We hypothesized that overexpression of cGMP-dependent protein kinase type 1α (PKG1α) could mimic the effect of tadalafil on the survival of bone marrow derived mesenchymal stem cells (MSCs) contributing to regeneration of the ischemic heart.
Methods and Results
MSCs from male rats were transduced with adenoviral vector encoding for PKG1α (PKG1αMSCs).Controls included native MSCs (NatMSCs) and MSCs transduced with an empty vector (NullMSCs). PKG1α activity was increased approximately 20, 5 and 16 fold respectively in PKG1αMSCs. PKG1αMSCs showed improved survival under oxygen and glucose deprivation (OGD) which was evidenced by lower LDH release, caspase-3/7 activity and number of positive TUNEL cells. Anti-apoptotic proteins pAkt, pGSK3β, and Bcl-2 were significantly increased in PKG1αMSCs compared to NatMSCs and NullMSCs. Higher release of multiple prosurvival and angiogenic factors such as HGF, bFGF, SDF-1 and Ang-1 was observed in PKG1αMSCs before and after OGD. In a female rat model of acute myocardial infarction, PKG1αMSCs group showed higher survival compared with NullMSCs group at 3 and 7 days after transplantation as determined by TUNEL staining and sry-gene quantitation by real-time PCR. Increased anti-apoptotic proteins and paracrine factors in vitro were also identified. Immunostaining for cardiac troponin I combined with GFP showed increased myogenic differentiation of PKG1αMSCs. At 4 weeks after transplantation, compared to DMEM group and NullMSCs group, PKG1αMSCs group showed increased blood vessel density in infarct and peri-infarct areas (62.5±7.7; 68.8±7.3 per microscopic view, p<0.05) and attenuated infarct size (27.2±2.5%, p<0.01). Heart function indices including ejection fraction (52.1±2.2%, p<0.01) and fractional shortening (24.8%±1.3%, p<0.01) were improved significantly in PKG1αMSCs group.
Overexpression of PKG1α transgene could be a powerful approach to improve MSCs survival and their angiomyogenic potential in the infarcted heart.
Citation: Wang L, Pasha Z, Wang S, Li N, Feng Y, Lu G, et al. (2013) Protein Kinase G1 α Overexpression Increases Stem Cell Survival and Cardiac Function after Myocardial Infarction. PLoS ONE 8(3): e60087. doi:10.1371/journal.pone.0060087
Editor: Piero Anversa, Brigham and Women's Hospital, United States of America
Received: January 24, 2013; Accepted: February 23, 2013; Published: March 25, 2013
Copyright: © 2013 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by National Institutes of Health grants HL095375, HL087246. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Transplantation of bone marrow-derived mesenchymal stem cells (MSCs) has been extensively used to treat cardiovascular ischemic disease due to MSCs' regenerative potential in both experimental animal model and human , , , , , , , . Despite of promising results, myriad problems including massive cell death after transplantation have limited the efficacy of this cell therapy , , . One strategy to improve MSCs survival is to precondition MSCs before transplantation , , . There are different ways to precondition MSCs including exposing cells to physical treatments (e.g. hypoxia, heat shock), pharmacological agents, “priming” with growth factors, and genetic modification by over-expression of anti-apoptotic proteins, growth factors or pro-survival genes , , , .
cGMP-dependent protein kinase (PKG), a critical mediator of cGMP signaling in cardiovascular system, phosphorylates many intracellular proteins to regulate important physiological functions such as cell differentiation and proliferation, and cell survival , , , . PKG has two isozymes, PKG1 and PKG2. Only PKG1 is detected in cardiac myocytes and vascular cells. The N-terminus of PKG1 is encoded by two different exons resulting in the production of two isoforms, PKG1α and PKG1β. Both isoforms are present in heart and vessels , , . However, PKG1β is activated at ∼10-fold higher cGMP concentrations than PKG1α. Previous studies have revealed a direct role for PKG1 in cardioprotection. Overexpression of PKG1α by adenoviral vectors inhibits necrosis and apoptosis in rat cardiomyocytes undergoing simulated ischemia-reoxygenation. PKG1-deficient mice showed increased myocardial structural and functional damage after trans-aortic constriction (TAC) than their wild-type controls . PKG is also a critical mediator in phosphodiesterase (PDE) inhibitor induced cardioprotection. Treatment with PDE-5 inhibitors, sildenafil or tadalafil, effected cardioprotection both in vitro and in vivo through PKG1 , , . In a previous study we showed that PDE5a inhibition with adenoviral short hairpin RNA could protect cardiomyocytes against anoxia, attenuate infarction size and improve cardiac remodeling and dysfunction .
Interestingly, the PDE-5 inhibitors can protect MSCs and adipose-derived stem cells (ASCs) in ischemic rat hearts both in vitro and in vivo via promoting PKG activity , . As PKG1α is more active than PKG1β at cGMP each found in the cardiovascular system , , , , we hypothesized that overexpression of PKG1α via adenovirus could prolong the survival of MSCs both in vitro and in vivo in rats with regional myocardial ischemia and improve the cardiac function.
Materials and Methods
All animal experimental procedures conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication #85-23, revised 1996) and were conducted according to a protocol approved by the Institutional Animal Care and Use Committee, University of Cincinnati.
In vitro studies
Construction of Adenoviral vector of PKG1α.
pShuttle-IRES-hrGFP-1 vector containing PKG1α cDNA amplified from mouse cardiomyocytes (Ad-PKG1α) was constructed based on AdEasy™ XL Adenoviral Vector System from Strategene (Agilent Technologies, USA). To prevent restriction enzymes from inappropriately digesting DNA, we introduced site-specific mutation into PKG gene T→C change at position 1773 by using QuickChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies). This is a non-sense mutation coding for isoleucine. A vector without the therapeutic gene (Ad-null) was routinely made to use as a control. Ad-PKG1α and Ad-null viral vectors were propagated in AD-293 cells using Dulbecco's modified Eagle's medium (DMEM; GIBCO Invitrogen) supplemented with 10% fetal bovine serum. The cell suspension was collected, purified, and used in further experiments.
Isolation of MSCs and Adenoviral Transduction.
Bone marrow-derived MSCs were isolated from 8–10 week male Fisher rats, as described previously by us . The cells were cultured for no more than 5 passages before using for both in vitro and in vivo studies. MSCs were transduced with Ad-null or Ad-PKG1α or treated with DMEM for 7 h followed by maintenance in the viral vector-free DMEM for 72 h. Transfection efficiency was about 45%–50%. The successful transduction was judged by the presence of green cells. Randomly selected microscopic fields (n>8; 400×) at 72 h after transduction were evaluated to calculate the ratio of green cells to the total number of cells in several independent experiments. These cells were further confirmed by immunostaining of GFP in either Ad-null transduced MSCs (NullMSCs) or Ad-PKG1α transduced MSCs (PKG1αMSCs). PKG1α transgene overexpression in PKG1αMSCs was confirmed by reverse transcription polymerase chain reaction (RT-PCR), Western blot and PKG activity assay.
Reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR.
We used RT-PCR to detect PKG1α gene overexpression and real-time PCR to detect pro-survial and angiogenic cytokines eg. HGF, bFGF, SDF-1, Ang-1 and expression of cardiac transcription factors. Isolation of total RNA from the different groups of MSCs and subsequent first-strand cDNA synthesis was performed using an RNeasy mini kit (Qiagen) and an Omniscript Reverse Transcription kit (Qiagen), respectively following the instructions of manufacturer. The thermocycle profile for PCR was set for initial denaturation at 94°C for 3 min, 30 cycles of denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec and extension at 72°C for 30 sec, and a final extension at 72°C for 10 min. Real-time PCR was performed using iQ SYBR-Green supermix (Bio-Rad) in a Bio-Rad iQ5 optical module. The cycling conditions were set for 3 min at 95°C 15 min for initial denaturation; 40 cycles of denaturation at 94°C for 15 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec; and a final extension at 55°C for 15 sec. The data was acquired during the extension step. Melting curves were obtained at the end of the reaction by gradually raising the temperature by 1°C/min from 55°C to 90°C over a time period of 35 minutes. The primer sequences used are shown in Table-1.
PKG activity assay.
PKG activity in MSCs was measured by using the Cyclex Cyclic GMP-dependent protein kinase Assay Kit (catalog no. CY-1161; MBL) following the manufacturer instructions.
Oxygen glucose deprivation (OGD).
MSCs were seeded at a cell density of 2×104 cells/cm2. The cells were divided into three groups, including nontreated native MSCs (NatMSCs), transduced with Ad-null (NullMSCs), and transduced with Ad-PKG1α (PKG1αMSCs). At 72 h after transduction, MSCs in all groups were rinsed twice with serum-free, glucose, and sodium pyruvate-free DMEM and were cultured in the same medium at 37°C in a anoxia chamber (InVivo 500; Ruskinn Life Science) saturated with 95% N2/5% CO2 for up to 8 h.
LDH release assay.
The release of lactate dehydrogenase (LDH) from cells with a damaged membrane after OGD was measured by using Homogeneous Membrane Integrity Assay (Promega) to determine cell viability. The fluorescence readings were obtained with excitation and emission filters at ∼550 and ∼590 nM, respectively. Percentage of cell death was calculated as 100× (ReadingExperimental-ReadingBackground)/(ReadingTotal Release-ReadingBackground).
Caspase-3/7 activity assay.
Caspase-3/7 activity was measured by Apo-ONE Homogeneous Caspase-3/7 Assay Kit (Promega) according to manufacturer's instruction. MSCs w ere seeded in a 96-well white-walled plate at a concentration of 104 cells/well in 100 µl medium. After 8 h OGD, a 100 µl of detection buffer was added to a well and the plate was read with a spectrofluorometer at excitation wavelength 485 nm and emission wavelength 535 nm (Plate Chameleon, HIDEX).
In vitro apoptosis assay.
Terminal dUTP nick-end labeling (TUNEL) assay was performed in three groups of MSCs described above to evaluate OGD induced apoptosis. After exposure of OGD for 8 h, the cells were fixed in 4% paraformaldehyde. TUNEL was performed by using an in situ cell death detection kit (TMR Red; Roche Applied Science) according to manufacturer's instructions. The nuclei were visualized by 4′6-diamidino-2-phenylindole (DAPI) in mounting medium. Randomly selected microscopic fields (n = 8) were evaluated to calculate the ratio of TUNEL+ cells to the total number of cells in three independent experiments.
Western blot analysis.
Protein lysate samples from cultured cells were obtained with ice cold lysis buffer (50 mM Tris-HCl, PH 7.6, 120 Mm NaCl, 0.5% NP-40, 1 mM PMSF, 2 µg/ml Leupeptin, and 2 µg/ml Aprotinin). Protein concentration was measured by using the Bio-Rad DC-Protein Assay Reagent (Bio-Rad). 20 µg of protein samples were loaded onto 4%-12% gradient precast gel (Invitrogen) and electrophoresed for 2 h at 100 Volts. Transblotting was performed onto a Polyvinylidene Fluoride (PVDF) membrane as described before . The membrane was blocked with 5% nonfat milk in Tris-buffered saline with Tween (TBST) for 1 h and subsequently incubated with specific primary antibodies (diluted in 5% nonfat milk or bovine serum albumin (BSA) in TBST) for overnight at 4°C. After washing with TBST 3 times, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. After washing with TBST, membranes were incubated with ECL or Femto supersensitive detection reagent, and their signals were exposed to Kodak Light film (Fisher Scientific). The primary antibodies used in Western blot included anti PKG1α (Santa Cruz, USA, 1∶200), anti-phosphorylated Akt (pAkt) (Cell Signaling Technology, USA 1∶2000), anti Akt (Cell Signaling Technology, USA 1∶2000), anti-phosphorylated GSK3β (pGSK3β) (Cell Signaling Technology, USA 1∶2000), anti GSK3β (Cell Signaling Technology, USA 1∶2000), anti Bcl2 (Cell Signaling Technology, USA 1∶2000).
Cell proliferation assay.
We used Ki67 immunostaining to evaluate cell proliferation in MSCs. After 8 h OGD, the cells were fixed in 4% paraformaldehyde. Immunostaining was routinely performed. The cells were then mounted on slides which were exposed to the medium containing (DAPI). Randomly selected microscopic fields (n = 8) were evaluated to calculate the ratio of Ki67 positive cells to the total number of cells in three independent experiments.
In vivo studies
Experimental rat model of acute myocardial infarction and cell transplantation.
MSCs from GFP transgenic male rats were used to study the fate of transplanted cells. An experimental rat model of acute myocardial infarction in young female Fischer rats (180–200 g) was developed by permanent ligation of the coronary artery, as described before .The number of stem cells injected varied in different studies. Massive donor cell death after transplantation is one of the key problems which need to be resolved in stem cell therapy. The number of cells to be given to the animal depends on the route of injection in order to maximize the protective effect. As previously reported from other research groups, 5-6×106 ,  were successfully injected in the rat hearts to determine their effects. We have previously injected 3×106 MSCs at multiple sites in and around the infarct area to maximize the efficacy of cell therapy. In the current study, we determined that 2×106 was the most suitable number for transplantation without any side effects.
Rats (n = 16/group) received intramyocardial injections of either 70 µl of DMEM without cells (DMEM group), or containing 2×106 NullMSCs (NullMSCs group), or containing 2×106 PKG1αMSCs (PKG1αMSCs group). The injections were given at multiple sites (4–5 sites/animal) in and around the infarcted area. The animals were euthanized at stipulated time points (n = 4 per group on day 3 and n = 8 per group at 4 weeks) for histological and molecular studies.
For molecular analysis, the animals were euthanized at day-3 after stem cell transplantation. Left ventricle was cut and processed for PKG1α expression and activity assay, paracrine and cardiac transcription factors expression and caspase-3/7 activity assay. pAkt, Akt, pGSK3β, GSK3β and Bcl-2 were measured by Western blot. To determine the survival of transplanted male MSCs, sry-gene analysis was performed at day-7 after stem cell transplantation by using isolated gDNA from left ventricle tissues.
TUNEL staining was performed on heart tissue sections for transplanted MSCs (combined with GFP) and residual cardiomyocytes (combined with desmin) at day 3 after stem cell transplantation. Double immunostaining of cardiac troponin I (TnI; 1∶100, Santa Cruz, USA) and GFP (1∶800, Abcam, USA) was performed at 7 days after stem cell transplantation.
At 4 weeks after transplantation, infarction size was measured in paraffin embedded heart tissue sections with Mason's trichome staining by using computer-based planimetry with Image-J analysis software (version 1.6065; NIH).
Blood vessels were identified by immunostaining of vonWillebrand Factor-VIII (vWF-VIII, 1∶ 200; Dako, Denmark) and detected with fluorescently labeled secondary antibody (Molecular Probes). The number of blood vessels were counted in both infarct and peri-infarct regions. At least 48 microscopic fields each in infarct and peri-infarct regions (n = 8) were randomly selected and counted in each group. Blood vessel density was expressed as the number of vessels per microscopic view at 200× magnification.
Heart function measurement.
At 4 weeks after stem cell transplantation, transthoracic echocardiography was used to measure the heart function as described before , . Indices of left ventricle (LV) systolic function including LV fractional shortening (LVFS) and LV ejection fraction (LVEF) were calculated respectively. The results were expressed as percentage. LV chamber dimensions (end-diastolic dimension [EDD] and end-systolic dimension [ESD]), and anterior wall thickness were measured.
All data were expressed as mean ± SE. Student's t-test or one-way ANOVA was performed to analyze statistical differences in each response variable. A value of p<0.05 was defined as statistically significant.
In vitro studies
Transgenic overexpression of PKG1α in MSCs.
Adenovirus transduction was evidenced by GFP (green fluorescence) expression in NullMSCs and PKG1αMSCs (Fig. 1A). Compared to NatMSCs and NullMSCs, PKG1αMSCs had significantly higher PKG1α gene, protein expression and PKG activity (Fig.1B–D, p<0.01).
(A) Fluorescence microscopic images (green) of MSCs successfully transducted with Ad-null and Ad-PKG1á 48 h after transduction; (B) RT-PCR showed enhanced mRNA level of PKG1á in PKG1áMSCs; (C) western blot showed higher PKG1á and PKG1á-flag fusion proteins in PKG1áMSCs. (D) PKG activity increased 16 fold in PKG1áMSCs compared to NatMSCs.
PKG1α promoted MSCs survival and proliferation under OGD.
Compared to controls (NatMSCs and NullMSCs), PKG1αMSCs had a reduced cell damage after 8 h OGD shown by low level of LDH release (PKG1αMSCs 13.7±1.3% vs NatMSCs 34.6±1.6% and NullMSCs 29.6±1.4%, p<0.01) (Fig. 2A), decreased caspase-3/7 activity (p<0.01)(Fig. 2B) and reduced number of TUNEL positive cells (11.7±1.9% vs 22.5±2.5% and 26.6±2.9%, p<0.05) (Fig. 2C). These results clearly showed the cytoprotective effects of PKG1α transgene on MSCs under glucose and serum-free anoxic conditions. In terms of cell proliferation, there were more Ki67 positive cells seen in PKG1αMSCs (41.2%±3% and 24.5±2.6%) than in controls (NatMSCs 17.8%±2% and 8.6±1.1%; NullMSCs 18.6%±3% and 10.2±1.5%) both before and after OGD (p<0.05) (Fig. 2D).
(A) LDH release assay showed less cell death on PKG1α MSCs at 8 h after exposure to oxygen glucose deprivation (OGD); (B) Parallel experiments were performed to show that caspase-3/7 activity was significantly decreased in PKG1αMSCs with concomitant increase in cell survival as determined by LDH release assay; (C–D) TUNEL and Ki67 staining on MSCs showed decreased number of TUNEL(+) cells and increased Ki67(+) cells in PKG1α MSCs compare with NatMSCs and NullMSCs after 8 h OGD. The nuclei were visualized by staining with DAPI (blue). Magnification ×400.
PKG1α overexpression reduced apoptosis.
In order to delineate the pro-survival pathway in PKG1αMSCs, we isolated and analyzed the cell lysate from different cell treatment groups for Western blot. PKG1α overexpression significantly increased phosphorylation of Akt (pAkt) and GSK3β (pGSK3β) without changing the total level of Akt and GSK3β in PKG1αMSCs after 8 h OGD compared to controls (NatMSCs and NullMSCs) (Fig. 3A). GSK3β is a downstream target in Akt signaling. Anti-apoptotic protein Bcl-2, was also significantly increased in PKG1αMSCs (Fig. 3A).
(A) Increased phosphorylation of Akt,GSK3â and high level of Bcl-2 expression were detected in PKG1á MSCs by western blot after 48 h OGD, protein expression of pAkt, pGSK3â and Bcl-2 are 2.4, 1.7, 2.8 fold higher respectively as compared to NatMSCs without changing the total level of Akt and GSK3â (p<0.01). (B and C) Real-time PCR based gene array analysis showed several pro-survival and angiogenic factors including HGF, TGFâ, FGF2, SDF-1 and Ang-1 as well as 2 cardiac transcription factors Nkx2.5 and GATA4 genes significantly increased as compared with the control cells (n = 3 experiments) before or after OGD (p<0.05).
PKG1α increased multiple pro-survival and angiomyogenic factors.
Compared to controls (NatMSCs and NullMSCs), PKG1αMSCs had significant pro-survival, angiogenic and cardiac transcription factors before and after OGD. Higher expression of paracrine factors including HGF, bFGF, SDF-1 and Ang-1 (p<0.01) and cardiac transcription factors Nkx2.5, GATA-4 (p<0.01) were documented before and after OGD (Fig. 3B). Moreover, PKG1αMSCs showed more than 2-fold increase in gene expression of these factors after OGD (Fig. 3C).
In vivo studies
PKG1α expression in PKG1αMSCs transplanted hearts.
All animals after their respective treatment in different groups survived the full length of the experiment (4 weeks), and there were no animal deaths related to PKG1α transgene overexpression in the infarcted heart. Analysis of the LV tissue samples by RT-PCR showed that the transplanted cells continued to overexpress PKG1α at the site of the cell graft in rat hearts. The level of PKG1α transgene expression on day 3 was elevated in PKG1αMSCs group compared to DMEM and NullMSCs group (Fig. 4A). PKG1α protein expression (Fig. 4B, p<0.01) and PKG activity were also significantly increased in PKG1αMSCs group (Fig. 4C).
(A) RT-PCR showed a significant increase in PKG1á mRNA level in group-3 (p<0.01); (B) PKG1á and transducted PKG1á-flag fusion proteins were detected by western blot and showed 2 fold higher expression in group-3 as compared to group-1 and group-2 (p<0.01). (C) PKG activity was enhanced 1.6 fold after PKG1áMSCs transplantation (p<0.01). Rat hearts injected with DMEM (group-1); injected with NullMSCs(group-2); injected with PKG1áMSCs (group-3).
PKG1α improved survival of transplanted MSCs and cardiomyocytes in rats with acute myocardial infarction.
DMEM group did not show any amplification of sry-gene as there were no transplanted male MSCs. Compared to NullMSCs group, PKG1αMSCs group had a significantly higher survival rate (Fig. 5A) based on the sry-gene analysis, and had fewer TUNEL+ cells amongst transplanted MSCs (Fig. 5B) and cardiomyocytes in or around infarct regions (Fig. 5C). Higher caspase-3/7 activity was detected in DMEM and NullMSCs group compared to PKG1αMSCs group (Fig. 5E). Expression of anti-apoptosis proteins including pAkt, pGSK3β and Bcl-2 (Fig. 5D), and pro-survival and angiogenic factors including HGF, bFGF, SDF-1 and Ang-1 (p<0.01) (Fig. 5F) were significantly increased.
(A) Real-time PCR for sry gene in female rat heart model of acute myocardial infarction at 7 days after sex-mismatched transplantation of NullMSCs (group-2) and PKG1αMSCs (group-3). Cell survival was significantly higher in PKG1αMSCs as compared with the NullMSCs group (n = 4/group). (B) TUNEL and GFP double staining on histological sections of rat heart tissue injected with NullMSCs (group-2) and PKG1αMSCs (group-3). The number of apoptotic transplanted MSCs (TUNEL+GFP+, red and green) was significantly reduced in group-3 (p<0.05 vs group-2). (C) TUNEL(red) combined with desmin immunostaining (green) on histological sections showed less apoptosis in residual myocardium in group-3 compared to group-1 and group-2 (p<0.05). (D) Western blot on protein lysate samples of the rat left ventricle showed increased phosphorylation of Akt,GSK3β, and high level of Bcl-2 expression at 3 days after transplantation in group-3 (p<0.05, n = 4/group). (E) Real-time PCR for growth factors expression showed HGF, TGFβ and FGF2 were significantly increased in left ventricle tissue at 3 days after transplantation. (F) Diminished caspase-3 activity was detected in heart tissues in group-3.
PKG1αMSCs promoted myoangiogenensis in the infarcted rat hearts.
At day 3 after transplantation, upregulation of cardiac transcription factors NKx2.5 and GATA-4 were observed in left ventricle tissue of rats treated with PKG1αMSCs compared to DMEM and NullMSCs (p<0.01) (Fig. 6A). Double-fluorescence immunostaining for cardiac troponin I (TnI, red) and GFP (green) at day 7 after transplantation showed more neomyofibers in transplanted hearts with PKG1αMSCs than in nullMSCs treated hearts (Fig. 6B–D).
(A) Real time PCR showed a significant increase in cardiac transcription factor Nkx2.5, GATA4 and pro-angiogenic factor SDF-1, Ang-1 genes expression in left ventricle tissue at 3 days after transplantation. (B–E) Double immunostaining for troponin I (red) and green fluorescent protein (GFP, green) on histological sections showed transplanted PKG1αMSCs differentiated into neofibers more actively as compared to nullMSCs at 7 days after transplantation. The nuclei were visualized by staining with DAPI. (E) Fluorescence immunostaining of histological sections from group-2 and group-3 for von Willebrand Factor VIII (green) at 4 weeks after transplantation, blood vessel density was significantly higher in both infarct and peri-infarct regions in group-3 as compared with group-1 and group-2(P<0.01).
Rat hearts transplanted with PKG1αMSCs also showed extensive angiogenic response as compared to DMEM and NullMSCs group. Blood vessel density was significantly higher in the infarct and peri-infarct areas in PKG1αMSCs group (62.5±7.7 and 68.8±7.3 per microscopic view, p<0.05) compared to DMEM group (21.5±5.4 and 27.5±7.0 per microscopic view) and NullMSCs group (36.3±4.1 and 46.3±6.6 per microscopic view) after 4 weeks of transplantation (Fig. 6F).
PKG1αMSCs improved cardiac function and attenuated infarction size.
At 4 weeks after transplantation, transthoracic echocardiography showed that the indices of LV heart function, including left ventricle ejection fraction (LVEF) and left ventricle fractional shortening (LVFS), were significantly preserved in PKG1αMSCs group (LVEF 52.1±2.2%; LVFS 24.8±1.3%) compared to DMEM group (LVEF 22.4±1.2%, p<0.01; LVFS 8.1±0.5%, p<0.01) and NullMSCs group (LVEF 38.4±2.4%, p<0.05; LVFS 15.2±1.5% p<0.01) (Fig. 7A–B). The baseline values of LVEF and LVFS were 91.4±1.3% and 60.5±1.5%. Both LV end-diastolic dimension (LVEDD) and end-systolic dimension (LVESD) (in millimeters) were smaller in PKG1αMSCs group (LVEDD 7.5±0.3, LVESD 5.8±0.3; p<0.01) compared to DMEM group (LVEDD 8.9±0.5, LVESD 8.1±0.3) and NullMSCs group (LVEDD 8.0±0.4, LVESD 6.8±0.4). Histological sections showed the infarction size was significantly reduced in PKG1αMSCs group (20.2±2.5%, p<0.01) compared to DMEM (44.8±4.3%) and NullMSCs group (30.1±3.7%). Improved LV wall thickness was also observed in PKG1αMSCs group (p<0.05 vs DMEM and NullMSCs group) (Fig.7C–F).
At 4 weeks after transplantation, (A and B) transthoracic echocardiography was performed to assess global heart function. Both LV ejection fraction (LVEF) and fractional shortening (LVFS) were significantly preserved in group-3 compared to group-1 and group-2 (p<0.05). (C–F) Quantitative assessment on the rat heart sections with mason trichrome staining showed a significant attenuation of infarct size in group-3 compared with group-1 and group-2 (p<0.01) (n = 8/group).
The major finding of the present study is that the overexpression of PKG1α transgene in MSCs significantly enhanced their resistance to ischemic stress and angiomyogenic potential in the infarcted heart. To our knowledge, this is the first time that the direct overexpression of the PKG1α transgene has been shown to effectively promote the survival of transplanted MSCs' via an anti-apoptotic mechanism and a coordinated increase of angiogenesis by paracrine factors in ischemic hearts.
One critical issue of stem cell therapy is how to reduce massive donor cell death after transplantation. In vitro preconditioning strategies have been used to enhance survival of transplanted MSCs in the infarcted heart. PKG1 was considered as a key effector in cardioprotection induced by PDE-5 inhibitors against ischemic injury in the infarcted heart and cardiomyocytes , , , , . In a previous study we demonstrated that the PDE-5 inhibitor tadalafil could promote MSCs survival in the infarcted heart via activating PKG1 . However, little is known about the direct effect of PKG1α on protecting MSCs and improving cardiac function. Our present study revealed that overexpression of PKG1α directly by adenoviral transduction could prolong the survival of MSCs against ischemic stress both in vitro and in infarcted hearts in vivo, and preserve cardiomyocyte survival and cardiac function following ischemic injury. Because of the prolonged survival of MSCs under ischemic stress both in vitro and in vivo, the cardiac function was preserved. These findings are consistent with previous results confirming tadalafil induced protection by PKG1α and suggests a new approach to improve stem cell therapy following MI in patients.
The potential mechanism of overexpression of PKG1α could include its anti-apoptotic effect as is evident by increased phosphorylation of Akt (pAkt) and GSK3β (pGSK3β), Bcl-2 expression, and prevention of caspase-3/7 activation. This is consistent with a previous report from Das et al, that showed higher upregulation of pAkt, pGSK3β and Bcl-2 played an essential role in the protective effect of PKG1α on cardiomyocytes in the infarcted heart , .
Although stem cell transplantation can protect ischemic cardiac muscle, reduce infarct size, and preserve cardiac function, the mechanisms are still unclear. Our present study demonstrated that overexpression of PKG1α in MSCs was responsible for the release of multiple paracrine factors. The significant increases in HGF, bFGF and SDF-1 detected at an early stage (day 3) of transplantation revealed their important role in angiogenesis, anti-apoptosis and sparing of damaged cardiomyocytes in cardiac repair , , , . The secreted paracrine factors were prominent within 72 h after transplantation and affected the cellular milieu for stem cell retention and survival. They provide the early benefit to the infarcted heart which may eventually trigger other pathways leading to protection and regeneration by endogenous stem cells . Our results are supported by the previous study where sildenafil preconditioned adipose-derived stem cells (ASCs) expressed higher growth factors including bFGF, HGF and Ang-1 and were associated with significant reduction in infarct size, cardiomyocytes apoptosis and improve heart function following ischemia . Taken together, our study provided multiple potential pathways that could explain the link between prolonged stem cell life, cardiac protection and functional recovery. Direct regulation of these cytokines could become another target of gene therapy for survival of stem cells.
Preconditioning is also believed to enhance the differentiation potential of stem cells. More neomyocytes (TnI and GFP double positive cells) were observed in PKG1αMSCs transplanted hearts than those transplanted with NullMSCs. In addition, elevated expression of cardiac transcription factors Nkx2.5 and GATA-4 was detected both in vitro and in vivo by real-time PCR. Although transplantation of PKG1α MSCs led to a significant angiomyogenic response, it could be the result of regeneration by both exogenous stem cells and endogenous stem cells activated by paracrine factors released by PKG1αMSCs in agreement with a previous report .This suggests that PKG1α gene delivery improved the myogenic potential of MSCs. The difference between NullMSCs and PKG1α MSCs in their myogenic differentiation post transplantation in the heart may also be due to the inhibition of GSK3β. Previous studies have shown that inhibition of GSK3β is sufficient to stimulate myogenic differentiation , . We observed increased phosphorylation of GSK3β as well as Akt in PKG1α MSCs after OGD and in PKG1αMSCs transplanted hearts following infarction compared to NullMSCs. From these observations we propose that PKG1α gene delivery promotes the myogenic potential of MSCs by phosphorylating and inactivating GSK3β activity via PI3K/Akt pathway and contributing to the cardiac recovery after ischemia.
Despite the positive data, our study has several limitations. First, we only studied the biomarker and paracrine factors in MSCs transplanted hearts till 7 days after transplantation. Further observation is necessary to determine the long term cell fate of PKG1α preconditioned MSCs and overexpression of cytokines, growth factors, anti-apoptotic and angiogenic factors as well as the formation of neomyocytes and blood vessles in the infarcted hearts. Second, an extensive and in-depth analysis of the secretable growth factors other than angiogenic and anti-apoptosis factors, which are up-regulated in PKG1α MSCs may be of value. Third, although transplantation of PKG1α MSCs led to a significant angiomyogenic response, we did not determine the functional status of the newly formed blood vessels and myofibers. Lastly, our previous study provided the evidence that PDE-5 inhibition with tadalafil prolonged survival of transplanted MSCs in ischemic heart via cGMP/PKG signaling. Future studies are needed to compare the effects of preconditioning with PDE5 inhibitors vs direct PKG1α gene delivering in the same experimental settings for cytoprotective and cardioprotective effects.
In conclusion, direct PKG1α transgene overexpression via adenoviral transduction significantly improves MSCs survival under conditions of ischemia via anti-apoptotic mechanism and promotes their angiomyogenic potential in ischemic heart. The preconditioned MSCs attenuated ischemia induced cardiomyocyte apoptosis, reduced the heart infarct size and improved the global cardiac function. Our findings suggest that direct PKG1α gene expression could be a new therapeutic target in stem cell-based therapy for the ischemic heart disease.
Conceived and designed the experiments: LW MA. Performed the experiments: LW SW NL YF GL. Analyzed the data: LW SW ZP YF NL GL RW MA. Contributed reagents/materials/analysis tools: MA. Wrote the paper: LW ZP RM MA.
- 1. Silva GV, Litovsky S, Assad JA, Sousa AL, Martin BJ, et al. (2005) Mesenchymal stem cells differentiate into an endothelial phenotype, enhance vascular density, and improve heart function in a canine chronic ischemia model. Circulation 111: 150–156. doi: 10.1161/01.cir.0000151812.86142.45
- 2. Quevedo HC, Hatzistergos KE, Oskouei BN, Feigenbaum GS, Rodriguez JE, et al. (2009) Allogeneic mesenchymal stem cells restore cardiac function in chronic ischemic cardiomyopathy via trilineage differentiating capacity. Proceedings of the National Academy of Sciences of the United States of America 106: 14022–14027. doi: 10.1073/pnas.0903201106
- 3. Makkar RR, Price MJ, Lill M, Frantzen M, Takizawa K, et al. (2005) Intramyocardial injection of allogenic bone marrow-derived mesenchymal stem cells without immunosuppression preserves cardiac function in a porcine model of myocardial infarction. Journal of cardiovascular pharmacology and therapeutics 10: 225–233. doi: 10.1177/107424840501000403
- 4. Yang ZJ, Ma DC, Wang W, Xu SL, Zhang YQ, et al. (2006) Experimental study of bone marrow-derived mesenchymal stem cells combined with hepatocyte growth factor transplantation via noninfarct-relative artery in acute myocardial infarction. Gene therapy 13: 1564–1568. doi: 10.1038/sj.gt.3302820
- 5. Pasha Z, Wang Y, Shiekh R, Zhang D, Zhao T, Ashraf M (2008) Preconditioning enhances cell survival and differentiation of stem cells during transplantation in infarcted myocardium. Cardiovasc Res. Jan 1 77(1): 134–42. doi: 10.1093/cvr/cvm025
- 6. Chen SL, Fang WW, Ye F, Liu YH, Qian J, et al. (2004) Effect on left ventricular function of intracoronary transplantation of autologous bone marrow mesenchymal stem cell in patients with acute myocardial infarction. The American journal of cardiology 94: 92–95. doi: 10.1016/j.amjcard.2004.03.034
- 7. Williams AR, Trachtenberg B, Velazquez DL, McNiece I, Altman P, et al. (2011) Intramyocardial stem cell injection in patients with ischemic cardiomyopathy: functional recovery and reverse remodeling. Circulation research 108: 792–796. doi: 10.1161/circresaha.111.242610
- 8. Trachtenberg B, Velazquez DL, Williams AR, McNiece I, Fishman J, et al. (2011) Rationale and design of the Transendocardial Injection of Autologous Human Cells (bone marrow or mesenchymal) in Chronic Ischemic Left Ventricular Dysfunction and Heart Failure Secondary to Myocardial Infarction (TAC-HFT) trial: A randomized, double-blind, placebo-controlled study of safety and efficacy. American heart journal 161: 487–493. doi: 10.1016/j.ahj.2010.11.024
- 9. Muller-Ehmsen J, Whittaker P, Kloner RA, Dow JS, Sakoda T, et al. (2002) Survival and development of neonatal rat cardiomyocytes transplanted into adult myocardium. Journal of molecular and cellular cardiology 34: 107–116. doi: 10.1006/jmcc.2001.1491
- 10. Zhang M, Methot D, Poppa V, Fujio Y, Walsh K, et al. (2001) Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies. Journal of molecular and cellular cardiology 33: 907–921. doi: 10.1006/jmcc.2001.1367
- 11. Freyman T, Polin G, Osman H, Crary J, Lu M, et al. (2006) A quantitative, randomized study evaluating three methods of mesenchymal stem cell delivery following myocardial infarction. European heart journal 27: 1114–1122. doi: 10.1093/eurheartj/ehi818
- 12. Yang YJ, Qian HY, Huang J, Li JJ, Gao RL, et al. (2009) Combined therapy with simvastatin and bone marrow-derived mesenchymal stem cells increases benefits in infarcted swine hearts. Arteriosclerosis, thrombosis, and vascular biology 29: 2076–2082. doi: 10.1161/atvbaha.109.189662
- 13. Jiang Y, Chen L, Tang Y, Ma G, Shen C, et al. (2010) HO-1 gene overexpression enhances the beneficial effects of superparamagnetic iron oxide labeled bone marrow stromal cells transplantation in swine hearts underwent ischemia/reperfusion: an MRI study. Basic research in cardiology 105: 431–442. doi: 10.1007/s00395-009-0079-2
- 14. Noort WA, Feye D, Van Den Akker F, Stecher D, Chamuleau SA, et al. (2010) Mesenchymal stromal cells to treat cardiovascular disease: strategies to improve survival and therapeutic results. Panminerva medica 52: 27–40.
- 15. Robey TE, Saiget MK, Reinecke H, Murry CE (2008) Systems approaches to preventing transplanted cell death in cardiac repair. Journal of molecular and cellular cardiology 45: 567–581. doi: 10.1016/j.yjmcc.2008.03.009
- 16. Haider H, Ashraf M (2008) Strategies to promote donor cell survival: combining preconditioning approach with stem cell transplantation. Journal of molecular and cellular cardiology 45: 554–566. doi: 10.1016/j.yjmcc.2008.05.004
- 17. Laflamme MA, Chen KY, Naumova AV, Muskheli V, Fugate JA, et al. (2007) Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nature biotechnology 25: 1015–1024. doi: 10.1038/nbt1327
- 18. Sauzeau V, Le Jeune H, Cario-Toumaniantz C, Smolenski A, Lohmann SM, et al. (2000) Cyclic GMP-dependent protein kinase signaling pathway inhibits RhoA-induced Ca2+ sensitization of contraction in vascular smooth muscle. The Journal of biological chemistry 275: 21722–21729. doi: 10.1074/jbc.m000753200
- 19. Soff GA, Cornwell TL, Cundiff DL, Gately S, Lincoln TM (1997) Smooth muscle cell expression of type I cyclic GMP-dependent protein kinase is suppressed by continuous exposure to nitrovasodilators, theophylline, cyclic GMP, and cyclic AMP. The Journal of clinical investigation 100: 2580–2587. doi: 10.1172/jci119801
- 20. Boerth NJ, Dey NB, Cornwell TL, Lincoln TM (1997) Cyclic GMP-dependent protein kinase regulates vascular smooth muscle cell phenotype. Journal of vascular research 34: 245–259. doi: 10.1159/000159231
- 21. Zhang R, Wang L, Zhang L, Chen J, Zhu Z, et al. (2003) Nitric oxide enhances angiogenesis via the synthesis of vascular endothelial growth factor and cGMP after stroke in the rat. Circulation research 92: 308–313. doi: 10.1161/01.res.0000056757.93432.8c
- 22. Lohmann SM, Vaandrager AB, Smolenski A, Walter U, De Jonge HR (1997) Distinct and specific functions of cGMP-dependent protein kinases. Trends in biochemical sciences 22: 307–312. doi: 10.1016/s0968-0004(97)01086-4
- 23. Wollert KC, Fiedler B, Gambaryan S, Smolenski A, Heineke J, et al. (2002) Gene transfer of cGMP-dependent protein kinase I enhances the antihypertrophic effects of nitric oxide in cardiomyocytes. Hypertension 39: 87–92. doi: 10.1161/hy1201.097292
- 24. Draijer R, Vaandrager AB, Nolte C, de Jonge HR, Walter U, et al. (1995) Expression of cGMP-dependent protein kinase I and phosphorylation of its substrate, vasodilator-stimulated phosphoprotein, in human endothelial cells of different origin. Circulation research 77: 897–905. doi: 10.1161/01.res.77.5.897
- 25. Das A, Smolenski A, Lohmann SM, Kukreja RC (2006) Cyclic GMP-dependent protein kinase Ialpha attenuates necrosis and apoptosis following ischemia/reoxygenation in adult cardiomyocyte. The Journal of biological chemistry 281: 38644–38652. doi: 10.1074/jbc.m606142200
- 26. Frantz S, Klaiber M, Baba HA, Oberwinkler H, Volker K, et al. (2011) Stress-dependent dilated cardiomyopathy in mice with cardiomyocyte-restricted inactivation of cyclic GMP-dependent protein kinase I. European heart journal doi: 10.1093/eurheartj/ehr445
- 27. Das A, Xi L, Kukreja RC (2008) Protein kinase G-dependent cardioprotective mechanism of phosphodiesterase-5 inhibition involves phosphorylation of ERK and GSK3beta. The Journal of biological chemistry 283: 29572–29585. doi: 10.1074/jbc.m801547200
- 28. Salloum FN, Chau VQ, Hoke NN, Abbate A, Varma A, et al. (2009) Phosphodiesterase-5 inhibitor, tadalafil, protects against myocardial ischemia/reperfusion through protein-kinase g-dependent generation of hydrogen sulfide. Circulation 120: S31–36. doi: 10.1161/circulationaha.108.843979
- 29. Salloum FN, Abbate A, Das A, Houser JE, Mudrick CA, et al. (2008) Sildenafil (Viagra) attenuates ischemic cardiomyopathy and improves left ventricular function in mice. American journal of physiology Heart and circulatory physiology 294: H1398–1406. doi: 10.1152/ajpheart.91438.2007
- 30. Li L, Haider H, Wang L, Lu G, Ashraf M (2012) Adenoviral short hairpin RNA therapy targeting phosphodiesterase 5a relieves cardiac remodeling and dysfunction following myocardial infarction. American journal of physiology Heart and circulatory physiology 302: H2112–2121. doi: 10.1152/ajpheart.00339.2011
- 31. Haider H, Lee YJ, Jiang S, Ahmed RP, Ryon M, et al. (2010) Phosphodiesterase inhibition with tadalafil provides longer and sustained protection of stem cells. American journal of physiology Heart and circulatory physiology 299: H1395–1404. doi: 10.1152/ajpheart.00437.2010
- 32. Hoke NN, Salloum FN, Kass DA, Das A, Kukreja RC (2012) Preconditioning by phosphodiesterase-5 inhibition improves therapeutic efficacy of adipose-derived stem cells following myocardial infarction in mice. Stem cells 30: 326–335. doi: 10.1002/stem.789
- 33. Feil R, Gappa N, Rutz M, Schlossmann J, Rose CR, et al. (2002) Functional reconstitution of vascular smooth muscle cells with cGMP-dependent protein kinase 1 isoforms. Circulation research 90: 1080–1086. doi: 10.1161/01.res.0000019586.95768.40
- 34. Aggarwal S, Rafikov R, Gross CM, Kumar S, Pardo D, et al. (2011) Purification and functional analysis of protein kinase G-1alpha using a bacterial expression system. Protein expression and purification 79: 271–276. doi: 10.1016/j.pep.2011.05.001
- 35. Feil R, Lohmann SM, de Jonge H, Walter U, Hofmann F (2003) Cyclic GMP-dependent protein kinases and the cardiovascular system: insights from genetically modified mice. Circulation research 93: 907–916. doi: 10.1161/01.res.0000100390.68771.cc
- 36. Lu G, Haider H, Porollo A, Ashraf M (2010) Mitochondria-specific transgenic overexpression of connexin-43 simulates preconditioning-induced cytoprotection of stem cells. Cardiovascular research 88: 277–286. doi: 10.1093/cvr/cvq293
- 37. Mangi AA, Noiseux N, Kong D, He H, Rezvani M, Ingwall JS, Dzau VJ (2003) Mesenchymal stem cells modified with Akt prevent remodelling and restore performance of infarcted hearts. Nat. Med 9: 1195–1201. doi: 10.1038/nm912
- 38. Li W, Ma N, Ong LL, Nesselmann C, Klopsch C, Ladilov Y, Furlani D, Piechaczek C, Moebius JM, Lützow K, Lendlein A, Stamm C, Li RK, Steinhoff G (2007) Bcl-2 engineered MSCs inhibited apoptosis and improved heart functions. Stem Cells. 2007 Aug 25(8): 2118–27. doi: 10.1634/stemcells.2006-0771
- 39. Pasha Z, Haider K H, M Ashraf (2011) Efficient non viral reprogramming of myoblasts to stemness with a single small molecule for generating cardiac progenitors cells. PLoS One 6(8): e23667 Epub 2011 Aug 17. doi: 10.1371/journal.pone.0023667
- 40. Yang ZJ, Zhang YR, Chen B, Zhang SL, Jia EZ, et al. (2009) Phase I clinical trial on intracoronary administration of Ad-hHGF treating severe coronary artery disease. Molecular biology reports 36: 1323–1329. doi: 10.1007/s11033-008-9315-3
- 41. Dong F, Harvey J, Finan A, Weber K, Agarwal U, et al. (2012) Myocardial CXCR4 expression is required for mesenchymal stem cell mediated repair following acute myocardial infarction. Circulation 126: 314–324. doi: 10.1161/circulationaha.111.082453
- 42. Jang YH, Kim JH, Ban C, Ahn K, Cheong JH, et al. (2012) Stromal Cell Derived Factor-1 (SDF-1) Targeting Reperfusion Reduces Myocardial Infarction in Isolated Rat Hearts. Cardiovascular therapeutics 30: 264–272. doi: 10.1111/j.1755-5922.2011.00301.x
- 43. House SL, Bolte C, Zhou M, Doetschman T, Klevitsky R, et al. (2003) Cardiac-specific overexpression of fibroblast growth factor-2 protects against myocardial dysfunction and infarction in a murine model of low-flow ischemia. Circulation 108: 3140–3148. doi: 10.1161/01.cir.0000105723.91637.1c
- 44. Padin-Iruegas ME, Misao Y, Davis ME, Segers VF, Esposito G, Tokunou T, UrbanekK, Hosoda T, Rota M, Anversa P, Leri A, Lee RT, Kajstura J (2009) Cardiac progenitor cells and biotinylated insulin-like growth factor-1 nanofibers improveendogenous and exogenous myocardial regeneration Circulation. Sep8 120(10): 876–87. doi: 10.1161/circulationaha.109.852285
- 45. van der Velden JL, Schols AM, Willems J, Kelders MC, Langen RC (2008) Glycogensynthase kinase 3 suppresses myogenic differentiation through negative regulationof NFATc3. The Journal of biological chemistry 283: 358–366. doi: 10.1074/jbc.m707812200
- 46. van der Velden JL, Langen RC, Kelders MC, Wouters EF, Janssen-Heininger YM (2006) etal (2006) Inhibition of glycogen synthase kinase-3beta activity is sufficient to stimulate myogenic differentiation. American journal of physiology Cell physiology 290: C453–462. doi: 10.1152/ajpcell.00068.2005