**1. Introduction**

Left ventricular (LV) hypertrophy in hypertension is considered to be a compensatory reaction to a chronically increased haemodynamic burden. LV mass enlargement supports the heart's performance without increasing wall tension. However, a hypertensive heart is associated with fibrotic rebuilding of the LV, resulting in a deterioration of cardiac function and a worsening prognosis. It is generally believed that curbing pathological cardiac

**Citation:** Simko, F.; Baka, T.; Stanko, P.; Repova, K.; Krajcirovicova, K.; Aziriova, S.; Domenig, O.; Zorad, S.; Adamcova, M.; Paulis, L. Sacubitril/Valsartan and Ivabradine Attenuate Left Ventricular Remodelling and Dysfunction in Spontaneously Hypertensive Rats: Different Interactions with the Renin–Angiotensin–Aldosterone System. *Biomedicines* **2022**, *10*, 1844. https://doi.org/10.3390/ biomedicines10081844

Academic Editor: Elena Kaschina

Received: 13 June 2022 Accepted: 27 July 2022 Published: 31 July 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

remodelling reduces the transition from a hypertensive heart to heart failure (HF). Thus, the search for novel therapeutic strategies against the consequences of haemodynamic overload-induced cardiac remodelling in various models of experimental hypertension and in clinical conditions is unremitting [1]. Hypertensive heart disease involves the structural remodelling of the musculature and collagenous and non-collagenous matrix. Myocardial hypertrophy is determined by pressure or volume overload, which induces the compensatory growth of cardiomyocytes. The structural homogeneity may be disturbed by the imbalance of two groups of substances: by increased levels of angiotensin II, aldosterone, endothelin, and catecholamines which represent stimulators of pathologic growth with fibrocyte proliferation and an overabundance of collagen; or by a reduced production of nitric oxide (NO), natriuretic peptides, bradykinin, and prostaglandins with the opposite effect on growth and proliferation. The absolute or relative overproduction of angiotensin (Ang) II and aldosterone governs the development of pathologic fibrosis associated with deteriorated heart function and rhythm disturbances [2,3]. Thus, blocking the renin–angiotensin–aldosterone system (RAAS) by angiotensin-converting enzyme (ACE) inhibitors, angiotensin II type 1 receptor (AT1R) blockers, or aldosterone receptor blockers enables the attenuation of the vasoconstrictor, pro-inflammatory, and pro-proliferative actions.

During the past decade, two novel approaches to HF management with different mechanisms of action have been introduced. Neprilysin is an enzyme expressed in the cell membrane of various tissues that splits atrial and brain natriuretic peptides (ANP and BNP, respectively). The inhibition of neprilysin by sacubitril enhances circulating ANP and BNP levels with vasodilative, diuretic, and antiproliferative actions. Since neprilysin's substrates include both natriuretic peptides (NP) and Ang II, its inhibition increases not only the level of beneficial NP but also the concentration of adverse Ang II, potentially counterbalancing the desirable vasodilative effects of NP. To avoid this, sacubitril, an inhibitor of neprilysin, was combined with the AT1R blocker valsartan to attenuate Ang II effects [4,5]. The PARADIGM-HF study involving heart failure patients with systolic dysfunction showed that the combination of neprilysin inhibition by sacubitril and the AT1R blocker valsartan, i.e., sacubitril/valsartan (ARNI), reduced morbidity and mortality more effectively than the ACE inhibitor enalapril [6]. Thus, ARNI is becoming the cornerstone of HF therapy. Moreover, in the PARALLAX trial comprising HF patients with a preserved LV ejection fraction, sacubitril/valsartan resulted in a significantly greater decrease in plasma N-terminal pro-brain natriuretic peptide levels compared with a standard treatment affecting the renin–angiotensin system [7]. Thus, the combination of neprilysin with a renin–angiotensin system blockade may be of potential benefit in hearts with not only systolic but also diastolic LV dysfunction.

Ivabradine is a selective inhibitor of the If current in the sinoatrial node, which is responsible for pacemaking. Ivabradine reduces the heart rate (HR) without the negative inotropic effect inherent to beta-blockers. In the SHIFT study, ivabradine decreased the composite end-point of mortality and hospitalisations for HF, and it is recommended for patients with systolic HF and a HR above 70 bpm despite treatment with or in case of intolerance of beta-blockers [8].

It is generally accepted that cardiovascular protection is achieved by interfering with the excessive neurohumoral activation seen in chronic HF. Indeed, modulation of the RAAS, whose chronic activation induces a pathologic remodelling of the target organs, is pivotal in HF management. Moreover, neprilysin activity is linked to RAAS modulation: while neprilysin participates in Ang I degradation, ANP and BNP inhibit the release of renin [9].

However, data regarding the complex interference of ARNI or ivabradine with the RAAS are sparse. Thus, the aim of this study was to show in a rat experimental model of spontaneous hypertension (spontaneously hypertensive rats, SHRs) whether ARNI or ivabradine are able to protect a hypertensive heart and whether this potential protection is due to their interaction with the deleterious classical ACE/Ang II/AT1R pathway and the protective alternative ACE2/Ang 1-7/Mas receptor (MasR) pathway of the renin– angiotensin system.

#### **2. Materials and Methods**

#### *2.1. Animals and Treatment*

Twelve-week-old male Wistar rats and age- and weight-matched male SHRs (Department of Toxicology and Laboratory Animals Breeding, Slovak Academy of Sciences, Dobra Voda, Slovak Republic) were randomly divided into five groups (15 per group) and treated for six weeks as follows: Wistar rats with no treatment (C); Wistar rats treated with ARNI (68 mg/kg/day; Novartis, Basel, Switzerland) (ARNI); SHRs with no treatment (SHR); SHRs treated with ARNI (68 mg/kg/day) (SHR + ARNI); and SHRs treated with ivabradine (10 mg/kg/day; Servier, Suresnes, France) (SHR + IVA). The therapeutics were dissolved in drinking water and their concentration was adjusted to daily water consumption. The natural water consumption was 12–13 mL per 100 g body weight. To ensure that all of the water-therapeutics solutions were drunk by a particular rat, only 10 mL per 100 g body weight of solution was offered. The solutions were prepared by dissolving the appropriate amount of therapeutics in water, while no additional substance was added. The rats were housed in individual cages, fed a regular pellet diet ad libitum and maintained under standard laboratory conditions (12:12-h light–dark cycle, 22 ± 2 ◦C temperature, and 55 ± 10% humidity). The study was conducted in conformity with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996). The protocol was approved by the Ethics Committee of the Institute of Pathophysiology, Faculty of Medicine, Comenius University, Bratislava, Slovak Republic (approval number: 809/19-221/3; approval date: 23 April 2019).

Systolic blood pressure (SBP) and HR were measured twice before treatment and once a week during treatment by non-invasive tail-cuff plethysmography (Hugo-Sachs Elektronik, Freiburg, Germany). After six weeks of treatment, the rats were euthanised by isoflurane inhalation. Body weight (BW), heart weight, and left ventricular weight (LVW) were measured, and the LVW/BW ratio was subsequently calculated. LV samples were frozen at −80 ◦C and, later, hydroxyproline concentrations were measured. Blood samples were collected from the abdominal aorta during euthanasia. Serum obtained by centrifuging the blood samples at 2000× *g* for 15 min was stored at −80 ◦C for subsequent angiotensin and aldosterone analysis.

#### *2.2. Determination of Hydroxyproline in the Left Ventricle*

Collagenous proteins in the LV were isolated by treating LV samples stepwise with different buffers, as described previously [10]. Briefly, CH3COOH-pepsin buffer (pH 1.4, 24 h at 4 ◦C) was used to extract soluble collagenous proteins, and 1.1 mol/L NaOH (45 min at 105 ◦C) was used to extract the remaining insoluble collagenous proteins. The hydrolysed samples were oxidised by chloramine T added to an acetate–citrate buffer at pH 6.0. After incubation for 20 min at room temperature, the reaction was stopped by adding 20 volumes of Ehrlich's reagent to the mixture. The samples were then incubated at 65 ◦C for 15 min, and the hydroxyproline concentration (a marker of fibrosis) in the LV was measured in both collagenous fractions using spectrophotometry at 550 nm. The hydroxyproline content in the LV was subsequently calculated and expressed as mg per total weight of the LV.

## *2.3. Determination of Serum Angiotensins and Aldosterone Concentration and the Markers of Renin and ACE Activities*

Serum samples from six animals per group that were not subject to prior echocardiography were used for angiotensin and aldosterone analyses. Equilibrium Ang peptide and aldosterone levels were determined by mass spectrometry, as described previously [11]. Briefly, the equilibrium peptide levels were stabilised by equilibration of the conditioned serum at 37 ◦C for 60 min. Thereafter, the stabilised samples were spiked with internal standards for each angiotensin metabolite (isotopes labelled Ang I, Ang II, Ang 1-7, Ang 1-5, Ang 2-8, and Ang 3-8) at concentrations of 200 pg/mL, and for aldosterone (deuterated aldosterone) at a concentration of 500 pg/mL. After a C18-based solid-phase extraction, the samples were analysed by LC–MS/MS using a reversed-phase analytical column (Acquity UPLC® C18, Waters Corp., Milford, MA, USA) operating in line with a XEVO TQ-S triple quadrupole mass spectrometer (Waters Corp.) in MRM mode. The peptide recovery of the sample preparation (for each Ang metabolite in each sample) was corrected using internal standards. The corresponding response factors determined with appropriate calibration curves in the original sample matrix, which integrated signals exceeding a signal-to-noise ratio of 10, were used to assess Ang peptide concentrations. The Ang 1-5/Ang 1-7 ratio, a marker of Ang 1-7 cleavage to Ang 1-5, was subsequently calculated.

The marker of renin activity (RA-S) was subsequently calculated as the sum of Ang I and Ang II. Indeed, in previous studies, the sum of Ang I and Ang II obtained from the above equilibrium analysis was shown to be closely correlated with the measured renin activity, independent of species or treatment [12].

The marker of ACE activity (ACE-S) was subsequently calculated as the Ang II/Ang I ratio. It provides information about the expected ACE activity [13].

The aldosterone/Ang II ratio (AA2 ratio) was calculated to assess adrenal responsiveness following Ang II signalling resulting in the release of aldosterone [14].

#### *2.4. Echocardiography*

After six weeks of treatment, transthoracic echocardiography was performed on seven animals per group using a 14-MHz matrix probe (M12L) coupled with a GE Medical Vivid 7 Dimension System (GE Medical Systems CZ Ltd., Prague, Czech Republic), as described previously [15]. Briefly, the animals were anesthetised throughout the protocol by applying isoflurane (2.5% inspiratory concentration at a flow rate of 2 L/min) during spontaneous breathing. After placing the rat in the supine position on a warming pad (38 ◦C), the thoracic wall was shaved. The HR and body temperature were monitored throughout the protocol. To assess the LV systolic function, the LV end-systolic and end-diastolic internal diameters were measured from the anatomical M-mode images in a long-axis view using the leading-edge method. Subsequently, the left ventricular fractional shortening (LVFS) and ejection fraction (LVEF, using the Teichholz formula) were determined. To assess the LV diastolic function, the diastolic transmitral peak early (E) and late (A) filling velocities were measured from the two-dimensionally guided Doppler spectra of mitral inflow in the apical four-chamber view, and the E/A ratio was then calculated. The maximal velocities of the early (Em) and late (Am) diastolic wall movement waves at the level of the septal mitral annulus were determined by tissue Doppler imaging from the apical four-chamber view; the E/Em ratio was subsequently calculated. Echocardiography was performed by an experienced echocardiographer blinded to the group identity. All measurements were averaged over three consecutive cardiac cycles.

## *2.5. Statistical Analysis*

The results are presented as means ± SEM. Data distribution was assessed by a Shapiro–Wilk normality test. A two-way, repeated-measures analysis of variance (ANOVA) followed by multiple comparisons with a Bonferroni post-hoc test was used for the statistical analysis of SBP and HR data. A one-way, two-tailed ANOVA followed by multiple comparisons with a Bonferroni post-hoc test was used for the statistical analysis of the remaining data, including the heart weights, LV hydroxyproline concentrations and contents, serum Ang and aldosterone levels, and echocardiography. The differences were considered significant if *p* < 0.05. The statistical analysis was conducted using GraphPad Prism 9 for Windows (GraphPad Software, La Jolla, CA, USA).

#### **3. Results**

*3.1. Haemodynamics and Heart Weights*

The SBP was 131.71 ± 3.71 mmHg in the control group, and ARNI decreased (*p* < 0.05) it by 13% after six weeks of treatment. In the SHR group, SBP was higher than in controls by 39% (182.89 ± 4.22 mmHg, *p* < 0.05 vs. C), and ARNI decreased (*p* < 0.05) it by 23%. Ivabradine did not affect SBP in SHRs (Figure 1A).

**Figure 1.** Effect of ARNI and ivabradine on systolic blood pressure (SBP) (**A**) and heart rate (HR) (**B**) throughout the experiment, and the relative weight of the left ventricle (left ventricular weight/body weight; LVW/BW) (**C**) 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* = 15 per group. Repeated measures ANOVA (**A**,**B**) or one-way, two-tailed ANOVA (**C**) followed by multiple comparisons with a Bonferroni post-hoc test; \* *p* < 0.05 vs. C; # *p* < 0.05 vs. SHR.

The HR was 375.93 ± 11.61 bpm in the control group, and ARNI did not affect it after six weeks of treatment. In the SHR group, the HR was higher than in controls by 26% (474.95 ± 10.53 bpm, *p* < 0.05 vs. C), and ARNI and ivabradine decreased it (*p* < 0.05) by 15% and 17%, respectively (Figure 1B).

The LVW/BW ratio was 1.04 ± 0.02 mg/g in the control group, and ARNI did not affect it after six weeks of treatment. In the SHR group, the LVW/BW ratio was higher than in controls by 75% (1.82 ± 0.04 mg/g, *p* < 0.05 vs. C), and ARNI decreased it (*p* < 0.05) by 13%. Ivabradine did not affect the LVW/BW ratio in SHRs (Figure 1C).
