Intermedin_1–53 enhances angiogenesis and attenuates adverse remodeling following myocardial infarction by activating AMP-activated protein kinase

Abstract

Adverse ventricular remodeling is a maladaptive response to acute loss of myocardium and an important risk factor for heart failure following myocardial infarction (MI). Intermedin (IMD) is a novel member of the calcitonin/calcitonin gene-related peptide family, which may possess potent cardioprotective properties. The aim of the present study was to determine whether IMD_1–53, a mature bioactive form of IMD, may promote therapeutic angiogenesis within the infarcted myocardium, therefore attenuating adverse ventricular remodeling post-MI. The present study observed that treatment with IMD_1–53 promoted proliferation, migration and tube formation of primary cultured myocardial microvascular endothelial cells (MMVECs). In a rat model of MI, chronic administration of IMD_1–53 increased capillary density in the peri-infarct zone, attenuated ventricular remodeling and improved cardiac performance post-MI. Treatment with IMD_1–53 also significantly increased the expression levels of phosphorylated-AMP-activated protein kinase (AMPK) and the subsequent activation of endothelial nitric oxide synthase in MMVECs and post-MI rat myocardium, without a significant influence on the expression of vascular endothelial growth factor. Notably, the in vitro effects of IMD_1–53 on angiogenesis and the in vivo effects of IMD_1–53 on post-MI ventricular remodeling were largely abrogated by the co-administration of compound C, an AMPK inhibitor. In conclusion, the present study demonstrated that IMD_1–53 could attenuate adverse ventricular remodeling post-MI via the promotion of therapeutic angiogenesis, possibly through the activation of AMPK signaling.

Introduction

Ventricular remodeling, which is characterized by progressive dilation and deterioration in cardiac performance, is a critical process underlying the progression to heart failure and subsequent mortality following myocardial infarction (MI). As a result, the attenuation of adverse myocardial remodeling is one of the most important aspects for improving prognosis following MI (1). However, despite previous advances in reperfusion therapy and optimized pharmacological treatments, left ventricular (LV) remodeling is still observed in a substantial proportion of patients, and progression to heart failure can still occur in up to one-third of patients who have had a MI (2,3). These data suggest that the current clinical treatments targeting myocardial remodeling post-MI remain inadequate. The promotion of therapeutic angiogenesis has been demonstrated to be a promising approach to ameliorate adverse myocardial remodeling (4).

Intermedin (IMD), also known as adrenomedullin 2, is a novel member of the calcitonin/calcitonin gene-related peptide (CGRP) family. The human IMD gene encodes a prepropeptide of 148 amino acids (preproIMD); proteolytic cleavage of preproIMD yields a series of biologically active C-terminal fragments. Previous studies have revealed that an endogenous degraded fragment termed IMD_1–53, which contains cleavage sites located between two basic amino acids at Arg93-Arg94, possessed potent cardioprotective activities in multiple physiopathological processes, including ischemia/reperfusion injury (5,6), cardiac fibrosis (7) and cardiac hypertrophy (8,9). However, the mechanisms underlying the cardioprotective effects of IMD require further elucidation. Notably, IMD was also reported to serve a role in the regulation of angiogenesis in vitro and in vivo (10,11). However, whether IMD_1–53 may promote therapeutic angiogenesis within the infarcted myocardium, and therefore attenuate adverse myocardial remodeling post-MI, has not been investigated. To test this hypothesis, the present study investigated the effects of recombinant IMD_1–53 on angiogenesis in primary cultured myocardial microvascular endothelial cells (MMVECs) and in a rat model of MI. The results suggested that IMD_1–53 attenuated adverse ventricular remodeling post-MI by promoting therapeutic angiogenesis, possibly through the activation of AMP-activated protein kinase (AMPK) signaling.

Materials and methods

Materials

IMD_1–53 peptide was purchased from Phoenix Pharmaceuticals, Inc. (Burlingame, CA, USA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA USA). Phosphorylated (p)-AMPKα^Thr-172 (cat. no. 2535), AMPKα (cat. no. 2532), p-Akt^Ser-473 (cat. no. 4060), and p-endothelial nitric oxide synthase (p-eNOS)^Ser-1179 (cat. no. 9570) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-Akt (cat. no. sc-8312), anti-eNOS (cat. no. sc-654), anti-GAPDH (cat. no. sc-25778) and anti-VEGF (cat. no. sc-152), anti-CD31 (cat. no. sc-1505) anti-CD34 (cat. no. sc-7045) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Compound C (Comp C) was purchased from Toronto Research Chemicals Inc. (North York, ON, Canada). Goat anti-rabbit (cat. no. sc-2030) and goat anti-mouse (cat. no. sc-3791) secondary antibodies were purchased from Santa Cruz Biotechnology, Inc.

For all experiments concerning IMD_1–53, the same volume of PBS was used as a vehicle. For experiments concerning Comp C, the same volume of DMSO was used as a vehicle.

Isolation and identification of MMVECs

Male Wistar rats (n=16; 4–6 weeks; 80–100 g) were used for the isolation of primary MMVECs. The rats were housed at room temperature with a 12/12 h light/dark cycle and free access to food and water. The animal protocol was approved by the Animal Care Committee of Shanghai Jiao Tong University (Shanghai, China). Rats were sacrificed with an overdose of sodium pentobarbital (180 mg/kg) and heparinized by intraperitoneal injection of sodium heparin (500 units/0.1 kg). Following thoracotomy, the heart was rapidly dislodged and washed in PBS. The atria, right ventricle, epicardial tissue and visible connective tissue were carefully removed, and the remaining myocardial tissue was washed in PBS prior to cutting into 1 mm³ sections without visible vessels. Myocardial tissues were seeded onto culture plates that were pre-coated with rat tail tendon gelatin and subsequently incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 30 min. Tissues were cultured in DMEM containing 4,500 mg/l D-glucose and supplemented with 20% FBS, 50 U/ml heparin, 100 U/ml penicillin and 100 µg/ml streptomycin. Tissue sections were discarded after the cells began to grow, and the medium was replaced at 72 h intervals. MMVECs were identified by typical ‘cobblestone’ appearance and positive CD31 and CD34 immunostaining. MMVECs at the second passage were used for experiments. The cells were grown to 80–90% confluence and were used in subsequent experimental analyses.

Immunostaining

MMVECs were plated on a 12×12 mm plate, fixed by methanol for 15 min at −20°C, blocked with 10% normal goat serum (cat. no. 31872; Gibco; Thermo Fisher Scientific, Inc.) for 30 min at room temperature, incubated with CD31 (cat. no. sc-1505; Santa Cruz Biotechnology, Inc.) and CD34 (cat. no. sc-7045; Santa Cruz Biotechnology, Inc.) antibodies at 1:200 overnight at 4°C, and then incubated with goat anti-mouse secondary antibody (cat. no. sc-3791; Santa Cruz Biotechnology, Inc.) at 1:1,500 for 1 h at room temperature. Finally, DAB working solution diluted by H₂O₂ was added and the MMVECs were observed under a light microscope (Olympus IX71; Olympus Corporation, Tokyo, Japan).

Cell proliferation assay

The proliferation rate of MMVECs was assessed using the MTT assay (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany). The cells were seeded into 96-well plates at a density of 2×10³ cells/well and incubated in DMEM containing 10% FBS at 37°C with 5% CO₂ for 24 h. IMD_1–53, Comp C or IMD_1–53+Comp C was added to the medium at different concentrations (0, 10, 20, 40, 80, 160 nm of IMD and/or 20 µmol compound C) at 0 h. Following 0, 8, 16, 24 and 48 h incubation, 10 µl of MTT was added to each well and the cells were incubated at 37°C for 4 h. Following incubation, the supernatant fluid was removed and 100 µl dimethyl sulfoxide was added to each well. The cells were incubated for 10 min and the absorbance at 490 nm was measured with an Epoch Microplate Spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA).

Migration assay

MMVEC migration rate was assessed using the Boyden Chemotaxis Chamber assay (Neuro Probe, Inc., Gaithersburg, MD, USA). The cells were trypsinized and resuspended in 10% DMEM containing 10% FBS. IMD_1–53 (80 nmol) and/or Comp C (20 µmol) were added to the wells in the lower chamber, and 1.5×10⁴ cells (200 µl/well) were added into the upper chamber. Cells migrating through the filter were fixed in 4% paraformaldehyde for 10 min and subsequently stained with 0.1% crystal violet stain solution (Merck Millipore) for 30 min. Five random microscopic fields per well were quantified using an Olympus IX71 fluorescence microscope (Olympus Corporation). Each experiment was repeated three times.

Tube formation assay

Tube formation of MMVECs was assessed using a Matrigel assay (BD Biosciences, Franklin Lakes, NJ, USA). Matrigel was chilled at 4°C overnight, melted prior to use and then quickly added (70 µl/well) to 96-well plates using a pre-chilled pipette. The plates were incubated at 37°C in 5% CO₂ for 1 h. The cells were seeded into the plates at a density of 1×10⁴ cells/well and incubated for 12 h in the presence or absence of IMD_1–53 (80 nmol) and/or Comp C (20 µmol) at 37°C. Tube formation was observed and images were captured using an Olympus 1X71 microscope (Olympus Corporation). Images were processed using Image-Pro Plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) to calculate the degree of tube formation by measuring the length of tubes from five randomly selected fields (magnification, ×200) from each well. Each experiment was repeated three times.

Western blot analysis

Cell or tissue samples were homogenized as follows: Freshly frozen myocardial tissue samples were ground into small pieces (~1×1×1 mm) in liquid nitrogen with a mortar and a pestle. The samples were transferred to microcentrifuges containing radioimmunoprecipitation lysis buffer (~150 µl per 10 mg tissue; Beyotime Institute of Biotechnology, Haimen, China) and 1 mM phenylmethylsulfonyl fluoride. The samples were then kept on ice for 1 h, vortexing every 10 min. The lysates were then centrifuged at 10,000 × g for 30 min at 4°C, and supernatants were removed for immediate western blot analysis. Protein samples (5 mg/ml) were loaded onto a 10% Bis-Tris gel; following electrophoresis, separated proteins were transferred to a polyvinylidene fluoride membrane for 1 h. The membranes were then incubated with blocking solution (5% skim milk) for 1–2 h at room temperature, followed by three washes with TBS-Tween-20 (TBST; 0.1% Tween; 10 min each). Membranes were incubated with one of the following primary antibodies: Anti-AMPK (1:800); anti-p-AMPK^Thr172 (1:1,000); anti-Akt (1:1,500); anti-p-Akt^ser473 (1:1,000); anti-eNOS (1:500); anti-p-eNOS^ser1117 (1:500); anti-vascular endothelial growth factor (VEGF; 1:1,000); and anti-GAPDH (1:8,000) at 4°C overnight. Primary antibodies were recovered and the membranes were washed three times with TBST (10 min each), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG; cat. no. sc-2030; 1:2,000) secondary antibody at room temperature for 1 h. Subsequently, the membranes were washed five times with TBST (5 min/wash), and the bands were detected using Western Blotting Luminol Reagent (Santa Cruz Biotechnology, Inc.) and quantified by densitometric analysis of digitized autoradiograms using Quantity One (version 4.6.2) software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Each immunoblotting experiment was repeated three times, and the averages of the results were calculated.

Animal model

A total of 70 male Sprague-Dawley rats (age, 8 weeks; weight, 250–280 g; Experimental Animal Center, Fudan University, Shanghai, China) were used in the present study, and were maintained at room temperature with a 12/12 h light/dark cycle and free access to food and water. The animal research study protocol was in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication no. 85–23, revised 1996) and approved by the Animal Care Committee of Shanghai Sixth Hospital, Shanghai Jiao Tong University. All rats were acclimated for 2 weeks prior to experimentation. All rats were anesthetized by intraperitoneal injection of pentobarbital sodium (60 mg/kg) and ventilated using a DW-200 animal respirator (Shanghai Alcott Biotech Co., Ltd., Shanghai, China) with room air following endotracheal intubation. Electrocardiogram (ECG) lead II was continuously monitored during the experiment. Heart exposure was performed by a thoracotomy at the left fourth intercostal space. The left anterior descending (LAD) coronary artery was ligated using a size 6–0 Prolene polypropylene suture, 1–2 mm below the tip of the left atrial appendage. LAD ligation was confirmed by a color change of the myocardium and an elevation of the ST segment on the ECG. A total of 4 weeks after LAD ligation, all the surviving rats were sacrificed by injection of 180 mg/kg pentobarbital sodium overdose.

Animal protocols

A total of 70 Sprague-Dawley rats were randomly divided and assigned to five treatment groups: i) Sham group (n=10), which underwent thoracotomy and incision of the pericardial sac without LAD ligation; ii) MI group (n=15), which underwent LAD ligation without any treatment; iii) IMD group (n=15), which contained MI rats that received subsequent intraperitoneal injections of IMD_1–53 (10 nmol/kg/day) starting 3 days after LAD ligation; iv) IMD+Comp C group (n=15), which contained MI rats that received subsequent intraperitoneal injections of IMD_1–53 (10 nmol/kg/day) and Comp C (5 mg/kg/day) starting 3 days after LAD ligation; and v) Comp C group (n=15), which contained MI rats that received subsequent intraperitoneal injections of Comp C (5 mg/kg) starting 3 days after LAD ligation.

Echocardiography

An echocardiographic examination was performed on day 28 following the induction of MI using a standard Acuson Sequoia 512 ultrasound system equipped with a 15 MHz probe (Siemens AG, Munich, Germany). Two-dimensional and motion-mode echocardiography were performed. The LV end-diastolic diameter (LVEDD) and the LV end-systolic diameter (LVESD) were measured. In addition, the LV ejection fraction (LVEF) and fractional shortening (FS) were calculated as previously described (12). All measurements from five consecutive cardiac cycles were averaged and analyzed by a single observer that was blinded to the treatment protocol.

Histological analysis

Under deep anesthesia with pentobarbital sodium (intraperitoneal injection at 100 mg/kg), the heart was excised and cut into 2-mm-thick transverse slices, which were then fixed in 10% formalin solution at room temperature for 24 h. The samples were subsequently embedded in paraffin and sectioned (4 µm). Immunohistochemical analysis was performed as previously described (13). Tissue sections were then deparaffinized using xylene and graded ethanol and were blocked in 10% goat serum (Gibco, Thermo Fisher Scientific, Inc.) for 30 min at 37°C. Samples were incubated at 4°C overnight with primary rabbit polyclonal antibodies against CD31 (ab28364) and VEGF (ab46154; Abcam, Cambridge). Sections were subsequently treated with secondary goat polyclonal secondary antibody against rabbit IgG (ab97047; Abcam). All sections were counterstained with hematoxylin. Immunoreactivity for CD31 and VEGF was measured using a fluorescence microscope and the Image-Pro Plus 4.0 analysis system (Media Cybernetics, Inc.). Capillaries were identified by positive staining for CD31. Results were expressed as the average amount of capillaries per ×10 field. Masson's trichrome stain was performed in the border area to evaluate collagen deposition.

Statistical analysis

Statistical analysis of the data was conducted using the statistical software SPSS version 13.0 (SPSS, Inc., Chicago, IL, USA). Experimental data were expressed as the mean ± standard deviation and analyzed using one-way ANOVA followed by a least-significant difference corrected multiple comparison test. P<0.05 was considered to indicate a statistically significant difference.

Results

IMD_1–53 increases proliferation of MMVECs in a dose-dependent manner

The effects of IMD_1–53 on the proliferation of MMVECs were examined using the MTT assay. The results indicated that IMD_1–53 was able to promote the proliferation of MMVECs in a dose-dependent manner with the maximum effect observed at 80 nmol (P<0.05 vs. 0 nmol control, Fig. 1A). In addition, co-administration of Comp C, a well-known inhibitor of AMPK, significantly attenuated the effects of IMD_1–53 on the proliferation of MMVECs (P<0.05 vs. IMD at 8, 16, 24 and 48 h; Fig. 1B), whereas treatment with Comp C alone had no significant influence on the proliferation of MMVECs (P>0.05, Fig. 1B).

Figure 1.

IMD_1–53 promotes the proliferation of MMVECs. Proliferation was assessed using the MTT assay. (A) MMVECs were seeded in 96-well dishes in the presence of different concentrations (0–160 nmol) of IMD_1–53 for 0, 8, 16, 24 and 48 h. (B) MMVECs were seeded in 96-well dishes in the presence of IMD_1–53 (80 nmol), Comp C (20 µmol), or both for 0, 8, 16, 24 and 48 h. All data are expressed as the mean ± standard deviation from three independent experiments. *P<0.05 vs. control group. Comp C, compound C; IMD, intermedin; MMVECs, myocardial microvascular endothelial cells.

IMD_1–53 promotes migration and tube formation in MMVECs

The effects of IMD_1–53 on endothelial cell migration were further evaluated by a modified Boyden chamber assay (Fig. 2). The results indicated that the optimal dose (80 nmol, as shown in the MTT assay) of IMD_1–53 treatment stimulated the migration of MMVECs (P<0.05 vs. control, Fig. 2A and C). Co-treatment with Comp C (20 µmol) partially suppressed the pro-migratory effect of IMD_1–53 (P<0.05 vs. IMD, Fig. 2A and C).

Figure 2.

IMD_1–53 promotes the migration of MMVECs and their differentiation into tube-like structures. (A) Representative images of the migration of MMVECs assessed by Boyden chamber analysis. Magnification, ×100. (B) Representative images of in vitro tube formation analysis of MMVECs on Matrigel. Magnification, ×200. Quantitative analysis of the (C) migration and (D) tube formation assays. Each treatment was performed in triplicate and five fields were randomly selected from each well for counting the number of migrated cells. All data are expressed as the mean ± standard deviation. *P<0.05 vs. control; ^#P<0.05 vs. IMD group. Comp C, compound C; IMD, intermedin; MMVECs, myocardial microvascular endothelial cells.

To examine the effects of IMD_1–53 on the differentiation of cardiovascular endothelial cells into vascular structures, MMVECs were plated at 1×10⁴ cells/well in the presence or absence of IMD_1–53 (80 nmol) and/or Comp C (20 µmol) and tube formation was detected microscopically. A total of 24 h after plating, the MMVECs treated with IMD_1–53 formed a significantly more extensive tube network compared with the control group (P<0.05). The effects of IMD_1–53 on tube formation were reduced in MMVECs that were also pretreated with Comp C (P<0.05 vs. IMD group). Treatment with Comp C alone significantly decreased tube formation as compared with the control group (P<0.05, Fig. 2B and D).

IMD_1–53 stimulates activation of the AMPK-Akt-eNOS signaling pathway in MMVECs

Since AMPK is involved in the regulation of angiogenesis (14) and IMD_1–53 was recently reported to increase AMPK activation in rat cardiomyocytes (9), the present study aimed to investigate whether AMPK is involved in IMD_1–53-induced angiogenesis in MMVECs. MMVECs were exposed to IMD_1–53 (80 nmol) for 2 h; subsequently, the total and phosphorylated protein levels of AMPKα, Akt and eNOS were analyzed by western blot analysis. As shown in Fig. 3, the expression levels of p-AMPKα, p-Akt and p-eNOS were all significantly increased by IMD_1–53 treatment (P<0.05 vs. control, Fig. 3). Co-treatment with the Comp C suppressed the ability of IMD_1–53 to activate either Akt or eNOS, as measured by the relative expression levels of p-Akt and p-eNOS (P<0.05 vs. IMD, Fig. 3), whereas treatment with Comp C alone slightly but significantly decreased the phosphorylated levels of Akt and eNOS (P<0.05, respectively; Fig. 3) and significantly decreased the phosphorylated levels of AMPKα (P<0.05; Fig. 3).

Figure 3.

Effects of IMD_1–53 on the level of expression and phosphorylation of AMPKα, Akt and eNOS in MMVECs. MMVECs were pretreated with either Comp C (20 µmol) or vehicle for 40 min and incubated with either IMD_1–53 (80 nmol) or vehicle for 2 h prior to harvest. Representative western blots demonstrating (A) p-AMPKα over total AMPKα protein expression, (B) p-Akt over total Akt protein expression and (C) p-eNOS over total eNOS protein expression. All data are expressed as the mean ± standard deviation; *P<0.05 vs. control group; ^#P<0.05 vs. IMD group. AMPKα, AMP-activated protein kinase α; Comp -C, compound C; eNOS, endothelial nitric oxide synthase; IMD, intermedin; MMVECs, myocardial microvascular endothelial cells; p, phosphorylated.

IMD_1–53 increases capillary density in post-MI rat heart

To assess the effects of IMB of angiogenesis in vivo, myocardium capillary density tests were performed. Compared with the sham group, induction of MI significantly increased the capillary density in the peri-infarct zone (P<0.05, Fig. 4A and C). Treatment with IMD_1–53 significantly increased capillary density in the peri-infarct zone of post-MI rat myocardium (P<0.05 vs. MI, Fig. 4A and C). Co-administration of Comp C mitigated the effects of IMD_1–53 on capillary density in the peri-infarct zone (P<0.05 vs. IMD, Fig. 4A and C). Compared with the MI group, administration of Comp C alone slightly decreased capillary density in the peri-infarct zone (P<0.05, Fig. 4A and C). Compared to the sham group, the expression levels of VEGF were increased following induction of MI (P<0.05, Fig. 4B and D). However, the administration of IMD_1–53, Comp C or a combination of the two had no significant effect on VEGF expression in the peri-infarct zone post-MI (P>0.05 vs. MI group, Fig. 4B and D).

Figure 4.

IMD_1–53 increases capillary density in post-infarct myocardium. Immunohistochemical staining for CD31 and VEGF were performed in each group 4 weeks post-MI induction. Representative slides of tissue from peri-infarct myocardium with (A) CD31 expression and (B) VEGF expression; magnification, ×200. Quantification of (C) CD31 (expressed as the number of capillaries per 10x field) and (D) VEGF expression in each group. All data are expressed as the mean ± standard deviation. n=4 for sham group, n=6 for other groups. *P<0.05 vs. MI group; ^#P<0.05 vs. IMD group. Comp C, Compound C; IMD, intermedin; MI, myocardial infarction; VEGF, vascular endothelial growth factor.

IMD_1–53 attenuates ventricular remodeling post-MI

To evaluate the effects of IMD_1–53 on ventricular remodeling post-MI, Masson's trichrome staining was performed on all groups 4 weeks post-MI induction (Fig. 5). The results demonstrated that treatment with IMD_1–53 markedly suppressed elongation of the infarct wall segment and dilation of the LV, as demonstrated by cross-section morphology (Fig. 5A) and calculated by infarct size post-MI (P<0.05 vs. MI group; Fig. 5B). The effects of IMD_1–53 on ventricular remodeling were abrogated by co-administration of Comp C (P<0.05 vs. IMD, Fig. 5), whereas Comp C alone had no significant effect on myocardial infarct size post-MI (P>0.05 vs. MI, Fig. 5).

Figure 5.

IMD_1–53 attenuates ventricular remodeling post-MI. (A) Representative Masson's trichrome-stained LV cross-sections obtained 4 weeks post-MI induction from each group; demonstrating the effects of IMD_1–53 on LV morphology. Fibrotic areas are stained blue and viable myocardium is stained red (x10 magnification). (B) Quantitative assessment of infarct size. Data are expressed as the mean ± standard deviation; n=4 for sham group, n=6 for other groups. *P<0.05 vs. MI group; ^#P<0.05 vs. IMD group. Comp C, compound C; IMD, intermedin; LV, left ventricle; MI, myocardial infarction.

IMD_1–53 improves cardiac function post-MI

A total of 4 weeks after MI induction, cardiac geometry and performance were evaluated using echocardiography (Table I). Compared with the sham group, significantly decreased LVEF and FS, along with increased LVESD and LVEDD, were observed in the MI group (P<0.05, Table I). In IMD-treated MI rats, LVEF and FS were significantly improved, whereas LVESD and LVEDD were significantly reduced, compared with the MI group (P<0.05, Table I). Co-administration of Comp C abrogated the effects of IMD_1–53 on LVEF, FS, LVESD and LVEDD (P<0.05 vs. IMD, Table I), whereas Comp C alone had no significant effects on cardiac geometry and performance (P>0.05, Table I). Neither IMD_1–53 nor Comp C administration had significant effects on survival rate of rats post-MI (P>0.05 vs. MI group; Table I).

Table I.

Representative motion-mode echocardiograms obtained 4 weeks post-MI induction.

Parameter	Sham	MI	IMD	IMD+Comp C	Comp C
Number	10	15	15	15	15
Survival	10	11	12	11	10
LVESD (mm)	3.47±0.28a	6.11±0.31	4.52±0.27a	5.84±0.33b	6.24±0.28
LVEDD (mm)	5.83±0.36a	8.28±0.44	6.42±0.42a	7.61±0.49b	8.48±0.47
LVEF (%)	81.3±5.38a	46.8±5.14	65.2±6.03a	53.7±5.93b	44.9±5.26
FS (%)	75.6±4.97a	44.9±3.71	66.5±4.89a	52.2±3.87b	43.7±3.54

Summary data of echocardiographic measurements in each group of animals. Data are expressed as the mean ± standard deviation.

^aP<0.05 vs. MI group

^bP<0.05 vs. IMD-treated group. Comp C, compound C; FS, fractional shortening; IMD, intermedin; LVEDD, left ventricular end-diastolic diameter; LVEF, left ventricular ejection fraction; LVESD, left ventricular end-systolic diameter; MI, myocardial infarction.

IMD_1–53 stimulates activation of the AMPK-Akt-eNOS signaling pathway in post-MI rat myocardium

Western blot analysis was used to evaluate the activation of AMPK, Akt and eNOS, and the expression levels of VEGF in rat myocardium (Fig. 6). The results indicated that chronic IMD_1–53 treatment resulted in significantly increased activation (as measured by phosphorylation) of AMPK, Akt and eNOS in post-MI rat myocardium (P<0.05 vs. MI, Fig. 6B-D), whereas it had no significant effect on the expression of VEGF (P>0.05 vs. MI, Fig. 6A). Co-administration of Comp C abrogated the effects of IMD_1–53 on AMPK, Akt and eNOS phosphorylation (P<0.05 vs. IMD, Fig. 6B-D), whereas Comp C alone slightly decreased levels of p-AMPK, p-Akt and p-eNOS (P<0.05 vs. MI, Fig. 6B-D).

Figure 6.

Effects of IMD_1–53 on expression and phosphorylation of VEGF, AMPK, Akt and eNOS in rat myocardium post-MI. The infarcted heart tissue from the LV free wall or the non-infarcted heart tissue at the same site was harvested 4 weeks after left anterior descending coronary artery ligation. Representative western blots demonstrating (A) VEGF protein expression, GAPDH is used as a loading control; (B) p-AMPK over total AMPK protein expression; (C) p-Akt over total Akt protein expression; and (D) p-eNOS over total eNOS protein expression. Data are expressed as the mean ± standard deviation; n=4 for sham group, n=6 for other groups. *P<0.05 vs. MI group; ^#P<0.05 vs. IMD group. AMPK, AMP-activated protein kinase; Comp C, compound C; eNOS, endothelial nitric oxide synthase; IMD, intermedin; MI, myocardial infarction; LV, left ventricle; p, phosphorylated; VEGF, vascular endothelial growth factor.

Discussion

Using in vitro and in vivo experiments, the present study demonstrated that IMD_1–53 promoted angiogenesis in primary cultured MMVECs, which was demonstrated by an increase in proliferation, migration and tube formation. In addition, experiments using an in vivo rat model of MI demonstrated that treatment with IMD_1–53 significantly increased the capillary density in ischemic myocardium, attenuated LV remodeling and improved post-MI cardiac function, as determined by CD31 staining, Masson trichrome staining and echocardiography, respectively. Furthermore, the addition of Comp C, an AMPK inhibitor, demonstrated that the in vitro and the in vivo effects of IMD_1–53 were at least partially dependent on the activation of AMPK. These results indicated that IMD_1–53 attenuates post-infarct myocardial remodeling via the promotion of AMPK-dependent angiogenesis. To the best of our knowledge, this is the first demonstration of the involvement of AMPK activation in IMD-induced angiogenesis and is one of only a few studies (8,10,15) to link the cardioprotective role of IMD to therapeutic angiogenesis.

The human IMD gene encodes a prepropeptide of 148 amino acids (preproIMD), which can generate a series of mature peptide fragments by proteolytic cleavage at the C-terminal in vivo (16), such as IMD_1–47, IMD_8–47 and IMD_1–53. Yang et al were the first to report that IMD_1–53 stimulated L-Arg transport and increased NOS activity in the rat aorta (15). Furthermore, using direct gene delivery, Smith et al established IMD as a novel angiogenic factor in a rodent hindlimb ischemia model and cultured human umbilical vein endothelial cells (10). Consistent with these findings, the present study demonstrated that IMD_1–53 increased proliferation of MMVECs in a dose-dependent manner with a maximal effect at around 80 nmol, and the optimal dose of IMD_1–53 treatment promoted migration and tube formation of MMVECs.

Cardiac hypertrophy is initiated as an adaptive response during post-infarction remodeling to compensate for increased load (17). However, the capillary network within the infarcted heart is unable to support the increased demands of the hypertrophied myocardium, resulting in a gradual loss of healthy tissue, extension of the infarct and fibrous replacement (18). Therefore, promotion of angiogenesis in post-infarct myocardium may be an effective approach to attenuate adverse ventricular remodeling following MI, as demonstrated by several previous studies (19–21). The present study demonstrated that the in vivo administration of IMD_1–53 promoted angiogenesis in post-infarct rat myocardium, which was indicated by increased capillary density in the peri-infarct zone. In addition, chronic treatment with IMD_1–53 preserved cardiac geometry and improved cardiac performance post-MI, as evidenced by Masson trichrome staining and echocardiography, suggesting that IMD_1–53 may attenuate adverse remodeling post-MI and prevent subsequent heart failure by promoting angiogenesis. IMD_1–53 has previously been reported to protect against myocardial ischemia/reperfusion injury (5) and to attenuate pressure-overload-induced cardiac hypertrophy (9) through other mechanisms (6,8); however, to the best of our knowledge, this is the first study to link the cardioprotective role of IMD to therapeutic angiogenesis.

AMPK is a key sensor and regulator of cellular energy homeostasis, which modulates various physiological processes that may favor the restoration of energy balance in several systems (22). In endothelial cells, AMPK was demonstrated to serve an essential role in angiogenesis in response to stress (14,23). In addition, a series of agents, including pravastatin, adiponectin and erythropoietin, and cytokines have been reported to increase angiogenesis through AMPK-dependent mechanisms (24–26). Notably, CGRP was also reported to promote angiogenesis by activating the AMPK-eNOS pathway in endothelial cells (27). The present study demonstrated that treatment with IMD_1–53 increased the expression levels of p-AMPK in cultured MMVECs and in post-MI rat myocardium, and notably, administration of Comp C, a known inhibitor of AMPK, abrogated the effects of IMD_1–53 on angiogenesis, as evidenced by reduced migration and tube formation of MMVECs in vitro, and lower capillary density at the peri-infarct zone in vivo. These results demonstrated that the angiogenic activity of IMD_1–53 is at least partially dependent on the activation of AMPK.

It has previously been reported that IMD_1–53 increased total NOS activity, but had little effect on mRNA levels of either inducible NOS or eNOS in the rat aorta (16). The results of the current study demonstrated that IMD_1–53 promoted the activation of eNOS in vitro and in vivo. In addition, the administration of Comp C significantly attenuated the effects of IMD_1–53 on the level of p-eNOS expression, suggesting that AMPK is involved in the IMD_1–53-induced activation of eNOS. It is also worth noting that, in the present study, treatment with IMD_1–53 had no significant effect on the expression of VEGF in post-infarct rat myocardium, indicating that IMD_1–53 may promote the activation of eNOS and subsequent angiogenesis through VEGF-independent mechanisms. Notably, the present study did not determine how IMD_1–53 regulated AMPK signaling. In addition, although the α subunit of AMPK was identified as the major target in the present study, further evaluation is required regarding the specific subunit (α1 or α2) involved.

In conclusion, the present study demonstrated that IMD_1–53 could attenuate adverse ventricular remodeling post-MI by promoting therapeutic angiogenesis, possibly through the activation of AMPK signaling.

Glossary

Abbreviations

Comp C

compound C

FS

fractional shortening

IMD

intermedin

LVEDD

left ventricular end-diastolic diameter

LVEF

left ventricular ejection fraction

LVESD

left ventricular end-systolic diameter; MMVECs;

MI

myocardial infarction