Exogenous hydrogen sulfide restores cardioprotection of ischemic post-conditioning via inhibition of mPTP opening in the aging cardiomyocytes

Drugs and reagents

Sodium hydrogen sulfide (NaHS), the anti-CSE antibody, PD98059 (an inhibitor of ERK),
LY294002 (a PI3K inhibitor), 5-hydroxydecanoate (5-HD, a mK
_ATP
inhibitor), Chelerythrine chloride (Che, a PKC inhibitor) were purchased from Sigma
Chemical Co. (St. Louis, MO, USA). The primary antibodies for anti-PKC-?, anti-cleaved
caspase-3 and -9, Bcl-2, cytochrome c (Cyt c), Na
⁺
/K
⁺
-ATPase, cyclin D1, p21
^Cip/WAF-1
and GAPDH were from Santa Cruz (Bergheimer, Germany). Hoechst 33342, JC-1 kit and
Calcein-AM were also from Santa Cruz. The anti-ERK1/2 and PI3K-Akt-GSK-3? antibodies
were obtained from Cell Signaling Technology (Danvers, USA). Senescence ?-galactosidase (?-gal) staining kit was purchased from Beyotime Institute of Biotechnology (Shanghai,
China). Rat advanced glycation end products (AGEs) ELISA kit was purchased from Proteintech
Group (Wuhan, China). All other chemicals were from Sigma or Santa Cruz.

Primary culture of cardiomyocytes

Primary cultures of neonatal cardiomyocytes were prepared as previously described
8], 27]. Newborn Wistar rats, aged 1–3 days and weighing 5–8 g, were used for this study.
All animal experiments were conducted in compliance with the Guide for the Care and
Use of Laboratory Animals published by the China National Institutes of Health and
approved by the Animal Care Committees of Harbin Medical University, China. Briefly,
cells were dissociated from minced hearts of 1- to 3-day neonatal Wistar rats with
a 0.25% solution of crude trypsin. Cells were cultured as monolayers at a density
of 5 × 10
⁴
cells/cm
²
in Dulbecco’s modified Eagle medium (DMEM) equilibrated with humidified air containing
5% CO
₂
at 37°C. The medium contained 10% calf serum and 2 µM fluorodeoxyuridine, the latter
to prevent proliferation of non-myocytes.

Aging of myocardial cells induced by D-galactose

The treatment for D-galactose induction was as previously described 28], 29]. Once the attached cardiomyocytes were beating spontaneously, the DMEM supplemented
with 20% fetal calf serum was removed, and DMEM supplemented with different concentrations
(0, 0.1, 1, 10, 100 g/L) D-galactose was added to the cardiomyocytes in the culture cluster for a further different
incubation period (0, 12, 24, 48, 72 h). The degree of cell aging was observed through
SA ?-Gal Staining and AGEs ELISA Assay. In the present study, we selected 10 g/L D-galactose concentrations for 48 h incubation period.

Established aging cardiomyocytes model of hypoxia/reoxygenation

A hypoxic condition was produced by D-Hank solution (in mM: 5.37 KCl, 0.44 KH
₂
PO
₄
, 136.89 NaCl, 4.166 NaHCO
₃
, 0.338 Na
₂
HPO
₄
, 5 D-glucose, pH 7.3–7.4 at 37°C) saturated with 95% N
₂
and 5% CO
₂
. The pH was regulated to 6.8 with lactate to mimic ischemic solution. The aging cardiomyocytes
were put into a hypoxic incubator that was equilibrated with 1% O
₂
/5% CO
₂
/94% N
₂
. After hypoxia, the culture medium was rapidly replaced with fresh DMEM with 10%
fetal bovine serum (normoxic culture solution) for initiating reoxygenation 9].

Experimental protocols

The aging cardiomyocytes were randomly divided into the following seven groups. Each
group included eight samples (n = 8) (Fig. 1): (1) control group (Control): the aging cardiomyocytes were cultured for 9 h with
10% fetal bovine serum-DMEM; (2) hypoxia/reoxygenation group (H/R): the aging cardiomyocytes
were exposed to hypoxic culture medium for 3 h and reoxygenated for 6 h by replacing
the hypoxic culture medium with fresh DMEM with 10% fetal bovine serum; (3) H/R + H
₂
S group: the procedure was similar to that for group 2, except that 100 ?M NaHS were
added in 6 h reoxygenation; (4) PC group: at the end of 3 h of hypoxia, the aging
cardiomyocytes were exposed to normoxic culture solution for 5 min, after which cells
were placed in hypoxic solution for 5 min. The PC cycle was repeated three times and
followed by 6 h of reoxygenation; (5) PC + H
₂
S group: at the end of 3 h of hypoxia, initiated immediately at the onset of reoxygenation,
100 ?M NaHS were given at the onset of reoxygenation for 5 min following with 5 min
hypoxia. This protocol was repeated for another two times. The cells were then treated
as those of group 3; (6) PC + PD98059 (or LY294002, or 5-HD, or Che) group: 10 µM
PD98059 (or 10 µM LY294002 or 100 µM 5-HD or 100 µM Che) were added to the medium
40 min before the end of hypoxia. The cells were then treated as those of group 4;
(7) PC + PD98059 (or LY294002, or 5-HD, or Che) + H
₂
S group: 10 µM PD98059 (or 10 µM LY294002 or 100 µM 5-HD or 100 µM Che) were added
to the medium 40 min before the end of hypoxia. The cells were then treated as those
of group 5.

Fig. 1. Summary of experimental treatments protocol. The aging cardiomyocytes were exposed
to hypoxic culture medium for 3 h and reoxygenated for 6 h by replacing the hypoxic
culture medium with fresh DMEM with 10% fetal bovine serum. For details of ischemic
postconditioning, NaHS, PD98059, LY294002, 5-HD and Che treatments see text.

The normal cultured cardiomyocytes (without D-galactose-treated cardiomyocytes) were randomly divided into three groups. Each group
included eight samples (n = 8): Control group; H/R group; PC group. The cells were
treated as those of group 1, 2 and 4, respectively.

AGEs ELISA Assay

The rat advanced glycation end products (AGEs) assay was performed with AGEs ELISA
kit according to the instructions from the manufacturer and was as previously described
29], 30]. The reagents of the test kit were placed at room temperature for 30 min and diluted
1:20 with distilled water. Aliquots of 100 µL of the standards and samples were added
to blank micropores and 50 µL enzyme marker solution was added. Microtiter plates
were incubated at 37°C for 60 min and then washed five times and put aside for 10–20 s
each time. The A and B substrate solutions (50 µL) were added into the microtiter
plates for 15 min dark reactions at 37°C. The reaction was terminated by the addition
of 50 µL stop solution, and the optical density (OD) at 450 nm was determined by an
ultra microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). An AGEs standard
curve was generated and the AGEs values of the samples were calculated from the standard
curve.

SA ?-Gal staining

Senescence-associated ?-gal (SA ?-gal) activity was measured with the ?-gal staining kit at pH 6.0 according to the instructions from the manufacturer 29], 30]. Briefly, the cells were washed in phosphate buffered saline (PBS), fixed for 10–15 min
at room temperature with 1 mL of fixative solution and incubated overnight at 37°C
with the staining solution mix. Cells were observed for development of the blue coloration
with a microscope at a magnification of 400×. Aging cardiomyocytes were assessed by
counting the number of cells that displayed blue coloration.

Observation of cell division index

Briefly, the cells were washed in phosphate buffered saline (PBS) for three times,
fixed for 30 min at room temperature with methanol:ice acetic acid (3:1) and incubated
for 10 min with Giemsa. Cells were observed with a microscope at a magnification of
400×. Five fields (at least 100 cells of each field) were randomly selected and the
percentage of cell division was calculated.

Detection of cell viability

Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay, as described previously 9], 25], 31]. Cells were cultured in 96-well plates. MTT (final concentration, 0.5 mg/mL) was
added to each well under sterile conditions, and the plates were incubated for 4 h
at 37°C. The supernatant was removed, and dimethyl sulfoxide (150 ?l/well) was added.
The plates were then agitated on a plate shaker. The absorbance of each well was measured
at 490 nm with a Bio-Rad automated EIA analyser (Bio-Rad Laboratories, Hercules, CA,
USA). The viability of control cells was considered 100%, and the others were expressed
as percentages of control.

Determination of H
₂
S production

H
₂
S production rate was measured as described previously 25]. In brief, after different treatments, the H9C2 cells (cardiomyocytes line) were
collected and homogenized in 50 mM ice-cold potassium phosphate buffer (pH 6.8). The
flasks containing the reaction mixture (100 mM potassium phosphate buffer, 10 mM L-cysteine, 2 mM pyridoxal 5-phosphate, and 10% cell homogenates) and center wells
containing 0.5 mL 1% zinc acetate and a piece of filter paper (2 × 2.5 cm) were flushed
with N
₂
gas and incubated at 37°C for 90 min. The reaction was stopped by adding 0.5 mL of
50% trichloroacetic acid, and the flasks were incubated at 37°C for another 60 min.
The contents of the center wells were transferred to test tubes, each containing 3.5 mL
of water. Then 0.5 mL of 20 mM N, N-dimethyl-p-phenylenediamine sulfate in 7.2 M HCl and 0.5 mL 30 mM FeCl
₃
in 1.2 M HCl was added. The absorbance of the resulting solution at 670 nm was measured
20 min later with a FLUOstar OPTIMA microplate spectrophotometer.

Apoptotic rate of cells by flow cytometry assay

The apoptotic rate was measured by flow cytometry as described previously 9], 28], 31]. Cells were washed three times with ice-cold PBS, and then stained with annexin V-fluorescein
isothiocyanate for 15 min at room temperature in 200 ?l binding buffer. Next, 300 ?l
binding buffer was added, and the cells were stained with propidium iodide for 30 min
at 4°C. The fluorescence of the cells was analyzed by flow cytometry. The percentage
of apoptotic cells was determined using Mod Fit LT software (Verity Software House
Inc., Topsham, ME, USA).

Hoechst 33342 staining

Cells were analyzed for apoptosis after visualization of nuclei morphology with fluorescent
DNA-binding dye Hoechst 33342, as described previously 9]. After treatment, cells were rinsed with PBS and incubated with 5 µg/mL Hoechst 33342
for 10 min. Nuclei were visualized at 400× magnification using fluorescent microscopy
(Nikon Corporation, Tokyo, Japan) at an excitation wavelength of 330–380 nm. Apoptotic
nuclei of cells were assessed by counting the number of cells that displayed nuclear
morphology changes, such as chromatin condensation and fragmentation.

Real-time PCR analysis

Total RNA was isolated using an RNeasy Mini Kit (Qiagen, Germantown, MD, USA) and
converted to cDNA with an iScriptTM cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA).
Real-time PCR was performed in an iCycler iQ5 apparatus (Bio-Rad) associated with
the iCycler optical system software (version 3.1) using SYBR Green PCR Master Mix.
The primers of Bcl-2 were 5?-GGCATCTTCTCCTTCCAG-3? (forward) and 5?-CATCCCAGCCTCCGTTAT-3?
(reverse). Caspase-3 primers were 5?-CAGACAGTGGAACTGACGATGA-3? (forward) and 5?-AACAGAAACATGCCCCTACCCC-3?
(reverse). Caspase-9 primers were 5?-CCCGTGAAGCAAGGATTT-3? (forward) and 5?-ACTGTGGGTCTGGGAAGC-3?
(reverse). P-ERK1/2 primers were 5?-ATCCCCCATGGAACGACCTG-3? (forward) and 5?-ACCCGCCAGGGACAAAAATG-3?
(reverse). The primers of p-PI3K were 5?-CCCTTCTGAACTGGCTTAAAGA-3? (forward) and 5?-GGACAGTGTAAATTCCTCAATGG-3?
(reverse). The primers for p-Akt were 5?-TGTGACCATGAACGAGTTTGA-3? (forward) and 5?-GTCGTGGGTCTGGAATGA-3?
(reverse). P-GSK-3? primers were 5?-CGGGACCCAAATGTCAAACA-3? (forward) and 5?-CGTGACCAGTGTTGCTGAGT-3?
(reverse). The primers of PKC-? were 5?-CATGGAAGGATAAGCGTTGGT-3? (forward) and 5?-CCCAAGTCCCGTGTTAAGA-3?
(reverse). The primers for GAPDH were 5?-CTCAACTACATGGTCTACATG-3? (forward) and 5?-TGGCATGGACTGTGGTCATGAG-3?
(reverse). The cycling conditions were: one cycle of 94°C for 2 min; 30 cycles of
94°C for 30 s, 60°C for 40 s and 72°C for 1 min; and 72°C for 4 min. Relative mRNA
quantification was calculated by using the arithmetic formula “2???CT”, where ?CT
is the difference between the threshold cycle of a given target cDNA and an endogenous
reference GAPDH cDNA.

Detection of Cyt c release from mitochondrial

Western blot analysis of Cyt c in the cytosolic fraction was performed as described previously 9], 25], 28], 31]. Briefly, cells were harvested, washed twice with ice-cold PBS, and incubated in
ice-cold Tris-sucrose buffer (0.35 mM sucrose, 10 mM Tris–HCl at pH 7.5, 1 mM EDTA,
0.5 mM dithiothreitol, 0.1 mM phenylmethylsulphonyl fluoride). After a 40 min incubation,
cells were centrifuged at 1,000×g for 5 min at 4°C and the supernatant was further centrifuged at 40,000×g for 30 min at 4°C. The supernatant was retained as the cytosolic fraction and analyzed
by Western blot with a primary rat anti-Cyt c monoclonal antibody and a secondary goat anti-rat immunoglobulin G (Promage). GAPDH
expression was used as the control.

Translocation of PKC-?

Cardiomyocytes were lysed immediately after different treatment by resuspending in
lysis buffer [50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.26%
sodium deoxycholate, 50 mM sodium fluoride, 10 mM ?-glycerophosphate, 0.1 mM sodium orthovanadate, 10 µg/mL leupeptin, and 1 mM phenylmethylsulfonyl
fluoride (PMSF)] and incubating on ice for 20 min. Cell debris and insoluble material
were cleared by centrifugation at 10,000×g for 10 min. The supernatant is called the detergent-solubilized cell lysate. For
the preparation of membrane-enriched fractions and subcellular fractions, cells were
resuspended in 25 mM Tris–HCl, pH 7.5, 250 mM sucrose, 5 mM MgCl
₂
, 100 mM KCl, 10 µg/mL each of aprotinin and leupeptin, and 1 mM PMSF. Cells were
disrupted by Dounce homogenization and fractionated by differential velocity centrifugation
as previously described 7], 9]. Cell membrane fractions were analyzed by Western blotting with primary rabbit polyclonal
isoform specific anti-PKC-? and secondary goat anti-rat IgG. Na
⁺
/K
⁺
-ATPase was used a membrane fractions loading control. The volume of protein bands
was quantified using a Bio-Rad Chemi DocTM EQ densitometer and Bio-Rad Quantity One
software (Bio-Rad Laboratories, Hercules, USA).

Measurement of mitochondrial membrane potential (??m)

Changes in mitochondrial transmembrane potential (??m) were measured with the dye
5,5?,6,6?-tetrachloro-1,1?,3,3?-tetraethylbenzimidazolcarbocyanine iodide (JC-1).
Mitochondrial membrane potential was determined as previously described 9]. Briefly, neonatal rat cardiomyocytes (2.5 × 10
⁵
cells/60 mm
²
dishes) were seeded, incubated, and treated for 48 h at 37°C. After experimentation,
cells were stained with JC-1 (2 µg/mL, Invitrogen) at 37°C in the dark for 15 min
and rinsed three times with cold PBS. Observations were made using a Zeiss LSM 510
inverted confocal scanning microscope. JC-1 monomer (green) fluorescence was observed
by excitation with 514 nm and examination of emission at 529 nm. JC-1 aggregate (red)
fluorescence was observed by examination of emission at 585/590 nm. At least 100 areas
were selected from each image, and the average intensity for each region was quantified.
The ratio of JC-1 aggregate to monomer (red/green) intensity for each region was calculated.
A decrease in this ratio was interpreted as decrease of ??m, whereas an increase in
this ratio was interpreted as gain in ??m.

Assay of mitochondrial permeability transition pore (mPTP) opening

Changes of mitochondrial permeability transition pore (mPTP) opening were measured
with coincubation of calcein-AM and cobalt chloride as previously described 9], 32]. Cardiomyocytes were plated in 24-well plates (0.5 × 10
⁶
cells/well). After different the treatments, the cells were loaded with calcein-AM
2 µM in presence of 5 mM of cobalt chloride in the dark for for 15 min at 37°C. Fluorescence
was measured in a Zeiss LSM 510 inverted confocal scanning microscope at 488 nm excitation
and 505 nm emission. Fluorescence intensity of individual cells was measured using
SigmaScan Pro 5 software. The fluorescence intensity in control group was considered
100% viable.

Western blotting analysis

Cells were homogenized in ice-cold lysis buffer containing 50 mM Tris–HCl (pH, 8.0),
150 Mm NaCl, 5 mM EDTA, 1% Triton X-100, 0.26% sodium deoxycholate, 50 mM sodium fluoride,
10 mM ?-glycerophosphate, 0.1 mM sodium orthovanadate, 10 µg/mL leupeptin, and 50 µg/mL phenylmethylsulfonyl
fluoride (PMSF), and incubated on ice for 40 min. The homogenate was centrifuged at
15,000×g for 15 min at 4°C to remove cellular debris and isolate total protein. Protein concentrations
were determined using a Bradford assay kit (Bio-Rad Laboratories; Hercules, USA).
Equal amounts of proteins were boiled and separated with SDS-PAGE and electrophoretically
transferred to a nitrocellulose membrane, as described previously 9], 25]. In each lane of a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis,
equal amounts of proteins were applied, electrophoresed and transferred to a polyvinylidene
fluoride membrane. Membranes were blocked with Tris-buffered saline containing 5%
non-fat milk at room temperature for 1 h, then incubated overnight at 4°C with primary
antibody. The primary antibody dilutions were 1:1,000 for CSE, phosphorylated or total
ERK1/2, PI3K, Akt or GSK-3?, and 1:500 for Bcl-2, cleaved caspase-3 and -9, GAPDH,
Na
⁺
-K
⁺
-ATPase, cyclin D1 and p21
^Cip/WAF-1
. The membrane was then washed three times with 1× Tris-buffer saline-Tween 20 (TBST)
buffer and incubated in TBST solution with horseradish peroxidase-conjugated secondary
antibody (diluted 1:500) for 1 h at room temperature on a shaker. Finally, the membrane
was washed with TBST solution for three times. Antibody–antigen complexes were detected
using Western Blue Stabilized Substrate for alkaline phosphatase. GAPDH expression
was used as the control. The intensities of the protein bands were quantified by a
Bio-Rad ChemiDocâ„¢ EQ densitometer and Bio-Rad Quantity One software (Bio-Rad Laboratories).

Statistical analysis

All data were expressed as the mean ± SE and represented at least three independent
experiments. Statistical comparisons were made using student’s t test or one-way ANOVA followed by a post hoc analysis (Tukey test) where applicable.
Significance level was set at p  0.05.