Sodium 4-Phenylbutyrate Attenuates Myocardial Reperfusion Injury by Reducing the Unfolded Protein Response

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Reperfusion Injury by Reducing the Unfolded Protein Response

著者鷹取治

著者別表示 Takatori Osamu journal or

publication title

博士論文本文Full 学位授与番号 13301乙第2080号

学位名博士（医学）

学位授与年月日 2017‑03‑22

URL http://hdl.handle.net/2297/00049317

doi: 10.1177/1074248416679308.

Creative Commons : 表示 ‑ 非営利 ‑ 改変禁止 http://creativecommons.org/licenses/by‑nc‑nd/3.0/deed.ja

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Article

Sodium 4-Phenylbutyrate Attenuates Myocardial Reperfusion Injury by

Reducing the Unfolded Protein Response

Osamu Takatori, MD

¹

, Soichiro Usui, PhD

¹

, Masaki Okajima, PhD

¹

,

Shuichi Kaneko, PhD

¹

, Hiroshi Ootsuji, PhD

¹

, Shin-ichiro Takashima, PhD

¹

, Daisuke Kobayashi, PhD

¹

, Hisayoshi Murai, PhD

¹

,

Hiroshi Furusho, PhD

¹

, and Masayuki Takamura, PhD

¹

Abstract

Background: The unfolded protein response (UPR) plays a pivotal role in ischemia–reperfusion (I/R) injury in various organs such as heart, brain, and liver. Sodium 4-phenylbutyrate (PBA) reportedly acts as a chemical chaperone that reduces UPR.

In the present study, we evaluated the effect of PBA on reducing the UPR and protecting against myocardial I/R injury in mice.

Methods:Male C57BL/6 mice were subjected to 30-minute myocardial I/R, and were treated with phosphate-buffered saline (as a vehicle) or PBA.Results:At 4 hours after reperfusion, mice treated with PBA had reduced serum cardiac troponin I levels and numbers of apoptotic cells in left ventricles (LVs) in myocardial I/R. Infarct size had also reduced in mice treated with PBA at 48 hours after reperfusion. At 2 hours after reperfusion, UPR markers, including eukaryotic initiation of the factor 2a-subunit, activating transcription factor-6, inositol-requiring enzyme-1, glucose-regulated protein 78, CCAAT/enhancer-binding protein (C/EBP) homologous protein, and caspase-12, were significantly increased in mice treated with vehicle compared to sham-operated mice.

Administration of PBA significantly reduced the I/R-induced increases of these markers. Cardiac function and dimensions were assessed at 21 days after I/R. Sodium 4-phenylbutyrate dedicated to the improvement of cardiac parameters deterioration including LV end-diastolic diameter and LV fractional shortening. Consistently, PBA reduced messenger RNA expression levels of cardiac remodeling markers such as collagen type 1a1, brain natriuretic peptide, andaskeletal muscle actin in LV at 21 days after I/R.

Conclusion:Unfolded protein response mediates myocardial I/R injury. Administration of PBA reduces the UPR, apoptosis, infarct size, and preserved cardiac function. Hence, PBA may be a therapeutic option to attenuate myocardial I/R injury in clinical practice.

Keywords

myocardial ischemia–reperfusion injury, unfolded protein response, apoptosis, sodium 4-phenylbutyrate

Introduction

Myocardial reperfusion therapy is essential for salvaging ischemic myocardium in clinical situations. Percutaneous coronary intervention for acute coronary syndrome (ACS) reduces the mortality rate and the incidence of recurrent ischemia.¹ However, reperfusion also has been reported to damage some of the myocytes that survive the ischemic insult, so-called lethal ischemia–reperfusion (I/R) injury.²Potential causes of this injury include generation of reactive oxygen species, distur- bances in intracellular calcium, rapid pH restoration, and inflammation. A potential new cause of I/R injury is the unfolded protein response (UPR). This response occurs in myocardial I/R in vitro and in vivo and induces myocardial apoptosis.^3-5

Under optimal conditions, the endoplasmic reticulum (ER) is the principal site of folding, synthesis, and modification of numerous proteins. The accumulation of unfolded or misfolded proteins and structural mutations of nascent proteins in the ER

is known as ER stress. When it presents, this stress demands the protein-folding capacity found in the ER and induces the UPR, which is composed of 3 signaling branches beginning by ER transmembrane protein sensors. These include protein kinase R-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1), which serve as the proximal effectors of the UPR.⁶The ER first responds to ER stresses by releasing molecular chaperones including

1Department of Disease Control and Homeostasis, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan

Manuscript submitted:February 10, 2014;accepted:September 06, 2016.

Corresponding Author:

Masaki Okajima, Department of Disease Control and Homeostasis, Kanazawa University Graduate School of Medical Science, Kanazawa 9200942, Japan.

Email: mmokaji@gmail.com

Journal of Cardiovascular Pharmacology and Therapeutics 1-10

ªThe Author(s) 2016 Reprints and permission:

sagepub.com/journalsPermissions.nav DOI: 10.1177/1074248416679308 cpt.sagepub.com

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glucose-regulated protein 78 (GRP78), one of the well-studied molecular chaperones, to maintain protein-folding quality. Upon removal of the chaperones, PERK oligomerizes and invites the phosphorylation of eukaryotic initiation of the factor 2a-subunit (eIF2a), leading to translational arrest and the suppression of protein synthesis. Activating transcription factor 6 relocates to the Goldi and is cleaved by proteases, resulting in activating gene expression that promotes transcription of protein-folding chaperones and enzymes. Inositol-requiring enzyme 1 oligomerizes and splices the messenger RNA (mRNA) that encodes active X box-binding protein 1, which is the transcription factor that induces numerous ER stress response genes.⁷

However, if these adaptive responses fail, the ER triggers apoptosis to protect the organism by eliminating the damaged cells. The apoptosis is mediated by the induction of C/EBP homologous protein (CHOP) or activation of c-Jun NH2- terminal kinases (JNK) and/or caspase-12-dependent pathway.^8,9 This apoptotic response has been demonstrated in various diseases including diabetes, cerebral infarction, hepatic I/R injury, and cardiovascular diseases.^10-15It is of interest that Miyazaki et al found that the oxidative stress caused by reperfusion activates the UPR pathway in myocardial I/R in CHOP- deficient mice.¹⁰They also demonstrated that apoptotic cardiomyocytes in myocardial I/R were most evident at 4 hours after reperfusion, and myocardial I/R activated UPR markers such as GRP78, CHOP, and phosphorylated eIF2a (P-eIF2a), which were elevated as early as 30 minutes to 2 hours after reperfusion.

Sodium 4-phenylbutyrate (PBA) is a low-molecular- weight fatty acid that is used for the treatment of urea cycle disorders in clinical settings.¹⁶Sodium 4-phenylbutyrate was known to act as a chemical chaperone by reducing UPR in cardiovascular disease, diabetes, cerebral infarction, and hepatic I/R injury.11-13,17-19

In the current study, we evaluated whether PBA decreases infarct size and number of apoptotic cells and improves cardiac function deterioration in mice by attenuating the UPR after myocardial I/R.

Methods Animal Care

All animal experiments were approved by the institutional and governmental animal research committees and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals in Kanazawa University, which strictly conforms to Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996).

Myocardial I/R Protocol

Male C57BL/6 mice (8-12 weeks of age; Charles River Labora- tories, Yokohama, Japan) were intraperitoneally administered either phosphate-buffered saline (PBS) as vehicle or PBA (100 mg/kg; Calbiochem, Billerica, Massachusetts) at 30 minutes before surgery.12,13,19,20

The mice were subjected to I/R or sham

operation as described elsewhere.²¹Briefly, a lateral thoracotomy was performed in anesthetized and ventilated mice to expose the heart, and a 7-0 suture was looped under the left coronary artery (LCA) for induction of a transient myocardial ischemia. After 30 minutes, the LCA was reperfused by releasing the ligature.

A sham operation included all procedures except ligation of the LCA.

At 2 hours after reperfusion, Western blot analysis of UPR markers was performed in the samples of LV (sham with vehicle, sham with PBA, I/R with vehicle, and I/R with PBA, n¼5 per group). At 4 hours after reperfusion, the blood samples of mice were collected and the serums were used for enzyme- linked immunosorbent assay (ELISA) of serum cardiac troponin I (cTnI; sham with vehicle, sham with PBA, I/R with vehicle, and I/R with PBA, n¼5 per group). At the same time, the left ventricles (LVs) of mice were used for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (I/R with vehicle and I/R with PBA, n¼5 per group). At 48 hours after reperfusion, triphenyltetrazolium chloride (TTC) staining was performed (I/R with vehicle and I/R with PBA, n¼8 per group). At 21 days after I/R, echocardiography was performed and lung weight was measured (sham with vehicle, sham with PBA, I/R with vehicle, and I/R with PBA, n¼10 per group). Half of their LVs (I/R with vehicle and I/R with PBA, n¼5 per group) were used for Azan staining, and another half (sham with vehicle, sham with PBA, I/R with vehicle, and I/R with PBA, n¼5 per group) were used for quantitative real-time polymerase chain reaction (PCR) assay to evaluate cardiac remodeling markers.

Cardiac Troponin I Measurements

We collected the blood samples 4 hours after reperfusion. The serum was measured by using a high-sensitive mouse cTnI ELISA kit (Life Diagnosis, West Chester, Pennsylvania), as an index of myocardial damage.

Triphenyltetrazolium Chloride Staining

Triphenyltetrazolium chloride staining was performed to evaluate the volume of myocardial infarction (MI). At 48 hours after reperfusion, mice were reanesthetized and LCA was occluded with same suture. Evans Blue (Sigma-Aldrich, St. Louis, Missouri) was injected to delineate area at risk (AAR). Then the hearts were removed and sectioned from apex to base (4-5 slices). All slices were incubated at 37C with 1% 2,3,4-triphenyltetrazolium chloride (Wako, Osaka, Japan) for 15 minutes. The volume of AAR, infarct area (IA), and LV were multiplied by the weight of the section and totaled from all sections, performed by a blinded observer.

Analysis of Myocardial Apoptosis

The mice were killed using pentobarbital sodium 4 hours after reperfusion. We qualitatively analyzed myocardial apoptosis using TUNEL assay (Takara, Shiga, Japan). Briefly, deparaffinized

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sections were incubated with proteinase K, and DNA fragments were labeled with fluorescein-conjugated dUTP using terminal deoxynucleotidyl transferase (TdT) (Roche Molecular Biochem- icals, Mannheim, Germany). Nuclear density was determined by counting of 4⁰-6-diamidino-2-phenylindole-stained nuclei in 10 fields for each animal using the 20objective, using BIOREVO BZ-9000 fluorescence microscopy (Keyence, Japan). The number of TUNEL-positive nuclei was counted by examination of the whole LV section, using the same power objective. The counting was performed by a blinded observer.²²

Western Blot Analysis

Western blot analysis was performed to evaluate UPR markers.

At 2 hours after reperfusion, the hearts of mice were removed.

The LV homogenates were prepared in radioimmunoprecipita- tion assay (RIPA) lysis buffer (Upstate, Lake Placid, New York) containing 50 mM Tris (pH 7.5), 150 mM NaCl, 1%

IGEPAL CA-630, 0.1%sodium dodecyl sulfate (SDS), deoxy- cholic acid, 10 mM Na4P2O7, 5 mM EDTA, 0.1 mM Na3VO4, 1 mM NaF, 0.5mg/mL aprotinin, and 0.5mg/mL leupeptin. Equal amount of proteins were loaded, separated by electrophoresis on SDS-polyacrylamide gels, and transferred onto a polyviny- lidene fluoride membrane. The following primary antibodies were used: immunoglobulin heavy-chain binding protein as GRP78, eIF2a(Cell Signaling Technology, Danvers, Massachu- setts), P-eIF2a (Abcam, Cambridge, Massachusetts), ATF6, IRE1, phosphorylated IRE1 (P-IRE1; Novus Biologicals, Little- ton, Colorado), caspase-12 (Sigma-Aldrich), growth arrest- and DNA damage-inducible gene 153 as CHOP, and glyceraldehyde- 3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnol- ogy, Santa Cruz, California). Quantification was performed using the ImageJ software [version 1.45] (NIH).

Echocardiographic Assessment of LV Function

Mice were lightly anesthetized at 21 days after operation, intraperitoneally administered with 30 mg/kg of 10%sodium pentobarbital. Transthoracic echocardiography was performed on the LV using a 15 to 8 MHz linear transducer interfaced with a Sonos 5500 (Agilent Technologies [Santa Clara, California], Philips [Amsterdam, Netherlands]) by an observer who was blinded to the experimental groups. Left ventricle end-diastolic diameters (LVEDDs) and LV end-systolic diameters (LVESDs) were assessed in all groups of mice. Left ventricle fractional shortening (LVFS) was calculated according to the following equation: LVFS¼([LVEDDþLVESD]/LVEDD)100.

Histological Examination

At 21 days after I/R, mice were reanesthetized and the hearts were removed. Serial horizontal sections of formalin-fixed, paraffin-embedded LV of the hearts at the level of the maximal diameter were subjected to Azan staining for detection of fibrosis. The fibrotic areas were quantified in the sections using the ImageJ software (NIH) performed by a blinded observer.²³

Isolation of mRNA and Quantitative Real-Time PCR

At 21 days after I/R, total RNA was isolated using RNeasy (Qiagen [Germantown, Maryland]) from the LV according to the manufacturer’s protocol. Total RNA (1mg) was used to generate complementary DNA using ReverTra Ace (Toyobo, Osaka, Japan), according to the manufacturer’s protocol.

Real-time quantitative PCR analysis was performed to evaluate cardiac remodeling markers, using the ABI PRISM 7300 sequence detection system (Applied Biosystems, Foster, Cali- fornia). The following primers and TaqMan probes (Applied Biosystems) were used: collagen type 1a1 (col1a1;

ID#Mm00801666_g1), brain natriuretic peptide (BNP; Nppb ID#Mm01255770_g1), and alpha skeletal muscle actin (aSMA; Acta1 ID#Mm00808212_g1); GAPDH (Gapdh ID#

Mm99999915_g1) was used as an endogenous control.

Statistical Analysis

The data are presented as the mean +standard error of the mean for each group and were assessed by analysis of variance with Bonferroni post hoc test for multiple comparisons. The significance of the differences between the 2 groups was determined using Student t test. P values < .05 were considered statistically significant. All statistical analyses were performed using GraphPad Prism v5.04 software.

Results

Sodium 4-Phenylbutyrate Did Not Affect Hemodynamic Parameters

Male C57BL/6 mice were intraperitoneally administered PBS as vehicle or 100 mg/kg of PBA without any operation. Sodium 4-phenylbutyrate administration did not affect heart rate and systolic/diastolic blood pressure at each time point between the 2 groups (Supplementary Table 1). These results indicate that there were no significant changes in hemodynamic parameters with PBA administration.

Sodium 4-Phenylbutyrate Reduced Infarct Size After Myocardial I/R

To investigate whether PBA administration reduces infarct size after I/R, we examined serum cTnI at 4 hours after reperfusion. The level of cTnI was significantly increased in I/R and lower in PBA-treated mice than in vehicle-treated mice (Figure 1A and Supplementary Table 2). We also assessed infarct size using TTC staining at 48 hours after reperfusion (Figure 1B). The AAR to LV ratio was similar in the PBA and vehicle groups (54.3%+4.4%vs 53.8%+ 2.0%, no significant difference; Figure 1C). This finding indicated that the LCA ligation levels were similar between the 2 groups. The IA to AAR ratio was significantly lower in the PBA group than in the vehicle group (17.0%+1.8%

vs 33.7%+2.3%,P< .01; Figure 1D). The IA to LV ratio was also lower in the PBA group than in the vehicle group

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(8.9% + 0.8% vs 17.9% + 0.9%, P < .01; Figure 1E).

These results suggest that PBA administration attenuates MI in myocardial I/R.

Sodium 4-Phenylbutyrate Reduced the Numbers of Apoptotic Cells After Myocardial I/R

We performed TUNEL staining on the myocardium of mice treated with PBA and vehicle at 4 hours after reperfusion for the evaluation of apoptosis (Figure 2A). There were significantly fewer TUNEL-positive cells in the PBA group than in the vehicle group (4.9% +0.4% vs 8.6% +1.6%,P < .05;

Figure 2B). These results suggest that apoptosis occurs at 4 hours after myocardial I/R and that PBA administration reduces I/R-induced apoptosis.

Sodium 4-Phenylbutyrate Attenuated the UPR After Myocardial I/R

To evaluate whether PBA was acting as a chemical chaperone, we first assessed the protein levels of GRP78, CHOP, and P-eIF2a in the whole homogenates of the LV, which were collected 2 hours after reperfusion. The protein levels of GRP78, CHOP, and P-eIF2a were elevated in I/R, and these markers were significantly lower in the PBA group than in the vehicle group (Figure 3A, B-D). Second, we assessed the protein levels of caspase-12, ATF6, and P-IRE1. These UPR markers were also elevated in I/R and were significantly lower in the PBA group than in the vehicle group (Figure 3A, E-G).

These results suggest that in myocardial I/R, the UPR is induced at 2 hours after reperfusion, and PBA administration generally reduces I/R-induced UPR.

Sodium 4-Phenylbutyrate Preserved Cardiac Function After Myocardial I/R

To investigate whether the reduction in infarct size and the number of apoptotic cells contributed to the improved cardiac function, we evaluated lung weight and echocardiography at 21 days after I/R. The increases in lung weight to body weight after I/R, an indicator of lung congestion, were significantly attenuated in PBA-treated mice compared with vehicle-treated mice (Figure 4A and Supplementary Table 3). Echocardiographic measurements showed that increases in LVEDD and decreases in LVFS after I/R were observed in I/R, and they were significantly reduced in PBA-treated mice at 21 days after I/R (Figure 4B, C and Supplementary Table 4). These findings suggested that the reduced infarct size in the acute phase of I/R by PBA administration contributes to improve cardiac function in the chronic phase of I/R.

Sodium 4-Phenylbutyrate Attenuated Cardiac Remodeling After Myocardial I/R

At 21 days after I/R, we performed Azan staining of the LV sections for the detection of fibrosis. The formation of necro- tic scar and perivascular cardiac fibrosis of remote area was observed (Figure 5A, B). We quantified the area of fibrosis in LV sections, and the areas were smaller in PBA-treated Figure 1.Sodium 4-phenylbutyrate (PBA) reduced infarct size after

myocardial ischemia–reperfusion (I/R). A, Cardiac troponin-I (cTnI) in the serum of mice treated with vehicle and PBA was measured 4 hours after reperfusion in myocardial I/R. The level of cTnI was significantly increased in I/R and lower in PBA-treated mice than in vehicle-treated mice (**P< .01), n¼ 5 for all groups. B, 2,3,5-Triphenyltetrazolium chloride (TTC) staining of myocardial tissue obtained from mice treated with vehicle and PBA 48 hours after reperfusion to determine the infarct area. The nonrisk area (NRA) is indicated in blue, area at risk (AAR) is indicated in red, and the infarct area (IA) is indicated in white.

C-E, Quantification of infarct size by TTC staining in mice treated with vehicle and PBA at 48 hours after reperfusion. AAR/left ventricle (LV), ratio of AAR to LV area (C). IA/AAR, ratio of infarct area to AAR (D).

IA/LV, ratio of infarct area to LV area (E). PBA administration reduced IA/AAR and IA/LV compared with vehicle administration (NS indicates no significant difference, **P< .01), n¼8 for both groups. All results are presented as mean+standard error of the mean (SEM).

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mice compared with vehicle-treated mice (5.3% +1.2%vs 9.9% +1.5%,P < .05; Figure 5C). The mRNA expression levels of cardiac remodeling markers, including collagen type 1a1, BNP, and aSMA, were elevated at 21 days after I/R.

Consistent with the results of echocardiographic and histological measurement, the mRNA expression levels of these cardiac remodeling markers were significantly decreased in PBA-treated mice compared with vehicle-treated mice (Figure 5D-F). These finding suggested that the reduced infarct size in the acute phase of I/R by PBA administration contributes to improve cardiac remodeling in the chronic phase of I/R.

Discussion

In the present study, we demonstrated that PBA protects against myocardial I/R injury. The major findings in this study are as follows: (1) PBA reduces apoptotic cell number and infarct size following myocardial I/R, (2) PBA suppresses the I/R-induced UPR, and (3) PBA improves cardiac dysfunction and remodeling after I/R. Thus, PBA reduced myocardial infarct size and inhibited apoptosis, resulting in improved cardiac dysfunction after myocardial I/R.

Evidence suggests that myocardial I/R and heart failure are accompanied by ER stress and the consequent UPR.10,20,24-26

Figure 2.Sodium 4-phenylbutyrate (PBA) reduced apoptotic cells after myocardial ischemia–reperfusion (I/R). A, Representative photographs of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and 4⁰-6-diamidino-2-phenylindole (DAPI)-stained area at risk from mice treated with vehicle and PBA 4 hours after reperfusion. B, Quantitative analysis of TUNEL-positive cells in whole left ventricle treated with vehicle and PBA 4 hours after reperfusion. The TUNEL-positive cells are expressed as the percentage of the total myocardium. Sodium 4-phenylbutyrate significantly reduced TUNEL-positive cells compared with vehicle (*P< .05), n¼5 for both groups. All results are presented as mean+standard error of the mean (SEM).

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Thuerauf et al demonstrated that simulating hypoxia in cultured rat myocytes increased the expression of GRP78 and other UPR markers. During hypoxia, protein levels of GRP78 increased in the border zone of the IA early on and declined after reoxygenation.²⁴ In mouse model, myocardial I/R activates UPR markers, such as GRP78, CHOP, and P-eIF2a, elevated as early as 30 minutes to 2 hours after reperfusion.¹⁰As important findings of GRP78, upregulation of GRP78 during ischemia preconditioning in cardiac myocytes is responsible for protecting against further ischemic damage.^20,27Activation of ATF6 in the hearts of transgenic mice was reported to protect the heart against I/R injury and with upregulation of GRP78.²⁶ These findings indicate that the UPR induces the upregulation of GRP78 to restore proper folding, reduce the accumulation of unfolded proteins caused by myocardial I/R,

and recover ER homeostasis. The functions of chemical chaperone are thought to stabilize unfolded proteins, reduce the aggregation, and partly assist the activity of endogenous chaperones including GRP78. This effect of chemical chaperone is considered to reduce broadly the UPR including the proximal 3 transmembrane proteins sensors.^11,28,29 In this study, we demonstrated that phosphorylation of eIF2aand IRE1, cleaved form of ATF6, and GRP78 were significantly increased at 2 hours after reperfusion in LV of myocardial I/R, and PBA- attenuated I/R induces increase of these UPR markers. These results suggest that PBA attenuates the I/R-induced UPR in hearts by acting as a chemical chaperone.

The proapoptotic pathway is activated upon continued or over- whelming ER stress. Terai et al demonstrated that the proapoptotic pathways of UPR, including CHOP expression and caspase-12 Figure 3.Sodium 4-phenylbutyrate (PBA) attenuated unfolded protein response (UPR) after myocardial ischemia–reperfusion (I/R). Western blot analyses were performed on whole left ventricular lysates from hearts of mouse 2 hours after reperfusion, treated with vehicle and PBA. A, Representative Western blots of glucose-regulated protein 78 (GRP78), activating transcription factor-6 (ATF6; cleaved-ATF6), C/EBP- homologous protein (CHOP), caspase-12 (procaspase-12 and cleaved-caspase-12), inositol-requiring enzyme-1 (IRE1), phosphorylated IRE1 (P-IRE1), eukaryotic initiation of the factor 2a-subunit (eIF2a), phosphorylated eIF2a(P-eIF2a), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B-G, Each expression levels were quantified. GRP78, ATF6, and CHOP were elevated in I/R (GAPDH was used as loading control).

Cleaved-caspase-12 to procaspase-12 ratio was similarly elevated and phosphorylation of IRE1 and eIF2awas invited. Sodium 4-phenylbutyrate attenuated the increase of these UPR markers compared with vehicle in I/R (*P< .05, **P< .01), n¼5 for all groups. All results are presented as mean+standard error of the mean (SEM).

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cleavage, are involved in cardiac myocytes death by hypoxic stimuli.³⁰Miyazaki et al found that the oxidative stress caused by reperfusion activates the induction of CHOP in myocardial I/R, and apoptotic cell number and infarct size following myocardial I/R were reduced in CHOP-deficient mice.¹⁰Caspase-12 cleaves procaspase-9, leading to caspase-9-dependent activation of caspase-3. The UPR-induced processing of procaspase-9 can occur in the absence of cytochrome c release, a finding that suggests that caspase-12 directly triggers caspase-9 activation and apoptosis independent of the mitochondrial cytochrome c/Apaf-1 pathway.³¹On the other hand, activation of CHOP by PERK/

eIF2aleads to the upregulation of proapoptotic Bcl-2-interacting mediator of cell death (Bim) and downregulation of antiapoptotic factor B-cell lymphoma 2 family.³²Our study showed that CHOP and cleaved caspase-12 were significantly increased after myocardial I/R and that PBA attenuated I/R-induced increases in them. Consistent with the result, PBA reduced the number of TUNEL-positive cells after myocardial I/R. The inhibition of I/R-induced upregulation of the 3 ER transmembrane protein sensors results in the reduction of UPR-induced apoptosis.

Evidence suggests that PBAs are useful with a reduction of UPR in various models of cardiovascular diseases such as MI, cardiac hypertrophy induced by transverse aortic constriction, cardiac fibrosis induced by isoproterenol, and vascular disease induced by exposure to saturated fatty acids.^17-19,33However, other evidences also suggest that the reduction of UPR by PBA is not useful in all cases. Cardioprotective effect of SO2pre- conditioning and local ischemic postconditioning was dimin- ished by low dose (20 mg/kg) of PBA, which abolished the upregulation of UPR markers such as GRP78 and ATF6.^20,34 But at this time, it is difficult to decide whether these therapies do not coexist with PBA administration. There remain several combinations within PBA administration, preconditioning therapy, and postconditioning therapy. And more, the effica- cies of them may be influenced by the magnitude of I/R injury and involved diseases such as diabetes, hypertension, and heart failure.^35,36This possibility has acquired further study.

The effects of PBA on UPR may be different in each patho- logical situations influenced by the dose and timing of PBA administration.17,19,20,34

It should be noted that excessive reduction of UPR may invite harmful results in some patholo- gical situations. For example, in CHOP-deficient mice, apoptotic cell number and infarct size were reduced after myocardial I/R.¹⁰But in MI model of CHOP-deficient mice, the acute-phase mortality was increased by cardiac rupture and the cardiac remodeling was not improved in the chronic phase.³⁷These findings may suggest that cell survival or death, in the UPR, is likely to depend on how much and how long cardiac myocytes are exposed to various ischemia forms.

On the other hand, PBA is also known as histone deacetylase (HDAC) inhibitor, and some HDAC inhibitors have cardioprotective effect in myocardial I/R.^38,39 Therefore, in our study, PBA may have partially acted as an HDAC inhibitor and protected against myocardial I/R injury. Sodium 4-phenylbutyrate may affect I/R injury in known ways and others that have yet to be proven, but such areas require further investigation.

We demonstrated that PBA administration not only reduces infarct size but improves LV remodeling after I/R. Left ventricle infarct size correlated with LV volume and LV ejection fraction (LVEF) in a rabbit model of MI.⁴⁰ Larger infarcts result in greater LV end-diastolic volume and LV end- systolic volume as well as lower LVEF occurred within 4 weeks after MI. In the present study, cardiac function was preserved and cardiac remodeling was prevented at 3 weeks after I/R in PBA-treated mice. These findings may show that the reduced infarct size in the acute phase by PBA administration contributes to improve cardiac function and attenuate cardiac remodeling in the chronic phase. In this regard, a single administration of PBA confers some long-term protection.

In conclusion, PBA protects against myocardial I/R injury, which is evidenced by reduced infarct volume and number of apoptotic cells, by suppressing excess ER stress. In addition, these effects contribute to the improvement of cardiac function Figure 4.Sodium 4-phenylbutyrate (PBA) preserved cardiac function at 21 days after myocardial ischemia–reperfusion (I/R). A. The ratio of lung weight to body weight was evaluated. Sodium 4-phenylbutyrate attenuated the increases in the ratio of lung weight to body weight compared with vehicle in I/R (*P< .05), n¼10 for all groups. B and C, Echocardiography was performed. Left ventricular end-diastolic diameter (LVEDD) and left ventricular fractional shortening (LVFS) were measured. Sodium 4-phenylbutyrate attenuated the enlargement of LVEDD and restored the reduction of LVFS in I/R (**P< .01), n¼10 for all groups. All results are presented as mean+standard error of mean (SEM).

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and remodeling after I/R in the chronic phase. We propose that PBA may be an additional therapeutic option to coronary reperfusion therapy for patients with ACS in clinical practice.

Acknowledgment

The authors thank Ayano Nomura for her excellent technical support.

Author Contributions

Osamu Takatori and Soichiro Usui contributed equally to this study.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was partially supported by a Grant-in-Aid for Scientific Research (C) awarded to S.U.

Figure 5. Sodium 4-phenylbutyrate (PBA) attenuated cardiac remodeling after myocardial ischemia–reperfusion (I/R). A, Representative photographs of Azan-stained horizontal sections of left ventricles (LVs) obtained from mice treated with vehicle and PBA. B, Perivascular fibrosis was occurred in remote area. C, Quantified data of cardiac fibrosis in LV sections. The fibrotic area was smaller in mice treated with PBA compared with mice treated with vehicle (*P< .05), n ¼5 for both groups. D-F, Messenger RNA (mRNA) expression levels of cardiac remodeling markers were quantified by real-time polymerase chain reaction. The expression levels of collagen type 1a1 (D), brain natriuretic peptide (BNP; E), and alpha skeletal muscle actin (aSMA; F) were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Sodium 4-phenylbutyrate attenuated mRNA expression of cardiac remodeling markers compared with vehicle in I/R (**P< .01), n¼5 for all groups. All results are presented as mean+standard error of the mean (SEM).

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Supplemental Material

The online supplemental tables are available at http://cpt.sagepub .com/supplemental.

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