Left ventricular hypertrophy (LVH) is causally related to increased morbidity and mortality following acute myocardial infarction (AMI) via still unknown mechanisms. Although rapamycin exerts cardioprotective effects against myocardial ischemia/reperfusion (MI/R) injury in normal animals, whether rapamycin-elicited cardioprotection is altered in the presence of LVH has yet to be determined. Pressure overload induced cardiac hypertrophied mice and sham-operated controls were exposed to AMI by coronary artery ligation, and treated with vehicle or rapamycin 10 min before reperfusion. Rapamycin produced marked cardioprotection in normal control mice, whereas pressure overload induced cardiac hypertrophied mice manifested enhanced myocardial injury, and was refractory to rapamycin-elicited cardioprotection evidenced by augmented infarct size, aggravated cardiomyocyte apoptosis, and worsening cardiac function. Rapamycin alleviated MI/R injury via ERK-dependent antioxidative pathways in normal mice, whereas cardiac hypertrophied mice manifested markedly exacerbated oxidative/nitrative stress after MI/R evidenced by the increased iNOS/gp91phox expression, superoxide production, total NO metabolites, and nitrotyrosine content. Moreover, scavenging superoxide or peroxynitrite by selective gp91phox assembly inhibitor gp91ds-tat or ONOO scavenger EUK134 markedly ameliorated MI/R injury, as shown by reduced myocardial oxidative/nitrative stress, alleviated myocardial infarction, hindered cardiomyocyte apoptosis, and improved cardiac function in aortic-banded mice. However, no additional cardioprotective effects were achieved when we combined rapamycin and gp91ds-tat or EUK134 in ischemic/reperfused hearts with or without LVH. These results suggest that cardiac hypertrophy attenuated rapamycin-induced cardioprotection by increasing oxidative/nitrative stress and scavenging superoxide/peroxynitrite protects the hypertrophied heart from MI/R.

Introduction

Several lines of studies have demonstrated that left ventricular hypertrophy (LVH) is an independent predictor of cardiovascular events [1,2] and increases the risk of acute myocardial infarction (AMI) [3,4]. LVH is present in approximately one-third patients with AMI and is associated with increased morbidity and mortality following AMI [5,6]. Moreover, preclinical studies have shown that animals with LVH have expanded myocardial infarct size and are refractory to cardioprotective treatments due to defective cytoprotective mechanisms following myocardial ischemia/reperfusion (MI/R) [7,8]. However, to date, the mechanisms underlying the exacerbated myocardial injury in the hypertrophied heart following MI/R are still incompletely understood.

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase and its role in regulating energy metabolism, oxidative stress, and cardiovascular disease has received intense attention [911]. mTOR links with other proteins and forms two distinct complexes, mTOR1 and mTOR2. The mTOR1 complex participates in the regulation of cellular growth, while mTOR2 is involved in the control of the cytoskeleton [12]. Previous studies have shown that chronic increased mTOR activation was related to diverse disorders, including diabetes [13,14], uremic cardiomyopathy [15,16], and cardiac hypertrophy [1719]. Therefore, therapeutic targeting of mTOR may provide promising prospects to develop potential treatment for LVH-related complications. Indeed, rapamycin, the mTOR1 inhibitor, was shown to mitigate pressure overload induced hypertrophic responses by rescuing the defective cytoprotective mechanisms in aortic-banded mice (2 mg/kg/day for 2 weeks) [17]. In addition, mTOR inhibition with rapamycin (0.25 mg/kg, once) was shown to induce preconditioning-like myocardial protection against ischemia/reperfusion injury in mice in vivo [20,21]. Moreover, Volkers et al. [22] demonstrated that increasing mTORC2 activation while inhibiting mTORC1 signaling reduced cardiomyocyte apoptosis and necrosis after MI. These exciting findings suggest that rapamycin would be a promising therapeutic strategy to rescue LVH-related cardiovascular impairment.

LVH is associated with increased phosphorylation of ERK and signal transducer and activator of transcription 3 (STAT3) expression, which was causally related to pathologic cardiac hypertrophy in animal studies [23,24]. Interestingly, the enhanced ERK/STAT3 phosphorylation was shown to play a crucial role in prompting cardiomyocyte survival and cardiac functional recovery by rapamycin after MI/R [20,21]. In addition, ischemic/pharmacologic preconditioning was proven to alleviate MI/R injury by ERK/STAT3 pathway-dependent mechanisms [2527]. However, to our knowledge, most studies investigating the cardioprotection by rapamycin were conducted in normal animals without LVH [20,21,28]. Whether the beneficial effects of rapamycin on MI/R would be altered in the presence of LVH, a pathologic condition with defective cytoprotective mechanisms including increased oxidative/nitrative stress as well as overactivated ERK/STAT3 pathway [23,24,29], has never been reported. Therefore, we conducted the present study to investigate whether the mTOR inhibitor rapamycin would recruit similar myocardial protective signalings in the hypertrophied heart and provided mechanistic insight involved.

Materials and methods

Animals

Male C57BL/6J mice were supplied by the Shanghai Laboratory Animal Center. All the protocols used conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (the 8th edition, NRC 2011), and they were approved by the Institutional Review Board of Zhongshan Hospital at Fudan University (number 2015-196).

Study groups and experimental protocol

To determine the role of ERK/STAT3 signalings in rapamycin-elicited cardioprotection against MI/R injury (Protocol A, Figure 1A), male C57BL6/J mice at 8 weeks of age were randomly assigned to the following experimental groups: (i) sham: mice given a sham operation (n=8, one mouse died after pentobarbital sodium anesthesia); (ii) vehicle: ischemic/reperfused mice treated with 0.5% DMSO (n=10, one mouse died from bleeding after coronary artery ligation, one mouse was excluded due to failed endotracheal intubation); (iii) RAPA: ischemic/reperfused mice treated with rapamycin (0.25 mg/kg) (n=10, one mouse was excluded due to bleeding after coronary artery ligation, one mouse died from arrhythmia during MI/R); (iv) RAPA-0.5: ischemic/reperfused mice treated with rapamycin (0.5 mg/kg) (n=10, two mice died from pneumothorax during MI/R, one mouse died from failed endotracheal intubation); (v) RAPA-1: ischemic/reperfused mice treated with rapamycin (1 mg/kg) (n=10, one mouse died from bleeding after coronary artery ligation, one mouse was excluded due to failed endotracheal intubation); (vi) RAPA-2: ischemic/reperfused mice treated with rapamycin (2 mg/kg) (n=10, two mice died from heart failure during MI/R, one mouse died from bleeding after coronary artery ligation); (vii) sham + PD98059: sham-operated mice treated with PD98059 (n=8, all mice survived after the surgical operation); (viii) PD98059: ischemic/reperfused mice pretreated with PD98059 (n=10, one mouse was excluded due to failed endotracheal intubation, one mouse died from bleeding after coronary artery ligation); (ix) RAPA + PD98059: ischemic/reperfused mice pretreated with rapamycin and PD98059 (n=10, one mouse was excluded due to failed endotracheal intubation, one mouse died from heart failure during MI/R); (x) sham + stattic: sham-operated mice pretreated with stattic (n=8, one mouse died from respiratory inhibition after pentobarbital sodium anesthesia); (xi) stattic: ischemic/reperfused mice pretreated with stattic (n=10, one mouse was excluded due to failed endotracheal intubation, one mouse died from bleeding after coronary artery ligation); (xii) RAPA + stattic: ischemic/reperfused mice pretreated with rapamycin and stattic (n=10, two mice were excluded due to bleeding after coronary artery ligation).

Experimental design: sham-operated C57 mice and aortic-banded mice were subjected to 40 min of coronary artery occlusion and 24-h reperfusion in vivo

Figure 1
Experimental design: sham-operated C57 mice and aortic-banded mice were subjected to 40 min of coronary artery occlusion and 24-h reperfusion in vivo

Rapamycin (RAPA) or vehicle (0.5% DMSO) was administrated intraperitoneally (i.p.) 10 min before reperfusion. PD98059 (1 mg/kg) or stattic (20 mg/kg) was administered (i.p.) 30 min before rapamycin treatment. gp91ds-tat (10 mg/kg) or scrambled sequence of gp91ds-tat (Scramb-tat, 10 mg/kg) or EUK134 (5 mg/kg) was injected into the caudal vein 10 min before reperfusion. The samples were collected after 3 h of reperfusion. Myocardial infarct size and cardiac function was measured after 24 h of reperfusion.(A) Protocoal A.(B) Protocol B.(C )Protocol C.(D) Protocol D.

Figure 1
Experimental design: sham-operated C57 mice and aortic-banded mice were subjected to 40 min of coronary artery occlusion and 24-h reperfusion in vivo

Rapamycin (RAPA) or vehicle (0.5% DMSO) was administrated intraperitoneally (i.p.) 10 min before reperfusion. PD98059 (1 mg/kg) or stattic (20 mg/kg) was administered (i.p.) 30 min before rapamycin treatment. gp91ds-tat (10 mg/kg) or scrambled sequence of gp91ds-tat (Scramb-tat, 10 mg/kg) or EUK134 (5 mg/kg) was injected into the caudal vein 10 min before reperfusion. The samples were collected after 3 h of reperfusion. Myocardial infarct size and cardiac function was measured after 24 h of reperfusion.(A) Protocoal A.(B) Protocol B.(C )Protocol C.(D) Protocol D.

To investigate the effects of LVH on rapamycin-elicited cardioprotection against MI/R injury (Protocol B, Figure 1B), male C57BL6/J mice were randomly assigned to the following experimental groups: (i) sham: mice given a sham operation (n=16, one mouse died after pentobarbital sodium anesthesia); (ii) vehicle: ischemic/reperfused mice treated with 0.5% DMSO (n=20, one mouse died after pentobarbital sodium anesthesia, three mice died from bleeding after coronary artery ligation, one mouse died from heart failure during MI/R); (iii) RAPA: ischemic/reperfused mice treated with rapamycin (0.25 mg/kg) (n=20, two mice were excluded due to bleeding after coronary artery ligation, one mouse died from arrhythmia during MI/R, one mouse died from heart failure during MI/R); (iv) transverse aortic constriction (TAC): mice given TAC for 2 weeks (n=20, two mice died after pentobarbital sodium anesthesia, four mice died from heart failure after aortic banding); (v) TAC + vehicle: aortic-banded mice were treated with 0.5% DMSO followed by MI/R (n=22, one mouse died from bleeding after aortic banding, two mice died from heart failure after aortic banding, three mice died from arrhythmia during MI/R, one mouse died from bleeding after coronary artery ligation); (vi) TAC + RAPA: aortic-banded mice were treated with RAPA 10 min before reperfusion (n=24, one mouse died from bleeding after aortic banding, two mice died from heart failure after aortic banding, one mouse died from bleeding after coronary artery ligation, three mice died from arrhythmia during MI/R, one mouse died from bleeding after coronary artery ligation).

To determine whether ROS suppression by selective gp91phox assembly inhibitor gp91ds-tat rescues the ability of rapamycin to reduce MI/R injury (Protocol C, Figure 1C), male C57BL6/J mice were randomly assigned to the following experimental groups: (i) Scramb-tat: ischemic/reperfused mice treated with scrambled gp91 sequence (a scrambled sequence of gp91ds-tat) (n=20, two mice were excluded due to failed endotracheal intubation, four mice died from bleeding after coronary artery ligation); (ii) gp91ds-tat: ischemic/reperfused mice treated with gp91ds-tat (a peptide inhibitor for gp91phox, 10 mg/kg) 10 min before reperfusion (n=20, two mice died from bleeding after coronary artery ligation, one mouse died from arrhythmia during MI/R, two mice died from heart failure during MI/R); (iii) RAPA: ischemic/reperfused mice treated with rapamycin (0.25 mg/kg) (n=20, two mice were excluded due to bleeding during MI/R, three mice died from heart failure during MI/R); (iv) RAPA + gp91ds-tat: ischemic/reperfused mice treated with rapamycin and gp91ds-tat (n=20, one mouse was excluded due to failed endotracheal incubation, one mouse was excluded due to bleeding after coronary artery ligation, one mouse died from arrhythmia during MI/R, two mice died from heart failure during MI/R); (v) TAC + Scramb-tat: aortic-banded mice treated with scrambled gp91 sequence followed by MI/R (n=24, three mice died from heart failure after aortic banding, two mice were excluded due to failed endotracheal incubation before MI/R, three mice died from arrhythmia during MI/R, two mice died from bleeding after coronary artery ligation); (vi) TAC + gp91ds-tat: aortic-banded mice treated with gp91ds-tat (n=24, three mice died from bleeding after aortic banding, one mouse died from heart failure after aortic banding, one mouse was excluded due to failed endotracheal incubation, three mice died from arrhythmia during MI/R, one mouse died from bleeding after coronary artery ligation); (vii) TAC + RAPA: aortic-banded mice treated with RAPA (n=24, one mouse died after pentobarbital sodium anesthesia, one mouse died from bleeding after aortic banding, three mice died from heart failure after aortic banding, one mouse was excluded due to failed endotracheal incubation, three mice died from arrhythmia during MI/R, one mouse died from bleeding after coronary artery ligation); (viii) TAC + RAPA + gp91ds-tat: aortic-banded mice treated with rapamycin and gp91ds-tat (n=24, two mice died from bleeding after aortic banding, two mice died from heart failure after aortic banding, four mice died from arrhythmia during MI/R, one mouse died from bleeding after coronary artery ligation).

To determine whether ONOO scavenger rescues the ability of rapamycin to reduce MI/R injury (Protocol D, Figure 1D), male C57BL6/J mice were randomly assigned to the following experimental groups: (i) vehicle: ischemic/reperfused mice treated with 0.5% DMSO (n=20, one mouse was excluded due to failed endotracheal intubation, four mice died from bleeding after coronary artery ligation); (ii) EUK134: ischemic/reperfused mice treated with EUK134 (5 mg/kg) (n=20, one mouse was excluded due to failed endotracheal intubation, two mice were excluded due to failed intravenous injection, two mice died from bleeding after coronary artery ligation); (iii) RAPA: ischemic/reperfused mice treated with rapamycin (0.25 mg/kg) (n=20, two mice were excluded due to bleeding during MI/R, two mice died from heart failure during MI/R); (iv) RAPA + EUK134: ischemic/reperfused mice treated with rapamycin and EUK134 (n=20, one mouse was excluded due to failed intravenous injection, three mice were excluded due to bleeding after coronary artery ligation); (v) TAC + vehicle: aortic-banded mice treated with 0.5% DMSO followed by MI/R (n=24, one mouse died from bleeding after aortic banding, three mice died from heart failure after aortic banding, one mouse was excluded due to failed endotracheal incubation before MI/R, three mice died from arrhythmia during MI/R, three mice died from bleeding after coronary artery ligation); (vi) TAC + EUK134: aortic-banded mice treated with EUK134 followed by MI/R (n=24, one mouse died after pentobarbital sodium anesthesia, two mice died from bleeding after aortic banding, three mice died from heart failure after aortic banding, three mice died from arrhythmia during MI/R, one mouse died from bleeding after coronary artery ligation); (vii) TAC + RAPA: aortic-banded mice treated with RAPA (n=24, two mice died after pentobarbital sodium anesthesia, one mouse died from bleeding after aortic banding, two mice died from heart failure after aortic banding, one mouse was excluded due to failed endotracheal incubation, three mice died from arrhythmia during MI/R, one mouse died from bleeding after coronary artery ligation); (viii) TAC + RAPA + EUK134: aortic-banded mice treated with rapamycin and EUK134 (n=24, one mouse died after pentobarbital sodium anesthesia, two mice died from bleeding after aortic banding, three mice died from heart failure after aortic banding, one mouse was excluded due to failed intravenous injection, three mice died from arrhythmia during MI/R, two mice died from bleeding after coronary artery ligation).

Rapamycin (0.25 mg/kg; Sigma–Aldrich, St. Louis, MO, U.S.A.) was administered intraperitoneally (i.p.) 10 min before reperfusion. PD98059 (1 mg/kg; Sigma–Aldrich, St. Louis, MO, U.S.A.) or stattic (20 mg/kg; Sigma–Aldrich, St. Louis, MO, U.S.A.) was administered i.p. 30 min before rapamycin treatment. gp91ds-tat or scrambled gp91 sequence (10 mg/kg; Absin Bioscience Inc., Shanghai, China) or EUK134 (5 mg/kg; Cayman Chemical, Ann Arbor, MI, U.S.A.) was injected to the caudal vein 10 min before reperfusion. The surgeon was blinded to the treatment allocation.

TAC

The minimally invasive TAC operation was performed in male C57BL/6 mice after anesthetizing with 1% pentobarbital sodium (50 mg/kg, i.p.). In brief, a longitudinal skin incision ~1 cm was made to locate the trachea along the suprasternal notch, then a horizontal sternum cut was made to locate the thymus and aorta. The aorta between the origin of the right innominate and left common carotid arteries was constricted with a 6-0 silk suture by ligating the aorta with a bent 27-gauge needle. Sham-operated animals underwent the same procedure except that the artery was not ligated. After the surgery, the mice were housed in standard animal housing conditions for 2 weeks. Then, the diastolic LV posterior wall thickness was assessed using echocardiography.

MI/R protocol

The surgical procedures were carried out as described previously [30]. Briefly, mice were anesthetized with 2% isoflurane and artificially ventilated. A PE-10 tube was placed on the surface of the left anterior descending artery (LAD), then the LAD was ligated by an 8-0 silk with the PE-10 tube. After 40 min of myocardial ischemia, the tie was removed. Epicardial cyanosis was apparent in the area at risk during 40 min of coronary artery occlusion, while successful reperfusion was confirmed by epicardial hyperemia. Mice that fully recovered from the surgery were housed in standard animal conditions for 24 h.

Determination of infarct size

The myocardial infarct size was assessed using 2,3,5-triphenyltetrazolium chloride (TTC, Sigma–Aldrich, St. Louis, MO, U.S.A.) staining after reperfusion for 24 h. Briefly, the coronary artery was re-ligated, and 0.2 ml 2% Evans Blue dye was injected into the right ventricular cavity to identify the unstained area as the area at risk. The hearts were harvested and frozen, sectioned into 2-mm slices, and stained in 1% TTC solution at 37°C for 10 min. The area of infarct (pale) and risk (red) was analyzed by planimetry using ImageJ 1.37 software (National Institutes of Health, Bethesda, MD, U.S.A.). The myocardial infarct size was expressed as a percentage of infarct area over ischemic area (area at risk).

Doppler echocardiography

After 24 h of reperfusion, the mice were anesthetized with 1% isoflurane. M-mode images of LV long-axis were obtained at the level of the papillary muscle tips using a Vevo 770 imaging system (VisualSonics, Toronto, Canada). The ejection fraction (EF) and fractional shortening (FS) were calculated using the Teichholz formula.

Detection of myocardial apoptosis

Myocardial apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining using a fluorescein in situ cell death detection kit (Roche, Indianapolis, IN) as we described elsewhere [31]. The green fluorescein staining indicates apoptotic nuclei. TUNEL-positive nuclei (green nuclei) was expressed as the percentage of total cell population.

Detection of caspase-3 activities in heart tissue

Myocardial Caspase-3 activity was assessed using a Caspase-3 colorimetric assay kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the instructions of the manufacturer. In brief, myocardial tissues from area at risk were harvested after 3 h of reperfusion, homogenized, and centrifuged (10000 g for 5 min, 4°C) to harvest the supernatants. Two hundred micrograms of total protein was incubated with 25 μg Ac-DEVD-pNA in a 96-well plate (37°C for 1.5 h). The pNA absorbance was determined by a SpectraMax plate reader at 405 nm.

Measurement of ROS generation

Dihydroethidium (DHE) staining was used to assess the in situ ROS levels. The hearts were harvested after 3 h of reperfusion and were cut into sections 5 µm thick. The slices were stained with DHE (5 μM) at 37°C for 30 min. The photographs were acquired using a fluorescence microscope. Fluorescent intensity was assessed by using ImageJ 1.37. Myocardial superoxide production was measured by lucigenin-ECL. The relative light units (RLU) emitted were recorded and integrated over 30-s intervals for 5 min. Superoxide production was normalized with the heart weight.

Determination of nitrotyrosine content in cardiac tissue

The hearts were harvested after 3 h of reperfusion and were cut into sections 5 µm thick after 4% paraformaldehyde fixation. The slices were by embedded by paraffin and stained with anti-nitrotyrosine antibody (1:100, Millipore, Billerica, MA, U.S.A.). The immunostaining was conducted by utilizing the Vectastain ABC kit (1:200, Vector Laboratories, Burlingame, CA, U.S.A.), and the images were acquired under light microscopy. The cardiac nitrotyrosine content was quantitated by utilizing the Nitrotyrosine ELISA Kit (Abnova, Taiwan, China). The nitrotyrosine content was expressed as microgram/milligram of protein.

Measurement of myocardial NO content

Myocardial NO content was assessed by determining nitrite using the Griess methods. The samples from ischemic area were harvested after 3 h of reperfusion. The NO content was detected by utilizing the Total Nitric Oxide Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China) following the manufacturer’s instructions.

Immunoblotting

Samples were harvested from the apex of the heart after reperfusion for 3 h. The expression levels of myocardial mTOR (Cell Signaling Technology (CST), Beverly, MA, U.S.A.; 1:1000), p-mTOR (Ser2448, CST, 1:1000), S6 ribosomal protein (CST, 1:1000), p-S6 ribosomal protein (Ser235/236, CST, 1:1000), Akt (CST, 1:1000), p-Akt (Ser473, CST, 1:1000), STAT3 (CST, 1:1000), p-STAT3 (Tyr705, CST, 1:1000), extracellular regulated protein kinase 1/2 (ERK1/2) (CST, 1:1000), p-ERK1/2 (Thr202/Tyr204, CST, 1:1000), glycogen synthase kinase 3β (GSK3β) (CST, 1:1000), p-GSK3β (Ser9, CST, 1:1000), eNOS (Abcam, 1:1000), p-eNOS (Ser1177, Abcam, 1:1000), gp91phox (Abcam, 1:1000), iNOS (Abcam, 1:1000); and GAPDH (CST, 1:5000) were determined by immunoblotting [31]. The blot density was assessed using ImageJ 1.37 software.

Statistical analysis

The data were presented as the mean ± S.E.M. Statistical analysis was performed with unpaired Student’s t test for two-group comparisons. For multigroup comparisons, one-way ANOVA following Bonferroni’s post hoc test was used. For the survival study, Kaplan–Meier analysis was used. A value of P<0.05 was considered to be statistically significant. All statistical analyses were performed using GraphPad Prism version 4.0 (GraphPad Prism Software, San Diego, CA, U.S.A.).

Results

Survival

A total of 588 mice were used in the present study. The overall survival rate after aortic banding was 79.4% (205 out of 258) and 98.5% (325 out of 330) in sham-operated mice before MI/R (P<0.05). Thirty-eight out of 50 mice survived in the vehicle-treated group (76%) as compared with 40 out of 50 in the rapamycin-treated group (80%) following MI/R (P>0.05). Post-MI/R survival rate was 61.1% (44 out of 72) in aortic-banded mice treated with rapamycin and 65.2% (30 out of 46) in aortic-banded mice treated with vehicle (P>0.05). Twenty-two out of 24 mice survived in the sham group (91.6%) as compared with 14 out of 20 in the TAC group (70%) without MI/R (P<0.05). The survival rate was from 70 to 80% in other sham-operated treatment groups and from 50 to 68.1% in other aortic-banded treatment groups followed by MI/R.

The physiological measurements after TAC in mice

The heart rate and EF were not significantly different in mice with TAC for 2 weeks than in age-matched mice given a sham operation (495 ± 25 beats/min compared with 506 ± 29 beats/min, P>0.05; 70.8 ± 3.2% compared with 71.2 ± 3.1%, P>0.05, respectively). The diastolic posterior wall thickness of the left ventricle assessed by echocardiography (0.64 ± 0.06 mm compared with 0.93 ± 0.08 mm, P<0.05) was significantly increased in mice with TAC for 2 weeks compared with age-matched mice subjected to a sham operation, indicating LVH (Table 1).

Table 1
The physiological measurements after TAC in mice
Group Sham TAC 
Heart rate 495 ± 25 506 ± 29 
LVEF (%) 70.8 ± 3.2 71.2 ± 3.1 
LVFS (%) 40.5 ± 2.2 40.8 ± 2.3 
LV posterior wall thickness (mm)   
Diastole 0.64 ± 0.06 0.93 ± 0.08* 
Systole 0.75 ± 0.06 1.13 ± 0.11* 
Body weight, g 24.5 ± 1.1 24.2 ± 1.1 
Heart weight, mg 101.6 ± 2.8 151.2 ± 4.3* 
Tibial length, mm 16.4 ± 0.2 16.5 ± 0.2 
Heart weight/body weight, mg/g 4.24 ± 0.11 6.16 ± 0.54* 
Heart weight/tibial length, mg/mm 6.4 ± 0.22 8.7 ± 0.33* 
Group Sham TAC 
Heart rate 495 ± 25 506 ± 29 
LVEF (%) 70.8 ± 3.2 71.2 ± 3.1 
LVFS (%) 40.5 ± 2.2 40.8 ± 2.3 
LV posterior wall thickness (mm)   
Diastole 0.64 ± 0.06 0.93 ± 0.08* 
Systole 0.75 ± 0.06 1.13 ± 0.11* 
Body weight, g 24.5 ± 1.1 24.2 ± 1.1 
Heart weight, mg 101.6 ± 2.8 151.2 ± 4.3* 
Tibial length, mm 16.4 ± 0.2 16.5 ± 0.2 
Heart weight/body weight, mg/g 4.24 ± 0.11 6.16 ± 0.54* 
Heart weight/tibial length, mg/mm 6.4 ± 0.22 8.7 ± 0.33* 

The physiological measurements after TAC 2 weeks in mice; n=6 per group; *P<0.05 compared with sham.

Rapamycin attenuated MI/R injury via ERK/STAT3-dependent mechanisms

To determine whether rapamycin would render the heart resistant to subsequent ischemic stress, rapamycin was administrated to male mice 10 min before reperfusion. Compared with vehicle group, mTOR inhibition by rapamycin significantly reduced MI/R-induced myocardial necrosis assessed by infarct size (21.81 ± 2.40% in RAPA-0.25 group, 22.81 ± 2.46% in RAPA-0.5 group, 22.98 ± 2.56% in RAPA-1 group, and 23.36 ± 3.74% in RAPA-2 group compared with 44.71 ± 4.21% in vehicle group, P<0.05, Figure 2A), whereas the area at risk (AAR) did not significantly differ amongst all the groups. These results demonstrated that the myocardial infarct-sparing effects by different doses of rapamycin, ranging from 0.25 to 2 mg/kg, did not markedly differ amongst all groups. Therefore, we used a low dose of rapamycin (0.25 mg/kg) in the current study. Furthermore, we found the infarct-limiting effect induced by rapamycin was blocked by the MEK-ERK1/2 inhibitor PD98059 or STAT3 inhibitor Stattic, demonstrating an ERK/STAT3-dependent mechanisms for rapamycin-elicited cardioprotection (Figure 2B,C).

Rapamycin reduces MI/R injury via ERK/STAT3 signaling

Figure 2
Rapamycin reduces MI/R injury via ERK/STAT3 signaling

(A) The myocardial infarct size expressed as a percentage of the total area of the myocardium in mice exposed to different doses of rapamycin. (B) The myocardial infarct size in mice pretreated with or without MEK-ERK1/2 inhibitor PD98059 (C) The myocardial infarct size in mice pretreated with or without STAT3 inhibitor Stattic. *P<0.05 compared with sham, #P<0.05 compared with vehicle, P<0.05 compared with RAPA; n=6–8 per group.

Figure 2
Rapamycin reduces MI/R injury via ERK/STAT3 signaling

(A) The myocardial infarct size expressed as a percentage of the total area of the myocardium in mice exposed to different doses of rapamycin. (B) The myocardial infarct size in mice pretreated with or without MEK-ERK1/2 inhibitor PD98059 (C) The myocardial infarct size in mice pretreated with or without STAT3 inhibitor Stattic. *P<0.05 compared with sham, #P<0.05 compared with vehicle, P<0.05 compared with RAPA; n=6–8 per group.

Hypertrophied heart was vulnerable to MI/R injury and refractory to rapamycin-induced cardioprotection

The basal cardiac function was similar between normal and aortic-banded mice. Compared with the vehicle group, aortic banding for 14 days remarkably increased myocardial infarct size in TAC + vehicle group (55.54 ± 4.09% in the TAC + vehicle group compared with 42.71 ± 3.39% in vehicle group, P<0.05, Figure 3A), while the AAR did not significantly differ amongst all groups. To assess LV performance, echocardiography was performed to measure cardiac function after 24 h of reperfusion. As shown in Figure 3B, MI/R markedly reduced LVEF (left ventricular ejection fraction) and LVFS (left ventricular fraction fractional shortening). Compared with the vehicle group, aortic-banding for 2 weeks markedly hindered cardiac functional recovery following MI/R evidenced by reduced LVEF and LVFS (31.28 ± 3.04% and 15.86 ± 2.16% in the TAC + vehicle group compared with 42.09 ± 4.06% and 22.29 ± 3.74% in the vehicle group, P<0.05, Figure 3B). Moreover, aortic constriction for 14 days remarkably increased MI/R-induced cardiomyocyte apoptosis evidenced by increased TUNEL-positive cell nucleus and Caspase-3 activity (P<0.05, Figure 3C). Taken together, these findings demonstrated that LVH prompted cardiomyocyte death and inhibited cardiac function following MI/R.

Hypertrophied heart was vulnerable to MI/R injury and refractory to rapamycin-induced cardioprotection

Figure 3
Hypertrophied heart was vulnerable to MI/R injury and refractory to rapamycin-induced cardioprotection

(A) The myocardial infarct size, determined by Evans Blue and TTC staining. (B) Cardiac function (LVEF and LVFS), determined by Doppler echocardiography following in situ MI/R. (C) Cardiomyocyte apoptosis assessed by TUNEL staining (400×) and Caspase-3 activity measurement following in situ MI/R. *P<0.05 compared with sham, #P<0.05 compared with vehicle, P<0.05 compared with RAPA; n=7–8 per group.

Figure 3
Hypertrophied heart was vulnerable to MI/R injury and refractory to rapamycin-induced cardioprotection

(A) The myocardial infarct size, determined by Evans Blue and TTC staining. (B) Cardiac function (LVEF and LVFS), determined by Doppler echocardiography following in situ MI/R. (C) Cardiomyocyte apoptosis assessed by TUNEL staining (400×) and Caspase-3 activity measurement following in situ MI/R. *P<0.05 compared with sham, #P<0.05 compared with vehicle, P<0.05 compared with RAPA; n=7–8 per group.

Having demonstrated that aortic constriction for 2 weeks increased myocardial vulnerability to MI/R injury, we next sought to investigate the myocardial effect of mTOR inhibition in normal and aortic-banded mice exposed to MI/R. Consistent with previous studies [21,32], treatment of normal animals with 0.25 mg/kg rapamycin markedly hindered MI/R-elicited injury evidenced by reduced myocardial infarct size (44.63 ± 4.86% in the vehicle group compared with 21.01 ± 1.86% in the RAPA group, P<0.05, Figure 3A), improved cardiac systolic function (LVEF and LVFS, 41.64 ± 3.66% and 21.14 ± 2.77% in the vehicle group compared with 50.94 ± 3.3% and 26.17 ± 1.96% in the RAPA group, P<0.05, Figure 3B), and reduced cardiomyocyte apoptosis (TUNEL staining and Caspase-3 activity, Figure 3C). However, the beneficial effects of rapamycin treatment was markedly attenuated in aortic-banded mice evidenced by augmented myocardial infarct size (56.43 ± 4.14% in the TAC + vehicle group compared with 57.35 ± 5.41% in the TAC + RAPA group, P>0.05, Figure 3A), worsening cardiac systolic function (LVEF and LVFS, 32.10 ± 2.81% and 16.58 ± 1.68% in the TAC + vehicle group compared with 31.41 ± 4.81% and 16.33 ± 2.45% in the TAC + RAPA group, P>0.05, Figure 3B), and increased cardiomyocyte apoptosis (TUNEL staining and Caspase-3 activity, Figure 3C). Next we sought to investigate whether a higher dose of rapamycin would rescue the detrimental effects of LVH on MI/R. Rapamycin (2 mg/kg) was administrated to sham-operated or aortic-banded mice by intraperitoneal injection 10 min before reperfusion. We found that such dose of rapamycin failed to rescue the detrimental effects of LVH on MI/R evidenced by exacerbated myocardial infarct and worsening cardiac function in aortic-banded mice followed by MI/R (Supplementary Figure S1). To determine whether direct intramyocardial injection of rapamycin would rescue the detrimental effects of ventricular hypertrophy on MI/R, rapamycin (0.25 mg/kg) was administrated to sham-operated or aortic-banded mice by direct intramyocardial injection. We found that direct intramyocardial injection of rapamycin reduced myocardial infarct size and improved cardiac function in ischemic/reperfused normal mice, however, the cardioprotective effects induced by direct intramyocardial injection of rapamycin was lost in aortic-banded mice followed by MI/R (Supplementary Figure S2). Taken together, these results indicate that LVH attenuates rapamycin-elicited myocardial protection against MI/R.

Aortic banding hindered rapamycin-induced mTOR inhibition and ERK/STAT3 phosphorylation in the myocardium following MI/R

mTOR inhibition by rapamycin exerts its cardioprotective effects via the ERK-STAT3 signaling axis [21,33], which is defective in persistent aortic-banded hearts. We observed the decreased phosphorylation of mTOR (Figure 4A) and S6 (Figure 4B) but not p-Akt (Figure 4C) in normal mice after rapamycin treatment. Moreover, we found the phosphorylation of mTOR, S6, and Akt was remarkably increased in aortic-banded mice in comparison with normal mice (Figure 4A–C), indicating increased mTOR activity in hypertrophied myocardium. However, rapamycin failed to inhibit myocardial mTOR and S6 phosphorylation in aortic-banded mice subjected to MI/R (Figure 4A–C). In parallel with previous findings [21,34], we observed the increased phosphorylation of ERK and STAT3 in normal mice after rapamycin treatment (Figure 4D,E). We also found that p-GSK3β (Figure 4F), a downstream target of ERK/STAT3 signaling, was markedly increased in normal mice after treatment with rapamycin. We further investigated whether LVH would disturb rapamycin-elicited ERK/STAT3 activation following MI/R. Interestingly, the level of phosphorylated ERK, STAT3, and GSK3β at baseline was markedly higher in the TAC group than in the sham group (Figure 4D,E). However, reperfusion-induced phosphorylation of ERK/STAT3 was suppressed in the TAC + vehicle group (Figure 4D,E). The inhibition of ERK/STAT3 phosphorylation upon reperfusion in the TAC + vehicle group was indicated also by the reduction in p-GSK3β, a kinase downstream of ERK/STAT3 signaling (Figure 4F). Moreover, rapamycin failed to further prompt myocardial ERK and STAT3 phosphorylation in aortic-banded mice subjected to MI/R (Figure 4D,E). In addition, the inactivation of GSK3β by rapamycin was also not observed in the presence of LVH, demonstrated by blunt GSKβ phosphorylation in aortic-banded mice (Figure 4F). Altogether, our findings demonstrated that the disturbed mTOR signaling and blunt ERK-STAT3 pathway may be related to the reduced cardioprotective actions by rapamycin in aortic-banded mice following MI/R.

Aortic banding hindered rapamycin-induced ERK/STAT3 phosphorylation in the myocardium following MI/R

Figure 4
Aortic banding hindered rapamycin-induced ERK/STAT3 phosphorylation in the myocardium following MI/R

The expression of p-mTOR (A), p-S6 (B), p-Akt (C), p-ERK1/2 (D), p-STAT3 (E), and p-GSK3β (F) in mice hearts subjected to MI/R with or without rapamycin. *P<0.05 compared with sham, #P<0.05 compared with vehicle, P<0.05 compared with RAPA; n=6 per group.

Figure 4
Aortic banding hindered rapamycin-induced ERK/STAT3 phosphorylation in the myocardium following MI/R

The expression of p-mTOR (A), p-S6 (B), p-Akt (C), p-ERK1/2 (D), p-STAT3 (E), and p-GSK3β (F) in mice hearts subjected to MI/R with or without rapamycin. *P<0.05 compared with sham, #P<0.05 compared with vehicle, P<0.05 compared with RAPA; n=6 per group.

Disturbed rapamycin elicited antioxidative protection in aortic-banded mice further proved cardiac resistance to rapamycin

Oxidative stress has been proven as an important upstream mediator of cardiomyocyte death [35], therefore, we investigated the roles of mTOR inhibition in regulating oxidative stress to provide mechanistic insights into the cardioprotection of rapamycin against cardiomyocyte death. We found that rapamycin treatment remarkably ameliorated MI/R-elicited gp91phox overexpression (Figure 5A) and superoxide production (Figure 5B–D) in normal mice. Nonetheless, administration of rapamycin failed to alleviate the increased myocardial oxidative stress in aortic-banded mice manifested by the failure to further attenuate myocardial gp91phox overexpression (Figure 5A) and superoxide production (Figure 5B–D). The expression of gp91phox (Figure 5A) and superoxide production (Figure 5B–D) were significantly increased in aortic-banding mice following MI/R, indicating increased oxidative stress in hypertrophied myocardium. Collectively, these findings imply that the disturbed rapamycin elicited antioxidative protection contributes to the reduced cardioprotective actions of rapamycin in hypertrophied myocardium.

Disturbed rapamycin elicited antioxidative protection in aortic-banded mice further proved cardiac resistance to rapamycin

Figure 5
Disturbed rapamycin elicited antioxidative protection in aortic-banded mice further proved cardiac resistance to rapamycin

(A) The myocardial expression of gp91phox, determined by immunobloting. (B) Superoxide production in ischemic/reperfused cardiac tissue, evaluated by lucigenin-ECL. (C) Myocardial superoxide at steady-state level, measured by in situ DHE staining, scale bar =50 μm. (D) Quantitative analyses of DHE fluorescence intensity. *P<0.05 compared with sham, #P<0.05 compared with vehicle, P<0.05 compared with RAPA; n=6 per group.

Figure 5
Disturbed rapamycin elicited antioxidative protection in aortic-banded mice further proved cardiac resistance to rapamycin

(A) The myocardial expression of gp91phox, determined by immunobloting. (B) Superoxide production in ischemic/reperfused cardiac tissue, evaluated by lucigenin-ECL. (C) Myocardial superoxide at steady-state level, measured by in situ DHE staining, scale bar =50 μm. (D) Quantitative analyses of DHE fluorescence intensity. *P<0.05 compared with sham, #P<0.05 compared with vehicle, P<0.05 compared with RAPA; n=6 per group.

Disturbed rapamycin elicited antinitrative protection in aortic-banded mice further demonstrated cardiac resistance to rapamycin

It is noteworthy that NO itself does not induce additional myocardial impairment under physiological conditions, however, NO interacts with superoxide and subsequently induces nitrative injury to mitochondria, protein, and lipids under pathological conditions and scavenging peroxynitrite ameliorates reperfusion injury [36]. We next sought to examine whether mTOR inhibition by rapamycin could affect myocardial NO generation. Therefore, we determined myocardial total NO content and eNOS activity in both normal and aortic-banded mice followed by MI/R. We found that the myocardial NO content was not altered by rapamycin treatment, whereas the eNOS activity was significantly enhanced, as evidenced by the unchanged myocardial NO metabolites (Figure 6A) and the increase in eNOS phosphorylation (Figure 6B,C) in normal mice. It is accepted that increased eNOS phosphorylation would induce NO generation, which seems to conflict with our present finding. Nonetheless, these paradoxical results indicate that other forms of NOS are involved in the increase in myocardial NO content. Therefore, we assayed myocardial iNOS expression by immunoblotting. Indeed, the myocardial iNOS expression was not affected by rapamycin (Figure 6D). Moreover, we observed a significant reduction in nitrotyrosine content in rapamycin-treated mice (Figure 6E,F), suggesting reduced nitrative stress by rapamycin. Nonetheless, administration of rapamycin failed to attenuate the increased myocardial nitrative stress in aortic-banded mice manifested by the failure to attenuate myocardial nitrotyrosine content (Figure 6E,F). The total NO content (Figure 6A), myocardial iNOS expression (Figure 6D), and nitrotyrosine content (Figure 6F) were significantly increased in aortic-banded mice compared with sham-operated mice, indicating augmented nitrative injury in hypertrophied myocardium. Collectively, these findings imply that disturbed rapamycin elicited antinitrative protection contributes to the attenuated cardioprotective actions of rapamycin in aortic-banded mice.

Disturbed rapamycin elicited antinitrative protection in aortic-banded mice further demonstrated cardiac resistance to rapamycin

Figure 6
Disturbed rapamycin elicited antinitrative protection in aortic-banded mice further demonstrated cardiac resistance to rapamycin

(A) NO content in ischemic/reperfused myocardial tissue, measured by using the Griess methods. (B) Representative immunobloting photographs for myocardial iNOS, p-eNOS, eNOS, and GAPDH expression. (C) Quantitative analyses of myocardial p-NOS expression, (D) iNOS. (E) Representative photographs for myocardial nitrotyrosine staining, scale bar =50 μm. (F) Quantitative analyses of myocardial nitrotyrosine content. *P<0.05 compared with sham, #P<0.05 compared with vehicle, P<0.05 compared with RAPA; n=6 per group.

Figure 6
Disturbed rapamycin elicited antinitrative protection in aortic-banded mice further demonstrated cardiac resistance to rapamycin

(A) NO content in ischemic/reperfused myocardial tissue, measured by using the Griess methods. (B) Representative immunobloting photographs for myocardial iNOS, p-eNOS, eNOS, and GAPDH expression. (C) Quantitative analyses of myocardial p-NOS expression, (D) iNOS. (E) Representative photographs for myocardial nitrotyrosine staining, scale bar =50 μm. (F) Quantitative analyses of myocardial nitrotyrosine content. *P<0.05 compared with sham, #P<0.05 compared with vehicle, P<0.05 compared with RAPA; n=6 per group.

gp91phox inhibition reduced myocardial superoxide production and ameliorated MI/R injury in aortic-banded mice

Having demonstrated that aortic constriction for 2 weeks increased myocardial vulnerability to MI/R injury and refractory to rapamycin-elicited cardioprotection in aortic-banded mice, we next sought to investigate whether scavenging overproduced superoxide by gp91phox inhibition would rescue the ability of rapamycin to reduce MI/R injury in aortic-banded mice. Scavenging superoxide by gp91phox inhibitor gp91ds-tat (10 mg/kg) 10 min before reperfusion markedly reduced myocardial superoxide content (Figure 7A), hindered myocardial nitrotyrosine production (Figure 7B), and alleviated MI/R evidenced by reduced myocardial infarct size (Figure 7C), improved cardiac function (Figure 7D), and inhibited cardiomyocyte apoptosis (Figure 7E) in both sham-operated and aortic-banded mice followed by MI/R. However, no additional cardioprotective effects were achieved when we combined rapamycin and gp91ds-tat in both sham-operated and aortic-banded mice exposed to MI/R (Figure 7A–E). These results demonstrated that rapamycin conferred cardioprotection against MI/R injury by inhibition of oxidative stress in normal heart and gp91phox inhibition rescued MI/R injury in hypertrophic hearts.

Scavenging superoxide by selective gp91phox assembly inhibitor gp91ds-tat markedly ameliorated MI/R injury in aortic-banded mice

Figure 7
Scavenging superoxide by selective gp91phox assembly inhibitor gp91ds-tat markedly ameliorated MI/R injury in aortic-banded mice

(A) Myocardial superoxide content and DHE fluorescence intensity. (B) Myocardial nitrotyrosine content. (C) Myocardial infarct size. (D) Cardiac function. (E) Apoptotic nuclei and Caspase-3 activity. *P<0.05 compared with Scramb-tat, #P<0.05 compared with TAC + Scramb-tat, P<0.05 compared with TAC + RAPA; n=6–8 per group.

Figure 7
Scavenging superoxide by selective gp91phox assembly inhibitor gp91ds-tat markedly ameliorated MI/R injury in aortic-banded mice

(A) Myocardial superoxide content and DHE fluorescence intensity. (B) Myocardial nitrotyrosine content. (C) Myocardial infarct size. (D) Cardiac function. (E) Apoptotic nuclei and Caspase-3 activity. *P<0.05 compared with Scramb-tat, #P<0.05 compared with TAC + Scramb-tat, P<0.05 compared with TAC + RAPA; n=6–8 per group.

Scavenging myocardial peroxynitrite by EUK134 attenuated MI/R injury in aortic-banded mice

Next we sought to determine whether scavenging overproduced peroxynitrite would rescue the detrimental effects of ventricular hypertrophy on MI/R injury in aortic-banded mice. The myocardial superoxide content did not markedly differ amongst all groups after ONOO scavenger EUK134 treatment (Figure 8A). Interestingly, we found that scavenging peroxynitrite by EUK134 (5 mg/kg) 10 min before reperfusion markedly reduced myocardial nitrotyrosine content (Figure 8B) and alleviated MI/R evidenced by reduced myocardial infarct size (Figure 8C), improved cardiac function (Figure 8D), and inhibited cardiomyocyte apoptosis in both sham-operated and aortic-banded mice followed by MI/R (Figure 8E). Nonetheless, no additional cardioprotective effects were achieved when we combined rapamycin and EUK134 in both sham-operated and aortic-banded mice exposed to MI/R (Figure 8A–E). These findings showed that rapamycin conferred cardioprotection against MI/R injury by inhibition of nitrative stress in normal hearts and scavenging peroxynitrite reduced MI/R injury in hypertrophic hearts. Altogether, these findings suggest that increased oxidative/nitrative stress is related to the aggravated MI/R and the attenuation of rapamycin-elicited cardioprotection in aortic-banded mice.

Scavenging peroxynitrite by EUK134 markedly attenuated MI/R injury in aortic-banded mice

Figure 8
Scavenging peroxynitrite by EUK134 markedly attenuated MI/R injury in aortic-banded mice

(A) Myocardial superoxide content and DHE fluorescence intensity. (B) Myocardial nitrotyrosine content. (C) Myocardial infarct size. (D) Cardiac function. (E) Apoptotic nuclei and Caspase-3 activity. *P<0.05 compared with vehicle, #P<0.05 compared with TAC + vehicle, P<0.05 compared with TAC + RAPA; n=6–8 per group.

Figure 8
Scavenging peroxynitrite by EUK134 markedly attenuated MI/R injury in aortic-banded mice

(A) Myocardial superoxide content and DHE fluorescence intensity. (B) Myocardial nitrotyrosine content. (C) Myocardial infarct size. (D) Cardiac function. (E) Apoptotic nuclei and Caspase-3 activity. *P<0.05 compared with vehicle, #P<0.05 compared with TAC + vehicle, P<0.05 compared with TAC + RAPA; n=6–8 per group.

Discussion

Several important contributions of present study can be summarized as follows. First, our findings demonstrated that LVH attenuated the myocardial protective effects of rapamycin in a clinically relevant animal model of in situ coronary artery ligation. Rapamycin provided beneficial cardiac actions against MI/R injury in normal mice, but it failed to do so in aortic-banded mice evidenced by aggravated cardiomyocyte apoptosis, incremental infarct size, and worsening cardiac function in aortic-banded mice. These results show the first direct evidence that the development of resistance to rapamycin-elicited beneficial cardiac effects in the hypertrophied myocardium in vivo. Second, we provided previously unknown mechanistic insights into this impaired cardiac response to rapamycin, which is related to the ERK-STAT3 activation resistance as well as reduced antioxidative action, at least in part. Last, we demonstrated that, although LVH resists rapamycin-elicited myocardial protection and enhances cardiac susceptibility to MI/R, scavenging peroxynitrite can still alleviate MI/R injury evidenced by attenuated myocardial infarction, ameliorated cardiomyocyte apoptosis, and inhibited cardiac dysfunction in the aortic-banded mice, suggesting a promising therapy for MI/R in the presence of LVH.

The effects of cardiac hypertrophy on MI/R are yet to be fully elucidated. Here, we provide first direct evidence that LVH markedly increased myocardial oxidative/nitrative stress in aortic-banded mice subjected to MI/R. Moreover, we observed markedly augmented myocardial infarct size, worsening cardiac function, and aggravated cardiomyocyte apoptosis in aortic-banded mice in comparison with that in normal ones following MI/R. Especially important, we demonstrated for the first time that aortic-banded mice resistant to rapamycin-elicited cardioprotection evidenced by increased myocardial infarct size and apoptosis during MI/R. Moreover, we found that scavenging peroxynitrite rescued cardiomyocyte fate against MI/R in hypertrophied myocardium. In parallel with the present study, the myocardial infarct size was markedly increased in spontaneously hypertensive rats subjected to MI/R [7,8]. However, other reports have shown conflicting results that hypertrophied myocardium is not specifically vulnerable to infarction during MI/R [37,38]. The reasons for these distinctly different findings are incompletely unknown but may be attributed to difference in models of cardiac hypertrophy (duration of pressure overload, species) and experimental setting (myocardial ischemic time).

Chronic increased mTOR activation was causally related to diverse disorders, including diabetes [13,34], uremic cardiomyopathy [15,16], and cardiac hypertrophy [1719]. Numerous studies have demonstrated that therapeutic target of mTOR alleviated LVH and LVH-related complications. For instance, rapamycin was shown to mitigate pressure overload induced hypertrophic responses by rescuing the defective cytoprotective mechanisms in aortic-banded mice [17]. In addition, mTOR inhibition with rapamycin (0.25 mg/kg, once) was shown to induce preconditioning like myocardial protection against ischemia/reperfusion injury in mice in vivo [20,21]. Moreover, Volkers et al. [22] demonstrated that increasing mTORC2 activation while inhibiting mTORC1 signaling reduced cardiomyocyte apoptosis and necrosis after MI [22]. Altogether, these exciting findings prove a vital role of rapamycin as a direct cardioprotectant against myocardial ischemic injury, implying that rapamycin might be a promising treatment in rescuing LVH-related cardiovascular impairment.

The role of rapamycin in prompting cardiomyocyte survival and cardiac functional recovery involved the ERK/STAT3 signaling, whereas its protective actions in eliciting vasodilatation/vasculoprotection occurs primarily via the STAT3/eNOS axis [20,21,32,33]. In parallel with these reports, our findings showed that rapamycin further prompted ERK activation and p-STAT3, thus elicited cardioprotective effects against MI/R injury in normal mice. Importantly, we observed LVH impaired this ERK/STAT3 phosphorylation by rapamycin and attenuated cardioprotection of rapamycin, showing novel mechanistic insights that the resistance to rapamycin in hypertrophied myocardium occurs through dysfunctional ERK activation, at least in part.

It has been proposed that GSK3β inhibition is an essential mechanism for cardioprotection [39]. In our study, we found that rapamycin enhanced the phosphorylation of GSK-3β at Ser9. A previous study showed that opioids conferred myocardial protection via the JAK2-STAT3 signals that regulates GSK3β signaling [40]. Moreover, the activated ERK1/2 leads to the phosphorylation of GSK3β and negatively regulates GSK3β activity [41]. Therefore, the deficit in GSK3β phosphorylation in hypertrophied hearts treated with rapamycin may, in part, be attributed to the observed deficit in the phosphorylation of STAT3 and ERK. And we reason that the loss of myocardial infarct sparing effect in the hypertrophied myocardium, at least in part, may be attributed to the dysfunction of GSK3β phosphorylation.

Although the ERK/STAT3 axis plays a crucial role in rapamycin’s prosurvival actions, we provided the first direct evidence of rapamycin-elicited myocardial protection via antioxidative/antinitrative injury in the intact animal. The overproduction of superoxide is a major contributor to endoplasmic reticulum and mitochondrial stress mediated apoptosis [35], and we demonstrated that rapamycin markedly hindered gp91phox activation, thus alleviated oxidative stress elicited cardiac injury. Moreover, we provided direct evidence of up-regulated gp91phox level and increased superoxide content in hypertrophied hearts following MI/R. Although rapamycin markedly reduced the overactivated gp91phox and inhibited superoxide production in normal mice following MI/R, the same dose of rapamycin failed to attenuate MI/R-induced oxidative stress, as evidenced by the increase in superoxide production and gp91phox expression in aortic-banded mice. Next we sought to determine whether scavenging superoxide by gp91phox inhibition would reduce myocardial susceptibility to MI/R in the hypertrophied myocardium, we observed markedly reduced MI/R injury after gp91ds-tat treatment manifested by reduced myocardial infarct size, improved cardiac function, and attenuated cardiomyocyte apoptosis. Nonetheless, no additional cardioprotective effects were achieved when we combined rapamycin and gp91ds-tat in aortic-banded mice exposed to MI/R. Altogether, these findings demonstrated the impaired rapamycin’s antioxidant action in the hypertrophied myocardium.

It is noteworthy that NO itself does not lead to additional myocardial impairment under physiological conditions, however, NO interacts with superoxide and subsequently induces oxidative/nitrative injury to mitochondria, protein, and lipids under pathological conditions [36]. In addition, previous studies have proved the harmful effects of nitrative stress and scavenging peroxynitrite ameliorates reperfusion injury [4244]. Although neither iNOS expression nor total NO metabolites were affected by rapamycin, we demonstrated that the myocardial nitrotyrosine content was markedly reduced by mTOR inhibition with rapamycin, suggesting reduced nitrative injury in normal mice. In addition, we demonstrated that the LVH increased myocardial iNOS expression, prompted NO production, and aggravated nitrotyrosine formation, thus increased myocardial susceptibility to MI/R. Next we sought to investigate whether scavenging peroxynitrite would reduce myocardial susceptibility to MI/R in the hypertrophied myocardium, we observed markedly reduced MI/R injury after EUK134 treatment evidenced by reduced myocardial infarct size, improved cardiac function, and attenuated cardiomyocyte apoptosis. However, we did not observe incremental cardioprotective effects when we combined rapamycin and EUK134 in aortic-banded mice exposed to MI/R. Taken together, these results suggest that LVH increased myocardial nitrative stress and rapamycin’s antinitrative injury was attenuated in mice with LVH.

mTOR inhibition is not only confined to modulate oxidative stress, but also related to cardioprotective molecules [32,45]. The present study has shown that mTOR inhibition by rapamycin markedly prompted the p-eNOS expression, and low concentration of NO produced by activated eNOS is well known to confer cardioprotective effects against necrosis and apoptosis during MI/R. Nonetheless, the role of mTOR in regulating NOS is still ambiguous. Recent studies have documented that ERK pathway is causally related to the cardioprotection by the activation of eNOS [21,46], and ERK activation has been demonstrated to attenuate oxidative/nitrative stress [47,48]. In our murine models, selective inhibiting cardiac mTOR1 increased p-ERK expression, implying that ERK signaling is involved in regulating eNOS activation by mTOR inhibition. Moreover, we show that the pathologic hypertrophied myocardium blocked rapamycin’s stimulation of eNOS phosphorylation, thus attenuated rapamycin-induced myocardial protection.

The findings provided here are translationally important in that they determined whether the cardioprotection induced by rapamycin occurs in animals with LVH. However, there are several limitations in our current work. First, the loss of myocardial protection by rapamycin in aortic-banded mice might also be due to changes in cardiac autophagy signaling, which were not explored in the present study. Second, although LVH may be associated with conditions other than hypertension, including myocardial infarction, anemia, aortic valve disease, hyperthyroidism, obesity, and renal disease, hypertension is the most common cause of LVH, which indicates that the hypertrophied murine heart model we used may not fully simulate the complex clinical setting of LVH. Third, the tissue for immunoblotting study was harvested from the apex of the heart and may have been mixtures of necrosis cells and salvaged cells. Therefore, cautious interpretation of the role of the protein in infarct size changes is needed and immunoblotting data normalized by percentage of viable mass in the sample may be more appropriate.

In summary, we demonstrated that LVH abrogated rapamycin’s cardioprotection and impaired ERK-dependent antioxidative mechanisms through which rapamycin protects the heart from MI/R. The detailed mechanisms underlying how LVH disturbs the cardioprotective actions of rapamycin involved in activation of ERK/STAT3 and its antioxidative/antinitrative effect is currently under investigation. Moreover, scavenging peroxynitrite reduced myocardial susceptibility to MI/R in aortic-banded animals. These findings may provide potential translation to make differential clinical treatment plans for patients with or without LVH in the presence of AMI.

Clinical perspectives

  • LVH is causally related to increased morbidity and mortality following AMI via still unknown mechanisms. Although rapamycin exerts cardioprotective effects against MI/R injury in normal animals, whether rapamycin-elicited cardioprotection is altered in the presence of LVH is yet to be determined.

  • We reported that ventricular hypertrophy abrogates rapamycin-elicited cardioprotection by increasing ERK-mediated antioxidative/nitrative stress in ischemic/reperfused mice hearts. Scavenging peroxynitrite rescued cardiomyocyte fate and ameliorated MI/R injury in hypertrophied myocardium.

  • Our findings may provide potential translation to make differential clinical treatment plans for patients with or without LVH in the presence of AMI.

Author contribution

L.-L.M., Y.L., F.-J.K., J.-J.G., H.-T.S., J.-B.Z., Y.-Z.Z., and J.-B.G. contributed to the initial experimental discussion and designs. L.-L.M. set up the animal model, analyzed the data, and wrote the manuscript. P.-P.Y., F.-J.K., J.-J.G., H.-T.S., and J.-B.Z. performed experiments. Y.-Z.Z. and J.-B.G. wrote or revised the manuscript. All the authors have reviewed the final manuscript and approved the submission to this journal.

Funding

This work was supported by the National Natural Science Foundation of China [grant numbers 81401633, 81521001].

Competing interests

The authors declare that there are no competing interests associated with the manuscript.

Abbreviations

     
  • AMI

    acute myocardial infarction

  •  
  • AAR

    area at risk

  •  
  • Akt

    protein kinase B

  •  
  • DHE

    dihydroethidium

  •  
  • EF

    ejection fraction

  •  
  • eNOS

    endothelial nitric oxide synthases

  •  
  • ERK1/2

    extracellular regulated protein kinase 1/2

  •  
  • FS

    fractional shortening

  •  
  • GSK3β

    glycogen synthase kinase 3β

  •  
  • iNOS

    inducible nitric oxide synthases

  •  
  • i.p.

    intraperitoneally

  •  
  • LV

    left ventricular

  •  
  • MI/R

    myocardial ischemia/reperfusion

  •  
  • mTOR

    mammalian target of rapamycin

  •  
  • ROS

    reactive oxygen species

  •  
  • S6

    S6 ribosomal protein

  •  
  • STAT3

    signal transducer and activator of transcription 3

  •  
  • TAC

    transverse aortic constriction

  •  
  • TTC

    2,3,5-triphenyltetrazolium chloride

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Author notes

*

These authors contributed equally to this work.

Supplementary data