|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Connexin43 promotes survival of mesenchymal stem cells in ischaemic heart
Deguo Wang, Wenzhi Shen, Fengxiang Zhang, Minglong Chen, Hongwu Chen and Kejiang Cao1
Department of Cardiology, The First Affiliated Hospital of Nanjing Medical University, Guangzhou Road 300, Nanjing, 210029, Peoples Republic of China
The involvement of connexins in regulating cell growth and death has recently been reported. We have investigated whether Cx43 (connexin43) contributes to MSC (mesenchymal stem cell) survival and improves therapeutic efficacy in MI (myocardial infarction). Genetically modified Cx43 MSCs were exposed to hypoxic conditions or injected intramyocardially into a rat MI model. MSCs overexpressing Cx43, with more Bcl-2 and phosphorylated Akt, but less Bax, were relatively tolerant to hypoxic injury. After transplantation, this Cx43 overexpression enhanced cell survival and reduced infarct size, improving contractile performance. Cx43 inhibition by SiRNA reversed the effects of Cx43 overexpression. Therefore, Cx43 may act as a potential target for improving the therapeutic efficacy of MSCs in ischaemic heart disease.
Key words: apoptosis, Bax, Bcl-2, connexin43, mesenchymal stem cells, myocardial infarction
Abbreviations: Ct, threshold cycle, Cx43, connexin43, EF, ejection fraction, FS, fractional shortening, GAPDH, glyceraldehyde-3-phosphate dehydrogenase, GJ, gap junctions, LAD, left anterior descending, MI, myocardial infarction, MSC, mesenchymal stem cells, PI, propidium iodide
1To whom correspondence should be addressed (email kJcao@njmu.edu.cn).
Cell transplantation shows great promise for repairing heart tissue and restoring heart function after MI (myocardial infarction) (Nygren et al., 2004; Caspi et al., 2007). However, poor survival of donor cells (Muller-Ehmsen et al., 2006) and lack of functional coupling (Leobon et al., 2003) with the host tissue compromise the efficacy of cell therapy. Cell-based gene therapy offers another approach that might enhance therapeutic efficacy.
Cx43 (connexin43) is a candidate target gene because of its dual role in intercellular communication and proliferation. It forms intracellular communication channels, called GJ (gap junctions), ensuring electrical and mechanical coupling between cells (Contreras et al., 2004). Deficiency of Cx43 in heart tissue results in slow conduction and ventricular arrhythmias (Danik et al., 2008). In addition, cell to cell communication through GJ is correlated with the pathophysiology of cell death in injury (Yasui et al., 2000). Cx43 located in the mitochondria may also regulate apoptosis (Giardina et al., 2007; Goubaeva et al., 2007). Therefore, Cx43-based cell therapy might promote cell survival and intracellular coupling.
Stem cells from bone marrow are widely recognized for use in therapy for heart failure and myocardial infarction (Nagaya et al., 2005; Tse et al., 2007). Generally, the Cx43 level in MSCs (mesenchymal stem cells) is very low, although it may increase progressively in the cardiac microenvironment (Pijnappels et al., 2006). Several interventions, including gene modification (Grauss et al., 2008), cytokine stimulation (Hahn et al., 2008) and hypoxic preconditioning (Rosova et al., 2008) seem to augment the survival, function and homing of MSCs in the hostile ischaemic environment. These enhanced MSCs express high levels of Cx43 (Grauss et al., 2008; Hahn et al., 2008; Rosova et al., 2008). Moreover, Cx43-overexpressing MSCs regenerate larger and more uniform volumes of tissue (Rossello et al., 2009). However, to our knowledge, the direct role of Cx43 in survival of MSCs in ischaemic hearts remains to be evaluated.
We therefore hypothesized that Cx43 affects MSC survival and therapeutic potential in ischaemic hearts. Using an in vitro model with deprivation of serum and oxygen, and a murine MI model with permanent ligation of the LAD (left anterior descending) coronary artery, we investigated whether Cx43 protects MSCs from apoptosis and enhances their therapeutic efficacy.
2. Materials and methods
2.1. Animal care
Sprague–Dawley rats were obtained from the Experimental Animal Center of Nanjing Medical University. All experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University.
2.2. Isolation, culture and identification of bone marrow MSCs
2.2.1. Isolation and culturing of bone MSCs
The MSCs were isolated from the femurs of 4-week male SD (Sprague–Dawley) rats as described previously (Muller-Ehmsen et al., 2006). Detailed protocols are described in the Supplementary material (at http://www.cellbiolint.org/cbi/034/cbi0340415add.htm).
2.2.2. FACS analysis and in vitro functional differentiation assay
MSCs from bone marrow were characterized by analysis of surface markers and induction of multiple differentiation pathways. Detailed protocols are described in the Supplementary material.
2.3. Cx43 genetic modification of MSCs
The full-length cDNA of rat Cx43 (kindly provided by Professor Xinbo Li, University of Manitoba, Winnipeg, MB, Canada) was cloned into the eukaryotic expression vector pIRES2-eGFP (Clontech). The siRNA (small interfering RNA) sequence 5′-CGTGGAGATGCACCTGAAGTTCAAGAGACTTCAGGTGCATCTCCACGTTTTTT-3′ was cloned into the pRNA-U6-neo-vector (GenScript Co.). Plasmids were delivered using Fugene HD transfection reagent (Roche) according to the manufacturer’s protocol (www.roche-applied-science.com). In brief, 105 cells per well were plated on six-well dishes 24 h before transfection. The transfection mixture, containing 2 μg plasmids and 8 μl Fugene HD in 100 μl Opti-MEM (Invitrogen), was added to the adherent MSCs. Two days later, the transfection efficiency was determined by FACS. Cx43 expression was quantified by Western blotting. The genetically modified MSCs were named MSCs-vector, MSCs-Cx43 and MSCs-SiCx43.
2.4. Cell death of MSCs induced by serum deprivation and hypoxia in vitro
Cells were plated in serum-free DMEM (Dulbecco's modified Eagle's medium) and incubated under anoxic conditions with an AnearoPack system (http://www.mgc-a.co; Mitsubishi Gas Chemical Co, Inc.) to scavenge free oxygen for 8 h. Oxygen was <0.1% within 1 h of transfer to the hypoxic chamber and was maintained throughout the experiment. After 8 h exposure to anoxia, apoptosis of MSCs was detected using a Vybrant Apoptosis Assay Kit (http://probes.invitrogen.com, Invitrogen) with Hoechst33342/ PI (propidium iodide) and analysed by flow cytometry.
2.5. Myocardial infarction model and MSC transplantation
A total of 100 female SD rats, ranging in age from 8 to 12 weeks and weighing 250–300 g, underwent LAD coronary artery ligation to create MI as previously described (Dai et al., 2005). Rats were anaesthetized by intraperitoneal injection of pentobarbital (50 mg/kg body weight; Roche), endotracheally intubated and mechanically ventilated. After thoracotomy, the LAD was ligated with a 6-0 silk suture 3–4 mm from the tip of the left atrium. Successful ligation of the LAD was verified by visual inspection of the left ventricular apex, which showed a pale discoloration. Electrocardiography indicated an elevated S-T. In the control group, rats underwent left thoracotomy without coronary artery ligation (n = 8). Seven days after ligation, 63 surviving animals were injected with a 30-μl volume containing 5×105 MSCs-vector (n = 18), MSCs-SiCx43 (n = 18), MSCs-Cx43 (n = 18) or PBS (n = 9) in three different peri-infarct regions of the heart, using a 31-gauge needle. Four days and two weeks after transplantation, surviving animals were killed for assay.
2.6. Western blotting
The protein concentration of the samples was determined by a BCA (bicinchoninic acid) protein assay. Total proteins (20 μg) were resolved using 14% SDS/PAGE before being transferred to a PVDF membrane. The membrane was blocked in PBS containing 0.2% Tween 20 and 5% skim milk for 2 h at 37°C and incubated overnight at 4°C with primary monoclonal antibodies (Cx43, Abcam, Akt and phosphorylated Akt, Bax and Bcl-2; Cell Signaling). The housekeeping protein GAPDH (glyceraldehyde-3-phosphate dehydrogenase) acted as a loading control. Antibody binding was detected with a HRP (horseradish peroxidase)-conjugated secondary antibody (1:2000; Sigma) and visualized using an ECL kit.
2.7. Functional assessment of the infarcted rat heart by echocardiography
Echocardiography was performed by two examiners “blinded” to the experimental groups 2 weeks after treatment to assess cardiac function using a two-dimensional system with a 15-MHz probe (Olivares et al., 2004). As a measure of LV (left ventricular) function, the EF (ejection fraction) and FS (fractional shortening) were calculated from M-mode recordings by the following equation:
LVEF = (LVEDV−LVESV)/LVEDV×100%;
LVSF = (LVDd−LVDs)/LVDd×100%.
2.8. Detection of Sry DNA of grafted donor cells by real-time PCR
The number of surviving male MSCs in female hearts treated with MSCs-vector, MSCs-SiCx43 or MSCs-Cx43 was quantified by real-time PCR of Sry. The survival ratio = number of surviving MSCs/total implanted cells. PCR of the rat Y-chromosome-specific Sry was performed on total left ventricular tissue 2 weeks after cell transplantation. The primers for rat Sry were: forward primer: 5′-CATCTCTGACTTCCTGGTTGC-3′ and reverse primer: 5′-ATGCTGGGATTCTGTTGAGCC-3′. The primers for rat GAPDH were: forward primer: 5′-AACCTGCCAAGTATGATGACATCA-3′ and reverse primer: 5′-TGTTGAAGTCACAGGAGACAACCT-3′.
PCR was carried out as previously described (Amsalem et al., 2007). Quantitative real-time PCR was performed with a Prism 7300 sequence detection system (Applied Biosystems, Agentek) using SYBR Green according to the manufacturer’s instructions. Female left ventricles 2 weeks after left coronary artery ligation were mixed with male BM cells (5×105), and a serial dilution series was used to generate a standard curve. All samples of DNA were tested by the Sry and GAPDH primers, and all running samples were composed of 4 μl of DNA, 1 μl of each primer (forward and reverse) and 10 μl of SYBR-green (Invitrogen). Each cycle (40 cycles in total) included denaturation for 3 s at 95°C, with annealing for 31 s at 60°C. The primary curve method was used to calculate the Ct (threshold cycle), which is defined as the cycle at which the fluorescence level reaches a predetermined threshold. Ct was measured for each reaction and used to calculate the cell number of each experimental sample according to the standard curve.
MSCs plated on coverslips were fixed with 4% paraformaldehyde in PBS for 20 min and permeabilized with 0.1% Triton X-100 (Sigma) for 10 min. Sections were deparaffinized, hydrated and antigen retrieved. Non-specific antibody binding sites were blocked by incubating with 5% FBS (fetal bovine serum) for 60 min. Cells or tissue sections were labelled overnight at 4°C with antibodies [Cx43, 1:200, Abcam; GFP (green fluorescent protein), 1:100; Santa Cruz]. Anti-rabbit and anti-mouse IgG conjugated with TRITC (tetramethylrhodamine isothiocyanate) were used as detection systems. Nuclei were stained with 10 μg/ml DAPI (4′,6-diamidino-2-phenylindole).
2.10. Infarct size measurement
The hearts were removed from the rats, washed, weighed and embedded in paraffin. Sections were cut into 5-μm slices and processed. Myocardial infarction size was determined from the heart sections cut at the mid-papillary muscle level after staining with Masson’s Trichrome. Infarct area is expressed as a percentage of the left ventricular area (Hahn et al., 2008).
2.11. Statistical analysis
The data are expressed as means±S.E.M. Two-way ANOVA (analysis of variance) and Student’s t test were performed to analyse statistical differences in each response variable. A value of P<0.05 was considered statistically significant.
3.1. Characteristics of MSCs
MSCs were confirmed by the presence of surface markers and induction of multiple differentiation pathways. The experimental results are described in detail in the Supplementary Figures S1 and S2 (at http://www.cellbiolint.org/cbi/034/cbi0340415add.htm).
3.2. Genetic modification of Cx43
A representative photomicrograph is shown in Figure 1(A). Over 40% of MSCs were transfected as scored by FACS analysis (Figure 1B). Cx43 expression increased significantly in MSCs-Cx43 (2.76±0.24, n = 3), but decreased very significantly in MSCs-SiCx43 (0.26±0.11, n = 3) compared with MSCs-vector (1.03±0.28, n = 3) and wild-type MSCs (Figure 1D) when analysed 2 days after transfection. Similar changes were visualized by immunostaining (Figure 1C). To evaluate the expression and duration of Cx43 protein in the genetically modified MSCs, MSC samples were analysed by immunobloting at various time-points (2, 5, 7 and 14 days) post-transfection. Changes in Cx43 expression lasted for 7 days and were the same as in MSCs-vector on day 14 (Figures 1E and 1F). In contrast, Cx43 expression was low in wild-type MSCs and MSCs-vector.
3.3. Cx43 expression protects against death of MSCs in vitro
Multiple changes, such as deprivation of nutrients, growth and survival factors, and oxygen, contribute to the death of grafted MSCs. To ascertain whether Cx43 participates in anoxia-induced MSC injury, genetically modified MSCs were subjected to hypoxia and stained with Hoechst33342/PI. After 8 h of deprivation of serum and oxygen (Figures 2B and 2C), the percentage of apoptotic cells was significantly lower in MSCs-Cx43 (9.82±2.98%), but higher in MSCs-SiCx43 (21.64±2.25%) than in MSCs-vector (17.38±1.97%). Cell death was assessed microscopically. Figure 2(A) shows that apoptotic nuclei had condensed chromatin, which also indicates that enhanced Cx43 expression in MSCs reduces cell death.
3.4. Cx43-associated cell signalling in MSCs under hypoxia and serum deprivation
Akt signalling plays a significant role in MSC survival after transplantation into ischaemic hearts (Mangi et al., 2003). There were no significant differences in the total Akt among MSCs-vector, MSCs-Cx43 and MSCs-SiCx43. However, compared with MSCs-vector, Phospho-Akt increased significantly in MSCs-Cx43 (1.79±0.12-fold), but was reduced in MSCs-SiCx43 (0.73±0.11-fold; all P-values<0.05, n = 6; see Figures 3A and 3B).
We also investigated the effects of Cx43 in MSCs on mitochondrion-related apoptotic factors, the anti-apoptotic protein Bcl-2 and the pro-apoptotic gene Bax. Western blotting showed that Bcl-2 was up-regulated (5.66±0.15-fold) and Bax down-regulated (0.87±0.1-fold) in MSCs-Cx43 compared with MSCs-vector. In contrast, Bcl-2 decreased (0.9±0.09-fold) and Bax increased (1.2±0.11 fold) in MSCs-SiCx43. (Again, all gave P-values of <0.05, n = 6; Figures 3C and 3D.)
3.5. Cx43 in MSCs affects graft survival in ischaemic hearts
After chest opening and cell or PBS injection, the numbers of surviving animals in the MSCs-vector, MSCs-SiCx43, MSCs-Cx43 and PBS groups were 14, 13, 14 and 8, respectively. There was no significant difference in mortality between the groups (P = 0.81), suggesting that intramyocardial cell injection did not increase morbidity.
Four days after cell transplantation, MSC engraftment was observed in the MSCs-vector (n = 3), MSCs-SiCx43 (n = 2) and MSCs-Cx43 (n = 3) groups. GFP+ cells could be seen at the border zone of the infarcted myocardium (Figures 4A–4C). Engraftment of male donor-derived cells in the ischaemic hearts of female transplant recipients was quantified by real-time PCR. Engrafted MSC survival was significantly higher in the MSCs-Cx43 than in the MSCs-vector (9.3±0.8% compared with 2.7±0.5%, P<0.001, n = 6 hearts/group; Figure 4D). In contrast, cell survival was significantly reduced in the MSCs-SiCx43 (2.1±0.4% compared with 2.7±0.5%, P = 0.036, n = 6 hearts/group).
3.6. Infarct size and cardiac fibrosis
LAD ligation for 3 weeks consistently resulted in transmural myocardial infarction, exhibiting typical histological changes, including thinning of the free wall of the left ventricle and extensive collagen deposition. The infarct area stained blue, and a minor viable island of myocardium stained red (Figure 5). Quantitative analysis revealed ∼50% infarcted myocardium in the PBS group (48±4%, n = 5). Animals treated with MSCs-Cx43 (26±3%, n = 5) had smaller infarcts than those treated with MSCs-SiCx43 (35±3%, n = 5) and MSCs-vector (34±2%, n = 5) (all differences were significant, P<0.05). However, the difference of infarct size between the MSCs-vector and MSCs-SiCx43 groups did not reach statistical significance (P = 0.123).
3.7. Cardiac function after MSC transplantation
Echocardiography showed a significant improvement in the ejection and shortening fractions in all the MSC groups compared with the PBS group (52.1±4.9% and 22.9±3.2%; Figures 6A–6C). Furthermore, the improvement of EF and SF was greater in the MSCs-Cx43 group (71.4±4.5% and 38.9±3.6%) than in the MSCs-vector (66.0±4.7% and 33.9±3.6%) and MSCs-SiCx43 (63.8±4.7% and 30.4±3.4%) groups.
Both laboratory experiments and clinical trials suggest that the transplantation of MSCs from adult bone marrow can restore infarcted heart function (Nagaya et al., 2005; Tse et al., 2007), but the poor survival of implanted cells hampers therapeutic efficacy. A high level of engrafted cell death occurs within 1 week after transplantation into ischaemic hearts (van der Bogt et al., 2009). The molecular mechanism of stem cell death can be attributed to the hostile environment of the ischaemic heart, which includes harmful factors from the inflammatory response, oxygen and nutritional deficiency, and pro-apoptotic or cytotoxic factors (van der Bogt et al., 2009). Therefore protection of MSCs from death during the first week is critical for improving the success of the engraftment. We have demonstrated two key findings: (i) Cx43 plays an important role in the survival of donor MSCs in ischaemic hearts, and (ii) Cx43-overexpressing MSCs reduce infarct size and improve contractile performance.
Any strategies that promote stem cell engraftment, survival and efficacy in post-infarct hearts have to overcome hypoxia and ischaemia. Previous studies showed that Cx43 expression is up-regulated in pretreated MSCs (Grauss et al., 2008; Hahn et al., 2008; Rosova et al., 2008) and seems to be associated with cytoprotective effects. This is because Cx43 knock-down results in loss of its beneficial effects (Lu et al., 2009). Our in vitro data show that apoptosis of MSCs is reduced in MSCs-Cx43 but increased in MSCs-SiCx43 under anoxic conditions, suggesting that Cx43 plays an important role in MSC apoptosis.
The Akt signalling pathway regulates MSC survival in both hypoxic conditions and ischaemic hearts (Mangi et al., 2003). Activation of the Akt pathway provides cells with a survival signal that allows them to withstand apoptotic stimuli (Mangi et al., 2003), and furthermore, Akt genetically engineered MSCs give better protection to the infarcted heart (Mangi et al., 2003). Also, the phosphorylation of Akt is related to gap junction communication (Hahn et al., 2008). Although the mechanism by which Cx43 confers cytoprotection requires further in-depth studies, our data show that Cx43 levels in MSCs are associated with the activation of Akt.
The mitochondria-mediated intrinsic cascade is a major apoptotic pathway (Elmore, 2007). A large amount of Cx43 located in the mitochondrion has been associated with cardiomyocyte apoptosis (Rodriguez-Sinovas et al., 2006; Goubaeva et al., 2007). Lu et al. (2009) found that Cx43 in stem cells was mainly distributed in the mitochondria rather than membranes in general. Cx43 inhibition reduced cell survival under oxygen and glucose deprivation and after transplantation into an infarcted heart. The balance between the pro-apoptotic Bax and the anti-apoptotic Bcl-2 regulates apoptosis under ischaemic conditions (Elmore, 2007). We found that the Bcl-2/Bax ratio was increased in MSCs-Cx43, but lower in MSCs-SiCx43, when MSCs were exposed to hypoxic and serum deprivation, implying that MSC Cx43 might be related to mitochondria-mediated cell protection.
As mentioned above, Cx43 up-regulation in MSCs must persist for several days in order to prevent ischaemic injury. Plasmids can deliver a target gene to cells for transient expression of deficient proteins. For example, MSCs modified with Bcl-2 by pcDNA3.1 plasmids protect against apoptosis under hypoxic conditions and reduce cardiac ischaemia (Li et al., 2007). A hypoxia-regulated HO-1 vector modification enhances the tolerance of engrafted MSCs to hypoxia-reoxygenation injury in vitro and improves their viability in ischaemic hearts (Tang et al., 2005). These engineered MSCs give better functional recovery of infarcted hearts for over 2 weeks after MI, even though the genetic up-regulation is probably transient (Tang et al., 2005; Li et al., 2007). We found that Cx43 protein expression in MSCs could be regulated by plasmid-mediated genetic modification and that their activity lasts for at least 7 days. Four days after implantation, GFP+ cells were clearly observed in the border zones of infarcted areas of the heart. Two weeks after transplantation, more sry gene was expressed in MSCs-Cx43-treated ischaemic hearts. However, knocking down Cx43 in MSCs resulted in poor cell survival. Our results support the idea that Cx43 participates in the survival of donor MSCs under ischaemic conditions.
Theoretically, the therapeutic efficacy of MSCs was closely linked to the in situ survival of cells implanted into the hostile environment of ischaemic hearts (Tang et al., 2005; Li et al., 2007). Our in vivo study shows that MSCs-Cx43 in the ischaemic heart increased cardiac function, resulting in smaller infarct size, which strongly supports the notion that enhanced Cx43 allows MSCs to exert better myocardial protection. However, further studies are required to evaluate the effects of Cx43 on paracrine signals in MSCs; a paracrine effect of MSCs has been reported as the major mechanism for the functional recovery of the infarcted heart (Gnecchi et al., 2005).
In conclusion, although additional studies are clearly required, we believe our results directly demonstrate that Cx43 is involved in apoptosis and survival of MSCs in the ischaemic heart. MSCs with high Cx43 expression improve heart function and reduce infarct size. Therefore, Cx43 might be a target gene for enhancing the protective efficacy of MSCs in ischaemic heart disease.
Deguo Wang was responsible for model creating, cell transplantation, Western blotting, immunohistochemistry and PCR. Wenzhi Shen Cell was responsible for the culture identification. Fengxiang Zhang was responsible for the cloning. Minglong Chen was responsible for echocardiography and infarct size measurement. Hongwu Chen was responsible for echocardiography and infarct size measurement. Kejiang Cao was responsible for the design of the study.
This work was supported by a grant from
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Received 12 August 2009/19 December 2009; accepted 12 January 2010
Published as Cell Biology International Immediate Publication 12 January 2010, doi:10.1042/CBI20090118
© The Author(s) Journal compilation © 2010 Portland Press Limited
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)
Figure 3 Cx43 regulates the expression of phospho-Akt, Bcl-2 and Bax in MSCs under hypoxic conditions
Figure 4 Cx43 genetic modification affects MSC survival after transplantation in myocardial infarction