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Introduction
Cellular senescence has been defined by Hayflick and Moorehead as the ultimate and irreversible loss of replicative
capacity occurring in primary somatic cell
culture[1]. Senescence was generally studied in culture, different markers of the
senescent phenotype, as mitochondrial
dehydration[2], senescence-associated b-gal
activity[3], and senescence-associated
gene expression[4,5]. Senescence has been identified in human
fibroblasts[6],
keratinocytes[7], endothelial
cells[8], smooth muscle
cells[9], tumor cells[10], hematopoietic
cells[11], and
cardiomyocytes[12,13].
The mammalian heart is an obligate aerobic organ.
Oxygen, beyond its indispensable role in cardiac energy metabolism,
plays a central role in other biological processes that can be determinants of cardiac function, including the determination of
cardiac gene expression patterns[14]. The possibility that cardiac aging is an independent determinant of morbidity and
mortality has faced opposition, and emphasis has been placed on age-associated changes, which increase the chances of
cardiovascular events in the elderly. Treatment of cardiac diseases in elderly patients has resulted in a prolongation of the
average lifespan. However, the maximum lifespan has not increased in the last 70
years[15], suggesting that cellular aging may
play a more important role than generally expected.
Accumulating evidence suggests that telomerase plays a role in cellular senescence
in vitro and in
vivo[16,17]. The enzyme telomerase, a ribonucleoprotein, is crucial for maintaining telomere length. Telomerase reverse transcriptase (TERT) is an
important component of telomerase. It can counteract telomere shortening and prevent
senescence[18]. Senescence in human fibroblasts can be overcome by over-expression of
TERT[19]. Telomerase elongates short telomere, and the cells become
immortalized.
Oxidative stress has been implicated in aging and numerous
diseases[20,21]. Nevertheless, whether premature senescence
could be induced and blocked by TERT in neonatal Sprague-Dawley (SD) rat cardiomyocytes exposed to hypoxia reoxygenation
remains unknown. In this study, we investigated the effect of hypoxia reoxygenation on neonatal SD rat cardiomyocytes.
Materials and methods
Experimental animals All experiments were performed in accordance with the protocols approved by Institutional Animal
Care and Use Committee and conformed to the Guide for the Care and Use of Laboratory Animals of Nanjing Medical
University, China.
Cardiomyocytes culture Neonatal SD rat hearts (1_2
dold) were removed under sterile conditions and washed 3 times in
phosphate buffered saline (PBS) to remove excess blood cells. The ventricles were minced to small fragments and then
agitated gently in a solution of 0.1% trypsin. The mixture was centrifuged at
1000×g for 15 min. The supernatant phase was
discarded, and the cells were resuspended in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100
U/mL of penicillin and 100 µg/mL of streptomycin. Isolated cells were cultured in a flask for 1 h at 37 °C in order to remove
fibroblasts. The cultures were incubated in a humidified atmosphere of 5%
CO2 and 95% air at 37 °C.
Hypoxia reoxygenation The suspension of cells was diluted to
1.0×106 cells/mL, and 1.5 mL was seeded in 6-well plastic
plate and cultured for 72 h. Then, the control cultures were continuously incubated in 5%
CO2 and 95% air at 37 °C. The hypoxic cultures were placed within a modular incubator chamber (BioSpherix, Redfield, NY, USA), filled with 1%
O2, 5% CO2, and balance
N2 for 6 h. The reoxygenated cultures were subjected to 1%
O2 and 5% CO2 for 6 h, then 21% oxygen for 4, 8, 12,
24, and 48 h, respectively.
Electron microscopy For electron microscopy, cardio-myocytes were digested with 0.1% trypsin, centrifuged at
1000×g for 10 min and fixed in 2.5% glutaraldehyde; they were postfixed in 1% osmium tetroxide in 0.1 mol/L phosphate buffer,
dehydrated through a graded series of ethanol and propylene oxide, and then embedded in epoxy resin. Ultrathin sections
of cardiomyocytes were stained with uranyl acetate and observed using an electron microscopy (JEM-1 010, JEOL, Tokyo,
Japan).
Cell proliferation assay Cell proliferation was determined using bromodeoxyuridine (BrdU) (Sigma, St Louis, MO, USA)
labeling. Experimental cells were pretreated with 10 µmol/L BrdU for 30 min before hypoxia treatment and continued until time
of hypoxia; normal cells were incubated with BrdU with an equivalent dose and
time. The labeled cells were observed by immunocytochemistry using a mouse monoclonal antibody directed against BrdU 1:200 (Sigma, USA) and rabbit anti-mouse
IgG as the secondary reagent. The labeled cells were viewed with the high-resolution Pathological Image Analysis System
1000 (High Resolution Pathological Image and Word Analysis System, Beijing, China). Stained BrdU and non-stained cells
were counted in randomly selected areas on the sections to a total number of 500 cells per fish.
RNA isolation and real-time quantitative
PCR Total RNA was isolated using the Trizol (Invitrogen, Life Technologies,
Carlsbad, California, USA) method from the
cells. RNA was treated with DNase to prevent contamination of genomic DNA.
Total RNA (2 µg) was subjected to reverse transcription using reverse transcriptase (TaKaRa Biotechnology Co Ltd, Dalian,
China) as described previously[22]. Expression levels of
p16INK4a and TERT mRNA were quantified by real-time quantitative
PCR, using the ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster city, CA, USA). With the use of
standard curves, the amount of copy numbers of target genes in each sample was calculated and expressed as a ratio to the
b-actin. Primers and probes of target genes were designed using Primer Express software (Applied Biosystems, USA) and
they are listed in Table 1.
b-galactosidase staining Cell cultures were washed twice in PBS (pH 7.4) and fixed for 10 min at room temperature with
20% formaldehyde and 2% glutaraldehyde in PBS. After 3 washes in PBS, the cultures were incubated for 24 h at 37 °C in
freshly prepared SA-b-galactosidase staining solution containing 1 mg/mL
5-bromo-4-chloro-3-indolyl-b-D-galacto-pyrano
side [X-gal; Sigma, USA (pH 6.0)] 400 mmol/L potassium ferrocyanide, 400 mmol/L potassium ferricyanide, and 200 mmol/L
MgCl2. The percent of SA-b-galactosidase positive cells was determined by counting the number of blue cells under bright
field illumination, and the total number of cells in the same field under phase as a contrast.
Immunohistochemical
procedures Slide chambers with cardiomyocytes were treated in 0.5%
H2O2 for 5 min and washed in PBS. Non-specific binding was blocked with normal goat serum. Free-floating sections were incubated for
2 h at 37 °C in monoclonal antibodies myosin light chain antibody (Sigma, USA) and TERT (Acris, Hiddenhausen, Germany),
all diluted at 1:100 in PBS. The sections were then washed in PBS and incubated for 30 min at 37 °C with the second antibody
goat anti-mouse IgG. The protein contents were analyzed using the high-resolution Pathological Image Analysis System
1000.
Telomerase activities assay Cardiomyocytes were washed in PBS, and the pellet was lysed with 200 µL lysis buffer for 30
min at 4 °C as previously
described[23]. The proteins were centrifuged for 20 min at
16 000×g, and protein concentrations were
determined in the supernatant using the Bradford assay. Telomerase activities were measured with 2 µg protein by the Telo
TAGGG Telomerase PCR ELISAplus kit according to the manufacturer's instructions (Roche Molecular Biochemicals, Mannheim,
Germany).
Statistical analysis Experiments were performed at least 3 times. Data were expressed as mean±SD. The
t test and c2 were used to determine significant differences between2 groups. P values less than 0.05 were considered to be significant.
Results
Identification of isolated cells as
cardiomyocytes No spindle-shaped fibroblast could be seen. Immunocytochemical
analysis revealed that most (>95%) cells in the isolated cultures were positive for sarcomeric myosin light chain (Figure 1).
Electron morphology of
cardiomyocytes Cardiomyocytes showed normal morphology and equal nuclear chromatin
distribution in the control group (Figure 2A). Most cells appeared to have cytoplasmic concentration, nuclear chromatin
condensation and margination in the hypoxia group (Figure 2B). The cells approached mitochondrial dehydration with
enlarged, flattened nuclear morphology in the hypoxia reoxygenation group (Figure 2C).
Cell proliferation assay We examined cardiomyocyte proliferation in the hypoxia reoxygenation-treated group by
measuring BrdU incorporation. The proportion of BrdU positive cells was 15.32% in the control group. It was reduced
significantly in the hypoxia reoxygenation-treated group
(P<0.01; Figure 3). These results indicated that cardiomyo-cyte proliferation could be blocked by hypoxia reoxygenation.
p16INK4a and TERT mRNA expression
We investigated whether hypoxia and reoxygenation treatment altered
theexpression of the p16INK4a and TERT. With the use of
real-time quantitative PCR, significant increases
inp16INK4a and TERT mRNA levels were observed in the hypoxia
reoxygenation-treated group compared with the untreated group
(P<0.01), respectively (Figures 4, 5).
b-galactosidase staining We investigated whether the hypoxia reoxygenation-treated cells were
b-galactosidase-positive. With the use of a senescence
b-galactosidase staining kit, a small part of the cells were positive for
b-galactosidase in the control group, the hypoxia 6 h group, and the reoxygenation 4 and 8 h group, but
b-galactosidase-positive cells significantly increased in the reoxygenation 12, 24, and 48 h group
(P<0.05 and P<0.01; Figure 6A, 6B).
TERT protein In our study, TERT protein expression was determined by immunohistochemistry. The
immunohistochemical results showed that the positive cardiomyocytes were stained brown in the intranucleus and cytoplasm; the
degree of color in each group was different. TERT protein expression in the group of hypoxia 6 h, reoxygenation 4, 8, 12, 24,
and 48 h was significantly increased compared with control group
(P<0.01; Figure 7).
Telomerase activity
assay With use of the Telo TAGGG Telomerase PCR
ELISAplus kit, relative telomerase activity
increased significantly in the hypoxia reoxygenation-treated group compared with the control group
(P<0.01; Figure 8).
Discussion
In our study, most cardiomyocytes demonstrated cytoplasmic concentration, nuclear chromatin condensation and margination (Figure 1B), then chromatin gathered at the center of cell nucleus in the hypoxia reoxygenation-treated group (Figure
1C). Mitochondrial dehydration, enlarged, flattened nuclear morphology could be observed in the reoxygenation-treated
group (Figure 1C). The results were in accordance with a previously published
study[24].
Unlike other cells, the proliferation of cardiomyocytes is so limited after birth; the rate was only 15.32% in the control
group. It was reduced significantly with hypoxia reoxygena-tion treatment (Figure 3). At or around birth, cardiomyocytes
nearly lose their ability to divide. Cardiomyocyte DNA synthesis is associated with cell proliferation during fetal life, and a
second DNA synthesis phase occurring after birth (up to approximately neonatal d 3) is associated only with
binucleation[25]. After birth, cardiac growth involves increasing the size of the myocytes without substantial increases in cell number.p16INK4a mRNA copies in the hypoxia reoxygenation-treated cells were significantly increased compared with the
untreated cells (Figure 4). p16INK4a, an inhibitor of the cell
cycle[26], encoded potent tumor suppressor
proteins[27], and was recognized as the key regulator of premature senescence. Its expression levels were found to increase dramatically in
premature senescent cells[28] whose overexpression in mammalian cells could induce
G1 phase arrest[29].b-galactosidase activity has recently been used as a histochemical marker of premature senescence in human fibroblasts,
keratinocytes, endothelial and smooth muscle
cells[30-32]. In our study, few cardiomyocytes were stained for
b-galactosidase in the control group, the hypoxia 6 h group, and the reoxygenation 4 and 8 h group, but the staining intensity increased
significantly in the reoxygenation 12, 24, and 48 h group (Figure 6A, 6B). Our findings are similar with a previous study on
bone marrow cells[21]. b-Galactosidase activity also correlated with replicative capacity in human mammary epithelial
cells[32]. It provided a simple assay for chromosomes that induced
senescence[33]. b-Galactosidase activity increased in combination
with the expression of senescence-associated proteins p53 and
p21WAF1 as well[34].
TERT mRNA and protein expression, relative telomerase activities, were all significantly increased in the hypoxia
reoxygenation-treated cells compared with the untreated cells (Figure 5, 7, 8), but they could not inhibit senescence. It has
been previously reported that the exposure of human cells to hypoxic conditions results in the induction of telomerase
activity through human telomerase reverse transcriptase (hTERT)
expression[35,36]. TERT mRNA levels were reported to
correlate with telomerase activity and to be implicated in the regulation of telomerase activity in cancer
cells[37]. Furthermore, telomerase activity in telomerase-negative cells can be restored by ectopic expression of TERT, suggesting that in certain
cases, TERT is the only limiting factor for telomerase
activation[38]. The relationship between hypoxia and the induction of
telomerase activity should be considered. Hypoxic conditions could induce a DNA damage response by causing telomere
damage. In response to this damage, hypoxia-inducible
factor-1a may induce telomerase in order to heal the damaged
chromosome ends[39].
The cells in the culture
underwent senescence. This phenotype was attributable to unsuitable
conditions (culture shock, oxidative stress) or to growth arrest mediated by p38 MAPK, and
p19Arf, not to telomere shortening
per se[40]. hTERT transduction also failed to prevent premature senescence in 21%
O2[41]. Maybe the activation of p38 MAPK contributed to
the onset of senescence induced by oxidative
stress[42]. p38 MAPK contributed to p53 accumulation in Ataxia
Telangiectasia/TERT fibroblasts during oxidative stress-induced
senescence[43]. p38 MAPK phosphorylated p53 on Ser33 and Ser46 in
the NH2 terminal activation domain and thereby regulated the transactivation activity
ofp53[43,44].
In summary, these data indicate that premature senescence could be induced in neonatal SD rat cardiomyocytes exposed
to hypoxia reoxygenation. Although TERT significantly increased, it could not block senescence.
Acknowledgement
We gratefully acknowledge helpful support from Prof Ji-nan ZHANG.
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