Sacubitril/Valsartan Decreases Cardiac Fibrosis in Left Ventricle Pressure Overload by Restoring PKG Signaling in Cardiac Fibroblasts

Abstract

Background Heart failure (HF) is invariably accompanied by development of cardiac fibrosis, a form of scarring that increases muscular tissue rigidity and decreases cardiac contractility. Cardiac fibrosis arises from a pathological attempt to repair tissue damaged during maladaptive remodeling. Treatment options to block or reverse fibrosis have proven elusive. Neprilysin is an endopeptidase that degrades vasoactive peptides, including atrial natriuretic peptide. Thus, neprilysin inhibition reduces hypertension, ultimately limiting maladaptive cardiac remodeling. LCZ696, which consists of an angiotensin receptor blocker (valsartan [VAL]) and a neprilysin inhibitor (sacubitril [SAC]), was shown to be well tolerated and significantly reduced the risk of death and hospitalization in HF patients with reduced ejection fraction. We hypothesized that SAC/VAL directly inhibits fibroblast activation and development of pathological fibrosis. Methods and Results We used a mouse model of left ventricle pressure overload coupled to in vitro studies in primary mouse and human cardiac fibroblasts (CFs) to study the impact of SAC/VAL on CF activation and cardiac fibrosis. SAC/VAL significantly ameliorated pressure overload-induced cardiac fibrosis by blocking CF activation and proliferation, leading to functional improvement. Mechanistically, the beneficial impact of SAC/VAL at least partially stemmed from restoration of PKG (protein kinase G) signaling in HF patient-derived CF, which inhibited Rho activation associated with myofibroblast transition. Conclusions This study reveals that SAC/VAL acts directly on CF to prevent maladaptive cardiac fibrosis and dysfunction during pressure overload-induced hypertrophy and suggests that SAC/VAL should be evaluated as a direct antifibrotic therapeutic for conditions such as HF with preserved ejection fraction.

Keywords: angiotensin II; fibrosis; heart failure; hypertrophy; neprilysin; valsartan.

Figure 1:. SAC/VAL prevents cardiac hypertrophy and loss of function associated with pressure overload-induced heart failure more effectively than equimolar valsartan.

A. Heart weight/tibia length ratio reveals a decrease in pressure overload-induced cardiac hypertrophy with SAC/VAL, which significantly reduces hypertrophy relative to saline or equimolar valsartan alone. n = 10, 10, 12, 14. One-way ANOVA with Tukey’s multiple comparisons test. B. LV mass by echo confirms the significant effect of SAC/VAL relative to equimolar valsartan. For A, B, D-I: n = 10, 10, 12, 14. Two-way ANOVA with repeated measures (time). * = p < 0.05 compared to saline; & = p < 0.05 for SAC/VAL compared to equimolar valsartan. C. H&E staining of transverse sections through the papillary muscle further confirms SAC/VAL’s significant effect against hypertrophic remodeling in response to pressure overload. Scale bar = 1mm. D-E. Systolic/diastolic LV internal diameter (ID) increase is prevented by SAC/VAL but not the molar equivalent of valsartan. F-G. Systolic/diastolic volume increase is prevented by SAC/VAL but not the molar equivalent of valsartan. Two-way ANOVA with repeated measures (time). H. Fractional shortening is significantly improved by SAC/VAL, but not the molar equivalent of valsartan. Two-way ANOVA with repeated measures (time). I. Ejection fraction is significantly improved by SAC/VAL but not the molar equivalent of valsartan. Two-way ANOVA with repeated measures (time). J. Myocyte cross sectional area (CSA) is visualized by wheat germ agglutinin (WGA) staining. Scale bar = 100 μm. K. CSA is increased due to concentric hypertrophic remodeling induced by pressure overload (saline), and is decreased in SAC/VAL-treated mice significantly more so than equimolar valsartan. One-way ANOVA, Tukey’s multiple comparisons test. n = 5, 5, 6, 7. * = p < 0.05 compared to saline; & = p < 0.05 for SAC/VAL compared to equimolar valsartan.

Figure 2:. SAC/VAL treatment more effectively reduces cardiac fibrosis in response to pressure overload than equimolar valsartan.

A. E/A ratio is not statistically distinct between SAC/VAL- and valsartan-treated animals (28 days post-TAC). n = 12, 14. Student’s unpaired t-test with Welch’s correction. B. E/E’ ratio (measured at mitral valve and intraventricular septum), trends lower (IVS: p = 0.11, MV: p = 0.06) in SAC/VAL-treated animals as compared to equimolar valsartan-treated animals. n = 12, 14. Student’s unpaired t-test with Welch’s correction. C. Heart sections were stained with PicroSirius Red and imaged with polarized light to visualize the deposition of fibrotic extracellular matrix. SAC/VAL significantly ameliorates fibrosis relative to saline, while the equivalent dose of valsartan does not. Scale bar = 200 μm D-F. Quantification of fibrosis in the epicardial, perivascular, and interstitial areas. Both green (thin/new) and red (thick/established) fibers were analyzed. n = 5, 5, 6, 7. Two-way ANOVA (independent variables green/new and red/established) with Tukey’s multiple comparisons test. * = p < 0.05 compared to saline; & = p < 0.05 for SAC/VAL compared to equimolar valsartan.

Figure 3:. SAC/VAL attenuates accumulation of vimentin⁺ and PDGFRα⁺ cell populations, and isolated CF from SAC/VAL-treated hearts show significantly altered gene expression.

A. Staining for PDGFRα and WGA reveals that SAC/VAL significantly decreases CF load post-pressure overload relative to equimolar valsartan. Scale bar = 50μm. B. Sections stained for vimentin revealed that DAPI⁺/vimentin⁺ cell population expansion is significantly prevented by SAC/VAL as well as the equivalent dose of valsartan. Scale bar = 100 μm. B. Quantification of PDGFRα staining. One-way ANOVA with Tukey’s multiple comparisons test. * = p < 0.05 compared to saline; & = p < 0.05 for SAC/VAL compared to equimolar valsartan. D. Quantification of vimentin staining, which confirms that while equimolar valsartan decreases fibroblast number, SAC/VAL is significantly more effective. n = 5, 5, 6, 7. One-way ANOVA with Tukey’s multiple comparisons test. * = p < 0.05 compared to saline; & = p < 0.05 for SAC/VAL compared to equimolar valsartan. E. qRT-PCR for gene expression in fibroblasts isolated by Langendorff perfusion of mouse hearts reveals that SAC/VAL effectively targets proliferation, activation, and extracellular matrix-associated gene expression. The equivalent dose of valsartan has no statistically significant effect on any of these genes, while SAC/VAL confers significant changes that reflect a less pathologic gene program. n = 5, 5, 6, 7. Two-way ANOVA, Tukey’s multiple comparisons test. * = p < 0.05 compared to saline; & = p < 0.05 for SAC/VAL compared to equimolar valsartan.

Figure 4:. Treatment of CF with exogenous ANF causes dose-dependent decreases in myofibroblast gene expression.

A-B. Pre-treatment of neonatal mouse CF with ANF significantly and dose-dependently attenuates TGF-β1 dependent induction of Acta2 (A) and Postn (B) gene expression. C. ANF pre-treatment trends toward recovery of Tcf21 expression. For all experiments, n = 3 each condition. One-way ANOVA with repeated measures (ANF concentration). * = p < 0.05 relative to 0ng/mL TGF-β1 / 0 M ANF. & = p < 0.05 relative to 10ng/mL TGF-β1 / 0 M ANF. D. ANF pre-treatment does not significantly alter cell cycle gene expression in neonatal CF that are stimulated with TGF-β1 alone. One-way ANOVA for each gene with repeated measures (concentration). * = p < 0.05 relative to vehicle control. E. ANF significantly ablates TGF-β1 / AngII-induced changes in cell cycle gene expression that are associated with proliferation. Two-way ANOVA with Tukey’s multiple comparisons test. * = p < 0.05 compared to vehicle control. & = p < 0.05 relative to TGF-β1/AngII condition.

Figure 5:. Sacubitrilat ameliorates expression of myofibroblast markers in TGF-β1/AngII/ANF-stimulated nCF.

A. Pretreatment with Sacubitrilat significantly decreases activation- and proliferation-associated gene expression in response to TGF-β1/AngII/ANF stress stimulus in neonatal CF. n = 3 each condition for all experiments. Two-way ANOVA with Tukey’s multiple comparisons test. * = p < 0.05 relative to vehicle control. & = p < 0.05 relative to the TGF-β1/AngII condition. ! = p < 0.05 relative to the TGF-β1/AngII/ANF condition. B. Pretreatment with valsartan alone only significantly alters proliferation-associated gene expression in neonatal CF, while valsartan + sacubitrilat also significantly inhibits some activation-associated genes. Two-way ANOVA with Tukey’s multiple comparisons test. * = p < 0.05 relative to vehicle control. & = p < 0.05 relative to the TGF-β1/AngII/ANF/valsartan condition. C. Valsartan and Sacubitrilat significantly inhibit ACTA2 induction by TGF-β1/AngII/ANF. One-way ANOVA with Tukey’s multiple comparison test. * = p < 0.05 relative to TGF-β1/AngII/ANF.

Figure 6:. Sacubitrilat stabilizes PKG signaling and inhibits RhoA in activated CF.

A. Sacubitrilat alone or in combination with valsartan promotes the PKG-dependent phosphorylation of VASP at S239. B. Quantification of (A). n = 3 for each condition for all experiments. One-way ANOVA with Tukey’s multiple comparisons test. * = p < 0.05 relative to TGF-β1/AngII/ANF, & = p < 0.05 relative to TGF-β1/AngII/valsartan. C. Use of the PKG inhibitor KT5822 confirms that Sacubitrilat effects are transduced primarily through PKG. D. Quantification of (C). Two-way ANOVA with Tukey’s multiple comparisons test. * = p < 0.05 relative to vehicle/ANF, & = p < 0.05 relative to TGF-β1/AngII/ANF; ND, not detected. E. Sacubitrilat promotes the PKG-dependent phosphorylation of RhoA at S188, which inactivates RhoA (a major transducer of myofibroblast activation). F. Quantification of (E). One-way ANOVA with Tukey’s multiple comparisons test. * = p < 0.05 compared to vehicle.

Figure 7:. SAC/VAL is more efficient at inactivating Rho than is SAC or VAL alone in CF derived from heart failure patients.

A. Pulldown of Rho with beads conjugated to the Rho-binding domain of rhotekin (specific for activated Rho) reveals that for all four patient-derived primary CF lines tested, the combination of Sacubitrilat and valsartan significantly ameliorates Rho activation. B-E. Quantification of data for each line isolated as shown in (A). n = 3 each condition. One-way ANOVA for each patient-derived line, Tukey’s multiple comparisons test. * = p < 0.05 relative to vehicle. & = p < 0.05 relative to TGF-β1/AngII/ANF.