-29b-1 precursor, *p < 0.05 vs. AMI group, **p < 0.01 vs. AMI group.
Figure 3.
ECM-related Col1a1, Col3a1, and α-SMA expression and miR-29b expression in carvedilol-treated rat cardiac fibroblasts.
A. Col1a1, Col3a1, and α-SMA mRNA expression by quantitative real-time PCR assay. *p < 0.05, **p < 0.01 vs. control group, N = 4. B. Col1a1, Col3a1, and α-SMA protein expression by Western-blot assay. C. Mature miR-29b expression by quantitative real-time PCR assay. *p < 0.05 vs. control group, N = 4. D. miR-29b-1 and miR-29b--2 precursor expression by quantitative real-time PCR assay. #p < 0.01 vs. miR-29b-1 in control group; *p < 0.05 vs. control group, N = 4.
Figure 4.
Smad3 signaling pathway, ECM-related genes and miR-29b expression in rat cardiac fibroblasts.
A. Measurement of intracellular ROS by DCFH-DA staining (400× magnification). The figures are representative of three independent experiments. #p <0.001 vs. Blank, & p <0.001 vs. Ang II, N=4. B. Inactivation of smad3 in NAC- and carvedilol-treated rat cardiac fibroblasts. #p <0.05 vs. Blank, *p <0.05 vs. Ang II, N=4. C. Downregulation of Col1a1, Col3a1, and α-SMA in smad3 knockdown rat cardiac fibroblasts. *p < 0.05, **p < 0.01 vs. control group, N = 4. D. Upregulation of mature miR-29b in smad3 knockdown rat cardiac fibroblasts. *p < 0.05, **p < 0.01 vs. control group, N = 4. E. Upregulation of miR-29b-2 precursor in smad3 knockdown rat cardiac fibroblasts. ※p < 0.01 vs. miR-29b-1 in control group, # p < 0.01 vs. control group, N = 4. F. Downregulation of Col1a1, Col3a1, and α-SMA in SIS-3 or Nar-treated rat cardiac fibroblasts. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Blank group, N = 4. G. Upregulation of mature miR-29b in SIS-3 or Nar-treated rat cardiac fibroblasts. *p < 0.05, **p < 0.01 vs. control group, N = 4. H. Upregulation of miR-29b-2 precursor in SIS-3 or Nar-treated rat cardiac fibroblasts. ※p < 0.01 vs. miR-29b-1 in blank control group, *p < 0.05, **p < 0.01 vs. miR-29b-2 in blank control group, N = 4.
Figure 5.
Enforced expression of miR-29b decreased Col1a1, Col3a1, and α-SMA expression.
A. Col1a1, Col3a1, and α-SMA mRNA expression by quantitative real-time PCR assay. *p < 0.05, # p < 0.01, # # p < 0.001 vs. control group, N = 3–5. B. Col1a1, Col3a1, and α-SMA protein expression by Western-blot assay. NC, scrambled oligonucleotide.
Figure 6.
Schematic diagram of the mechanism whereby carvedilol attenuates AMI-induced myocardial fibrosis.
Inactivation of ROS-induced smad3 signaling by carvedilol results in suppression of ECM genes, including Col1a1, Col3a1, and α-SMA. Additionally, de-suppression of miR-29b by smad3 inactivation contributes to ECM-related gene expression.