TBX3 deficiency accelerates apoptosis in cardiomyoblasts through regulation of P21 expression

Journal Pre-proof TBX3 deficiency accelerates apoptosis in cardiomyoblasts through regulation of P21 expression Meiling Cao, Binlu Zhu, Yuanyuan Sun, Xueqi Zhao, Guangrong Qiu, Weineng Fu, Hongkun Jiang PII:

S0024-3205(19)30967-1

DOI:

https://doi.org/10.1016/j.lfs.2019.117040

Reference:

LFS 117040

To appear in:

Life Sciences

Received Date: 22 July 2019 Revised Date:

23 October 2019

Accepted Date: 31 October 2019

Please cite this article as: M. Cao, B. Zhu, Y. Sun, X. Zhao, G. Qiu, W. Fu, H. Jiang, TBX3 deficiency accelerates apoptosis in cardiomyoblasts through regulation of P21 expression, Life Sciences, https:// doi.org/10.1016/j.lfs.2019.117040. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

1

TBX3 deficiency accelerates apoptosis in cardiomyoblasts through regulation of

2

P21 expression

3

Meiling Cao1, Binlu Zhu2, Yuanyuan Sun3, Xueqi Zhao2, Guangrong Qiu3, Weineng

4

Fu3, Hongkun Jiang2, *

5

1

6

University, Shenyang 110001, People’s Republic of China

7

2

8

Shenyang 110001, People’s Republic of China

9

3

Department of Neonatology, The First Affiliated Hospital of China Medical

Department of Pediatrics, The First Affiliated Hospital of China Medical University,

Department of Medical Genetics, College of Basic Medical Sciences, China Medical

10

University, Shenyang 110122, People’s Republic of China

11

*

12

Affiliated Hospital of China Medical University, 155 North Nanjing Street, Shenyang

13

110001, People’s Republic of China

14

Tel/Fax: +86-24-83282527

15

16

17

18

19

20

Corresponding author: Dr. Hongkun Jiang, Department of Pediatrics, The First

21

Abstract

22

Congenital heart disease (CHD) is the most common birth defect in newborns.

23

There is increasing evidence that apoptosis and remodeling of the cardiomyoblasts are

24

the major pathology of CHD. Previous research found that T-box transcription factor

25

3 (TBX3) was compulsory for the regulation of proliferation, cell cycle arrest and

26

apoptosis in various cells. Hence, TBX3 might be involved in the treatment of CHD.

27

The primary aim of this study was to study the effects of TBX3 on apoptosis in aged

28

cardiomyoblasts and investigate the latent mechanism. In the present study, we found

29

TBX3 knockdown induced proliferation inhibition, cell cycle arrest and apoptosis

30

accompanied by mitochondrial dysfunction in cardiomyoblasts at passage 10 to 15.

31

Apoptosis-inducing effects of TBX3 silence could be neutralized by silencing P21

32

using specific siRNA. In addition, the mRNA and protein expression levels of TBX3

33

in the heart tissues of sporadic type CHD donors were obviously down-regulated. In

34

conclusion, we demonstrated that TBX3 deficiency accelerated apoptosis via directly

35

regulating P21 expression in senescent cardiomyoblasts.

36

Keywords: Congenital heart disease; TBX3; Cell cycle arrest; Apoptosis

37

Abbreviations: ATPase, adenosine triphosphatase; CDK, cyclin dependent kinase;

38

CHD, congenital heart disease; ROS, reactive oxygen species; TBX3, T-box

39

transcription factor 3; UMS, ulnar-mammary syndrome.

40

1. Introduction

41

Congenital heart disease (CHD), the leading non-infectious cause of morbidity

42

and mortality in newborn children in the world, is a life-threatening congenital

43

malformation associated with genetic backgrounds and environmental risk factors.

44

According to the clinical manifestation, CHD can be classified into two types,

45

including acyanotic congenital heart disease and cyanotic congenital heart disease

46

[1-2]. An increasing number of epidemiological investigations found that the

47

incidence of CHD was 19 to 75 of every 1000 living newborns [3]. If the infants who

48

did not survive were calculated, CHD incidence would be higher [4-5]. Despite

49

simple CHD can be completely remitted at present, early surgical management that

50

has high risk is the necessary therapeutic strategy for complex CHD. As known,

51

although multi-gene abnormalities have been confirmed in the occurrence of CHD,

52

the accurate mechanism of CHD is remained to be investigated. Programmed cell

53

death plays a crucial part in maintaining normal cardiac function under physiological

54

conditions and apoptosis-related protein is expressed at the basal level in the postnatal

55

hearts [6-7]. A clinical assessment indicated that aggravated apoptosis could be found

56

in the cardiac tissue samples from CHD patients compared with the normal heart

57

tissues from the aged-matched subjects [8]. In addition, promoting proliferation and

58

suppressing apoptosis exerted protective effects in H9C2 cardiomyoblasts,

59

highlighting that apoptosis inhibition might be a potential therapeutic strategy for

60

CHD [9-10]. Hence, we believe that inhibition of apoptosis in cardiomyoblasts might

61

be a promising therapeutic strategy to inhibit CHD development.

62

T-box transcription factor 3 (TBX3) belongs to the T-box factor family that is

63

composed of evolutionary conserved transcription factors regulating early embryonic

64

development [11]. TBX3 has established role in the development of the heart,

65

mammary glands, limbs and genitalia in the vertebrates [12]. In addition, human

66

TBX3 gene mutation leads to ulnar-mammary syndrome (UMS), which is

67

characterized by upper limb malformation, mammary hypoplasia, as well as

68

deficiency in hair, tooth and reproductive system [13-14]. On the contrary, the

69

oncogenic effects of TBX3 overexpression have emerged in a wide variety of cancer,

70

including gastric cancer, breast cancer and pancreatic cancer [15-17]. A recent study

71

showed that the occurrence and development of conotruncal heart defect could be

72

attributed to the variants of TBX3 [18]. Hence, the effects of TBX3 in

73

cardiomyoblasts are worth to be emphasized in CHD. Except for the definite

74

regulation role on the development of multiple organs, TBX3 expression possesses

75

the function on modulating cell cycle. The previous experiments have shown that the

76

protein expression of TBX3 is elevated at S phase and TBX3 silence results in S

77

phase arrest, indicating the regulation of TBX3 in cell cycle [19]. Moreover, the

78

previous study also has illustrated that the function of TBX3 on overriding cell cycle

79

to inhibit apoptosis and enhance proliferation is achieved by suppressing cell cycle

80

regulatory protein P21 (also named as P21 waf1/cip1 and P21/CDKN1A) [19]. As the

81

downstream signaling molecule of P53, P21 serves as the inhibitor of CDK family in

82

both P53-dependent and P53-independent manners, which is necessary for adjusting

83

cell cycle and proliferation [20]. Cell cycle transitions (G1 phase to S phase and G2

84

phase to mitosis) is depended on continuous activation and inactivation of CDK

85

proteins that are attached to serine/threonine protein kinases [21]. For instance, the

86

activation of CDK4 and CDK6 by cyclin D1 senses the mitogenic signals and serves

87

as the growth factor sensors in G1 phase, cyclin E1 and E2-induced CDK activation

88

triggers the transition from G1 phase to S phase [22]. Plus, the first identified CKI

89

protein P21 suppresses diverse cell cycle progression through disrupting the

90

interactions between CDKs and cyclin proteins [23]. Several lines of evidence

91

demonstrate that the mitochondrion involves in regulation of apoptosis in mammals.

92

Mitochondria are the cellular bioenergy centers in a physiological manner [24]. In

93

addition to participating in regulation of energy generation, intercellular

94

communication and metabolism, the importance of mitochondria in apoptosis has

95

been

96

inter-membrane space to the cytoplasm and triggers the apoptosis-related events at

97

molecular levels [26]. Cytosolic cytochrome C interacts with Apaf-1, recruits

98

procaspase-9, assembles cytochrome c/Apaf-1/caspase-9 apoptosomes, and thereby

99

initiating downstream apoptotic cascade [27]. These events above suggested that

100

TBX3 might be the important bond between the pathogenesis of CHD and

101

pathological apoptosis as well as cell cycle arrest in cardiomyoblasts. Therefore, the

102

effects of TBX3 in the pathogenesis of CHD are worthy to be investigated.

well

established

[25].

Cytochrome

C

migrates

from

mitochondrial

103

Here we aimed to investigate the effects of TBX3 on mitochondrial function,

104

apoptosis and cell cycle arrest in aged H9C2 cardiomyoblasts and explore the

105

potential mechanism of CHD occurrence.

106

2. Materials and methods

107

2.1 Cell treatment

108

2.1.1 Cell culture

109

Rat cardiomyoblast H9C2 cell line was obtained from Zhong Qiao Xin Zhou

110

Biotechnology Co., Ltd. (Shanghai, China). H9C2 cardiomyoblasts at passage 10 to

111

15 were employed. H9C2 cardiomyoblasts were grown in Dulbecco’s modifed

112

Eagle’s medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum

113

(FBS) (Hyclone, USA) at 37 °C with 5% CO2 in a humidified incubator.

114

2.1.2 Transfection

115

H9C2 cardiomyoblasts at passage 10 to 15 were transfected with TBX3-specific

116

small interfering RNA (siRNA) 1-5 or control siRNA (Wanlei Biological Technology

117

Co., Ltd., Shenyang, China) according to the instructions. Then, the transfected H9C2

118

cardiomyoblasts were maintained in fresh culture medium for 48 h before the

119

transfection efficiency detection.

120 121 122 123 124 125 126 127 128 129

The sequences for TBX3 siRNA1-5 and control siRNA were listed as follows: TBX3 siRNA-1: 5’-CCAUGAGAGAUCCGGUUAUTT-3’ 5’-AUAACCGGAUCUCUCAUGGTT-3’ TBX3 siRNA-2: 5’-GCGAAUGUUCCCUCCAUUUTT-3’ 5’-AAAUGGAGGGAACAUUCGCTT-3’ TBX3 siRNA-3: 5’-GCUGAUGACUGUCGAUAUATT-3’ 5’-UAUAUCGACAGUCAUCAGCTT-3’ TBX3 siRNA-4: 5’-CCAACAACAUUUCGGAUAATT-3’ 5’-UUAUCCGAAAUGUUGUUGGTT-3’ TBX3 siRNA-5: 5’-GCAGUCCAUGAGGGUGUUUTT-3’

5’-AAACACCCUCAUGGACUGCTT-3’

130

Control siRNA: 5’-UUCUCCGAACGUGUCACGUTT-3’

131

5’-ACGUGACACGUUCGGAGAATT-3’

132

133

2.1.3 Transduction

134

H9C2 cardiomyoblasts at passage 10 to 15 were transduced with TBX3-expressing

135

lentivirus or negative control lentivirus (Wanlei Biological Technology Co., Ltd.,

136

Shenyang, China) and all the transduction protocols were carried out according to the

137

manufacturer’s instruction. Afterwards, the transduced H9C2 cardiomyoblasts were

138

maintained in fresh culture medium for 48 h before harvest.

139

2.2 Real-time polymerase chain reaction (RT-PCR)

140

After the corresponding treatment, the total RNA of aforementioned H9C2

141

cardiomyoblasts or heart samples was extracted by using Trizol reagent. After TBX3

142

cDNA was synthesized by using the M-MLV reverse transcriptase (TianGEN, China),

143

RT-PCR analysis was conducted with SYBR Green master mix kits in the Exicycler

144

96 System (Bioneer, Korea). TBX3 mRNA level was normalized to that of GAPDH

145

and calculated by 2 -△△CT method.

146

The primers for RT-PCR were as follows:

147

Rat

148

5’-GTTGGCTCTGACGATGTGG-3’

149

Rat

150

5’-TAGGAGCCAGGGCAGTAA-3’

151

Human

152

5’-TCGCCTTCCCGACTTGGT-3’

153

Human

TBX3

forward:

GAPDH

TBX3

GAPDH

5’-TCGGATAAACACGGATTTACTT-3’;

forward:

forward:

forward:

5’-ACGTTGACATCCGTAAAGAC-3’;

5’-GGCATCCCGTTCTCCTCCCT-3’;

5’-GACCTGACCTGCCGTCTAG-3’;

reverse:

reverse:

reverse:

reverse:

154

5’-AGGAGTGGGTGTCGCTGT-3’

155

2.3 Cell counting kit-8 (CCK-8) assay

156

To assess the viability of aged H9C2 cardiomyoblasts following treatment, H9C2

157

cardiomyoblasts at passage 10 to 15 were seeded in plates at a density of 5×103

158

cells/well. After the transfection or transduction described above, the H9C2

159

cardiomyoblasts were incubated for another 0, 24 or 48 h before the exposure to

160

CCK8 solution (KeyGEN BioTECH, China) according to the manufacturer’s

161

instructions. The absorbance at 450 nm was measured by a microplate reader.

162

2.4 Cell cycle analysis

163

Cell cycle distribution of the aforementioned H9C2 cardiomyoblasts was

164

analyzed by using flow cytometry. To be brief, H9C2 cardiomyoblasts at passage 10

165

to 15 were harvested 48 h after the respective treatment, fixed in 70% ethanol at 4˚C

166

for 2 h, treated with propidium iodide (PI) dye (Beyotime Institute of Biotechnology,

167

China) at 37˚C for 30 min, and subsequently analyzed by using a flow cytometer

168

(NovoCyte, United States).

169

2.5 Hochest 33342 staining

170

Hoechst 33342 staining was implemented to observe the apoptotic morphological

171

characteristics of aged H9C2 cardiomyoblasts. Simply, H9C2 cardiomyoblasts at

172

passage 10 to 15 were plated in a 6-well plate at a density of 1×105 cells/well.

173

Following the transfection or transduction protocol, the H9C2 cardiomyoblasts were

174

incubated for another 48 h, washed with PBS, and stained by Hoechst 33342

175

according to the manufacturer’s instruction. Finally, apoptotic morphological

176

alterations in the stained H9C2 cardiomyoblasts were observed under a fluorescence

177

microscope at 400 × magnification.

178

2.6 Apoptosis analysis

179

The influence of TBX3 expression on apoptosis in aged H9C2 cardiomyoblast

180

was examined using flow cytometry. Simply, H9C2 cardiomyoblasts at passage 10 to

181

15 were plated in a 6-well plate at a density of 1×105 cells/well. Following the

182

transfection or transduction protocol, the H9C2 cardiomyoblasts were stained by

183

adding Annexin V-fluorescein isothiocyanate (FITC) and PI in dark at room

184

temperature for 10-20 min. finally, apoptosis rate of the treated H9C2

185

cardiomyoblasts was detected by using a flow cytometer (NovoCyte, United States).

186

2.7 Mitochondrial membrane potential detection

187

The changes of mitochondrial membrane potential in aforementioned H9C2

188

cardiomyoblasts were detected by using specific fluorescent probe JC-1 according to

189

the requirement of mitochondrial membrane potential detection kit (Beyotime

190

Institute of Biotechnology, China). The relative fluorescence intensity (excitation and

191

emission wavelengths of 485 and 525 nm, excitation and emission wavelengths of

192

525 and 590 nm, respectively) represented the change of mitochondrial membrane

193

potential.

194

2.8 Adenosine triphosphatase (ATPase) activity

195

Treated H9C2 cardiomyoblasts were lysed with PBS and the protein

196

concentrations of lysis were determined by BCA protein quantification kit (Beyotime

197

Institute of Biotechnology, China). In short, the activity of ATPase in H9C2

198

cardiomyoblasts was quantified by corresponding commercial kits (Nanjing Jiancheng

199

Bioengineering Institute, China). The activity of ATPase was expressed as U per

200

milligram protein (U/mg prot).

201

2.9 Mitochondrial reactive oxygen species (ROS) production

202

Mitochondria of the H9C2 cardiomyoblasts at passage 10 to 15 were separated by

203

centrifugation as recommended by the manufacturer (Beyotime Institute of

204

Biotechnology, China). The separated mitochondria were incubated with 10 µM

205

fluorescence probe DCFH-DA for 30 min at 37 °C followed by fluorescence intensity

206

detection according to the instructions of ROS detection kit (Beyotime Institute of

207

Biotechnology, China).

208

2.10 Immunofluorescence

209

H9C2 cardiomyoblasts at passage 10 to 15 were treated as described in cell

210

treatment, fixed with 4% paraformaldehyde for 15 min, and then incubated with 0.1%

211

tritonX-100 (Beyotime Institute of Biotechnology, China) for 30 min at room

212

temperature. After wash with PBS for three times, the treated H9C2 cardiomyoblasts

213

were incubated with primary Ki67 antibody (Cell Signaling Technology, United States,

214

dilution: 1:100) overnight at 4 °C followed by Cy3-labeled goat anti-rabbit IgG (H +

215

L) (Solarbio, China). Afterwards, 4’, 6-diamidino-2-phenylindole (DAPI, Beyotime

216

Institute of Biotechnology, China) incubation was utilized to stain the nuclei. Finally,

217

immune-positive H9C2 cardiomyoblasts were observed under a fluorescence

218

microscope at 400 × magnification.

219

2.11 Chromatin immunoprecipitation (ChIP) assay

220

ChIP assay was implemented to detect the targeted relationship between TBX3

221

and P21. Briefly, the aforementioned H9C2 cardiomyoblasts were fixed in 1%

222

formaldehyde solution at room temperature for 10 min, washed with PBS, and

223

harvested by centrifugation at 2000rpm for 5 min. The chromatin was isolated and

224

exposed to sonication to obtain DNA fragments (fractionated size: 500-1000 bp) that

225

were in turn immunoprecipitated with TBX3 antibody. Afterwards, the TBX3

226

protein-bound DNA was purified by a DNA purifcation kit (Wanlei Biological

227

Technology Co., Ltd., Shenyang, China). Finally, the purified DNA was utilized to

228

analyze the combination between TBX3 and P21.

229

2.12 Western blot

230

The mitochondrial and total protein samples were isolated from the

231

aforementioned H9C2 cardiomyoblasts. Briefly, the same amount of protein samples

232

were separated by 8%, 10% or 14% sodium dodecyl sulfate polyacrylamide gel

233

electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore, USA)

234

that were subsequently blocked by 5% non-fat milk for 1 h at the room temperature.

235

Afterwards, the PVDF membranes were subjected to the relevant primary antibody at

236

4 °C overnight, respective horseradish peroxidase (HRP) labeled secondary antibody

237

(Solarbio, China) for 45 min at 37 °C, and ultimately the ECL kit (Solarbio, China).

238

The quantification of the protein expression was implemented by Gel-Pro-Analyzer

239

software.

240

The primary antibodies were as follows: TBX3 (Abcam, UK), cyclin A (Abcam,

241

UK), cyclin D1 (Cell Signaling Technology, United States), cyclin B1 (Cell Signaling

242

Technology, United States), caspase-3 (Boster, China), cleaved caspase-3 (Cell

243

Signaling Technology, United States), caspase-9 (Abcam, UK), cleaved caspase-9

244

(Cell Signaling Technology, United States), Bcl-2 (proteintech, China), Bax

245

(proteintech, China), cleaved-PARP (Cell Signaling Technology, United States),

246

cytochrome C (Cell Signaling Technology, United States), P21 (Abclonal, China).

247

Patient samples

248

Eight heart samples from CHD fetuses (24-25 weeks) were recruited in the First

249

Affiliated Hospital of China Medical University. The sporadic types of CHD included

250

ventricular septal defect, atrioventricular septal defect and atrial septal defect. Eight

251

healthy hearts came from fetuses (24-25 weeks) with other congenital defects. Heart

252

tissue collection was implemented after abortion. RT-PCR and TBX3 ELISA (USCN,

253

China) were used to detect heart TBX3 mRNA and protein levels. The procedures

254

were in accordance with the Declaration of Helsinki and the protocols were

255

accordance with the Ethics Committee of the First Affiliated Hospital of China

256

Medical University.

257

2.13 Statistical analysis

258

Data were presented as mean ± standard derivations (SD) and calculated by

259

GraphPad Prism 7.0 (GraphPad Software, United States). Significant differences

260

between two groups were analyzed by t-test and data from three or more groups was

261

determined using one way analysis of variance (ANOVA) followed by a Tukey’s

262

multiple comparison test. Value of p less than 0.05 was considered statistically

263

significant. All in vitro experiments were performed three times (n=3).

264

3. Results

265

3.1 Effect of TBX3 on proliferation in H9C2 cardiomyoblasts

266

CCK-8 assay was utilized to inspect the influence of TBX3 expression on

267

proliferation of H9C2 cardiomyoblasts. We used TBX3 specific siRNA to silence

268

CXCR2 gene in H9C2 cardiomyoblasts, meanwhile, TBX3-expressing lentivirus was

269

used to induce TBX3 overexpression (Supplement Fig. S1-2). As shown in Fig. 1A,

270

TBX3 knockdown significantly decreased H9C2 cardiomyoblast viability in a time

271

dependent manner. On the contrary, H9C2 cardiomyoblast proliferation could be

272

elevated with TBX3 overexpression. Similar to the results of CCK8, the fluorescence

273

targeting the proliferation markers Ki67 was whittled with TBX3 silence while

274

enhanced with TBX3 overexpression in H9C2 cardiomyoblasts (Fig. 1B). The above

275

results indicated that TBX3 silence accelerated proliferation deficiency in H9C2

276

cardiomyoblasts.

277

3.2 Effect of TBX3 on cell cycle arrest in H9C2 cardiomyoblasts

278

To investigate whether TBX3 expression was involved in cell cycle arrest, we

279

analyzed the distribution of H9C2 cardiomyoblast cell cycle following TBX3

280

expression interference. As exhibited in Fig. 2A, compared with the control group,

281

there was a significant increase in the percentage of H9C2 cardiomyoblasts at G1

282

phase (p < 0.05) while an obvious decrease at S phase (p < 0.05) after TBX3 specific

283

siRNA treatment. However, TBX3 overexpression did not markedly affected cell

284

cycle distribution in H9C2 cardiomyoblasts. In addition, TBX3 silence inhibited the

285

expression of cyclin protein family, including cyclin D1, cyclin A and cyclin B1 (Fig.

286

2B-D, p < 0.05). Interestingly, TBX3 overexpression could aggrandize cyclin protein

287

expression while did not regulate cell cycle in H9C2 cardiomyoblasts. The data

288

indicated that TBX3 knockdown accelerated cell cycle arrest at S phase in H9C2

289

cardiomyoblasts.

290

3.3 Effect of TBX3 on apoptosis in H9C2 cardiomyoblasts

291

Since apoptosis is a consequence of cell cycle arrest, we employed Hoechst

292

33342 staining to observe apoptosis morphology and Annexin V FITC/PI double

293

staining to quantify cell apoptosis rate in H9C2 cardiomyoblasts after TBX3

294

expression interference. As shown in Fig. 3A, TBX3 siRNA-treated H9C2

295

cardiomyoblasts exhibited typical morphological characteristics of apoptosis,

296

including nuclear fragmentation and chromatin condensation. Afterwards, the

297

percentage of apoptotic H9C2 cardiomyoblasts was assessed by Annexin V FITC/PI

298

double staining and the data showed that TBX3 silence dramatically induced

299

apoptosis in H9C2 cardiomyoblasts (Fig. 3B-C, p < 0.05). Subsequently, western blot

300

was also conducted to investigate the levels of Apoptosis related proteins, such as

301

cleaved caspase-9, cleaved caspase-3, cleaved PARP, Bcl-2 and Bax. The results

302

demonstrated

303

PARP and Bax, and a decrease in Bcl-2 after TBX3 knockdown in H9C2

304

cardiomyoblasts (Fig. 3D-H, p < 0.05). However, there was no significant change in

305

apoptotic state of TBX3-overexpressing H9C2 cardiomyoblasts. The results indicated

306

that TBX3 knockdown was involved in H9C2 cardiomyoblast apoptosis.

307

3.4 Effect of TBX3 on mitochondrial function in H9C2 cardiomyoblasts

an increase in the levels of cleaved caspase-9, cleaved caspase-3, cleaved

308

To further examine the effects of TBX3 expression on mitochondrial function in

309

H9C2 cardiomyoblasts, we first detected the alterations of mitochondrial membrane

310

potential by utilizing the JC-1 fluorescent dye. As shown in Fig. 4A, the ratio between

311

the red and green fluorescence in H9C2 cardiomyoblasts loaded with JC-1 staining

312

was significantly down-regulated after TBX3 knockdown (p < 0.05). Then

313

mitochondrial ATPase activity and ROS generation were also analyzed. The results

314

showed that mitochondrial ATPase activity was down-regulated (Fig. 4B, p < 0.05)

315

and ROS generation was up-regulated (Fig. 4C, p < 0.05) in H9C2 cardiomyoblasts

316

with TBX3 knockdown, which indicated the existence of mitochondrial dysfunction.

317

Besides, as exhibited in Fig. 4D-E, the cytochrome C content in the cytoplasm was

318

prominently increased while decreased in the mitochondria after TBX3 siRNA

319

treatment, indicating TBX3 knockdown accelerated cytochrome C release from the

320

mitochondria into the cytoplasm in H9C2 cardiomyoblasts. The results indicated that

321

TBX3 knockdown induced mitochondrial disorder in in H9C2 cardiomyoblasts.

322

3.5 Effect of TBX3 on p21 expression in H9C2 cardiomyoblasts

323

To investigate the underlying mechanism of TBX3 expression on cell cycle and

324

apoptosis, we detected cell cycle regulatory protein P21 levels in H9C2

325

cardiomyoblasts after TBX3 expression interference. As shown in Fig. 5A-B, both the

326

mRNA and protein levels of P21 were increased in H9C2 cardiomyoblasts with TBX3

327

knockdown (p < 0.05). Moreover, the interaction between TBX3 protein and P21

328

DNA was verified by ChIP assay (Fig. 5C). The results indicated that P21 gene

329

expression was directly regulated by TBX3 protein.

330

3.6 Effect of TBX3/p21 pathway on proliferation in H9C2 cardiomyoblasts

331

To verify the effect of TBX3/p21 pathway on proliferation in H9C2

332

cardiomyoblasts, TBX3 siRNA and p21 siRNA were co-transfected into H9C2

333

cardiomyoblasts. As shown in Fig. 6A, P21 expression was obviously suppressed

334

after the co-transfection compared with TBX3 siRNA transfection alone (p < 0.05).

335

Additionally, the decreased proliferation capacity induced by TBX3 silence could be

336

observably promoted by TBX3 siRNA and p21 siRNA co-transfection in H9C2

337

cardiomyoblasts (Fig. 6B), manifesting that TBX3/p21 pathway might participate in

338

the regulation of proliferation in H9C2 cardiomyoblasts.

339

3.7 TBX3 expression in CHD samples

340

Finally, we examined the mRNA and protein expression of TBX3 in the hearts of

341

CHD donors to verify its potential in CHD treatment. As shown in Fig. 7A and B, the

342

mRNA and protein expression levels of TBX3 were obviously decreased in donors

343

with sporadic type CHD compared with the control ones (p < 0.05), indicating the

344

importance of TBX3 in CHD treatment.

345

4. Discussion

346

Pathological cardiomyoblast loss is a major histopathological hallmark of CHD,

347

but the accurate molecular mechanisms still need future investigation. In the present

348

study, we investigated whether TBX3 expression deficiency was the key point of

349

accelerating mitochondrial dysfunction, cell cycle arrest and apoptosis in aged H9C2

350

cardiomyoblasts. We found that TBX3 silence by specific siRNA inhibited

351

proliferation and blocked G1/S phase transition in aged H9C2 cardiomyoblasts. TBX3

352

knockdown up-regulated aged H9C2 cardiomyoblast apoptosis accompanied by

353

mitochondrial dysfunction. Besides, the apoptosis induction of TBX3 deletion in aged

354

H9C2 cardiomyoblasts could be abolished by P21 knockdown. Interestingly,

355

exorbitant proliferate was not observed in H9C2 cardiomyoblast with TBX3

356

overexpression. Hence, our results manifested that TBX3 was an important mediator

357

that regulated proliferation and apoptosis by targeting P21 in aged H9C2

358

cardiomyoblasts.

359

TBX3 is a critical member of the highly conserved T-box family of transcription

360

factors that possess explicit regulatory role on development in vertebrates [28].

361

Moreover, TBX3 involves in cell fate decision, cell cycle regulation, organ

362

development and even oncogenesis [29]. In addition, a strong relationship between

363

TBX3 insufficiency and congenital developmental disorders has been reported in the

364

literature [30]. As mentioned in the aforementioned literature, TBX3 variants could be

365

observed in patients with conotruncal heart defects, a heterogeneous congenital heart

366

malformation [18]. In our study, we found TBX3 deficiency in the hearts of donors

367

with sporadic types of CHD, which indicated its clinical importance in CHD

368

treatment. Afterwards, in order to further verify the effects of TBX3 expression on the

369

cell proliferative potential in aged H9C2 cardiomyoblasts, we employed TBX3

370

specific siRNA to silence TBX3 and TBX3-expressing lentivirus to increase TBX3 in

371

H9C2 cells at passage 10 to 15. After the corresponding treatment, the mRNA and

372

protein levels of TBX3 were respectively up-regulated and down-regulated in H9C2

373

cardiomyoblasts (Supplement Fig. S1-2). Besides, the inhibiting effects of TBX3

374

silence did not appear until 48 h after TBX3 specific siRNA employment. Similarly,

375

the effects of promoting growth were detected 48 h after TBX3-expressing lentivirus

376

transduction. It is well understood that Ki67 protein expressed at all stages of the cell

377

cycle other than resting phase is an approved cellular marker labeling proliferative

378

potential in several cell types [31]. Hence, we utilized immunofluorescence targeting

379

Ki67 to verify cellular proliferation in aged H9C2 cardiomyoblasts 48 h after

380

treatment and the results were in line with CCK8 analysis. Based on these data, we

381

confirmed that the abnormal expression of TBX3 was involved in the regulation of

382

proliferation in aged H9C2 cardiomyoblasts.

383

It is well accepted that transition from G1 phase to S phase and G2 phase to M

384

phase are the two critical stage of the cell cycle. In addition, cell growth can be

385

inhibited, if the cell cycle is blocked at any phase. Prior studies that have suggested

386

the importance of TBX3 expression on inducing cell cycle arrest through blocking

387

G1/S transition [19]. Subsequently, the effects of TBX3 expression on the distribution

388

of cell cycle in H9C2 cardiomyoblasts were investigated by using flow cytometry. We

389

also observed that the percentage of aged H9C2 cardiomyoblast at G1 phase was

390

increased while decreased in S phase, indicating the diminished DNA synthesis after

391

TBX3 knockdown. Since cell cycle was triggered by CDKs and their regulatory

392

subunit cyclins, we found cyclin D1, cyclin A and cyclin B1 levels were decreased in

393

TBX3 siRNA-treated H9C2 cardiomyoblasts. Wondrously, TBX3 knockdown

394

inhibited cell proliferation and triggered cell cycle arrest, but TBX3 over-expression

395

is ineffective for the cell cycle in aged H9C2 cardiomyoblasts, the mechanism of

396

which needed further investigation.

397

Since cell cycle arrest is an induction factor of apoptosis, which is considered to

398

participate in pathological changes of diverse cardiac disorders, such as myocarditis

399

and cardiomyopathy [7]. Although apoptosis is occurred at base levels under the

400

physiological circumstance, excessive cardiomyoblast apoptosis can be detected in

401

CHD patients with volume overload [32]. Recently, inhibiting apoptosis and elevating

402

survival of cardiomyoblasts have been proposed as the promising treatment strategies

403

targeting CHD. Based on the above, we tried to evaluate changes in apoptosis

404

morphology, apoptosis index and apoptosis-related protein expression in modified

405

H9C2 cardiomyoblasts. The data of current study demonstrated that apoptosis was

406

promoted after TBX3 silence in aged H9C2 cardiomyoblasts. Moreover,

407

mitochondrial dysfunction characterized by decreased mitochondrial membrane

408

potential, suppressed ATPase activity and elevated ROS generation was also detected

409

in TBX3-silenced H9C2 cardiomyoblasts. Since apoptosis could be affected by

410

multifarious intracellular transduction cascades, we only explored the pathway

411

associated with cell cycle arrest. As an inhibitor of CDKs, P21 is known to possess

412

multiple biological functions, such as regulating cell cycle, differentiation and

413

apoptosis [33]. Nonetheless, the effect of P21 on apoptosis is bidirectional. This is

414

mainly because P21 expression induces cell cycle blockage to repair DNA damage

415

when the cell suffers mild DNA lesions, whereas P21 triggers apoptotic response once

416

the DNA damage exceeds the repair ability [34]. In order to investigate the role of P21

417

in modified H9C2 cardiomyoblasts, we co-transfected TBX3 siRNA and P21 siRNA

418

into aged H9C2 cardiomyoblasts. In our study, P21 silence abolished the

419

apoptosis-inducing effects of TBX3 deficiency. Combined the CHIP assay, we found

420

P21 was the direct downstream signaling molecule of TBX3. Based on the data above,

421

we summarized TBX3/P21 signaling was involved in apoptotic occurrence in aged

422

H9C2 cardiomyoblasts.

423

H9C2 cardiomyoblasts at passage 10 to 15 were employed in the present study.

424

Prior studies have suggested that morphology changes and sensitivity to toxicity are

425

increased with the number of passages in H9C2 cardiomyoblasts [35]. However, the

426

literature results also indicated that the influence of cell line passage number on the

427

reliability of study results was intricate and depended on multi-factor, such as culture

428

conditions [35]. Hence, we will investigate the effect of TBX3 deficiency on

429

apoptosis in cardiomyoblasts at different passage in the future.

430

In conclusion, the present study showed that TBX3 expression deficiency

431

accelerated apoptosis in aged cardiomyoblasts, which was accompanied by

432

mitochondrial dysfunction, cell cycle arrest, growth suppression and apoptosis.

433

Although more research should be implemented to verify the accurate mechanism of

434

TBX3 expression on cardiomyoblast function, our work suggests TBX3 maybe one of

435

the potential therapeutic targets of CHD.

436

Acknowledgement

437

This study was supported by grants from the National Natural Science

438

Foundation of China (No. 81300130) and the Basic Scientific Research Projects of

439

Liaoning Higher Education Institutions (No. LFWK201701).

440

References

441

1.

442

BJM. The changing epidemiology of congenital heart disease. Nature Reviews

443

Cardiology. 2011;8(1):50–60.

444

2.

445

Epidemiological study of congenital heart defects in children and adolescents:

446

analysis of 4,538 cases. Arq Bras Cardiol. 2003;80(3): 269-278.

Van Der Bom T, Zomer AC, Zwinderman AH, Meijboom FJ, Bouma BJ, Mulder

Miyague NI, Cardoso SM, Meyer F, Ultramari FT, Araújo FH, Rozkowisk I, et al.

447

3.

Zhou Y, Jia WK, Jian Z, Zhao L, Liu CC, Wang Y, et al. Downregulation of

448

microRNA-199a-5p protects cardiomyoblasts in cyanotic congenital heart disease by

449

attenuating endoplasmic reticulum stress. Mol Med Rep. 2017;16(3):2992–3000.

450

4.

451

Pediatr Cardiol. 1995;16(4):155–165.

452

5.

453

Cardiol. 2002;39(12):1890–900.

454

6.

455

of graded hypoxia on the embryonic chick heart. Teratology. 1985;32(3):463–472.

456

7.

457

2000;45(3):603–620.

458

8.

459

might play a role in the onset of congenital heart disease by inducing cardiomyocyte

460

apoptosis, Circ. J. 74 (2010) 2410–2418.

461

9.

462

congenital heart disease by suppressing HES1, Eur. J. Pharmacol. 836 (2018) 18–24.

463

10. L. Wang, D. Tian, J. Hu, H. Xing, M. Sun, J. Wang, Q. Jian, H. Yang,

464

MiRNA-145 Regulates the Development of Congenital Heart Disease Through

465

Targeting FXN, Pediatr. Cardiol. 37 (2016) 629–636.

466

11. Sarangapani KK. A, Asbury CL. Trends Genet. Trends Genet. 2012;37(1):62–70.

467

12. Carlson H, Ota S, Song Y, Chen Y, Hurlin PJ. Tbx3 impinges on the p53 pathway

468

to suppress apoptosis, facilitate cell transformation and block

469

differentiation. Oncogene. 2002;21(24):3827–3835.

Hoffman JIE. Incidence of Congenital Heart Disease: II. Prenatal Incidence J.I.E.

Hoffman JIE, Kaplan S. The incidence of congenital heart disease. J Am Coll

Ruckman RN, Rosenquist GC, Rademaker DA, Morse DE, Getson PR. The effect

van den Hoff M. Programmed cell death in the developing heart. Cardiovasc Res.

H.K. Jiang, G.R. Qiu, J. Li-Ling, N. Xin, K.L. Sun, Reduced ACTC1 expression

Y. Zhang, B. Peng, Y. Han, MiR-182 alleviates the development of cyanotic

myogenic

470

13. Willmer T, Cooper A, Peres J, Omar R, Prince S. The T-Box transcription factor 3

471

in development and cancer. BioScience Trends. 2017;11(3):254–266.

472

14. Renard CA, Labalette C, Armengol C, Cougot D, Wei Y, Cairo S, et al. Tbx3 is a

473

downstream target of the Wnt/β-catenin pathway and a critical mediator of β-catenin

474

survival functions in liver cancer. Cancer Res. 2007;67(3):901–910.

475

15. Miao ZF, Liu XY, Xu HM, Wang ZN, Zhao TT, Song YX, et al. Tbx3

476

overexpression in human gastric cancer is correlated with advanced tumor stage and

477

nodal status and promotes cancer cell growth and invasion. Virchows Arch.

478

2016;469(5):505–513.

479

16. Aliwaini S, Lubbad A, Shourfa A, Hamada H, Ayesh B, Abu Tayem H, et al.

480

Overexpression of TBX3 transcription factor as a potential diagnostic marker for

481

breast cancer. Mol Clin Oncol. 2019; 10(1): 105-112.

482

17. Perkhofer L, Walter K, Costa IG, Carrasco MCR, Eiseler T, Hafner S, et al. Tbx3

483

fosters

484

activin/nodal-dependent induction of stemness. Stem Cell Res. 2016;17(2):367–378.

485

18. Xie H, Zhang E, Hong N, Fu Q, Li F, Chen S, et al. Identification of TBX2 and

486

TBX3 variants in patients with conotruncal heart defects by target sequencing. Hum

487

Genomics. 2018;12(1):44.

488

19. Knaapen MWM, Davies MJ, De Bie M, Haven AJ, Martinet W, Kockx MM.

489

Apoptotic versus autophagic cell death in heart failure. Cardiovasc Res.

490

2001;51(2):304–312.

491

20. Roninson IB. Oncogenic functions of tumour suppressor p21Waf1/Cip1/Sdi1:

492

Association with cell senescence and tumour-promoting activities of stromal

493

fibroblasts. Cancer Letters. 2002;179(1):1–14.

pancreatic

cancer

growth

by

increased

angiogenesis

and

494

21. Yagi A, Hasegawa Y, Xiao H, Haneda M, Kojima E, Nishikimi A, et al. GADD34

495

Induces p53 Phosphorylation and p21/WAF1 Transcription. J Cell Biochem.

496

2003;90(6):1242–1249.

497

22. Lee EW, Lee MS, Camus S, Ghim J, Yang MR, Oh W, et al. Differential

498

regulation of p53 and p21 by MKRN1 E3 ligase controls cell cycle arrest and

499

apoptosis. EMBO J. 2009;28(14):2100–2113.

500

23. Wang Y, Fisher JC, Mathew R, Ou L, Otieno S, Sublet J, et al. Intrinsic disorder

501

mediates the diverse regulatory functions of the Cdk inhibitor p21. Nat Chem Biol.

502

2011;7(4):214–221.

503

24. Zhang H, Menzies KJ, Auwerx J. The role of mitochondria in stem cell fate and

504

aging. Development. 2018;145(8):dev143420.

505

25. Clavier A, Rincheval-Arnold A, Colin J, Mignotte B, Guénal I. Apoptosis in

506

Drosophila: Which role for mitochondria? Apoptosis. 2016;21(3):239–251.

507

26. Liang S, Sun K, Wang Y, Dong S, Wang C, Liu LX, et al. Role of

508

Cyt-C/caspases-9,3, Bax/Bcl-2 and the FAS death receptor pathway in apoptosis

509

induced by zinc oxide nanoparticles in human aortic endothelial cells and the

510

protective effect by alpha-lipoic acid. Chem Biol Interact. 2016;258:40–51.

511

27. Ruffolo SC, Breckenridge DG, Nguyen M, Goping IS, Gross A, Korsmeyer SJ, et

512

al. BID-dependent and BID-independent pathways for BAX insertion into

513

mitochondria. Cell Death Differ. 2000;7(11):1101–1108.

514

28. Papaioannou VE. T-box genes in development: From hydra to humans. Int Rev

515

Cytol. 2001;207:1–70.

516

29. Wansleben S, Peres J, Hare S, Goding CR, Prince S. T-box transcription factors in

517

cancer

biology.

Biochimica

et

Biophysica

Acta

-

Reviews

on

Cancer.

518

2014;1846(2):380–391.

519

30. Packham

520

2003;12(90001):37R–44.

521

31. Sundara Rajan S, Hanby AM, Horgan K, Thygesen HH, Speirs V. The potential

522

utility of geminin as a predictive biomarker in breast cancer. Breast Cancer Res Treat.

523

2014;143(1):91–98.

524

32. Kajstura J, Mansukhani M, Cheng W, Reiss K, Krajewski S, Reed JC, et al.

525

Programmedcell death and expression of the protooncogene bcl-2 in myocytes during

526

postnatal maturation of the heart. Exp Cell Res. 1995;219(1):110–121.

527

33. Blundell RA. The Biology of p21Waf1/Cip1 - Review Paper. Am J Biochem

528

Biotechnol. 2009;2(1):33–40.

529

34. Jackson JG, Pant V, Li Q, Chang LL, Quintás-Cardama A, Garza D, et al.

530

P53-Mediated Senescence Impairs the Apoptotic Response to Chemotherapy and

531

Clinical Outcome in Breast Cancer. Cancer Cell. 2012;21(6):793–806.

532

35. P. Witek, A. Korga, F. Burdan, M. Ostrowska, B. Nosowska, M. Iwan, J. Dudka,

533

The effect of a number of H9C2 rat cardiomyocytes passage on repeatability of

534

cytotoxicity study results, Cytotechnology. 68 (2016) 2407–2415.

535

Disclosure: The authors declared no conflict of interest.

536 537 538 539 540

EA.

T-box

genes

in

human

disorders.

Hum

Mol

Genet.

541

Figures and legends

542

Figure.1

543

544

Fig. 1. Effect of TBX3 on proliferation in H9C2 cardiomyoblasts. (A) CCK-8 was

545

used to evaluate the effect of TBX3 expression on viability in H9C2 cardiomyoblasts.

546

(B) Immunofluorescence targeting Ki67 in treated H9C2 cardiomyoblasts. Data were

547

represented as mean ± SD at least three independent experiments. The data from three

548

or more groups were analyzed by one-way analysis of variance (ANOVA) followed

549

by Tukey’s multiple comparison test.

550

551

552

553

554

555

556

557

558

Figure.2

559

560

Fig. 2. Effect of TBX3 on cell cycle arrest in H9C2 cardiomyoblasts. (A) Flow

561

cytometry was used to analyze cell cycle distribution in H9C2 cardiomyoblasts.

562

Representative western blot for (B) Cyclin D1, (C) Cyclin A and (D) Cyclin B1 in

563

H9C2 cardiomyoblasts. Data were represented as mean ± SD at least three

564

independent experiments. The data from three or more groups were analyzed by

565

one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison

566

test.

567

568

569

Figure.3

570 571

Fig. 3. Effect of TBX3 on apoptosis in H9C2 cardiomyoblasts. (A) Representative

572

images of Hoechst 33342 staining at 400× magnification in H9C2 cardiomyoblasts.

573

(B) Annexin V FITC/PI double staining and (C) apoptosis rate of H9C2

574

cardiomyoblasts were detected by using a flow cytometer. Representative western blot

575

for (D) cleaved caspase-9, (E) cleaved caspase-3 and (F) Bcl-2, (G) Bax and (H)

576

cleaved PARP in H9C2 cardiomyoblasts. Data were represented as mean ± SD at least

577

three independent experiments. The data from three or more groups were analyzed by

578

one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison

579

test.

580

581

582

583

584

585

586

587

588

589

590

591

592

593

594

Figure.4

595

596

Fig. 4. Effect of TBX3 on mitochondrial function in H9C2 cardiomyoblasts. (A) The

597

alteration of mitochondrial membrane potential in H9C2 cardiomyoblasts. (B) The

598

activity of ATPase in H9C2 cardiomyoblasts. (C) Fluorescence intensity of ROS in

599

H9C2 cardiomyoblasts. Representative western blot for (D) cytochrome C in the

600

cytoplasm and (E) cytochrome C in the mitochondria of H9C2 cardiomyoblasts. The

601

data from three or more groups were analyzed by one-way analysis of variance

602

(ANOVA) followed by Tukey’s multiple comparison test.

603

604

605

606

Figure.5

607

608

Fig. 5. Effect of TBX3 on p21 expression in H9C2 cardiomyoblasts. The relative (A)

609

mRNA and (B) protein levels of P21 in H9C2 cardiomyoblasts. (C) ChIP was used to

610

verify the targeted combination between TBX3 and p21 in H9C2 cardiomyoblasts.

611

Negative control: control IgG; Positive control: anti-RNA Polymerase II;

612

PCR-negative control: distilled H2O; RT-Input 1: P21; RT-Input 2: GAPDH. The data

613

from three or more groups were analyzed by one-way analysis of variance (ANOVA)

614

followed by Tukey’s multiple comparison test.

615

616

617

618

619

620

621

Figure.6

622

623

Fig. 6. Effect of TBX3/p21 pathway on proliferation in H9C2 cardiomyoblasts. (A)

624

The relative protein levels of P21 in H9C2 cardiomyoblasts. (B) Immunofluorescence

625

targeting Ki67 in H9C2 cardiomyoblasts. The data from three or more groups were

626

analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple

627

comparison test.

628

629

630

631

632

633

634

635

Figure.7

636

637

Fig. 7. TBX3 expression in CHD samples. TBX3 mRNA (A) and protein (B) levels

638

in the heart tissues of CHD donors. The data from two groups was determined by t

639

test.

640

641

642

643

644

645

646

647

648

Supplement Figure. 1

649 650

Supplement Fig. 1. Silencing TBX3 expression by specific siRNA in H9C2

651

cardiomyoblasts. (A) The mRNA expression levels of TBX3 after TBX3 siRNA

652

transfection in H9C2 cardiomyoblasts. (B) The protein expression levels of TBX3

653

after TBX3 siRNA transfection in H9C2 cardiomyoblasts. *p< 0.05 vs. control siRNA

654

group

655

656

657

658

659

660

661

662

Supplement Figure. 2

663

664

Supplement Fig. 2. Elevating TBX3 expression by lentivirus in H9C2

665

cardiomyoblasts. (A) The mRNA expression levels of TBX3 after TBX3-expressing

666

lentivirus transduction in H9C2 cardiomyoblasts. (B) The protein expression levels of

667

TBX3 after TBX3-expressing lentivirus transduction. *p< 0.05 vs. control lentivirus

668

group.

Disclosure: The authors declared no conflict of interest.