LCZ696 Ameliorates Oxidative Stress and Pressure Overload-Induced Pathological Cardiac Remodeling by Regulating the Sirt3/MnSOD Pathway

Aims We aimed to investigate whether LCZ696 protects against pathological cardiac hypertrophy by regulating the Sirt3/MnSOD pathway. Methods In vivo, we established a transverse aortic constriction animal model to establish pressure overload-induced heart failure. Subsequently, the mice were given LCZ696 by oral gavage for 4 weeks. After that, the mice underwent transthoracic echocardiography before they were sacrificed. In vitro, we introduced phenylephrine to prime neonatal rat cardiomyocytes and small-interfering RNA to knock down Sirt3 expression. Results Pathological hypertrophic stimuli caused cardiac hypertrophy and fibrosis and reduced the expression levels of Sirt3 and MnSOD. LCZ696 alleviated the accumulation of oxidative reactive oxygen species (ROS) and cardiomyocyte apoptosis. Furthermore, Sirt3 deficiency abolished the protective effect of LCZ696 on cardiomyocyte hypertrophy, indicating that LCZ696 induced the upregulation of MnSOD and phosphorylation of AMPK through a Sirt3-dependent pathway. Conclusions LCZ696 may mitigate myocardium oxidative stress and apoptosis in pressure overload-induced heart failure by regulating the Sirt3/MnSOD pathway.


Introduction
Cardiac hypertrophy is a pathological remodeling process of the heart characterized by hypertrophied cardiomyocytes, interstitial fibrosis, perivascular fibrosis, and decreased cardiac compliance. The process can eventually lead to malignant arrhythmias, heart failure, and even sudden cardiac death [1,2]. Heart failure is the final stage of all the cardiovascular diseases and is currently a heavy burden for national medical and health services. Although several treatment options, such as CRT (Cardiac Resynchronization Therapy), exist, heart failure-associated morbidity and mortality are still on the rise. Thus, there is a need to identify novel therapeutic targets against the condition [3].
LCZ696 is an angiotensin-receptor-neprilysin inhibitor (ARNI) consisting of the neprilysin inhibitor sacubitril (AHU377) and angiotensin-receptor blocker (ARB) valsartan [4]. Recent clinical trials have revealed that LCZ696 is superior to enalapril in reducing mortality and rehospitalization rate in patients with chronic heart failure [5,6]. Moreover, LCZ696 can reduce both sudden cardiac death and deaths from progressive heart failure compared to angiotensin-converting enzyme inhibitors (ACEI), such as enalapril [5]. Although LCZ696 is effective in the treatment of heart failure [7,8], the specific mechanism underlying its role in long-term pressure overload-induced cardiac hypertrophy and sequential heart failure remains unknown.
The intracellular generation and scavenging of reactive oxygen species (ROS) are usually in a homeostasis state under normal physiological conditions. However, under various pathogenic stimuli, such as irradiation, anticancer agents, and aromatic hydrocarbons, the accumulation of ROS far exceeds the clearance capacity of cells. Consequently, various macromolecules suffer from oxidative stress damage, including lipid peroxidation, oxidative damage to DNA, protein oxidation, and monosaccharide oxidation [9]. Reactive oxygen species can enhance the progression of cardiac hypertrophy and heart failure [10][11][12][13]. Cardiac hypertrophy causes elevated mitochondrial ROS levels, which in turn induces myocardium apoptosis and further aggravates hypertrophy, ending up in a vicious circle [14]. ROS can activate multiple signaling cascades during cardiac hypertrophy. As such, impeding ROS and breaking the vicious cycle may hold promise for improving cardiac hypertrophy [15].
Sirtuins are a family of nicotinamide adenine nucleotide-(NAD + -) dependent class III histone deacetylases (HDACs) consisting of seven homologues. Among the homologues, sirtuin 3 (Sirt3) is the most extensively studied because of the decisive role it plays in a variety of diseases. Sirt3 is highly expressed in tissues requiring high energy metabolisms, especially in the heart tissues. The role of Sirt3 in mitochondrial dysfunction and redox homeostasis has been extensively demonstrated [16,17]. Recent studies have shown that Sirt3 plays a crucial role in defending mitochondria from oxidative damage [18]. Also, it has been elaborated that Sirt3 deficiency exacerbates cardiac hypertrophy and heart failure in transverse aortic constriction mice, whereas Sirt3 overexpression protects against maladaptive ventricular remodeling [19,20]. Given this, we hypothesized that the potential mechanisms underlying the beneficial effects of LCZ696 on pathological cardiac remodeling could be mediated by the Sirt3-dependent pathway.
The results of the present study showed that LCZ696 upregulated Sirt3 expression levels both in hypertrophied cardiac myocytes with transverse aortic constriction and cardiomyocytes induced with phenylephrine (PE). Also, we observed that the cardioprotective effects of LCZ696 were partly mediated by the Sirt3-dependent pathway.

Animals and Treatments.
Eight-week-old male C57BL/6 mice and neonatal Sprague-Dawley (SD) rats were obtained from GemPharmatech Co. Ltd. (Nanjing, China). The animals had free access to food and drinking water. All the animal experiments were conducted as per the guidelines of the care and use of laboratory animals of the Shanghai Jiao Tong University School of Medicine.
Transverse aortic constriction surgery was performed as previously described [21]. In brief, mice were anaesthetized with a mixture of ketamine (100 mg/kg) and xylazine (5 mg/kg) through intraperitoneal injection. The lack of toe pinching reflex indicated successful anaesthesia. The chest cavity was opened to the second intercostal space, and the adipose and connective tissue around the aortic arch was gently separated. The constriction was applied between the left common carotid artery and the innominate artery, by tying a 6-0 nylon suture ligature against a 27-gauge needle. The thorax was closed using a 6-0 suture, and mice were left to rest on a warming pad until they were fully awake. The sham-operated mice underwent the same operation procedure devoid of aortic ligation. After the ligation, the mice were randomly divided into four groups: sham+vehicle group, sham+LCZ696 group, TAC+vehicle group, and TAC+LCZ696 group. To determine the role of LCZ696 in pathological cardiac remodeling, mice were administered with LCZ696 at a dose of 20 mg/kg/d through gavage. The dosage of LCZ696 was selected according to previous studies [22,23]. The vehicle group was given the same volume of corn oil. After 4 weeks, hearts were snap-frozen in liquid nitrogen, then kept at -80°C for subsequent analyses.

Hemodynamic and Echocardiographic Measurements.
The VEVO 2100 echocardiography system (Visual Sonics Inc., Toronto, Canada) was used to perform transthoracic echocardiography as per the methods described previously. Briefly, 4 weeks after TAC surgery, the mice were anaesthetized with 2.5% isoflurane. Subsequently, M-mode echocardiography was used to obtain left ventricular internal diameter at end-systole (LVIDs), left ventricular internal diameter at end-diastole (LVIDd), left ventricular enddiastolic volume (LVEDV), and end-systolic volume (LVESV). After taking the measurements, the following calculations were made: 2.6. Western Blot Analysis. Hearts and primary cardiomyocyte lysate homogenates were prepared as previously described [24]. Briefly, proteins of whole heart homogenates or cell lysates from different treatments were separated using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes (PVDF membranes, Millipore, Billerica, MA, USA). After blocking with 5% nonfat powder milk for 1 h, the membranes were incubated with primary antibodies overnight at 4°C. The primary and secondary antibodies used in our experiments were listed in the Supplementary materials Table S4. The next day, the membranes were washed with the TBST buffer, then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The bands were detected using an ImageQuant LAS 4000 Imager (General Electric Co.), and gray-scale value analysis was performed using the Gel-Pro analyzer.  Figure S1). Cells transfected with Sirt3 siRNA were then treated with or without PE (50 μM) for 24 h.
3 Oxidative Medicine and Cellular Longevity 2.13. Statistical Analysis. Data analysis was performed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, United States). All values are presented as mean ± standard error of the mean (SEM). All the data acquired from our experiments are in accordance with normal distribution. Differences between two groups were analyzed using unpaired Student's t-test. One-way ANOVA followed by Bonferroni post hoc test was used for multiple-group comparisons. p value < 0.05 was considered statistically significant.

LCZ696 Improves Cardiac Function and Alleviates
Cardiac Hypertrophy in Pressure Overload-Induced Cardiac Remodeling. To determine the effect of LCZ696 on pathological cardiac hypertrophy, wild type mice were administered with LCZ696 via oral gavage at a dose of 20 mg/kg/d for 4 weeks. After 4 weeks of aortic constriction, the mice were anaesthetized, and echocardiography was performed. Administration of LCZ696 increased ejection fraction and fractional shortening (Figures 1(a) and 1(b)), which were both dramatically decreased following TAC surgery. Similarly, TAC caused marked pathological cardiac hypertrophy as indicated by increased ratios of heart weight (HW) to body weight (BW), lung weight (LW) to BW, and HW to tibia length (TL). LCZ696 treatment reduced these ratios, suggesting improved cardiac hypertrophy contractile dysfunction. General view under the microscope and HE staining of the cardiac cross-section also confirmed that TAC-induced cardiac dilation was significantly inhibited by LCZ696 (Figure 1(d)). Furthermore, HE and WGA staining showed that LCZ696 substantially reduced the cross-sectional area of cardiomyocytes (Figures 2(a) and 2(b)). Also, the mRNA markers of cardiac hypertrophy, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and β-myosin heavy chain (β-MHC), also referred to as fetal genes, were overtly surged in TAC-primed hearts compared with the sham group. Expectedly, LCZ696 treatment significantly decreased the expression levels of these fetal genes ( Figure 2(c)).

LCZ696
Attenuates TAC-Induced Cardiac Fibrosis. Myocardial fibrosis is a crucial feature in pathological cardiac remodeling, which results in impaired cardiac compliance and a decline in the pumping function of the heart. These together promote the progression of hypertrophy to heart failure. Herein, we estimated the effect of LCZ696 in TACinduced cardiac fibrosis, a classic feature of pathological cardiac hypertrophy, to further elucidate the role of LCZ696 in maladaptive cardiac hypertrophy. Picric Sirius red (PSR) staining and Masson's trichrome staining showed that no significant collagen deposition occurred in the sham group. However, the hearts of TAC mice exhibited severe collagen deposition both in interstitial and in perivascular areas (Figures 3(a) and 3(b)). As expected, LZC696 treatment significantly attenuated TAC-induced cardiac fibrosis (Figure 3(d)). Furthermore, immunohistochemistry staining revealed that LCZ696 treatment markedly alleviated α-SMA deposition, which is the hallmark of the transformation of fibroblasts to myofibroblasts. Moreover, we detected the transcription levels of fibrotic markers, including collagen I, collagen III, TGF-β, and CTGF. TAC-induced upregulation of these genes was overtly reversed by LCZ696 treatment (Figure 3(e)).

LCZ696 Mitigates TAC-Induced Myocardium Oxidative
Stress and Cellular Apoptosis. Given that oxidative stress can deteriorate cardiac hypertrophy and heart failure [11][12][13], we examined the effect of LCZ696 on TAC-induced hearts for oxidative stress damage. Pathological hypertrophic stimuli remarkably triggered the production of peroxide byproducts, such as nitrotyrosine (NT) and 4 hydroxynonenal (4-HNE) and superoxide accumulation, as indicated by DHE staining (Figures 4(a)-4(c)). Consistent with our speculations, LCZ696 reduced the deposition of ROS in the myocardium as a result of pressure overload. Furthermore, we assessed myocardial apoptosis using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. LCZ696 attenuated cardiomyocyte apoptosis, as revealed by TUNEL staining (Figure 5(a)). In addition, we examined Bax and Bcl-2 protein levels in different groups, which are both protein markers for apoptosis detection. As expected, LCZ696 remarkably decreased the level of Bax and Bcl-2 expression induced by TAC (Figures 5(b) and 5(c)).
Given that LCZ696 can exert antihypertrophic and antifibrosis effects and attenuate oxidative stress, we further explored the antioxidant reagents in the TAC-induced mice. Pathological hypertrophic stimuli overtly reduced the expression level of MnSOD and Sirt3, whereas LCZ696 treatment distinctly reversed the downregulation of MnSOD and Sirt3 (Figures 5(d)-5(g)). Moreover, we found that LCZ696 upregulated the phosphorylation of AMPK, which was inhibited by pressure overload (Figures 5(h) and 5(i)). Given this, we hypothesized that LCZ696 might exert an antihypertrophic effect by modulating Sirt3 in an AMPK-dependent manner. Thus, we conducted in vitro experiments to confirm our speculations.

LCZ696 Blocks the Hypertrophic Response and Alleviates
Oxidative Stress and Apoptosis in NRCMs Stimulated with Phenylephrine. Here, we verified the effect of LCZ696 on NRCMs. Firstly, we conducted a cell viability assay to evaluate effect of different concentrations of LCZ696 on cardiomyocyte viability (Supplementary Figure S2), and we used 20 μM LCZ696 to incubate with NRCMs in the following in vitro experiments. Then, phenylephrine (PE, 50 μmol/L) was used to stimulate NRCMs for 24 h to imitate the process of cardiac hypertrophy in vitro. As expected, LCZ696 reduced the surface area of hypertrophied cardiomyocytes, as demonstrated by α-actinin staining (Figures 6(a) and 6(b)). Likewise, transcription levels of other hypertrophic markers, such as atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and βmyosin heavy chain (β-MHC), were significantly increased in the PE-treated cardiomyocytes compared with the control group. LCZ696 overtly reduced the expression of these fetal genes (Figure 6(c)). 4 Oxidative Medicine and Cellular Longevity We also monitored the oxidative damage induced by phenylephrine in NRCMs. LCZ696 reduced the intensity of DCFH fluorescence in NRCMs following PE treatment. Furthermore, we detected the levels of superoxide in the mitochondria via MitoSOX staining (Life Technologies, Darmstadt, Germany). As anticipated, LCZ696 significantly reduced the oxidative stress in the mitochondria ( Figure 6(e)). Besides, LCZ696 reduced cardiomyocyte apoptosis, as witnessed by TUNEL staining (Figure 6(f)).   (Figure 7(c)). We then determined the cellular localization of Sirt3 in cardiomyocytes. Sirt3 was colocalized with mitochondrial marker COX IV and decreased in response to PE stimulation (Figure 7(e)). LCZ696 upregulated Sirt3 expression following PE treatment. Notably, LCZ696 did not upregulate the level of Sirt3 basal expression.

LCZ696 Represses
3.6. Sirt3 Knockdown Abolishes the Protective Effect of LCZ696 on Cardiomyocyte Hypertrophy. Sirt3 deficiency impaired the cardioprotective effect of LCZ696 in alleviating hypertrophic response of cardiomyocytes following PE treatment (Figure 8(a)). In addition, LCZ696 did not influence the expression of Bax and Bcl-2 under Sirt3 deficiency conditions (Figures 8(c) and 8(d)). Furthermore, Sirt3 knockdown abolished the alleviation of oxidative stress mediated by LCZ696 (Figure 8(e)). To verify the involvement of Sirt3 in the LCZ696-induced antioxidant property, we performed Mito-SOX staining to examine the generation of superoxide by the mitochondria. Sirt3 deficiency aggravated PE-induced oxidative stress and abrogated the protective effect of LCZ696 in mitochondrial ROS generation. Phenylephrine (PE) stimulation decreased the ratio of p-AMPK/AMPK, which was reversed by LCZ696. However, Sirt3 knockdown impeded this process, indicating that the cardioprotective effect of LCZ696 was partly involved in the upregulation of phospho-AMPK.

Discussion
In recent decades, heart failure-associated deaths have increased drastically, prompting researchers to focus on conducting studies that can solve the problem. Nevertheless, heart  7 Oxidative Medicine and Cellular Longevity failure is the last battlefield in cardiovascular therapy and has always been challenging to tackle [25,26]. Many pathological conditions, such as acute myocardial infarction, chronic uncontrolled hypertension, and diabetes, can contribute to the occurrence of heart failure. Among them, cardiac hypertrophy, which is usually caused by pressure or volume overload, is the most common cause of heart failure [1,2,27].
LCZ696, also referred to as sacubitril/valsartan, is currently the standard treatment option against the progression of heart failure with reduced ejection fraction [4][5][6][7]. Numerous studies have examined the cardioprotective effects of LCZ696 on left ventricular remodeling after myocardial infarction. LCZ696 improves cardiac function by attenuating cardiac fibrosis and MMP-9 expression after ischemia-reperfusion (IR) injury [22,28,29]. Moreover, LCZ696 can attenuate cardiac remodeling and cardiac inflammation by alleviating dynamin-related protein 1 (Drp-1) expression in a doxorubicin-induced cardiomyopathy animal model [30]. Although there are several reports on the basic mechanisms underlying the effectiveness of LCZ696, candidate mechanisms responsible for the direct protective effect of LCZ696 against cardiomyocyte hypertrophy remain unexplored.
Several studies have confirmed that reactive oxygen species (ROS) can cause adverse effects by oxidizing proteins, lipids, and nucleic acids. Cardiac hypertrophy and oxidative stress are mutually causal, forming a vicious circle. Therefore, breaking the vicious circle could be a vital breakthrough in the treatment of heart failure. However, antioxidant therapy has not been developed. Recent studies have confirmed that impairment of catalase, a major antioxidant enzyme, can aggravate diabetes-induced cardiac toxicity. On the contrary, cardiac-specific overexpression of catalase alleviated agingrelated cardiac dysfunction and cardiac hypertrophy [31,32]. Superoxide dismutases (SOD) are the major antioxidant enzymes responsible for the scavenger of superoxide in the mitochondria. Among them, manganese SOD (MnSOD, SOD2) in the mitochondria plays a pivotal role in redox homeostasis [33]. Manganese superoxide dismutase (MnSOD) is a major superoxide-scavenging enzyme, converting superoxide to hydrogen peroxide, which is in turn hydrolyzed to water by catalase [34].     Oxidative Medicine and Cellular Longevity Sirt3 belongs to CLASS III histone deacetylases (HDACs) and localizes to the mitochondrial matrix, where it may function as a primary stress-responsive protein deacetylase [35]. Many studies have focused on examining the cardioprotective role of Sirt3 in cardiac hypertrophy and heart failure [36]. For example, Sirt3 knockout mice exhibited severe cardiac hypertrophy and fibrosis induced by TAC, and nevertheless, cardiomyocyte-specific overexpression of Sirt3 resisted cardiac fibrosis and oxidative damage following angiotensin II infusion [20]. A study also reported that Sirt3-deficient mice developed dilated cardiomyopathy, and Sirt3 knockout mice exacerbated angiotensin II-induced cardiac hypertrophy [19]. SIRT3 deficiency can aggravate diabetic cardiomyopathy by inactivating Foxo3A-mediated mitophagy and accelerate hypertensive cardiac remodeling by impairing angiogenesis [35,37]. Besides, Sirt3 silencing can abolish the protective effects of NaHS to reverse Ang II-induced cardiomyocyte hypertrophy and mitochondrial dysfunction as a result of the decline in FOXO3a and SOD2 expression [38,39]. Studies have shown that many small molecules with Sirt3-activating property can protect against cardiac hypertrophy and heart failure. Honokiol increases Sirt3 activity, thereby alleviating the severity of cardiac hypertrophy [40]. It also reduces oxidative stress induced by melatonin and improves cardiac function by interacting with the Sirt3-dependent pathway [41,42]. Resveratrol, known as a potent activator of SIRT1, also ameliorates cardiac fibrosis by slightly activating Sirt3 [43]. Sirt3 plays an anticarcinogenic role and functions as a tumor suppressor protein [44]. Clinical studies have shown that the Sirt3 level decreases by 40% in 65-year-old people [45], which may be closely related to the occurrence of many multiple senile diseases, such as hypertension, atherosclerosis, and heart failure.
Given that Sirt3 can play such a crucial role in pathological cardiac remodeling by deacetylating MnSOD [20,44,46], we hypothesized that LCZ696 might regulate the expression of Sirt3, mitigate oxidative stress, and thus improve cardiac hypertrophy and heart failure. To verify our hypothesis, we conducted an in vivo experiment with transverse aortic constriction, as described earlier. Laparoscopic Transabdominal Cerclage (TAC) mice exhibited deteriorated cardiac dysfunction as evidenced by echocardiography and the ratios of HW/BW, HW/TL, and LW/TL (Figure 1). Moreover, TAC markedly promoted the fibrosis deposition and transdifferentiation from fibroblast to myofibroblast, and as a result, cardiac distensibility declined. Fortunately, LZC696 alleviated myocardium hypertrophy and fibrosis, thereby improving cardiac function. With that, we investigated the mechanisms underlying the role of LCZ696 in cardiac hypertrophy. Pressure overload repressed the expression level of Sirt3 and MnSOD, whereas LCZ696 rescued the decline of Sirt3 and MnSOD. Also, we found that LCZ696 upregulated the phosphorylation of AMPK, whose deficiency could aggravate pressure overload-induced cardiac hypertrophy. Therefore, we hypothesized that LCZ696 might exert an antihypertrophic effect by modulating Sirt3 in an AMPKdependent manner. To verify this hypothesis, we conducted in vitro experiments. Phenylephrine (PE) was used to stimulate neonatal rat cardiomyocytes (NRCMs) to imitate an in vitro cardiac hypertrophy model. As explained earlier, LCZ696 blocked the hypertrophic response and alleviated oxidative stress and apoptosis in NRCMs following PE stimulation. In addition, LCZ696 upregulated Sirt3 and MnSOD expression, as well as the ratio of p-AMPK/AMPK. However, Sirt3 silencing abolished the ability of LCZ696 to reverse the PE-induced cardiomyocyte hypertrophy, mitochondrial oxidative stress, and apoptosis, along with the decline in MnSOD expression. Also, Sirt3 deficiency hampered the capacity of LCZ696 to activate the phosphorylation of AMPK. Collectively, these results demonstrate that Sirt3 could be an endogenous negative regulator of cardiac hypertrophy, which protects hearts by suppressing cellular levels of ROS. In addition, LCZ696 might exert antihypertrophic effect by ameliorating oxidative stress via the Sirt3/MnSOD pathway.

Conclusion
Our results show that Sirt3 can act as a therapeutic target in the treatment of heart failure. Also, the application of specific small molecules for activating Sirt3 can be a novel strategy to hinder the progression of pathological hypertrophy into heart failure. These findings provide new insights into the molecular mechanism of LCZ696 and its novel therapeutic role in the treatment of pathological cardiac remodeling and heart failure.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that they have no conflicts of interest.