**4. Discussion**

The effects of the neprilysin inhibitor/AT1R blocker sacubitril/valsartan (ARNI) and ivabradine on SBP, HR, myocardial remodelling, LV systolic and diastolic function, and the RAAS were investigated in SHRs.

The SHR, a commonly employed rat experimental model of spontaneous hypertension, mimics primary hypertension with target organ damage in humans. The mechanisms underlying the development of primary hypertension are complex and comprise several potential players. Endothelial dysfunction in conduit and resistance arteries is frequently considered to contribute to the BP increase in the SHR. However, disturbed endothelial function has been described mainly in aged but not young SHRs, suggesting that endothelial dysfunction is more a consequence than a cause of elevated BP [16]. Data regarding the participation of the RAAS in hypertension pathophysiology in the SHR are

driven mainly by the impact of targeted inhibition of the presumably deleterious classical ACE/Ang II/AT1R pathway [17] or by activation of the supposedly beneficial alternative ACE2/Ang 1-7/MasR pathway [18,19]. Data characterising the RAAS in the untreated SHR varies considerably in different laboratories. The serum level of renin, Ang II [20], and aldosterone [20,21], AT1R expression in mesenteric and coronary arteries [19], and heart expression of Mas-related G protein-coupled receptor D (MrgD) were higher in SHRs compared to Wistar rats, while the MasR expression in arteries was lower in SHRs [19]. Moreover, SHRs showed an altered circadian gene expression affecting the transcriptional regulation of clock-controlled genes for aldosterone and corticosterone [22]. In our experiment with three-month-old male SHRs, the levels of Ang I (Ang 1-10), Ang II (Ang 1-8), Ang III (Ang 2-8), Ang IV (Ang 3-8), Ang 1-7, and Ang 1-5 did not show significant changes compared to Wistar controls, corresponding with the unchanged marker of renin activity (RA-S). The trend towards reduced Ang II levels, albeit non-significant, corresponded with the decreased marker of ACE activity (ACE-S), calculated as the Ang II/Ang I ratio, and with the trend toward serum aldosterone concentration reduction. These data suggest that SHR is a normal-to-low renin and normal-to-low angiotensin/aldosterone model of hypertension. Thus, the underlying mechanism of hypertension and target organ damage in the SHR remains elusive. Importantly, increased renal sympathetic activity has been previously reported in the SHR [23]. Indeed, in our experiment, HR was significantly elevated in SHRs by approximately 100 bpm during the entire course of the six-week experiment, indicating activation of the sympathetic nervous system. In previous experiments, renal sympathetic denervation reduced intrarenal norepinephrine, the renal tissue protein of Ang II, aldosterone, and AT1R [24], and ameliorated renal fibrosis and dysfunction along with the delayed onset of hypertension in the SHR [25]. These results correspond with the findings of the higher activity of tyrosine hydroxylase, the rate-limiting enzyme in the synthesis of catecholamines, observed in the heart and kidney of SHRs compared with Wistar rats [26]. The above data suggest that sympathetic system activation plays a crucial role in BP elevation in the SHR. Along with sympathetic activation, the local renin–angiotensin system may also be activated in the kidney [25], brain [21], or other organs. In addition, other neurohumoral alterations, such as an elevated endothelin 1 level [20] or reduced endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) bioavailability [16,25], may also contribute to BP elevation in the SHR. Furthermore, hypertensive encephalopathy due to a higher mineralocorticoid receptor expression and their activation by endogenous corticosterone may participate in hippocampal neuroinflammation, which may potentially contribute to BP dysregulation and hypertension [27].

In our study, the dual inhibitor of the endopeptidase neprilysin and the AT1R, sacubitril/valsartan (ARNI), significantly reduced systolic BP as well as LV mass in SHRs after six weeks of treatment. Moreover, ARNI significantly reduced the LV concentration of insoluble collagen, and numerically also the total collagen, and significantly decreased their LV contents. ARNI completely prevented the deterioration of LV systolic function and attenuated the deterioration of diastolic function in SHRs. This anti-remodelling nature of ARNI is in agreement with data from other laboratories and large clinical studies. Sacubitril/valsartan prevented myocardial fibrosis and remodelling and improved cardiac function after myocardial infarction in mice [28] and rats [29,30], and in streptozotocin-induced diabetic hearts in mice [31]; it reduced cardiomyocyte size in Ang II-induced cardiac hypertrophy in mice [32], attenuated LV fibrosis and dysfunction in high-salt diet-induced diastolic dysfunction in rats [33], and reduced BP and prevented stroke in stroke-prone hypertensive rats [34]. A meta-analysis of clinical studies from 2010 to 2019 revealed that ARNI exerted reverse remodelling in terms of reduced LV size and hypertrophy compared with ACE inhibitors or AT1R blockers in patients with HF with a reduced LV ejection fraction [35].

While the classical ACE/Ang II/AT1R pathway is considered to be deleterious when chronically activated by stimulating vasoconstriction, proliferation, and inflammation, the alternative ACE2/Ang 1-7/Ang 1-5/MasR seems to be a counterbalancing pathway by reducing oxidative stress and inflammation, inducing vasodilatation, and inhibiting

myocyte growth and fibrotic proliferation [36–39]. In our experiment, ARNI, via AT1R blockade by valsartan, enhanced the levels of Ang II and Ang I and increased Ang 1-7 and Ang 1-5, along with Ang III and Ang IV. The marker of ACE activity (ACE-S), calculated as the Ang II/Ang I ratio, provides information about the expected ACE activity. ACE-S was lower in SHR and remained unaffected by ARNI. On the other hand, Ang 1-7 was remarkably increased by ARNI in SHRs and also stabilised, as shown by a trend to a reduced Ang 1-5/Ang 1-7 ratio. Ang 1-7 is considered to be a decisive player in damaged heart protection [40]. The increase in Ang 1-7 levels has to be considered with regard to the simultaneous AT1R blockade by valsartan, rendering Ang II ineffective and Ang 1-7 as the dominant effector in the RAAS. The results on serum aldosterone levels are puzzling. Although the AT1R blocker moiety of ARNI effectively blocked AT1R signalling in the adrenal glands, as indicated by a decreased aldosterone/Ang II ratio (AA2 ratio), the actual aldosterone level was increased by ARNI in SHRs, suggesting an important role of stimuli (such as potassium levels, NO availability, ACTH release or sympathetic activity) [41,42] for aldosterone production different from Ang II. Thus, in SHRs, the presumably protective effect of increased Ang 1-7 may be partly counterbalanced by elevated aldosterone levels. The increased serum levels of aldosterone with ARNI treatment might have determined the only mild-to-moderate antifibrotic effect of ARNI in this study. One could hypothesise that the potential combination of ARNI with a mineralocorticoid receptor antagonist would be a beneficial strategy in the treatment of hypertensive hearts. Additionally, the inhibition of neprilysin by sacubitril, with the subsequent enhancement of ANP and BNP exerting vasodilative and antiproliferative effects [43], might have also contributed to the protection by ARNI in SHRs.

Elevated HR is a risk factor in healthy individuals and various cardiovascular pathologies [44]. Ivabradine selectively inhibits the If current of the pacemaker cells in the sinoatrial node, thus reducing HR without negative inotropy [45]. Although HR reduction coming with an improvement in myocardial energy balance seems to be the principal factor underlying the protection offered by ivabradine in HF patients, a number of pleiotropic effects, including antioxidative, anti-inflammatory, and neurohumoral action modulation, may contribute to heart protection [45]. Indeed, in aortic constriction-induced LV hypertrophy in mice, ivabradine attenuated LV hypertrophy, fibrosis, and dysfunction [46]. In isoproterenol-induced heart damage, ivabradine reduced LV fibrotic remodelling and improved survival [47]. In our previous experiment with L-NAME-induced hypertension, ivabradine improved the systolic and diastolic function of the remodelled LV [48]. In line with the above data, in this experiment, ivabradine reduced fibrosis of the LV and improved systolic and diastolic function in SHRs. However, ivabradine did not affect the serum concentrations of Ang II and Ang 1-7 or other downstream products of the classical and alternative pathways of the renin–angiotensin system. Interestingly, even the serum levels of aldosterone, which were reduced by ivabradine in L-NAME-induced hypertension [48] and supposedly contributed to the hypertensive heart protection by ivabradine, were unchanged in SHRs. There are two potential factors underlying the apparent protection by ivabradine in SHRs to consider. First, HR reduction associated with an improvement of myocardial energy balance in a haemodynamically overloaded hypertensive heart could result in improved LV contractility and relaxation. Second, ivabradine may provide a benefit via its presumable pleiotropic sympatholytic effect. Indeed, the pre-treatment of rats exposed to handling stress with ivabradine was associated with a reduced release of adrenaline and noradrenaline into the blood stream [49], and changes to heart rate variability (HRV) in a rat model of doxorubicin-induced HF indicated an improved autonomic imbalance by ivabradine [50]. The HRV analysis in a SHIFT Holter sub-study showed an ivabradine-mediated shift toward a more prominent parasympathetic tone [51].

Limitations: For the pathophysiological implications, the local tissue concentration of RAAS peptides remains important. It seems, however, that the local RAAS remains largely dependent on the circulating RAAS peptides, as previously discussed for the brain RAAS [52]. In fact, the concentration of renin and angiotensinogen in the heart tissue is very low compared to plasma [53]. Additionally, we have recently shown [48] that apart from Ang 1-10 and Ang 1-8, the tissue concentrations of other RAAS components remain low and the LV concentration of Ang II correlates with the circulating Ang II levels.

#### **5. Conclusions**

We conclude that the SHR is a normal-to-low RAAS model of experimental hypertension. Recent drugs from the portfolio of HF management—ARNI and ivabradine—exerted the attenuation of LV remodelling and dysfunction in the SHR. Considering the changes to the RAAS, the cardiovascular protection by ARNI may be related to the Ang II blockade and the protective nature of Ang 1-7, while the cardiovascular protection by ivabradine was not associated with the modification of RAAS in the SHR.

**Author Contributions:** Conceptualization, F.S.; methodology, F.S., T.B., P.S., O.D. and M.A.; validation and formal analysis, F.S., T.B., S.Z., M.A. and L.P.; investigation, resources, data curation, T.B., P.S., K.R., K.K., S.A., O.D. and M.A.; writing—original draft preparation, F.S.; writing—review and editing, all authors; visualization, T.B., P.S., K.R. and O.D.; supervision, F.S., K.K., M.A. and L.P.; project administration, F.S. and K.K.; funding acquisition, F.S. and S.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by research grants VEGA 1/0035/19 and APVV-20-0421.

**Institutional Review Board Statement:** The animal study 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).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data supporting the reported results are available from the corresponding author per request.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

#### **References**

