cardioprotective effect of apelin-13 on cardiac performance

Circulation JournalOfficial Journal of the Japanese Circulation Societyhttp://www.j-circ.or.jp

pelin is a recently described ligand for the G-protein-coupled receptor APJ (APLNR).1,2 The apelin and APJ constitute a signaling pathway implicated in

numerous cardiac and vascular functions. In addition, apelin is synthesized as a pre-propeptide consisting of 77 amino acids with shorter biologically active forms.3 Given the cell and developmental specific patterns of expression of apelin and APJ in vascular and cardiac structures and the initial studies in developmental model organisms, it is likely that this pathway has a fundamental role in the embryogenesis of the cardiovas-cular system.4 In the adult cardiovascular system, both apelin and APJ are expressed in the endothelium of heart, kidney, and lung, and APJ is expressed by myocardial cells and some vas-cular smooth muscle cells.5,6 Recent studies have suggested that the apelin/APJ pathway is a pivotal regulator of cardiovas-cular function. Apelin has been shown to have a positive effect on contractility, and experiments using myocardial injury models have suggested that apelin also has a positive inotropic effect on failing myocardium.7,8 Apelin causes endothelium-

dependent vasorelaxation by triggering the release of nitric oxide (NO), and this effect can be almost completely abolished in the presence of endothelial NO synthase (eNOS) inhibitor, suggesting that the apelin/APJ pathway exerts a vasorelaxation effect via activation of serine/threonine Akt and eNOS phos-phorylation.9–11 Moreover, previous studies have demonstrated that treatment with the NOS inhibitor, L-NG-nitroarginine methyl ester (L-NAME), enhances the expression of NAD(P)H oxidase subunit, which activates the production of reactive oxygen species (ROS).12

Recently, a reduction in myocardial apelin/APJ expression has been reported in experimental models of heart failure (HF). In a pressure overload-induced HF model, apelin knock-out (KO) mice developed severely impaired heart contractil-ity.13 Moreover, Iwanaga et al reported that the cardiac apelin/APJ pathway is markedly downregulated in Dahl salt-sensitive hypertensive (DS) rats with HF.14 Recently, we also showed a similar reduction in cardiac apelin/APJ protein expression seen and these suppressive expressions were restored by

A

Received July 3, 2011; revised manuscript received September 22, 2011; accepted September 26, 2011; released online November 12, 2011 Time for primary review: 19 days

Department of Hypertension and Cardiorenal Medicine, Dokkyo Medical University School of Medicine, Tochigi, JapanMailing address: Naohiko Kobayashi, MD, PhD, Department of Hypertension and Cardiorenal Medicine, Dokkyo Medical University

School of Medicine, Mibu, Tochigi 321-0293, Japan. E-mail: [email protected] doi: 10.1253/circj.CJ-11-0689All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: [email protected]

Cardioprotective Effect of Apelin-13 on Cardiac Performance and Remodeling

in End-Stage Heart FailureWataru Koguchi, MD; Naohiko Kobayashi, MD, PhD; Hiroshi Takeshima, MD; Mayuko Ishikawa, MD;

Fumihiro Sugiyama, MD; Toshihiko Ishimitsu, MD, PhD

Background: Apelin and its cognate G protein-coupled receptor, APJ, constitute a signaling pathway with a positive inotropic effect on cardiac function. Recently, we and other investigators demonstrated that a reduction in myocardial apelin/APJ expression might play a critical role in experimental models of end-stage heart failure (HF). Therefore, we evaluated whether exogenous apelin infusion restores apelin/APJ expression and improves cardiac function in the failing heart of Dahl salt-sensitive hypertensive (DS) rats.

Methods and Results: High salt-loaded DS rats were treated with vehicle and pyroglutamylated apelin-13 (Pyr-AP13; 200 μg · kg–1 · day–1, IP) from the age of 11 to 18 weeks. Decreased end-systolic elastance and percent frac-tional shortening in failing rats was significantly ameliorated by Pyr-AP13. Pyr-AP13 effectively inhibited vascular lesion formation and suppressed expression of inflammation factors such as tumor necrosis factor-α and interleukin-1β protein. Downregulation of apelin and APJ expression, and phosphorylation of endothelial nitric oxide synthase at Ser1177 and Akt at Ser473 in failing rats was significantly increased by Pyr-AP13. Upregulation of NAD(P)H oxidase p22phox, p47phox, and gp91phox in DS rats was significantly suppressed by Pyr-AP13.

Conclusions: Exogenous apelin-13 may ameliorate cardiac dysfunction and remodeling and restore apelin/APJ expression in DS rats with end-stage HF. Thus, apelin-13 may have significant therapeutic potential for end-stage HF.

Key Words: Apelin; Cardiac function; Heart failure; Remodeling

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KOGUCHI W et al.

angiotensin II (Ang II) type 1 receptor (AT1R) blocker in end-stage HF with severe left ventricular (LV) dysfunction.15 How-ever, the effect of exogenous apelin on the apelin/APJ pathway in end-stage HF remain unknown. Accordingly, we investi-gated whether exogenous apelin-13 restores expression of apelin and APJ and improves cardiac function and cardiovas-cular remodeling in DS rats with end-stage severe HF.

MethodsAll experimental procedures and protocols in this study were in accordance with the Dokkyo Medical University School of Medicine institutional guidelines for animal research and with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Animal Models and Experimental DesignsMale, inbred DS rats and Dahl salt-resistant (DR) rats (Eisai, Tokyo, Japan) were weaned and fed a diet containing 0.3% NaCl until the age of 6 weeks. Thereafter, they were fed a diet containing 8% NaCl until the age of 18 weeks. Systolic blood pressure (SBP) was measured by the tail-cuff method at the start of the 8% NaCl diet and at 1-week intervals thereafter. Transthoracic echocardiography evaluating the LV end-dia-stolic diameter (LVEDD), and percent fractional shortening (%FS) were performed at 18 weeks, as described previously.16–18 At the age 11 weeks, when LV hypertrophy developed, DS rats were randomly divided into 2 groups: treated with vehicle (DSHF-V), and automatic infusion of pyroglutamylated ape-lin-13 (Pyr-AP13, 200 μg · kg–1 · day–1; DSHF-AP) via an Alzet osmotic minipump implanted intraperitoneally in the abdomi-nal cavity followed by closure of the abdominal wall in layers. This common posttranslational modification preserves bio-logical activity by rendering the peptide more resistant to enzymatic cleavage. Therefore, we used Pyr-AP13 for this chronic study.19 Age-matched male DR rats served as a con-trol group (DR-C).

LV Pressure-Volume RelationThe chest was opened via a midline sternotomy, and the peri-cardium was dissected to expose the heart. The LV end-sys-tolic pressure – volume relationship (contractility: end-systolic elastance (Ees)) was modified for the conductance catheter technique as described previously.20–22 Briefly, the conductance catheter was inserted into the LV through the apex and pushed until the distal tip was placed in the ascending aorta along the longitudinal axis of the LV. A 3F catheter-tip micromanometer (SPR-524, Millar instruments) was also inserted into the LV from the apex. To change the preload, a snare was placed around the inferior vena cava. We recorded conductance vol-ume and LV pressure simultaneously during gradual inferior vena cava occlusion. Electrical signals were digitized through an analog-to-digital converter (AD12-8, Contec) at a sampling frequency of 1,000 Hz with 12-bit resolution and stored on a personal computer (Dynabook SS 330, Toshiba). The points of the end-systolic pressure – volume relation were determined by an iterative technique reported previously.23 In several con-secutive pressure – volume loops, the points of each cardiac cycle with maximum pressure-to-volume ratio were first deter-mined. Linear regression of these points with expression:

Pes=Ees(Ves – V0)yielded estimates for the slope, or Ees, and the volume-axis intercept (V0), where Pes and Ves are end-systolic pressure and volume, respectively.

Western Blot AnalysisApelin, APJ, Akt, NAD(P)H oxidase p22phox, p47phox, gp91phox, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), mono-cyte chemoattractant protein-1 (MCP-1), angiotensin-converting enzyme (ACE), AT1R, and nuclear factor-κ B (NF-κB) protein expressions were measured as described previously.21,22,24 LV was homogenized (25% W/vol) in 10 mmol/L HEPES buffer, pH 7.4, containing 320 mmol/L sucrose, 1 mmol/L EDTA, 1 mmol/L DTT, 10 μg/ml leupeptin, and 2 μg/ml aprotinin at 0°C to 4°C with a polytron homogenizer. Protein concentrations were determined with bovine serum albumin as a standard protein. Equal amounts of protein were loaded in each lane of SDS-PAGE using 13% gels. The proteins in the gels were trans-ferred electrophoretically to PVDF sheets for 1 h at 2 mA/cm2. The sheets were immunoblotted with an anti-apelin, anti-APJ, anti-Akt, anti-NAD(P)H oxidase subunits, anti-TNF-α, anti-IL-1β, and anti-MCP-1 antibody (Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA) in a buffer containing 10 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, 0.1% Tween 20, and 5% skim milk. The proteins transferred to the sheets were detected using the ECL immunoblotting detection system (Amersham Life Science Inc).

Phosphorylation of eNOS at Ser1177, Akt at Ser473, and NF-κB at Ser536

Phosphorylation of eNOS at Ser1177, Akt at Ser473, and NF-κB at Ser536 was measured in detail as described previously.22,24,25

Detection of Superoxide Anion in the LVHistological detection of superoxide anion in the LV was performed using dihydroethidium (DHE) as described previ-ously.20,22,24

Nitrite ProductionNitrite production was measured as described in detail previ-ously.26,27 The LV was used for the assay of nitrite produc-tion within 24 h. Three 50-μm sections of myocardium from each rat were cut on a vibratome and incubated in a buffer (pH 7.2) containing 25 mmol/L N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) (Sigma, St Louis, MO, USA); 140 mmol/L NaCl, 5.4 mmol/L KCl, 1.8 mmol/L CaCl2, 1 mmol/L MgCl2, and 5 mmol/L glucose for 48 h at 37°C. The supernatant was used for the assay of NO2− production, and the amount of NO2− was corrected via the protein amount measured using the Bradford method (Bio-Rad, Richmond, CA, USA). Nitrite was measured with an autoanalyzer (TCI-NOX 1000 m; Tokyo Kasei Kogyo, Tokyo, Japan) using the Griess method.

Histologic Examination and Evaluation of Cardiovascular RemodelingHistologic examination was performed as described in detail previously.15–17,20–22,24 The wall-to-lumen ratio (the area of the vessel wall divided by the area of the total blood vessel lumen) was determined. The area of fibrosis immediately surrounding the blood vessels was calculated, and perivascular fibrosis was determined as the ratio of the area of fibrosis surrounding the vessel wall to the total area of the vessel. To assess the area of myocardial interstitial fibrosis, the area of pathological colla-gen deposition was measured in the microscopic field of each Masson’s trichrome-stained section. The ratio of the total area of fibrosis within the LV myocardial sample to the total area of the LV myocardium in each heart was calculated and used for analysis.


Effect of Apelin-13 in CHF

Statistical AnalysisAll values are expressed as mean ± SEM. Mean values were compared among the 3 groups by ANOVA and the Bonferroni post hoc test for multiple comparisons. A value of P<0.05 was considered statistically significant.

ResultsPhysiological Profiles After 7 Weeks’ Treatment With Pyr-AP13 in DS RatsBody weight (BW), SBP, LV weight (LVW) to BW ratio, and heart rate (HR) in the 3 groups are presented in Table. BW was significantly lower in DSHF-V than in DR-C rats. Long-term Pyr-AP13 therapy in DS rats significantly increased BW. In contrast, DSHF-V rats had a higher LVW/BW than did DR-C rats. Long-term Pyr-AP13 therapy in DS rats significantly decreased LVW/BW.

Time-related changes in SBP among the 3 groups are shown in Figure 1. Before feeding with 8% NaCl diet, SBP was 114±3 mmHg in DR-C, 117±4 mmHg in DSHF-V and 118±4 mmHg in DSHF-AP. As shown in Figure 1 and Table, SBP in DSHF-V and DSHF-AP rats was similar and signifi-cantly higher than that in DR-C rats. HR was significantly higher in DSHF-V than in DR-C rats. Long-term Pyr-AP13 therapy in DS rats significantly decreased HR (Table, Figure 1).

LV Ees in End-Systolic Pressure-Volume Relation and Cardiac Function for LVEDD and %FSLVEDD was significantly higher in DSHF-V than in DR-C rats. Long-term Pyr-AP13 therapy in DS rats significantly decreased LVEDD. In contrast, DSHF-V rats had lower Ees and %FS than did DR-C rats. Long-term Pyr-AP13 therapy in DS rats significantly improved Ees and %FS (Table).

Cardiovascular RemodelingThe wall-to-lumen ratio, perivascular fibrosis, and area of interstitial fibrosis of the heart in the 3 groups are shown in Figure 2F and Table (n=10 tissue sections per group). The wall-to-lumen ratio was significantly increased in DSHF-V rats compared with DR-C. Long-term Pyr-AP13 treatment caused significant amelioration of this ratio. Perivascular and intersti-tial fibrosis was significantly greater in DSHF-V rats than in DR-C rats. Long-term Pyr-AP13 treatment also caused signifi-

cant improvement in perivascular and interstitial fibrosis.

Apelin and APJ ExpressionApelin and APJ protein expressions in the LV of DS rats were significantly lower in DSHF-V rats than in DR-C rats. Long-term Pyr-AP13 significantly increased these expressions in DS rats (Figure 3).

Phosphorylation of eNOS at Ser1177 and Akt at Ser473 and Nitrite ProductionPhosphorylation of eNOS at Ser1177 and Akt at Ser473 and

Table. General Characteristics and Cardiac Function and Remodeling in DS Rats and Apelin-Treated DS Rats

Parameter DR-C DSHF-V DSHF-AP

N 10 9 8

BW, g 474±6　 296±5*　　　　 391±10*,†　　

SBP, mmHg 130±3　 249±6*　　　　 241±7*　　　　　　

LVW/BW, mg/g 1.92±0.04 4.49±0.09* 2.74±0.07*,†

Lung weight/BW, mg/g 3.2±0.1 12.6±1.4*　　 5.5±0.5*,†

HR, beats/min 409±11 487±13*　　 414±12†　　　　

Ees, mmHg/ml 2,393±201 1,064±94*　　　　　 2,301±113†　　　　　

%FS 53.9±0.8 26.1±1.1*　　 45.4±0.9*,†　　

LVEDD, mm 7.2±0.1 9.4±0.3* 7.5±0.2†　　

Wall-to-lumen ratio 0.13±0.01 0.40±0.03* 0.26±0.02*,†

Perivascular fibrosis 0.22±0.02 0.79±0.05* 0.51±0.04*,†

Area of interstitial fibrosis, % 2.0±0.2 13.1±0.8*　　 6.1±0.5*,†

Data are expressed as mean ± SEM.*P<0.01 vs. DR-C; †P<0.01 vs. DSHF-V.DS, Dahl salt-sensitive hypertensive; DR-C, Dahl salt-resistant control rats; DSHF-V, Dahl salt-sensitive hypertensive rats treated with vehicle; AP, apelin-13; BW, body weight; SBP, systolic blood pressure; LVW, left ventricular weight; HR, heart rate; Ees, contractility; FS, fractional shortening; LVEDD, left ventricular end-diastolic diameter.

Figure 1. Time-related changes in systolic blood pressure in the DR-C, DSHF-V, and DSHF-AP groups. DR-C, Dahl salt-resistant rats; DSHF-V, Dahl salt-sensitive hypertensive rats treated with vehicle; DSHF-AP, Dahl salt-sensitive hypertensive rats treated with apelin-13. Values are mean ± SEM. **P<0.01 vs. DR-C.


KOGUCHI W et al.

nitrite production in the LV was significantly lower in DSHF-V rats than in DR-C rats. Long-term Pyr-AP13 significantly increased eNOS and Akt phosphorylation and nitrite produc-tion in DS rats (Figure 4).

NAD(P)H Oxidase Subunits Expression and Superoxide Anion ProductionNAD(P)H oxidase p22phox, p47phox, and gp91phox protein lev-els, and superoxide anion production by DHE in the LV were significantly higher in DSHF-V rats than in DR-C rats. Long-term Pyr-AP13 therapy significantly decreased these expres-sions in DS rats (Figure 5).

TNF-α, IL-1β, and MCP-1 ExpressionTNF-α, IL-1β, and MCP-1 protein levels in the LV were sig-nificantly higher in DSHF-V rats than in DR-C rats. Long-term Pyr-AP13 therapy significantly decreased these expres-sions in DS rats (Figure 6).

ACE and AT1R Expression and Phosphorylation of NF-κB at Ser536

ACE and AT1R protein levels, and phosphorylation of NF-κB at Ser536 in the LV were significantly higher in DSHF-V rats than in DR-C rats. Long-term Pyr-AP13 therapy significantly decreased ACE and AT1R expression and NF-κB phosphory-

Figure 2. Effects of chronic apelin-13 infusion on histological evaluation of (A–C) cardiovascular remodeling and (D–F) interstitial fibrosis after Masson’s trichrome staining in the microcoronary artery. (A,D) DR-C; (B,E), DSHF-V; (C,F) DSHF-AP. Bar=100 μm. DR-C, Dahl salt-resistant control rats; DSHF-V, Dahl salt-sensitive hypertensive rats treated with vehicle; DSHF-AP, Dahl salt-sen-sitive hypertensive rats treated with apelin-13.

Figure 3. Effects of chronic apelin-13 infusion on apelin and APJ protein expression. Values are mean ± SEM. n=5–6 per group. *P<0.05 vs. DR-C;†P<0.05 vs. DSHF-V. APJ, cognate G protein-coupled receptor of apelin, DSHF-V, Dahl salt-sensitive hyper-tensive rats treated with vehicle; DR-C, Dahl salt-resistant control rats.



lation in DS rats (Figure 7).

DiscussionWe administered Pyr-AP13 to DS rats with end-stage HF and observed an inhibition of cardiac dysfunction and cardiovas-cular remodeling, and expression of inflammation factors such

as TNF-α, IL-1β, and MCP-1. In addition, we demonstrated that the cardiac apelin/APJ, Akt/eNOS, oxidative stress path-way had deteriorated in end-stage HF with severe LV dysfunc-tion, but was restored by Pyr-AP13. These findings suggest that Pyr-AP13 may improve cardiac dysfunction and remodel-ing and the apelin/APJ expression associated with Akt/eNOS and oxidative stress pathways in the failing heart of DS rats.

Figure 4. Effects of chronic apelin-13 infusion on phosphorylation of eNOS at Ser1177 and Akt at Ser473 and nitrite production. Values are mean ± SEM, n=5–6 per group. *P<0.05 vs. DR-C; †P<0.05 vs. DSHF-V. DSHF-V, Dahl salt-sensitive hypertensive rats treated with vehicle; DR-C, Dahl salt-resistant control rats; eNOS, endothelial nitric oxide synthase.

Figure 5. Effects of chronic apelin-13 infusion on NAD(P)H oxidase p22phox, p47phox, and gp91phox protein expression (A) and superoxide anion production (B–D). Values are mean ± SEM, n=5–6 per group. *P<0.05 vs. DR-C; †P<0.05 vs. DSHF-V. (B) DR-C; (C) DSHF-V; (D) DSHF-AP. Bar=500 μm. DSHF-V, Dahl salt-sensitive hypertensive rats treated with vehicle; DSHF-AP, Dahl salt-sensitive hypertensive rats treated with apelin-13; DR-C, Dahl salt-resistant control rats.


KOGUCHI W et al.

Thus, the apelin/APJ pathway may play a pivotal role in end-stage HF with severe LV dysfunction, and apelin-13 may be a potential therapeutic target in end-stage severe HF.

Enormous effort has been directed toward identifying new therapeutic strategies with long-term efficacy in HF.28–35 Recent studies have confirmed that the endogenous apelin/APJ signal-ing pathway contributes to cardiac function in the failing heart. Although apelin-deficient mice display normal or only slightly impaired basal cardiac function, they have marked reductions in exercise capacity and maximum oxygen consumption. They manifest progressive cardiac dysfunction from 6 months of age and develop severe HF when subjected to chronic pressure overload via surgical aortic banding.13 Iwanaga et al demon-strated that although apelin mRNA was unchanged in the com-pensatory LH hypertrophy phase, it decreased together with the

progression of LV dysfunction, and the expression levels APJ also decreased.14 These findings indicated that myocardial ape-lin expression might be downregulated, accompanied by a decrease in APJ expression, in end-stage HF. Recently, we also reported that a similar reduction in cardiac apelin/APJ protein expression was seen in end-stage HF with severe LV dysfunc-tion.15 Moreover, exogenous apelin potently enhances myocar-dial contractility. In isoproterenol-induced myocardial injury in rats, apelin and APJ genes were downregulated and apelin infu-sion ameliorated the effects of cardiac injury.36 In vivo, acute apelin infusion restores ejection fraction, increases cardiac output and reduces LV end-diastolic pressure in rats with chronic HF.37 Kuba et al showed that apelin KO mice exhibited normal cardiac development in adulthood, but which deterio-rated with aging or with chronic pressure overload, and a con-

Figure 6. Effects of chronic apelin-13 infusion on TNF-α, IL-1β, and MCP-1 protein expressions. Values are mean ± SEM. n=5–6 per group. *P<0.05 vs. DR-C; †P<0.05 vs. DSHF-V. DSHF-V, Dahl salt-sensitive hypertensive rats treated with vehicle; DR-C, Dahl salt-resistant control rats; IL, interleukin; MCP, monocyte chemoattractant protein; TNF, tumor necrosis factor.

Figure 7. Effects of chronic apelin-13 infusion on ACE and AT1R protein expression and phosphorylation of NF-κB at Ser536. Values are mean ± SEM. n=5–6 per group. *P<0.05 vs. DR-C; †P<0.05 vs. DSHF-V. ACE, angiotensin-converting enzyme; AT1R, angiotensin II type 1 receptor; DSHF-V, Dahl salt-sensitive hypertensive rats treated with vehicle; DR-C, Dahl salt-resistant control rats; NF, nuclear factor.



tinuous infusion of apelin-13 in these elderly mice reversed the decreased contractility because of aging.13 In addition, Ashley et al reported that acute administration of apelin in vivo caused a reduction in LV end-diastolic area and an increase in LV elastance, whereas chronic apelin infusion increased cardiac output without causing hypertrophy.7 In a recent important report, Japp et al38 demonstrated that short-term apelin infusion caused peripheral and coronary vasodilatation, reduced cardiac preload and afterload, and increased cardiac output in vivo in humans. These important hemodynamic effects appeared to be preserved in patients with HF. They concluded that APJ ago-nism had potential therapeutic benefits in patients with HF maintained on optimal contemporary medical therapy. In the present study, we showed that apelin-13 infusion ameliorated cardiac function and cardiovascular remodeling, and restored apelin/APJ expression in the LV of DS rats with end-stage HF. These findings suggest that the apelin/APJ pathway is main-tained or even augmented in conditions of chronic pressure overload and the early stages of HF, but is substantially down-regulated in severe HF, and that the beneficial effects of apelin-13 infusion may at least in part be explained by restoration of the apelin/APJ signaling pathway in end-stage HF with severe LV dysfunction.

The vasodilatory response to apelin is endothelium-depen-dent because vasoconstriction occurs in endothelium-denuded vessels.39 This endothelium-dependent vasodilatation appears to be mediated predominantly through eNOS-dependent path-ways.9 The current study found diminished phosphorylation of Akt and eNOS in aortic tissues form diabetic db/db mice. In addition, an abnormal Akt/eNOS phosphorylation pathway might serve to dampen endothelial NO signaling in aortic rings, thereby leading to abnormal vasoactive responses to Ang II and acetylcholine. In aortic rings from db/db mice, apelin treatment significantly enhanced phosphorylation of Akt and eNOS, which is consistent with previously published find-ings.10,40 Apelin can facilitate the activity of eNOS in endothe-lial cells and APJ is crucially involved in apelin-induced phos-phorylation of eNOS, which promotes the release of NO.9,10 In addition, the apelin/APJ pathway mediated amelioration of atherosclerosis might be related to increased NO production, and the NOS inhibitor, L-NAME, blocks apelin-mediated ame-lioration of Ang II-exacerbated vascular disease in the ApoE-KO model.41 These results suggest that chronic apelin infusion potentiates the activity of the apelin/APJ signaling pathway, which may, at least in part, contribute to increased Akt/eNOS phosphorylation. On the other hand, ROS act as intercellular and intracellular messengers that play an important patho-physiologic role in vascular biology.42 Increased oxidative stress resulting from enhanced production of ROS and/or inad-equate mechanisms of antioxidant defense has been recognized as a critical mechanism in the initiation of cardiac dysfunction and the transition from hypertrophy to HF.43 Moreover, numer-ous studies have reported that the failing heart is subjected to increased oxidative stress, and antioxidant strategies have been proposed to prevent the progression and development of chronic HF.44,45 Recently, Foussal et al46 showed that chronic treatment of mice with apelin attenuated pressure-overload-induced LV hypertrophy, and that the prevention of hypertro-phy by apelin was associated with increased myocardial cata-lase activity and decreased plasma lipid hydroperoxide, as an index of oxidative stress. Apelin preserved cardiac function by inhibiting oxidative stress and stimulating catalase activity, supporting the premise that apelin is a potent regulator of car-diomyocyte antioxidant reserve against oxidative stress during hypertrophic remodeling of the heart. In the present study, we

demonstrated that apelin significantly prevented the progres-sion of cardiac dysfunction and cardiovascular remodeling in HF, and inhibited NAD(P)H oxidase subunit expression and ROS production. These findings suggest that chronic apelin infusion may play a pivotal role in activation of the antioxidant system in DS rats with end-stage severe HF.

In the present study, we showed that long-term apelin-13 infusion ameliorated LV weight, cardiovascular remodeling, and cardiac function, and inhibited inflammation and oxidative stress, and restored apelin/APJ expression. However, the under-lying cardioprotective mechanism of apelin-13 is unclear. The apelin/APJ pathway has opposing actions to the renin – angio-tensin system (RAS) in a number of physiologic and patho-physiologic settings.5,9 There is some evidence for direct coun-terregulation of the RAS by the apelin/APJ pathway. Ishida et al10 have demonstrated differences in blood pressure response in mice lacking both AT1R and APJ compared with mice lacking only AT1R. In addition, Ang II stimulation produces superoxide via AT1R and activation of NAD(P)H oxidases in endothelial and vascular smooth muscle cells. Moreover, chronic activation of NF-κB may induce expression of inflam-matory cytokines and produce detrimental consequences. Therefore, to evaluate the mechanism, we performed experi-ments to determine whether long-term apelin-13 infusion inhibited ACE and AT1R expression and NF-κB phosphory-lation. We found that ACE and AT1R protein expressions and NF-κB phosphorylation were upregulated in DS rats, and apelin-13 infusion suppressed these changes. These findings suggest that the cardioprotective mechanism of apelin-13 may in part be explained by inhibition of the RAS and NF-κB signaling pathway.

ConclusionsWe administered long-term apelin infusion DS rats with severe end-stage HF and observed an inhibition of both cardiovascu-lar remodeling and cardiac dysfunction. Moreover, we showed that the cardiac apelin/APJ pathway is suppressed in end-stage HF with severe LV dysfunction, and that this signaling path-way is restored by apelin infusion. In addition, apelin infusion stimulated Akt/eNOS phosphorylation, and inhibited expres-sion of NAD(P)H oxidase subunits and ROS production. These findings suggest that the apelin/APJ pathway may play a critical role in the pathophysiology of HF, and that apelin infusion may be a therapeutic strategy for severe HF.

AcknowledgmentsWe thank Yasuko Mamada, Keiko Fukuda, Hisato Hirata, Yoshifumi Machida, Noriko Suzuki, Fumie Yokotsuka, Kyoko Tabei, and Machiko Sakata for technical assistance.

DisclosureAll the authors declared no conflicts of interest.

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cardioprotective effect of apelin-13 on cardiac performance

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