Sacubitril/Valsartan Improves Diastolic Function But Not Skeletal Muscle Function in a Rat Model of HFpEF

Figures

• 

  Figure 1. Baseline characterization of 20-week-old lean and obese Zucker fatty and spontaneously hypertensive (ZSF1) rats—myocardial fibrosis and vasodilative properties. (A) Brightfield microscopy of perivascular fibrosis in representative picrosirius-stained heart sections. (B,C) mRNA expression of Col1a1 (B) and Col3a1 (C) in LV tissue. (D) Endothelial-dependent vasodilation of sections of left carotid arteries in response to increasing concentrations of acetylcholine (AcH). (E) Endothelial-independent vasodilation of sections of left carotid arteries in response to increasing concentrations of sodium nitroprusside (SNP). Myocardial fibrosis: n = 10 for each group. Vascular function: n = 15 (lean and obese, respectively) and n = 13 (Sac/Val). Lean- and obese animals were compared by 2-sided Student’s t-test of equal variances. * p < 0.05, ** p < 0.01 vs. lean, respectively.

• 

  Figure 2. Basic characterization of 20-week-old lean and obese ZSF1 rats—skeletal muscle weight and function. Muscle weight of (A) TA muscle, (B) Soleus muscle, and (C) EDL muscle. (D,E) Absolute force measured for EDL (D) and Soleus (E) and specific force measured in EDL (F) and Soleus (G). n = 10 for each group. Lean and obese animals were compared by 2-sided Student’s t-test of equal variances. * p < 0.05, ** p < 0.01 vs. lean, respectively.

• 

  Figure 3. Development of cardiac function during 12 weeks of Sac/Val treatment. Systolic (A) and diastolic function (B) of lean, obese, and Sac/Val treated obese ZSF1 rats indicated by the LVEF and the ratio E/é. Comparison of representative pulsed wave (PW) Doppler and Tissue Doppler sequences after 12 weeks of Sac/Val or sedentary life style (C). n = 15 (each, lean and obese) and n = 13–15 (Sac/Val). To test the effect of Sac/Val two-way one-way analysis of variance (ANOVA) with repeated measures followed by Bonferroni post hoc was used to compare echo data between the groups over time. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. lean, respectively. Ɨ p < 0.05, ƗƗƗ p < 0.001 vs. obese.

• 

  Figure 4. LV hemodynamic conditions and compliance after 12 weeks of Sac/Val treatment. Representative baseline Pressure-Volume (PV) loops of lean, obese, and Sac/Val treated rats at an age of 32 weeks (A). Representative PV loop shifts in response to a transient occlusion of the inferior vena cava of a lean (B), an obese (C), and a Sac/Val treated obese rat (D). (E) Comparison of LV end diastolic pressure (LVEDP) and (F) LV end systolic pressure (LVESP) at baseline conditions. Comparison of the LV contractility indicated by the slope LV-Ees obtained by the end-systolic pressure-volume relationships (ESPVR) (G). Comparison of the LV chamber stiffness indicated by the LV stiffness constant βW (H) obtained by the end-diastolic pressure-volume relationships (EDPVR). n = 10–12. To test the effect of Sac/Val, one-way analysis of variance (ANOVA) followed by Bonferroni post hoc was used to compare groups.

• 

  Figure 5. LV fibrosis and remodeling after 12 weeks of Sac/Val treatment. Brightfield microscopy of perivascular fibrosis in representative picrosirius-stained heart sections (A). Quantification of LV perivascular fibrosis (B). LV mRNA expression of Col1a1 (C) and Col3a1 (D). MMP-2 activity measured in LV tissue (E). LV mRNA expression of ANP (F) and BNP (G). cGMP concentration measured in LV tissue (H). LV protein expression of phosphorylated titin (I). n = 13–14. To test the effect of Sac/Val one-way analysis of variance (ANOVA) followed by Bonferroni post hoc was used to compare groups.

• 

  Figure 6. Comparison of LV fat deposits at the site of the mitral valve annulus after 12 weeks of Sac/Val. Brightfield microscopy (A,C,E) and nonlinear optical microscopy (NLOM) (B,D,F) of representative heart sections of a lean (A,B), an obese (C,D), and a Sac/Val treated obese rat (E,F). Arrows point to mitral valve structures. Arrow heads indicate the mitral valve annulus. NLOM visualizes collagen fibers (appearing in blue), myofilaments (appearing in green), and lipid structures (appearing in red).

• 

  Figure 7. Contractile and vasodilative properties of left arterial carotid sections after 12 weeks of Sac/Val. Comparison of tension (A), endothelial-dependent (B), and endothelial-independent (C) vasodilation of left arterial carotid sections of lean, obese and Sac/Val treated obese ZSF1 rats after 12 weeks of Sac/Val or sedentary lifestyle. n = 13–15. To test the effect of Sac/Val, two-way ANOVA with repeated measures followed by Bonferroni post hoc was performed. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. lean. Ɨ p < 0.05, ƗƗ p < 0.01 vs. obese.

• 

  Figure 8. Skeletal muscle weight and function after 12 weeks of Sac/Val. Muscle weight (normalized to tibia length (TL)) (A) and cross-sectional area (CSA) of the TA muscle (B). Muscle weight of the Soleus (C) and the EDL (D), each normalized to tibia length. Absolute (E,F) and specific force (G,H) of the EDL (E,G) and Soleus (F,H). n = 13–15. To test the effect of Sac/Val, two-way ANOVA with repeated measures followed by Bonferroni post hoc was performed. ** p < 0.01, *** p < 0.001 vs. lean, respectively.

• 

  Figure 9. Study design. In the present study, a total number of 25 ZSF1-lean and 40 ZSF1-obese rats was included. At an age of 20 weeks, 10 lean and 10 obese rats were randomly selected for baseline characterization. The remaining obese rats were randomly divided into two equal sized groups with n = 15 receiving a daily oral gavage of Sac/Val (60 mg/kg/day, LCZ696, Novartis, Germany) over a time period of 12 weeks (Sac/Val) and n = 15 serving as sedentary obese control. The 15 lean rats served as sedentary lean control.