Sacubitril/valsartan reverses cardiac structure and function in experimental model of hypertension-induced hypertrophic cardiomyopathy

This study evaluated the effect of sacubtril/valsartan on cardiac remodeling, molecular and cellular adaptations in experimental (rat) model of hypertension-induced hypertrophic cardiomyopathy. Thirty Wistar Kyoto rats, 10 healthy (control) and 20 rats with confirmed hypertension-induced hypertrophic cardiomyopathy (HpCM), were used for this study. The HpCM group was further subdivided into untreated and sacubitril/valsartan-treated groups. Myocardial structure and function were assessed using echocardiography, Langendorff’s isolated heart experiment, blood sampling and qualitative polymerase chain reaction. Echocardiographic examinations revealed protective effects of sacubitril/valsartan by improving left ventricular internal diameter in systole and diastole and fractional shortening. Additionally, sacubitril/valsartan treatment decreased systolic and diastolic blood pressures in comparison with untreated hypertensive rats. Moreover, sacubitril/valsartan treatment reduced oxidative stress and apoptosis (reduced expression of Bax and Cas9 genes) compared to untreated rats. There was a regular histomorphology of cardiomyocytes, interstitium, and blood vessels in treated rats compared to untreated HpCM rats which expressed hypertrophic cardiomyocytes, with polymorphic nuclei, prominent nucleoli and moderately dilated interstitium. In experimental model of hypertension-induced hypertrophic cardiomyopathy, sacubitril/valsartan treatment led to improved cardiac structure, haemodynamic performance, and reduced oxidative stress and apoptosis. Sacubitril/valsartan thus presents as a potential therapeutic strategy resulted in hypertension-induced hypertrophic cardiomyopathy.


Introduction
Hypertension represents one of the leading causes of cardiovascular morbidity and mortality worldwide, with its increasing prevalence warranting public health concern [1,2].Untreated chronic systemic hypertension leads to cardiac structural and functional abnormalities which may result in hypertensive hypertrophic cardiomyopathy (HpCM), manifested through left ventricular (LV) hypertrophy, diastolic or systolic dysfunction [3].Initially, an expansion in the LV wall thickness is a compensatory reaction to increased afterload to maintain cardiac output and reduce wall stress [4].However, persistently increased pressure in aorta and increased afterload result in transition from physiological to pathophysiological hypertrophy and potentially to heart failure [5].Crucial approach in the prevention of cardiac dysfunction is timely management of hypertension [3].
There is strong evidence linking irregularities in the renin-angiotensin-aldosterone system (RAAS) to cardiac hypertensive complications such as the development of HpCM [6].The RAAS is associated with the myocardial hypertrophic response, and angiotensin II levels affect myocardial fibrosis [3,7].Therefore, RAAS-suppressing agents, such as angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs), constitute a cornerstone in the management of hypertension and hypertension-related heart damage.Significant effort has been invested in finding efficient therapeutic agents with the potential to simultaneously control blood pressure and forestall heart intricacies.A potential helpful procedure involves the inhibition of neprilysin, a neutral endopeptidase, which corrupts natriuretic peptides, bradykinin, and angiotensin II [8].Neprilysin inhibition might exert significant protective effects in hypertensive conditions.Considering these facts, a dual-acting drug containing sacubitril and valsartan in a 1:1 molar ratio has been shown to be effective in effective in neprilysin inhibition due to its diuretic and natriuretic effect, as well as inhibition of RAAS, which significantly contribute to the improvement of cardiac function.
Previous data suggested protective effects of sacubitril/ valsartan in prevention of left ventricular remodeling by angiotensin II blockade and RAAS modulation in spontaneously hypertensive rat model [9].Additional preclinical studies in animal models of hypertension also revealed potential of this medication in attenuation of cardiac hypertrophy [10,11].Importantly, sacubitril/valsartan was shown to exert greater benefits in suppression of hypertrophy in hypertensive conditions in comparison with valsartan and enalapril monotherapy [12].The cardioprotective effects of this double mix were also confirmed in several clinical trials, with the main benefits observed in patients with cardiovascular breakdown with diminished discharge portion [8].Moreover, sacubitril/valsartan appeared to be superior in lowering blood pressure in hypertensive patients compared to ARB monotherapy [13].Nevertheless, the effect of sacubitril/valsartan and the underlying molecular and cellular mechanisms have not been investigated in hypertrophic cardiomyopathy.
Therefore, the aim of the present study was to evaluate the effects of sacubitril/valsartan on cardiac functional, structural, molecular and cellular adaptations in experimental model of hypertension-induced hypertrophic cardiomyopathy.

Methods
This study was conducted on 10 male normotensive Wistar Kyoto rats (control; CTRL group) and 20 male spontaneously hypertensive (SHR) Wistar Kyoto rats with hypertrophic cardiomyopathy.Hypertension (systolic blood pressure up to 200 mm Hg) developed in the SHR group during 12 to 14 weeks of age [4].Hypertrophic cardiomyopathy was confirmed by echocardiography, and SHR rats were haphazardly partitioned into 10 untreated rats (HpCM group) and 10 rats treated with a fixed combination of sacubitril/valsartan (HpCM + sac/val group).A proper mix of sacubitril/valsartan 103/97 mg in the HpCM + sac/val group was applied per os (by gavage) at a dose of 2.6 mg/kg twice a day for 4 weeks.
All rats were sourced from the Institute for Medical Research, University of Belgrade and housed under controlled environmental conditions throughout the experimental period: temperature (22 ± 2 °C), humidity (40-70%), illumination (12/12 h light/darkness cycle), and free access to food and water.
The preset study complies with the Declaration of Helsinki, and the protocol was approved by the ethical committee for experimental animals' well-being of the Faculty of Medical Sciences, University of Kragujevac, Serbia.The study was performed according to EU Directive (86/609/ EEC), principles of Good Laboratory Practice and ARRIVE guidelines.

In vivo examinations in HpCM rats-Echocardiographic examination and evaluation of blood pressure and heart rate
A special ultrasound, the Hewlett-Packard Sonos 5500 ultrasound (Andover, MA, USA), equipped with a 15.0 MHz phased-array transducer for small animals, was used for in vivo investigation of rat hearts [14].Prior to measurements, rats were anaesthetized with a combination of ketamine and xylazine (75:5 mg/kg), shaved from the neck area to the center chest and placed on the warming cushion.Ultrasound measurements were taken from the parasternal long axis view in two-layered mode.Images were obtained in the M-mode and used to measure interventricular septal wall thickness at end-diastole (IVSd), LV internal dimension at end-diastole (LVIDd), LV posterior wall thickness at end-diastole (LVPWd), interventricular septal wall thickness end-systole (IVSs), LV internal diameter end-systole (LVIDs) and LV posterior wall thickness at end-systole (LVPWs).The percentage of fractional shortening (FS%) was additionally determined from the M-mode.
Systolic and diastolic blood pressures (SBP and DBP), as well as heart rate (HR), were estimated utilizing a painless tail-cuff (Mouse and Rat Tail Cuff Blood Pressure Systems IITC Life Science Inc., Los Angeles, CA, USA).Eight to ten measurements were made over a period of five minutes and the mean values calculated [15].

Ex vivo examinations on isolated hearts of HpCM rats-Cardiodynamic parameters
Langendorff model of an isolated retrogradely perfused heart was used for the ex vivo examination of rat heart capability.
After short-term anesthesia caused by intraperitoneal application of ketamine (10 mg/kg) and xylazine (5 mg/kg) and premedication with heparin as an anticoagulant, animals were killed.The thoracic cavity was opened via midline thoracotomy, and the hearts were immediately removed and drenched in cold saline.A short time later, the aortas were cannulated and retrogradely perfused according to the Langendorff method, under gradually increasing coronary perfusion pressure (CPP) from 40 to 120 cm H 2 O.
After placing the sensor (transducer BS473-0184, Experimetria Ltd., Budapest, Hungary) in the left ventricle, the following parameters of myocardial function were measured: maximum and minimum rate of pressure development (dp/ dt max and dp/dt min respectively) in the left ventricle, systolic and diastolic left ventricular pressure (SLVP and DLVP respectively), and heart rate (HR).Coronary flow (CF) was estimated flowmetrically.Following the establishment of heart perfusion, the hearts were stabilized within 30 min with a basal coronary perfusion pressure of 70 cm H 2 O.To analyze the impact of sacubitril/valsartan chronic treatment on cardiac function, the CPP was gradually decreased to 60 cm H 2 O, and then increased to 80, 100 and 120 cm H 2 O and decreased to 40 cm of H 2 O tube (pressure changing protocol 1; PCP 1).To examine cardiac autoregulation, CPP was again slowly exposed to pressures from 40 to 120 cm H 2 O (pressure changing protocol 2, PCP 2).Response of the heart was assessed by expressing percentage differences between PCP1 and PCP2 protocol at every CPP, i.e. the lower percentage difference at certain CPP indicates preserved cardiac function [16].

Assessment of metabolic and lipid profile
After treatment with sacubitril/valsartan (the day before killing animals), an oral glucose tolerance test (OGTT) was performed on both groups of animals.Following an overnight fast (12-14 h), a baseline blood sample was taken by tail bleeding to determine the fasting blood glucose and insulin levels.Subsequently, glucose was administered orally at a portion of 2 g/kg body weight, and blood glucose levels were evaluated utilizing a glucometer (Accu-Chek, Roche Diagnostics, Indianapolis, USA) at 30, 60, 120 and 180 min thereafter.Insulin levels were also assessed at baseline and 180 min using enzyme-linked immunosorbent assay (ELISA) method, as previously described [17].Total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) levels were examined in serum, using spectrophotometry on a biochemical analyzer (Dimension Xpand, Siemens, IL, USA).

Relative gene expression-(anti)oxidative, (anti) inflammatory, (anti)apoptotic and cardiac-specific parameters
Quantitative polymerase chain reaction (qPCR) assays of relative gene expression were performed in the Bioengineering Laboratory, Institute of Information Technologies Kragujevac according to laboratory protocols following the Minimum Information for the Publication of Quantitative Real-Time PCR Experiments (MIQE) Guidelines [18] and the GLP standards for PCR detection.All methods and consumables used were applied according to manufacturer instructions, and qPCR reactions were performed in triplicates.The heart samples were firstly homogenized using the IKA® ULTRA-TURRAX® tube disperser workstation system (IKA Werke GmbH & Co. KG, Staufen im Breisgau, Deutschland).Samples immersed in 5 mL of pH-neutral pure PBS solution were placed in disperser tubes with glass balls and homogenized 120 s at 5000 rpm.Fresh samples (5 mg) were used for RNA extractions using a spin-column kit for purification of total RNA (Quick-RNA™ Miniprep Kit, Zymo research, R1054, Irvine, CA, US).The RNA concentration was estimated on an Eppendorf BioPhotometer® D30 (Hamburg, Germany) at 260 nm using an Eppendorf® UVette® cuvette (ref.nr.Z605050).The yield was optimal with an A 260 /A 280 ratio between 1.75 and 2.0.For each group of isolated RNA, reverse transcription was performed immediately by using FastGene Scriptase Basic cDNA Kit (Cat.No. LS62, NIP-PON Genetics EUROPE, Düren, Germany).cDNA was stored at -20 °C until all samples were prepared and ready for qPCR relative gene expression detection.Primers were DSL purified and C18 desalinated, no-end modified.Relative gene expression was determined by FastGene 2 × IC Green Universal with Fluorescein kit (Cat.No. LS41, NIPPON Genetics EUROPE, Düren, Germany) based on FastGene® IC Green as an intercalating dye MIC qPCR Cycler is controlled by intuitive software package micPCR v2.10.3 for obtaining Ct values and subsequent melting analyses.Since there is no single gene constitutively expressed in all cell types [19], we chose glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as reference genes.

Pathohistological analysis
Cardiac tissue samples were fixed in 4% paraformaldehyde buffer solution and submerged in paraffin.Segments of 4 mm thick were then placed on slides and stained with hematoxylin/eosin (HE) and Masson's trichrome [20].

Statistical analysis
The data were expressed as mean ± standard error (SE), and statistical analysis was performed using the IBM SPSS Statistics 20.0.Data distribution was checked by Shapiro-Wilk test, and in the normal distribution, statistical comparisons were performed using the one-way analysis of variance (ANOVA) tests with a Tukey's post hoc test for multiple comparisons.For data not following a normal distribution, the Kruskal-Wallis test was used.Values of P < 0.05 were statistically significant.All qPCR reactions were performed in triplicates.Relative gene expression was calculated using the delta-delta Ct formula according to Livak and Schmittgen [21].

Cardiac effects of sacubitril/valsartan
In vivo echocardiographic examination and assessment of blood pressure Parameters of in vivo cardiac function such as IVSd, IVSs, LVIDd, LVIDs, LVPWd, LVPWs and FS are presented in Fig. 1A.In HpCM rats, IVSd and IVSs were significantly increased, while LVIDd and LVIDs were decreased in comparison with the CTRL group.Sacubitril/valsartan treatment significantly increased LVIDs and LVIDd and decreased FS compared to untreated HpCM rats.It's important to note that most of observed parameters of cardiac function didn't significantly differ in HpCM + sac/val and CTRL rats, except IVSs that were significantly higher in treated rats (Fig. 1A).
Additional in vivo examinations involved monitoring systolic and diastolic blood pressures, as well as heart rate (Fig. 1B).Systolic and diastolic blood pressures were significantly increased, while heart rate was decreased  Nevertheless, in the treated rats, the values of dp/dt min at the highest CPP (120 cm H 2 O), as well as SLVP in all CPPs and DLVP in higher CPPs (80-120 cm H 2 O), were similar to the CTRL group and significantly higher than values observed in the HpCM group, thus suggesting recovery of heart function (Fig. 2A).Hearts of all rats were subjected to gradually increasing CPPs twice, marked as PCP1 and PCP2.Percentage differences in all cardiac parameters between PCP1 and PCP2 at all CPPs are presented in Fig. 2B.The lowest percentage changes between cardiac parameters in PCP1 and PCP2 were observed in the CTRL group.In fact, the greatest percentage changes in the values of almost all monitored cardiodynamic parameters in PCP1 and PCP2 were noticed in the HpCM group.On the other hand, chronic sacubitril/ valsartan administration was associated with lower percentage changes in dp/dt max (at CPP 60 cm H 2 O), dp/dt min (at CPPs 80 and 120 cm H 2 O), SLVP (at CPPs 80-120 cm H 2 O), DLVP (at CPPs 60 and 100 cm H 2 O), HR (at CPPs 60, 80 and 120 cm H 2 O), CF (at CPPs 60-120 cm H 2 O) compared to the HpCM group (Fig. 2B).

Effects of sacubitril/valsartan on metabolic and lipid profile
Glucose levels after 30, 60, 120 and 180 min during OGTT in all groups were presented in Fig. 3A.Glucose levels at all observed time points were significantly increased in HpCM rats compared to CTRL.Sacubitril/valsartan treatment did not affect glucose levels during OGTT compared to HpCM rats.Generally observed, blood glucose values in all groups were within physiological range (Fig. 3A).Additionally, serum levels of TC, HDL and LDL were presented in Fig. 3B.All parameters of lipid status were significantly reduced in both treated and untreated HpCM rats compared to CTRL rats.There was no difference between HpCM and HpCM + sac/val rats.Importantly, followed parameters of lipid status in all groups were within physiological range (Fig. 3B).

Effects of sacubitril/valsartan treatment on oxidative stress parameters
Pro-oxidant markers in blood samples are presented in Fig. 4A.In the HpCM group, NO 2 − level was significantly decreased, while the level of H 2 O 2 was significantly increased compared to CTRL.Additionally, level of TBARS didn't differ in HPCM and CTRL rats.Nevertheless, sacubitril/valsartan-treated rats showed reduced H 2 O 2 levels compared to HpCM rats.Comparison of the pro-oxidant markers in HpCM + sac/val and CTRL rats revealed decrease in the level of NO ; superoxide anion radical, SOD; superoxide dismutase, TBARS; thiobarbituric acid reactive substances.Statistical analyses were calculated using one-way ANOVA, P < 0.01 **CTRL vs. HpCM, P < 0.01 § §CTRL vs. HpCM + sac/val, P < 0.05 #HpCM vs. HpCM + sac/val, P < 0.01 ##HpCM vs. HpCM + sac/val group (Fig. 4A).Markers of antioxidant defense system in blood samples are presented in Fig. 4B.In the HpCM group, CAT activity and GSH level were significantly decreased compared to CTRL.Moreover, these parameters were significantly increased in HpCM + sac/val compared to HpCM.In treated rats, GSH level was lower compared to CTRL level (Fig. 4B).
Relative gene expression of antioxidant markers SOD1 and SOD2 is presented in Fig. 4C.In the HpCM group, the relative expression of SOD1 and SOD2 genes was increased compared to the CTRL group.However, treatment with sacubitril/valsartan reduced the relative gene expression of these genes compared to HpCM rats, while relative expression of SOD1 and SOD2 was higher compared to CTRL (Fig. 4C).

Effects of sacubitril/valsartan treatment on apoptosis and pathological changes in the myocardium
The relative gene expression of Bax, Bcl2 and Cas9 is presented in Fig. 5A.Increased expression of those markers of apoptosis was noticed in HpCM compared to the CTRL group.Moreover, sacubitril/valsartan treatment decreased the relative gene expression of Bax and Cas9 and increased expression of Bcl2 compared to HpCM rats.The relative gene expression of Bcl2 and Cas9 was increased in HpCM + sac/val compared CTRL groups (Fig. 5A).
Pathological changes in myocardium of all groups are presented in Fig. 5B.In the CTRL group, there is a regular distribution of cardiac myofibers.Cardiomyocytes are striated with moderate nuclear polymorphism.The nuclei have a vesicular appearance, with prominent nucleoli.Interstitium is without signs of dilatation, elements of fibrosis or lipomatosis.In the HpCM group, there is a presence of irregular arrangement of cardiac myofibers, with shortened, hypertrophic cardiomyocytes, which are showing a tendency to create "vortices".The nuclei are large, irregular, and bizarre.The interstitium is dilated with signs of fibrosis.On the other hand, in HpCM + sac/val, there is a regular arrangement of cardiomyocytes, regular interstitium, blood vessels of normal histomorphology, and no hypertensive changes of the vascular wall (Fig. 5B).

Discussion
Considering that hypertension is the most frequent risk factor for hypertrophic cardiomyopathy and heart failure, there is an urgent need for finding an optimal therapeutic modality that will control blood pressure and prevent cardiac complications [22].Since the approval of sacubitril/ valsartan in 2014 for the treatment of chronic heart failure, several researchers have highlighted its benefits in the reduction of cardiovascular morbidity and mortality [23].
The present study explored the therapeutic influence of this pharmacological agent on cardioprotection in hypertensioninduced cardiomyopathy.The primary focus was to evaluate sacubitril/valsartan effects on functional, structural and biochemical markers of this cardiac pathology.The principal findings indicate that sacubitril/valsartan combination has a great potential in triggering cardioprotection.To the best of our knowledge, this is the first study to utilize in vivo and ex vivo techniques to assess sacubitril/valsartan effects in a hypertension-induced hypertrophic cardiomyopathy rat model.
In vivo measurements indicate that sacubitril/valsartan treatment resulted in improvement in heart function, confirmed by increased LVIDs and LVIDd and decreased FS compared to untreated hypertensive rats.These findings were expected since previous studies revealed that sacubitril/valsartan as neprilysin and angiotensin receptor inhibitor led to improvement of cardiac reverse remodeling and left ventricular ejection fraction [24].The positive effects of sacubitril/valsartan on LV diameters, volumes and systolicdiastolic performance were reported previously [24,25].However, due to conflicting results, it is still unclear which echocardiographic parameter is the most likely to change after sacubitril/valsartan treatment.Sacubitril/valsartaninduced alterations in echocardiographic markers in our study are of great importance since they reflect the heart's ability to contract and relax effectively and represent the main prognostic markers of heart failure in hypertensive individuals [23].Moreover, sacubitril/valsartan treatment in the current study was associated with a reduction in both SBP and DBP compared to HpCM, thus confirming potential of this fixed drug combination to preserve blood pressure values in hypertensive conditions.Similar observations were reported in the previously conducted studies in different models of experimental hypertension [9,10].
The special focus of our research was based on ex vivo cardiac examinations, which reflect the capability of the heart to respond adequately to changes in CPPs.The highest percentage difference between PCP1 and PCP2 in most of cardiodynamic parameters in HpCM rats represents an indicator of disturbed cardiac function in conditions of hypertensive cardiomyopathy.Depression of inotropic and lusitropic response, verified by dp/dt max and dp/dt min alterations, can lead to systolic and diastolic dysfunction, thus confirming deleterious effects of long-lasting hypertension on the heart.On the other hand, markers of cardiac function remained almost unchanged (manifested by the slight difference between PCP1 and PCP2) in rats treated with sacubitril/valsartan after exposure to various perfusion pressures.Based on these observations, it is reasonable to conclude that sacubitril/valsartan treatment might preserve the inotropic and lusitropic heart response to the pressure stimulants.Previous data also suggest the capacity of sacubitril to restore impaired myocyte contractility through phosphatase and tensin homolog inhibition.Additionally, it has been reported that activation of the serine/threonine-protein kinases, AKT1 and AKT3, which suppress the endothelial nitric oxide synthase are responsible for benefits of this drug on cardiac contractility force.It is important to note that the protective action of valsartan on cardiac remodeling might be partially mediated by inhibiting guanine nucleotide-binding proteins, which is synergized by the addition of sacubitril [26].Ex vivo measurements are in correlation with in vivo markers, thus confirming cardioprotective potential of sacubitril/valsartan treatment in a rat model of hypertrophic cardiomyopathy.
Since previous studies suggested an association between hypertension and metabolic changes, i.e. impairment of glucose and lipid homeostasis in hypertensive conditions, we aimed to follow the influence of this drug on serum glucose and lipid profile [27,28].Literature data suggest a correlation between increased myocardial glucose uptake and oxidation and myocardial metabolic abnormalities in spontaneously hypertensive rat hearts with hypertrophy [29,30].Our findings showed that sacubitril/valsartan treatment did not affect serum glucose or lipid profile in comparison to hypertensive untreated rats (HpCM rats).Although values in these two groups differed from the CTRL group, animals in all groups showed normal lipid status and glucose levels during OGTT, since all values were maintained within the physiological range.We might assume that the influence of sacubitril/valsartan on serum TC and TG would be achieved by a longer duration of the treatment.
Given the fact that oxidative stress stands out as one of the fundamental factors contributing to hypertension and development of hypertensive complications, our goal was to assess the influence of sacubitril/valsartan treatment on systemic redox balance [31].Systemic redox homeostasis was assessed by monitoring levels of pro-oxidants as well as capacity of antioxidant defense system.Elevated H 2 O 2 and decreased NO 2 − levels were noticed in hypertensive rats (HpCM group) compared to control, thus confirming that hypertensive state is associated with disturbed redox balance.Systemic levels of index of lipid peroxidation measured as TBARS did not significantly differ between groups.Although previous data suggested association of lipid peroxidation with hypertension, the absence of systemic alterations in TBARS levels in our study might be explained by the duration of the protocol.We may assume that lipid peroxidation during this duration of the study protocol occurred locally in heart tissue, while systemic disturbances and increase in TBARS levels in blood would occur if untreated hypertension would last longer.Precise influence of this drug combination on the level of lipid peroxidation should be thoroughly examined in the future [32].Moreover, sacubitril/valsartan treatment led to a reduction in H 2 O 2 and increase in GSH value and CAT activity compared to the HpCM group, while other observed systemic pro-oxidant and antioxidant markers remained unchanged.
To provide insight into the cardiac redox status, we determined gene expression of SOD1 and SOD2 which are significant in mitigating the deleterious effects of pro-oxidants [33].SOD1 and SOD2 gene expression was reduced in treated rats compared to hypertensive control.Considering that elevated SOD expression is expected during cardiomyocytes response to oxidative stress exposure [34], our findings indicate that the drug combination prevented cardiac oxidative damage via reduced expression of SOD1 and SOD2.This is in line with previous studies which reported that sacubitril/valsartan therapy exert cardioprotection via reducing oxidative stress [35,36].
Furthermore, sacubitril/valsartan treatment reduced gene expression of the pro-apoptotic markers Bax and Cas9 and elevated expression of anti-apoptotic Bcl2 in comparison with untreated hypertensive rats.The anti-apoptotic potential of sacubitril/valsartan has also been previously demonstrated in other cardiac pathologies [37].
HE and Masson's trichrome tissue staining revealed that hypertension was linked to the presence of hypertrophic cardiomyocytes, with polymorphic nuclei and moderately dilated interstitium.Nevertheless, chronic use of sacubitril/valsartan alleviated hypertension-induced structural alterations of cardiomyocytes and the vascular wall.Our results are compatible with the previous studies that identified structural pathological changes in HpCM [38], which were markedly attenuated after receiving sacubitril/valsartan treatment.The proposed mechanisms responsible for the protective influence of this drug combination in cardiac fibrosis were previously assessed in heart failure patientderived cardiac fibroblasts.In fact, the beneficial effects of sacubitril/valsartan in amelioration of cardiac fibrosis and diastolic dysfunction are mediated by the prevention of pathological suppression of protein kinase G signaling and inhibition of Rho kinase activation [39].Several data support the fact that dual drug combination exerts synergistic effects in prevention and attenuation of cardiomyocyte cell death, hypertrophy and left ventricular remodeling [26].

Limitations
The presented study reports critical observations in the physiology of the heart muscle in response to sacubitril/valsartan administration.However, the present study is limited as it does not provide adequate mechanistic rational for the findings of the study.

Conclusion
Based on our findings, we could presume that molecularcellular perturbations in HpCM were greatly reversed in rats treated with sacubitril/valsartan. Cardioprotection was accomplished through progress in heart construction and capability, attenuation of oxidative stress and apoptosis, thus suggesting significant role of sacubitril/valsartan in HpCM.Such integrative results, encompassing morphological, functional, and biochemical analysis of sacubitril/valsartan therapy, may indicate rationale for further investigation and potentially translation of the use of sacubitril/valsartan in treatment of patients with hypertension-induced hypertrophic cardiomyopathy.

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max (% change in values between PCP1 and PCP2) min (% change in values between PCP1 and PCP2)Ex vivo examinations on isolated hearts of rats with HpCM-Cardiodynamic parametersValues of ex vivo examined cardiodynamic parameters in all groups during exposure to perfusion pressures 40-120 cmH 2 O are presented in Fig.2A.Cardiodynamic parameters, such as dp/dt max, dp/dt min, SLVP, DLVP as well as CF, were significantly reduced in untreated HpCM compared to healthy rats at CPPs from 40 to 120 cmH 2 O.Moreover, HR didn't significantly differ in HpCM compared to CTRL rats.