A novel small molecule compound VCP979 improves ventricular remodeling in murine models of myocardial ischemia/reperfusion injury

Figures

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  Figure 1. Schematic representation of the experimental protocol for murine models. The four groups: Sham+N.S, sham+VCP979, MI/R+N.S and MI/R+VCP979 (n=6 per group). MI/R injury was established by 45 min ischemia and continuous reperfusion until day 28. VCP979 and N.S were injected intraperitoneally from days 1 to 14 following surgery. The experiments were repeated three times. MI/R, myocardial ischemia/reperfusion; N.S, normal saline.

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  Figure 2. Safety of VCP979 treatment in mice and cultured cells. (A) The body weight of mice in the sham groups was recorded on days 0, 7, 14, 21 and 28 following surgery (n=6 per group). (B) Representative hematoxylin and eosin-stained histological sections of the lung, liver, kidney, spleen and heart of mice after 28 days. Scale bar=100 µm. (C) The AST, ALT and LDH levels in serum were measured in sham groups. LDH leakage percent of the (D) NCFs and (E) NCMs following treatment with or without different concentrations of VCP979 (0.1, 1, 3 and 9 µM) for 24 h. NCF, neonatal cardiac fibroblast; NCM, neonatal cardiac myocyte; LDH, lactate dehydrogenase; AST, aspartate transaminase; ALT, alanine aminotransferase.

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  Figure 3. VCP979 administration improved cardiac function in mice post-MI/R injury. (A) Cardiac function was detected on days 1 before and 1, 7, 14 and 28 after surgery (n=6 per group). (B) Representative images of M-mode echocardiography from the sham+N.S, sham+VCP979, MI/R+N.S and MI/R+VCP979 groups on day 28. (C) The survival curves of the sham and MI/R groups injected with N.S or VCP979 were observed for 28 days (n=12 per group). Results are expressed as the mean ± standard error of the mean. *P<0.05 and **P<0.01, sham+N.S vs. MI/R+N.S group. #P<0.05 and ##P<0.01, MI/R+N.S vs. MI/R+VCP979. N.S, normal saline; MI/R, myocardial ischemia/reperfusion; LVEF, left ventricular ejection fraction; LV vol, left ventricular volume; LVFS, left ventricular fractional shortening; LVIDd/s, left-ventricular internal dimension; diastolic/systolic.

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  Figure 4. VCP979 administration of attenuated myocardial fibrosis following MI/R injury. (A) Representative Masson's Trichrome-stained histological sections of hearts from the sham+N.S, sham+VCP979, MI/R+N.S and MI/R+VCP979 groups. Scale bar=1 mm. (B) Representative collagen I staining pictures. Scale bar=200 µm. (C) The fibrosis area of heart tissues and the (D) positive rate of collagen I were measured using ImageJ software. (E) mRNA levels of α-SMA, collagen I and Ang II in heart tissues of different groups. Results are expressed as the mean ± standard error of the mean. **P<0.01 and ***P<0.001, sham+N.S vs. MI/R+N.S. #P<0.05 and ##P<0.01, MI/R+N.S vs. MI/R+VCP979. SMA, smooth muscle actin; MI/R, myocardial ischaemia/reperfusion; N.S, normal saline; Ang II, angiotensin II; ns, not significant.

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  Figure 5. VCP979 treatment can alleviate pathological hypertrophy of the heart after day 28 in vivo. (A) Representative WGA immunofluorescence-stained sections. Green, WGA; Blue, nucleus. Scale bar=25 µm. (B) The cross-sectional areas of the heart sections were estimated using ImageJ software. (C) The ratio of heart weight (g) to body weight (g) post-MI/R injury on day 28. (D) mRNA expression of ANP and BNP in hearts. Results are expressed as the mean ± standard error of the mean. *P<0.05 and ***P<0.001, sham+N.S vs. MI/R+N.S. #P<0.05, ##P<0.01 and ###P<0.001. MI/R+N.S vs. MI/R+VCP979. WGA, wheat germ agglutinin; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; MI/R, myocardial ischemia/reperfusion; N.S, normal saline; ns, not significant.

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  Figure 6. VCP979 treatment prevents inflammatory cell infiltration and suppresses the p38-MAPK signaling pathway on day 14. (A) Representative CD4, CD8 and F4/80 staining pictures. Scale bar=50 µm. (B) The relative positive cells of the sections were determined by ImageJ software. (C) Reverse transcription-quantitative PCR analysis of mRNA levels of Arg1, CD206, iNOS, IL-1β, IL-6 and TNF-α in the heart tissues. (D) Western blotting analysis and (E) quantitative analysis of p-p38 and p38 protein levels in heart tissues from the MI/R+N.S and MI/R+VCP979. Results are shown as mean ± standard error of the mean. #P<0.05 and ##P<0.01, MI/R+N.S vs. MI/R+VCP979 group. MAPK, mitogen-activated protein kinase; iNOS, inducible nitric oxide synthase; p-, phosphorylated-; IL, interleukin; TNF, tumor necrosis factor; CD, cluster of differentiation; MI/R, myocardial ischemia/reperfusion; N.S, normal saline; ns, not significant; Arg1, arginase 1.

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  Figure 7. Addition of VCP979 inhibits the Ang II-induced proliferation and collagen synthesis of NCFs and hypertrophy of NCMs and suppresses the p38-MAPK signaling pathway in vitro. (A) Ang II-induced proliferation percentage in NCFs following treatment with different concentrations of VCP979. (B) Collagen I mRNA level in NCFs and (C) ANP mRNA level in NCMs following treatment with VCP979 for 24 h. (D) Representative Phalloidin immunofluorescence staining pictures of NCMs. Phalloidin is highly integrated with F-actin. Red, F-actin; Blue, nucleus. Scale bar=100 µm. (E) The CSAs of the NCMs were measured using ImageJ software. (F) Western blotting and (G) analysis of p-p38 proteins in Ang II-induced NCFs, with or without the addition of VCP979. The mRNA expression of IL-1β, IL-6 and TNF-α in (H) NCFs and (I) NCMs. Results are presented as the mean ± standard error of the mean. & P<0.05, &&P<0.01 and &&&P<0.001, control vs. Ang II-induced group. *P<0.05, **P<0.01 and ***P<0.001, Ang II-induced vs. VCP979 treatment group. #P<0.05, ##P<0.01 and ###P<0.001, significant differences between various concentrations of VCP979 in (A-C). NCFs, neonatal cardiac fibroblasts; NCMs, neonatal cardiac…