Inhalation of Hydrogen Attenuates Progression of Chronic Heart Failure via Suppression of Oxidative Stress and P53 Related to Apoptosis Pathway in Rats

Background: Continuous damage from oxidative stress and apoptosis are the important mechanisms that facilitate chronic heart failure (CHF). Molecular hydrogen (H2) has potentiality in the aspects of anti-oxidation. The objectives of this study were to investigate the possible mechanism of H2 inhalation in delaying the progress of CHF. Methods and Results: A total of 60 Sprague-Dawley (SD) rats were randomly divided into four groups: Sham, Sham treated with H2, CHF and CHF treated with H2. Rats from CHF and CHF treated with H2 groups were injected isoprenaline subcutaneously to establish the rat CHF model. One month later, the rat with CHF was identified by the echocardiography. After inhalation of H2, cardiac function was improved vs. CHF (p < 0.05), whereas oxidative stress damage and apoptosis were significantly attenuated (p < 0.05). In this study, the mild oxidative stress was induced in primary cardiomyocytes of rats, and H2 treatments significantly reduced oxidative stress damage and apoptosis in cardiomyocytes (p < 0.05 or p < 0.01). Finally, as a pivotal transcription factor in reactive oxygen species (ROS)-apoptosis signaling pathway, the expression and phosphorylation of p53 were significantly reduced by H2 treatment in this rat model and H9c2 cells (p < 0.05 or p < 0.01). Conclusion: As a safe antioxidant, molecular hydrogen mitigates the progression of CHF via inhibiting apoptosis modulated by p53. Therefore, from the translational point of view and speculation, H2 is equipped with potential therapeutic application as a novel antioxidant in protecting CHF in the future.


INTRODUCTION
Because chronic heart failure (CHF), the end-stage of various heart diseases, continues to cause substantial morbidity and mortality, the ideally treatment for CHF needs to improve. CHF is a complex syndrome characterized by defecting bioenergetics, altering signal transduction pathways, and abnormal calcium homeostasis. In addition, apoptosis serves as another mechanism for the aggravation of CHF (Aubert et al., 2013;Li J. et al., 2014;Gorski et al., 2015). Acute oxidative stress causes severe injury to tissues (Kotur-Stevuljevic et al., 2015), and continuous oxidative stress is one of the reasons of abundant chronic diseases, tumor and senility (Sundar et al., 2013;Granados-Principal et al., 2014;Miller and Sadeh, 2014). A majority of intracellular reactive oxygen species (ROS) are by-products of mitochondrial metabolism. Bioenergetic activity and mitochondrial dysfunctions result in the generation of excess amounts of oxidant stress and further enhance cardiomyocytes apoptosis (Okonko and Shah, 2015).
C-Jun N-terminal kinase (JNK)-P53 is an important signaling pathway in oxidative stress. It is activated by ROS and then mediates apoptosis which related to the Bax-Bcl-2 complex in colistin-treated PC-12 cells (Lu et al., 2017). P53, as a gene of tumor suppression, plays an important role mainly through transcription of target genes referring to some processes, such as apoptosis, redox reaction, and cell cycle (Krifka et al., 2013). P53 is able to cause cell apoptosis by activating Bax directly or activate puma to upregulating Bax and Bak indirectly (Zhang et al., 2016). However, studies on p53 of cardiomyocytes in the failing heart are rare.
Molecular hydrogen (H 2 ) has many potential therapeutic applications as a novel antioxidant. In 2007, the first article reported H 2 keeps tissues and cells safe from oxidative stress injury by eliminating ROS. As regulatory signaling molecules, ROS plays an important role in a mass of signal transduction cascades and also modulates bioprocess-apoptosis, for instance (Ohsawa et al., 2007). From then on, accumulating evidence has shown the protective action of H 2 in various disease models (Guo et al., 2015;Kishimoto et al., 2015), including the infarct size of heart and brain by reducing ischemia-reperfusion injury without changing hemodynamic index, and protection against multiple-organ damage arising from generalized inflammation (Hayashida et al., 2008;Zhai et al., 2013;Miller and Sadeh, 2014). Some primary clinical trials also were performed in recently years (Ostojic et al., 2014;Yuan and Shen, 2016). About cardiovascular diseases, most H 2 -delivery ways were hydrogen-rich saline injection, which is not convenient. However, there was not reported about inhalation administration of H 2 in the cardiovascular system.
Previous studies have demonstrated that hydrogen-containing saline treatment ameliorates the deterioration of doxorubicintriggered heart failure in rats, which was likely mediated by its anti-autophagic or anti-apoptotic biological effects (Wu et al., 2014;Gao et al., 2016Gao et al., , 2017. However, the molecular mechanism underlying H 2 -induced anti-apoptosis FIGURE 1 | The time course of the study on each animal. The protocols and interventions on each animal and when they were performed were indicated. effect in CHF was still unknown. In order to further clarify the aforementioned mechanism, the aims of this study were to determine to use much more convenient therapy-inhalation of H 2 , to completely investigate changes in the cardiac function and structure. Furthermore, signaling pathways mediated H 2 -induced inhibition of apoptosis in cardiomyocytes were explored, especially effects of p53 in rats with CHF. Our results provided new insights into molecular mechanisms that H 2 gas inhibited apoptosis in CHF both in vivo and in vitro. We expect that in the near future H 2 will be used clinically through inhalation as a novel antioxidant to protect against deterioration in CHF patients.

Animals
Sixty Sprague-Dawley (SD) rats (male, 180-220 g; 6-7 weeks) were purchased from Liaoning Changsheng Biotechnology Co. Ltd. (China). The animals were fed in the room with natural light. The rats had food and water ad libitum and were food-deprived for 12 h before surgery. The study was conducted in accordance with the "Guidelines for the Care and Use of Laboratory Animals" (Fletcher and Crossgrove, 2011). All protocols were approved by the Animal Ethics Screening Committee (The First Affiliated Hospital of Harbin Medical University, China, clearance number 2017006). Rats were separated into four groups at random (15/group): Sham, rats treated with H 2 , CHF and CHF treated with H 2 (Figure 1).

Establishment of Heart Failure Model
Large doses of isoprenaline lead to injury of myocardium. The damaged heart gradually progress to heart failure. Isoprenaline was diluted in a sterile environment in saline immediately before the injection (Grimm et al., 1998). To confirm the most effective dose which could cause heart failure and along with an acceptable rate of survivors, three different doses of isoprenaline (I5627, Sigma-Aldrich, United States) were tested: 85, 170, and 340 mg/kg body weight (b.w.) (n = 10 for each group) were given subcutaneously once a day for two consecutive days. One month later, the 340 mg/kg was turned up to be the most ideal dose to beat the targets ( Table 1). CHF was defined as LVEDD > 6.0 mm and EF < 60% by echocardiography (Wu et al., 2007). Animals that did not meet the criteria of CHF were excluded from the study. The rats without heart failure were executed in the manner of spinal dislocation.

Echocardiography
Echocardiography was performed using an animal-specific Doppler ultrasound system (S12-4, Philips CX50, Holland). Animals were intraperitoneally anesthetized by thiobutabarbital sodium (T133, Sigma-Aldrich, United States; 100 mg/kg b.w.), and M-mode images were recorded when the heart rates of the rats were maintained at 350-450 beats per minute. Left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD), right ventricle (RV), left atrium (LA), right atrium vertical diameter (RAVD), right atrium transverse diameter (RATD), interventricular septum (IVS), left ventricular posterior wall (LVPW), thickness of RV, E peak/mitral A peak (E/A), ejection fraction (EF), and fractional shortening (FS) were measured in this study. Three experienced technicians executed the measurement which was averaged for five consecutive cardiac cycles. The technicians were unaware of the identities of the grouping of animals.

Hydrogen Treatment of Rats
Hydrogen was prepared by the hydrogen generator (HA-300, SCDEALL, China; Output pressure: 0.4 MPa, Flow rate: 80 ml/min). Hydrogen was fed to a ventilated box which contained rats (ROX, 44 cm × 74 cm × 23 cm, Tenma, Japan). The concentration of hydrogen was maintained at 2% monitored by hydrogen sensor (SDH-B101, Honeywell, United States). Another ventilated box containing rats was filled with air. For successive 14 days, rats were treated in the two boxes for 12 h everyday (Figure 2).

FIGURE 2 |
The H 2 and air intake device for the rats. The fan in the ventilated box contributes to flowing of the air or H 2 . The box contains food, water, and bedding. Therefore, the rats could live in the box for a long time.

Hydrogen and Oxygen Measurement
H 2 and oxygen (O 2 ) dissolved in the solution and blood was measured with the method described before (Ohsawa et al., 2007). H 2 and O 2 concentrations were determined by hydrogen sensor (SDH-B101, Honeywell, United States) and oxygen detector (AR8100, Smart Sensor, Hong Kong, China).

Histology
Heart tissues were fixed with 4% paraformaldehyde and imbedded in paraffin; At the left ventricle (LV) papillary muscle level, tissue sections (4-µm thickness) of the hearts were sliced and stained with Masson trichrome, terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL, In Situ Cell Death Detection Kit, POD, 11684817910, Roche, Switzerland), and immunohistochemical methods using anti-p53 (OP33, Millipore, United States) and 8-OHdG (ab10802, Abcam, United States). The immunostaining of p53 was performed according to the immunohistochemical protocol from Millipore using p53 antibody (1:100 dilution). Digital photomicrographs were taken at magnification of 200×, and five random fields from each section were selected and analyzed with unawareness of animal groups. Size of fibrosis, apoptosis cardiomyocytes, p53 and 8-OHdG positive cells were measured in five sections from each heart, and the mean value was calculated.

Measurement of Brain Natriuretic Peptide (BNP)
Blood samples from abdominal aorta of all survived rats were collected into heparin-containing tubes, centrifuged at 3000 × g, 4 • C for 15 min. Plasma was collected and tested within 2 h. The plasma concentrations of BNP were measured with ELISA kits according the manufacturers' instructions (H166, Nanjing Jiancheng Bioengineering Institute, China). The experiment mentioned above was replicated for three times and intra-assay coefficients of variation were <10% for all assays.

Cell Culture
Neonatal rats (0-3 days) were acquired from the Laboratory

Cell Death Assay
The death of cardiomyocyte was evaluated by the staining of trypan blue (T6146, Sigma-Aldrich, United States). Cells (5.0 × 10 5 ) were incubated with 0.4% trypan blue solution for 10 min at room temperature. The blue dead cells and all the cells were counted, respectively. The percentage of dead cells was calculated (%). Each experiment was performed in triplicate.

Overexpression of P53 in H9c2 Cells
H9c2 cells from rat embryonic ventricular myocardium were acquired from Cell Bank of Chinese Academy of Sciences [H9c2 (1-2), China]. Full-length p53 was obtained from liver tissue of rat, then subcloned into plasmid for lentivirus expression vector (pLVX, Clontech, United States) between EcoRI and BamHI sites. Lentivirus for overexpressing rat P53 was produced in 293T cells. The sense and antisense primers for rat p53 were as follows: 5 -TATGTCTAAGGGACCTGCGGTTGGCATTGATCTTG-3 and 5 -GTGCCAAGATCAATGCCAACCGCAGGTCCCGGAG ACA-3 . H9c2 cells from matched group were infected with pLVX. Lentivirus with Enhanced Green Fluorescent Protein (EGFP) was applied to infect H9c2 cells to confirm the optimum concentration and to assess the efficacy of infection. H9c2 cells were infected with 0, 5, 10, 20, or 30 µl/ml lentivirus for 6 h. The day after infection, the cells were selected with 0, 0.1, 0.2, 0.4, 0.8, 1.0, and 1.5 µg/ml puromycin. The cells were visualized by fluorescence microscopy at 24 h post infection (Vert. A1, HBO 50, ZEISS, Germany). After puromycin screening for 48 h, EGFP-stable clones were screened. The percentages of cells with EGFP were determined from four separate visual fields. The optimal doses were 20 µl/ml lentivirus and 0.8 µg/ml puromycin. The process related to the lentivirus were handled following the guideline of biosafety level 2. The selected cells were starved before VK 3 and H 2 treatment for 16 h. The expression of p53, Bax and cleaved caspase-3 were determined by Western blotting.

Inhibition of P53 in H9c2 Cells
For inhibition of p53 in response to H 2 , H9c2 cells were treated with Pifithrin-α (PFTα, S1816, Beyotime, China), which was a p53 inhibitor that dissolved in dimethyl sulfoxide (DMSO). Chu et al. (2006) have reported that expression of p53 in H9c2 cells was inhibited after the cells were treated with 20 µM PFTα and doxorubicin for 8 h. However, we observed that after treatment  with 20 µM PFTα and 10 µM VK 3 , H9c2 cells all died 2 days later. Therefore, the final concentration of PFTα was 10 µM in the DMEM in this study. DMSO as a control was given the same volume to PFTα to H9c2 cells. After 16 h, the culture medium was changed to DMEM with 1% FBS, 1% penicillin-streptomycin and 10 µM PFTα and then the treatments of VK 3 and H 2 were continued.

TUNEL Staining
The TUNEL assay was carried out to label the 3 -end of fragmented DNA in tissue slices or cardiomyocytes in accordance with the manufacturer's directions (Roche, Switzerland) and stained with DAB kit (ZLI-9018, ZSGB-BIO, China). The TUNEL-positive area was expressed as the percentage of the total area. Finally, slides were observed by a light microscope (DP72, Olympus Co., Japan) and fluorescence microscope, and quantitative statistical analysis was performed with the Medical Image Analysis System (Motic Med 6.0, China).

Measurement of Malondialdehyde (MDA)
Myocardial tissues from survived rats or neonatal rat cardiomyocytes were homogenized in ice-cold phosphate buffer saline. MDA, a marker of lipid peroxidation, was detected with a commercial kit (KGT003, KeyGEN Biotech. Co. Ltd., China). The MDA concentration was represented as the ratio of MDA content and the concentration of proteins which was tested with bicinchoninic acid (BCA) method (P0012, Beyotime Biotechnology, China). The experiment mentioned above was triplicated.

Statistical Analysis
The data were expressed as mean ± SD or mean ± SE. The normal frequency distribution was analyzed with Kolmogorov-Smirnov test and the homogeneity of variances was analyzed with Levene statistics. The multiple group comparison was carried out with one-way analysis of variance (ANOVA) and least significant differences t-test. The tests were two-sided and p < 0.05 was regarded as statistical significance, beta-error was 0.2. A rank sum test was used when there was heterogeneity of variance or abnormal frequency, and values of p < 0.0083 (α = 0.0083) were considered as statistically significant (α = α/N, N = C 2 n = n × (n − 1)/2, n = 4, α = 0.05). All of statistical analyses were carried out with Statistical Product and Service Solutions (SPSS) 24.0 software (IBM, New York, NY, United States) (Chi et al., 2014).

H 2 Improved Cardiac Function in Rats
To elucidate the role of H 2 in CHF, myocardial injury was induced using isoprenaline in rats. One month later, 12-14 rats/group subject to the echocardiography, and 8-12 rats/group developed CHF. The rats with CHF were treated with H 2 for 14 days. After treatment, cardiac function of rats was reexamined with echocardiography ( Figure 3A). The LVESD, LVEDD, RAVD, RATD, IVS, and RV dramatically reduced when compared to the CHF group, however, there were no significant differences between CHF and CHF with H 2 group in LVPW and left atrium (LA). In addition, LVEDD of the treatment group was still greater than matched group (Figures 3B-E). Meanwhile, EF, FS, and mitral E/A in rats with CHF were much lower than those in the control group. Besides, E/A in normal rats increased significantly after treating with H 2 in comparison with untreated normal group (Figures 3F,G). Moreover, BNP, as an indicator of CHF in serum, showed similar results to the results from the echocardiography ( Figure 3H). Furthermore, myocardial fibrosis was reduced after inhalation of H 2 (Figures 4A,B). These results collectively suggest that H 2 may prevent the development of CHF and protect the cardiac function in rats.

H 2 Released Oxidative Stress Injury in CHF
H 2 -mediated molecular changes of CHF was detected, by incubating heart slices with the antibody of 8-OHdG to evaluate the level of nucleic acid oxidation. As the oxidative marker, 8-OHdG staining was substantially reduced after H 2 -treatment in both normal and CHF rats (Figures 4C,D). Furthermore, the level of MDA was examined in myocardial tissues, which reflected the degree of lipid peroxidation. As expected, the results showed that the level of MDA was significantly decreased after treated with H 2 when compared to untreated rats which subjected to CHF ( Figure 4E). All in all, these results indicated that H 2 is capable of attenuating oxidative stress markedly and decreasing damage to the myocardium.

H 2 Decreased Apoptosis in CHF
The effects of H 2 modulation on apoptosis were also determined in rats with CHF. After treatment with H 2 , the number of apoptotic cells was significantly reduced in cardiac tissues (Figures 5A,C). Meanwhile, karyopyknosis was mitigated after the rats were treated with H 2 (Figure 5B). Moreover, the expression of Bax which promotes apoptosis was attenuated by H 2 (Figure 5D). Likewise, cleaved-caspase-3 as a marker of executing apoptosis was significantly reduced after H 2  100×). Scale bar = 500 µm. (C-E) Confirmation by Western blot analyses for the expression of cardiac cleaved caspase-3, phospho-p53, p53, and Bax in primary cardiomyocytes. Representative photographs are shown. GAPDH was used as a loading control. Expressions of cleaved-caspase-3, phospho-p53, p53 and Bax were, respectively, quantified as folds of GAPDH (n = 3-5). The columns indicate the mean, and the bars indicate the SD (MDA and cell viability) or SE (Western blotting). * p < 0.05 vs. VK 3 ; * * p < 0.01 vs. VK 3 ; && p < 0.01 vs. VK 3 +H 2 .  100×). Scale bar = 500 µm. Inset: Magnified region illustrating dead cardiomyocytes (blue staining). The columns indicate the mean, and the bars indicate the SD. * p < 0.05 vs. VK 3 ; * * p < 0.01 vs. VK 3 ; && p < 0.01 vs. VK 3 +H 2 . treatment ( Figure 5E). These results collectively suggest that H 2 protects heart against exhaustion partly by reducing apoptosis of cardiomyocytes.

P53 Plays a Vital Role in the Effects of H 2 on CHF
It is known that the tumor suppressor p53 regulates apoptotic process of cardiomyocyte and is profoundly involved in the development of CHF (Sun et al., 2014). Therefore, whether modulation of H 2 could affect the expression and phosphorylation of p53 was further examined. The level of p53 protein was significantly increased in failing hearts and decreased in the H 2 -treated CHF (Figures 5F,G). Besides, the expression and phosphorylation levels of p53 in cardiomyocytes were significantly increased in rats with CHF and decreased after H 2 -treated CHF (Figures 5H,I). Meanwhile, as found in rat primary cardiomyocyte, the inhibitory effect of H 2 on VK 3 was also found in H9c2 cells, however, overexpression of p53 restrained this effect (Figures 6A-D). As found in rat primary cardiomyocyte, the expression of p53, Bax and cleavedcaspase-3 was increased by the stimulation of VK 3 in H9c2 cells, however, inhibition of p53 restrained the stimulative effect (Figures 6E-H). Taken together, the results above illustrated that p53 plays a vital role in the modulation of H 2 in CHF.

H 2 Reduced Apoptosis Caused by Oxidative Stress in Primary Cardiomyocytes
Modulatory effects of molecular hydrogen on apoptosis were also determined in cultured primary cardiomyocytes that underwent oxidative stress injury (Figures 7A,B). The expression of cleavedcaspase-3, p53 and phosphor-p53 was down-regulated by H 2 treatment after oxidative stress injury (Figures 7C,D), which was similar to the results from in vivo. However, Bax transcribed by p53 did not appear significant difference between oxidative stress and hydrogen treatment groups ( Figure 7E). Furthermore, H 2 treatment also attenuated the apoptosis caused by oxidative stress in cardiomyocytes (Figures 8A-C). In general, these results suggest that H 2 could be a major player in protection of cardiomyocytes from oxidative stress-induced apoptosis.

H 2 Protected Primary Cardiomyocytes From Damage of Oxidative Stress
As is well-known that VK 3 can be used for inhibiting complex I or complex III of the mitochondrial electron transport chain. Therefore, the leakage of electrons was accelerated and O 2 − was produced. In order to test the protective effect of H 2 on cardiomyocytes with oxidative stress, cardiomyocytes were exposed to 75% H 2 for 8 h. After H 2 treatment, FIGURE 9 | Schematic depicting the mechanisms by which molecular hydrogen mediates apoptosis. H 2 eliminated oxygen free radicals which led p53 phosphorylation. As a transcription factor, phosphorylated p53 increased expression of Bax which increased expression of caspase-3. Cleaved-caspase-3 executed apoptosis of cardiomyocytes. Hence, apoptosis was suppressed by inhibition of p53 in response to molecular hydrogen.
the level of MDA in cardiomyocytes was examined in this study. The results showed that H 2 could also reduce damage of oxidative stress in primary cardiomyocytes ( Figure 8D). In addition, H 2 also significantly reduced cell death of primary cardiomyocytes (Figures 8E,F).

Key Findings of the Study
Our findings revealed that molecular hydrogen could markedly reduce oxidative stress and suppress myocardium injury in vivo and in vitro. Inhalation of H 2 might prevent CHF from progressing and protect the cardiac function of rats partly by reducing apoptosis of cardiomyocyte. Meanwhile, H 2 could also be confirmed effective effects on protection of cardiomyocytes from oxidative stress-induced apoptosis. Notably, the expression and phosphorylation of p53 play an important role in CHF when treated with hydrogen.

Hydrogen Alleviated Continuous and Mild Oxidative Stress in Cardiomyocytes
Many studies from in vitro and in vivo have demonstrated that ROS are generated partly in the circulatory system, including the failing heart (Hafstad et al., 2013;Chouchani et al., 2014;Brown and Griendling, 2015). Continuous and mild oxidative stress may lead to injury in various organs and tissues (Jourdan et al., 2014;Li Y. et al., 2014). It has been shown that inhalation of H 2 ameliorated oxidative myocardial injury during cardiopulmonary resuscitation and after returning of spontaneous circulation (Hayashida et al., 2012). Our previous study also indicated that the serum MDA level was decreased after injection of hydrogen-rich saline (Gao et al., 2017). In this study, the levels of MDA and 8-OHdG were examined in myocardial tissue, and found that inhalation of H 2 ameliorated DNA damage of cardiomyocytes in rats with CHF. The experiments using cultured cardiomyocytes also showed that H 2 -treated myocardial lipid were more resistant to the VK 3 -induced oxidant stress. Interestingly, the level of 8-OHdG in cardiomyocytes also significantly decreased after inhalation of H 2 in the normal rats, which needs a larger sample size to verify. Results from trypan blue assay indicated that H 2 reduced cell death of cardiomyocytes, which has not been reported before. These findings suggest that inhalation of H 2 alleviate the oxidative stress injury of cardiomyocytes both in neonatal rats and adult rats with CHF.

Effects of Inhalation H 2 on CHF Were Observed Completely for the First Time
Dysfunction of LV is related to oxidative stress in the myocardium and plasma of patients with heart failure (Maack et al., 2003). The treatment of CHF has been a worldwide puzzle on account of irreversible fibrosis, remodeling, and apoptosis. The anti-fibrosis effect of H 2 has been verified in overload-induced cardiac dysfunction in rats recently . In this study, our findings found that inhalation of H 2 may decrease the area of fibrosis in isoprenaline induced CHF. Moreover, our findings also confirmed that inhalation of H 2 was able to improve cardiac function which was also verified by hemodynamic measurement. Furthermore, the safety of molecular hydrogen to cardiomyocytes has been confirmed as reported before (Tao et al., 2015). However, in our previous study, LVEDD in saline, doxorubicin and doxorubicin+hydrogen-rich saline groups was not found differences (Gao et al., 2017). Meanwhile, LVEDD, LVPW and LA from the treatment group did not decline to the baseline. Maybe a universal standard of CHF in rats should be established in the future. Additionally, the improvement of E/A indicates that the diastolic function may be improved by H 2 . Ventricular remodeling is a primary reason of deterioration of heart failure and death after myocardial damage (Ahmad et al., 2014). ROS may participate in the remodeling processes in various ways, such as activating matrix metalloproteinases (MMPs) that correlate with reconstitution of the extracellular matrix; functioning as signaling factors in the progress of vicarious hypertrophy; and resulting in cardiomyocytes death via apoptosis or other mechanisms (Liu et al., 2012;Yang et al., 2013;Li Y. et al., 2014;Zhang et al., 2014;Kessler et al., 2015). In addition, the result that E/A increased in normal rats compared with the matched group treated with H 2 indicates that inhalation of H 2 is not only harmless to myocardium but also helpful for diastolic function. Further clinical studies are needed to verify whether inhalation of H 2 would be used in clinic.

Suppression of Apoptosis Was Further Observed in Cardiomyocytes of CHF Treated With H 2
Apoptosis of cardiomyocytes may be a cause and effect of CHF, and inhibition of this cell death process may contribute to the exploration of new treatment (Melman et al., 2015). ROS activate the JNK, p38 mitogen-activated protein kinases (MAPKs) and Akt and induces apoptosis (Hao et al., 2015;Santabárbara-Ruiz et al., 2015). In this study, we observed alleviated karyopyknosis by electron microscope and the number of apoptotic cardiomyocytes was decreased after treated with H 2 . In addition, studies of apoptosis-related protein in vivo and in vitro suggest that molecular hydrogen may inhibit apoptosis, which is in line with the studies before (Guo et al., 2015;Song et al., 2015;Gao et al., 2017). However, our present study revealed that after treated with VK 3 , the expression of Bax in primary cardiomyocytes was not suppressed by H 2 , but was suppressed in H9c2 cells. Therefore, a larger sample size is also needed to verify this result in the future. Moreover, Song et al. (2013) found that hydrogen-rich water protected activity of endothelial cells from apoptosis. In our study, a similar phenomenon on activity of cardiomyocytes was also found. In addition, our study also verified that molecular H 2 decreased VK 3 induced apoptosis in H9c2 cells. Therefore, we confirm that H 2 may protect CHF from progressing in apoptosis pathway much more completely than before, especially in inhalation manner.

P53 Mediated Apoptosis Pathway Intervened by H 2 in Cardiomyocytes
As an important transcription factor, p53 plays a vital role in the signaling pathway of apoptosis induced by ROS (Li et al., 2015). P53 protein is not novel in apoptosis, however, it is an important factor in cardiomyocytes and is rarely studied in CHF in especial. Our present study revealed that molecular hydrogen reduced the expression and phosphorylation of p53 in cardiomyocytes for the first time. However, inhalation of H 2 could not decrease the expression of p53 to the normal level. Our data suggest that molecular hydrogen may inhibit oxidative stress in CHF. Consequently, H 2 might provide a suppressive signal to p53 leading to the attenuated p53 activity. The relative contributions of H 2 in the ROS/p53/apoptosis signaling pathway and preferential protection of CHF were a meaningful subject to study. Hence, knockdown p53 expression or overexpressed p53 to observe the impact of H 2 in protecting cardiomyocytes. When knockdown the expression of p53, H9c2 cells treated with VK 3 could not increase the expressions of p53, Bax or cleaved-caspase-3, which was in line with the result of the study that doxorubicin induced apoptosis in H9c2 cells (Chu et al., 2006). When p53 was overexpressed in H9c2 cells, the protective effect of H 2 on oxidative stress injury was weakened. Therefore, in H 2 protecting process of cardiomyocytes, the modulating effect of p53 was firstly confirmed. We believe that the decreasing p53 protein modulated by H 2 is important because it targets apoptosis in the treatment of CHF.

FUTURE DIRECTION AND CONCLUSION
Our study indicates that H 2 protected cardiomyocytes and myocardial tissues of the failing heart against apoptosis by inhibiting oxidative stress. This process was carried out by inhibiting phosphorylation and transcription of p53 to Bax (Figure 9). Applying H 2 in CHF may open up a new way for researching. Inhalation of H 2 may be a safe and effective therapy for CHF, and may be applied to patients with CHF. The clinic trail is ongoing. Besides, it is possible that H 2 may protect the failing heart against progression through direct or indirect mitochondrion pathways, and our relevant studies are also ongoing. For instance, H 2 may inhibit the generation of adenosine triphosphate (ATP) from mitochondrion (data not shown), which has been confirmed in PC12 cells (Ohsawa et al., 2007).
Continuous damage from oxidative stress is an important mechanism that facilitates CHF. We summarize that molecular hydrogen as a safe antioxidant could mitigate progression of CHF via inhibiting apoptosis modulated by p53. Therefore, we propose that inhalation of H 2 as a safe, convenient and inexpensive therapy may serve as a new antioxidant in the prevention and treatment of CHF in clinic in the future.

AUTHOR CONTRIBUTIONS
JC, WY, and JL conceived and designed the study. JC, TZ, YB, WZ, JxY, ZF, ZY, QX, LZ, WL, and JmY performed the experiments. JC, XH, and LZ organized the database. JC, YG, and HY performed the statistical analysis. JC and ZL drafted and revised the work. All authors contributed to manuscript revision, read and approved the submitted version.