Histone Deacetylase 1 Inhibition Protects Against Hypoxia-Induced Swelling in H9c2 Cardiomyocytes Through Regulating Cell Stiffness

Abstract

Background: The process of cardiomyocyte swelling involves changes of biomechanical properties and profiles of cellular genes. Although many genes have been proved to regulate cell edema of cardiomyocyte, the mechanisms involved in this event, as well as the biomechanical properties of swelling cell, remain unknown.

Methods and Results: Whether histone deacetylase 1 (HDAC1) inhibition protects against hypoxia-induced H9c2 cardiomyocyte swelling is examined in this study. Hypoxia-induced changes in the biomechanical properties and cytoskeletal structure that are relevant to cell swelling were also determined. H9c2 cells were treated under a chemical hypoxia situation (cobalt chloride) with HDAC1 inhibition (chemical inhibitor or siRNA) for 5 h, followed by in vitro biological and mechanical characterization. The results showed that expression of HDAC1 instead of HDAC4 was upregulated by chemical hypoxia. HDAC1 inhibition protects H9c2 cells against chemical hypoxia-induced hypoxic injury and cell swelling. HDAC1 inhibition improved cell viability, decreased lactate dehydrogenase leakage, cell apoptosis, malondialdehyde concentration, cell volume, and particles on the cell surface, and increased superoxide dismutase activity. Moreover, chemical hypoxia induced a decrease of Young’s modulus, accompanied by alterations in the integrity of acetylated histone and organization of the cytoskeletal network. HDAC1 inhibition significantly reversed these processes.

Conclusions: Based on the ideal physical model, HDAC1 inhibition protects against hypoxia-induced swelling in H9c2 cardiomyocytes through enhancing cell stiffness. Overall, HDAC1 is a potential therapeutic target for myocardial edema.

Myocardial edema is a frequently detectable incident in the acute phase of myocardial ischemia and other pathological situations.^1–3 Previous studies have revealed that there are many elements involved in regulating cell volume in the cardiomyocyte, including osmotic forces, channels, cell volume sensing and intracellular water distribution.^4–8 However, swelling in cardiomyocytes has not been completely elucidated in these aspects. The mechanism of cellular edema has been subjected to debate.^9,10

Through influence on chromatin modification of histone deacetylases (HDACs) and histone acetyltransferases, epigenetic modifications to histone and non-histone proteins affect gene expression.^11,12 Modifications in the structure of chromatin result in the relaxing or condensing of the nucleosome and changes in transcriptional activation. Previous studies indicate that class II HDACs play a significant role in the process of development of cardiac hypertrophy.^13,14 Du et al showed that HDAC4 inhibition increased the resistance of cardiomyocytes in response to hypoxia/reoxygenation, as evidenced by an increased cell survival and reduction of cytotoxicity, and an anti-apoptotic effect.¹⁵ The class II HDACs are cytoplasmic in the absence of an activating signal; however, class I HDACs are predominantly nuclear proteins and ubiquitously expressed, which may be important for regulating the expression of certain genes. Under hypoxic situations, the expression level and activity of HDAC1 are obviously increased in tumor cell lines.^16,17 Whether HDAC1 possesses the capacity to control the volume of cardiomyocyte has not been investigated.

Cellular mechanical properties are crucial for their biological processes. Under pathological conditions, there are changes in the profile of mechanical properties, and thus the modifications have been recognized as a pathological marker.^18–20 Mechanical properties, associated with cell adhesion and cytoskeletal organization, are regarded as biomarkers for cellular phenotypic events.^21–23 Recent studies have indicated that the cell cytoskeleton undergoes dynamical rearrangements in response to the environmental and/or mechanical condition alterations, including the hypoxic environment.^24,25 Several lines of evidence have convincingly shown that mechanical properties of cells are largely determined by the actin cytoskeleton.^26–29 Thus, mechanical properties of cardiomyocytes in hypoxia condition may change drastically, and consequently affect biological processes of natural cells.

As stated above, either modification of mechanical properties alone or HDAC1 of cells alone may be involved in cell swelling. However, the process of change in cellular phenotype involves at both physical layer and biological level with interacting with each other. Thus, it is crucial to study the biological and mechanical coupling mechanism in cardiomyocyte swelling, which is induced by hypoxia. In the present study, we established the first study to gain an insight into the biological and mechanical coupling mechanism of cell swelling induced by chemical hypoxia, as well as the effect of HDAC1 inhibition in swelling H9c2 cells.

Methods

Cell Culture and Treatments

The rat cardiomyoblast cell line, H9c2, was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) containing 10% (v/v) fetal bovine serum (FBS; Invitrogen Corp., Carlsbad, CA, USA), penicillin (100 U/mL) and streptomycin (100 μg/mL). The cells were maintained at 37℃ in a humidified atmosphere with 5% CO₂. The medium was replaced every 2–3 days, and cells were then subcultured or subjected to experimental procedures. The cells were passaged at a ratio of 1:4 when they grew to 80% confluence, and discarded after 10 passages. For hypoxia treatment, H9c2 cells were incubated for 5 h in DMEM (no glucose; Gibco) in the presence of different concentrations (0, 100, 200 and 400 μmol/L) of Cobalt chloride (CoCl₂) without FBS.

Targeted HDAC1 Inhibition

For HDAC1 inhibition, cells were incubated in a chemical hypoxia situation supplemented with 0.5 μmol/L MGCD0103 (Selleck Chemicals, Houston, TX, USA), a HDAC1-selective inhibitor (but not for HDAC4)³⁰ for 5 h.

In addition, to verify the effect of HDAC1 inhibition in H9c2 cells under chemical hypoxia, siRNA-mediated knockdown of HDAC1 was performed. H9c2 cells were transfected with either siRNA-targeting HDAC1 (siHDAC1) or a negative control siRNA (siControl), by using Lipofectamine^® 2000 (Life Technologies, Gaithersburg, MD, USA). The specific target HDAC1 siRNA gene sequence was 5'-UGGCUGAUAUUACCCAAGCTT-3'. siControl was purchased from GenePharma (GenePharma, Shanghai, China). Transfected H9c2 cells were incubated for 6 h at 37℃, then replaced with fresh cell culture medium, and assessed for changes in mRNA and protein expression 48 h later by using real-time PCR and western blotting.

Cell Viability Assay

Lactate Dehydrogenase (LDH) Release Assay

Antioxidant and Lipid Peroxide Assay

Evaluation of Apoptotic Cells

See Supplementary Methods for more information.

Measurement of Cellular Volume

After CoCl₂ and/or HDAC1 inhibition treatments, H9c2 volumes were estimated by measuring the intracellular water space.³¹ In brief, 1 mmol/L 3-O-methylglucose (3-OMG) and 0.5 µCi/ml [3H]-3-OMG were added to the culture for 5 h, in the beginning of chemical hypoxia procedure. Radioactivities in the medium and cell extracts were measured, and an aliquot of the cell extract was used for protein determination using the bicinchoninic acid (BCA) assay.

Detection of Granularity of H9c2 Cell Surface

Cells were seeded on culture slides (5×10⁵ cells per well) for 24 h and then the CoCl₂-induced hypoxia protocol with or without MGCD0103 was performed. Then, cells were collected to analyze with flow cytometry. Data of side scatter (SSC) was obtained to reflect granularity of the H9c2 cell surface.

Fluorescence Analysis of Acetylated Histones and Cytoskeleton

H9c2 cells fixed on coverslips using 3.7% paraformaldehyde were incubated with Anti-acetyl-Histone H3 (Lys9/Lys14) antibody (CST, MA, USA) and anti-acetyl-Histone H4 antibody (Millipore, MA, USA) (1:250) overnight at 4℃, and then incubated with FITC-labeled IgG for 1 h at room temperature. After immunostaining, the cells were incubated with phalloidine-rhodamine (Sigma-Aldrich, MO, USA) (10 μg/mL) for 2 h at room temperature. Fluorescence images were obtained by using a confocal laser scanning microscope (Olympus, Tokyo, Japan).

Western Blot Analysis

Western blot analysis was performed, as described previously.³² Blotted membranes were subsequently incubated with the hypoxia-inducible factors 1α (HIF-1α), HDAC1, HDAC4 (CST), and histone acetyltransferase 1 (HAT1) (Abcam, Cambridge, UK) antibodies (1:1,000 dilution) overnight at 4℃. The detection was performed using the ECL system. The quantification of band intensity was performed by using Quantity One software (Bio-Rad, CA, USA).

AFM Indentation System

For detection of mechanical properties of H9c2 cells in a hypoxia situation with or without HDAC1 inhibition, a NanoWizard III AFM (JPK Instruments, Germany) was used, as previously described.³³ H9c2 cells in physiological medium were imaged and measured under contact mode. During the indentation test, 70–100 force-displacement curves were obtained at the centre of the cells. All of the AFM data were acquired and analyzed with JPK instruments data processing software 4.2.61.

Elasticity Modulus Measurement

The elastic modulus of the H9c2 cell was calculated by applying the modified Hertz model contact theory to the force curves.³⁴ The formula for the sphere mechanical model is expressed as:

where F is the applied force, R is the radius of the sphere, δ is the indentation and E* is the relative Young’s modulus term.

Statistical Analysis

Data are expressed as mean±standard deviation (SD). Statistical comparisons of the results were made by using analysis of variance (ANOVA). P-values <0.05 were considered statistically significant. Calculations and statistical tests were performed by using SPSS 18.0 (SPSS, Chicago, IL, USA).

Data Fitting and Ideal Physical Model Structuring Describing the Process of Cell Swelling

Data from elasticity modulus measurement and cellular volume were non-linear or linear fitting, and analyzed by using NonlinearRegress statements in the Mathematica 10.3 software (Wolfram Research, IN, USA) environment. Then we set up an ideal physical model to describe the process of cell swelling and find out possible elements involved in cell swelling induced by CoCl₂, as well as the effect of HDAC1 inhibition (by using MGD) in the swelling of the H9c2 cell.

Results

CoCl₂ Upregulated the Expression Level of HIF-1α and HDAC1

CoCl₂ treatment significantly upregulated the level of HIF-1α in a dose-dependent manner (Figure 1A,B), whereas it did not change the expression level of HAT1 (Figure 1A,C). The results of HIF-1α suggest that CoCl₂ treatment did induce cardiomyocyte hypoxia. In addition, chemical hypoxia upregulated the level of HDAC1, and did not affect the HDAC4 expression level (Figure 1A,C,D). In comparison with the CoCl₂ treatment group, MGCD0103 treatment with chemical hypoxia downregulated levels of HDAC1 significantly (Figure 1F,G). Compared with the siControl group, siControl with CoCl₂ treatment upregulated the level of HIF-1α and HDAC1 significantly (Figure 1I,J,K), and siHDAC1 downregulated the level of the proteins (Figure 1I,J,K).

Figure 1.

Expression level of histone deacetylases (HDACs), hypoxia-inducible factor (HIF)-1α and histone acetyltransferase (HAT)1 after concentrations of cobalt chloride (CoCl₂) treatment with MGCD0103 for 5 h. Expression of HDACs, HIF-1α and HAT1 in H9c2 cells incubated in chemical hypoxia situation alone (A) or in combination with MGCD0103 (F) or siHDAC1 knockdown (I) for 5 h. Representative images of the Western blots are shown. Parts B–E represent relative expression levels of HIF-1α, HAT1, HDAC1 and HDAC4, respectively, in H9c2 cells exposed to different concentrations of CoCl2. Parts G and H represent the relative expression levels of HDAC1 and HDAC4 in cells exposed to 400 μmol/L CoCl₂ with MGCD0103, respectively. Parts J and K represent relative expression levels of HDAC1 and HIF-1α in cells exposed to 400 μmol/L CoCl₂ with siHDAC1 knockdown, respectively. *P<0.05 vs. control, ^#P<0.05 vs. hypoxia group. MGD, MGCD0103.

HDAC1 Inhibition Protects Cardiomyocytes Against Chemical Hypoxia-Induced Injury

A MTT assay demonstrated that, compared with the control, chemical hypoxia significantly suppressed the growth of H9c2 cells in a dose-dependent manner after cells were treated with CoCl₂ at doses of 100–400 μmol/L for 5 h (Figure 2A). Compared with the hypoxia groups, HDAC1 inhibition protected H9c2 cells from hypoxia damage (Figure 2A,C).

Figure 2.

CoCl₂ induced cell death in H9c2 cells and a protective effect of HDAC1 inhibition against chemical hypoxia-induced injury. H9c2 cells were exposed to different concentrations of CoCl₂ for 5 h, with MGCD0103 or siHDAC1 knockdown, and cell viability was analyzed by a MTT assay (A) and (C). *P<0.05 vs. control, ^#P<0.05 vs. hypoxia group. Lactate dehydrogenase (LDH) assay (B) and (D). Cells were incubated in chemical hypoxia situation with MGCD0103 treatment or siHDAC1 for 5 h. *P<0.05 vs. control (incubated in a normoxia situation without MGCD0103 treatment), ^#P<0.05 vs. hypoxia group (400 μmol/L CoCl₂). Caspase 3 activity assay (E) and (F): cells were incubated in chemical hypoxia situation with MGCD0103 treatment or siHDAC1 for 5 h. *P<0.05 vs. control, ^#P<0.05 vs. hypoxia group. MGD, MGCD0103. Other abbreviations as in Figure 1.

HDAC1 Inhibition Reverses Chemical Hypoxia-Induced Necrotic Death and Apoptosis of H9c2 Cells

A LDH release (a marker of necrotic cellular death) assay, Hoechst 33342 staining, and Caspase 3 activity were performed. As shown in Figure 2B and 2D, there were significant increases in LDH leakage after CoCl₂ treatment compared to normoxia controls in H9c2 cardiomyocytes. Upregulated LDH release was attenuated by MGCD0103 or siHDAC1 in H9c2 cells, suggesting that inhibition of HDAC1 protects cardiomyocytes against chemical hypoxia injury. Results of Hoechst 33342 staining showed that apoptotic changes, such as the formation of apoptotic bodies, appeared in cells that underwent 5 h of hypoxia with chemical treatment (data not shown). MGCD0103 treatment restrained the occurrence of apoptosis induced by radiation (data not shown). In line with results of Hoechst 33342 staining, chemical hypoxia significantly upregulated the activity of Caspase 3 in H9c2 cells, and HDAC1 inhibition treatment depressed the activity of Caspase 3 in the cells treated with CoCl₂ (Figure 2E,F).

HDAC1 Inhibition Protects Against Chemical Hypoxia-Induced Oxidative Stress in H9c2 Cells

The data demonstrated that chemical hypoxia significantly increased malondialdehyde (MDA) concentration but reduced superoxide dismutase (SOD) activity (Figure 3A,B). Compared with all hypoxia groups, HDAC1 inhibition treatment clearly ameliorated the oxidative abnormalities appearing in H9c2 cells, as manifested by the downregulated level of MDA and the upregulated level of SOD (Figure 3).

Figure 3.

HDAC1 inhibition protects against chemical hypoxia-induced imbalance of oxidative stress in H9c2 cells. The effects of chemical hypoxia alone or in combination with HDAC1 inhibition on MDA (A) (C) and SOD (B), (D) in H9c2 cells were determined using commercial kits, as described in the Methods section. *P<0.05 vs. control, ^#P<0.05 vs. hypoxia group. MDA, malondialdehyde; SOD, superoxide dismutase. Other abbreviations as in Figures 1,2.

Morphological Distortion of H9c2 Cardiomyocytes Induced by Chemical Hypoxia and the Protective Effect of HDAC1 Inhibition

Under normoxia conditions, cells were regular shuttle-like in shape and flat, and filaments were spread all over the surface of the cells (Figure 4A,E). In contrast, in hypoxia cells, dose-dependent changes in the surface morphology were observed, with becoming inflate, fusion of filaments and thickening of edge of cells (Figure 4A). Notably, fine particles appeared on the surface of H9c2 cardiomyocytes, which were treated with CoCl₂ (Figure 4A,E). When a MGCD0103 treatment protocol or HDAC1-targeted RNAi was performed on H9c2 cardiomyocytes throughout chemical hypoxia, morphological distortion of cells was inapparent, except fine particles (Figure 4A,E). The cells cultured in normoxia condition with MGCD0103 (without CoCl₂) had different morphological characteristics. Compared with normoxia cardiomyocytes, there were thick fibers on the smoother surface of cells (Figure 4A).

Figure 4.

Effect of CoCl₂ alone or in combination with MGCD0103 on the physical property of H9c2 cells. Representative images obtained by AFM showing H9c2 cell exposure to chemical hypoxia with HDAC1 inhibition (A,E). The effects of chemical hypoxia alone or in combination with HDAC1 inhibition on cell volume (B,F), particles on the cell surface (C,G) and Young’s modulus (D,H) were measured. *P<0.05 vs. control, ^#P<0.05 vs. hypoxia group. AFM, atomic force microscopy; 3-D, three dimensions. Other abbreviations as in Figures 1,2.

HDAC1 Inhibition Attenuates Cardiomyocyte Swelling and Decreases Particles in H9c2 Cell Surface

The volumes of normoxia cardiomyocyte were significantly increased after 5 h of CoCl₂ treatment (Figure 4B,F). Treatment with MGCD0103 or siHDAC1 throughout the chemical hypoxia injury significantly abolished the increase in cell volume (Figure 4B,F). Data of flow cytometry showed the chemical hypoxia-induced increase of particles on the H9c2 cell surface, and that HDAC1 inhibition decreased particles on the H9c2 cell surface (Figure 4C,G). Control cardiomyocyte cultures treated with 0.5 μmol/L MGCD0103 did not change in cell volume and decrease particles on cell surface (Figure 4C).

Elasticity Measurements of Swelling H9c2 Cardiomyocytes With HDAC1 Inhibition by AFM

Chemical hypoxia reduced Young’s modulus in H9c2 cardiomyocytes relative to normoxia cells in a dose-dependent manner, and a minimum value was attained at 400 μmol/L CoCl₂ (Figure 4D,F). Compared with hypoxia groups, Young’s modulus in H9c2 cardiomyocytes with HDAC1 inhibition throughout 400 μmol/L CoCl₂ treatment was increased drastically (Figure 4D,F). Data from H9c2 cells with HDAC1 inhibition in normoxia condition showed that 0.5 μmol/L MGCD0103 did not change Young’s modulus (Figure 4D).

Observation of Actin Cytoskeleton and Acetylated Histone in Anoxic H9c2 Cardiomyocytes With or Without HDAC1 Inhibition by Confocal Microscope

In normoxia cells, actin fibers formed an isotropic network throughout the cell body and this was organized into parallel filamentous structures, and the fibers of actin were homogeneous and continuous (Figure 5). In contrast, under chemical hypoxia conditions, actin fibers of H9c2 cells were discontinuous, heterogeneous and more slender (Figure 5). Breakage points also emerged on the actin fibers (Figure 5). HDAC1 inhibition (by using MGD) throughout the process of hypoxia increased breakage of actin fiber and the density of the fiber arrangement (Figure 5). CoCl₂ treatments downregulated expression levels of H3 and H4 of H9c2 cardiomyocytes (Figure 5A,B). During chemical hypoxia, MGCD0103 treatment improved expression level of H3 and H4 (Figure 5A,B). In normoxia condition, MGCD0103 upregulated the expression levels of H3 and H4 in H9c2 cells (Figure 5A,B).

Figure 5.

Chemical hypoxia-induced morphological changes in F-actin (red) organization and a protective effect of HDAC1 inhibition. (A) Images of acetyl-histone H3 K9/K14 (green) and (B) acetyl-histone H4 (green) after a 5-h CoCl₂ treatment with HDAC1 inhibition. H3, acetyl-histone H3 K9/K14; H4, acetyl-histone H4. Other abbreviations as in Figures 1,2.

Empirical Formula and Ideal Physical Model for Young’s Modulus and HDAC1 Inhibition in Cardiomyocyte Swelling

To determine the relationship between Young’s modulus and cardiomyocyte swelling induced by chemical hypoxia, data that were non-linear or linear fitting were analyzed (Figure 6). Equations for the relationship between concentrations of CoCl₂ and either cellular volume or Young’s modulus were obtained by data fitting (Figure 6, Supplementary Results). From the equations, we obtained the empirical equation for the relationship between Young’s modulus and cellular volume of H9c2 cardiomyocytes in chemical hypoxia (Estimated Variance=0.00760).

Figure 6.

Data-fitting analysis of volume changes and Young’s modulus changes of H9c2 cells incubated in chemical hypoxia situation. The relationship between volume changes of H9c2 cells incubated in chemical hypoxia situation and concentration of cobalt chloride (CoCl₂) is demonstrated by data fitting analysis (A). Relationship between Young’s modulus changes of H9c2 cells incubated in chemical hypoxia situation and concentration of CoCl₂ is demonstrated by data fitting analysis (B). Relationship between volume changes and Young’s modulus changes of H9c2 cells incubated in chemical hypoxia situation is demonstrated by data fitting analysis (C). Solid lines, results of non-linear fitting; dotted lines, results of linear fitting.

Simplified formula:

V∝[c/E] (2b)

Where V is the cellular volume of H9c2 cardiomyocytes incubated in chemical hypoxia situation for 5 h, and E is Young’s modulus of the cells.

Equation (2b) shows that the volume of H9c2 cardiomyocytes incubated in chemical hypoxia situation for 5 h was inversely proportional to Young’s modulus of the cells, and equation (2a) is completely compatible with the results from experiments.

We assumed that cardiomyocyte swelling may be a slow, homogeneous and isotropous sphere enlargement process. In the process, we hypothesized expansive pressure, which was sustained by cytolemma was constant, and change of intracellular osmotic pressure could be negligible. The cytolemma would become thinner and broader than normal. According to Hooke’s law, the process of calculability could be expressed by numerical equations (Supplementary Results). Then, in consideration of bulk moduli, we could get

Equation (3) described the cell swelling process of H9c2 cardiomyocytes cultured in chemical hypoxia (0–400 μmol/L CoCl₂ treatments) without HDAC1 inhibition. When we choose μ=1 (because of the hypothesis that expansive pressure, which was sustained by the cytolemma, was constant, and the change in intracellular osmotic pressure may be negligible), ideal physical model (3) is approximately equal to that of the empirical equation (2a), suggesting that the assumptions of process of cell swelling are rational, and, in this case, the value of Young’s modulus is inversely proportional to the cell volume.

When we enhanced Young’s modulus of H9c2 cells incubated in chemical hypoxia situation by exerting HDAC1 inhibiting, it changed sharply (Figures 4D,7). Young’s modulus of H9c2 cells did not decrease along with increasing CoCl2 concentration (Figure 7). Data fitting results are not to match equation (2a). HDAC1 inhibition does increase Young’s modulus of H9c2 cells during the process of chemical hypoxia, but Young’s modulus is not the unique element that was accountable for cell swelling during chemical hypoxia treatment with HDAC1 inhibition (Figure 7). Results of oxidative stress assays, flow cytometry and images by AFM demonstrated that there was an increase in the number of microscopic states of cardiomyocyte system during chemical hypoxia. In this case, Equations (2a, 3) may be false. According to Boltzmann relation and formulas (Supplementary Results), we rebuilt an ideal physical model to describe the process of H9c2 cardiomyocyte swelling with low HDAC1 expression level, which is as follows:

Figure 7.

Data-fitting analysis of volume changes and Young’s modulus changes of H9c2 cells incubated in chemical hypoxia situation with MGCD0103. Data-fitting analysis of volume changes of H9c2 cells incubated in chemical hypoxia situation with HDAC1 inhibition and a concentration of CoCl₂ (A). Data-fitting analysis of Young’s modulus changes of H9c2 cells incubated in chemical hypoxia situation with HDAC1 inhibition and a concentration of CoCl₂ (B). Data-fitting analysis of volume changes and Young’s modulus changes of H9c2 cells incubated in chemical hypoxia situation with HDAC1 inhibition (C). Solid lines, data-fitting analysis result of H9c2 cells incubated in chemical hypoxia situation alone; data-fitting analysis result of H9c2 cells incubated in 400 μmol/L CoCl₂ with HDAC1 inhibition. Abbreviations as in Figure 1.

Discussion

The process of cell swelling involves physical transformation and changes of gene profile, which interact with each other. This important feature prompts us to investigate the relation among cellular phenotype, mechanical property, and biological mechanism.

Data obtained from western blotting showed that CoCl₂ markedly upregulated the expression level of HIF-1α and HDAC1 in H9c2 cells after a 5-h incubation under chemical hypoxia situation, and did not affect the expression level of HDAC4 and HAT1. The results indicate that HDAC1 plays an important role in cardiomyocyte swelling. A previous study by Du et al suggests that HDAC4 is crucial to injury induced by hypoxia/reoxygenation with physical hypoxia for 50 h (hypoxia 48 h, reoxygenation 2 h), not HDAC1.¹⁵ There are several possible reasons accounting for the observed differences between the aforementioned and our studies. One major factor is due to 2 different types of HDAC inhibitors, trichostatin A and MGCD0103, which was used in the study by Du et al and the present study, respectively. In addition, based on the results of siHDAC1, we assume that HDAC1 is significantly important to gene response in the early phase of chemical hypoxia, and that gene response induced by a different hypoxia protocol is distinct. Therefore, MGCD0103, a HDAC1-selective inhibitor (but not for HDAC4), was selected for subsequent experiments.

Both MTT and Hoechst 33342 staining showed that CoCl₂ significantly suppressed cell viability and induced apoptosis in H9c2 cells, whereas HDAC1 inhibition treatment significantly reduced the chemical hypoxia-induced proliferation inhibition of H9c2 cells, suggesting that HDAC1 inhibition may be a protective method against chemical hypoxia-induced injury to cardiomyocytes. We determined LDH leakage, which may induce cell growth suppression and death.³⁵ Results from the LDH assay were consistent with findings from the MTT assay. These findings suggest that HDAC1 inhibition may decrease chemical hypoxia-induced necrotic death in cardiomyocytes.

Oxidative stress plays a central role in the pathogenesis of hypoxia/ischemia-induced injuries because of the lipid peroxidation production of MDA and ROS.³⁶ Kang et al demonstrated that oxidative stress could upregulate expression and increase the activity of HDAC1.³⁷ Our results showed that MGCD0103 treatment and siHDAC1 significantly promoted the production of SOD and blocked MDA production in swelling H9c2 cells induced by chemical hypoxia. These results indicate that the HDAC1 inhibition contributed to the protection of cells against chemical hypoxia-induced oxidative stress disequilibrium.

Several previous studies have investigated the effects of swelling induced by hypoxia on the damage of cardiomyocytes by assessing variation in the expression or status of proteins and signaling pathways.^15,38 However, it remains unclear about how hypoxia-induced cell swelling affects the biomechanics of the cell and consequently alters cell behavior. Cellular ultrastructure changed markedly upon chemical hypoxia exposure, accompanied with the decrease of Young’s modulus and the increase of cell volume. Based on these results, we obtained empirical equations (2a, 2b) and an ideal model equation (3), suggesting that the volume of swelling H9c2 cell is inversely proportional to Young’s modulus values in 5 h. Thus, Young’s modulus may be a key factor for early phase therapy of myocardial edema.

The results from AFM, cell volume, and confocal microscope demonstrated that HDAC1 inhibition reduces cellular volume via upregulating Young’s modulus, and the observed decrease in Young’s modulus in cells exposed to chemical hypoxia was in accordance with a reduction in the organization of the actin cytoskeleton and a decrease in signal intensity of actin fibers stained, whereas HDAC1 inhibition with chemical hypoxia treatment attenuated disorganization and signal intensity decrease. Cytoskeleton confers tensile strength and resistance to deformation on cell and determines the shape of a cell.³⁹ There is greater disorganization of the cytoskeletal structure in the cells with lower Young’s modulus values.⁴⁰ HDAC1 inhibition results in aberrant gene expression, including cell structure and motility-related genes.⁴¹ Therefore, the mechanical mechanism of HDAC1 inhibition that induced a decrease in cell volume may be mediated through increasing Young’s modulus of the cell, and its molecular mechanism could be through changing expression profile of genes related to cell structure and motility.

However, results of data fitting analysis about Young’s modulus and cellular volume of swelling H9c2 cells with HDAC1 inhibition showed that the empirical equations (2a, 2b) and the ideal model equation (3) are no longer workable. The assumption that intracellular osmotic pressure of swelling H9c2 cells induced by chemical is constant seems no longer rational. These findings suggest that HDAC1 inhibition not only results in a change of Young’s modulus. Meanwhile, equation (3) fails to describe the systematic physical process of cell swelling induced by chemical hypoxia. We assumed that the swelling process of H9c2 cardiomyocytes is not only homogeneous and isotropous, but also heterogeneous. Results of flow cytometry and AFM scanning showed chemical hypoxia increased granularity on the surface of swelling H9c2 cells with or without HDAC1 inhibition. The volume of particles on the edema cell surface may contribute to the total amount of expansion (Figure 8).

Figure 8.

Proposed model for the HDAC1 inhibition protecting against hypoxia-induced swelling in the H9c2 cell. (A) H9c2 cells in normoxia. (B) H9c2 cells in chemical hypoxia situation. CoCl₂ induces H9c2 cardiomyocyte swelling through upregulating the expression level of HIF1α and HDAC1, thereby distorting the organization and expression level of the actin cytoskeleton, leading to a decrease in Young’s modulus. (C) HDAC1 inhibition protects against hypoxia-induced swelling in H9c2 cardiomyocytes through increasing Young’s modulus of the cell, regulating cell structure and motility-related genes, and thereby reversing disorganization and signal intensity decrease. Abbreviations as in Figure 1.

Thus, we built a new ideal physical model equation (4) to describe the physical process of cell swelling and reveal the biological and mechanical coupling mechanism of HDAC1 inhibition that is protective effect against cell swelling. It suggests that HDAC1 is able to regulate the intracellular osmotic pressure of the H9c2 cell, and that the intracellular osmotic pressure is of great importance for myocardial edema. Previous studies suggest that the expression of channels controls cell volume in cardiomyocytes through altering osmotic pressure in the cell, and HDAC1 inhibition results in expression change of ion channels.^41,42 Thus, channel-related genes may be ideal targets for edema treatment.^5,43,44 From equation (4), maintaining cell system stability is another effective method to protect cells against hypoxia-induced swelling. However, we cannot determine which biological process induced entropy change contributing to swelling of H9c2 cell. Moreover, by enhancing Young’s modulus through HDAC1 inhibition, cell stiffness is still a key element for protecting cardiomyocytes from edema.

In the present study, results from experiments and the ideal physical model indicated that Young’s modulus plays a significantly important role in H9c2 cell swelling induced by chemical hypoxia. HDAC1 inhibition protects against hypoxia-induced swelling in H9c2 cardiomyocytes through increasing Young’s modulus of the cell, regulating cell structure and motility-related genes, and thereby reversing disorganization and signal intensity decrease (Figure 8). Entropy of cell and intracellular osmotic pressure are also crucial for maintaining cell volume. In conclusion, cell stiffness may be a vital element involved in the process of myocardial edema, and HDAC1 is a promising target for regulating cell stiffness.

Acknowledgments

This study was supported by the International Cooperative Research of Translational Medicine for Congenital Heart Defects, Ministry of Science and Technology of China (2011DFA33120) and the Fundamental Research Funds from Medical Genetic and Biological Medicine Collaborative Innovation Center of Yunnan Province, and the Medical Scientific Research Project, Health and Family Planning Commission of Lanzhou (LZWSKY2014-1-02).

Disclosures

None.

Supplementary Files

Supplementary File 1

Supplementary Methods

Supplementary Results

Please find supplementary file(s);

http://dx.doi.org/10.1253/circj.CJ-17-0022

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