Thymosin Beta 4 Prevents Oxidative Stress by Targeting Antioxidant and Anti-Apoptotic Genes in Cardiac Fibroblasts

Rationale Thymosin beta-4 (Tβ4) is a ubiquitous protein with diverse functions relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory responses. The effecter molecules targeted by Tβ4 for cardiac protection remains unknown. The purpose of this study is to determine the molecules targeted by Tβ4 that mediate cardio-protection under oxidative stress. Methods Rat neonatal fibroblasts cells were exposed to hydrogen peroxide (H2O2) in presence and absence of Tβ4 and expression of antioxidant, apoptotic and pro-fibrotic genes was evaluated by quantitative real-time PCR and western blotting. Reactive oxygen species (ROS) levels were estimated by DCF-DA using fluorescent microscopy and fluorimetry. Selected antioxidant and antiapoptotic genes were silenced by siRNA transfections in cardiac fibroblasts and the effect of Tβ4 on H2O2-induced profibrotic events was evaluated. Results Pre-treatment with Tβ4 resulted in reduction of the intracellular ROS levels induced by H2O2 in the cardiac fibroblasts. This was associated with an increased expression of antioxidant enzymes Cu/Zn superoxide dismutase (SOD) and catalase and reduction of Bax/Bcl2 ratio. Tβ4 treatment reduced the expression of pro-fibrotic genes [connective tissue growth factor (CTGF), collagen type-1 (Col-I) and collagen type-3 (Col-III)] in the cardiac fibroblasts. Silencing of Cu/Zn-SOD and catalase gene triggered apoptotic cell death in the cardiac fibroblasts, which was prevented by treatment with Tβ4. Conclusion This is the first report that exhibits the targeted molecules modulated by Tβ4 under oxidative stress utilizing the cardiac fibroblasts. Tβ4 treatment prevented the profibrotic gene expression in the in vitro settings. Our findings indicate that Tβ4 selectively targets and upregulates catalase, Cu/Zn-SOD and Bcl2, thereby, preventing H2O2-induced profibrotic changes in the myocardium. Further studies are warranted to elucidate the signaling pathways involved in the cardio-protection afforded by Tβ4.


Introduction
Reactive oxygen species (ROS) plays an important role in regulating a variety of cellular functions, including gene expression, cell growth and cell death and has been implicated as one of the major contributors of cardiac damage in various cardiac pathologies [1,2,3]. Increased ROS levels can cause damage to nucleic acids, lipids, and proteins and can directly damage the vascular cells, cardiac myocytes and cardiac fibroblasts [3,4]. Oxidative stress has been shown to be a precursor of cardiac apoptosis and has also been implicated in cardiac hypertrophy and fibrosis [5,6,7]. The declining protective enzymes and the reduced adaptive capacity to counter the oxidative stress cause activation of apoptotic death pathways [8]. Although, cells, tissues and organs utilize multiple layers of antioxidant defenses and damage removal, the heart is particularly more vulnerable to oxidative damage as it has a weak endogenous antioxidant defense system [9].
It has been suggested that an increased level of oxidative stress heart is primarily due to the functional uncoupling of the respiratory chain caused by inactivation of complex I in the mitochondria [10] or due to impaired antioxidant capacity, such as reduced activity of Cu/Zn superoxide dismutase (Cu/Zn-SOD) and catalase [6], or stimulation of enzymatic sources, including xanthine oxidase, cyclo-oxygenase, nitric oxide synthase, and nonphagocytic NAD(P)H oxidases [11]. Irrespective of the source of the stress stimuli involved, oxidative damage remains the main challenge and numerous efforts have been made to devise strategies to protect the heart against oxidative damage. Considerable attempts have been made in the recent decades to discover an ''ideal'' cardio-protective agent, which can abrogate the oxidative damage and maladaptive changes in the heart. In this pursuit, thymosin b4 (Tb4) emerged as a powerful candidate.
Tb4, a 43 amino acids, ubiquitous intracellular protein, bind to and sequester G-actin to modulate cell migration [12]. Recent studies implicated that Tb4 had multiple diverse physiological and pathological functions, such as wound healing, angiogenesis, preserved cardiac function after myocardial infarction which essentially depends on cell migration [13,14,15]. Essentially, Tb4 possesses cardiac tissue repair properties that cause epicardial cell migration, neovascularization and revascularization, and activation of cardiac progenitor cells in the heart [16,17]. Apart from the cardiac repair properties, it has been demonstrated that Tb4 protects human corneal epithelial cells and conjunctival cells from apoptosis after ethanol and hydrogen peroxide (H 2 O 2 ) injury [18,19,20]. A few reports exhibit the role of Tb4 in the myocardial infarction settings where it promotes endothelial and myocardial cell survival, cardiac cell migration, activation of integrin linked kinase (ILK) and activation of Akt/Protein Kinase B that resulted in improved cardiac function and reduction in scarring [14,16,21]. In this context, we have shown that Tb4 also plays a crucial role in cardiac protection by enhancing the levels of PINCH-1-ILK-aparvin components and promotes Akt activation while substantially suppressing NF-kB activation [22]. A recent report showed that Tb4 upregulates anti-oxidative enzymes in human corneal cells against oxidative stress [23].
In this study, we used rat neonatal cardiac fibroblasts to study the effects of Tb4 under oxidative stress. The rationale for using cardiac fibroblasts in this study originates from the fact that apart from being the most abundant cell type in the mammalian heart [24,25,26], the fibroblasts are intricately involved in myocardial development and cardiac protection under various stimuli [27,28]. Recently, it has been shown that cardiac fibroblasts also undergo apoptosis under myocardial damage which can further aggravate the myocardial injury [29] and this can occur independent of the myocyte death [30]. The action of Tb4 on cardiac fibroblasts under oxidative stress and its effects are largely unknown. It was for this reason we utilized the cardiac fibroblasts in the in vitro model to study the cardioprotective effects of Tb4 under oxidative stress.
Here, we showed the effects of Tb4 on ROS act ivy, antioxidant enzymes, anti-apoptotic and pro-fibrotic gene expression in preventing H 2 O 2 induced oxidative stress in rat cardiac fibroblasts.

Cell culture and treatment
Primary cultures of cardiac fibroblasts were prepared from ventricles of 1-3 day-old Wistar rats as described [31,32] and were plated at a field density of 2.5610 4 cells per cm 2 on coverslips, 6well plates, 60 mm culture dishes, or 100 mm dishes as required with DMEM containing 10% FBS. After 24 h, cells were serum deprived overnight before stimulation. The dose of 100 mM H 2 O 2 did not show any toxic effect and damage to the cells and was therefore used throughout the study. Tb4 was pretreated 2 h before the H 2 O 2 challenge at a final concentration of 1 mg/mL which was based on the previous report [23]. The cardiac cell culture experiments were approved by Institutional Biosafety Committee (IBC) and IACUC, Texas A & M University, Temple, TX.

Standardization of H 2 O 2 dose on cardiac fibroblasts
To determine the optimal sub-lethal working concentration of H 2 O 2 that is sufficient to induce oxidative stress but does not cause considerable cell death, the cell viability of cardiac fibroblasts under oxidative stress using a dose course of H 2 O 2 (1 to 250 mM) was evaluated by MTT assay as described [33]. The absorbance was measured at 570 nm using a microplate reader (Molecular Devices, SpectraMax 250). The effect of Tb4 (1 mg/mL) was assessed on the H 2 O 2 challenge and the cytotoxicity curve was constructed and expressed as percentage cell viability compared to control. To rule out the possible direct interaction between MTT with H 2 O 2 or Tb4, the treatment media was aspirated before the addition of MTT solution.

Quantification of intracellular ROS levels
Cardiac fibroblasts after respective treatments were incubated with 50 mM H 2 DCFH-DA at 37uC in the dark for 30 min as described [33]. To rule out the possible direct cleavage of nonfluorescent DCFH-DA to fluorescent DCF by the nonspecific intracellular esterases and extracellular esterases in the serum, the treatment media was completely removed before H 2 DCFH-DA staining. Cells were then harvested, washed with phosphatebuffered saline (PBS), and re-suspended in 50 mM HEPES buffer (5 mM HEPES, pH 7.4; 5 mM KCl, 140 mM NaCl, 2 mM CaCl 2 , 1 mM MgCl 2 and 10 mM glucose) before their fluorescence intensities were acquired by fluorimetry (SpectraMaxPro, USA).

Confocal Microscopy
For qualitative estimation of the levels of intracellular ROS, cells were seeded on coverslips in 6-well plates, treated and subsequently incubated with 50 mM H 2 DCFH-DA at 37uC in the dark for 30 min as previously described [33]. Cells were then fixed with 2% paraformaldehyde, washed 3 times with PBS, and mounted using anti-fade 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 (Olympus, Tokyo, Japan). Since, ROS induces generation of other ROS species and loss of mitochondrial membrane potential, we estimated the levels of other free radicals like superoxide and nitric oxide using fluorescent probes. For this, DHE, DAF-2DA and MitoTracker Red were used for estimation of superoxide (O 2 .-) radicals, nitric oxide (NO) and mitochondrial membrane potential (Dym), respectively [33]. Image intensities from 15-20 fields of at least 3-4 confocal images of the same treatment group were quantified using ImageJ version 1.34 (NIH, Bethesda, USA) as described previously by Elia et. al. [34] Western blot analysis For western blot analysis, cardiac fibroblasts were treated with or without Tb4 for 2 h before treatment with 100 mM of H 2 O 2 . Cells were washed with PBS and cytosolic protein extracts were prepared using 1X Cell Lysis buffer (Santa Cruz Biotechnology, CA, USA) supplemented with protease inhibitor cocktail. Protein concentrations were determined using the Bradford assay (Bio-Rad) as per manufacturer's protocol. Aliquots of protein lysates (40 mg/lane) were separated on sodium dodecyl sulfate-10% polyacrylamide gels and western blotting was performed as described previously [31]. The primary antibodies used in this study include Mn-SOD, Cu/Zn-SOD, catalase, Bcl 2 , Bax, Caspase-3, Col-I, Col-III, CTGF and GAPDH. The detection was performed using chemiluminescence assay (Cell Signaling Technology, Beverly, MA). Membranes were exposed to x-ray film to observe the bands (Kodak, Rochester, NY). The quantification of each western blot was measured by densitometry as described previously [31]. The changes observed as a result of H 2 O 2 stimulation were expressed as fold change while the changes observed as a result of Tb4 treatment were depicted as percent increase or decrease unless otherwise noted.

RNA isolation and quantitative real-time PCR (q-RT-PCR) analyses
Total RNAs from the cardiac fibroblasts were extracted using RNEasy kit (Qiagen, Stanford, Valencia, CA) as per the manufacturer's instructions. For real-time RT-PCR, 200 ng to 1 mg of total RNAs was reverse transcribed to cDNA using high capacity cDNA synthesis kit (Applied Bio systems, Foster City, CA, USA) following the manufacturer's protocols. Quantitative PCR was then carried out in a MX-3005 real-time PCR equipment (Stratagene, Cedar Creek, USA), using 2 ml (20 ng) of cDNA template, 1 nmol of primers, and 12.5 ml of iQ SYBR green supermix (Bio-Rad, Hercules, CA) in a total volume of 25 ml reaction. The primers used for the quantitative PCR are shown in Table 1. The cDNA were amplified with initial denaturation at 95uC for 10 min, followed PCR by 40 cycles of: 95uC 30 s, 60uC 30 s, 72uC 40 s and finally 1 cycle of melting curve following cooling at 40uC for 10 s. To confirm amplification specificity the PCR products from each primer pair were subjected to a melting curve analysis. Analysis of relative gene expression was done by evaluating q-RT-PCR data by 2 (-DDCt) method as described by others [35,36]. Each experiment was repeated at least three times and GAPDH or 18S was used as housekeeping gene for internal control. The changes observed as a result of H 2 O 2 stimulation were expressed as fold change while the changes observed as a result of Tb4 treatment were depicted as percent increase or decrease unless otherwise noted.

RNA interference and siRNA transfection
Silencing of Cu/Zn-SOD, catalase and Bcl 2 proteins was achieved by using small interfering RNA (si-RNA) transfections. Pre-designed double-stranded si-RNA against Cu/Zn-SOD (SA-SI_Rn01_00112871), catalase (SASI_Rn01_00053417) and Bcl 2 (SASI_Rn01_00062026) were purchased from Sigma Life Science (Saint Louis, MO, USA). As a negative control, non-targeting control si-RNA (scrambled si-RNA), verified to have no significant effect on most essential mammalian genes, was obtained from Sigma. Rat neonatal fibroblasts were seeded into 6-well plates with 3 mL of complete DMEM. After 24 h, the medium was replaced with 2 mL of Opti-MEM (Life Technologies). Cells were then transfected with 200 pmol of the siRNAs for catalase, Cu/Zn-SOD, Bcl 2 or negative control siRNA using N-TER TM nanoparticle siRNA transfection system (Sigma) as per the manufacturer's protocol. After 24 h of transfection, cells were treated and harvested to determine the transfection efficiency and effect of Tb4 in presence and absence of H 2 O 2 treatment. Quantification of caspase-3 gene expression by q-RT-PCR was done to study the extent of pro-apoptotic gene expression.

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). Briefly, 2610 5 fibroblasts were seed on the coverslips in the 6-well plates, followed by siRNA transfections and treatment. The TUNEL staining was done using deoxyneucleotidyl transferase (TdT) and FITC-labeled 29-deoxyuridine 59-triphosphate (dUTP) as per manufacturer's instructions. The Analysis was done using 3-5 images from hi-power field (40X objective) and the TUNEL index (%) was calculated as the number of TUNEL-positive cells divided by the total number of DAPI-stained nuclei.

Statistical analyses
All experiments were performed at least three times for each determination. Data are expressed as means6Standard Error (SE) and were analyzed using student t-test or one-way analysis of variance (ANOVA) and secondary analysis for significance with Tukey-Kramer post tests using Prism 5.0 Graph Pad software (Graph Pad, San Diego, CA, USA). A p value less than 0.05 was considered statistically significant.

Pretreatment with Tb4 improves cardiac fibroblasts survival
The viability of cardiac fibroblasts cells was assessed after treating the cells with various concentrations of H 2 O 2 for 24 h by MTT assay. We observed that the 50% lethal dose (LD 50 ) of H 2 O 2 on these cells was between 150 and 250 mM ( Figure 1A). Pretreatment with Tb4 (1 mg/mL) resulted in an improved cell survival count (19.2%; p,0.05) compared to the untreated group. This beneficial effect of pretreatment was observed until 100 uM

Tb4 protects cardiac fibroblasts against H 2 O 2 induced oxidative stress
Oxidative stress leads to detrimental changes in the cell signaling process. To determine whether Tb4 has any effect on ROS activity under oxidative stress, intracellular ROS levels in the cardiac fibroblast were estimated after H 2 O 2 treatment by confocal microscopy and fluorimetry. Cardiac fibroblasts treated with H 2 O 2 showed enhancement of fluorescence intensity of DCF-DA by 9.9-fold at 12 h (p,0.01) and 13.8fold at 24 h (p,0.01), respectively, compared to the untreated controls ( Figure 1B). Pretreatment with Tb4 showed a 50.2% decrease at 12 h (p,0.05) and 75.1% at 24 h (p,0.05), respectively, compared to H 2 O 2 treated cardiac fibroblasts. Tb4 treatment reduced the DCF-DA fluorescence by 32.2% (p,0.01) in the unstimulated cardiac fibroblast indicating that Tb4 treatment was effective in preventing oxidative stress in cardiac fibroblasts. The confocal microscopy images matched with the quantitative fluorimetry results ( Figure 1C). Our data indicated that Tb4 abrogated the ROS activity significantly in cardiac fibroblasts.

Tb4 reduces the formation of superoxide radicals and nitric oxide
Oxidative stress generated superoxide and nitric oxide radicals. These can lead to production of more noxious free radicals, like peroxynitrite (ONOO -). To evaluate the effect of Tb4 in superoxide and nitric oxide radical generation in cardiac fibroblasts, we estimated the levels of those free radical generations from H 2 O 2 treatment by using confocal microscopy. The confocal microscopy data showed that H 2 O 2 treated cells enhanced the fluorescence intensity of DHE and DAF-2DA, an indicator of increased O 2 .and NO radicals, compared to the controls ( Figure 1D and E). The enhanced fluorescence intensity of DHE and DAF-2A was prevented by Tb4 pretreatment, compared to the H 2 O 2 treated cells ( Figure 1 D and E). The quantifications of image intensities were tabulated in Table 2. Together, our data suggest that Tb4 attenuated H 2 O 2 induced superoxide and nitric oxide radical generation in cardiac fibroblasts.

Tb4 treatment prevents the loss of mitochondrial membrane potential (Dym)
Oxidative stress impacts the mitochondrial function leading to disruption of electron transport and loss of mitochondrial transmembrane potential. We evaluated the effect of Tb4 on the mitochondrial membrane potential in the cardiac fibroblast under H 2 O 2 induced oxidative stress using MitoTrackerH Red by confocal microscopy. Our data showed that there was loss of Dym as indicated by a decrease in the fluorescence intensity of MitoTrackerH Red in H 2 O 2 -treated cells. This decrease in the fluorescence intensity was prevented by the pretreatment with Tb4 (Figure 1 F) suggesting that Tb4 protected mitochondrial function by preventing the loss of Dym in the cardiac fibroblast. The quantifications of image intensities were tabulated in Table 2. Our data suggest that Tb4 treatment significantly reduced mitochondrial potential in H 2 O 2 treated cardiac fibroblasts.  Figure 2B). In unstimulated cell, the mRNA expression of catalase was upregulated by 1.3-fold after Tb4 treatment. H2O2 treatment reduces the catalase mRNA expression by 50% (p,0.05) and 89% (p,0.05) at 12 and 24 h treatment, respectively, compared to the control. Pretreatment with Tb4 upregulated the catalase mRNA expression by 1.5-fold (p,0.05), and 6.4-fold (p,0.05) at 12 h and 24 h, respectively, compared to the H 2 O 2 treated cells ( Figure 2C).
To examine the status of the above antioxidant genes at the translational levels, western blots were performed. Our data showed that the expression level of Mn-SOD was relatively unchanged at protein level at 12 h and 24 h, respectively. Treatment with Tb4 did not alter the expression of Mn-SOD ( Figure 2D). Compared to the untreated cells, H2O2 treatment showed a decline of Cu/Zn-SOD protein expression by 23% (p,0.05) and 24.6% (p,0.05) at 12 h and 24 h, respectively, compared to the controls. Cells pretreated with Tb4 showed a 2.0-fold (p,0.05) and 1.4-fold (p,0.05) increased in the expression of Cu/Zn-SOD at 12 h and 24 h treatment, respectively, compared to the H 2 O 2 treated cells ( Figure 2D). The expression of catalase declined by 25% (p,0.05) at 12 h and 15% (p,0.05) at 24 h, respectively, with H 2 O 2 treatment compared to untreated cells. Tb4 pretreatment increased catalase expression by 1.3-fold (p,0.05) and 1.4-fold (p,0.05) increase at 12 h and 24 h, respectively, compared to the H 2 O 2 treated cells ( Figure 2D). The normalized quantification for Mn-SOD, Cu/ Zn-SOD and catalase expression by densitometry is shown in the Figure 2E. Tb4 reduced the expression of pro-fibrotic genes in cardiac fibroblast under oxidative stress We next determine the pro-fibrotic gene expression under oxidative stress condition. Our data showed that H 2 O 2 treatment increased the mRNA expression of CTGF by 2.3-fold (p,0.05) and 2.3-fold (p,0.05) at 12 h and 24 h, respectively, compared to the controls. Tb4 treatment showed reduction in the mRNA expression of CTGF by 17% (p,0.05) and 38% (p,0.05) at 12 h and 24 h, respectively (Figure 4 A). The mRNA expression of collagen-1 was increased by 4.0-fold (p,0.05) and 5.7-fold (p,0.05) at 12 h and 24 h, respectively, in H 2 O 2 -treated cells compared to the controls. Tb4 pretreatment decreased collagen-I by 46% (p,0.05) and 42% (p,0.05) at 12 h and 24 h, respectively, compared to the H 2 O 2treated cells (Figure 4 B). The mRNA expression of fibronectin was increased by 2.1-fold (p,0.05) and 3.3-fold (p,0.05) at 12 h and 24 h, respectively, in the H 2 O 2 -treated cells. The mRNA expression of fibronectin in the Tb4 treatment was decreased by 9% (p = ns) and 34% (p,0.05) at 12 h and 24 h, respectively, compared to the H 2 O 2 -treated cells ( Figure 4C).
At translational level, we did not observe any significant change in the CTGF protein level under oxidative stress. However, pretreatment with Tb4 reduced the expression of CTGF by 27% (p,0.05) and 43% (p,0.05) at 12 h and 24 h, respectively, compared to the H 2 O 2 treated cells. The collagen-I protein expression also increased in H 2 O 2 treatment by 24% (p,0.05) and 21% (p,0.05) at 12 h and 24 h treatment, respectively, compared to the controls. Tb4 treatment reduced collagen-I protein  (Figure 4 D). The normalized quantification of each western blot profile is shown (Figure 4 E).

Effect of knocking down of Cu/Zn-SOD, catalase and Bcl 2 in cardiac fibroblasts
We first determine the efficacy of siRNA mediated transfection of Cu/Zn-SOD, catalase and Bcl 2 in cardiac fibroblasts. Western blots were performed to confirm the specific knock-down of Cu/ Zn-SOD, catalase and Bcl 2 in cardiac fibroblasts.
To determine whether Tb4 has target for Cu/Zn-SOD, catalase and Bcl 2 , we tested the effect of Tb4 in the cardiac fibroblasts either transfected with scrambled siRNA or siRNA of catalase, Cu/Zn-SOD, and Bcl 2 in the presence and absence of H 2 O 2 induced oxidative stress. Treatment with scrambled siRNA did not alter the expression of catalase, Cu/Zn-SOD, and Bcl 2 ; therefore, scrambled siRNA treated group was taken as control for this experiment. Cardiac fibroblasts transfected only with scrambled siRNA showed 15.565.4%, 12.666.4% and 18.160.9% (p,0.05) increased protein expression of catalase, Cu/Zn-SOD and Bcl 2 , due to Tb4 treatment (Fig 5 B, D   The down regulation in the levels of catalase, Cu/Zn-SOD and Bcl 2 proteins was abrogated by pre-treatment of Tb4 (Fig 5 A, C and E, 2 nd lane). We observed that the expression of catalase, Cu/ Zn-SOD, and Bcl 2 was upregulated by 84.565.4%, 45.6611.2%, 39.866.4%, respectively, p,0.05, compared to their control group (Fig 5 A, C and E, 1 st lane). Furthermore, to evaluate the effect of Tb4 under more stringent conditions, the siRNA-treated fibroblasts were subjected to H 2 O 2 challenge, to further enhance the levels of oxidative stress. Our data showed that H 2 O 2 treatment further depleted the levels of catalase, Cu/Zn-SOD and Bcl 2 ( Figure 5A, C, E (3 rd Lane). This dual stress ultimately reduced the expression of catalase, Cu/Zn-SOD and Bcl 2 to 23.363.8%, 17.264.1% and 12.762.1% (p,0.05) of the endogenous levels ( Figure 5A, C, E; 3 rd Lane). Tb4 pretreatment resulted in a significant upregulation of catalase and Cu/Zn-SOD (142.467.3% and 36.564.1%, respectively, p ,0.05) but not for Bcl 2 (12.568.1%, p.0.05), compared to respective controls ( Figure 5A, C, E (4 th Lane). This implicates that under conditions Tb4 restored the levels of catalase and Cu/Zn-SOD but failed to restore the expression of Bcl 2 . GAPDH was used as an internal  These results indicate that Tb4 selectively targets antioxidant and anti-apoptotic genes to provide protection under oxidative stress.

Discussion
The current study was aimed to determine the target molecules modulated by Tb4 that enables it to prevent oxidative stress in an in vitro system using cardiac fibroblasts. H 2 O 2 induced a marked increase in intracellular ROS in the cardiac fibroblasts. This increase in the intracellular ROS led to altered expression of antioxidant enzymes like the SODs and catalase. Increased ROS also led to the loss of mitochondrial membrane potential and subsequently an increase in the Bax/Bcl 2 ratio favoring apoptosis. This study shows for the first time that Tb4 reduced the ROS accumulation by modulating the expression of antioxidant enzymes especially catalase and Cu/Zn-SOD and thereby preventing the loss of mitochondrial membrane potential. Additionally, Tb4 upregulated the expression of anti-apoptotic protein Bcl 2 and prevented apoptosis of cardiac fibroblasts in response to oxidative stress. Tb4 failed to alleviate the oxidative stress and prevent cardiac apoptosis when these molecules were knocked down in the cells. Thus, Tb4 possibly targets antioxidant and anti-apoptotic genes to provide protection under oxidative stress in cardiac fibroblasts.
The myocardium is composed of cardiomyocytes, fibroblasts, endothelial cells and other cell types along with surrounding extracellular matrix (ECM). Apart from the myocytes, cardiac fibroblasts make up the bulk of the myocardium and important cell type as they can play crucial role in cardiac protection by providing sustenance, ECM support and defense against various stress conditions. Therefore, in this study, we used cardiac fibroblasts, so as to study the effect of Tb4 on this cell type and to study how oxidative stress affects them in the in vitro system.
In the cardiac cells, H 2 O 2 is known to induce oxidative stress and apoptosis. This system has been used on various occasions as a model to study the regulation of stress signaling stimuli in cell death [37,38]. Here, we used this model system to show the effects of Tb4 on H 2 O 2 induced fibroblast cell death. Our results indicate that 2 h pretreatment with Tb4 is able to significantly decrease the apoptotic effects of H 2 O 2 in cardiac fibroblasts and thus protecting   [39,40,41]. Also, 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 .can yield a strong biological oxidant, peroxynitrite that is more detrimental to the cells [42]. Oxidative stress causes altered expression of antioxidant enzymes SOD and catalase which prevents effective scavenging of the ROS formed as a result of H 2 O 2 treatment in the fibroblasts. Our results show that Tb4 restored the levels of Cu/Zn-SOD and catalase close to normal physiological level even under oxidative stress and thus scavenging the extra H 2 O 2 -induced ROS from the cellular system. One of the traditional hallmarks of ROS initiated cell death is mitochondrial dysfunction and energy depletion [43]. This is manifested by opening of the mitochondrial permeability transition pore (MPTP), the collapse of the mitochondrial membrane potential (Dym) and a concomitant drop in ATP production [44]. These events lead to cascade of cell destruction and apoptosis. Increased production of ROS in the failing heart leads to mitochondrial permeability transition [45], which causes loss of mitochondrial membrane potential, swelling of mitochondrial matrix, release of apoptotic signaling molecules, such as cytochrome c, from the inter-membrane space, and irreversible injury to the mitochondria [46]. Increased ROS in our system led to a decrease in the Dym as evident by the staining with MitoTracker Red. This loss of Dym was prevented by pretreatment of Tb4. At this point, it is beyond our scope to investigate how Tb4 modulate mitochondrial membrane potential under oxidative stress and, therefore, warranted further investigation.
Tb4 was extremely effective in reducing intracellular ROS in H 2 O 2 treated cardiac fibroblasts (Figure 2 A-C). Tb4 acts via upregulation of selected antioxidant genes like Cu/Zn-SOD and catalase. In fact, the intracellular ROS level was markedly reduced when the cells were pretreated with Tb4 at least 2 h prior to H 2 O 2 exposure, suggesting that it might activate the key molecules that play an important role in the enzymatic antioxidant defense system. Another particularly relevant protein that loses function upon oxidation is Mn-SOD; its loss of function would further compromise antioxidant capacity and lead to further oxidative stress [47]. 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 [48]. In our experimental system, we found that Tb4 upregulated the expression levels of Cu/Zn-SOD and not Mn-SOD in cardiac fibroblast thus affording cardiac protection which is in contrast to the previous report by Ho et al [23]. This could be probably due to the different cell type used in the study. Catalase, which was directly responsible for H 2 O 2 clearance, was upregulated by Tb4 both at protein and gene level in the presence of H 2 O 2, indicating that Tb4 preferentially targets catalase which enables effecting scavenging of the H 2 O 2 from the system. The mechanism of Cu/Zn-SOD and catalase upregulation by Tb4 is currently unknown but, a transcription factor mediator activity has been postulated [49]. Tb4 has been reported to translocate into the nucleus by an active transport mechanism or possibly through its cluster of positively charged amino acid residues (KSKLKK) but the exact function is still obscure. Alternatively, it might be the similar event like nuclear localization of actin where it is postulated that it might involve in chromatin remodeling [50], mRNA processing and transport [51].
Improved fibroblast survival during oxidative stress is important because increased fibroblast survival belittles the cardiac injury and prevents overall damage to the heart. Oxidative stress is known to trigger apoptotic cell death by up regulating the expression of pro-apoptotic protein like Bax which then dimerises and translocate to the outer mitochondrial membrane and aggregates to form pores that permit cytochrome c from mitochondria to cytosol [52]. Oxidative stress also leads to activation of caspase-3 cleavage and initiation of intrinsic apoptotic pathway [52]. Therefore, reduction in the production and accumulation of intracellular ROS is important to protect the cells from apoptosis triggered by the intrinsic pathway. Our data further showed that Tb4 treatment inhibits excessive Bax expression and caspase-3 activation and enhance Bcl 2 expression in fibroblast suggesting its protective effects under oxidative stress. As Tb4 reduces the intracellular ROS levels, it would be reasonable to explain that Tb4 probably promote the Bcl 2 /Bax ratio to protect the cells from apoptosis. To best of our knowledge, this is the first report that elucidates the beneficial effect of Tb4 in cardiac fibroblast by up regulating the Bcl 2 /Bax ratio in favor of cell survival. This finding is in contrast to the previous report by Sosne et al, where they did not observe any change in the Bax/ Bcl 2 expression [18]. This could be probably due to the different cell type used in the study. Although, there is no definitive mechanism by which Tb4 exerts this anti-apoptotic effects, but, an internalization of Tb4 has been proposed as one of the possible mechanism in human corneal epithelial cell under oxidative stress [19]. Alternatively, Tb4 may use its methionine residues to react with intracellular oxygen at the post-translational level to generate sulfoxide, reducing the ROS level and eventually protects the cells from apoptosis [53].
Increased oxidative stress leads to pro-fibrotic changes in the fibroblasts [54,55]. We observed that there was an increase in the expression of pro-fibrotic genes like CTGF, Collagen-I, Collgen-III and fibronectin in cardiac fibroblasts treated with H 2 O 2 . Decreased expression of fibrotic genes in the fibroblasts is beneficial because it prevents cardiac fibrosis and stiffening by reducing the ECM deposition. Our study showed that the increase in the pro-fibrotic gene expression was prevented by pretreatment of fibroblasts with Tb4. These results corroborated with our previous observation in myocardial infarction (MI) model [22]. We showed that Tb4 abrogated cardiac fibrosis in post-MI period by attenuating collagen type I and type III gene expression. This is the first report that elucidates the down regulation of pro-fibrotic gene expression by Tb4 under oxidative stress.
Interestingly, we found that Tb4 prevented apoptotic cell death by specifically targeting these antioxidant and anti-apoptotic molecules and when these antioxidant and apoptotic molecules were knocked down by si-RNA treatment; Tb4 was not able to contribute its cardio-protective effects. These findings led us to suggest that Tb4 provide cardiac protection by reducing the intracellular ROS levels and enhancing the expression of antioxidant enzymes (Cu/Zn-SOD and catalase) and antiapoptotic protein (Bcl 2 ) under oxidative stress.
In conclusion, we demonstrated that Tb4 protects the cardiac fibroblasts against apoptosis by reducing intracellular oxidative stress through enhancing the expression of selected anti-oxidative enzymes and anti-apoptotic proteins. Given the preference of cardio-protective effects of Tb4, it is still not clear how Tb4 exerts its beneficial effects, is still under investigation. Although, Tb4 is internalized by cells but the cell surface receptors are still not known. Furthermore, high concentration and ubiquitous presence of Tb4 in the organs/tissues, it is reasonable to advocate that Tb4 functions as an important intracellular mediator when either release from the cells or exogenously added, acts as a moonlighting peptide for repairing the damages tissues or cells [56,57]. 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 peptide in protecting the myocardium against oxidative damage in variety of disease condition where ROS has been implicated to play a damaging role. Also, further investigations are needed to explore the antifibrotic properties of Tb4 which could be used to alleviate detrimental conditions like cardiac fibrosis, hypertrophy and heart failure.

Clinical Implications
Many studies have shown that there is a depletion of antioxidant levels due to aging especially in the heart, which makes it more vulnerable to damage especially under ischemia and under high pro-oxidant condition. We believe that the identified molecules (Cu/Zn-SOD, catalase and Bcl 2 ) are important players under oxidative stress to mitigate the damage. Our findings indicated that the beneficial role of Tb4 and its effects on upregulation of anti-oxidant genes under oxidative stress in the cardiac fibroblasts might be of clinical relevance. This makes Tb4 a better therapeutic target and could have wider implications once translated from bench to bedside.

Limitation of the Study
In this study, we validated the effect of Tb4 by selectively knocking down the anti-oxidant and apoptotic genes targeted by Tb4 in cardiac fibroblasts. However, the possibility of interaction between these genes and other molecular pathways cannot be ignored. Also other off target effects of our anti-oxidative strategies with Tb4 in conditions like cardiac fibrosis, cardiomyopathies and heart failure needs to be further investigated.