M E T A B O L I S M C L I N I C A L A N D E X P E R I M E N T A L 6 2 ( 2 0 1 3 ) 1 3 8 7 – 1 3 9 3

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Metabolism

Thyroid hormone improves the mechanical performance of thepost-infarcted diabetic myocardium: A response associatedwith up-regulation of Akt/mTOR and AMPK activation

www.metabo l i sm jou rna l . com

Iordanis Mourouzis, Irini Giagourta, Georgios Galanopoulos, Polixeni Mantzouratou,Erietta Kostakou, Alexandros D. Kokkinos, Nikolaos Tentolouris, Constantinos Pantos⁎

Department of Pharmacology, University of Athens, 75 Mikras Asias Ave., 11527 Goudi, Athens, Greece

A R T I C L E I N F O

Abbreviations: TH, thyroid hormone; T4, Lhormone receptor alpha1; STZ, streptozotociinfarction; LV, left ventricle; RV, right ventriinternal diameter at the systolic phase; LVPWindex; SI, sphericity index; KCl, potassium chphenylmethanesulfonylfluoride; DTT, dithiextracellular signal-regulated kinase; AKT,kinase; HRP, horseradish peroxidase; S.E.M, s⁎ Corresponding author. Fax: +30 210 7462560

E-mail address: cpantos@med.uoa.gr (C. P

0026-0495/$ – see front matter © 2013 Elsevihttp://dx.doi.org/10.1016/j.metabol.2013.05.00

A B S T R A C T

Article history:Received 15 March 2013Accepted 11 May 2013

Objective. Thyroid hormone (TH) is shown to be protective against cardiac and pancreaticinjury. Thus, this study explored the potential effects of TH treatment on the functionalstatus of the postinfarcted diabetic myocardium. Diabetic patients have worse prognosisafter acute myocardial infarction (AMI).

Materials/Methods. AMI was induced by left coronary ligation in rats previously treatedwith 35 mg/kg streptozotocin (STZ), (DM-AMI). TH treatment was initiated at 2 weeks afterAMI and continued for 6 weeks (DM-AMI + TH), while sham-operated animals served ascontrol (DM-SHAM).

Results. TH treatment increased cardiac mass, improved wall stress and favorablychanged cardiac geometry. TH significantly increased echocardiographic left ventricularejection fraction (LVEF%): [54.2 (6.5) for DM-AMI + TH vs 37 (2.0) for DM-AMI, p < 0.05]. THtreatment resulted in significantly increased insulin and decreased glucose levels in serum.The ratios of phosphorylated (p)-Akt/total Akt and p-mTOR/total mTOR were increased 2.0fold and 2.7 fold in DM-AMI + TH vs DM-AMI respectively, p < 0.05. Furthermore, the ratio ofp-AMPK/total AMPKwas found to be increased 1.6 fold in DM-AMI + TH vs DM-AMI, p < 0.05.

Conclusion. TH treatment improved the mechanical performance of the post-infarctedmyocardium in rats with STZ-induced diabetes, an effect which was associated with Akt/mTOR and AMPK activation.

© 2013 Elsevier Inc. All rights reserved.

Keywords:Thyroid hormoneDiabetesMyocardial infarctionInsulinKinase signaling

-thyroxine; T3, 3,5,3′ tri-iodothyronine; TSH, thyroid stimulating hormone; TRα1, thyroidn; i.p, intraperitoneal; DM, diabetes mellitus; ECG, electrocardiogram; AMI, acute myocardialcle; LVIDd, left ventricular internal diameter at the diastolic phase; LVIDs, left ventricular, posterior wall thickness at the diastolic phase; EF%, ejection fraction; WTI, wall tension

loride; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol tetraacetic acid; PMSF,othreitol; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; ERK,protein kinase B; mTOR, mammalian target of rapamycin; AMPK, AMP-activated proteintandard error of mean; ANOVA, one-way analysis of variance; NS, non-significant..antos).

er Inc. All rights reserved.8

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1. Introduction

Myocardial infarction results in high mortality in diabeticpatients, despite current available therapies [1,2]. Accordingly,experimental studies show that cardiac remodeling followingmyocardial infarction is accelerated in the presence ofdiabetes [3–5]. Diabetic hearts fail to develop compensatoryhypertrophy after myocardial infarction due to the defect instress-induced growth kinase signalling [3]. In this context,induction of physiologic growth by thyroid hormone (TH)treatment, early after the index event, appears to improvemechanical function of the post-infarcted diabetic myocardi-um [4]. However, this beneficial effect may be lost, when THtreatment is initiated at later stages in the course ofmyocardial infarction, in which cardiac remodeling hasprogressed [3]. This issue, although of clinical relevance, hasnot been previously addressed. In fact, recovery from end-stage heart failure was shown to be facilitated with increasingcirculating TH levels [6,7]. On the basis of this evidence, in thepresent study, we explored whether TH treatment, initiatedlate in the process of cardiac remodeling, can improve cardiacfunction in animals with myocardial infarction and strepto-zotocin (STZ)-induced diabetes.

2. Materials and methods

2.1. Animals

Male Wistar rats, 300–380 g, were maintained on a 12 h light/dark cycle and fed with a standard chow ad libitum. Therats were handled in accordance with the Guide for theCare and Use of Laboratory Animals published by the USNational Institutes of Health Guide (NIH Pub. No. 83-23,Revised 1996). Approval was also granted by the universityethics review board.

2.2. Induction of diabetes mellitus (DM)

DM was induced by a single injection of STZ (35 mg/kg;intraperitoneally). STZ was prepared in 0.1 mol/L sodiumcitrate buffer, pH 4.5 (Sigma, Munich, Germany) [4]. Insulinlevels in serum were 1651 (205) pg/ml in treated animalsversus 5100 (741) pg/ml in age-matched control rats, p < 0.05.Rats were subjected to surgical procedure 30 days afterSTZ injection.

2.3. Experimental model of myocardial infarction

Acute myocardial infarction (AMI) was induced by ligation ofthe left coronary artery as previously described [8,9]. Rats wereanesthetized with an intraperitoneal injection of ketamine(70 mg/kg) and midazolam (0.1 mg/kg), intubated and venti-lated via a tracheal cannula using a constant-volume rodentventilator (Harvard Apparatus, Inspira, 50 breaths/min, 1 ml/100 g tidal volume). Anesthesia was maintained by inhalationof small doses of sevoflurane (1%–2%). Left thoracotomy wasperformed at the fourth intercostal space followed by pericar-diotomy. Left coronary artery was then ligated with a 6-0 silkround-bodied suture. The heart was quickly returned to the

chest cavity, the chest was closed and rats were allowed torecover using assist mode ventilation. Atelectasis was pre-vented by using positive end-expiratory pressure at the end ofthe surgical procedure. Continuous electrocardiogram (ECG)recording was used to monitor heart rate and ECG ischaemicchanges after coronary artery ligation. Body temperature wasmaintained at 37 °C by using a heating blanket (HarvardHomeothermic Blanket, 50-7061). The mortality was approx-imately 30% in the first 24 h following the surgical procedure.The animals were left to recover for 8 weeks after myocardialinfarction. The same procedure was followed for sham-operated animals without coronary artery ligation.

2.4. Thyroid hormone administration

Two weeks after the operation, diabetic rats were randomlydivided in two groups. The first group received standard ratchow (DM-AMI), while the second group received foodcontaining thyroid powder 0.05% (Sigma, T1251, containing0.42 μg/mg T3 and 1.7 μg/mg T4) for 6 weeks (DM-AMI + TH)as previously described [9,10]. Mean daily intake of thyroidhormone per rat was estimated to be 3.0 μg T3 and 12 μg T4

respectively. Diabetic sham-operated rats receiving standardrat chow were designated as DM-SHAM.

2.5. Echocardiography

At 8 weeks after surgery, rats were sedated with ketaminehydrochloric acid (100 mg/kg) and heart function was evalu-ated by echocardiography as previously described [9,11]. Shortand long-axis images were acquired using a digital ultrasoundsystem (Vivid 7 version Pro, GE Healthcare) with the 14.0-MHzi13L probe. A large number of consecutive measurementswere performed and analysed by two independent operators.

Left ventricular (LV) internal diameter at the diastolicphase (LVIDd), LV internal diameter at the systolic phase(LVIDs), posterior wall thickness at the diastolic phase (LVPW)and the ejection fraction (EF%) were measured. EF% wascalculated using the Simpson equation. EF% was used toassess global contractile LV function.

Wall tension index (WTI) was defined as the ratio (LVIDd/2*Posterior Wall thickness). WTI was measured in order toassess myocardial wall stress. In addition, sphericity index(SI), defined as the ratio of maximum long axis (in mm) tomaximum short axis (in mm) of the left ventricle, wasdetermined in order to assess LV geometry. All measurementswere averaged for at least 3 consecutive cardiac cycles.

2.6. Protein isolation, sodium dodecyl sulfate-proteinpolyacrylamide (SDS-PAGE) gel electrophoresis andimmunodetection

LV tissue was homogenized in ice-cold buffer (A) containing10 mmol/L 4-(2-hydroxyethyl)piperazine-1-ethanesulfonicacid (Hepes, pH: 7.8), 10 mmol/L Potassium chloride (KCl),0.1 mmol/LEthylenediaminetetraacetic acid (EDTA), 0.1 mmol/L ethylene glycol tetraacetic acid (EGTA), 0.5 mmol/L phenyl-methanesulfonylfluoride (PMSF), 1 mmol/L Dithiothreitol(DTT) and 10 μg/ml leupeptin. 200 μl of 10% Igepal was addedand samples were left in ice for 30 min. Homogenization was

Table 1 – Thyroxine (T4), triiodothyronine (T3), thyroidstimulating hormone (TSH), insulin and glucose levels inserum in sham-operated diabetic rats (DM-SHAM), post-infarcted diabetic rats (DM-AMI) and post-infarcteddiabetic rats treated with thyroid hormone (DM-AMI + TH) are presented in this table.

DM-SHAM,n = 11

DM-AMI,n = 13

DM-AMI + TH,n = 11

T4 (ng/ml) 306 (12) 267 (20) 454 (40) †

T3 (ng/ml) 3.1 (0.06) 2.77 (0.05) ⁎ 3.9 (0.23) †

TSH (pg/ml) 1.83 (0.25) 2.6 (0.24) 0.15 (0.03) †

Insulin (pg/ml) 1651 (305) 2280 (900) 7160 (940) †

Glucose (mg/dL) 291 (21) 329 (45) 182 (12) †

The values are mean (S.E.M). Variables without normal distribution(T3, insulin and glucose) were analyzed with Kruskal–Wallis Test.Variables with normal distribution (T4, TSH) were analyzed withOne-way ANOVA.⁎ P<0.05 vs DM-SHAM.† P < 0.05 vs DM-SHAM and DM-AMI.

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repeated, the homogenate was centrifuged at 1000 g for 5 min,4 °C and the supernatant containing the cytosolic fraction wasstored at −80 °C. Protein concentrations were determined bythe Bicinchoninic Acid method.

Samples were prepared for sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) by boiling for 5 minin Laemmli sample buffer containing 5% 2-mercaptoethanol.30 μg (cytosolic fraction) of total protein was loaded onto 7.5%or 10% (w/v) acrylamide gels and subjected to SDS-PAGE in aBio-Rad Mini Protean gel apparatus. For Western blotting,proteins were transferred electrophoretically to a nitrocellu-losemembrane (Hybond ECL) at 100 V and 4 °C, for 1.5 h usingTowbin buffer. Filters from cytosolic protein extracts wereprobed with specific antibodies against total and phosphory-lated (p)-ERK (Extracellular signal-regulated kinase), total andp(Ser473)-AKT (also known as Protein kinase B), total andp(Ser2448)-mTOR (mammalian target of rapamycin) (CellSignaling Technology, dilution 1:1000) and total AMPK andp(Thr172)-AMPK (AMP-activatedprotein kinase) (Cell SignalingTechnology, dilution 1:1000) overnight at 4 °C. Filters wereincubatedwith appropriate anti-rabbit horseradishperoxidase(HRP) secondary antibodies (Cell Signaling). Immunoreactivitywas detected by enhanced chemiluminescence using Lumigloreagents (New England Biolabs). Chemiluminescence wasdetected by FluorChem HD2 (AlphaInnotech, 14743, CatalinaStreet, San Leandro, CA), an image analysis system equippedwith a CCD camera. Five samples fromeach groupwere loadedon the same gel.

2.7. Measurement of thyroid hormones and insulin levels

Serum L-thyroxine (T4), 3,5,3′ tri-iodothyronine (T3) andthyroid stimulating hormone (TSH) quantitative measure-ments were performedwith Luminex XMAP technology, usingMilliplex assay kit RTHY-30K according to manufacturer'sinstructions. T4 and T3 levels were expressed in ng/ml, whileTSH was expressed in pg/ml. Insulin levels (in pg/ml) weredetermined in serum by Luminex XMAP technology, usingMilliplex rat metabolic magnetic bead panel according to themanufacturer's instructions. Measurements were performedwith Luminex 200™ instrument.

2.8. Experimental procedure

Eight weeks after the surgical procedure, rats were anaes-thetized with ketamine hydrochloride and subjected toechocardiographic analysis. Thereafter, the heart was re-moved and washed in ice-cold Krebs buffer. Left ventricle (LV)and right ventricle (RV) of the heart were separated, scar LVtissue was dissected out and the non-infarcted remote areawas frozen in liquid nitrogen for further analysis. Blood wascollected from the right atrium in order to measure thyroidhormones and insulin levels in serum. The area of the scartissue was measured in mm2 and the weight in mg.

Rats were separated into the following experimentalgroups; diabetic sham-operated rats (DM-SHAM, n = 11);diabetic rats subjected to myocardial infarction (DM-AMI,n = 13) and diabetic rats subjected to myocardial infarctionand treatedwith thyroid hormone after the secondweek post-ligation (DM-AMI + TH, n = 11).

2.9. Analysis of data and statistics

Results are presented as mean (standard error of mean,S.E.M.). Normal distribution of variables was estimated withKolmogorov–Smirnov test. Levene's test was used to estimatehomogeneity of variance. One-way analysis of variance(ANOVA) with Bonferroni or Dunnett's correction was usedfor multiple comparisons of variables with normal distribu-tion. Kruskal–Wallis non parametric test was used for vari-ables without normal distribution. When the Kruskal–Wallistest resulted in statistical significance, post-hoc analysis wasfurther carried out with Mann–Whitney test with Bonferronicorrection. Significance was set at 0.05. Non-significant re-sults are designated as P = NS.

3. Results

3.1. Thyroid hormone levels

T3 levels were significantly lower in DM-AMI rats as comparedto DM-SHAM, P = 0.002. A trend towards an increased TSH wasobserved inDM-AMI rats as compared toDM-SHAM,but this didnot reach statistical significance, P = NS. T4 levels were notdifferent between DM-AMI and DM-SHAM rats, P = NS. Table 1.

T4 and T3 serum levels were significantly increased by 70%and 40% respectively in DM-AMI + TH rats as compared toDM-AMI rats (P < 0.001 for both T4 and T3), while TSH levelswere significantly reduced (P < 0.001) Table 1.

3.2. Insulin and glucose levels

No difference was found in the glucose and insulin levelsbetween DM-SHAM and DM-AMI, P = NS. Insulin levels weresignificantly increased in DM-AMI + TH rats in comparison toDM-SHAM and DM-AMI rats, P = 0.001 and P = 0.016 respec-tively Table 1. In addition, glucose levels were significantlyreduced in DM-AMI + TH rats versus DM-SHAM and DM-AMIrats, P = 0.001 for both. Table 1.

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3.3. Extent of injury, reactive cardiac hypertrophyand geometry

Scar area and weight were not significantly different betweengroups subjected tomyocardial infarction. In fact, scar area andweight were 81 (8) mm2 and 112 (7) mg in DM-AMI vs 60(12) mm2 and 75 (13) mg in DM-AMI + TH rats respectively, P =NS. Left ventricular weight (LVW) and the ratio of LVW to bodyweight were similar between DM-SHAM and DM-AMI [800(28) mg and 1.8 (0.06) versus 752 (34) mg and 1.7 (0.03)], P = NS.

Echocardiographic analysis showed that LVPWwas similarbetween DM-AMI and DM-SHAM hearts, P = NS. LV diastolicdiameter (LVIDd) was significantly increased in DM-AMIversus DM-SHAM hearts, P < 0.05 resulting in a markedincrease in WTI in DM-AMI versus DM-SHAM hearts, P < 0.05Table 2. Furthermore, sphericity Index (SI) was decreased inDM-AMI as compared to DM-SHAM hearts, P < 0.05 Table 2.

After TH treatment, LVW and the ratio of LVW to bodyweight were significantly increased [921 (33) mg and 2.2 (0.08)]in DM-AMI + TH as compared to [752 (34) mg and 1.7 (0.03)] inDM-AMI hearts, P < 0.05]. In addition, LVPW was increased inDM-AMI + TH vs DM-AMI hearts, indicating the developmentof cardiac hypertrophy, Table 2. LVIDd was significantlyreduced in DM-AMI + TH as compared to DM-AMI hearts,P < 0.05. As a consequence, WTI was nearly normalized inDM-AMI + TH and was found to be significantly reduced incomparison to DM-AMI hearts, P < 0.05, Table 2. Furthermore,sphericity index was nearly normalized and was significantlyincreased in comparison to DM-AMI hearts, P < 0.05.

Body weight was comparable in all groups, P = NS. Table 2.

Table 2 – Body weight (in g), heart rate (in beats per min)and echocardiographic measurements of posterior wallthickness at diastolic phase (LVPW), wall tension index(WTI, LVIDd/2 * LVPW) and sphericity index, leftventricular internal diameter at diastolic phase (LVIDd)and at systolic phase (LVIDs), ejection fraction (EF%) andsystolic velocity of LV posterior wall (SVPW) in sham-operated diabetic rats (DM-SHAM), post-infarcted diabeticrats (DM-AMI) and post-infarcted diabetic rats treatedwith thyroid hormone (DM-AMI + TH) are shown inthis table.

DM-SHAM,n = 11

DM-AMI,n = 13

DM-AMI + TH,n = 11

Body weight (g) 460 (14) 456 (13) 425 (12)Heart rate (bpm) 280(34) 275 (24) 393 (29) †

LVIDd (mm) 6.7(0.15) 9.2 (0.25) ⁎ 8.0 (0.3) †

LVIDs (mm) 4.1(0.4) 7.8 (0.27) ⁎ 6.0 (0.56) †

LVPW (mm) 1.8 (0.05) 1.82 (0.07) 2.1 (0.08) †

WTI 1.75 (0.05) 2.6 (0.15) ⁎ 2.09 (0.08) ‡

Sphericity Index 2.3 (0.08) 1.75 (0.05) ⁎ 2.1 (0.09) ‡

SVPW (mm/s) 27 (3.0) 21 (1.4) ⁎ 31 (3.4) ‡

EF% 75.4 (2.8) 37 (2.0) ⁎ 54.2 (6.5) ‡

The values are mean (S.E.M). Variables without normal distribution(Body weight) were analyzed with Kruskal–Wallis Test, while therest of the variables (with normal distribution) were analyzed withOne-way ANOVA.⁎ P < 0.05 vs DM-SHAM.† P < 0.05 vs DM-SHAM and DM-AMI.‡ P < 0.05 only vs DM-AMI.

3.4. Left ventricular function

LVEF% was significantly reduced in DM-AMI hearts ascompared to DM-SHAM group, P < 0.05. Regional systolicvelocity of non-infarcted LV posterior wall was significantlyreduced in DM-AMI hearts in comparison to DM-SHAM group,P < 0.05, Table 2. Heart rate was similar between DM-AMI andDM-SHAM. TH treatment increased heart rate by nearly 40% inDM-AMI + TH hearts as compared to DM-AMI and DM-SHAMhearts, P < 0.05. EF% and SVPW were found to be significantlyimproved in DM-AMI + TH in comparison to DM-AMI hearts,P < 0.05, Table 2.

3.5. Changes in the pattern of intracellularkinases activation

The ratio of p-Akt to total Akt was similar between DM-AMIand DM-SHAM hearts, P = NS, while the ratio of the p-mTORto total mTORwas reduced 1.5 fold in DM-AMI as compared toDM-SHAM hearts, P < 0.05, Fig. 1. Furthermore, the ratio of p-p44 ERK to total ERK was shown to be reduced 2.0 fold in DM-AMI as compared to DM-SHAMhearts, P < 0.05, while the ratioof p-p42 ERK to total ERK did not change, Fig. 2. The ratio of p-AMPK to total AMPK was similar between DM-AMI and DM-SHAM hearts, P = NS, Fig. 3.

After TH treatment, the ratio of p-Akt to total Akt wasincreased 1.8 fold and 2.0 fold in DM-AMI + TH as compared toDM-SHAM and DM-AMI hearts respectively, P < 0.05. More-over, the ratio of p-mTOR to total mTORwas increased 1.8 foldand 2.7 fold in DM-AMI + TH as compared to DM-SHAM andDM-AMI hearts respectively, P < 0.05, Fig. 1. The ratio of p-p44ERK to total ERK was reduced 2.0 fold in DM-AMI + TH ascompared to DM-SHAM hearts, P < 0.05, while there was nodifference in the ratio of p-p42 ERK to total ERK between DM-AMI + TH and DM-AMI hearts, Fig. 2. The ratio of p-AMPK tototal AMPK was increased 1.6 fold in DM-AMI + TH incomparison to DM-SHAM and DM-AMI hearts, P < 0.05, Fig. 3.

4. Discussion

Recent clinical and experimental research provides substan-tial evidence that TH may play an important role in theresponse of the living organism to stress and post-stressadaptation [12]. In this context, TH is shown to possess areparative action after injury in almost every tissue [13–15]. Inaccordance to this evidence, the present study showed thatTH could facilitate recovery of function of the post-infarcteddiabeticmyocardium and rescue pancreatic tissue from injuryinduced by STZ administration. This may be of importantphysiologic and therapeutic relevance. In fact, myocardialischemia and diabetes frequently co-exist [1,2].

Acute myocardial infarction was induced in rats bycoronary ligation, while diabetes was induced by STZ admin-istration. In this experimental model, cardiac dysfunctionappears to be accelerated in the diabetic as compared to nondiabetic rats [3]. Diabetic hearts seem to lose the ability toincrease cardiac mass and normalize cardiac mechanics, asearly as twoweeks aftermyocardial infarction. In this context,we showed that early administration of TH could prevent this

Fig. 1 – Summary of the densitometry ratio (mean ± SEM) ofexpression and their representative immunoblots of p-Akt tototal of Akt (A) and p-mTOR to total mTOR (B) in sham-operated diabetic rats (DM-SHAM), post-infarcted diabeticrats (DM-AMI) and post-infarcted diabetic rats treated withthyroid hormone (DM-AMI + TH). *P < 0.05 vs DM-SHAM,†P < 0.05 vs DM-SHAM and DM-AMI.

Fig. 2 – Summary of the densitometry ratio (mean ± SEM) ofexpression and their representative immunoblots of p-p44and p-p42 ERK to total p44 and p42 ERK in sham-operateddiabetic rats (DM-SHAM), post-infarcted diabetic rats (DM-AMI) and post-infarcted diabetic rats treated with thyroidhormone (DM-AMI + TH). *P < 0.05 vs DM-SHAM.

ig. 3 – Summary of the densitometry ratio (mean ± SEM) ofxpression and their representative immunoblots of p-AMPKtotal AMPK in sham-operated diabetic rats (DM-SHAM),

ost-infarcted diabetic rats (DM-AMI) and post-infarctediabetic rats treated with thyroid hormone (DM-AMI + TH).P < 0.05 vs DM-SHAM and DM-AMI.

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response by the induction of physiologic growth [3]. In thepresent study, we provided further evidence that thisbeneficial effect can be preserved, when TH treatment isinitiated at later stages in the course of cardiac remodeling.Thus, TH was administered two weeks after the coronaryligation and continued for six weeks thereafter and changes inleft ventricular structure and function were assessed byechocardiography. TH increased cardiac mass and alteredcardiac geometry. In fact, the sphericity index (the ratio ofmaximum long axis to maximum short axis of the leftventricle) was nearly normalized. These changes were trans-lated into a marked reduction of wall tension index (an indexof wall stress). Furthermore, TH significantly increased leftventricular systolic function, as this was evident by thesignificant increase in the systolic velocity of the leftventricular posterior wall. Consequently, left ventricularejection fraction was found to be significantly increased intreated as compared to untreated hearts. TH also resulted inlower scar area, but this effect was not significant. TH canprotect against ischemia–reperfusion injury [16–18]. However,this actionmay be weak in the case of TH treatment, late after

myocardial infarction induced by permanent coronaryocclusion.

The present study further showed that TH rescued injuredpancreatic tissue, as this was indicated by the increase incirculating insulin levels. This novel finding has previouslybeen reported in STZ-induced diabetes and in experimentalmodels of type II diabetes [19,20]. TH appears to regulateinsulin secretion via its thyroid hormone receptor alpha1(TRα1) [21,22]. Consistent with this experimental evidence, T3levels in plasma were shown to be positively associated withinsulin secretion in euthyroid individuals [23]. However,excess in circulating TH levels may result in insulin resistanceand impaired glucose homeostasis [24,25]. In fact, adminis-

Fetopd†

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tration of high doses of TH, after experimental myocardialinfarction, resulted in pathological remodelling with in-creased activation of ERK kinase [26]. Interestingly, ERK kinaseis implicated in both pathologic growth development andinsulin resistance [27–31].

In the present study, TH treatment did not result inimpaired glucose homeostasis. In fact, glucose levels inserum were significantly lower after TH treatment. Further-more, at the tissue level, insulin regulated kinases, Akt andmTOR, were activated without any change in the activation ofERK. Along this line, AMPK activation in the myocardium wasalso found to be significantly increased. This pattern of kinaseactivation is consistent with physiologic remodelling[26,27,32–34]. Controlled Akt over-expression in mice resultsin moderate cardiac hypertrophy with preserved systolicfunction [35]. Furthermore, AMPK, which is regulated by TH,serves important physiologic functions [36–38]. AMPK regu-lates contractile function and links metabolism to myofila-ment energetics [39,40], while suppressing proliferation ofcardiac fibroblasts via its negative regulation of ERK [41].

4.1. Clinical implications

The present study is of important clinical and therapeuticrelevance. There is emerging evidence showing that changesin TH in acute and chronic illnesses may impact on patients’morbidity andmortality. Thus, despite early revascularizationafter AMI, functional recovery was found to be poor in patientswho failed to recover T3 levels in plasma [42]. Furthermore,low T3 levels were associated with increased mortality in AMIpatients less than 75 years-old [43]. More recently, TH isshown to be an independent predictor of cardiovascularevents in patients with type 2 diabetes [44]. This accumulatingclinical evidence has been confirmed by several experimentalstudies [12] and prompts for clinical evaluation of TH as apotential treatment for heart failure [45].

4.2. Limitations of the study

The present study has identified novel changes in stress-induced signaling pathways, which were associated withthe beneficial effect of TH on the injured diabetic myocar-dium. However, cause–effect relationships have not beenestablished. Further studies are probably needed to eluci-date this issue.

In conclusion, TH can improve mechanical performance ofthe post-infarcted myocardium in animals with STZ-induceddiabetes and this response is associated with Akt/mTOR andAMPK activation.

Author contributions

I. Mourouzis: Diabetes model, myocardial infarction model,echocardiographic analysis, measurements of T3, T4, TSH,insulin, molecular analysis, statistical analysis, writing ofmanuscript.

I. Giagourta: Diabetes model, myocardial infarction model,thyroid hormone treatment, measurements of T3, T4, TSHand insulin, statistical analysis.

G. Galanopoulos: Diabetes model and myocardial infarc-tion model.

P. Mantzouratou: Echocardiographic analysis and molecu-lar analysis.

E. Kostakou: Molecular analysis.A. D. Kokkinos: Diabetes model, measurements of T3, T4

and TSH.N. Tentolouris: Diabetes model, measurements of insulin

and glucose, writing of manuscript.C. Pantos: Design of the study, director of study imple-

mentation, statistical analysis, writing of manuscript.

Conflict of interest

The authors declare no conflict of interests.

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