Cardiac stem cells transplantation enhances the expression of connexin 43 via the ANG II/AT1R/TGF-beta1 signaling pathway in a rat model of myocardial infarction

https://doi.org/10.1016/j.yexmp.2015.11.013Get rights and content

Abstract

Background

In this study, we hypothesized that CSCs mediated the expression of Cx43 after transplantation post MI via the ANG II/AT1R/TGF-beta1 signaling pathway.

Methods

Myocardial infarction (MI) was induced in twenty male Sprague–Dawley rats. The rats were randomized into two groups and were then received the injection of 5 × 106 CSCs labeled with PKH26 in phosphate buffer solution (PBS) or equal PBS alone into the infarct anterior ventricular free wall two weeks after MI. Six weeks later, relevant signaling molecules involved were all examined.

Results

In the CSCs group, an increased expression of Cx43 could be observed in different zones of the left ventricle (P < 0.01). There was a significant reduction of the angiotensin II (ANG II) level in plasma and different regions of the left ventricular cardiac tissues (P < 0.05; P < 0.01). The angiotensin II type I receptor (AT1R) was decreased accompanied with an enhanced expression of angiotensin II type II receptor (AT2R) (P < 0.01). Transforming growth factor beta-1(TGF-beta1) was downregulated (P < 0.01). The expression of mothers against decapentaplegic homolog (SMAD) proteins including SMAD2 and SMAD3 was attenuated whereas SMAD7 was elevated (P < 0.01, P < 0.01, P < 0.05). In addition, the expression of mitogen-activated protein kinases (MAPKs) including extracellular kinases 1/2 (ERK1/2) and p38 was also found to be reduced (P < 0.01).

Conclusion

CSCs transplantation could enhance the level of Cx43 after MI. They might function through intervening the ANGII/AT1R/TGF-beta1 signaling pathway to regulate the expression of Cx43.

Introduction

Myocardial infarction (MI) with resultant congestive heart failure and malignant arrhythmia continues to be a major cause of death and disability worldwide (Mozaffarian et al., 2015). Various approaches have been taken to improve the survival of patients with MI over the past decade, however, the morbidity and mortality remain significant (Hausenloy et al., 2013). In recent years, cell therapy has emerged as a potentially novel strategy for the treatment of MI. Series of preclinical and clinical studies have displayed the benefits of cell interventions in MI (Sanganalmath and Bolli, 2013, Pavo et al., 2014). Substantial evidence has already revealed that stem cell transplantation can reduce infarct size, preserve cardiac function and improve clinical outcomes in patients with MI (Schutt et al., 2015, Heldman et al., 2014). Several types of stem cells, such as embryonic stem cells (ESCs), bone marrow stem cells (BMSCs) and induced pluripotent stem cells (iPSCs) have been used in the therapy studies of MI. In spite of this, the adverse effects and ethical issues brought about by them have limited their further clinical applications (Hou et al., 2013).

Cardiac stem cells (CSCs) represent a logical candidate for cardiac regeneration therapy after MI. they participate in the turnover of myocytes in the absence of pathological state and they are intrinsically programmed to form cardiac tissues rapidly upon activation (Hou et al., 2013, Bergmann et al., 2009, Davis et al., 2010). Our previous work has demonstrated that CSCs transplantation can improve the electrophysiological stability in MI (Zheng et al., 2011). One further study shows that CSCs are superior to MSCs in modulating the electrophysiological abnormality and improving the VFT in rats with MI (Zheng et al., 2013). They can express connexin 43 (Cx43) in the infarct zone and border zone after cells implantation. However, it is still unknown how CSCs may regulate the expression of Cx43 in the procedure.

Cx43 is, by far, the most predominant type of gap junction proteins expressed in the conductive and working myocardial cells (Akar et al., 2004). The downregulation or heterogeneous redistribution of Cx43 causes the structural and electrical changes that impair electrical stability (Kim and Fishman, 2013). The expression and remodeling of Cx43 post MI is dominated by numerous factors. Specific molecular mechanisms involved are intricate and not completely understood.

The renin-angiotensin system (RAS) exerts a pivotal role in the pathogenesis of myocardial infarction (MI) (Morales-Suárez-Varela et al., 2011). Angiotensin II (ANG II) is a key component of the RAS and has been implicated in the development of various cardiovascular ailments including cardiac hypertrophy, remodeling and heart failure (Lang and Struthers, 2013). Most of the classical and biological functions of angiotensin II are mediated by the angiotensin II type 1 receptor (AT1R), whereas the angiotensin II type 1 receptor (AT2R) plays a counter-regulatory role. Blockade of AT1R has been reported to reduce the incidence of ventricular arrhythmias (Jiao et al., 2012). Accumulating evidence has indicated that the activation of ANG II is closely linked with the loss of Cx43 in vivo (Hussain et al., 2010, Yasuno et al., 2013).

ANG II performs its functions via activating several signaling pathways. TGF-β1 is a crucial downstream molecule of ANG II. It mediates a variety of biological processes such as cell proliferation, immune suppression, and inflammation (Shi and Massagué, 2003). It is also vital in the pathogenesis of infarct healing, cardiac remodeling, and interstitial fibrosis (Vivar et al., 2013). TGF-β1 expression has been shown to be markedly increased in experimental models of myocardial infarction and in patients with cardiomyopathy (Dobaczewski et al., 2011). Recent studies manifest that TGF-β1 modulates the formation of Cx43 (Neuhaus et al., 2009). Nevertheless, relevant signaling pathways involved are still undetermined.

In this study, we hypothesized that CSCs transplantation promoted the expression of Cx43 via the inhibition of ANGII and its downstream signaling molecules including AT1R and TGFβ1 signaling pathways after MI.

Section snippets

Ethics statement

Twenty adult male Sprague–Dawley (SD) rats weighing 250 g–350 g were obtained from the Animal Experimental Center of the Sun Yat-sen University and housed in a standard animal facility with 12-hour on/off light conditions. All animals were acclimatized for at least one week prior to surgery and allowed free access to standard food and water. All animal handling and procedures were performed in accordance with protocols approved by the Animal Ethics Committee of Sun Yat-sen University

CSCs transplantation enhanced the expression of Cx43

CSCs marked with PHK26 were found to be surviving in the infarct zone and border zone and expressed Cx43 by immunofluorescence staining (Fig.1A, c and e), which was accorded with the results of our previous work. In the CSCs group, the expression of Cx43 was detected in different zones of the left ventricle (Fig.1A; a, c and e) whereas the PBS group rarely expressed Cx43 (Fig.1A, b, d and f). Western blot analysis and qRT-PCR showed that the expression of Cx43 was distinctly increased not only

Discussion

The present study revealed that CSCs transplantation could enhance the expression of Cx43 after MI. They could probably function through the blockade of ANGII/AT1R/TGF-β1 signaling pathway during the process.

Malignant ventricular arrhythmia is the main cause of sudden cardiac death following MI (Ersbøll et al., 2013). Over the past half-century, remarkable progresses in pharmacological and interventional therapeutic approaches have improved the survival and prognosis of patients with MI (

Conclusions

CSCs transplantation enhanced the expression of Cx43. They might function via ANGII/AT1/TGF-β1 signaling pathway to regulate the expression of Cx43.

Competing interests

The authors declare that there are no competing interests.

Authors' contributions

Dr. Jingying Hou and Dr. Ping Yan carried out the experiments and drafted the manuscript; Yue Xing, Tianzhu Guo, Shaoxin Zheng and Changqing Zhou participated in the preparation of the animal model, tissue staining, and molecular assay; Hui Huang and Jingfeng Wang provided the technical assistance; Huibao Long, Tingting Zhong and Quanhua Wu participated and statistical analysis; Tong Wang conceived the study and participated in the study design. All authors read and approved the final

Acknowledgments

This study was supported by Grant [2013]163 from Key Laboratory of Malignant Tumor Molecular Mechanism and Translational Medicine of Guangzhou Bureau of Science and Information Technology; Grant KLB09001 from the Key Laboratory of Malignant Tumor Gene Regulation and Target Therapy of Guangdong Higher Education Institutes; National Natural Science Foundation of China (Nos. 81070125, 81270213), Science and Technology Foundation in Guangdong Province (No: 2010B031600032, 2014A020211002) and the

References (62)

  • D.T. Nguyen et al.

    Pirfenidone mitigates left ventricular fibrosis and dysfunction after myocardial infarction and reduces arrhythmias

    Heart Rhythm

    (2010)
  • Y. Oishi et al.

    AT2 receptor mediates the cardioprotective effects of AT1 receptor antagonist in post-myocardial infarction remodeling

    Life Sci.

    (2006)
  • N. Pavo et al.

    Cell therapy for human ischemic heart diseases: critical review and summary of the clinical experiences

    J. Mol. Cell. Cardiol.

    (2014)
  • Y. Shi et al.

    Mechanisms of TGF-beta signaling from cell membrane to the nucleus

    Cell

    (2003)
  • A.A. Sovari et al.

    Dudley SC. Inhibition of c-Src tyrosine kinase prevents angiotensin II-mediated connexin-43 remodeling and sudden cardiac death

    J. Am. Coll. Cardiol.

    (2011)
  • S.A. Thompson et al.

    Engraftment of human embryonic stem cell derived cardiomyocytes improves conduction in an arrhythmogenic in vitro model

    J. Mol. Cell. Cardiol.

    (2012)
  • W. Tung et al.

    A novel high throughput approach to screen for cardiac arrhythmic events following stem cell treatment

    Med. Hypotheses

    (2015)
  • S.K. Vanamala et al.

    Effect of human umbilical cord blood cells on Ang-II-induced hypertrophy in mice

    Biochem. Biophys. Res. Commun.

    (2009)
  • R. Vivar et al.

    TGF-β1 prevents simulated ischemia/reperfusion-induced cardiac fibroblast apoptosis by activation of both canonical and non-canonical signaling pathways

    Biochim. Biophys. Acta

    (2013)
  • Y. Zhao et al.

    Down-regulation of connexin43 gap junction by serum deprivation in human endothelial cells was improved by (−)-epigallocatechin gallate via ERK MAP kinase pathway

    Biochem. Biophys. Res. Commun.

    (2011)
  • I. Agarwal et al.

    Fibrosis-related biomarkers and incident cardiovascular disease in older adults: the cardiovascular health study

    Am. J. Epidemiol.

    (2014)
  • F.G. Akar et al.

    Mechanisms underlying conduction slowing and arrhythmogenesis in nonischemic dilated cardiomyopathy

    Circ. Res.

    (2004)
  • W. Altarche-Xifró et al.

    Cardiac c-kit + AT2 + cell population is increased in response to ischemic injury and supports cardiomyocyte performance

    Stem Cells

    (2009)
  • O. Bergmann et al.

    Evidence for cardiomyocyte renewal in humans

    Science

    (2009)
  • L. Bocchi et al.

    Growth factor-induced mobilization of cardiac progenitor cells reduces the risk of arrhythmias, in a rat model of chronic myocardial infarction

    PLoS ONE

    (2011)
  • R. Bolli et al.

    Intracoronary delivery of autologous cardiac stem cells improves cardiac function in a porcine model of chronic ischemic cardiomyopathy

    Circulation

    (2013)
  • H.S. Chen et al.

    Electrophysiological challenges of cell based myocardial repair

    Circulation

    (2009)
  • P. Dai et al.

    Cx43 mediates TGF-beta signaling through competitive SMADs binding to microtubules

    Mol. Biol. Cell

    (2007)
  • D. D'Amario et al.

    Insulin-like growth factor-1 receptor identifies a pool of human cardiac stem cells with superior therapeutic potential for myocardial regeneration

    Circ Res

    (2011)
  • Y.F. Ding et al.

    Gualou Xiebai decoction prevents myocardial fibrosis by blocking TGF-beta/Smad signalling

    J. Pharm. Pharmacol.

    (2013)
  • S.A. Fisher et al.

    Meta-analysis of cell therapy trials for patients with heart failure

    Circ. Res.

    (2015)
  • Cited by (11)

    • Long noncoding RNAs: Novel molecules in cardiovascular biology, disease and regeneration

      2016, Experimental and Molecular Pathology
      Citation Excerpt :

      Previous studies have revealed that implantation of these cells results in cardiomyocytes differentiation and neovascularization. ( Carvalho et al., 2015; Hou et al., 2015). However, optimizing their differentiation into desired cell types for cardiac repair may be a tough task due to their pluripotency and unique origins.

    • Experimental induction of reparative morphogenesis and adaptive reserves in the ischemic myocardium using multipotent mesenchymal bone marrow-derived stem cells

      2016, Pathophysiology
      Citation Excerpt :

      We used an aliquot of 1–5 × 106 cells, which was applied to the MCS transplantation, as this is the amount recommended as ideal for experimental and clinical research [14,29,30]. Clinical investigations have shown that intravenous injection of 1–5 × 106 MCS (isolated from bone marrow aspirate and after 5–6 passages in culture) effectively replaces damaged non-hematopoietic tissue function (myocardium, skeletal muscles, nervous tissue) without complications or side effects [31–33]. After administration of narcotics, the animal was fixed on its back.

    View all citing articles on Scopus

    This work was done by the investigators of the Sun Yat-sen Memorial Hospital of Sun Yat-sen University. The authors took responsibility for all aspects of the reliability and had no differences in data presentation and interpretation.

    1

    Dr. Jingying Hou and Dr. Ping Yan played equally important roles in the development of the experimental protocol, in the interpretation of the results, and in the texture of the present article.

    View full text