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Introduction

Ischemic heart disease is a leading cause of morbidity and mortality (1,2). Reperfusion of coronary arteries in response to thrombolytic treatment or percutaneous coronary intervention is crucial for reducing ischemia-induced heart damage (3–5). However, reperfusion may induce additional myocardial injury, including cardiomyocyte death and loss of cardiac function; this is known as myocardial ischemia/reperfusion (I/R) injury (6–8). The cellular mechanisms underlying I/R injury remain to be completely elucidated. Increasing evidence has suggested that ischemia initiates myocardial apoptosis, which is amplified by reperfusion, thus contributing to cardiac cell death (9,10). Conversely, suppressing apoptotic processes can minimize I/R-induced cardiac damage (11).

Transient receptor potential vanilloid 1 (TRPV1) is a ligand-gated nonselective cation channel, which is primarily expressed in sensory nerves that innervate cardiovascular tissues, including the heart and blood vessels (12,13). TRPV1 has been considered to act as a molecular integrator of numerous chemical and physical mediators, including noxious heat, low pH, capsaicin and lipid metabolites (14–16). TRPV1 expressed in the cardiac sensory nerves, which conduct angina pain signals (17,18), may function as a molecular sensor for the detection of tissue ischemia and the modulation of cardiac function (12). Pharmacological studies have suggested that activation of TRPV1 with exogenous agonists protects the heart from I/R injury (19,20), whereas TRPV1 gene deletion may impair cardiac recovery following I/R (21). Furthermore, activation of TRPV1 inhibits hypoxia/reoxygenation-induced apoptosis in rat hippocampal neurons via activation of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) signaling pathways (22), which are key regulators of cell growth and survival (23,24). During I/R, both signaling pathways are activated and confer cardioprotective effects through the recruitment of downstream anti-apoptotic molecules (25); however, the role of TRPV1 in myocardial apoptosis in response to I/R injury remains to be fully characterized. Furthermore, it is currently unknown whether the PI3K/Akt and ERK1/2 signaling pathways are involved in TRPV1-mediated myocardial apoptosis in I/R. The present study aimed to determine the effects of TRPV1 activation on myocardial apoptosis in response to I/R injury and explored the downstream signaling mechanism of TRPV1 activation.

Materials and methods

Animals and reagents

Male TRPV1 knockout (TRPV1−/−) and wild-type (WT) C57BL/6J mice (n=54 each; weight, 25–30 g; age, 10–12 weeks) were provided by the Experimental Animal Center of Chongqing Medical University (Chongqing, China) and were maintained under specific pathogen-free conditions (temperature, 22°C; humidity, 60%), with a 12-h light/dark cycle and with free access to food and water. All surgical procedures performed on mice were conducted under sodium pentobarbital anesthesia [50 mg/kg, intraperitoneal (IP) injection], and all efforts were made to minimize their suffering. The present study was approved by the Ethics Committee of Chongqing Medical University. The mice were treated in accordance with the recommendations listed in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH) (26).

LY294002 (a PI3K inhibitor) and 2,3,5-triphenyl tetrazolium chloride (TTC) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany); antibodies against phosphorylated (p)-Akt (Ser473; cat. no. 9271), Akt (cat. no. 9272), p-ERK1/2 (Thr202/Thr204; cat. no. 4370), ERK1/2 (cat. no. 9102), B-cell lymphoma-2 (Bcl-2; cat. no. 2870) and Bcl-2-associated X protein (Bax; cat. no. 14796) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-GAPDH antibody (cat. no. AG019) and the bicinchoninic acid (BCA) protein assay kit were purchased from Beyotime Institute of Biotechnology (Haimen, China). The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) reaction mixture was purchased from Roche Diagnostics (Laval, QC, Canada).

Langendorff heart preparation

Mice were treated with heparin (500 U/kg, IP) and anesthetized with pentobarbital sodium (50 mg/kg, IP) prior to thoracotomy. The hearts were rapidly excised, placed into ice-cold Krebs-Henseleit (K-H) buffer (composition in mM: NaCl 118, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, CaCl2 2.5, NaHCO3 25, Na-EDTA 0.5 and glucose 11) and perfused in a Langendorff apparatus within 2 min under a constant pressure of 80 mmHg. The perfusion fluid was oxygenated with a mixture of 95% O2 and 5% CO2, and was maintained at pH 7.4. In addition, temperature of the K-H buffer was maintained at 37°C throughout the experiment. A fluid-filled balloon connected to a pressure transducer was inserted into the left ventricle (LV) via the mitral valve to monitor LV pressure. The volume of the balloon was adjusted to maintain a stable LV end-diastolic pressure of 5–8 mmHg during initial equilibration.

Experimental protocol

The isolated mouse hearts were randomly divided into the following six groups (n=9/group): i) WT Sham group; ii) TRPV1−/− Sham group; iii) WT I/R group; iv) TRPV1−/− I/R group; v) WT I/R + LY294002 group; and vi) TRPV1−/− I/R + LY294002 group. To induce I/R, the perfused hearts were stabilized for 30 min and subjected to global normothermic (37°C) ischemia (no flow) for 30 min, followed by 60 min of reperfusion. Conversely, hearts in the sham groups were perfused with K-H solution continuously until the end of the experiment. Hearts in the LY294002 treatment group were perfused for 10 min with LY294002 (50 µM) in K-H buffer prior to induction of global ischemia, the hearts were then subjected to global ischemia followed by reperfusion without LY294002. LY294002 was initially dissolved in dimethyl sulfoxide and then in K-H buffer to reach a final concentration of 50 µM. The concentration of LY294002 used in the present study has been reported to specifically abolish PI3K activity and inhibit Akt phosphorylation, but not the phosphorylation of other protein kinases, including phosphatidylinositol 4-kinase, protein kinase C, mitogen-activated protein kinase or c-Src (27). The experimental protocol is presented in Fig. 1.

Figure 1 Experimental protocol. Isolated hearts from WT and TRPV1−/− mice were subjected to 30 min no-flow global ischemia followed by 60 min of reperfusion. The sham-operated group underwent time-matched perfusion without ischemia. LY (50 µM) was perfused for 10 min prior to ischemia. I/R, ischemia/reperfusion; LY, LY294002; TRPV1−/−, transient receptor potential vanilloid 1 knockout; WT, wild-type.

Determination of myocardial infarct size

At the end of reperfusion, the hearts were frozen at −20°C for 15 min and cut into five pieces along the longitudinal heart axis. Heart sections were incubated for 10 min in 1% TTC at 37°C in the dark. Subsequently, the sections were soaked in 4% paraformaldehyde in phosphate buffer overnight at 4°C to enhance the contrast of the stain. Viable myocardium exhibited red staining, whereas the infarcted area exhibited white staining. Each image was digitally photographed and the infarct size was analyzed using ImageJ software version 1.49v (NIH, Bethesda, MD, USA). The infarct size was expressed as a percentage of the total area of the heart.

Measurement of myocardial cell apoptosis

For tissue TUNEL staining, the heart samples were fixed in 4% paraformaldehyde at 25°C for 24 h, embedded in paraffin and cut into transverse sections (5 µm). Myocardial apoptosis was assessed using a TUNEL staining kit according to the manufacturer's protocol. The numbers of TUNEL-positive myocyte nuclei and total myocyte nuclei were counted in 10 different fields for each stained section at high magnification (objective, ×400). The number of TUNEL-positive nuclei (brown staining) was calculated using ImageJ software (NIH) according to the following formula: Percentage of TUNEL-positive myocyte nuclei = TUNEL-positive myocyte nuclei/total myocyte nuclei × 100%.

Western blot analysis

Total proteins were extracted from mouse heart tissues and protein concentrations were assessed using a BCA protein assay kit. The mouse heart tissues were homogenized using radioimmunoprecipitation lysis buffer (RIPA; Beyotime Institue of Biotechnology) and then centrifuged at 4°C. Equal amounts of protein (4 µg/µl) were separated by 10–12% SDS-PAGE and were transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The PVDF membranes were blocked in 5% non-fat milk in Tris-buffered saline containing 0.05% Tween-20 for 2 h at room temperature, and were then incubated overnight at 4°C with primary antibodies against GAPDH (1:1,000 dilution), p-Akt (Ser473) (1:1,000 dilution), Akt (1:1,000 dilution), p-ERK1/2 (Thr202/Thr204) (1:2,000 dilution), ERK1/2 (1:1,000 dilution), Bcl-2 (1:1,000 dilution) and Bax (1:1,000 dilution). Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000 dilution; ZB-5305; Beijing Zhongshan Golden Bridge Biotechnology Co., Beijing, China) for 2 h at room temperature. The immunoreactive proteins were visualized using an enhanced chemiluminescence detection system (Pierce; Thermo Fisher Scientific Inc., Waltham, MA, USA). The band density was analyzed by ImageJ software (NIH).

Statistical analysis

SPSS 17.0 statistical software (SPSS, Inc., Chicago, IL, USA) was used for all statistical analyses. Data are expressed as the means ± standard deviation. Significance was determined using either an unpaired Student's t-test (differences in TUNEL-positive cells and infarct size in TRPV1−/− hearts compared with in WT hearts) or one-way analysis of variance followed by the Tukey-Kramer multiple comparison test. P<0.05 was considered to indicate a statistically significant difference.

Results

Myocardial apoptosis is increased in TRPV1−/− hearts following I/R

To investigate the role of TRPV1 in apoptosis, WT and TRPV1−/− hearts were subjected to I/R, and myocardial apoptosis was detected using TUNEL staining (Fig. 2). The percentage of TUNEL-positive cardiomyocytes was markedly increased in the TRPV1−/− and WT hearts following I/R compared with in the respective sham control groups (P<0.01). Furthermore, the percentage of TUNEL-positive cardiomyocytes in TRPV1−/− hearts subjected to I/R was significantly greater than in WT hearts (25.10+1.03 vs. 18.20+2.79%; P<0.01). In addition, treatment with the PI3K inhibitor LY294002 prior to I/R, increased myocardial apoptosis in WT (P<0.01) but not in TRPV1−/− hearts (P>0.05) (Fig. 2).

Figure 2 I/R injury induced myocardial apoptosis, as determined by TUNEL staining. (A) Representative photomicrographs of TUNEL staining (scale bars, 50 µm). (B) Bar graph showing the quantified results of TUNEL staining analysis. TUNEL-positive cells are shown at ×400 magnification. Black arrows indicate TUNEL-positive cells. Data are presented as the means ± standard deviation of three independent experiments. *P<0.01 vs. the WT Sham group; **P<0.01 vs. the TRPV1−/− Sham group; #P<0.01 vs. the WT I/R group. I/R, ischemia/reperfusion; LY, LY294002; TRPV1−/−, transient receptor potential vanilloid 1 knockout; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; WT, wild-type.

Infarct size is larger in TRPV1−/− hearts following I/R

Myocardial infarct size was assessed using TTC staining (Fig. 3). Myocardial infarct size was markedly increased in the TRPV1−/− and WT groups following I/R compared with in the corresponding sham groups (P<0.01) (data not shown). Notably, infarct size in TRPV1−/− hearts was markedly increased compared with in the WT hearts (49.58+4.83 vs. 26.32+4.57%; P<0.01). In addition, treatment with LY294002 significantly increased infarct size in WT (P<0.01) but not in TRPV1−/− hearts (P>0.05) (Fig. 3).

Figure 3 Infarct size, as measured by TTC staining following 30 min of global ischaemia and 60 min of reperfusion. Upper panels are the representative cross-sections of TTC-stained hearts. White staining indicates the infarcted region, and red staining indicates viable myocardium. Data are presented as the means ± standard deviation of three independent experiments. *P<0.01 vs. the WT I/R group. I/R, ischemia/reperfusion; LY, LY294002; TRPV1−/−, transient receptor potential vanilloid 1 knockout; TTC, I/R, ischemia/reperfusion; WT, wild-type.

PI3K/Akt signaling is involved in the anti-apoptotic effects of TRPV1

The PI3K/Akt and ERK1/2 survival signaling pathways serve key roles in protecting cardiomyocytes from apoptosis during I/R injury. To determine the downstream signaling pathway associated with the effects of TRPV1, Akt and ERK1/2 phosphorylation was detected in heart samples exposed to I/R (Fig. 4). Myocardial I/R significantly increased p-Akt and p-ERK1/2 expression in TRPV1−/− and WT hearts (P<0.05), without any significant changes in total ERK1/2 and Akt protein levels. The ratios of p-AKT/AKT and p-ERK1/2/ERK1/2 were significantly higher following I/R in WT hearts compared with in TRPV1−/− hearts (P<0.01); however, these ratios were decreased following LY294002 treatment (P<0.01) (Fig. 4). These results suggested that the PI3K/Akt signaling pathway may be involved in the beneficial effects of TRPV1 during I/R injury.

Figure 4 Phosphorylation of Akt and ERK1/2 following I/R. (A) Protein expression levels of p-Akt and total Akt, and (B) p-ERK1/2 and total ERK1/2 were assessed by western blotting with specific antibodies. Protein signals were semi-quantified by densitometry, and relative levels are presented in graphs. Data are presented as the means ± standard deviation of three independent experiments. *P<0.05 vs. the WT Sham group; **P<0.05 vs. the TRPV1−/− Sham group; #P<0.01 vs. the WT I/R group. Akt, protein kinase B; ERK1/2, extracellular signal-regulated protein kinase 1/2; I/R, ischemia/reperfusion; LY, LY294002; p-, phosphorylated; TRPV1−/−, transient receptor potential vanilloid 1 knockout; WT, wild-type.

TRPV1 activation increases Bcl-2/Bax ratio by activating the PI3K-Akt signaling pathway

To further examine the mechanism of apoptosis, the protein expression levels of Bax and Bcl-2 were determined (Fig. 5). Bcl-2 and Bax have major roles in determining cell survival or death in response to apoptotic stimuli (28,29). The present study demonstrated that I/R resulted in a significant decrease in Bcl-2/Bax protein ratio compared with in the sham group in TRPV1−/− and WT hearts (P<0.05). In addition, the ratio of Bcl-2/Bax was lower in the of TRPV1−/− hearts compared with in WT hearts following I/R (P<0.01). Furthermore, treatment with LY294002 markedly decreased Bcl-2/Bax ratio in WT hearts (P<0.01) but not in TRPV1−/− hearts (Fig. 5).

Figure 5 Bcl-2/Bax ratio following I/R. Bcl-2 and Bax protein levels were determined by western blot analysis. Data are presented as the means ± standard deviation of three independent experiments. *P<0.05 vs. the WT Sham group; **P<0.05 vs. the TRPV1−/− Sham group; #P<0.01 vs. the WT I/R group. Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; I/R, ischemia/reperfusion; LY, LY294002; p-, phosphorylated; TRPV1−/−, transient receptor potential vanilloid 1 knockout; WT, wild-type.

Discussion

The present study demonstrated that TRPV1 gene knockdown significantly increased myocardial apoptosis and infarction during I/R. In addition, treatment with the PI3K inhibitor LY294002 increased infarct size and number of TUNEL-positive cardiomyocytes in WT but not in TRPV1−/− hearts. These results indicated that TRPV1 may protect the heart against I/R injury, possibly through its anti-apoptotic effects via activating the PI3K/Akt signaling pathway.

The importance of the TRPV1 channel in regulating heart function has recently been highlighted. TRPV1 can be activated by numerous metabolites that are accumulated during myocardial ischemia (12). Prior induction of TRPV1 may confer a benefit to the myocardium against further severe damage. This concept is supported by evidence that suggests that short episodes of sub-lethal ischemia may induce ischemic preconditioning, and that TRPV1 knockout abrogates the effects of ischemic preconditioning (30). In addition, TRPV1 activation promotes recovery of cardiac systolic/diastolic function during I/R (21). Apoptosis is a type of programmed cell death, which significantly contributes to myocardial I/R injury (9,10); therefore, inhibition of myocardial cell apoptosis may prevent cell loss and attenuate cardiac injury during myocardial I/R (11). Apoptosis-associated proteins, including Bcl-2 and Bax serve pivotal roles in apoptosis (28,29). In particular, apoptosis is regulated by the ratio of Bcl-2 and Bax protein expression (31,32). Previous studies have indicated that TRPV1 is involved in the regulation of apoptosis (22,33–35). Capsaicin has been reported to significantly reduce reperfusion-induced liver injury by reducing apoptosis due to activation of TRPV1 (34). Therefore, the present study investigated the role of TRPV1 in myocardial apoptosis during I/R. The results demonstrated that the Bcl-2/Bax ratio was significantly reduced in TRPV1−/− compared with WT hearts following I/R. Furthermore, I/R increased the percentage of TUNEL-positive cells and infarct size in TRPV1−/− hearts compared with in WT hearts. These findings suggested that TRPV1 activation protects cardiomyocytes from I/R injury by suppressing myocardial apoptosis. Although cardiac function was not assessed in the present study, our previous study demonstrated that TRPV1 deficiency results in increased mortality, aggravated inflammatory response, enhanced cardiac fibrosis and exaggerated progression of LV remodeling 7 days after myocardial infarction (36). However, further studies are required to investigate the role of TRPV1 in regulating cardiac function following I/R in vivo.

Previous studies have revealed that capsaicin activates the PI3K/Akt and ERK1/2 pathways in dorsal root ganglion neurons (37) and human HepG2 cells (38) through activation of the capsaicin receptor TRPV1. The PI3K/Akt and ERK1/2 signaling pathways, when specifically activated at the time of myocardial reperfusion, may inhibit cardiomyocyte apoptosis and attenuate I/R injury through targeting downstream molecules, including Bcl-2 and Bax (39,40). Therefore, the present study investigated the role of TRPV1 in phosphorylation of Akt and ERK1/2 in hearts subjected to I/R. The results indicated that the ratios of p-AKT/AKT and p-ERK1/2/ERK1/2 were upregulated in TRPV1−/− and WT hearts following I/R compared with in the sham groups. In addition, the ratios of p-AKT/AKT and p-ERK1/2/ERK1/2 were lower in TRPV1−/− hearts compared with in WT hearts. Notably, treatment with LY294002 decreased Bcl-2/Bax ratio, and increased infarct size and TUNEL-positive cardiomyocytes in WT but not in TRPV1−/− hearts. These results suggested that TRPV1 may inhibit I/R-induced cardiomyocyte apoptosis via PI3K/Akt activation. In addition, treatment with LY294002 significantly inhibited p-ERK1/2 levels in WT but not TRPV1−/− hearts following I/R. Previous evidence has suggested that PI3K inhibition suppresses ERK1/2 activation induced by capsaicin and nerve growth factor in primary sensory dorsal root ganglion neurons (41), which is supported by the present study. However, in a previous study, PI3K has been reported to inhibit, rather than increase, ERK1/2 activation (42). It has been suggested that the ability of PI3K inhibitors to suppress ERK1/2 activation depends on cell type, the type of stimuli and the strength of signals (43,44). In the present study, PI3K inhibition decreased I/R-induced ERK1/2 activation in WT mice, thus suggesting that ERK1/2 activation is PI3K-dependent. Further studies are required to explore the precise role of the ERK1/2 signaling pathway in TRPV1-induced cardiac protection.

As an ex vivo model, the Langendorff preparation has its limitations. For example, the isolated and perfused heart is denervated and its performance is not regulated by neurohumoral factors. In addition, crystalloid-perfused hearts are prone to tissue edema, which has a negative impact on cardiac function, particularly in I/R study protocols >2 h, which is not the case in the present study. However, the preparation is simple, reproducible and enables the study of the heart without other organ systems, which may confound physiological assessment. Due to these advantages, the Langendorff model has been used for >100 years to generate insightful data.

In conclusion, the present study demonstrated that TRPV1 may exert anti-apoptotic effects against myocardial I/R injury via PI3K/Akt signaling activation in isolated mouse hearts. These data suggested that TRPV1 may be considered a potential target for pharmacological intervention to reduce cardiac damage and improve clinical outcomes following cardiac I/R.

Glossary

Abbreviations

Abbreviations:

Acknowledgments

The present study was funded by the National Natural Science Foundation of China (grant nos. 81170188 and 30971212), the Natural Science Foundation of Chongqing (grant no. CSCT2009BB5069) and the National Key Subject Construction Project (grant no. 2011170).

References