Thymosin Beta 4 Protects Cardiomyocytes from Oxidative Stress by Targeting Anti-Oxidative Enzymes and Anti-Apoptotic Genes

Background Thymosin beta-4 (Tβ4) is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. The mechanism by which Tβ4 modulates cardiac protection under oxidative stress is not known. The purpose of this study is to dissect the cardioprotective mechanism of Tβ4 on H2O2 induced cardiac damage. Methods Rat neonatal cardiomyocytes with or without Tβ4 pretreatment were exposed to H2O2 and expression of antioxidant, apoptotic, and anti-inflammatory genes was evaluated by quantitative real-time PCR and western blotting. ROS levels were estimated by DCF-DA using fluorescent microscopy and fluorimetry. Selected antioxidant, anti-inflammatory and antiapoptotic genes were silenced by siRNA transfections in neonatal cardiomyocytes and effect of Tβ4 on H2O2-induced cardiac damage was evaluated. Results Pre-treatment of Tβ4 resulted in reduction of the intracellular ROS levels induced by H2O2 in cardiomyocytes. Tβ4 pretreatment also resulted in an increase in the expression of antiapoptotic proteins and reduction of Bax/BCl2 ratio in the cardiomyocytes. Pretreatment with Tβ4 resulted in stimulating the expression of antioxidant enzymes copper/zinc SOD and catalase in cardiomyocytes at both transcription and translation levels. Tβ4 treatment resulted in the increased expression of anti-apoptotic and anti-inflammatory genes. Silencing of Cu/Zn SOD and catalase gene resulted in apoptotic cell death in the cardiomyocytes which was prevented by treatment with Tβ4. Conclusion This is the first report that demonstrates the effect of Tβ4 on cardiomyocytes and its capability to selectively upregulate anti-oxidative enzymes, anti-inflammatory genes, and antiapoptotic enzymes in the neonatal cardiomyocytes thus preventing cell death thereby protecting the myocardium. Tβ4 treatment resulted in decreased oxidative stress and inflammation in the myocardium under oxidative stress.


Introduction
Adverse cardiac remodeling is a detrimental process accountable for the development of various cardiac diseases including myocardial infarction, cardiac hypertrophy and heart failure. Although the mechanisms underlying the cardiac remodeling are multi-factorial, current evidences suggest that oxidative stress plays a critical role in the process. Oxidative stress is defined as an imbalance in antioxidant defense mechanism that elicits the production of reactive oxygen species (ROS) [1][2][3][4]. ROS are primarily characterized as oxygen based free chemical particles, if present in excess, causes contractile dysfunction and structural damage in the myocardium [5]. Therefore the balance between ROS production and removal of excess ROS are essential in maintaining the redox state and, homeostasis balance in the cell [6]. At the subcellular level, increased ROS levels can cause damage to nucleic acids and proteins leading to programmed cell death or apoptosis [7][8][9]. Thus, ROS mediated oxidative damage in cardiomyocytes is responsible for structural integrity of the myocardium.
It has been reported that increase in the levels of oxidative stress in the failing heart is primarily due to the functional uncoupling of the respiratory chain caused by inactivation of complex I in the mitochondria and considered to be a good source for ROS production [10,11]. Another source would consider is the impaired antioxidant capacity that include superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) and considered as such as the first line of cellular defense against oxidative injury [12]. Accumulating evidences indicate that cardiac overexpression of Mn-SOD or CAT protects the heart from ischemic insult or myocardial infarction [13,14].
Oxidative stress triggers pro-inflammatory signaling pathways that activate nuclear factor kappa B (NF-kB) and AP-1 transcription factors [15]. Previously, we and others have shown that NF-kB activation is associated with cardiac dysfunction, ventricular hypertrophy, and maladaptive cardiac growth [16][17][18][19][20]. The biochemical nexus between oxidative stress and inflammation represent an integral part in the pathophysiology of myocardial damage.
Thus, it is evident from our literature that oxidative damage remains a great challenge to promote significant myocardial damage and, numerous efforts have been made in the search of strategies to protect the heart against oxidative damage. In search of an ideal cardio-protective agent, Thymosin b4 (Tb4) emerged as powerful candidate.
Tb4, a G-actin sequestering molecule is primarily implicated in reorganizing actin cytoskeleton that needed for cell mobility [21]. Moreover, Tb4 is present in all cells and body fluids and, has diverse biological function that includes tissue development, repair and pathology [21,22]. Importantly, Tb4 contributes a significant cardiac repair mechanism by activating integrin link kinase [23][24][25] and, has further shown to promote cardiac regeneration, epicardial cell migration and neovascularization [26,27]. Our previous study demonstrated that treatment of Tb4 restored the adverse cardiac remodeling (due to ischemic insult) by reducing inflammation, fibrosis and, activating ILK, PINCH and a-Parvin [27]. In the case of oxidative stress, Tb4 has been shown to protect the cells by enhancing antioxidant enzymes and reducing caspase 9 activation in human corneal epithelial cells [28][29][30]. Under this setting, we recently have shown in cardiac fibroblast that Tb4 has the target for SOD and catalase and thereby protect the cell from oxidative stress [31]. But the exact mechanism by which Tb4 functions in the myocardium under oxidative stress and its effects on the cardiac myocytes is largely unknown.
The present study elucidates the protective mechanism of Tb4 under oxidative stress using rat neonatal cardiac myocytes. We hypothesize that Tb4 protect myocytes under oxidative stress by modulating antioxidant enzymes, apoptotic genes and proinflammatory genes. As for the limitation of our study, we used neonatal cardiomyocytes to study the protective effect of Tb4 under oxidative stress conditions that may not mimic the changes in clinical conditions, and thus results using cultured cardiomyocytes should be interpreted carefully. An advantage of neonatal cardiomyocytes is the easy procedure for their isolation in contrast to adult cardiomyocytes, which are very sensitive to the concentration of Ca2+ in the medium. Moreover, the phenotype of cultured neonatal cardiomyocytes is very stable and their contractile profile very closely mimicking the adult cardiomyocytes. Experiments in isolated neonatal cardiomyocytes have generally reproduced the results on adult cardiomyocytes with a wide variety of interventions exploring the cellular and molecular mechanisms in oxidative stress.

Cell culture and treatment
Primary cultures of cardiac myocytes were prepared from ventricles of 1-3-day-old Wistar rats as described previously [32]. In brief, cardiomyocytes were plated at a field density of 2.5610 4 cells per cm 2 on coverslips, 6-well plates, 60-mm culture dishes, or 100-mm dishes as required with DMEM containing 10% FBS and supplemented with insulin, transferrin and selenium and bromodeoxy-uridine. After 24 h, cells were serum deprived overnight before stimulation. A standardized dose of 100 mM H 2 O 2 was used to induce oxidative stress in the in vitro system. To study the protective effects of Tb4, cells were pretreated with Tb4 2 hours prior to H 2 O 2 challenge. The final concentration of Tb4 used in this study was 1 mg/ml which was based on previous reports [28,32].

Detection of the cell viability
Cell viability of cardiac myocytes was measured quantitatively using MTT as described previously [31]. The absorbance was measured at 570 nm using a microplate reader (Molecular Devices, SpectraMax 250). The effect of Tb4 was assessed on the H 2 O 2 treated myocyte and the cytotoxicity curve was made and, expressed as percentage cell viability compared to control.

Measurement of intracellular ROS levels
For measuring the levels of intracellular ROS, cardiac myocytes after treatments were incubated with 50 mM 29,79-dichlorodihydrofluorescein diacetate (H 2 DCFH-DA, Molecular Probes, Eugene, OR) at 37uC in the dark for 30 min as described previously [31].

Confocal microscopy
For measuring the levels of intracellular ROS, cells were seeded on coverslips in 6-well plates and after treatments were incubated with 50 mM 29,79-dichlorodihydrofluorescein diacetate (H 2 DCFH-DA, Molecular Probes, Eugene, OR) at 37uC in the dark for 30 min as previously described [31]. Cells were then fixed and mounted on glass slides and observed under confocal laser scanning microscope (Fluoview FV1000) fitted with a 488 nm argon ion laser. Images were acquired using the F10-ASW 1.5 Fluoview software.

Western blot analysis
Cardiac myocytes were treated with or without Tb4 for 2 h before stimulated with 100 mM of H 2 O 2 . The cell lysate preparation, western blot analysis and image quantification were performed as described previously [31].

RNA isolation and quantitative RT-PCR (q RT-PCR) analysis
Cardiomyocytes were treated with or without Tb4 for 2 h followed by stimulation with H 2 O 2 (100 mM) for up to 24 h. The preparation of RNA, 1 st strand cDNA synthesis and q RT-PCR was performed as described previously [31]. Analysis of relative gene expression was done by evaluating the real-time quantitative PCR data by 2 (2DDCt) method as described previously by others [33,34]. GAPDH or 18S was used as housekeeping gene.

RNA interference and siRNA transfection
The gene silencing experiment using small interfering (si) RNA of Cu/Zn-SOD and Bcl 2 was performed using predesigned double-stranded siRNA of the above from Sigma Life Science, Saint Louis MO, USA as described previously [31]. A scramble siRNA was used for negative control was also obtained from Sigma. In brief, cells were then transfected with 200 pmol of the siRNAs for Cu/Zn-SOD and Bcl 2 or negative control siRNA using N-TER TM nanoparticle siRNA transfection system (Sigma) in accordance with the manufacturer's protocol. After 24 h of transfection, cells were treated and harvested to determine the transfection efficiency and effect of Tb4 treatment on H 2 O 2 treatment in the transfected cells.

TUNEL staining
Quantification of TUNEL staining was done to study the extent of apoptotic cell death on transfected fibroblasts by in situ cell death detection kit (Roche Applied Science, Indianapolis, IN) as described previously [31].

Statistical analysis
All experiments were performed at least three times for each determination. Data are expressed as means 6 standard error (SE) and were analyzed using one-way analysis of variance and secondary analysis for significance with Tukey-Kramer post tests using Prism 5.0 GraphPad software (GraphPad, San Diego, CA, USA). A p value less than 0.05 was considered statistically significant.

Tb4 protects cardiomyocytes cells against H 2 O 2 -induced cell death
The viability of cardiomyocytes was determined by MTT assay. Cardiomyocytes were treated with increasing doses of H 2 O 2 and, cell viability was determined over a period of 24 hours. Our data showed that the 50% lethal dose (LD 50 ) of H 2 O 2 was between 150 and 250 mM ( Figure 1A). Pretreatment with Tb4 (1 mg/mL) prevented the myocyte cell death by 23.4% (p,0.05), compared to the H 2 O 2 -treated group indicating a protective role of Tb4 in cardiomyocytes. The optimal sub-lethal concentration of H 2 O 2 was determined and 100 mM H 2 O 2 was used for the entire study.

Tb4 protects cardiomyocytes in H 2 O 2 -induced oxidative stress
Intracellular ROS levels in myocytes for 12 and 24 h post-H 2 O 2 (100 mM) treatment were subsequently measured by fluorimetry and confocal microscopy analyses. There was an increase in ROS  Table 1.
Tb4 reduces the formation of superoxide radicals and nitric oxide in H 2 O 2 -induced oxidative stress in cardiomyocytes H 2 O 2 treatment induces a cascade of biochemical reaction in the cell leading to generation and accumulation of a variety of free radicals in the cells. We estimated the levels of superoxide and nitric oxide by using confocal microscopy. Our data revealed that there was an increase in the fluorescence intensity of DHE and DAF-2DA in H 2 O 2 treated cells, an indicator of O 2 .2 and NO radicals, compared to unstimulated cells ( Figure 1D and 1E). This increase in the fluorescence intensity of DHE and DAF-2A was significantly prevented by Tb4 pretreatment. The quantifications of image intensities have been tabulated in Table 1.

Tb4 treatment protects mitochondrial membrane potential (DYm) in oxide in H 2 O 2 -induced oxidative stress in cardiomyocytes
Oxidative stress is known to elicit depolarization of mitochondrial membrane potential. We evaluated the effect of Tb4 on the mitochondrial membrane potential in H 2 O 2 stimulated cardiomyocytes using MitoTracker Red by confocal microscopy. Our data revealed that there was loss of mitochondrial membrane potential as indicated by a decrease in the fluorescence intensity of MitoTracker Red H 2 O 2 stimulated cell. Tb4 treatment significantly restored the phenomenon ( Figure 1F). The quantifications of image intensities have been tabulated in Table 1.   (Figure 3 C). The reduced mRNA expression of Bcl 2 under oxidative stress was reversed by pretreatment with Tb4 by 1.14-fold (p,0.05) and 1.

Effect of Tb4 treatment and analysis of NF-kB target genes by RT 2 PCR array
To gain further insight into NF-kB-target genes, we performed q RT-PCR array. The data showed alteration of NF-kB family genes in H 2 O 2 treated cardiomyocytes, compared to unstimulated cells. Furthermore, Tb4 treatment restored those altered genes significantly. The list of NF-kB genes are shown in Table S1. Our data showed that H 2 O 2 treatment induced upregulation of several NF-kB target genes, importantly, the following: TNFa, Irak1, Stat1, Tgfbr1, IkBa, IKKb, Casp1, Rel, Egr1, NF-kB1, Tgfbr2, Rela, Ifnc, Ccl2, Fasl, Il1b, IL-6 and Fadd. A list of selected NF-kB family genes is provided in Table S2.

Validation of NF-kB target genes in cardiomyocytes
The expression of NF-kB target genes, FasL, TNFa, c-Fos, c-Jun and ICAM-1 were analyzed in H 2 O 2 treated cardiomyocytes in the presence and absence of Tb4. Our data showed that the expression of FasL, TNFa, c-Fos, c-Jun and ICAM-1 genes were increased by 1 (Figure 4).

Tb4 selectively upregulates Cu/Zn-SOD and Bcl 2 genes in cardiac myocytes
We took knock-down approach to further validate the target molecule of Cu/Zn-SOD and Bcl 2 by Tb4. Both genes were knock-down in cardiomyocytes using their specific siRNAs and, were subsequently challenged with H 2 O 2 in the presence and absence of Tb4. The scramble siRNA were used as a control. Pretreatment with Tb4 in scramble transfection enhanced the expression of Cu/Zn-SOD and Bcl 2 under normal conditions (Fig. 5 A and 5 C). H 2 O 2 treatment significantly downregulated the Cu/Zn-SOD and Bcl 2 protein to 0.6760.01 and 0.4560.08fold (p,0.05), respectively, compared to control. Tb4 pretreatment for 24 h partly restored the expression of both Cu/Zn-SOD  (Fig. 5E, middle panel). The caspase3 gene expression was determined in Cu/Zn-SOD depleted cells. The expression of caspase3 was increased by 2.3261.1 fold in unstimulated cells (Fig. 5E, right panel). The expression of caspase3 was further increased to 4.1961.52 fold (p,0.05) with the knockdown of Cu/ Zn-SOD gene by siRNA transfection. Tb4 pretreatment showed 2.360.70-fold (p,0.05) reduction of caspase3 expression in H 2 O 2 treated cells (Fig. 5E, right panel). The

Discussion
The present study showed for the first time that Tb4 protects cardiomyocytes under oxidative stress by upregulating antioxidant enzymes and reducing pro-apoptotic and pro-inflammatory genes. H 2 O 2 elicits marked increment in intracellular ROS that promotes degradation of antioxidant enzymes (Cu/Zn-SOD and catalase) and activates pro-apoptotic (Bax and caspase3) and pro-inflammatory genes in cardiomyocytes. Increased ROS further advocate detrimental changes in cardiomyocytes leading to the loss of mitochondrial membrane potential and, subsequently increases the Bax/Bcl 2 ratio favoring apoptosis. Pretreatment with Tb4 showed significant attenuation of ROS activity and restoration of the above molecules and protecting cardiomyocytes from oxidative stress. Finally, we showed that knocking down of either Cu-Zn-SOD or Bcl 2 in cardiomyocytes failed to protect the cells from oxidative stress in presence of Tb4.
The myocardium has a complex mechanism to maintain the oxygen supply demand in response to diverse physiological and pathological stresses and, control the contractile function. The major pathological manifestation of oxidative stress is the generation of ROS that damage the cellular activity and function. It has become more apparent that the effect of oxidative stress in cardiac cells predisposes the condition that lead to adverse cardiac remodeling including cell death, myocardial hypertrophy and contractile dysfunction [6,35,36]. Cardiomyocytes are the major ''bulk'' in the myocardium and primarily governs the contractile function. Any sort of stress will have a serious impact on cardiomyocytes and affect various signaling cascades that ultimately lead to dysfunction. In an attempt to protect these cells under oxidative stress, we tested the efficacy of Tb4 in cardiomyocytes which is currently undetermined. Our results indicate that cardiomyocytes pretreated for 2 h with Tb4 increases the cell viability under oxidative stress suggest that Tb4 contributes a crucial role in the cardio-protection under oxidative stress.
Oxidative stress and ROS have been implicated in triggering cell death. Following a one-, two-or three-electron reduction, O 2 may generate successively O 2 .2 (superoxide radical), H 2 O 2 or OH 2 (hydroxyl radical). ROS are able to oxidize biological macromolecules such as DNA, protein and lipids [37,38]. Superoxide dismutase (SOD) converts O 2 .2 into H 2 O 2 and the latter can generate OH 2 in the presence of Fe 2+ cations (Fenton reaction). It should be noted that nitric oxide (NO) can also be oxidized into reactive nitric oxide species, which may show behavior similar to that of ROS. In particular the combination of NO and O 2 .2 can yield a strong biological oxidant, peroxynitrite that is more detrimental to the cells [10,39]. In our study, we showed that treatment of Tb4 restored all H 2 O 2 induced free radical generation in cardiomyocytes suggesting a protective role in this setting. One of the traditional hallmarks of ROS-initiated cell death is mitochondrial dysfunction and energy depletion [40,41]. Several mechanisms can impair energy production in cardiac mitochondria, including damage to the electron transport chain and phosphorylation apparatus, mtDNA injury, opening of the mitochondrial permeability transition pore (MPTP), the loss of the mitochondrial membrane potential (DYm) and, the concomitant drop in ATP production [42,43]. Dysfunction of mitochondrial machinery in the heart releases apoptotic signaling molecules e.g. cytochrome c and may cause an irreversible injury to the mitochondria [44]. Our data showed significant decrease in DYm which was prevented by pretreatment with Tb4.
Tb4 is very effective in reducing intracellular ROS in H 2 O 2treated cardiomyocytes. Our study is the first to show that the attenuation of ROS is mediated by restoring Cu/Zn-SOD and catalase, the two important antioxidant enzymes. Another relevant antioxidant that loses function upon oxidation is Mn-SOD. Although, both Mn-SOD and Cu/Zn-SOD have been reported to play a crucial role in protecting the cardiac cells from oxidative damage by scavenging ROS [13,45] but, we found that Tb4 upregulated the expression levels of Cu/Zn-SOD in cardiomyocytes. Catalase, which was directly responsible for H 2 O 2 clearance, was upregulated by Tb4 both at mRNA and protein level in the presence of H 2 O 2 stimulus indicating that Tb4 preferentially targets catalase in the cardiomyocytes which enable effecting scavenging of the H 2 O 2 from the system. Also it was worth notice that even though the protein and gene expression levels of both catalase and Cu/Zn-SOD were increased by Tb4, this peptide upregulated the gene encoding the former more efficiently in cardiomyocytes. Furthermore, oxidative stress promotes apoptotic cell death by lowering Bax/Bcl 2 ratio. In our study, we showed that Tb4 reduced the intracellular ROS levels in cardiomyocytes and prevents cell death by restoring Bax/Bcl 2 ratio and inhibiting the activation of caspase3. This observation supports our previous observation using cardiac fibroblast [31] but, contrast to the previous reported by Sosne G et al where they did not observe any change in Bax/Bcl 2 expression [30]. We did not know the reason for this but, the use of different cell type may accountable for this altered phenomenon.
To confirm the target of Cu/Zn-SOD and Bcl 2 by Tb4 in order to protect the cardiomyocytes from oxidative stress, we selectively knocked down these molecules and determined the efficacy of Tb4 under oxidative stress. We found that Tb4 prevented cell death by specifically targeting Cu/Zn-SOD and Bcl 2 molecules in H 2 O 2 treated cardiomyocytes. But, when these molecules were knocked down in the cell, Tb4 failed to protect the cells from apoptosis. These data led us to convey the message that Tb4 may provide cardiac protection under oxidative stress by restoring Cu-Zn SOD and Bcl 2 levels in the myocardium.
Our study also indicates that Tb4 protects the cardiomyocytes from oxidative stress by attenuating pro-inflammatory genes regulated by NF-kB. It is evident that ROS activation often triggers NF-kB translocation and thereby promotes pro-inflammatory response [46,47] As mentioned previously, ROS are toxic in cells and damage the cellular integrity, it is therefore, critical to make a balance of ROS production in order to prevent further oxidative damage. In this setting, our study further indicates that Tb4 protects the cardiomyocytes from oxidative stress by attenuating the pro-inflammatory genes regulated by NF-kB. Taken together, our data validate and re-established a potential role Tb4 as an anti-inflammatory molecule which may provide a new therapeutic module for cardiac protections under oxidative stress. Future studies may aim to delineate the interaction or association between NF-kB and Tb4 in the context of NF-kB transcriptional regulatory circuit and anti-inflammatory properties in the cardiac cells.
In conclusion, we demonstrated that Tb4 protects the myocardium from oxidative stress by reducing ROS activity via re-establishing the antioxidant enzyme levels, Cu/Zn-SOD and catalase and, further attenuating Bax and caspase3 levels and restoring Bcl 2 as well. Our results not only offered more mechanistic explanation about the protective mechanism of Tb4 but also supported the need to further investigate the use of this small molecule in protecting the myocardium against oxidative damage in variety of disease condition where ROS has been implicated to play a damaging role like cardiac hypertrophy and heart failure.

Therapeutic implication
Our findings are relevant in the clinical settings as many studies have shown that depletion of anti-oxidants in the heart makes it more vulnerable to damage especially under ischemia and under high pro-oxidant condition. Although, we did not investigate adult rat cardiomyocytes, but, many studies have shown that primary cultured neonatal rat cardiomyocytes were useful models to investigate cardio-protective effects. Future studies are, therefore, warranted to examine the effect of Tb4 under the similar setting. We believe that Tb4 is a better therapeutic target as it has the ability to enhance the expression of the selected antioxidant and anti-inflammatory genes, thereby, alleviating the damage to the myocytes under oxidative stress. These possibilities regarding the mechanisms whereby Tb4 modulates the above molecules need to be further tested experimentally in future studies.

Supporting Information
Table S1 NF-kB RT 2 PCR array Cells were treated with H 2 O 2 in the presence and absence of Tb4 and NF-kB RT 2 PPCR array was performed using a kit from SA Bioscience according to the manufacturer's protocol. (DOCX)

Author Contributions
Conceived and designed the experiments: SG. Performed the experiments: CW SK IKK. Analyzed the data: SG CW SK IKK. Wrote the paper: SG. .