*3.3. Serum Concentration of Angiotensins and Aldosterone, and the Markers of Renin and ACE Activities*

The mean serum angiotensin and aldosterone concentrations in the study groups after six weeks of treatment are schematically depicted in Figure 3.

**Figure 3.** Schematic depicting the serum angiotensin and aldosterone concentrations in the study groups after six weeks of treatment. Results are presented as means. ACE, angiotensin-converting enzyme; Aldo, aldosterone; Ang, angiotensin; AP, aminopeptidase; ARNI, sacubitril/valsartan; AT1R, angiotensin II type 1 receptor; NEP, neprilysin (neutral endopeptidase); SHRs, spontaneously hypertensive rats. *n* = 6 per group.

The serum equilibrium level of Ang 1-10 (Ang I) was 247.15 ± 40.13 pmol/L and 223.32 ± 47.4 pmol/L in the control and SHR groups, respectively (ns); ARNI increased it by 217% (ns) and 645% (*p* < 0.05) in the control and SHR groups, respectively (Figure 4A).

The level of Ang 1-8 (Ang II) was 595.77 ± 87.89 pmol/L and 349.22 ± 79.1 pmol/L in the control and SHR groups, respectively (ns); ARNI increased it by 279% (*p* < 0.05) and 588% (*p* < 0.05) in the control and SHR groups, respectively, after six weeks of treatment (Figure 4B).

The level of Ang 2-8 (Ang III) was 21.88 ± 3.22 pmol/L and 10.63 ± 3.88 pmol/L in the control and SHR groups, respectively (ns); ARNI increased it by 285% (ns) and 697% (*p* < 0.05) in the control and SHR groups, respectively (Figure 4C).

The level of Ang 3-8 (Ang IV) was 28.27 ± 4.49 pmol/L and 17.72 ± 4.0 pmol/L in the control and SHR groups, respectively (ns); ARNI increased it by 329% (ns) and 637% (*p* < 0.05) in the control and SHR groups, respectively (Figure 4D).

The level of Ang 1-7 was 20.33 ± 2.66 pmol/L and 19.07 ± 4.69 pmol/L in the control and SHR groups, respectively (ns); ARNI increased it by 279% (ns) and 763% (*p* < 0.05) in the control and SHR groups, respectively (Figure 4E).

The level of Ang 1-5 was 55.75 ± 6.95 pmol/L and 46.97 ± 11.54 pmol/L in the control and SHR groups, respectively (ns); ARNI increased it by 230% (ns) and 642% (*p* < 0.05) in the control and SHR groups, respectively (Figure 4F).

None of the Ang levels in SHR were significantly affected by ivabradine after six weeks of treatment (Figure 4A–F).

The Ang 1-5/Ang 1-7 ratio was 2.78 ± 0.23 and 2.73 ± 0.32 in the control and SHR groups, respectively (ns); ivabradine decreased it by 32% (*p* < 0.05) in the SHR group. ARNI had no significant effect on the Ang 1-5/Ang 1-7 ratio in either controls or SHRs (Figure 4G).

The marker of renin activity (RA-S; Ang I + Ang II) was 842.88 ± 125.38 pmol/L and 572.5 ± 125.96 pmol/L in the control and SHR groups, respectively (ns); ARNI increased it by 261% (ns) and 610% (*p* < 0.05) in the control and SHR groups, respectively. Ivabradine had no significant effect on the marker of renin activity in SHRs after six weeks of treatment (Figure 4H).

The marker of ACE activity (ACE-S; Ang II/Ang I ratio) was 2.47 ± 0.20 and 1.53 ± 0.08 in the control and SHR groups, respectively (*p* < 0.05). ARNI and ivabradine had no significant effect on ACE-S in SHRs (Figure 4I).

**Figure 4.** Effect of ARNI and ivabradine on the serum level of angiotensin 1-10 (Ang I) (**A**), angiotensin 1-8 (Ang II) (**B**), angiotensin 2-8 (Ang III) (**C**), angiotensin 3-8 (Ang IV) (**D**), angiotensin 1-7 (**E**), angiotensin 1-5 (**F**), angiotensin 1-5/angiotensin 1-7 ratio (**G**), marker of renin activity (RA-S; Ang I + Ang II) (**H**), and marker of angiotensin-converting enzyme activity (ACE-S; Ang II/Ang I) (**I**) in SHRs after six weeks of treatment. Ang, angiotensin; ARNI, sacubitril/valsartan; C, Wistar controls; IVA, ivabradine; SHRs, spontaneously hypertensive rats. Results are presented as means ± SEM. *n* = 6 per group. One-way, two-tailed ANOVA followed by multiple comparisons with a Bonferroni post-hoc test; \* *p* < 0.05 vs. C; # *p* < 0.05 vs. SHR.

The serum concentration of aldosterone was 312.73 ± 66.99 fmol/mL and 163.63 ± 29.02 fmol/mL in the control and SHR groups, respectively (ns); ARNI increased it by 10% (ns) and 203% (*p* < 0.05) in the control and SHR groups, respectively (Figure 5A). The aldosterone/Ang II ratio (AA2 ratio) was 0.55 ± 0.14 and 0.52 ± 0.08 in the control and SHR groups, respectively (ns); ARNI decreased it by 61% (ns) and 52% (ns) in the control and SHR groups, respectively (Figure 5B). Ivabradine had no effect on the serum concentration of aldosterone and the AA2 ratio in SHRs after six weeks of treatment (Figure 5A,B).

**Figure 5.** Effect of ARNI and ivabradine on the serum level of aldosterone (**A**) and the aldosterone/angiotensin II ratio (AA2 ratio) (**B**) in SHRs after six weeks of treatment. ARNI, sacubitril/valsartan; C, Wistar controls; IVA, ivabradine; SHRs, spontaneously hypertensive rats. Results are presented as means ± SEM. *n* = 6 per group. One-way, two-tailed ANOVA followed by multiple comparisons with a Bonferroni post-hoc test; # *p* < 0.05 vs. SHR.

#### *3.4. Echocardiography*

The LVEF was 72.52 ± 1.55% in the control group, and ARNI had no effect after six weeks of treatment. In the SHR group, the LVEF was lower than controls by 13% (63.0 ± 2.12%, *p* < 0.05 vs. C), and ARNI and ivabradine increased it (*p* < 0.05) by 12% and 10%, respectively (Figure 6A).

The LVFS was 37.38 ± 1.29% in the control group, and ARNI had no effect after six weeks of treatment. In the SHR group, the LVFS was lower than controls by 20% (30.0 ± 1.39%, *p* < 0.05 vs. C), and ARNI and ivabradine increased it (*p* < 0.05) by 17% and 14%, respectively (Figure 6B).

The E/A ratio was 1.42 ± 0.1 in the control group, and ARNI had no effect after six weeks of treatment. In the SHR group, the E/A ratio was higher than controls by 53% (2.17 ± 0.12%, *p* < 0.05 vs. C), and ivabradine decreased it (*p* < 0.05) by 24%. ARNI had no significant effect on the E/A ratio in SHRs after six weeks of treatment (Figure 6C).

The E/Em ratio was 10.58 ± 0.76 in the control group, and ARNI had no effect after six weeks of treatment. In the SHR group, the E/Em ratio was higher than controls by 132% (24.58 ± 2.01, *p* < 0.05 vs. C), and ARNI and ivabradine decreased it (*p* < 0.05) by 28% and 42%, respectively (Figure 6D).

**Figure 6.** Effect of ARNI and ivabradine on left ventricular ejection fraction (LVEF) (**A**), left ventricular fractional shortening (LVFS) (**B**), the ratio of the diastolic transmitral peak early and late filling velocities (E/A ratio) (**C**), and the ratio of the diastolic transmitral peak early filling velocity and the maximal velocity of early diastolic wall movement wave at the level of mitral annulus (E/Em ratio) (**D**) in SHRs after six weeks of treatment. ARNI, sacubitril/valsartan; C, Wistar controls; IVA, ivabradine; SHRs, spontaneously hypertensive rats. Results are presented as means ± SEM. *n* = 7 per group. One-way, two-tailed ANOVA followed by multiple comparisons with a Bonferroni post-hoc test; \* *p* < 0.05 vs. C; # *p* < 0.05 vs. SHR.
