Attenuation of Endoplasmic Reticulum Stress–Related Myocardial … · 2019. 8. 15. · IHD is a chronic and complex process, which may involve abnormalities in en-ergy metabolism,

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INTRODUCTIONChronic myocardial ischemia has be-

come the leading cause of heart failure.In China, more than half of the patientswith heart failure also have coronary ar-tery heart disease (1). These patients suf-fer from ischemic heart disease (IHD),which leads to the further deteriorationof cardiac function. The pathogenesis ofIHD is a chronic and complex process,which may involve abnormalities in en-ergy metabolism, altered expression orfunction of contractile proteins, ventricu-lar remodeling and myocardial apoptosis

(2). Growing evidence suggests that a de-fect of myocardial Ca2+ transport systemwith cytosolic Ca2+ overload is a majorcontributor to ischemic myocardial in-jury (3). Sarco/endoplasmic reticulum Ca2+

ATPase 2a (SERCA2a), which is primarilyresponsible for sarcoplasmic reticulumCa2+ uptake, was reported to be de-creased in protein level or activity in theischemic myocardium in various animalsand humans (4–7). Additionally, vector-mediated gene transfer to increaseSERCA2a expression or the use of trans-genic animals with SERCA2a overexpres-

sion have clearly established the poten-tial beneficial effects in ischemic heart,and thus promoted the field of potentialSERCA2a gene therapy in IHD (8–11).Recently, evidence from our group andothers suggests that the beneficial effectsof SERCA2a gene transfer to animalswith heart failure may involve a decreaseof myocardial apoptosis (12,13). How-ever, the exact mechanisms are still notclear.

Endoplasmic reticulum (ER) is recog-nized as an organelle that participates inthe folding of secretory and membraneproteins (14). Recent evidence suggeststhat another important function of theER is apoptotic regulation (15–17). Vari-ous stimuli, such as disturbance of Ca2+

homeostasis, ischemia, hypoxia, expo-sure to free radicals, oxidative stress, ele-vated protein synthesis and gene muta-tion—all of which can potentially cause

Attenuation of Endoplasmic Reticulum Stress–RelatedMyocardial Apoptosis by SERCA2a Gene Delivery inIschemic Heart Disease

Wei Xin,1,2* Xiaochun Lu,1* Xiaoying Li,1 Kun Niu,1 and Jimei Cai1

From the 1First Department of Geriatric Cardiology, Chinese PLA General Hospital, Beijing, China; and the 2Medical College ofNankai University, Tianjin, China

Previous studies suggested that endoplasmic reticulum (ER) stress–associated apoptosis plays an important role in the patho-genesis of ischemic heart disease. Gene transfer of sarco/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) attenuates myo-cardial apoptosis in a variety of heart failure models. This study is to investigate the effects of SERCA2a gene delivery on the my-ocardial apoptosis and ER stress pathway in a porcine ischemic heart disease model. Eighteen pigs were either subjected toameroid implantation in the coronary artery or sham operation. Eight wks after gene delivery, the protein level and activity ofSERCA2a were measured. Myocardial apoptosis was determined using terminal deoxynucleotidyl transferase–mediated DNAnick-end labeling assay. Regional myocardial perfusion and function were evaluated by 99m Tc-sestamibi (99m Tc-MIBI) single pho-ton emission computed tomography and echocardiography. The ER stress signaling was assessed by Western blot. SERCA2a pro-tein level and activity were significantly decreased in the ischemic myocardium and restored to normal after SERCA2a genetransfer. Restoration of SERCA2a expression significantly improved the cardiac function, although no improvement of regionalmyocardial perfusion was detected. Restoration of SERCA2a significantly attenuated myocardial apoptosis and reversed the ac-tivation of unfolded protein response (UPR) pathway and the ER stress–associated apoptosis pathways. These findings demon-strate a robust role of SERCA2a in attenuation of ischemic myocardial apoptosis, correlating with reverse activation of the ERstress–associated apoptosis pathways, suggesting that the beneficial effects of SERCA2a gene transfer may involve the attenu-ation of ER stress–associated myocardial apoptosis.© 2011 The Feinstein Institute for Medical Research, www.feinsteininstitute.orgOnline address: http://www.molmed.orgdoi: 10.2119/molmed.2010.00197

*WX and XL contributed equally to this work.

Address correspondence and reprint requests to Xiaoying Li, 28 Fuxing Road, First Depart-ment of Geriatric Cardiology, Chinese PLA General Hospital, Beijing 100853, PR China.

Phone: 86-010-66876271; Fax: 86-010-68212494; E-mail: [email protected].

Submitted October 11, 2010; Accepted for publication December 3, 2010; Epub

(www.molmed.org) ahead of print December 8, 2010.

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S E R C A 2 A A T T E N U A T E S M Y O C A R D I A L A P O P T O S I S

ER dysfunction—are designated as ERstress (14,17,18). To prevent deleteriouseffects of ER stress, cells have variousprotective strategies such as the unfoldedprotein response (UPR) through the me-diation of ER transmembrane receptors:protein kinase R–like ER kinase (PERK),activating transcription factor 6 (ATF6)and inositol-requiring enzyme (IRE) (19).These transmembrane receptors are main-tained in an inactive state by glucose- regulated protein 78 (GRP78). However,if the stress cannot be resolved, signalingswitches from prosurvival to proapo -ptotic through the mediation of down-stream molecules such as CCAAT/ enhancer binding protein homology pro-tein (CHOP), c-Jun N-terminal kinases(JNK) and caspase-12 (20–22). Accumu-lating evidences have demonstrated thatapoptosis initiated by excessive ER stressis involved in the ischemic injury of car-diomyocytes in vitro and pathogenesis ofIHD in vivo (23–25).

Early observations showed that alter-ation of sarcoplasmic reticulum/ER Ca2+

levels below functional acceptable limitsenhances the ER stress, and SERCA2 ex-pression and activity could be inducedduring ER stress in cardiomyocytes andother cells, suggesting a potential role ofSERCA2a protein in ER stress (26–28). Be-cause the major function of SERCA2a is toreplenish the sarcoplasmic reticulum Ca2+

load during the contraction-relaxationcycle of the heart (29), which is fundamen-tal to the ER function of protein folding,we hypothesized that restoration ofSERCA2a expression by recombinantadeno-associated virus 1 (rAAV1)- mediated gene delivery could maintainthe ER function and attenuate ERstress–associated myocardial apoptosis inan IHD pig model. The effects of SERCA2agene transfer on regional myocardial func-tion and perfusion were also investigated.

MATERIALS AND METHODS

Construction and Production of rAAV1Vectors

The rAAV1 vectors carrying humanSERCA2a or the enhanced green fluorescent

protein (EGFP) reporter gene were con-structed and produced by AGTC GeneTechnology Company (Beijing, China). Ineach vector, gene expression was undercontrol of the cytomegalovirus promoterand polyadenylation signal provided bysimian virus 40. The vector preparationsused in this study were diluted to titersof 1 × 1012 vector genomes (v.g.)/mL inphosphate buffered saline (PBS, pH 7.4)and were stored at 4°C.

Animal Experiment and GeneDelivery

Eighteen purebred male pigs (AnimalExperimental Center of Chinese PLAGeneral Hospital, Beijing, China), weigh-ing 24.5–28.5 kg, 3 months of age, werehoused at the institution for a minimumof 1 wk before use. Standard swine foodwas used for feeding. The animals wererandomized into three groups. Four nor-mal pigs underwent sham operation andserved as the control group. The remain-ing 14 pigs served as 2 viral-administeredgroups and underwent ameroid constric-tor implantation around the proximal ofleft anterior descending (LAD) coronaryartery and received intramyocardial viralinjection 4 wks later.

Ameroid-induced progressive coronaryocclusion was performed as describedpreviously (30). Briefly, initial sedationwas achieved with intramuscularly keta-mine hydrochloride (20 mg/kg), di-azepam (0.05 mg/kg) and atropine (0.05 mg/kg). An ear vein was then can-nulated for administration of an infusionof pentobarbital as needed to maintainanesthesia. The mini-pig was intubatedand ventilated. A left lateral thoracotomywas performed through the fifth inter-costal space. After opening the peri-cardium, an ameroid constrictor (2.5 mm,Research Instruments, Lebanon, OR,USA) was implanted around the proximalLAD for the viral-administered groups,but was not for the control group. Chronicmyocardial ischemia was induced by pro-gressive coronary artery occlusion withameroid coronary constriction. The chestwas closed after a stable position of theconstrictor was confirmed.

Four weeks after ameroid placement,second thoracotomies were performed onthe animal of the viral-administeredgroups, and rAAV1-SERCA2a andrAAV1-EGFP (1 × 1012 v.g. per pig, in 1 mL PBS solution, pH 7.4) were injecteddirectly into the ischemic area along thetwo sides of the constricted LAD coro-nary artery (10 sites for each animal and5 for each side with a 1-cm interval). Theinjection sites were marked with Indiaink for subsequent identification and pro-tein analysis. Experimental protocolscomplied with the Guide for the Care andUse of Laboratory Animals of Chinese PLAGeneral Hospital and were approved bythe Chinese Academy of Sciences.

Assessment of Regional MyocardialPerfusion

Regional myocardial perfusion wasevaluated at 4 wks (before viral adminis-tration) and 12 wks (8 wks after viral ad-ministration) after ameroid implantationby means of 99m Tc-sestamibi (99m Tc-MIBI)single photon emission computed tomog-raphy (SPECT). The pigs were placed inthe dorsal (supine) position, and PETscans were performed with 15-cm axialfield of view (Discovery LS, GE, Milwau-kee, WI, USA) over the cardiac region.PET images were acquired in two- dimensional mode, starting 20 min afterintravenous injections of a 740-MBq (20-mCi) bolus of 99m Tc-MIBI. During thePET scans, animals were maintainedunder general anesthesia as describedabove. Transaxial cardiac images werethen reoriented into horizontal long-axis,vertical long-axis and short-axis imageswith a thickness of 4.25 mm by an experi-enced expert in nuclear medicine who wasblinded to the treatment group. Three axisviews were analyzed further.

Echocardiographic Assessment ofRegional Myocardial Function

The same zones for each animal wereanalyzed by echocardiography at baseline,4 wks and 12 wks after ameroid implanta-tion. The pigs were sedated and placed inthe left lateral decubitus position, andstandard two-dimensional and M-mode

R E S E A R C H A R T I C L E


transthoracic images were obtained with aVivid 7 Dimension echocardiographic ma-chine (GE Healthcare, Waukesha, WI,USA) and a 1.5- to 4.0-MHz frequencytransthoracic transducer. From the rightparasternal approach, short-axis, midpap-illary views were obtained at rest for 3min. Regional wall thickening, myocardialcontractile function and wall motion weredetermined by a single experienced inves-tigator in a blinded fashion. These imagesand parameters were recorded for subse-quent review and analysis.

Measurement of Serum BrainNatriuretic Peptide Concentration

A blood sample was collected fromeach animal and put into plastic tubescontaining anticoagulant (1:9, 0.129 mol/Ltrisodium citrate) at baseline, 4 wks and12 wks after ameroid implantation. Thecitrated blood was immediately cen-trifuged at 1,760g for 10 min at roomtemperature to obtain plasma. The serumbrain natriuretic peptide (BNP) concen-trations were assayed using radioim-munoassay kits (Radioimmunity Insti-tute, Chinese PLA General Hospital,Beijing, China) according to the proce-dure described by the manufacturer.

SERCA2a Activity MeasurementsTo investigate the effects of rAAV1-

SERCA2a treatment on myocardialSERCA2a activity, proteins were ex-tracted and quantified as previously de-scribed from heart tissue samples takenfrom the region of the left ventriculararound the previously marked sites 8wks after viral administration. Tissues inthe same areas were taken in the controlgroup for subsequent analysis (31).SERCA2a activity was measured using aCa2+- ATPase assay kit (Jiancheng Bio-engineering Institute, Nanjing, China) ac-cording to the manufacturer’s instruc-tions. SERCA2a activity was normalizedto protein concentration.

TUNEL and ImmunohistochemistryStaining

Heart tissue samples were taken fromthe region of the left ventricle around the

previously marked sites 8 wks after viraladministration and cut into 4-μm-thicksections. Tissues in the same areas aretaken in the control group. Some sectionswere stained with hematoxylin andeosin. Some sections were used for apo-ptotic assessment with a terminal de-oxynucleotidyl transferase (TDT)- mediated DNA nick-end labeling(TUNEL) assay. Some sections were usedfor immunohistochemistry staining withGRP78 antibody.

Assessment for apoptosis was con-ducted using a commercially availableTUNEL assay kit (Promega, Madison, WI,USA). Briefly, sections were deparaffinized,digested with proteinase K (20 mg/mL)at room temperature for 15 min andsoaked in PBS for 5 min. Each sectionwas covered with a TDT enzyme solutionand incubated for 1 h at 37°C in a humid-ified chamber. The sections were im-mersed in stop buffer to terminate the en-zymatic reaction and then gently rinsedwith PBS. Streptavidin–horseradish per-oxidase solution was applied to each sec-tion and then incubated at room temper-ature for 30 min in the darkness. Slideswere washed in PBS and exposed to 3,3-diaminobenzidine (Golden BridgeBiotechnology, Beijing, China) for 5–7 min. The slides were then rinsed inwater and counterstained with hema-toxylin. The number of TUNEL-positivecells was counted in 10 randomly se-lected fields (400× magnifications) foreach animal under ocular micrometers(Olympus Optical, Tokyo, Japan) by ablinded investigator without knowledgeof groups.

The tissue expression of GRP78 was as-sessed immunohistochemically using anantibody (Santa Cruz, CA, USA). Afterdeparaffinization, endogenous peroxidaseactivity was quenched with 30%methanol and 0.3% hydrogen PBS. Theslides were then boiled in citrate bufferwith microwaves. After blocking nonspe-cific binding with 5% BSA, the slides wereincubated with primary antibodiesovernight at 4°C (dilutions for anti-GRP781:400). The following day, the sectionswere thoroughly washed in PBS and incu-

bated with a peroxidase-conjugated poly-mer that carries antibodies to goat (1:200)immunoglobulin (Golden Bridge Biotech-nology) for 30 min. After rinsing withPBS, the sections were exposed to 3,3-diaminobenzidine for 7 min. Theslides were rinsed in water and counter-stained with hematoxylin. The sectionswere examined using light microscopyand analyzed with a computer-assistedcolor image analysis system (Image- ProPlus 7.0, Media Cybernetics, MD,USA). The positive areas were assessed inat least 16 randomly selected tissue sec-tions from each group studied.

Western BlottingAll five samples were taken from the

ischemic heart region around the previ-ously marked sites in one pig 8 wksafter vector administration, and tissuesin the same areas were taken in the con-trol group for protein analysis. To inves-tigate the effects of rAAV1-SERCA2atreatment on myocardial tissue proteinexpression, proteins were extracted,quantified and separated by sodium dodecyl sulfate–polyacrylamide gel elec-trophoresis (SDS-PAGE) as describedpreviously (31). Blots were incubatedwith primary antibodies: anti-SERCA2aand anti-phospho-IRE1α (SER 724)(from Abcam, Cambridge, MA, USA)and anti-Bip/GFP78, anti-XBP1, anti-phospho PERK (Thr 981), anti-ATF6α,anti-phospho eIF2α (Ser 52), anti-PERK,anti-IRE1α, anti-CREB-2, anti-GADD 153,anti-caspase-12, anti-JNK, anti-phosphoJNK (Thr183/Tyr185), anti-caspase-9,anti-Bcl-2, anti-Bax and anti-β-actin (allfrom Santa Cruz, CA, USA). The intensi-ties of the various protein bands werequantified by densitometry.

Statistical AnalysisData were expressed as the mean ±

SEM. Comparisons of parameters wereperformed by one-way analysis of vari-ance, followed by the Newman-Keuls testfor unpaired data. Comparisons of param-eters between two groups were made byunpaired Student t test. P values of



RESULTS

Overall AssessmentOf the 18 pigs in the study, 16 (control

group, n = 4; IHD+EGFP group, n = 6;IHD+SERCA2a, n = 6) survivedthroughout the study, without any clini-cal signs of toxicity; 2 pigs died duringthe experimental surgery (1 died of ven-tricular fibrillation and the other died ofuncontrollable hemorrhage caused byvessel injury during the procedure). Thetwo viral administered groups (ameroidconstriction of LAD coronary artery) ex-hibited evidence of chronic myocardial

ischemia, as demonstrated by (a) a fixeddefect in the LAD coronary artery zoneof the 99m Tc-MIBI SPECT images; (b) athinned, akinetic region of the left ven-tricular and decreased cardiac systolicand diastolic function during echocar-diography; and (c) myocardial fibrosisand extensive focal coalescent areas ofischemic cardiomyocyte degeneration, inthe hematoxylin and eosin staining of is-chemic myocardial tissue.

SERCA2a Gene Delivery Restores theExpression and Activity of SERCA2aProteins

The in vivo expression and activity ofSERCA2a proteins were significantly de-creased in the IHD+EGFP group com-pared with the control group. However,

there were no significant differences inSERCA2a protein expression or activitiesbetween the control and IHD+SERCA2agroup (Figure 1A, B). These results indi-cate that rAAV1-mediated SERCA2a genedelivery restores both the expression andactivities of SERCA2a proteins in myo-cardial tissue.

rAAV1-SERCA2a Attenuates Apoptosisin Ischemic Myocardium

Apoptosis of myocardial cells was eval-uated by TUNEL staining assay 8 wksafter the viral transduction (Figure 2A, C).The SERCA2a gene, transferred to ische-mic myocardium, attenuates the apopto-sis of ischemic myocardium comparedwith the IHD+EGFP group, although thenumber of apoptotic myocardial cells is

Figure 1. Myocardial expression and activ-ity of SERCA2a protein after rAAV1-SERCA2a gene delivery. (A) Western blotand quantitative analysis of SERCA2a pro-tein expression. β-Actin was used as an in-ternal control. (B) Analysis of myocardialSERCA2a activity. Results show substantialdecreases of SERCA2a protein and activ-ity in the IHD+EGFP group compared withthe control group, but activity was re-stored in the IHD+SERCA2a group. Dataare presented as mean ± SEM (n = 4~6per group).

Figure 2. Restoration of SERCA2a expression attenuates myocardial apoptosis and re-verses the increase of GRP78. (A) TUNEL staining of apoptotic myocardial cells(pointed by the arrows). (B) Immunohistochemical staining of GRP78 protein. TheGRP78 proteins are stained in brown. (C) Quantitative analysis of TUNEL-positive cells.(D) Quantitative analysis of GRP78 density. Data are presented as mean ± SEM (n =4~6 per group). IOD, integrated optical density.



still greater in the IHD+SERCA2a groupthan in the control group.

Effects of rAAV1-SERCA2a Delivery onMyocardial Perfusion

The 99m Tc-MIBI SPECT image datawere used to display the myocardialperfusion and the severity of myocardialischemia. The control group for myocar-dial perfusion in sham-operated pigs ex-hibited no detectable defect (Figure 3A).In the two viral transduced groups,SPECT images obtained at 4 wks afterameroid implantation displayed similarmyocardial perfusion with a characteris-tic perfusion defect, mainly at apical, an-terior and some septal regions. At 8 wksafter viral administration, no significantimprovement of myocardial perfusioncould be detected in IHD+EGFP orIHD+SERCA2a groups (Figure 3B, C).Together, these data indicate thatrAAV1-SERCA2a treatment has no obvi-ous effect on myocardial blood perfusionafter chronic myocardial ischemia.

Effects of rAAV1-SERCA2a Delivery onMyocardial Function and Serum BNPLevels

The effects of rAAV1-SERCA2a deliv-ery on the myocardial function were ex-amined by echocardiography. Cardiacsystolic and diastolic function, regionalwall motion and ventricular wall thick-ness were reduced 4 wks after ameroidimplantation in the two viral administra-tion groups compared with the controlgroup (Figure 4A–F).

In contrast, by 8 wks after vector ad-ministration (12 wks after ameroid im-plantation), the increase of left ventricu-lar ejection fraction and the ratiobetween early and atrial peak filling ve-locities (Ev/Av) were found in theIHD+SERCA2a group compared withthe IHD+EGFP group, indicating im-provement in both systolic and diastolicmyocardial function (Figure 4A, B). Theregional wall motion, verified by the an-terior lateral wall systolic thickeningfraction and the interventricular septalsystolic thickening fraction, showed fur-ther improvement in the IHD+SERCA2a

group than in the IHD+EGFP group (Fig-ure 4C, D). The septal and anterior lat-eral wall diastolic thickness, according toanterior lateral wall diastolic thicknessand interventricular septal diastolic

thickness, were increased in theIHD+SERCA2a group (Figure 4E, F).These results demonstrate that rAAV1-SERCA2a transfer results in enormousimprovement of cardiac function and

Figure 3. Effects of rAAV1-SERCA2a transfer in regional myocardial perfusion. (A) 99m Tc-MIBISPECT images of control group. (B and C) 99m Tc-MIBI SPECT images of the IHD+EGFP andthe IHD+SERCA2a groups. The control group exhibited no detectable defect. At 4 wksafter ameroid implantation, the two viral-administered groups exhibited a significant myo-cardial perfusion defect in the LAD coronary artery–dependent zone. No significant im-provement of myocardial perfusion was detected at 8 wks after rAAV1-SERCA2a transferin the IHD+SERCA2a group.



wall motion and a significant increase inwall thickness.

Serum levels of BNP, a biomarker ofcardiac dysfunction, were also found toincrease significantly 4 wks afterameroid implantation in the two viraladministration groups compared with

the control group (Figure 4G). However,by 8 wk after vector administration,serum BNP levels were significantly re-duced in the IHD+SERCA2a group, par-alleling an improvement of cardiac func-tion compared with the IHD+EGFPgroup.

Effects of rAAV1-SERCA2a Delivery onER Stress Pathways

The effects of rAAV1-SERCA2a deliv-ery on the UPR pathway and ERstress–related apoptosis pathways wereexamined. Western blot and immuno-histochemistry staining results showedthat the levels of the ER chaperonesGRP78 and the components of the threearms of UPR pathway, such as ATF6α(50 kDa), phospho-IRE1α XBP1, phos-pho-PERK, phospho-eIFα and ATF4were significantly increased in the IHD+EGFP group compared with thecontrol group (Figure 2B, D; Figure 5).Moreover, the expressions of ERstress–related apoptotic proteins, in-cluding CHOP, cleaved caspase-12 andphospho-JNKs, were also enhanced inthe IHD+EGFP group compared withthe control group (Figure 6). However,changes of the above proteins were allreversed in the IHD+SERCA2a groups.Taken together, these results suggestthat restoration of SERCA2a protein lev-els by rAAV1-SERCA2a treatment re-verses the activation of UPR signals andER stress–related apoptosis pathways inthe ischemic myocardial tissue.

DISCUSSIONIn the present study, with a porcine

IHD model induced by chronic occlu-sion of LAD coronary artery, we foundthat the protein level and activity ofSERCA2a in the ischemic myocardiumwas decreased. Furthermore, restorationof SERCA2a expression by intramyocar-dial rAAV1-SERCA2a gene delivery im-proved regional myocardial functionand cardiac systolic and diastolic func-tion. Further investigation demonstratedthat all three arms of UPR and the ER stress–associated apoptosis pathwayswere activated in the myocardium ofIHD pigs, and enhanced myocardialapoptosis was observed in the ischemicmyocardium. However, restoration ofSERCA2a expression reversed the activa-tion of UPR and the ER stress–associatedapoptosis pathways and attenuated myocardial apoptosis in IHD pigs, suggesting that the beneficial effects

Figure 4. Restoration of SERCA2a protein expression improves myocardial function. (A and B)Left ventricular ejection fraction (LVEF) and the ratio between early and atrial peak fillingvelocities (Ev/Av) showed that cardiac systolic and diastolic function was improved in theIHD+SERCA2a group 8 wks after viral administration. (C and D) Anterior lateral wall systolicthickening fraction (ΔALWT) and anterior lateral wall systolic thickening fraction (ΔIVST)showed the regional motion of anterior lateral and interventricular septal wall was im-proved in the IHD+SERCA2a group 8 wks after viral administration. (E and F) Anterior lat-eral wall diastolic thickness (ALWTd) and interventricular septal diastolic thickness (IVSTd)showed the thickness of anterior lateral and interventricular septal wall was increased inthe IHD+SERCA2a group 8 wks after viral administration. (G) Serum BNP level was de-creased in the IHD+SERCA2a group 8 wks after viral administration. Data are presented asmean ± SEM (n = 4~6 per group). #P < 0.05 versus control group; *P < 0.05 versus IHD+EGFPgroup.



of SERCA2a gene transfer to IHD myocardium are associated with the re-duction of ER stress–related myocardialapoptosis.

Abnormal calcium handling associatedwith a decrease in the expression and ac-tivity of SERCA2a is suggested to be oneof the key abnormalities in a variety ofcardiovascular disorders, including heartfailure, acute myocardial ischemia and ischemia/perfusion, and chronic myocar-

dial ischemia (3,29,32). A large number ofstudies in isolated cardiomyocytes andanimal models of heart failure showedthat restoring SERCA2a expression bygene transfer corrects the contractile dys-function and energetic and electrical re-modeling (29,32). After a long line of in-vestigation, two clinical trials areunderway using rAAV-SERCA2a to re-store SERCA2a expression in patientswith heart failure (33). However, the ef-

fects of SERCA2a gene delivery on IHDare still unknown. In this study, withameroid constrictor–induced progressiveocclusion of the LAD coronary artery, wemade a chronic ischemic heart diseasemodel in large animals to examine the ef-fects of SERCA2a gene transfer. The re-sults showed that intramyocardial injec-tion of rAAV1 can lead to the efficientlocal gene delivery in a patchy pattern,with the highest level in the area of theinjection sites, as indicated by EGFP fluo-rescence (data not shown), (consistentwith our previous study in dogs with thesame vectors [31]). Restoration ofSERCA2a expression significantly im-proved the regional myocardial contrac-tile function and wall thickness in the is-chemic area and the overall cardiacsystolic and diastolic function, indicatingpro-hypertrophy may involve in the ben-eficial effects of SERCA2a. However, theheart weight/body weight (HW/BW)was not significantly different among thethree groups 8 wks after gene delivery(data not shown), and perhaps more ani-mals and longer observational periodsare needed to show the difference. Recentevidence suggests that SERCA2a genetransfer increases the expression and ac-tivity of the endothelial isoform of nitricoxide synthase (eNOS) and improvesvascular activity and coronary flow in thesetting of mitral regurgitation–inducedheart failure (34). We went on to examinethe effects of SERCA2a gene transfer onregional myocardial perfusion. However,no significant improvement of myocar-dial perfusion could be detected by 99m Tc-MIBI SPECT at 8 wks after genedelivery. Because the LAD coronary ar-tery in our model was occluded eventu-ally, it was not a surprise to find thateven if SERCA2a gene transfer could po-tentially improve the vascular activity, lit-tle beneficial effects could be detected onthe regional myocardial perfusion.

It has been suggested that apoptosisplays an important role in the pathogen-esis of IHD (35,36). Ischemia inducesmyocardial apoptosis, which causes theloss of cardiomyocytes, leading to theimpairment of cardiac systolic and dias-

Figure 5. Restoration of SERCA2a protein expression reverses the activation of UPR signal-ing pathways. (A) The protein levels of the ATF6α branch of UPR: ATF6α (90 kDa) andATF6α (50 kDa). (B) The protein levels of the IRE1α branch of UPR: IRE1α, phospho-IRE1αand XBP-1. (C) The protein levels of the PERK branch of UPR: PERK, phospho-PERK, phospho-eIF2α and ATF4. (D) The protein levels of GRP78. Data are presented as mean ±SEM (n = 4~6 per group). *P < 0.05 versus control group; #P < 0.05 versus IHD+EGFP group.



tolic functions. A few studies demon-strated that ischemia, along with depri-vation of oxygen, nutrition and energysupply, led to the activation of UPR-and ER stress–associated pathways incultured cardiomyocytes or different an-imal models (23–25). In this study, wefound that apoptosis was significantlyenhanced in the ischemic myocardium,and the three arms of the UPR pathwaymediated by the three transmembranereceptors, namely PERK, ATF6 andIRE1, were also activated as shown by

the detection of their target molecules(37). We detected marked induction ofXBP1 protein, a marker for the coordi-nated action of active ATF6 and IRE1 inthe ischemic myocardium, suggestingthe activation of these two branches.The PERK branch was also activated be-cause the expression of phosphorylatedeIF2α and subsequent ATF4 proteinswere also enhanced. Additionally,GRP78, the central regulator of ER func-tion and the master modulator for theUPR network by binding to the above

three ER stress sensors, were also in-duced in the ischemic myocardium.

Although the UPR is primarily anadaptive response, if the stress persists,the ER stress receptors can also triggerpro-apoptotic pathways to initiate celldeath (18). CHOP, caspase-12 and JNKare three well-defined apoptotic path-ways related to ER stress. In our study,these apoptotic pathways were activatedin the myocardium of IHD models, asshown by the induction of CHOP andcleaved caspase-12 proteins and en-hanced phosphorylation of JNKs. TheCHOP protein belongs to the C/EBPfamily of transcription factors, and it istranscriptionally induced during the de-velopment of ER stress by ATF6, PERKand IRE1 signaling (38,39). Our resultsare consistent with the earlier findingsthat showed that CHOP sensitized thecells to ER stress–induced apoptosis viadownregulation of Bcl-2 expression(40,41). Caspase-12–mediated apoptosiswas a specific apoptotic pathway of ER,and apoptosis that occurred as a result ofmembrane- or mitochondrial-targetedsignals did not activate it (42). Cleavedcaspase-12 reportedly activates caspase-9,followed by activation of caspase-3 (43).JNKs were activated during ER stressthrough phosphorylation mediated by theformation of the IRE1–tumor necrosis fac-tor receptor–associated factor 2–apoptosissignal–regulating kinase 1 complex (44).Activation of JNKs is a common responseto many forms of stress and is known toinfluence the cell-death machinerythrough the regulation of BCL2 familyproteins (45). In our study, the aboveapoptotic pathways were all activatedwith the enhanced myocardial apoptosis,suggesting ER stress–associated apopto-sis may be involved in the pathogenesisof IHD.

Interestingly, we found that SERCA2agene delivery reversed the activation ofthe above UPR- and ER stress–relatedapoptotic pathways, with significant at-tenuation of apoptosis in the ischemicmyocardium. These results suggestedthat the beneficial effects of SERCA2agene transfer on IHD maybe involved

Figure 6. Restoration of SERCA2a protein expression reverses the activation of ER stress–re-lated apoptosis pathways. (A) The protein levels of CHOP, Bcl-2 and Bax. (B) The proteinlevels of procaspase 9, 12 (CASP9 and CASP12) and cleaved caspase 9, 12. (C) The pro-tein levels of total and phospho-JNKs. Data are presented as mean ± SEM (n = 4~6 pergroup). *P < 0.05 versus control group; #P < 0.05 versus IHD+EGFP group.



the attenuation of ER stress–associatedmyocardial apoptosis. It has been re-ported that the expression and activityof SERCA2b, the other spliced protein of the SERCA2 gene, could be inducedby the UPR pathway in the PC12 cellline during ER stress (26,27). In the car-diomyocytes, SERCA2a expressioncould be upregulated as a potentialphysiologically important compensa-tory mechanism by ATF6 in response tosarcoplasmic reticulum calcium deple-tion–induced ER stress (28). In our study,although the ATF6 branch of UPR wasactivated, we did not find an increasedSERCA2a protein level in the IHD+EGFPgroup. This is not surprising, mainly be-cause ischemia is a more complex patho-physiological process compared with invitro sarcoplasmic reticulum calcium de-pletion, and many other mechanismsmay affect SERCA2 expression besidesthe activation of ATF6. However, previ-ous evidence suggests that SERCA2aprotein may be an ER stress–inducedprotein, which may contribute to therestoration of ER calcium homeostasis toattenuate ER stress. Considering its im-portant role in calcium uptake to the ER,it is likely that intra myocardial deliveryof SERCA2a in our IHD model restoredthe ER calcium homeostasis and poten-tially attenuated ER stress–associatedmyocardial apoptosis. Further studiesare needed to detect the direct connec-tion of cellular calcium disturbance andER stress–associated apoptosis, and themechanisms and signal pathways in-volved in the beneficial effects ofSERCA2a gene transfer in this setting areto be investigated.

Although the effects of SERCA2a genetransfer on the other apoptotic pathwaysstill need to be determined, it can be con-cluded on the basis of our study thatSERCA2a gene delivery in the myo -cardium of the porcine IHD model im-proves the regional myocardium func-tion and overall cardiac systolic anddiastolic function. The beneficial effectsof SERCA2a to ischemic myocardiummay involve the attenuation of ERstress–associated myocardial apoptosis.

Our findings suggest the potential valueof SERCA2a gene delivery as a treatmentof chronic ischemic heart disease. Furtherstudies are needed to determine the doseand time effects and the safety of rAAV1-SERCA2a gene transfer on ischemic heartdisease.

ACKNOWLEDGMENTSThis study was supported by the Na-

tional Basic Research Development Pro-gram of China, namely “973” Program(number 2007CB512004), and the Na-tional Science Foundation of China(numbers 30600236 and 30770900).

DISCLOSUREThe authors declare that they have no

competing interests as defined by Molecu-lar Medicine, or other interests that mightbe perceived to influence the results anddiscussion reported in this paper.

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Attenuation of Endoplasmic Reticulum Stress–Related Myocardial … · 2019. 8. 15. · IHD is a chronic and complex process, which may involve abnormalities in en-ergy metabolism,