Transforming Growth Factor‐β Receptor III is a Potential Regulator of Ischemia‐Induced Cardiomyocyte Apoptosis
Background Myocardial infarction (MI) is often accompanied by cardiomyocyte apoptosis, which decreases heart function and leads to an increased risk of heart failure. The aim of this study was to examine the effects of transforming growth factor‐β receptor III (TGFβR3) on cardiomyocyte apoptosis during MI.
Methods and Results An MI mouse model was established by left anterior descending coronary artery ligation. Cell viability, apoptosis, TGFβR3, and mitogen‐activated protein kinase signaling were assessed by methylthiazolyldiphenyl‐tetrazolium bromide assay, terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling assay, immunofluorescence, electron microscopy, and Western blotting. Our results demonstrated that TGFβR3 expression in the border region of the heart was dynamically changed during MI. After stimulation with H2O2, TGFβR3 overexpression in cardiomyocytes led to increased cell apoptosis and activation of p38 signaling, whereas TGFβR3 knockdown had the opposite effect. ERK1/2 and JNK1/2 signaling was not altered by TGFβR3 modulation, and p38 inhibitor (SB203580) reduced the effect of TGFβR3 on apoptosis, suggesting that p38 has a nonredundant function in activating apoptosis. Consistent with the in vitro observations, cardiac TGFβR3 transgenic mice showed augmented cardiomyocyte apoptosis, enlarged infarct size, increased injury, and enhanced p38 signaling upon MI. Conversely, cardiac loss of function of TGFβR3 by adeno‐associated viral vector serotype 9–TGFβR3 short hairpin RNA attenuated the effects of MI in mice.
Conclusions TGFβR3 promotes apoptosis of cardiomyocytes via a p38 pathway–associated mechanism, and loss of TGFβR3 reduces MI injury, which suggests that TGFβR3 may serve as a novel therapeutic target for MI.
What is New?
The expression of transforming growth factor‐β receptor III (TGFβR3) changes during MI and we defined TGFβR3 as a novel and potential regulator of ischemia‐induced cardiomyocyte apoptosis.
p38 mitogen–activated protein kinase pathway is critical for TGFβR3‐mediated cardiomyocyte apoptosis induced by MI.
Cardiac‐specific transgenic overexpression of TGFβR3 accelerates myocardial injury during ischemia.
What are the Clinical Implications?
TGFβR3‐p38 mitogen–activated protein kinase pathway plays an important role in cardiomyocyte apoptosis induced by MI; our findings identified TGFβR3 as a potential therapeutic target for the treatment of MI.
Myocardial infarction (MI) is caused by coronary artery stenosis, which leads to the termination of blood flow to the corresponding myocardium. During MI, pathological changes occur in the heart, including inflammatory reactions, production of reactive oxygen species, and wall stress, resulting in cardiomyocyte apoptosis.1 Cardiomyocyte apoptosis, which is characterized by the compaction and segregation of chromatin, the fragmentation of nuclei, and the formation of apoptotic bodies, may lead to arrhythmias, cardiac remodeling, and eventually heart failure. Thus, novel targets of cardiomyocyte apoptosis are important for developing therapies for preventing and treating MI.
The transforming growth factor‐β (TGF‐β) signaling pathway plays an important role in regulating proliferation, migration, invasion, and apoptosis through Smad‐dependent and ‐independent pathways. TGF‐β receptor III (TGFβR3) is a transmembrane proteoglycan that functions as a coreceptor of the TGF‐β superfamily.2 As the most abundant TGF‐β receptor, in many cases, TGFβR3 binds and presents TGF‐β to TGFβR1 or TGFβR2 in a cell type–specific manner, which is referred to as the ligand‐dependent pathway. Recent research suggests that TGFβR3 also plays a critical role in ligand‐independent pathways. In L6 myoblasts, for example, TGFβR3 is reported to induce p38 phosphorylation through a ligand‐independent manner.3 However, the precise role of TGFβR3 in ligand‐independent pathways remains to be investigated. Notably, recent studies show that TGFβR3 is critical in the cardiac pathological process. In cardiac fibroblasts, a synthetic peptide from TGFβR3 (P144) and full‐length TGFβR3 both reduced collagen production.4, 5 Notably, we recently demonstrated that TGFβR3 is an important regulator in cardiac hypertrophy.6 Moreover, we determined that TGFβR3 prevents apoptosis of cardiac fibroblasts exposed to hypoxia and established a functional link between ERK1/2 and JNK1/2 signaling pathways and TGFβR3.5, 7 However, the changes in the expression level, functional role, and molecular mechanism of TGFβR3 in cardiomyocyte apoptosis during MI are still largely unclear.
Accordingly, in this study we investigated the potential role of TGFβR3 as a novel target for regulation of cardiomyocyte apoptosis. Our results show that TGFβR3 induces apoptosis of cardiomyocytes during MI via the p38 mitogen–activated protein kinase (MAPK) pathway. Furthermore, knockdown of TGFβR3 reduces the heart injury resulting from MI, which suggests a therapeutic approach for the treatment of heart failure.
Animal care and experimental protocols in this study conformed to the Institutional Animal Care approval by the ethics committee of the Harbin Medical University and the Guide for the Care and Use of Laboratory Animals by the US National Institutes of Health. In total, 117 adult male wild‐type (WT) mice (C57BL/6), 1003 neonatal WT mice (C57BL/6), and 46 cardiac‐specific transgenic (Tg) TGFβR3 overexpression mice were used for this study.
Mouse Model of MI
Adult male C57BL/6 mice (25–30 g) were provided by the Experimental Animal Center of Harbin Medical University (grade II) and were fed with food and water freely throughout the experiments.
The mice were anaesthetized with sodium pentobarbitone (30 mg/kg IP) and xylazine (10 mg/kg IP), and the chest skins were shaved and disinfected. Intubation was applied with an artificial respiration machine (UGO Bsile S.R.L. Biological Research Apparatus). An incision was made through the 4th intercostal space, and the heart was exposed. In the MI group, the left anterior descending artery (LAD) was ligated with 7/0 silk thread; infarction was indicated by a significant ST‐segment elevation in the ECG. In the sham group, 7/0 silk thread was twined around the LAD and was not tightened.
Isolation of Cardiomyocytes
One‐ to 3‐day‐old C57BL/6 mice were anaesthetized by 4% to 5% isoflurane inhalation. The hearts were removed and cut into small pieces. The heart tissues with equirotal shape were digested in 0.25% trypsin, and the isolated cells were collected in 6‐well plates with DMEM (HyClone) containing 10% fetal bovine serum (HyClone). Cardiomyocytes were separated from the fibroblasts after 90 minutes of incubation. Then the nonadherent cardiomyocytes were moved into 6‐well or 96‐well plates with DMEM containing 10% fetal bovine serum and cultured at 37°C in 95% air with 5% CO2 for 48 hours.
Drug Administration and Transfection of Cardiomyocytes
To assess the effect of TGFβR3 on cardiomyocytes, pc‐DNA3.1‐mTGFβR3 plasmid (GeneChem) or small interfering RNAs targeting TGFβR3 (GenePharma) were transfected into cells with X‐tremeGENE siRNA Transfection Reagent (Roche Molecular Biochemicals). The target sequence of TGFβR3 was 5′GGGA GGUU CACA UCCU AAATT3′. Twenty‐four hours after transfection, H2O2 (150 μmol/L) was added to the medium to induce cell apoptosis. A p38 MAPK inhibitor (SB203580, Sigma‐Aldrich; 10 μmol/L) was added to block p38 pathway activation.
Generation of TGFβR3 Tg Mice
TGFβR3‐overexpressing Tg mice were generated as previously described.6, 8 A transgene construct was generated by subcloning the TGFβR3 coding sequence (GenBank: NM_011578.3) into the pRP.Des3d vector backbone, which contains a murine cardiac α‐myosin heavy chain gene promoter. The final targeting vector was electroporated into C57 oosperms, which were then injected into C57 blastocysts, giving rise to a germ‐line transmission resulting in TGFβR3+/+ Tg mice. Genomic DNA prepared from tails was subjected to polymerase chain reaction for genotyping.
Generation of Adeno‐Associated Viral Vectors and In Vivo Vector Delivery
As previously described by our group, adeno‐associated viral vector (AAV) serotype 9 (AAV9)–mediated shRNA for type III TGF‐β coreceptor was purchased from Biowit Technologies.6 Sh‐TGFβR3 (5′‐GGGAGGTTCACATCCTAAA‐3′)‐GFP was cloned into a self‐complementary AAV vector backbone. Iodixanol gradient ultracentrifugation was used to purify the recombinant AAV9 vectors.6, 9 Briefly, a thoracotomy was performed on mice to open the chest through the 4th intercostal space under a general anesthetic. The ascending aortic artery and the main pulmonary artery were occluded. The AAV9 was injected into the left ventricular cavity via the tip of the heart as a 100‐μL bolus (1×109 pfu). Then the arteries were clamped for 10 seconds after AAV9 injection. LAD ligation was performed on the 2nd week after AAV9 injection.
Measurement of Cell Viability
According to the manufacturer's instructions, cells were seeded at a concentration of 10 000 per well. After experimental treatment, the cells were incubated with 10 μL of methylthiazolyldiphenyl‐tetrazolium bromide solution (Sigma‐Aldrich) (0.5 mg/mL) for 4 hours at 37°C. One‐hundred microliters of dimethyl sulfoxide were added into each well, and the plates were shaken for 10 minutes to completely dissolve the formazan. A microplate spectrophotometer (Tecan) was used to read the absorbance value for each well at 490 nm.
Measurement of Cell Apoptosis
To measure apoptotic activity of cardiomyocytes indicated by DNA stand breaks, terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling (TUNEL; In Situ Cell Death Detection FITC Kit or TMR red, Roche) assays were performed on cardiomyocytes or heart tissues, which were immunostained with anti‐α‐actinin antibody (Sigma‐Aldrich). Cells planted on cover slips or cryosections of mouse heart were fixed in 4% paraformaldehyde for 1 hour, blocked with methanol with 3% H2O2 for 10 minutes, and permeabilized with 0.1% Triton X–100 in 0.1% sodium citrate for 2 minutes. Cryosections were incubated with anti‐α‐actinin antibodies (1:200 dilution; Sigma‐Aldrich) in a humidified chamber for 1 hour, followed by TUNEL staining for 1 hour and 4’,6‐diamidino‐2‐phenylindole staining for nuclei for 5 minutes. Laser scanning confocal microscopy (FV300, Olympus) was used to detect TUNEL‐positive cells in randomly selected fields.
The collected cardiomyocytes or heart tissues were fixed in 2% glutaraldehyde and immersed in 2% osmium tetroxide. Samples were then dehydrated by a graded series of ethanol (30%, 50%, 70%, 80%, and 90%) and pure acetone, embedded in Araldite (Serva) and cut into ultrathin sections using an FCR Reichert Ultracut ultramicrotome (Leica Microsystems). Sections were contrasted with uranyl acetate and lead acetate. A JEOL 1200 electron microscope (JEOL Ltd) was used to observe the micromorphological changes in cardiomyocytes.
In brief, the quantities of protein in samples extracted from cardiomyocytes or the peri‐infarct region of mice were determined using a bicinchoninic acid kit. Equivalent amounts of protein were loaded in equal volumes and fractionated by SDS‐PAGE (10–15% polyacrylamide gels). GAPDH was used as the internal control. The antibodies were as follows: anti‐TGFβR3, anti‐phospho‐p38, anti‐phospho‐ERK1/2, anti‐phospho‐JNK1/2, anti‐p38, anti‐ERK1/2, anti‐JNK1/2 (1:1000 dilution; Cell Signaling Technology), and anti‐GAPDH (1:500 dilutions; Research Diagnostics).
Cryosections of the border region (4 μm) were fixed in 4% paraformaldehyde for 2 hours, permeabilized with 0.1% Triton X–100 in 0.1% sodium citrate for 10 minutes, and blocked with 5% bovine serum albumin in PBS with Tween 20 for 1 hour. Cryosections were incubated with anti‐α‐actinin antibody (1:500; Sigma‐Aldrich) and anti‐TGFβR3 antibody (1:100; Sigma‐Aldrich) in a humidified chamber overnight at 4°C, followed by 4’,6‐diamidino‐2‐phenylindole staining for nuclei for 5 minutes. Laser scanning confocal microscopy (FV300, Olympus) was used to image slides in randomly selected fields.
Measurement of Infarction Size
Infarct size was measured by the triphenyl tetrazolium chloride (Sigma‐Aldrich) assay as previously described.6 In brief, hearts were excised and dissected into 1 mm‐thick sections. The sections were immersed in 1% triphenyl tetrazolium chloride for 25 minutes at 37°C and fixed with 3.5% methanol overnight. Viable tissues stained red and infarct tissues remained uncolored. The infarct size was calculated with image analysis software (Image‐Pro Plus v4.0; Media Cybernetics) as previously described.6
Transthoracic echocardiography using an echocardiogram (Vivid 7, GE Medical) with a 10‐MHz linear transducer was performed to determine left ventricular function as previously described.6
The data were processed with GraphPad Prism 5.0 analysis software and are presented as means±SEM. Multiple comparisons were determined by 1‐way ANOVA with the Bonferroni's post hoc test. We considered a 2‐tailed value of P<0.05 as statistically significant.
Dynamic Expression of TGFβR3 in the Border Region During MI and in Cardiomyocytes Stimulated With H2O2
Several reports have substantiated the loss of TGFβR3 expression in human prostate cancer and in epithelial‐derived ovarian cancer and have identified a suppressive role for TGFβR3 in the migration and invasion of these tumors. To characterize the possible role of TGFβR3 in MI, we removed the hearts from mice that had received LAD ligation for 3, 6, 9, and 12 hours, and we examined the expression of TGFβR3 in the border region by Western analysis (Figure 1A). The protein levels of TGFβR3 were elevated initially and peaked at 6 hours postinfarction, gradually decreasing to baseline levels by 12 hours postinfarction. Findings from immunostaining images further confirmed that TGFβR3 was mainly expressed in membrane of cardiomyocytes and showed increased TGFβR3 protein in the border region of the heart after 6 hours infarction (Figure 1B).
Consistent with the in vivo data, TGFβR3 levels were increased in cardiomyocytes exposed to short‐term treatment with H2O2 (3 and 6 hours) and decreased to baseline from 6 to 12 hours (Figure 1C). The dynamic TGFβR3 expression, which undergoes transient elevation both in vivo and in vitro suggests the possibility that TGFβR3 might regulate the response to MI.
TGFβR3 is Involved in Regulating Apoptosis of Cardiomyocytes Exposed to H2O2
TGFβR3 is known to regulate cell proliferation, migration and invasion, and apoptosis in tumor cells.10 To determine whether it also plays a role in apoptosis in cardiomyocytes, we overexpressed TGFβR3 or a negative control (NC) vector in cardiomyocytes for 24 hours (Figure S1A) and also treated them with or without H2O2 (150 μmol/L) for 6 hours. Western blotting showed that H2O2 increased the expression of TGFβR3 by 1.9‐fold and TGFβR3 plasmid increased the expression of TGFβR3 by 2.1‐fold; a combination of TGFβR3 plasmid and H2O2 treatment increased expression of TGFβR3 by 4‐fold (Figure 2A). Methylthiazolyldiphenyl‐tetrazolium bromide and TUNEL assays revealed that overexpression of TGFβR3 directly impaired cell viability and led to cell apoptosis. Furthermore, compared with the damage in the H2O2+NC group, the damage was accumulatively enhanced in cardiomyocytes that both overexpressed TGFβR3 and were treated with H2O2 for 24 hours (Figure 2B through 2D). To verify these findings, we performed electron microscopy. Distinct subcellular morphological changes of apoptosis (chromosome condensation and apoptotic bodies) were evident in H2O2‐treated and TGFβR3‐overexpressing cells, while cells with both TGFβR3 overexpression and H2O2 treatment had the most dramatic effects (Figure 2E). These data suggest that H2O2 and TGFβR3 overexpression mediate increases in both the TGFβR3 protein level and apoptosis, and that the increased level of TGFβR3 tended to parallel the degree of apoptosis.
To further confirm the protective role of TGFβR3, we prepared knockdown cardiomyocytes (Figure S1B). Compared with cardiomyocytes exposed to H2O2, TGFβR3 knockdown cells with H2O2 treatment showed decreased injury as evidenced by the effects on cell viability (Figure 3A) and apoptosis (Figure 3B and 3C). Furthermore, knockdown of TGFβR3 in H2O2‐treated cells alleviated changes in the microstructure (Figure 3D). Notably, TGFβR3 knockdown alone did not alter cell apoptosis. Thus, loss of TGFβR3 partially reduced the effect of H2O2 on cardiomyocytes apoptosis, suggesting that TGFβR3 is involved in regulation of cell apoptosis.
TGFβR3 Mediates Cardiomyocyte Apoptosis via p38 Signaling Upon H2O2 Treatment
The MAPK signaling pathway has a well‐established role in the pathogenesis of cardiovascular diseases, including MI.11, 12 The ERK1/2, JNK1/2, and p38 signaling pathways are activated in the infarcted myocardium zone and border regions upon MI.13 Furthermore, the p38 signaling pathway is activated in the basal state in the heart, and the p38 inhibitor RWJ‐67657 can restrict the infarction size in the rat following MI.14 To explore the effects of TGFβR3 on the MAPK pathway, we performed Western analysis of cardiomyocytes with or without TGFβR3 overexpression and before and after H2O2 exposure. Our results suggest that under both the normal and H2O2 conditions, TGFβR3 enhances p38 signaling but does not influence ERK1/2 or JNK1/2 signaling (Figure 4A through 4C). In addition, while H2O2 and TGFβR3 overexpression activated p38 signaling separately, p38 phosphorylation was further activated by the combination of TGFβR3 and H2O2. The converse effect was observed when TGFβR3 was knocked down (Figure 4D). These findings suggest that TGFβR3 is critical for the activation of p38 signaling in cardiomyocytes treated with H2O2. To determine whether the p38 pathway is involved in regulation by TGFβR3 of apoptosis, we used a p38 inhibitor (SB203580, 10 μmol/L) to block the p38 pathway. Methylthiazolyldiphenyl‐tetrazolium bromide and TUNEL assays demonstrated that blocking p38 signaling inhibits the effect of TGFβR3 on apoptosis, confirming the key role of the p38 pathway in TGFβR3‐dependent induction of apoptosis (Figure 5A through 5C). Thus, our results provide further verification of the role of the TGFβR3‐p38 pathway in regulating cardiomyocyte apoptosis.
Overexpression of TGFβR3 Augments MI Injury
To study the in vivo function of TGFβR3, we next generated TGFβR3+/+ Tg mice driven by cardiac specific α‐myosin heavy‐chain promoter (Figure 6A and 6B). After LAD ligation for 24 hours, the infarct size was greater for TGFβR3+/+ Tg mice than for WT mice (Figure 6C). In addition, the Tg mice exhibited a deterioration in heart function after 24 hours of MI: the fractional shortening was decreased to 9.5±1.4% and the ejection fraction was decreased to 21.5±2.5% (P<0.05) in TGFβR3+/+ Tg mice subjected to LAD ligation (Figure 6D and 6E).
To reveal the changes in apoptosis and the microstructure of the myocardium that are associated with TGFβR3 overexpression, the border regions of mice were examined by TUNEL staining and electron microscopy. Compared with WT mice, TGFβR3+/+ Tg mice had more apoptotic cells (red) and the sarcomere structures were disordered with condensation of chromatin after 24 hours of MI (Figure 6F through 6H). Furthermore, the phosphorylation of p38 was enhanced in TGFβR3+/+ mice compared with WT mice at 6 hours after MI, although the 2 groups of mice had similar levels of p38 activation before MI (Figure 6I). These data suggest that overexpression of TGFβR3 enhances the sensitivity of the myocardium to MI injury, indicating the possibility that loss of TGFβR3 may mitigate the effects of MI on the heart.
Knockdown of TGFβR3 Alleviates MI Injury
To determine whether loss of TGFβR3 mitigates the effects of MI on the heart, we delivered AAV9 vectors carrying a short hairpin RNA of TGFβR3 (AAV‐shTGFβR3) by injection. AAV9 is a cardiac‐specific AAV with high efficiency of transduction in the heart and coronary artery, and injection of AAV has successfully delivered genes into the myocardium of rats, pigs, and hamsters.15, 16 The effectiveness of cardiac infection with AAV‐shTGFβR3‐GFP is shown (Figure 7A). Knockdown of TGFβR3 in cardiomyocytes was confirmed by Western blotting (Figure 7B). The infarct size after 24 hours of MI was decreased to 32.5±3.0% (P<0.05) in AAV‐shTGFβR3 mice (Figure 7C). We examined the heart function after 4 weeks of MI because several reports have shown that the muridae ischemia heart shows characteristics of heart failure at this time point.5, 17 Fractional shortening and ejection fraction were significantly improved in AAV‐shTGFβR3 mice compared with AAV‐scramble mice (Figure 7D and 7E).
To verify the in vivo protective effects of TGFβR3 shRNA, we examined the number of apoptotic cells and the microstructure of heart tissue from AAV‐shTGFβR3 mice. Fewer apoptotic cells were observed in AAV‐shTGFβR3 mice compared with AAV‐NC mice after 24 hours of MI. Furthermore, consistent with the in vitro observations, silencing of TGFβR3 attenuated the damage to the microstructure at 24 hours after MI (Figure 7F and 7G). AAV‐shTGFβR3 also partially restored the sarcomere disruption and chromatin condensation observed in AAV‐shNC mice after MI (Figure 7H). Finally, p38 activation after 6 hours of MI was significantly suppressed in AAV‐shTGFβR3 mice but not in AAV‐shNC mice (Figure 7I). Collectively, these data suggest that loss of function of TGFβR3 partially protects the heart from MI injury.
We demonstrated that TGFβR3 expression is dynamically regulated in the heart during MI and in cardiomyocytes stimulated with H2O2. Furthermore, using both in vitro and in vivo assays with both gain‐of‐function and loss‐of‐function experiments, we demonstrated that TGFβR3 potentiates cell apoptosis, increases infarct size, and impairs heart function by regulating the p38 pathway during MI. Our findings provide novel insight for the development of therapeutic strategies for MI.
Previous findings have shown that changes in TGFβR3 affect cell homeostasis.2, 18 In the early stage of some tumors, the expression of TGFβR3 is suppressed, and replenishment of TGFβR3 inhibits tumor metastasis, invasion, and growth.19 TGFβR3 mRNA is also induced by glucocorticoids, especially aldosterone, dexamethasone, and hydrocortisone, in hepatic stellate cells, fibroblasts, and osteoblasts, which suggests a critical role for TGFβR3 in controlling pleiotropic cellular effects.20 During coronary vessel development, TGFβR3 is dynamically regulated and is required; TGFβR3‐null mice cannot survive after embryonic day 14.5.21 In addition, β‐arrestin2 promotes the endocytosis of TGFβR3, and TGFβR3 and β‐arrestin2 coordinately function to regulate epithelial and cancer cell migration.22 Interestingly, TGFβR3 expression is significantly suppressed in rat lungs during long‐term anoxia (day 14).23 In contrast, our results suggest that TGFβR3 is upregulated in mice during MI. This difference may be explained by the acute course of MI injury as compared with the long‐term course of anoxia. In our study, the expression of TGFβR3 peaked at 6 hours after MI and then fell to baseline levels at 12 hours after MI. We also demonstrated that targeting TGFβR3 at an early stage of MI (within 6 hours) may ameliorate the injury, which suggests that TGFβR3 participates in the regulation of cardiomyocyte behavior during the early stage of MI. Consistently, the expression of TGFβR3 was upregulated by H2O2 and was further increased by the combination of H2O2 and TGFβR3 overexpression plasmid. In cultured cardiomyocytes, TGFβR3 directly leads to cell apoptosis and enhances the cell ultrastructural changes under normal conditions. Under H2O2 conditions, an increase in TGFβR3 levels exerts a cumulative effect on the degree of apoptosis. Consistently, TGFβR3 is also known to enhance apoptosis in some tumor cells; however, TGFβR3 suppresses the apoptosis of cardiofibroblasts,7, 10 potentially by deactivating the TGFβR1/TGFβR2 complex to downregulate cell apoptosis.24 According to our data, TGFβR3 promotes cardiomyocyte apoptosis induced by myocardial infarction. The induction of apoptosis by TGFβR3 is observed in some tumor cells, while TGFβR3 is also reported to suppress apoptosis of cardiofibroblasts. And the discrepant effect of TGFβR3 on apoptosis in various context may be explained by the difference of disease models or cell type‐specific signaling pathways.
To examine the signaling pathways that mediate TGFβR3‐dependent apoptosis in cardiomyocytes, we evaluated the activation of MAPKs after MI. In most cases, TGFβR3 serves as a coreceptor in TGF‐β signaling and binds and presents ligands such as TGF‐β1 to specific receptors, leading to the activation of downstream Smad‐dependent and Smad‐independent signaling.21 The Smad‐independent activation of MAPK signaling by TGF‐β has been extensively studied. For example, in mouse mesangial cells, the p38 pathway is activated by TGF‐β1 to induce collagen and fibronectin expression. We demonstrated that overexpression of TGFβR3 during MI activates the p38 signaling pathway but does not alter the JNK1/2 or ERK1/2 pathway. Interestingly, TGFβR3‐induced activation of p38 signaling has also been detected in colon cancer cells, fibroblasts, and L6 myoblasts.24, 25 In the infarct heart, cytokines such as tumor necrosis factor and interleukin 6 and injury of ischemia‐reperfusion activate the p38 signaling pathway.26 Furthermore, as a critical part of the response to MI in the heart, several investigations have confirmed that the p38 pathway sensitizes the caspase cascade to induce cell apoptosis, which supports our findings.27, 28 We observed that the activation of p38 was attenuated by knockdown of TGFβR3 under H2O2 or MI conditions. As a target of H2O2, TGFβR3 is involved in the regulation of H2O2 on cardiomyocyte apoptosis, which is also influenced by some other factors. Thus, loss of TGFβR3 in knockdown cells partially impaired the effect of H2O2. Moreover, p38 inhibitor abolished the effects of TGFβR3 on apoptosis, suggesting that TGFβR3 mediates cardiomyocyte apoptosis at least partially via p38 signaling. This is consistent with previous reports demonstrating that p38 signaling mediates cell apoptosis during ischemia reperfusion injury.29 In our recent study, calmodulin‐dependent protein kinase II, a regulator of the p38 pathway, was confirmed to function downstream of TGFβR3.6 However, whether calmodulin‐dependent protein kinase II also functions in the response to MI warrants further investigation.
To dissect the in vivo role of TGFβR3, we generated cardiac‐specific Tg mice overexpressing TGFβR3. Notably, the infarct size of TGFβR3+/+ Tg mice was significantly increased compared with that of WT mice. In addition, forced expression of TGFβR3 impaired heart function after 24 hours of MI, as evidenced by the reduced ejection fraction and fractional shortening, suggesting the increased sensitivity to MI injury by Tg mice. Although p38 signaling was upregulated in mice after MI, in this study, p38 signaling was not altered in TGFβR3+/+ Tg mice compared with WT mice before MI induction, which is different from the in vitro results. This difference may be attributed to the following factors: first, the expression of TGFβR3 was increased less in the in vivo study (1.5‐fold increase) than in the in vitro study (2‐fold increase); second, the complex in vivo microenvironment makes it difficult to eliminate interference; finally, the activation of p38 in TGFβR3+/+ Tg mice during MI is less dramatic than the activation in cardiomyocytes after H2O2 exposure, which is consistent with the suppressed heart function and augmented infarct size. Nevertheless, p38 activation was statistically enhanced in both TGFβR3‐overexpression cardiomyocytes and in TGFβR3+/+ mice, and the link between TGFβR3 expression and p38 was verified in loss‐of‐function experiments, which supports the role for p38 in mediating the effects of MI injury.
Loss‐of‐function experiments in mice were performed by left ventricular cavity injection of cardiac‐specific AAV expressing shTGFβR3 rather than producing TGFβR3 null mice because TGFβR3 plays a critical role in heart growth. Coronary artery development is blocked in TGFβR3 null mice, which cannot survive after E14.5 during embryonic development.30 The high efficiency for myocardial transduction of AAV9 makes it a strong tool in Tg research of the heart, and it has been successful in previous investigations.31 Our results show that AAV‐shTGFβR3–mediated knockdown of TGFβR3 in the myocardium alleviates the injury of MI on the heart, as exemplified by improved heart function and decreased apoptosis, which was verified by echocardiography. During the development of MI, the ventricular wall became thicker as a result of myocardial hypertrophy and collagen deposition. The inflexible myocardium impaired the systolic and diastolic function of the heart.32, 33 Thus, we confirmed that knockdown TGFβR3 in the myocardium protects the heart from injury caused by MI, providing a novel target of MI therapy.
We present evidence of the critical role of TGFβR3 in the pathogenesis of MI. We show that TGFβR3 may mediate heart ischemic injury by inducing apoptosis, which may be promoted in part by hyperactivated p38 signaling. Although the underlying mechanisms of p38 in mediating the effects of MI need to be further investigated, our present work identifies TGFβR3 as a potential therapeutic target for MI.
Sources of Funding
This study was supported by the National Natural Science Foundation of China (grant numbers 81370244, 91639202, 31400983, and 81503070); the Funds for Creative Research Groups of the National Natural Science Foundation of China (grant number 81421063); the Heilongjiang Province Outstanding Youth Foundation (grant number JC201315); the China Postdoctoral Science Foundation Funded Project (grant numbers 132986 and 2014M560269); and the Harbin Medical University Scientific Research Innovation Fund (grant number 2016JCZX54).
Figure S1. Efficacy of transforming growth factor‐β receptor III (TGFβR3) overexpression or knockdown in cardiomyocytes and heart tissues. A, Protein expression of TGFβR3 in cardiomyocytes transfected with pc‐DNA3.1‐m TGFβR3 plasmid or negative control (NC) plasmid (*P<0.05; n=6). B, Protein expression of TGFβR3 in cardiomyocytes transfected with TGFβR3 small interfering RNA (siRNA) or NC siRNA (**P<0.01; n=6). Data are shown as means±SEM. CTRL indicates control.
- ↵Turley RS, Finger EC, Hempel N, How T, Fields TA, Blobe GC. The type III transforming growth factor‐beta receptor as a novel tumor suppressor gene in prostate cancer. Cancer Res. 2007;67:1090–1098.
- ↵Hermida N, Lopez B, Gonzalez A, Dotor J, Lasarte JJ, Sarobe P, Borras‐Cuesta F, Diez J. A synthetic peptide from transforming growth factor‐beta1 type III receptor prevents myocardial fibrosis in spontaneously hypertensive rats. Cardiovasc Res. 2009;81:601–609.
- ↵Lou J, Zhao D, Zhang LL, Song SY, Li YC, Sun F, Ding XQ, Yu CJ, Li YY, Liu MT, Dong CJ, Ji Y, Li H, Chu W, Zhang ZR. Type III transforming growth factor‐beta receptor drives cardiac hypertrophy through beta‐arrestin2‐dependent activation of calmodulin‐dependent protein kinase II. Hypertension. 2016;68:654–666.
- ↵Muller FU, Lewin G, Baba HA, Boknik P, Fabritz L, Kirchhefer U, Kirchhof P, Loser K, Matus M, Neumann J, Riemann B, Schmitz W. Heart‐directed expression of a human cardiac isoform of cAMP‐response element modulator in transgenic mice. J Biol Chem. 2005;280:6906–6914.
- ↵Werfel S, Jungmann A, Lehmann L, Ksienzyk J, Bekeredjian R, Kaya Z, Leuchs B, Nordheim A, Backs J, Engelhardt S, Katus HA, Muller OJ. Rapid and highly efficient inducible cardiac gene knockout in adult mice using AAV‐mediated expression of Cre recombinase. Cardiovasc Res. 2014;104:15–23.
- ↵Margulis V, Maity T, Zhang XY, Cooper SJ, Copland JA, Wood CG. Type III transforming growth factor‐beta (TGF‐beta) receptor mediates apoptosis in renal cell carcinoma independent of the canonical TGF‐beta signaling pathway. Clin Cancer Res. 2008;14:5722–5730.
- ↵Zhang Q, Deng Y, Lai W, Guan X, Sun X, Han Q, Wang F, Pan X, Ji Y, Luo H, Huang P, Tang Y, Gu L, Dan G, Yu J, Namaka M, Zhang J, Deng Y, Li X. Maternal inflammation activated ROS‐p38 MAPK predisposes offspring to heart damages caused by isoproterenol via augmenting ROS generation. Sci Rep. 2016;6:30146.
- ↵See F, Thomas W, Way K, Tzanidis A, Kompa A, Lewis D, Itescu S, Krum H. P38 mitogen‐activated protein kinase inhibition improves cardiac function and attenuates left ventricular remodeling following myocardial infarction in the rat. J Am Coll Cardiol. 2004;44:1679–1689.
- ↵Pan Z, Sun X, Shan H, Wang N, Wang J, Ren J, Feng S, Xie L, Lu C, Yuan Y, Zhang Y, Wang Y, Lu Y, Yang B. MicroRNA‐101 inhibited postinfarct cardiac fibrosis and improved left ventricular compliance via the FBJ osteosarcoma oncogene/transforming growth factor‐beta1 pathway. Circulation. 2012;126:840–850.
- ↵Hempel N, How T, Dong M, Murphy SK, Fields TA, Blobe GC. Loss of betaglycan expression in ovarian cancer: role in motility and invasion. Cancer Res. 2007;67:5231–5238.
- ↵Compton LA, Potash DA, Brown CB, Barnett JV. Coronary vessel development is dependent on the type III transforming growth factor beta receptor. Circ Res. 2007;101:784–791.
- ↵Mythreye K, Blobe GC. The type III TGF‐beta receptor regulates epithelial and cancer cell migration through beta‐arrestin2‐mediated activation of Cdc42. Proc Natl Acad Sci USA. 2009;106:8221–8226.
- ↵Vicencio AG, Eickelberg O, Stankewich MC, Kashgarian M, Haddad GG. Regulation of TGF‐beta ligand and receptor expression in neonatal rat lungs exposed to chronic hypoxia. J Appl Physiol. 2002;93:1123–1130.
- ↵You HJ, Bruinsma MW, How T, Ostrander JH, Blobe GC. The type III TGF‐beta receptor signals through both Smad3 and the p38 MAP kinase pathways to contribute to inhibition of cell proliferation. Carcinogenesis. 2007;28:2491–2500.
- ↵Liu H, Pedram A, Kim JK. Oestrogen prevents cardiomyocyte apoptosis by suppressing p38alpha‐mediated activation of p53 and by down‐regulating p53 inhibition on p38beta. Cardiovasc Res. 2011;89:119–128.
- ↵Kyoi S, Otani H, Matsuhisa S, Akita Y, Tatsumi K, Enoki C, Fujiwara H, Imamura H, Kamihata H, Iwasaka T. Opposing effect of p38 MAP kinase and JNK inhibitors on the development of heart failure in the cardiomyopathic hamster. Cardiovasc Res. 2006;69:888–898.
- ↵Chen Z, Zhang X, Liu Y, Liu Z. Morphine postconditioning protects against reperfusion injury via inhibiting JNK/p38 MAPK and mitochondrial permeability transition pores signaling pathways. Cell Physiol Biochem. 2016;39:61–70.
- ↵Konkalmatt PR, Beyers RJ, O'Connor DM, Xu Y, Seaman ME, French BA. Cardiac‐selective expression of extracellular superoxide dismutase after systemic injection of adeno‐associated virus 9 protects the heart against post‐myocardial infarction left ventricular remodeling. Circ Cardiovasc Imaging. 2013;6:478–486.
- ↵Tijsen AJ, Pinto YM, Creemers EE. Non‐cardiomyocyte microRNAs in heart failure. Cardiovasc Res. 2012;93:573–582.
- ↵Landmesser U, Wollert KC, Drexler H. Potential novel pharmacological therapies for myocardial remodelling. Cardiovasc Res. 2009;81:519–527.