Cardioprotective Effects of LCZ696 (Sacubitril/Valsartan) After Experimental Acute Myocardial Infarction

Figures

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  Figure 1. Preliminary Study Using Echocardiography, Biomarker Measurement, and Radiotelemetry (A) Correlation curve for serum troponin I level and fractional shortening obtained from 18 mice 1 day after myocardial infarction (MI), with a correlation coefficient of −0.710 (p = 0.001). (B, C) At 1 day after MI, fractional shortening in infarcted mice was significantly lower, and the serum troponin I level in infarcted mice was significantly higher, compared with sham-operated mice (p < 0.05 and p < 0.05, respectively). There were no differences in these parameters between MI mice treated with vehicle, enalapril, and LCZ696. Results were mean ± SD. n = 5 to 6 per group. ∗p < 0.05 vs. sham. (D) 24-h average SBP in mice treated with enalapril and LCZ696. Each drug was administered in an incremental manner every week by gastric gavage once daily. The dose of 20 mg/kg BW/day LCZ696 and 4 mg/kg BW/day enalapril were the maximum that did not lower baseline blood pressure. Results were mean ± SD. n = 4 per group. (E) Circadian rhythm of SBP in mice treated with LCZ696. There were no differences among 20 mg/kg BW/day LCZ696, 4 mg/kg BW/day enalapril, and baseline during the 12-h dark and 12-h light…

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  Figure 2. LCZ696 Treatment Improves Survival and LV Dysfunction After MI (A, B) Kaplan-Meier survival curves for overall and cardiac rupture-free survival for MI mice treated with vehicle, enalapril, and LCZ696. n ≥75 mice per group. ∗p < 0.01 for log-rank test, †p < 0.05 for log-rank test. (C) Number of mice of the vehicle (24 of 77), enalapril (21 of 79), and LCZ696 (10 of 75) groups that died with cardiac rupture within 1 week after MI. (D to F) Serial measurements of (D) fractional shortening, (E) LV end-diastolic dimension, and (F) LV end-systolic dimension in MI mice treated with vehicle, enalapril, and LCZ696. Results were mean ± SD. n ≥ 34 per group. ∗p < 0.01 LCZ696 versus vehicle at 14 days and 28 days after MI, Bonferroni’s multiple comparison test. LV = left ventricular; other abbreviation as in Figure 1.

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  Figure 3. Characterization of Inflammatory Response in the Border Area of the Infarcted Region 3 Days After MI (A) Representative images of LV cross-sections with Masson’s trichrome staining (M-T) and immunohistochemistry (FA-11 and Gr-1) in the border of infarcted region 3 days after MI in the vehicle, enalapril, and LCZ696 groups. Scale bar = 200 μm (B and C). Immunohistochemical staining showed macrophages (FA-11), and granulocytes (Gr-1) in the infarcted region 3 days after MI between the vehicle, enalapril, and LCZ696 groups. Results were mean ± SD. n = 7 per group.

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  Figure 4. Beneficial Effects of LCZ696 on Gene Expression 3 Days After MI Real-time reverse transcriptase PCR results for (A) TNFα, (B) IL-1β, (C) IL-6, (D) MCP-1, (E) TGFβ-1, (F) Col1α1, (G) Col3α1, (H) MMP-2, (I) MMP-9, (J) TIMP-1, (K) ANP, and (L) BNP mRNA levels 3 days after MI. The mRNA level was normalized by the level of endogenous control 18S ribosomal RNA. n = 26 per group. In these box-and-whisker plots, lines within the boxes represent median values; the upper and lower lines of the boxes represent the 75th and 25th percentiles, respectively; and the upper and lower bars outside the boxes represent maximum and minimum values within 1.5 times the interquartile range from the 75th and 25th percentile, respectively. *p < 0.05, Bonferroni’s multiple comparison test. ANP = atrial natriuretic peptide; BNP = brain natriuretic peptide; Col = collagen; IL = interleukin; IR = infarcted region; MCP = monocyte chemotactic protein; MMP = matrix metalloproteinase; PCR = polymerase chain reaction; TGF = tumor growth factor; TIMP = tissue inhibitor of metalloproteinase; TNF = tumor necrosis factor; other abbreviation as in Figure 1.

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  Figure 5. LCZ696 Suppresses MMP-9 Activity in the Infarcted Region 3 Days After MI (A) Representative images of LV cross-sections with in situ zymography in the border area of the infarcted region 3 days after MI of the vehicle, enalapril, and LCZ696 groups. Scale bar = 200 μm (B). Semi-quantitative analysis of gelatinolytic activity showed significantly lower activity in the LCZ696 compared to the vehicle and enalapril groups. n = 7 per group. *p < 0.05, Bonferroni’s multiple comparison test. (C, D) Specific MMP-9 activity assay in the infarcted region 3 days after MI, showing no differences in (C) total MMP-9 among the vehicle, enalapril, and LCZ696 groups. However, (D) active MMP-9 was significantly lower in the LCZ696 compared to the vehicle and enalapril group. In these box-and-whisker plots, lines within the boxes represent median values; the upper and lower lines of the boxes represent the 75th and 25th percentiles, respectively; and the upper and lower bars outside the boxes represent maximum and minimum values within 1.5 times the interquartile range from the 75th and 25th percentile, respectively. n ≥ 24 per group. *p < 0.05, Bonferroni’s multiple comparison test. (E) Double…

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  Figure 6. Combined Effects of Valsartan With LBQ657 in In Vitro Assay Using Peritoneal Macrophages (A) Gelatin zymography on the supernatants of peritoneal macrophages stimulated with LPS (10 ng/ml) for 48 h. (B) Semi-quantitative analysis of gelatinolytic activity showed significantly lower activity in VAL + LBQ compared to the VAL-alone and LBQ-alone groups. n = 6 per group. Quantification of (C) IL-1β, and (D) IL-6 concentrations in the supernatants of peritoneal macrophages stimulated with LPS (10 ng/ml) for 48 h. n = 6 per group. (E to G) Real-time reverse transcriptase PCR result for (E) MMP-9, (F) IL-1β, and (G) IL-6 mRNA level in peritoneal macrophages stimulated with LPS (10 ng/ml) for 48 h. The mRNA level was normalized by the level of endogenous control beta-2-microglobulin RNA. n = 8 per group. Results were mean ± SD. Ctrl = control; LBQ = LBQ657; LPS = lipopolysaccharide; VAL = valsartan; other abbreviations as in Figure 4.

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  Figure 7. Beneficial Effects of the Dual Action of Neurohumoral System in LCZ696 Treatment Quantification of plasma (A) aldosterone and (B) cGMP levels by ELISA 3 days after MI. (C) The ratio of plasma aldosterone to plasma cGMP. n = 28 per group. In these box-and-whisker plots, lines within the boxes represent median values; the upper and lower lines of the boxes represent the 75th and 25th percentiles, respectively; and the upper and lower bars outside the boxes represent maximum and minimum values within 1.5 times the interquartile range from the 75th and 25th percentile, respectively. The inter-group comparisons were corrected by the Bonferroni’s multiple comparison test. cGMP = cyclic guanosine monophosphate; ELISA = enzyme-linked immunosorbent assay; other abbreviation as in Figure 1.

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  Figure 8. Schematic Diagram of the Proposed Model of the Mechanism of the Protective Effect of LCZ696 Against Post-MI Cardiac Rupture Proposed model of the mechanism that LCZ696 (sacubitril/valsartan), which simultaneously regulate RAAS and NPS, can protect against cardiac rupture after MI through the suppression of pro-inflammatory cytokines and ECM degradation. ECM = extracellular matrix; NPS = natriuretic peptide system; RAAS = renin-angiotensin-aldosterone system; other abbreviations as in Figures 1 and 4.