Protein Kinase G1 α Overexpression Increases Stem Cell Survival and Cardiac Function after Myocardial Infarction

Background 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. Conclusion Overexpression of PKG1α transgene could be a powerful approach to improve MSCs survival and their angiomyogenic potential in the infarcted heart.


Introduction
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 [1,2,3,4,5,6,7,8]. Despite of promising results, myriad problems including massive cell death after transplantation have limited the efficacy of this cell therapy [9,10,11]. One strategy to improve MSCs survival is to precondition MSCs before transplantation [4,12,13]. 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 [14,15,16,17].
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 [18,19,20,21]. PKG has two isozymes, PKG1 and PKG2. Only PKG1 is detected in cardiac myocytes and vascular cells. The Nterminus of PKG1 is encoded by two different exons resulting in the production of two isoforms, PKG1a and PKG1b. Both isoforms are present in heart and vessels [22,23,24]. However, PKG1b is activated at ,10-fold higher cGMP concentrations than PKG1a. Previous studies have revealed a direct role for PKG1 in cardioprotection. Overexpression of PKG1a by adenoviral vectors inhibits necrosis and apoptosis in rat cardiomyocytes undergoing simulated ischemia-reoxygenation [25]. PKG1-deficient mice showed increased myocardial structural and functional damage after trans-aortic constriction (TAC) than their wild-type controls [26]. 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 [27,28,29]. 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 [30].
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 [31,32]. As PKG1a is more active than PKG1b at cGMP each found in the cardiovascular system [25,33,34,35], we hypothesized that overexpression of PKG1a 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.

Ethics Statement
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 PKG1a. pShuttle-IRES-hrGFP-1 vector containing PKG1a cDNA amplified from mouse cardiomyocytes (Ad-PKG1a) was constructed based on AdEasy TM 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 TRC 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-PKG1a 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 [5]. 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-PKG1a 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; 4006) 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 ( Null MSCs) or Ad-PKG1a transduced MSCs ( PKG1a MSCs). PKG1a transgene overexpression in PKG1a MSCs 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 PKG1a 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 firststrand 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 94uC for 3 min, 30 cycles of denaturation at 94uC for 30 sec, annealing at 58uC for 30 sec and extension at 72uC for 30 sec, and a final extension at 72uC 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 95uC 15 min for initial denaturation; 40 cycles of denaturation at 94uC for 15 sec, annealing at 55uC for 30 sec, extension at 72uC for 30 sec; and a final extension at 55uC 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 1uC/min from 55uC to 90uC 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 2610 4 cells/cm 2 . The cells were divided into three groups, including nontreated native MSCs ( Nat MSCs), transduced with Ad-null ( Null MSCs), and transduced with Ad-PKG1a ( PKG1a MSCs). At 72 h after transduction, MSCs in all groups were rinsed twice with serum-free, glucose, and sodium pyruvatefree DMEM and were cultured in the same medium at 37uC in a anoxia chamber (InVivo 500; Ruskinn Life Science) saturated with 95% N 2 /5% CO 2 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 Table 1. Sequence of the primers used in the study. 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 10 4 cells/well in 100 ml medium. After 8 h OGD, a 100 ml 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 496-diamidino-2phenylindole (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 mg/ml Leupeptin, and 2 mg/ml Aprotinin). Protein concentration was measured by using the Bio-Rad DC-Protein Assay Reagent (Bio-Rad). 20 mg 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 [36]. 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 4uC. 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 PKG1a ( 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 [5].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, 5-6610 6 [37,38] were successfully injected in the rat hearts to determine their effects. We have previously injected 3610 6 MSCs [31]at multiple sites in and around the infarct area to maximize the efficacy of cell therapy. In the current study, we determined that 2610 6 was the most suitable number for transplantation without any side effects.
Rats (n = 16/group) received intramyocardial injections of either 70 ml of DMEM without cells (DMEM group), or containing 2610 6 Null MSCs ( Null MSCs group), or containing 2610 6 PKG1a MSCs ( PKG1a MSCs 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.
Molecular Analyses. For molecular analysis, the animals were euthanized at day-3 after stem cell transplantation. Left ventricle was cut and processed for PKG1a expression and activity assay, paracrine and cardiac transcription factors expression and caspase-3/7 activity assay. pAkt, Akt, pGSK3b, GSK3b 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.
Immunohistological studies. 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 2006 magnification.
Heart function measurement. At 4 weeks after stem cell transplantation, transthoracic echocardiography was used to measure the heart function as described before [5,39]. 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 endsystolic dimension [ESD]), and anterior wall thickness were measured.

In vitro studies
PKG1a overexpression reduced apoptosis. In order to delineate the pro-survival pathway in PKG1a MSCs, we isolated and analyzed the cell lysate from different cell treatment groups for Western blot. PKG1a overexpression significantly increased phosphorylation of Akt (pAkt) and GSK3b (pGSK3b) without changing the total level of Akt and GSK3b in PKG1a MSCs after 8 h OGD compared to controls ( Nat MSCs and Null MSCs) (Fig. 3A). GSK3b is a downstream target in Akt signaling. Anti-apoptotic protein Bcl-2, was also significantly increased in PKG1a MSCs (Fig. 3A).

PKG1a
increased multiple pro-survival and angiomyogenic factors. Compared to controls ( Nat MSCs and Null MSCs), PKG1a MSCs 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, PKG1a MSCs showed more than 2-fold increase in gene expression of these factors after OGD (Fig. 3C).  there were no animal deaths related to PKG1a transgene overexpression in the infarcted heart. Analysis of the LV tissue samples by RT-PCR showed that the transplanted cells continued to overexpress PKG1a at the site of the cell graft in rat hearts. The level of PKG1a transgene expression on day 3 was elevated in PKG1a MSCs group compared to DMEM and Null MSCs group (Fig. 4A). PKG1a protein expression (Fig. 4B, p,0.01) and PKG activity were also significantly increased in PKG1a MSCs group (Fig. 4C).  MSCs-PKG1a Improves Cardiac Repair PLOS ONE | www.plosone.org 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 Null MSCs group compared to PKG1a MSCs group (Fig. 5E). Expression of antiapoptosis proteins including pAkt, pGSK3b 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.

PKG1a
MSCs 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 PKG1a MSCs compared to DMEM and Null MSCs (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 PKG1a MSCs than in null MSCs treated hearts (Fig. 6B-D).
Rat hearts transplanted with PKG1a MSCs also showed extensive angiogenic response as compared to DMEM and Null MSCs group.

Discussion
The major finding of the present study is that the overexpression of PKG1a 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 PKG1a transgene has been shown to effectively promote the survival of transplanted MSCs' via an antiapoptotic 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 [27,28,29,31,32]. In a previous study we demonstrated that the PDE-5 inhibitor tadalafil could promote MSCs survival in the infarcted heart via activating PKG1 [31]. However, little is known about the direct effect of PKG1a on protecting MSCs and improving cardiac function. Our present study revealed that overexpression of PKG1a 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 PKG1a and suggests a new approach to improve stem cell therapy following MI in patients.
The potential mechanism of overexpression of PKG1a could include its anti-apoptotic effect as is evident by increased phosphorylation of Akt (pAkt) and GSK3b (pGSK3b), 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, pGSK3b and Bcl-2 played an essential role in the protective effect of PKG1a on cardiomyocytes in the infarcted heart [25,27].
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 PKG1a 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 [40,41,42,43]. 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 [44]. 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 [32]. 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 PKG1a MSCs transplanted hearts than those transplanted with Null MSCs. 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 PKG1a MSCs led to a significant angiomyogenic (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). doi:10.1371/journal.pone.0060087.g006 response, it could be the result of regeneration by both exogenous stem cells and endogenous stem cells activated by paracrine factors released by PKG1a MSCs in agreement with a previous report [44].This suggests that PKG1a gene delivery improved the myogenic potential of MSCs. The difference between Null MSCs and PKG1a MSCs in their myogenic differentiation post transplantation in the heart may also be due to the inhibition of GSK3b. Previous studies have shown that inhibition of GSK3b is sufficient to stimulate myogenic differentiation [45,46]. We observed increased phosphorylation of GSK3b as well as Akt in PKG1a MSCs after OGD and in PKG1a MSCs transplanted hearts following infarction compared to Null MSCs. From these observations we propose that PKG1a gene delivery promotes the myogenic potential of MSCs by phosphorylating and inactivating GSK3b 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 PKG1a 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 PKG1a MSCs may be of value. Third, although transplantation of PKG1a 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 PKG1a gene delivering in the same experimental settings for cytoprotective and cardioprotective effects.

Conclusions
In conclusion, direct PKG1a 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 PKG1a gene expression could be a new therapeutic target in stem cell-based therapy for the ischemic heart disease.   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). doi:10.1371/journal.pone.0060087.g007