The PTEN/Akt Signaling Pathway Mediates Myocardial Apoptosis in Swine After Coronary Microembolization

Abstract

Background/Aims:

Phosphatase and the tensin homolog deleted on chromosome ten (PTEN) has been recognized as a promoter of apoptosis in various tissues and has been shown to be upregulated in circumstances of coronary microembolization (CME). We hypothesized that the upregulation of PTEN correlates with CME-induced myocardial apoptosis.

Methods:

Swine CME was induced by an intracoronary injection of inert plastic microspheres (diameter of 42 μm) into the left anterior descending coronary, with or without pretreatment of the PTEN small-interfering RNA (siRNA). Echocardiological measurements, a pathological examination, Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL) staining, and Western blotting, were performed to assess their functional, morphological, and molecular effects in CME.

Results:

PTEN was aberrantly upregulated in cardiomyocytes following CME. Downregulation of PTEN in vivo via siRNA was associated with improved cardiac function and attenuated myocardial apoptosis; concomitantly inhibited the expression of key proapoptotic proteins, such as phosphorylated Bad (p-Bad); cleaved caspase-3; and enhanced the expression of key antiapoptotic proteins, such as phosphorylated protein kinase B (p-Akt). However, there was no difference in the Akt-regulated downstream protein IκB kinases (IKKα, IKKβ, and IKKγ) among the sham, CME, and control siRNA groups.

Conclusion:

This study demonstrates, for the first time, that the PTEN/Akt signaling pathway contributes to cardiomyocyte apoptosis. The data generated from this study provide a rationale for the development of PTEN-based therapeutic strategies for CME-induced myocardial injury.

Introduction

Coronary microembolization (CME), which is caused by the erosion or rupture of a vulnerable atherosclerotic plaque, occurs spontaneously in acute coronary syndromes and iatrogenically during percutaneous coronary interventions. ^1,2 The typical consequences of CME include microinfarcts with an inflammatory response, contractile dysfunction, and reduced coronary reserve. ³ Statins, antiplatelet agents, and coronary vasodilators protect against microembolization when their administration is initiated prior to percutaneous coronary interventions, and distal protection devices could retrieve atherothrombotic debris and prevent its embolization into the microcirculation; however, its effect on the clinical outcome has been disappointing to date, except for saphenous vein bypass grafts. ^1,4 Because of the grave consequences of CME, many investigations have aimed to determine the underlying mechanisms of CME-induced myocardial injury and alleviate the deleterious effects of CME. There have been major advances in identifying the factors of the myocardial inflammatory response and apoptosis that are involved in CME-induced myocardial injury ^5,6 ; however, the overall complexity of myocardial injury suggests that additional regulatory mechanisms remain to be elucidated. Our previous studies demonstrated that cardiomyocyte apoptosis plays a vital role in the mechanism of CME-induced myocardial injury. ^7,8

Phosphatase and the tensin homolog deleted on chromosome ten (PTEN), a negative regulator of the phosphatidylinositol 3 kinase (PI3K)-Akt pathways, has recently been found to be upregulated in ischemic myocardium and involved in a wide variety of myocardial apoptotic responses. ⁹ Growing evidence has revealed that PTEN increases myocardial contractility and is involved in post-MI remodeling by inhibiting the activation of the PI3K/Akt signaling pathway. The PTEN/PI3K/Akt pathway has been involved in the pathogenesis of many cardiovascular diseases, such as cardiac hypertrophy, myocardial contractile dysfunction, coronary angiogenesis, heart failure, and ischemia–reperfusion injury. ¹⁰ Recently, our research revealed that PTEN is highly expressed in myocardial tissues that are subjected to CME-induced myocardial injury and that the inhibition of PTEN reduces myocardial damage by attenuating myocardial inflammation. ¹¹ Regardless of whether PTEN is involved in the pathological process of CME-induced myocardial injury, its effect remains to be explored.

In this study, we focused on the potential role of PTEN in the regulation of cardiomyocyte apoptosis and its possible mechanism.

Materials and Methods

Animal Preparation

Healthy swine (25-30 kg) were purchased from the Animal Center of the Agriculture College of Guangxi University (Nanning, People’s Republic of China), and throughout all of the experimental stages, the animals were maintained under controlled temperature, humidity, and light conditions, with pig feed and water provided ad libitum. This investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH publication No. 85-23, revised 1996). The Clinical and Animal Research Ethics Committees of Guangxi Medical University approved all of the procedures.

Generation of the CME Model

The CME model was induced by the manual unremitting injection of microspheres into the left anterior descending (LAD) artery, as described previously by Dorge et al, ¹² Breuckmann et al, ¹³ and Carlsson et al. ¹⁴ The swine were initially sedated via an intramuscular injection of a combination of ketamine and atropine (10-15 mg/kg and 2 mg, respectively). After endotracheal intubation, anesthesia was maintained via an intravenous drip of diazepam into the ear vein. The right femoral artery was separated, and a 6F (Cordis, Hialeah, Florida) vascular sheath was placed. Prior to the coronary cannulation, the animals were anticoagulated via an intravenous injection of 200 U/kg heparin followed by 100 U/kg/h to maintain heparinization. A 6F JL 4.0 guiding catheter was used for the coronary angiography. After the coronary angiography, a 1.8F infusion catheter (Cordis) was placed into the LAD artery with the tip located between the second and the third diagonal branches. Microspheres with a diameter of 42 μm (Dynospheres; Dyno Particles; Lillestrøm, Norway) at a mean dosage of 100 000 were selectively infused into the LAD within 40 minutes followed by a flush with 10 mL of saline. The sham-operated swine (sham group) were subjected to the same procedures, except that the injection was saline rather than microspheres. The systemic blood pressure and heart rate were continuously monitored during the procedure.

In Vivo PTEN Small-Interfering RNA Administration

The swine were randomly distributed into 4 groups as follows: the sham surgery group (control, n = 8), CME group (CME, n = 8), PTEN small-interfering RNA (siRNA)-treated before CME group (PTEN siRNA, n = 8), and control siRNA-treated group (control siRNA, n = 8). In vivo transfer was performed according to the method described previously. ¹⁵ After coronary angiography, a 1.8F infusion catheter (Cordis) was placed in the LAD artery with the tip located between the second and third diagonal branches. A total of 100 μg of PTEN siRNA (PTEN-sus-368, Genepharma) or control siRNA (Genepharma) was diluted in the same volume of the in vivo transfection reagent (EntransterTM-in vivo; Engreen, Beijing, China). After mixing the solution gently by pipetting up and down, the siRNA solutions were selectively infused into the LAD. Then, 72 hours after the PTEN siRNA or control siRNA administration, CME surgery was performed. In our previous study, we found that the protein level of PTEN was obviously upregulated at 12 hours post-CME, ¹¹ so all of the swine were killed at 12 hours after the CME or sham surgery in this study.

Statistical Analysis

Statistical analyses were performed using SPSS 19.0 statistical software (IBM Corporation, Chicago, IL). The quantitative data are presented as the mean ± standard deviation. Unpaired Student’s t test and analysis of variance were used for comparisons between 2 groups and among multiple groups, respectively, followed by the Student Newman Keuls test for the post hoc analysis. The P < .05 level indicated statistical significance.

Results

Animal Groups

No significant differences in the body weight or heart rate were observed among the 4 groups (Supplemental Table S1).

PTEN siRNA Pretreatment Improved Cardiac Function Following CME

The results of the echocardiographic examination (Table 1) showed that 12 hours after the CME modeling, the CME group exhibited significantly decreased cardiac systolic function compared to the sham group, as indicated by significantly reduced left ventricular ejection fraction, fractional shortening, and cardiac output as well as increased left ventricular end-diastolic diameter in the CME group (P < .05). Additionally, PTEN siRNA pretreatment was associated with improved cardiac function in the CME swine.

Table 1.

Effect of PTEN siRNA on Cardiac Function Following Coronary Microembolization (CME).^a

Groups	LVEF (%)	FS (%)	LVEDd, mm	CO, L/min
Sham (N = 8)	68.70 (2.73)	41.05 (2.08)	32.32 (1.53)	4.19 (0.29)
CME (N = 8)	48.81 (4.00)b	23.44 (3.54)b	39.28 (1.23)b	2.50 (0.23)b
PTEN siRNA (N = 8)	59.32 (4.26)b,c	32.30 (3.63)b,c	33.85 (1.20)	3.50 (0.22)c
Control siRNA (N = 8)	50.02 (3.57)b	24.61 (3.03)b	39.56 (1.35)b	2.57 (0.28)b

Abbreviations: CME, coronary microembolization; LVEF, left ventricular ejection fraction; FS, fractional shortening; LVEDd, left ventricular end-diastolic diameter; CO, cardiac output; PTEN, phosphatase and tensin homolog deleted on chromosome ten; Sham, sham group; CME, CME group; PTEN siRNA, CME + PTEN siRNA group; Control siRNA, CME+ control siRNA group; siRNA, small-interfering RNA; SD, standard deviation.

^aData are presented as the mean (SD).

^bP < .05 compared with sham.

^cP < .05 compared with CME or control siRNA.

The Release of the Myocardial Injury Marker

The serum cTnI concentration was significantly higher in the CME and control siRNA groups than in the sham-operated group after surgery, 0.251 ± 0.038 ng/mL and 0.245 ± 0.042 ng/mL versus 0.044 ± 0.009 ng/mL, P < .05. Moreover, the administration of PTEN siRNA reduced the level of cTnI to 0.135 ± 0.024 ng/mL (Figure 1).

Figure 1.

The serum c-troponin I level was increased following coronary microembolization (CME). Serum cTnI expression was significantly increased in the CME and control siRNA-treated groups compared to that of the sham group (P < .05). The administration of phosphatase and the tensin homolog deleted on chromosome ten (PTEN) small-interfering (siRNA) reduced the cTnI level. ^aP < .05 versus sham; ^bP < .05 vs CME and control siRNA-treated .N = 8 for each group. Mean ± standard deviation (SD). Sham indicates sham group; CME, CME group; PTEN siRNA, CME+ PTEN siRNA group; Control siRNA: CME + Control siRNA group.

Histopathology of CME

As revealed by Mayer hematoxylin and eosin (H&E) and hematoxylin-basic fuchsin-picric acid (HBFP) staining, the sham control animals exhibited subendocardial ischemia without infarction foci, whereas the CME animals exhibited multiple microinfarction foci (Supplemental Figure S1a-A). However, the administration of PTEN siRNA reduced the microinfarct volume and inflammatory cell infiltration (Supplemental Figure S1a-B). Most of the foci were wedge-shaped, locally distributed, and nontransmural, and the foci were primarily found in the subendocardium, left ventricular anterior wall, and heart apex. Staining with HBFP revealed myocardial karyolysis or hypochromatosis based on the red cytoplasmic staining of the microinfarction foci. In addition, peripheral cardiac muscle edema and denaturation, peripheral inflammatory cell infiltration, and erythrocyte effusion were detected (Supplemental Figure S1b-A-E). The infarct area of each group after CME was 0.00 (0)%, 5.64 (2.27)%, and 6.17 (2.57)% in the sham, CME, and control siRNA groups, respectively. However, the administration of PTEN siRNA reduced the microinfarct volume to 3.28 (1.46)% (P < .05).

TUNEL Assay

Using the Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL) assay, the dead cells in the myocardial tissue were stained yellow–brown, whereas the normal cells were stained light blue. Myocardial apoptosis after CME was primarily detected in the myocardial microinfarction foci and in the peripheral zones. In the sham animals, we occasionally detected myocardial apoptosis in the subendocardium and the papillary muscles. The myocardial apoptotic index (AI) of the sham group, CME group, and control siRNA group were 0.12 (0.08)%, 8.89 (2.85)%, and 7.25(2.23)%, respectively. The administration of PTEN siRNA reduced the myocardial apoptosis to 3.85 (1.84)% (Supplemental Figure S2).

The Messenger RNA Levels of PTEN Each Group

The PTEN messenger RNA (mRNA) expression was significantly higher in the CME and control siRNA groups than in the sham-operated group after surgery, 12 hours: 3.28 (0.31) and 3.19 (0.25) versus 0.44 (0.09), P < .05.The administration of PTEN siRNA reduced the mRNA level of PTEN to 1.34 (0.12; Figure 2).

Figure 2.

The phosphatase and the tensin homolog deleted on chromosome ten (PTEN) messenger RNA (mRNA) level was significantly increased following coronary microembolization (CME). The PTEN mRNA level was significantly increased in the CME and control small-interfering RNA (siRNA)-treated groups compared to that of the sham group (P < .05). However, the administration of PTEN siRNA reduced the levels of PTEN mRNA. ^aP < .05 versus sham; ^bP < .05 versus CME and control siRNA-treated. N = 8 for each group. Mean ± SD. Sham indicates sham group; CME, CME group; PTEN siRNA: CME + PTEN siRNA group; Control siRNA: CME + Control siRNA group.

The PTEN siRNA Pretreatment Inhibited Myocardial PTEN/Akt Signaling in CME Swine

Western blotting showed significant upregulation of the PTEN, p-Bad, and cleaved caspase-3 proteins as well as a remarkable downregulation of p-Akt following CME modeling. Western blot analysis of the infarcted myocardial tissue in each group showed that the expression of PTEN (Figure 3A), Bad (Figure 3C), and cleaved caspase-3 (Figure 3D) was significantly increased in the myocardium of swine from the CME groups compared to those from the sham group (P < .05). However, PTEN siRNA pretreatment was associated with reduced levels of the PTEN, cleaved caspase-3, and p-Bad proteins as well as the enhanced p-Akt (Figure 3B) protein compared to the CME or control siRNA groups (P < .05). However, the Akt-regulated downstream protein IκB kinases (IKKs: IKKα, IKKβ, an IKKγ) were not different in the sham, CME, and control siRNA groups (Figure 3E). The inhibition of Akt/Bad rather than Akt/IKK reduced cardiomyocyte apoptosis. These results indicate that CME induces cardiomyocyte apoptosis via the PTEN/Akt/Bad signaling pathway (Figure 4).

Figure 3.

Coronary microembolization (CME) induces cardiomyocyte apoptosis via the phosphatase and the tensin homolog deleted on chromosome ten (PTEN)/Akt/Bad signaling pathway. The effects of CME and PTEN small-interfering RNA (siRNA) on the protein levels of PTEN and the Akt pathways as well as Bad and caspase 3 in the myocardial tissues in swine 12-hour post-CME modeling are as follows: the results of the Western blotting analyses. The expression levels of PTEN, p-Bad, and cleaved caspase-3 were significantly increased in the CME group compared to those of the sham group (P < .05). The expression level of p-Akt (phosphorylation sitesThreonine308) was significantly decreased in the CME group compared to that of the sham group (P < .05). The expression levels of Akt, Bad, and caspase-3 were not different among the sham, CME, and control siRNA groups. However, PTEN siRNA reduced the levels of PTEN, p-Bad (phosphorylation sites Serine112), and cleaved caspase-3 and enhanced the level of p-Akt compared to that of the CME group or the control siRNA group. However, the Akt-regulated downstream protein IκB kinase (IKKs; IKKα, IKKβ, IKKγ) were not different in the sham, CME, and control siRNA groups. The inhibition of Akt/Bad rather than Akt/IKK reduced cardiomyocyte apoptosis. These results indicate that CME induces cardiomyocyte apoptosis via the PTEN/Akt/Bad signaling pathway.^aP < .05 versus sham; ^bP < .05 versus CME or control siRNA. N = 8 for each group. Sham indicates sham group; CME, CME group; PTEN siRNA, CME + PTEN siRNA group; Control siRNA: CME + Control siRNA group.

Figure 4.

The molecular cascade after coronary microembolization (CME). CME-induced apoptosis via upregulation of phosphatase and the tensin homolog deleted on chromosome ten (PTEN) with Akt and cooperating with other Akt-downstream apoptotic signals, such as Bad and caspase 3. After using PTEN small-interfering RNA (siRNA), Akt and other downstream genes were affected, followed by the inhibition of the apoptotic cell death process. Sham indicates sham group; CME, CME group; PTEN siRNA, CME + PTEN siRNA group; Control siRNA: CME + Control siRNA group. Signal transduction of CME-induced cardiomyocyte apoptosis.

Discussion

The novel findings of this study are summarized as follows. We showed that the PTEN/Akt signaling pathway is involved in the pathogenesis of CME-induced myocardial apoptosis and dysfunction. The results of this study highlighted the important role of PTEN/Akt in the pathogenesis of CME-related myocardial dysfunction and apoptosis. These findings help to elucidate the mechanisms by which PTEN mediates myocardial injury and supports our hypothesis that PTEN might represent a potential target for intervention.

The CME is widely observed in acute coronary syndrome and is considered to be an iatrogenic complication following coronary interventions. ⁴ The CME, which is caused by the embolization of thrombotic material and debris or the rupture of an atherosclerotic plaque, is hypothesized to generate a transient decrease in the coronary blood flow, followed by reactive hyperemia and myocardial systolic dysfunction. Rioufol et al ¹⁶ demonstrated that the formation of atherosclerosis frequently presents with the rupture and repair of plaques. Therefore, our data further confirm the pathophysiological manifestations of CME.

In this study, we applied a catheterization interventional technique to inject 100 000 microspheres into the LAD artery to fabricate the CME model. The model was validated in the following aspects. First, the pathological examination showed microinfarcts foci, inflammatory cell infiltration, and cardiomyocyte apoptosis. In addition, the peripheral circulation of cTnI was increased, indicating a cardiomyocyte injury. Progressive deterioration of cardiac function was dynamically observed during the follow-up period. These changes were consistent with the pathophysiological characteristics revealed by previous studies. ^17,18 To the best of our knowledge, this study is the first to indicate that the PTEN/Akt apoptosis-signaling pathway is involved in the pathogenesis of CME-induced myocardial injury. Our study revealed a novel molecular pathway involved in the pathogenesis of CME-related cardiac dysfunction.

The PTEN has been identified as a tumor suppressor gene that is associated with maintaining a balance between cell survival and cell death. ¹⁹ Increasing evidence has indicated that PTEN acts as a crucial regulatory factor in the development of various cardiovascular diseases. ^20,21 The PTEN was found to be highly expressed in myocardial tissues subjected to ischemia–reperfusion injury, which inhibits the activation of Akt as a result of myocardial apoptosis. However, the silencing of PTEN expression increases Akt activation, reduces cardiomyocyte apoptosis, and improves cardiac function. ^{22 –24} Activated Akt might exert its antiapoptotic effect via the regulation of caspase-3, Bcl-2-antagonist of cell death (Bad), forkhead transcription factor as well as IKK. ^25,26 Although early investigations suggested that activation of the PI3K/Akt signaling pathway could effectively inhibit cardiomyocyte apoptosis and reduce the extent of myocardial injury, ^27,28 a recent study by Oudit et al ²⁹ showed that inhibition of the PTEN/PI3K/Akt pathway could delay the process of ventricular remodeling and heart failure, and another recent study by Parajuli et al ³⁰ showed that PTEN is critically involved in post-MI remodeling through the Akt/IL-10 signaling pathway. A study by Li et al ³¹ showed that the PTEN/Akt pathway played a major role in neuronal apoptosis induced by ischemia and hypoxia in rats. Finally, a recent study indicated that microRNA-1 might attenuate myocardial apoptosis by regulating the PTEN/Akt signaling pathway in infarcted myocardial tissue. The results of our study suggested that PTEN was induced in the myocardium of animals with CME. Compared with those of the control group, the CME myocardial levels of antiapoptotic protein p-Akt expression were sharply decreased, whereas the proapoptotic protein p-Bad and cleaved caspase-3 were evidently increased, indicating that apoptosis was induced during the CME process. Inhibiting PTEN expression by PTEN siRNA could partially reverse these changes in the myocardium of CME animals. The Akt-regulated downstream protein IKKs (IKKα, IKKβ, IKKγ) were not different in the sham, CME, and control siRNA groups. The inhibition of Akt/Bad rather than Akt/IKK reduced cardiomyocyte apoptosis. Therefore, these results further confirmed that the PTEN/Akt/Bad pathway was activated in CME and involved in the pathogenesis of myocardial apoptosis.

The rupture of an atherosclerotic plaque in an epicardialcoronary artery, with the subsequent occlusive coronary thrombosis, has been established as the decisive event in the pathogenesis of acute myocardial infarction. Milder forms of plaque rupture might result in the subsequent embolization of atherosclerotic and thrombotic debris into the coronary microcirculation. The immediate consequences of CME are a transient decrease in coronary blood flow with subsequent reactive hyperemia and a moderate reduction in the regional myocardial function, which partially recovers within minutes. Subsequently, progressive contractile dysfunction develops in the presence of normal or increased blood flow, and there is a perfusion/contraction mismatch. Many investigations have sought to determine the underlying mechanisms of CME-induced myocardial injury and to alleviate the deleterious effects of CME. There have been major advances in identifying the factors of the myocardial inflammatory response and apoptosis that are involved in CME-induced myocardial injury. In this study, we revealed an important role of cardiac-specific PTEN in CME-induced myocardial apoptosis and highlighted that PTEN could be viewed as a potential interventional target for the treatment of CME-related myocardial apoptosis and dysfunction. Further research, especially translational research in humans, is needed to evaluate whether the PTEN/Akt pathway could become a promising treatment strategy for CME-related cardiac dysfunction in clinical scenarios.