Alogliptin, a dipeptidyl peptidase-4 inhibitor, regulates the atrial arrhythmogenic substrate in rabbits

Alogliptin, a dipeptidyl peptidase-4 inhibitor, regulates the atrial arrhythmogenic substrate in rabbits Toshihiko Yamamoto, MD,* Masayuki Shimano, MD, PhD,*† Yasuya Inden, MD, PhD,* Mikito Takefuji, MD, PhD,* Satoshi Yanagisawa, MD,* Naoki Yoshida, MD, PhD,* Yukiomi Tsuji, MD, PhD,‡ Makoto Hirai, MD, PhD,§ Toyoaki Murohara, MD, PhD* From the *Department of Cardiology, Nagoya University Graduate School of Medicine, Nagoya, Japan, † Nagoya First Red Cross Hospital, Nagoya, Japan, ‡Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan, and §Nagoya University Graduate School of Health Science, Nagoya, Japan. BACKGROUND Dipeptidyl peptidase-4 (DPP-4) inhibitors were recently reported to have cardioprotective effects via amelioration of ventricular function. However, the role of DPP-4 inhibition in atrial remodeling, especially of the arrhythmogenic substrate, remains unclear. OBJECTIVE We investigated the effects of a DPP-4 inhibitor, alogliptin, on atrial fibrillation (AF) in a rabbit model of heart failure caused by ventricular tachypacing (VTP). METHODS Rabbits subjected to VTP at 380 bpm for 1 or 3 weeks, with or without alogliptin treatment, were assessed using echocardiography, electrophysiology, histology, and immunoblotting and compared with nonpaced animals. RESULTS VTP rabbits exhibited increased duration of atrial burst pacing-induced AF, whereas administration of alogliptin shortened this duration by 73%. The extent of atrial fibrosis after VTP was reduced by 39% in the alogliptin-treated group. VTP rabbits treated with alogliptin displayed a 1.6-fold increase in left atrial myocardial capillary density compared with nontreated rabbits. A 2-fold increase in endothelial nitric oxide synthase (eNOS) phosphorylation was observed in the left atrium of alogliptin-treated rabbits

Introduction Atrial fibrillation (AF) is the most common cardiac arrhythmia. It occurs more frequently in older people, and its prevalence is increasing in developed countries.1 Congestive heart failure (HF) is an important clinical cause of AF, which occurs in 13%–27% of congestive HF patients; these conditions exacerbate each other, and AF patients with HF show increased mortality.2 Experimental HF caused by ventricular tachypacing (VTP) can produce a substrate that maintains AF caused by atrial interstitial fibrosis and local disturbance of conduction.3 Moreover, atrial fibrosis causes structural remodeling, which creates an AF substrate.4 Address reprint requests and correspondence: Dr. Masayuki Shimano, Department of Cardiology, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa, Nagoya, Aichi466-8550, Japan. E-mail address: [email protected].

1547-5271/$-see front matter B 2015 Heart Rhythm Society. All rights reserved.

compared with nontreated rabbits. Moreover, a nitric oxide synthase inhibitor (Nω-nitro-L-arginine methyl ester) blocked the beneficial effects of alogliptin on AF duration, fibrosis, and capillary density. CONCLUSION Alogliptin shortened the duration of AF caused by VTP-induced fibrotic atrial tissue by augmenting atrial angiogenesis and activating eNOS. Our findings suggest that DPP-4 inhibitors may be useful in the prevention of heart failure-induced AF. KEYWORDS Atrial fibrillation; DPP-4 inhibitor; Atrial arrhythmogenic substrate; Atrial fibrosis; Atrial neovascularization ABBREVIATIONS AF ¼ atrial fibrillation; CV ¼ conduction velocity; DPP-4 ¼ dipeptidyl peptidase-4; eNOS ¼ endothelial nitric oxide synthase; EPS ¼ electrophysiological study; ERP ¼ effective refractory period; HF ¼ heart failure; LA ¼ left atrium; LAA ¼ left atrial appendage; LV ¼ left ventricular; NOS ¼ nitric oxide synthase; RAA ¼ right atrial appendage; VTP ¼ ventricular tachypacing (Heart Rhythm 2015;12:1362–1369) I 2015 Heart Rhythm Society. All rights reserved.

Because atrial remodeling is sometimes irreversible, AF worsens over time.5 These findings demonstrate that experimental HF models can be used to develop novel therapeutic approaches for AF, and such models are particularly useful for the development of therapeutic vectors that target the underlying substrate,6 such as angiotensin-modulating agents.7,8 AF can produce life-threatening complications such as systemic embolism, and control of AF is therefore required clinically. AF can occur in patients with diabetes mellitus, which is increasing in prevalence globally.9 Diabetes increases risk for AF, and AF is associated with a substantially increased risk of death and cardiovascular events in patients with diabetes.10 Conversely, patients hospitalized for HF with AF are less likely to have a history of diabetes than patients with sinus rhythm.11 A potential reason for this phenomenon is that AF can exacerbate HF conditions, leading to worse http://dx.doi.org/10.1016/j.hrthm.2015.03.010

Yamamoto et al

Alogliptin Attenuates Atrial Fibrillation

outcomes despite a lower prevalence of diabetes. Recently, dipeptidyl peptidase-4 (DPP-4) inhibitors have come to be widely used as novel oral drugs for the treatment of type 2 diabetes mellitus. DPP-4 is a ubiquitously distributed glycoprotein peptidase with various biological roles.12–15 Although DPP4 inhibition by saxagliptin may increase clinical HF risk,16 a number of studies demonstrate that in addition to their glucose-lowering effects, DPP-4 inhibitors have cardiovascular pleiotropic effects, including reduction of mortality and cardiac remodeling after myocardial infarction,12 modulation of vascular tone,13 promotion of neovascularization in limb ischemia,14 and enhancement of angiogenesis and reduction of myocardial fibrosis in nonischemic diastolic left ventricular (LV) dysfunction.15 Collectively, the cardioprotective effects of DPP-4 inhibitors stem from their amelioration of ventricular and vascular function; however, the role of DPP-4 inhibition in atrial remodeling and particularly on the arrhythmogenic substrate remains unclear. Therefore, we investigated the effects of alogliptin, a commercially available and highly selective inhibitor of DPP-4,17 on AF associated with HF caused by VTP in rabbits.

1363 -2 weeks

+1 week

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Echo, EPS and sampling

PMI

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PMI

Animal model and experimental protocol Male New Zealand White rabbits (2.7–3.2 kg) were obtained from Kitayama Labs (Kyoto, Japan). Alogliptin was provided by Takeda Pharmaceutical Company Limited (Osaka, Japan). Animal studies were approved by the Institutional Animal Care and Use Committee of Nagoya University School of Medicine. Programmable right ventricular pacemakers were implanted as described previously.18 A unipolar pacing electrode (Streamline 6491, Medtronic, Minneapolis, MN) was fixed onto the free wall of the right ventricle by a right thoracotomy and connected to a pacemaker (Kappa or Sensia, Medtronic), which was implanted subcutaneously in the back. The pacemakers were programmed to pace at 380 bpm for 1 or 3 weeks after the rabbits recovered from the surgical procedure. The rabbits were divided into 6 groups. Three groups of nontreated rabbits were subjected to no pacing (n ¼ 15), VTP for 1 week (n ¼ 10), or VTP for 3 weeks (n ¼ 15) (NP/ non, 1w/non, and 3w/non, respectively), and 3 groups of alogliptin-treated rabbits were subjected to no pacing (n ¼ 15), VTP for 1 week (n ¼ 10), or VTP for 3 weeks (n ¼ 15) (NP/alo, 1w/alo, and 3w/alo, respectively). Administration of alogliptin mixed with the feed (20 mg  kg1  d1) was started 2 weeks before VTP initiation and continued throughout the study (Figure 1). Subgroups of animals were administered Nω-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor. L-NAME was dissolved in saline and administered intraperitoneally (10 mg/ kg) to rabbits in the 3w/non (n ¼ 5) and 3w/alo (n ¼ 5) groups twice per week from the initiation of VTP, and these groups were designated 3w/non þ LN and 3w/alo þ LN, respectively.

Iniaon of VTP

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Figure 1 Experimental schedule. Ventricular tachypacing (VTP) was initiated 2 weeks after pacemaker implantation (PMI) in nontreated rabbits and in rabbits treated with alogliptin and maintained for 1 week (1w/non, 1w/alo) or 3 weeks (3w/non, 3w/alo). A nitric oxide synthase inhibitor, Nωnitro-L-arginine methyl ester (L-NAME), was applied to the nontreated and treated rabbits subjected to 3 weeks of VTP (3w/non þ LN, 3w/alo þ LN). Alogliptin was administered to the nonpaced/alogliptin-treated (NP/alo), 1w/alo, 3w/alo, and 3w/alo þ LN groups by mixing it with animal feed throughout the experiment. The rabbits were examined at the indicated time points by echocardiography (Echo) and open chest electrophysiological study (EPS).

After 1 or 3 weeks of VTP, we performed echocardiographic analysis. The 3w/non and 3w/alo groups were subjected to open chest electrophysiological study (EPS). After EPS, the rabbits were euthanized, and the left atrial (LA) tissues were collected, frozen immediately in liquid nitrogen, and stored at 801C. Small pieces of LA tissue were immersed in 10% formaldehyde for subsequent histologic studies.

Echocardiography We performed transthoracic echocardiography with a SONOS 7500 instrument (Philips, Amsterdam, the Netherlands) to measure chamber dimension, wall thickness, and cardiac function on the day of VTP initiation and before animals were euthanized. The data were obtained with the M-mode image and a Doppler pulse wave.

Electrophysiological study On the day after completion of 3 weeks of VTP, we performed open-chest EPS under anesthesia with ketamine

1364 hydrochloride and isoflurane to evaluate the electrophysiological properties of the hearts of the rabbits subjected to VTP. The rabbits were mechanically ventilated and their pacemakers deactivated. Median sternotomy was performed, and custom-made bipolar electrodes were attached to the right atrial appendage (RAA) and LA appendage (LAA) for recording and stimulation. A programmable stimulator (Nihon Kohden, Tokyo, Japan) was used to deliver 3- or 4-fold-threshold currents with 2-ms pulse duration. Effective refractory period (ERP) was measured at the RAA and LAA with basic cycle lengths of 150, 200, and 250 ms with a train of 10 basic stimuli (S1), followed by a premature extra stimulus (S2) in 5-ms decrements. The ERP was defined as the longest S1–S2 interval that failed to capture the atria. The conduction velocity (CV) was calculated as the distance between the LAA and RAA divided by the time from the LAA pacing spike to RAA activation (CV ¼ distance/ conduction time). AF induction was tested by application of burst pacing (cycle length 80–120 ms). AF was defined as a rapid (4500/min) irregular atrial rhythm lasting longer than 1 second and observed as fractionated potential on the intra-atrial electrocardiogram; the periods of AF from all sessions were recorded and averaged for each rabbit.19,20

Histology and immunofluorescence staining Transverse sections of LA muscle were cut at 5-mm intervals and stained with Masson trichrome stain to evaluate the extent of fibrosis. To quantify the fibrotic area in the LA muscle, the blue pixel content of the digitized images was measured relative to the total tissue area by use of the image analyzer in Adobe Photoshop CS6 (Adobe Systems, San Jose, CA).19–21 Seven-micrometer–thick frozen sections of the LA were used for immunofluorescence staining. After fixing and blocking, the sections were incubated overnight at 41C with anti-CD31 antibodies (sc-1506, Santa Cruz Biotechnology Inc, Santa Cruz, CA) and anti-dystrophin antibodies (NCLDYS1, Leica Biosystems, Newcastle upon Tyne, United Kingdom). The secondary antibodies (Alexa Fluor 488 or Alexa Fluor 594 for the anti-CD-31 and anti-dystrophin primary antibodies, respectively; Life Technologies, Carlsbad, CA) were applied, and the stained sections were mounted with VECTASHIELD Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA). The slides were visualized with a laser scanning confocal microscope (Biozero, Keyence Corp, Osaka, Japan), which was used to measure the number of CD31-positive cells and the cardiomyocyte surface area.15

Enzyme-linked immunosorbent assay To quantify the amount of type I collagen in the rabbit LA, we performed an enzyme-linked immunosorbent assay using a rabbit type I collagen detection kit (6016, Chondrex, Inc, Redmond, WA). To solubilize the collagen in the tissue samples, they were digested with pepsin and elastase according to the manufacturer’s protocol.

Heart Rhythm, Vol 12, No 6, June 2015

Western blots Western blotting was performed to assess the effects of pharmacologic intervention on the expression of endothelial NOS (eNOS), phospho-eNOS (p-eNOS), Akt, and phosphoAkt (p-Akt), as described previously.20,21 The primary antibodies were eNOS (ab5589, Abcam, Cambridge, United Kingdom), p-eNOS (pS1177, BD Biosciences, Franklin Lake, NJ), Akt (9272, Cell Signaling Technology, Danvers, MA), and p-Akt (Ser 473) (9271, Cell Signaling Technology). After incubation with the primary antibodies, the membranes were incubated with the appropriate horseradish peroxidase–conjugated secondary antibodies (GE Healthcare, Little Chalfont, United Kingdom), developed with an enhanced chemiluminescence substrate kit (Clarity, BioRad, Hercules, CA), and exposed to X-ray film (Fujifilm, Tokyo, Japan). The bands were quantified with ImageJ software (National Institutes of Health, Bethesda, MD).

Statistical analysis Data are expressed as mean ⫾ standard error of the mean. Statistical analysis was performed by 1-way analysis of variance with Bonferroni correction using SPSS (IBM Corp, Armonk, NY). P o .05 was considered statistically significant.

Results VTP-induced chamber dilatation and systolic dysfunction The echocardiographic parameters of the 6 experimental groups (NP/non, NP/alo, 1w/non, 1w/alo, 3w/non, and 3w/ alo) are summarized in Table 1. In nontreated and alogliptintreated rabbits, 1 week of VTP significantly decreased systolic function (measured as fractional shortening and ejection fraction) and increased LA diameter and LV endsystolic diameter. LA diameter, LV end-diastolic diameter, and LV end-systolic diameter were enlarged after 3 weeks of VTP compared with 1 week of VTP. Diastolic function, evaluated by peak E-wave velocity and early mitral flow deceleration time, was similar in all study groups. Failing hearts were extremely enlarged compared with normal hearts in the nontreated and alogliptin-treated groups (Figure 2).

Alogliptin prevented VTP-related prolongation of AF We performed open chest EPS to explore arrhythmic features in response to VTP-induced HF and studied the effects of alogliptin administration on these arrhythmic features. When the chest was opened, we noticed some pleural effusions in the rabbits with HF. VTP tended to prolong ERP and decrease the CV from the LA to the right atrium, but alogliptin treatment did not affect these parameters (Figure 3A and 3B). AF was initiated more easily and sustained longer after VTP (Figure 3C). AF duration in VTP rabbits was shortened significantly by alogliptin treatment compared with nontreated VTP rabbits. Figure 3D shows representative images of AF induced by LAA burst pacing.

Yamamoto et al Table 1

Alogliptin Attenuates Atrial Fibrillation

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Echocardiographic parameters in 6 experimental groups NP/non

LAD (mm) LVDd (mm) LVDs (mm) IVST (mm) PWT (mm) FS (%) EF (%) pE (cm/s) DcT (ms)

7.2 ⫾ 12.3 ⫾ 8.2 ⫾ 2.7 ⫾ 2.7 ⫾ 33.7 ⫾ 59.1 ⫾ 41.1 ⫾ 92.9 ⫾

0.1* 0.2 0.2* 0.0 0.0 0.7* 0.9* 1.8 4.3

NP/alo

1w/non

7.6 ⫾ 0.1* 12.3 ⫾ 0.2 8.4 ⫾ 0.2* 2.7 ⫾ 0.0 2.7 ⫾ 0.0 32.4 ⫾ 0.6* 57.5 ⫾ 0.9* 42.2 ⫾ 1.0 90.6 ⫾ 2.8

10.4 ⫾ 13.5 ⫾ 10.8 ⫾ 2.7 ⫾ 2.7 ⫾ 20.0 ⫾ 38.1 ⫾ 37.9 ⫾ 83.3 ⫾

0.6† 0.1† 0.3† 0.0 0.0 1.7 2.9 2.2 4.2

1w/alo

3w/non

3w/alo

10.1 ⫾ 0.3† 14.0 ⫾ 0.3† 10.7 ⫾ 0.2† 2.7 ⫾ 0.1 2.8 ⫾ 0.1 23.4 ⫾ 1.3 43.8 ⫾ 2.1 41.2 ⫾ 1.6 81.0 ⫾ 3.4

12.3 ⫾ 0.3 18.5 ⫾ 0.4 15.3 ⫾ 0.4 2.9 ⫾ 0.1 2.8 ⫾ 0.1 17.6 ⫾ 1.0 33.6 ⫾ 1.7 34.7 ⫾ 0.3 86.5 ⫾ 13.5

12.3 ⫾ 0.7 18.1 ⫾ 0.6 14.8 ⫾ 0.9 2.7 ⫾ 0.1 2.7 ⫾ 0.1 19.0 ⫾ 2.1 35.6 ⫾ 3.5 42.0 ⫾ 2.8 85.2 ⫾ 7.5

Values are expressed as mean ⫾ SEM. 1w ¼ ventricular tachypacing for 1 week; 3w ¼ ventricular tachypacing for 3 weeks; alo ¼ groups treated with alogliptin; DcT ¼ deceleration time; EF ¼ ejection fraction; FS ¼ fractional shortening; IVST ¼ intraventricular septum thickness; LAD ¼ left atrial diameter; LVDd ¼ left ventricular end-diastolic diameter; LVDs ¼ left ventricular end-systolic diameter; NP ¼ nonpaced; non ¼ nontreated groups; pE ¼ peak E wave; PWT ¼ posterior wall thickness. * P o .05 vs 1w/non or 1w/alo. †P o .05 vs 3w/non or 3w/alo.

Alogliptin attenuated VTP-induced atrial fibrosis Representative LA tissue sections from each group are shown in Figure 4A. Interstitial fibrosis was increased over time in the LA myocardium of rabbits in response to VTP; however, atrial fibrosis was significantly attenuated in rabbits treated with alogliptin after 3 weeks of VTP (Figure 4B). The amount of type I collagen in the LA tissue of the 3w/non rabbits was significantly higher than that of the 3w/alo rabbits (Figure 4C), which corroborated the assessment of fibrosis area.

Alogliptin augmented neovascularization in LA muscle To investigate the mechanism involved in alogliptinmediated attenuation of atrial fibrosis, CD31 (an endothelial cell marker) was analyzed to examine cardiac microvessel formation. Representative images of CD31 and dystrophin immunofluorescence in LA tissue are presented in Figure 5A. Quantitative analysis of the CD31-positive cells showed that alogliptin-treated rabbits subjected to Non-paced

VTP for 1 week

VTP for 3 weeks displayed a 1.6-fold increase in capillary density per LA cardiomyocyte compared with nontreated rabbits; however, we found no significant differences in capillary density between the NP/non and NP/alo groups or between the 1w/non and 1w/alo groups (Figure 5B).

Alogliptin induced enhancement of eNOS activation in LA Representative immunoblots of eNOS, p-eNOS, Akt, and pAkt from each group are shown in Figure 6A and 6C. The eNOS phosphorylation ratio was notably higher in 1w/alo rabbits than in 1w/non rabbits; this difference corresponded with the increase in the number of CD31-positive cells in the 3w/alo group (Figure 6B). eNOS activation was similar in the NP/non, NP/alo, and 1w/non groups. In contrast, there were no differences in Akt phosphorylation in the NP/non, NP/alo, 1w/non, and 1w/alo groups (Figure 6D). Interestingly, 3 weeks of VTP reduced activation of eNOS and Akt, regardless of treatment. VTP for 3 weeks

Figure 2 Images of whole hearts from nonpaced animals and those that were subjected to ventricular tachypacing (VTP) for 1 or 3 weeks, as indicated. Left, nonpaced; middle, VTP for 1 week; right, VTP for 3 weeks.

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(ms)

LA ERP

100

† †

80

† †

†

60 40

Conducon velocity in atria

120

1.2 1.0 0.8

(s) 12

† †

† †

NP/non NP/alo 3w/non

0.6

3w/alo

0.4

150 200 250 Basic cycle length

AF duraon in LA

Discussion

(m/s)

150

200

250

Basic cycle length

PVHF

The present study was the first to identify beneficial effects of a DPP-4 inhibitor on AF in a rabbit model of VTP-induced HF. Administration of alogliptin, a highly specific DPP-4 inhibitor, shortened the duration of AF induced by atrial burst pacing and reduced atrial tissue fibrosis. Furthermore, we found that angiogenesis and eNOS expression modified atrial fibrosis. It has been reported that congestive HF caused by VTP in an animal model generated atrial interstitial fibrosis, leading to electrical conduction heterogeneity and disturbance, and eventually to formation of an AF substrate3,4,6–8; these reports are consistent with the findings of this study. Therefore, therapeutic interventions targeting atrial fibrosis may

,,

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8 6

/$$

non

alo

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Application of L-NAME blocked the beneficial effects of alogliptin on AF duration, atrial fibrosis, and capillary density L-NAME,

an NOS inhibitor, was administered to animals in the 3w/non and 3w/alo groups (3w/non þ LN and 3w/alo þ LN, respectively). Echocardiography revealed that VTP for 3 weeks (even with L-NAME) increased LA diameter and decreased ejection fraction, although there were no significant differences in LA diameter or ejection fraction between the 3w/non, 3w/alo, 3w/non þ LN, and 3w/alo þ LN groups (Figure 7A and 7B). EPS did not identify significant differences in AF duration between the 3w/non þ LN and 3w/alo þ LN groups. L-NAME blocked the shortening effect of alogliptin (Figure 7C) and alogliptinmediated attenuation of VTP-induced fibrosis and augmentation of neovascularization (Figure 7D and 7F). There were no significant differences in the amount of type I collagen between the 3w/non þ LN and 3w/alo þ LN groups. Moreover, type I collagen abundance was increased significantly by L-NAME compared with the other 6 groups (Figure 7E).22

1w

3w 100 μm

B

C

(%) 15

* N.S.

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1.5

Collagen I level

Figure 3 Electrophysiological study. A: Left atrium (LA) effective refractory period (ERP) at basic cycle lengths of 150, 200, and 250 ms for the nonpaced controls with (NP/alo) and without (NP/non) alogliptin treatment and rabbits subjected to ventricular tachypacing for 3 weeks with (3w/alo) and without (3w/non) treatment. B: Intra-atrial conduction velocity from the LA appendage (LAA) to the right atrial appendage (RAA) of the indicated study groups at basic cycle lengths of 150, 200, and 250 ms. C: Duration of induced atrial fibrillation (AF) in each group. D: Representative image of AF induced by burst pacing at LAA, recorded by the limb lead (II) and intracardiac leads at RAA and LAA. Values are mean ⫾ standard error (bars). *P o .05 for the comparison indicated; †P o .05 vs 3w/non; P o .05 vs NP/non; n ¼ 15 in each group. alo ¼ treated with alogliptin; non ¼ nontreated; NP ¼ nonpaced; N.S. ¼ nonsignificant.

% fibrosis area

NP

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5

0

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0

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3w

Figure 4 Analysis of atrial fibrosis and collagen I level. A: Representative left atrial tissue sections from each group subjected to Masson trichrome staining. Sections are shown from the nonpaced controls without alogliptin treatment (NP/non, left upper panel) and with alogliptin treatment (NP/alo, right upper panel), from rabbits subjected to ventricular tachypacing for 1 week without treatment (1w/non, left middle panel) and with alogliptin treatment (1w/alo, right middle panel), and from rabbits subjected to ventricular tachypacing for 3 weeks without alogliptin treatment (3w/non, left lower panel) and with alogliptin treatment (3w/alo, right lower panel). Magnification, 400. Scale bar ¼ 100 mm. B: Evaluation of the proportion of fibrotic area of each group. C: Evaluation of the amount of type I collagen quantified by enzyme-linked immunosorbent assay in each group. Values are mean ⫾ standard error (bars). *P o .05, P o .05 vs NP/non and NP/alo; n ¼ 15 (NP/non, NP/alo, 3w/non, and 3w/alo), n ¼ 10 (1w/non and 1w/alo). Other abbreviations as in Figure 3.

Yamamoto et al

A

Alogliptin Attenuates Atrial Fibrillation non

suppressed by microvasculature expansion.24 In this study, we found that administration of alogliptin increased LA capillary density in VTP rabbits, and this neovascularization may have contributed to alogliptin-mediated inhibition of atrial fibrosis. Recent research has shown that some proteins and enzymes play pivotal roles in cardiac fibrosis, including mitogen-activated protein kinase,7,19,20 angiotensin II,7 vascular endothelial growth factor,21 tumor necrosis factoralpha,19,20,25 AMP (adenosine monophosphate)-activated protein kinase,21 matrix-degrading metalloproteinases,25 and eNOS.26 Because the actions of DPP-4 inhibitors are too complex to resolve completely, various molecular changes may have contributed to the pharmacologic effects observed in the present study. DPP-4 is expressed abundantly in endothelial cells, and some reports showed that augmentation of neovascularization by DPP-4 inhibitors was correlated with endothelial function and activation of eNOS signaling.13,14,27 Therefore, we analyzed eNOS expression in atrial tissue. eNOS phosphorylation ratio was increased markedly in the 1w/alo group compared with the 1w/non group, whereas Akt activation did not differ significantly between these groups. Subsequently, we added L-NAME to inhibit NOS and to investigate the involvement of eNOS in the

alo

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B 0.4 0.3

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alo

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Figure 5 Assessment of capillary density. A: Representative images of CD31 and dystrophin immunofluorescence staining of left atrium tissue from nonpaced controls without alogliptin treatment (NP/non, left upper panel) and with alogliptin treatment (NP/alo, right upper panel), from rabbits subjected to ventricular tachypacing for 1 week without treatment (1w/non, left middle panel) and with alogliptin treatment (1w/alo, right middle panel), and from rabbits subjected to ventricular tachypacing for 3 weeks without treatment (3w/non, left lower panel) and with alogliptin treatment (3w/alo, right lower panel). Magnification, 800. Scale bar ¼ 50 mm. B: Quantitative analysis of CD31 signal per cardiomyocyte from each group. Values are mean ⫾ standard error (bars). *P o .05, P o .05 vs NP/non and NP/alo; n ¼ 15 (NP/non, NP/alo, 3w/non, and 3w/alo), n ¼ 10 (1w/non and 1w/alo). Other abbreviations as in Figure 3.

p-eNOS

p-Akt

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4

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3 1.0

2

0.5 1

effectively prevent AF. Echocardiographic investigation showed that VTP (even for 1 week) induced LV systolic dysfunction and dilation of the LA and LV, and alogliptin administration did not alter this effect. Furthermore, VTP and alogliptin did not affect diastolic function. VTP-induced HF is similar to the condition of tachycardia-induced cardiomyopathy, which is mainly associated with systolic dysfunction.23 EPS showed that VTP reduced the intra-atrial CV, although there were no significant differences between the 3w/non and 3w/alo groups. Atrial dilatation and intraatrial conduction delay were observed in both 3-week-VTP groups; however, AF duration and LA fibrosis were correlated with the amount of type I collagen and were significantly reduced in duration and area, respectively, in alogliptin-treated rabbits compared with nontreated rabbits. Some reports have indicated that cardiac fibrosis can be

0

0.0

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1w

3w

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1w

3w

Figure 6 Evaluation of endothelial nitric oxide synthase (eNOS) and Akt activation. A and C: Representative immunoblots of phosphorylated eNOS (p-eNOS) and eNOS (t-eNOS; A) and phosphorylated Akt (p-Akt) and Akt (t-Akt; C) in atrial tissue extracts. B and D: Densitometric analysis of relative changes in the phosphorylation ratio of eNOS (B) and Akt (D) in nonpaced controls (NP) without alogliptin treatment (non) and with alogliptin treatment (alo), rabbits subjected to ventricular tachypacing for 1 week (1w) without treatment and with alogliptin treatment, and rabbits subjected to ventricular tachypacing for 3 weeks (3w) without treatment and with alogliptin treatment. Values are mean ⫾ standard error (bars). *P o .05; n ¼ 15 (NP/non, NP/alo, 3w/non, and 3w/alo), n ¼ 10 (1w/non and 1w/ alo). N.S. ¼ nonsignificant.

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3w +LN

Figure 7 The effects of nitric oxide synthase inhibition. Left atrial diameter (A) and left ventricular ejection fraction (B) measured by echocardiography in the indicated study groups. Atrial fibrillation (AF) duration (C), percent fibrosis area (D), type I collagen level (E), and number of CD31-positive cells (F) in the indicated study groups. Values are mean ⫾ standard error (bars). *P o .05; n ¼ 15 (NP/non, NP/alo, 3w/non, and 3w/alo), n ¼ 10 (1w/non and 1w/alo), and n ¼ 5 (3w/non þ LN and 3w/alo þ LN). þLN ¼ rabbits given Nω-nitro-L-arginine methyl ester. Other abbreviations as in Figure 3.

beneficial effects of alogliptin. L-NAME blocked the favorable effects of alogliptin in VTP rabbits; L-NAME reversed the reduction in AF duration and fibrosis area by alogliptin and increased capillary density in alogliptin-treated animals. These results support the importance of eNOS activation in the prevention of AF, atrial fibrosis, and neovascularization by alogliptin. There were several limitations to this study. First, we only examined the Akt-eNOS axis in our investigation of suppression of AF by DPP-4 inhibitors. Anti-inflammatory actions are involved in the mechanism whereby proliferator-activated receptor-γ activator and statins suppress AF,19,28 and there are reports that DPP-4 inhibitors have anti-inflammatory effects.29,30 Therefore, the anti-inflammatory effects of DPP-4 inhibitors may have attenuated AF promotion in our study, and further detailed biochemical investigations are required to understand these mechanisms. Second, we did not assess DPP-4 activity or plasma glucagon-like peptide-1 level, although the dosage of alogliptin used was greater than the usual human dosage.31 Third, the duration of the induced AF was comparatively short, even in VTP rabbits, probably because of the small size of the rabbit atria; however, AF duration was reproducibly prolonged

in rabbits with VTP-induced HF and reproducibly reduced by treatment with alogliptin. Furthermore, AF duration was reproducibly increased by L-NAME, even in alogliptin-treated rabbits. Finally, we must be careful when translating these results to clinical practice, because there are species differences between rabbits and humans, and the rabbit model used in this study was not diabetic. There may be species differences in the fundamental mechanisms involved in AF caused by differences in heart rate, atrial size, and atrial ion channel expression.

Conclusion Alogliptin disrupted VTP-induced AF promotion caused by the accumulation of fibrotic atrial tissue by augmenting atrial angiogenesis and enhancing eNOS activation. Our findings suggest that DPP-4 inhibitors may be useful for the prevention of VTP-induced AF.

Acknowledgements We thank Takeda Pharmaceutical Co Ltd for providing alogliptin, Medtronic Japan for supplying the pacemakers,

Yamamoto et al

Alogliptin Attenuates Atrial Fibrillation

St. Jude Medical Japan for technical support, and Editage (www.editage.jp) for English language editing. Finally, the author would like to express heartfelt gratitude to Yoko Inoue, Hao Changning, and Wu Hongxian for technical advice and Fukiko Yamamoto for unconditional support.

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References 1. Heeringa J, van der Kuip DA, Hofman A, Kors JA, van Herpen G, Stricker BH, Stijnen T, Lip GY, Witteman JC. Prevalence, incidence and lifetime risk of atrial fibrillation: the Rotterdam study. Eur Heart J 2006;27:949–953. 2. Wang TJ, Larson MG, Levy D, Vasan RS, Leip EP, Wolf PA, D'Agostino RB, Murabito JM, Kannel WB, Benjamin EJ. Temporal relations of atrial fibrillation and congestive heart failure and their joint influence on mortality: the Framingham Heart Study. Circulation 2003;107:2920–2925. 3. Li D, Fareh S, Leung TK, Nattel S. Promotion of atrial fibrillation by heart failure in dogs: atrial remodeling of a different sort. Circulation 1999;100:87–95. 4. Burstein B, Nattel S. Atrial fibrosis: mechanisms and clinical relevance in atrial fibrillation. J Am Coll Cardiol 2008;51:802–809. 5. Allessie M, Ausma J, Schotten U. Electrical, contractile and structural remodeling during atrial fibrillation. Cardiovasc Res 2002;54:230–246. 6. Shinagawa K, Shi YF, Tardif JC, Leung TK, Nattel S. Dynamic nature of atrial fibrillation substrate during development and reversal of heart failure in dogs. Circulation 2002;105:2672–2678. 7. Li D, Shinagawa K, Pang L, Leung TK, Cardin S, Wang Z, Nattel S. Effects of angiotensin-converting enzyme inhibition on the development of the atrial fibrillation substrate in dogs with ventricular tachypacing-induced congestive heart failure. Circulation 2001;104:2608–2614. 8. Kumagai K, Nakashima H, Urata H, Gondo N, Arakawa K, Saku K. Effects of angiotensin II type 1 receptor antagonist on electrical and structural remodeling in atrial fibrillation. J Am Coll Cardiol 2003;41:2197–2204. 9. Aksnes TA, Schmieder RE, Kjeldsen SE, Ghani S, Hua TA, Julius S. Impact of new-onset diabetes mellitus on development of atrial fibrillation and heart failure in high-risk hypertension (from the VALUE Trial). Am J Cardiol 2008;101: 634–638. 10. Du X, Ninomiya T, de Galan B, et al; ADVANCE Collaborative Group. Risks of cardiovascular events and effects of routine blood pressure lowering among patients with type 2 diabetes and atrial fibrillation: results of the ADVANCE study. Eur Heart J 2009;30:1128–1135. 11. Mountantonakis SE, Grau-Sepulveda MV, Bhatt DL, Hernandez AF, Peterson ED, Fonarow GC. Presence of atrial fibrillation is independently associated with adverse outcomes in patients hospitalized with heart failure: an analysis of get with the guidelines-heart failure. Circ Heart Fail 2012;5:191–201. 12. Sauvé M, Ban K, Momen MA, Zhou YQ, Henkelman RM, Husain M, Drucker DJ. Genetic deletion or pharmacological inhibition of dipeptidyl peptidase-4 improves cardiovascular outcomes after myocardial infarction in mice. Diabetes 2010;59:1063–1073. 13. Shah Z, Pineda C, Kampfrath T, Maiseyeu A, Ying Z, Racoma I, Deiuliis J, Xu X, Sun Q, Moffatt-Bruce S, Villamena F, Rajagopalan S. Acute DPP-4 inhibition modulates vascular tone through GLP-1 independent pathways. Vascul Pharmacol 2011;55:2–9. 14. Huang C-Y, Shih CM, Tsao NW, et al. Dipeptidyl peptidase-4 inhibitor improves neovascularization by increasing circulating endothelial progenitor cells. Br J Pharmacol 2012;167:1506–1519. 15. Shigeta T, Aoyama M, Bando YK, Monji A, Mitsui T, Takatsu M, Cheng XW, Okumura T, Hirashiki A, Nagata K, Murohara T. Dipeptidyl peptidase-4

18.

19.

20.

21.

22.

23. 24. 25.

26.

27.

28.

29.

30.

31.

modulates left ventricular dysfunction in chronic heart failure via angiogenesisdependent and -independent actions. Circulation 2012;126:1838–1851. Scirica BM, Bhatt DL, Braunwald E, et al. SAVOR-TIMI 53 Steering Committee and Investigators. Saxagliptin and cardiovascular outcomes in patients with type 2 diabetes mellitus. N Engl J Med 2013;369:1317–1326. Feng J, Zhang Z, Wallace MB, et al. Discovery of alogliptin: a potent, selective, bioavailable, and efficacious inhibitor of dipeptidyl peptidase IV [published correction appears in J Med Chem 2008;51:4357]. J Med Chem 2007;50: 2297–2300. Tsuji Y, Opthof T, Yasui K, Inden Y, Takemura H, Niwa N, Lu Z, Lee JK, Honjo H, Kamiya K, Kodama I. Ionic mechanisms of acquired QT prolongation and torsades de pointes in rabbits with chronic complete atrioventricular block. Circulation 2002;106:2012–2018. Shimano M, Tsuji Y, Inden Y, Kitamura K, Uchikawa T, Harata S, Nattel S, Pioglitazone Murohara T. a peroxisome proliferator-activated receptor-gamma activator, attenuates atrial fibrosis and atrial fibrillation promotion in rabbits with congestive heart failure. Heart Rhythm 2008;5:451–459. Kitamura K, Shibata R, Tsuji Y, Shimano M, Inden Y, Murohara T. Eicosapentaenoic acid prevents atrial fibrillation associated with heart failure in a rabbit model. Am J Physiol Heart Circ Physiol 2011;300:H1814–H1821. Shimano M, Ouchi N, Shibata R, Ohashi K, Pimentel DR, Murohara T, Walsh K. Adiponectin deficiency exacerbates cardiac dysfunction following pressure overload through disruption of an AMPK-dependent angiogenic response. J Mol Cell Cardiol 2010;49:210–220. Sonia Z, Blanca AJ, Ruiz-Armenta MV, Miguel-Carrasco JL, Arévalo M, Vázquez MJ, Mate A, Vázquez CM. L-Carnitine protects against arterial hypertension-related cardiac fibrosis through modulation of PPAR-γ expression. Biochem Pharmacol 2013;85:937–944. Gallagher JJ. Tachycardia and cardiomyopathy: the chicken-egg dilemma revisited. J Am Coll Cardiol 1985;6:1172–1173. Zeisberg EM, Tarnavski O, Zeisberg M, et al. Endothelial-to-mesenchymal transition contributes to cardiac fibrosis. Nat Med 2007;13:952–961. Porter KE, Turner NA, O'Regan DJ, Ball SG. Tumor necrosis factor alpha induces human atrial myofibroblast proliferation, invasion and MMP-9 secretion: inhibition by simvastatin. Cardiovasc Res 2004;64:507–515. Kazakov A, Hall R, Jagoda P, Bachelier K, Müller-Best P, Semenov A, Lammert F, Böhm M, Laufs U. Inhibition of endothelial nitric oxide synthase induces and enhances myocardial fibrosis. Cardiovasc Res 2013;100:211–221. Ishii M, Shibata R, Kondo K, Kambara T, Shimizu Y, Tanigawa T, Bando YK, Nishimura M, Ouchi N, Murohara T. Vildagliptin stimulates endothelial cell network formation and ischemia-induced revascularization via an endothelial nitric-oxide synthase-dependent mechanism. J Bio Chem 2014;289: 27235–27245. Kumagai K, Nakashima H, Saku K. The HMG-CoA reductase inhibitor atorvastatin prevents atrial fibrillation by inhibiting inflammation in a canine sterile pericarditis model. Cardiovasc Res 2004;62:105–111. Dobrian AD, Ma Q, Lindsay JW, Leone KA, Ma K, Coben J, Galkina EV, Nadler JL. Dipeptidyl peptidase IV inhibitor sitagliptin reduces local inflammation in adipose tissue and in pancreatic islets of obese mice. Am J Physiol Endocrinol Metab 2011;300:E410–E421. Shah Z, Kampfrath T, Deiuliis JA, et al. Long-term dipeptidyl-peptidase 4 inhibition reduces atherosclerosis and inflammation via effects on monocyte recruitment and chemotaxis. Circulation 2011;124:2338–2349. Christopher R, Covington P, Davenport M, Fleck P, Mekki QA, Wann ER, Karim A. Pharmacokinetics, pharmacodynamics, and tolerability of single increasing doses of the dipeptidyl peptidase-4 inhibitor alogliptin in healthy male subjects. Clin Ther 2008;30(3):513–527.

CLINICAL PERSPECTIVES This experimental research suggests that DPP-4 inhibitors have new therapeutic applications, specifically via their suppressive effect on AF promotion in HF patients, which are mediated by suppression of atrial fibrosis, augmentation of atrial angiogenesis, and enhancement of eNOS activation. Furthermore, DPP-4 inhibitors may decrease the risk of AFrelated cardiovascular events.