Articles in PresS. Am J Physiol Heart Circ Physiol (December 5, 2014). doi:10.1152/ajpheart.00666.2014
1
Vasonatrin peptide attenuates myocardial ischemia/reperfusion injury in diabetic rats and underlying
2
mechanisms
3
Zhenwei Shi1,*, Feng Fu2,*, Liming Yu1,*, Wenjuan Xing2, Lele Ji1, Ru Tie1, Xiangyan Liang1, Miaozhang Zhu2,
4
Jun Yu3, Haifeng Zhang1
5
1
Experiment Teaching Center, 2Department of Physiology, Fourth Military Medical University, Xi’an 710032,
6
China, 3Experimental Center, The Second Affiliated Hospital, School of Medicine, Xi'an Medical University,
7
Xi’an 710038, China
8 9
*
These authors contributed equally to this study.
10 11
Running Title: VNP attenuates MI/R injury in diabetes
12
Word count: 7057
13 14
Correspondence to:
15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
Haifeng Zhang, M.D., Ph.D. Experiment Teaching Center Fourth Military Medical University 169 Changlexi Road Xi’an 710032, China E-mail: [email protected] Tel: 86-29-84779277 Fax: 86-29-84774764 OR Jun Yu, Ph.D. Experimental Center The Second Affiliated Hospital, School of Medicine, Xi'an Medical University 167 Fang East Road Xi’an 710038, China E-mail: [email protected] Tel: 86-29-84772334 Fax: 86-29-84774764
1
Copyright © 2014 by the American Physiological Society.
35
Abstract
36
Aims: Diabetes mellitus increases morbidity/mortality of ischemic heart disease. Although atrial natriuretic
37
peptide and C-type natriuretic peptide reduce the myocardial ischemia/reperfusion damage in non-diabetic rats,
38
whether vasonatrin peptide (VNP), the artificial synthetic chimera of atrial natriuretic peptide and C-type
39
natriuretic peptide, confers cardioprotective effect against ischemia/reperfusion injury, especially in diabetic
40
patients, is still unclear. This study was designed to investigate the effects of VNP on ischemia/reperfusion
41
injury in diabetic rats, and to further elucidate its mechanisms.
42
Methods and Results: The high-fat diet-fed streptozotocin induced diabetic Sprague-Dawley rats were
43
subjected to ischemia/reperfusion operation. VNP treatment (100 µg/kg, i.v., 10 min before reperfusion)
44
significantly improved ± LV dP/dtmax and LVSP and reduced LVEDP, apoptosis index, caspase-3 activity,
45
plasma CK and LDH activities. Moreover, VNP inhibited endoplasmic reticulum (ER) stress by suppressing
46
GRP78 and CHOP. These effects were mimicked by 8-Br-cGMP, a cGMP analogue, whereas inhibited by
47
KT-5823, the selective inhibitor of PKG. In addition, pretreatment with TUDCA, a specific inhibitor of ER
48
stress, couldn’t further promote the VNP’s cardioprotective effect in diabetic rats. In vitro H9c2 cardiomyocytes
49
were subjected to hypoxia/reoxygenation and incubated with or without VNP (10-8mol/L). Gene knockdown of
50
PKG1α with siRNA blunted VNP’s inhibition of ER stress and apoptosis, while overexpression of PKG1α
51
resulted in significant decreased ER stress and apoptosis.
52
Conclusions: VNP protects diabetic heart against ischemia/reperfusion injury by inhibiting ER stress via
53
cGMP-PKG signaling pathway. These results suggest that VNP may have potential therapeutic value for the
54
diabetic patients with ischemic heart disease.
55
Keywords: Vasonatrin peptide; Myocardial ischemia/reperfusion injury; Endoplasmic reticulum stress; PKG;
2
56
Diabetes
57
3
58
1. Introduction
59
Diabetes mellitus (DM) is closely related to cardiovascular morbidity and mortality. People with DM have a risk
60
of ischemic heart disease (IHD) two to five times greater than that in nondiabetic individuals (34). Importantly,
61
diabetic patients have more severe and fatal myocardial infarctions than nondiabetic patients (15, 44).
62
Identifying the cellular and molecular basis linking diabetes with increased IHD morbidity and mortality is not
63
only scientifically important, but may reveal new therapeutic targets against heart diseases in diabetes.
64
Recent animal and human studies have revealed that various factors interfere with endoplasmic reticulum
65
function and lead to accumulation of unfolded proteins, including ischemia/reperfusion, disturbance of calcium
66
homeostasis, and overexpression of normal and/or incorrectly folded proteins (2, 23). Considerable evidence
67
indicates that endoplasmic reticulum (ER) stress plays an essential role in cardiomyocyte death induced by
68
myocardial ischemia/reperfusion (MI/R) (35, 39). Furthermore, diabetic state enhances ER stress in
69
cardiovascular complications such as IHD (9). Improved therapeutic intervention on ER stress may attenuate
70
injury endured in myocardial ischemic diseases.
71
Natriuretic peptides (NPs) are a family of structurally related but genetically distinct peptides including atrial
72
natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C type natriuretic peptide (CNP), which
73
principally mediate natriuretic, diuretic, vasorelaxant, and antimitogenic effects via NPs-cGMP-PKG signaling
74
pathway (25-27). It is elucidated that NPs reduce the MI/R damage in normal rats (3, 12). However, their effects
75
on ischemic heart disease in diabetic state was rarely reported before. Vasonatrin peptide (VNP) is an artificial
76
synthetic chimera of ANP and CNP, which proved to be more potentially diuretic, natriuretic and vasorelaxant
77
compared with other NPs, such as ANP, CNP (37). Our previous studies have demonstrated that VNP induced a
78
dose-dependent vasorelaxation via natriuretic peptide receptors (NPR) A/B-cGMP-BKCa signaling pathway (43).
4
79
However, the effects of VNP on acute MI/R injury, especially in patients with diabetes, were still unclear.
80
Therefore, the aim of the present study was to (1) investigate the potential for exogenous VNP treatment to
81
attenuate MI/R injury in diabetic rats; (2) determine whether inhibition of the ER stress is involved in VNP’s
82
therapeutic effect; (3) elucidate the underlying mechanisms with a special focus on VNP-cGMP-PKG signaling
83
pathway.
5
84
2. Materials and Methods
85
2.1 High-fat diet-fed streptozotocin rat model
86
All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory
87
Animals published by the U.S. National Institutes of Health, NIH Publication No. 85–23, revised 1996 and were
88
approved by the Fourth Military Medical University Committee on Animal Care. The high-fat diet-fed
89
streptozotocin (HFD-STZ) rat model was developed as described previously with slight modifications (36).
90
Normal 6-week-old male Sprague-Dawley rats were fed with HFD diet containing 40% fat (as a percentage of
91
total kcal), 41% carbohydrate, and 18% protein or a normal chow diet consisting of 12% fat, 60% carbohydrate,
92
and 28% protein for 4 weeks. STZ (i.p., 25 mg/kg) (Sigma, St. Louis, MO) was injected once intraperitoneally
93
(IP) to the rats after 4 weeks of HFD feeding. HFD was continuously administrated after STZ injection, and then
94
rats with fasting plasma glucose level of above 11.1mmol/L 72h post STZ injection were considered diabetic
95
and were studied. The normal diet-fed rats were used as nondiabetic controls. Glucose tolerance of various
96
groups was further estimated by oral glucose tolerance test (OGTT) and intraperitoneal glucose tolerance test
97
(IPGTT). Glucose (2 g/kg) was administered to 12 h fasted rats either by gastric lavage or intraperitoneally and
98
blood samples were collected from the tail vein at 0 (before glucose load), 30, 60, 90 and 120 min for
99
measurement of plasma glucose.
100
2.2 Myocardial infarction protocol
101
Rats were anesthetized by 3% pentobarbital sodium (30 mg/kg, IP). Anaesthesia was then maintained with 2%
102
isoflurane. A microcatheter was inserted into the left ventricle through the right carotid artery to measure left
103
ventricular pressure. Hemodynamic data were continuously monitored as described before (17). Myocardial
104
ischemia was produced by exteriorizing the heart through a left thoracic incision and placing 6-0 silk suture and
6
105
making a slipknot around the left anterior descending coronary artery. After 30 min of ischemia, the slipknot
106
was released, and the heart was reperfused for 4 h (for analysis of myocardial apoptosis) and 6 h (for
107
quantification of myocardial infarct size). At the end of the reperfusion, all rats were anesthetized with sodium
108
pentobarbital (100mg/kg, IP) and euthanized by decapitation.
109
2.3 Experimental groups
110
Nondiabetic rats were randomly assigned to two experiment groups (n=8/group): Con+Sham—sham operation
111
(rats underwent the same operation, and the ligature was looped through the myocardium next to the LAD) and
112
receiving no treatment; Con+MI/R (vehicle)—MI/R operation and infusion of saline (0.5 ml, i.v., 10 min before
113
reperfusion). Diabetic rats were randomized to one of the following groups (n=8/group): DM+Sham; DM +
114
MI/R + saline; DM+MI/R+VNP (100 µg/kg, intravenous infusion at 25 µg/kg/h for 4 hours, 10 min before
115
reperfusion). We tested the effects of VNP at 2.5, 25, and 250 µg/kg/h on plasma cGMP level and arterial blood
116
pressure in the preliminary study. It was found that 25 µg/kg/h VNP induced an increase in plasma cGMP level
117
without significant effect on mean arterial pressure and this dose was used in this study; DM + MI/R +
118
8-Br-cGMP (1 mg/kg, i.p., 20 min before reperfusion,); DM + MI/R + VNP + KT-5823 (0.5 mg/kg, i.p., 20 min
119
before reperfusion); DM+MI/R+TUDCA (50 mg/kg, i.p., 10 min before reperfusion).
120
2.4 Determination of apoptosis and myocardial infarction
121
At the end of 4-h reperfusion, myocardial apoptosis was analysed by TUNEL (terminal deoxynucleoti-dyl
122
transferase dUTP nick end labelling) assay as described previously (33). Cardiac caspase-3 activity was
123
measured by using caspase colorimetric assay kits (Chemicon International, Temecula, CA, USA), as described
124
in the previous study (17). At the end of 6-h reperfusion, myocardial infarction was determined by means of a
125
double-staining technique (33). Evans blue-stained areas (area not at risk), triphenyltetrazolium chloride (TTC)
7
126
stained areas (red staining, ischemic but viable tissue), and TTC staining negative areas (infarcted myocardium)
127
in each slice were determined by planimetry on a computer with Sigma Scan software. The myocardial infarct
128
size was expressed as a percentage of infarct area (INF) over total AAR (INF/AAR × 100%).
129
2.5 Biochemical estimation
130
After the reperfusion of 4 h, blood samples were obtained from abdominal aorta, and then centrifugated at 3000
131
× g for 10 min, getting plasma for LDH, CK, MDA, SOD measurement. The procedures strictly followed
132
manufacturer’s instructions. All chemicals used for biochemical analysis were obtained from Nanjing
133
Jiangcheng Bioengineering Institute (Nanjing, China).
134
2.6 Cell culture and treatments
135
H9c2 cardiomyocytes were purchased from the Cell Bank of Chinese Academy of Science (Beijing, China).
136
Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO) supplemented with 10%
137
inactivated fetal bovine serum (FBS, GIBCO), 500µg/ml penicillin, and 500 µg/ml streptomycin (GIB-CO) at
138
37℃ in a humidified atmosphere with 5% CO2. As suggested by some laboratories, concentrations of glucose
139
approaching 10 mM are pre-diabetic levels, and concentrations of glucose above 10 mM are analogous to a
140
diabetic condition within the cell culture system. Accordingly, DMEM containing 25 mM levels of glucose have
141
been applied in several studies to explore the pathophysiology of diabetes as well as to test agents for the
142
treatment of diabetic complications, in which DMEM with 5.5 mmol/L glucose was used to culture H9c2 cells
143
as a control (4, 11, 20). In this study the cells were incubated with control (5.5 mmol/L) or high glucose/high fat
144
(HG/HF) medium containing glucose (25 mmol/L) and saturated FFA palmitate (16:0; 500 μmol/L) as
145
previously described (41). After 18 h of HG/HF incubation, cardiomyocytes were then exposed to
146
hypoxia/reoxygenation treatment as previously described (40). In brief, H9c2 cardiomyocytes were exposed to
8
147
glucose-free
148
(Billumps-Rothenberg) flushed with 5% CO2 and 95% N2 for 3 h of hypoxia. Then, the culture medium was
149
replaced with fresh oxygenated normal or VNP (10-8mol/L) cultured medium, and the dishes were transferred to
150
a normoxic incubator (95% air-5% CO2) for 6 h of reoxygenation. The number of viable H9c2 cells was
151
measured after 48 h by MTT cell viability assay.
152
2.7 Adenovirus infection
153
Adenoviral vectors for overexpression of PKG1α were used as previously described (38). Viruses were
154
propagated in 293 cells and kept at -80°C with a titre of 1011–12 pfu/ml before using. Gene transfer with
155
adenovirus encoding GFP was used as an internal control. Cardiomyocytes plated at a density of 0.5 to 1 ×
156
105/cm2 were infected by adenovirus at 50 multiplicity of infection (moi) for 2 h at 37°C in a humidified, 5%
157
CO2 incubator. Subsequently, the cells were cultured in serum-free DMEM media for an additional 24 h before
158
processing.
159
2.8 Transfection of siRNAs
160
Double-stranded RNAi for selective silencing of PKG1α with a final concentration of 100 nM was transfected
161
into cells (FuGENE Reagent, Roche Applied Science, Mannheim, Germany) according to the manufacturer’s
162
instructions. After transfection, cells were performed the same H/R treatment as described above. Scrambled
163
RNAi was used as control. Knockdown of gene expression was identified by Western blot analysis 48 h after
164
transfection.
165
2.9 Western blotting
166
Samples were lysed with lysis buffer. After sonication, the lysates were centrifuged, proteins were separated by
167
electrophoresis on SDS-PAGE and then transferred onto PVDF (polyvinylidenedifluoride)-Plus membrane
serum-free
culture
medium
and
transferred
9
into
a
Modular
Incubator
Chamber
168
(Micron Separations, Inc., Westborough, MA). After being blocked with 5% milk, the immunoblots were probed
169
with anti-GRP78, CHOP, PKG1α, Akt, pAkt (Ser 473), ERK1/2, pERK1/2 (Thr202/Tyr204) and β-actin (Cell
170
Signaling, Beverly, MA) overnight at 4°C followed by incubation with the corresponding secondary antibodies
171
at room temperature for 1 h. The blots were visualized with ECL-plus reagent.
172
2.10 Statistical analysis
173
All values are presented as mean ± SEM. Differences were compared by ANOVA followed by Bonferroni
174
correction for post hoc t test where appropriate; P
1
Vasonatrin peptide attenuates myocardial ischemia/reperfusion injury in diabetic rats and underlying
2
mechanisms
3
Zhenwei Shi1,*, Feng Fu2,*, Liming Yu1,*, Wenjuan Xing2, Lele Ji1, Ru Tie1, Xiangyan Liang1, Miaozhang Zhu2,
4
Jun Yu3, Haifeng Zhang1
5
1
Experiment Teaching Center, 2Department of Physiology, Fourth Military Medical University, Xi’an 710032,
6
China, 3Experimental Center, The Second Affiliated Hospital, School of Medicine, Xi'an Medical University,
7
Xi’an 710038, China
8 9
*
These authors contributed equally to this study.
10 11
Running Title: VNP attenuates MI/R injury in diabetes
12
Word count: 7057
13 14
Correspondence to:
15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
Haifeng Zhang, M.D., Ph.D. Experiment Teaching Center Fourth Military Medical University 169 Changlexi Road Xi’an 710032, China E-mail: [email protected] Tel: 86-29-84779277 Fax: 86-29-84774764 OR Jun Yu, Ph.D. Experimental Center The Second Affiliated Hospital, School of Medicine, Xi'an Medical University 167 Fang East Road Xi’an 710038, China E-mail: [email protected] Tel: 86-29-84772334 Fax: 86-29-84774764
1
Copyright © 2014 by the American Physiological Society.
35
Abstract
36
Aims: Diabetes mellitus increases morbidity/mortality of ischemic heart disease. Although atrial natriuretic
37
peptide and C-type natriuretic peptide reduce the myocardial ischemia/reperfusion damage in non-diabetic rats,
38
whether vasonatrin peptide (VNP), the artificial synthetic chimera of atrial natriuretic peptide and C-type
39
natriuretic peptide, confers cardioprotective effect against ischemia/reperfusion injury, especially in diabetic
40
patients, is still unclear. This study was designed to investigate the effects of VNP on ischemia/reperfusion
41
injury in diabetic rats, and to further elucidate its mechanisms.
42
Methods and Results: The high-fat diet-fed streptozotocin induced diabetic Sprague-Dawley rats were
43
subjected to ischemia/reperfusion operation. VNP treatment (100 µg/kg, i.v., 10 min before reperfusion)
44
significantly improved ± LV dP/dtmax and LVSP and reduced LVEDP, apoptosis index, caspase-3 activity,
45
plasma CK and LDH activities. Moreover, VNP inhibited endoplasmic reticulum (ER) stress by suppressing
46
GRP78 and CHOP. These effects were mimicked by 8-Br-cGMP, a cGMP analogue, whereas inhibited by
47
KT-5823, the selective inhibitor of PKG. In addition, pretreatment with TUDCA, a specific inhibitor of ER
48
stress, couldn’t further promote the VNP’s cardioprotective effect in diabetic rats. In vitro H9c2 cardiomyocytes
49
were subjected to hypoxia/reoxygenation and incubated with or without VNP (10-8mol/L). Gene knockdown of
50
PKG1α with siRNA blunted VNP’s inhibition of ER stress and apoptosis, while overexpression of PKG1α
51
resulted in significant decreased ER stress and apoptosis.
52
Conclusions: VNP protects diabetic heart against ischemia/reperfusion injury by inhibiting ER stress via
53
cGMP-PKG signaling pathway. These results suggest that VNP may have potential therapeutic value for the
54
diabetic patients with ischemic heart disease.
55
Keywords: Vasonatrin peptide; Myocardial ischemia/reperfusion injury; Endoplasmic reticulum stress; PKG;
2
56
Diabetes
57
3
58
1. Introduction
59
Diabetes mellitus (DM) is closely related to cardiovascular morbidity and mortality. People with DM have a risk
60
of ischemic heart disease (IHD) two to five times greater than that in nondiabetic individuals (34). Importantly,
61
diabetic patients have more severe and fatal myocardial infarctions than nondiabetic patients (15, 44).
62
Identifying the cellular and molecular basis linking diabetes with increased IHD morbidity and mortality is not
63
only scientifically important, but may reveal new therapeutic targets against heart diseases in diabetes.
64
Recent animal and human studies have revealed that various factors interfere with endoplasmic reticulum
65
function and lead to accumulation of unfolded proteins, including ischemia/reperfusion, disturbance of calcium
66
homeostasis, and overexpression of normal and/or incorrectly folded proteins (2, 23). Considerable evidence
67
indicates that endoplasmic reticulum (ER) stress plays an essential role in cardiomyocyte death induced by
68
myocardial ischemia/reperfusion (MI/R) (35, 39). Furthermore, diabetic state enhances ER stress in
69
cardiovascular complications such as IHD (9). Improved therapeutic intervention on ER stress may attenuate
70
injury endured in myocardial ischemic diseases.
71
Natriuretic peptides (NPs) are a family of structurally related but genetically distinct peptides including atrial
72
natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C type natriuretic peptide (CNP), which
73
principally mediate natriuretic, diuretic, vasorelaxant, and antimitogenic effects via NPs-cGMP-PKG signaling
74
pathway (25-27). It is elucidated that NPs reduce the MI/R damage in normal rats (3, 12). However, their effects
75
on ischemic heart disease in diabetic state was rarely reported before. Vasonatrin peptide (VNP) is an artificial
76
synthetic chimera of ANP and CNP, which proved to be more potentially diuretic, natriuretic and vasorelaxant
77
compared with other NPs, such as ANP, CNP (37). Our previous studies have demonstrated that VNP induced a
78
dose-dependent vasorelaxation via natriuretic peptide receptors (NPR) A/B-cGMP-BKCa signaling pathway (43).
4
79
However, the effects of VNP on acute MI/R injury, especially in patients with diabetes, were still unclear.
80
Therefore, the aim of the present study was to (1) investigate the potential for exogenous VNP treatment to
81
attenuate MI/R injury in diabetic rats; (2) determine whether inhibition of the ER stress is involved in VNP’s
82
therapeutic effect; (3) elucidate the underlying mechanisms with a special focus on VNP-cGMP-PKG signaling
83
pathway.
5
84
2. Materials and Methods
85
2.1 High-fat diet-fed streptozotocin rat model
86
All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory
87
Animals published by the U.S. National Institutes of Health, NIH Publication No. 85–23, revised 1996 and were
88
approved by the Fourth Military Medical University Committee on Animal Care. The high-fat diet-fed
89
streptozotocin (HFD-STZ) rat model was developed as described previously with slight modifications (36).
90
Normal 6-week-old male Sprague-Dawley rats were fed with HFD diet containing 40% fat (as a percentage of
91
total kcal), 41% carbohydrate, and 18% protein or a normal chow diet consisting of 12% fat, 60% carbohydrate,
92
and 28% protein for 4 weeks. STZ (i.p., 25 mg/kg) (Sigma, St. Louis, MO) was injected once intraperitoneally
93
(IP) to the rats after 4 weeks of HFD feeding. HFD was continuously administrated after STZ injection, and then
94
rats with fasting plasma glucose level of above 11.1mmol/L 72h post STZ injection were considered diabetic
95
and were studied. The normal diet-fed rats were used as nondiabetic controls. Glucose tolerance of various
96
groups was further estimated by oral glucose tolerance test (OGTT) and intraperitoneal glucose tolerance test
97
(IPGTT). Glucose (2 g/kg) was administered to 12 h fasted rats either by gastric lavage or intraperitoneally and
98
blood samples were collected from the tail vein at 0 (before glucose load), 30, 60, 90 and 120 min for
99
measurement of plasma glucose.
100
2.2 Myocardial infarction protocol
101
Rats were anesthetized by 3% pentobarbital sodium (30 mg/kg, IP). Anaesthesia was then maintained with 2%
102
isoflurane. A microcatheter was inserted into the left ventricle through the right carotid artery to measure left
103
ventricular pressure. Hemodynamic data were continuously monitored as described before (17). Myocardial
104
ischemia was produced by exteriorizing the heart through a left thoracic incision and placing 6-0 silk suture and
6
105
making a slipknot around the left anterior descending coronary artery. After 30 min of ischemia, the slipknot
106
was released, and the heart was reperfused for 4 h (for analysis of myocardial apoptosis) and 6 h (for
107
quantification of myocardial infarct size). At the end of the reperfusion, all rats were anesthetized with sodium
108
pentobarbital (100mg/kg, IP) and euthanized by decapitation.
109
2.3 Experimental groups
110
Nondiabetic rats were randomly assigned to two experiment groups (n=8/group): Con+Sham—sham operation
111
(rats underwent the same operation, and the ligature was looped through the myocardium next to the LAD) and
112
receiving no treatment; Con+MI/R (vehicle)—MI/R operation and infusion of saline (0.5 ml, i.v., 10 min before
113
reperfusion). Diabetic rats were randomized to one of the following groups (n=8/group): DM+Sham; DM +
114
MI/R + saline; DM+MI/R+VNP (100 µg/kg, intravenous infusion at 25 µg/kg/h for 4 hours, 10 min before
115
reperfusion). We tested the effects of VNP at 2.5, 25, and 250 µg/kg/h on plasma cGMP level and arterial blood
116
pressure in the preliminary study. It was found that 25 µg/kg/h VNP induced an increase in plasma cGMP level
117
without significant effect on mean arterial pressure and this dose was used in this study; DM + MI/R +
118
8-Br-cGMP (1 mg/kg, i.p., 20 min before reperfusion,); DM + MI/R + VNP + KT-5823 (0.5 mg/kg, i.p., 20 min
119
before reperfusion); DM+MI/R+TUDCA (50 mg/kg, i.p., 10 min before reperfusion).
120
2.4 Determination of apoptosis and myocardial infarction
121
At the end of 4-h reperfusion, myocardial apoptosis was analysed by TUNEL (terminal deoxynucleoti-dyl
122
transferase dUTP nick end labelling) assay as described previously (33). Cardiac caspase-3 activity was
123
measured by using caspase colorimetric assay kits (Chemicon International, Temecula, CA, USA), as described
124
in the previous study (17). At the end of 6-h reperfusion, myocardial infarction was determined by means of a
125
double-staining technique (33). Evans blue-stained areas (area not at risk), triphenyltetrazolium chloride (TTC)
7
126
stained areas (red staining, ischemic but viable tissue), and TTC staining negative areas (infarcted myocardium)
127
in each slice were determined by planimetry on a computer with Sigma Scan software. The myocardial infarct
128
size was expressed as a percentage of infarct area (INF) over total AAR (INF/AAR × 100%).
129
2.5 Biochemical estimation
130
After the reperfusion of 4 h, blood samples were obtained from abdominal aorta, and then centrifugated at 3000
131
× g for 10 min, getting plasma for LDH, CK, MDA, SOD measurement. The procedures strictly followed
132
manufacturer’s instructions. All chemicals used for biochemical analysis were obtained from Nanjing
133
Jiangcheng Bioengineering Institute (Nanjing, China).
134
2.6 Cell culture and treatments
135
H9c2 cardiomyocytes were purchased from the Cell Bank of Chinese Academy of Science (Beijing, China).
136
Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO) supplemented with 10%
137
inactivated fetal bovine serum (FBS, GIBCO), 500µg/ml penicillin, and 500 µg/ml streptomycin (GIB-CO) at
138
37℃ in a humidified atmosphere with 5% CO2. As suggested by some laboratories, concentrations of glucose
139
approaching 10 mM are pre-diabetic levels, and concentrations of glucose above 10 mM are analogous to a
140
diabetic condition within the cell culture system. Accordingly, DMEM containing 25 mM levels of glucose have
141
been applied in several studies to explore the pathophysiology of diabetes as well as to test agents for the
142
treatment of diabetic complications, in which DMEM with 5.5 mmol/L glucose was used to culture H9c2 cells
143
as a control (4, 11, 20). In this study the cells were incubated with control (5.5 mmol/L) or high glucose/high fat
144
(HG/HF) medium containing glucose (25 mmol/L) and saturated FFA palmitate (16:0; 500 μmol/L) as
145
previously described (41). After 18 h of HG/HF incubation, cardiomyocytes were then exposed to
146
hypoxia/reoxygenation treatment as previously described (40). In brief, H9c2 cardiomyocytes were exposed to
8
147
glucose-free
148
(Billumps-Rothenberg) flushed with 5% CO2 and 95% N2 for 3 h of hypoxia. Then, the culture medium was
149
replaced with fresh oxygenated normal or VNP (10-8mol/L) cultured medium, and the dishes were transferred to
150
a normoxic incubator (95% air-5% CO2) for 6 h of reoxygenation. The number of viable H9c2 cells was
151
measured after 48 h by MTT cell viability assay.
152
2.7 Adenovirus infection
153
Adenoviral vectors for overexpression of PKG1α were used as previously described (38). Viruses were
154
propagated in 293 cells and kept at -80°C with a titre of 1011–12 pfu/ml before using. Gene transfer with
155
adenovirus encoding GFP was used as an internal control. Cardiomyocytes plated at a density of 0.5 to 1 ×
156
105/cm2 were infected by adenovirus at 50 multiplicity of infection (moi) for 2 h at 37°C in a humidified, 5%
157
CO2 incubator. Subsequently, the cells were cultured in serum-free DMEM media for an additional 24 h before
158
processing.
159
2.8 Transfection of siRNAs
160
Double-stranded RNAi for selective silencing of PKG1α with a final concentration of 100 nM was transfected
161
into cells (FuGENE Reagent, Roche Applied Science, Mannheim, Germany) according to the manufacturer’s
162
instructions. After transfection, cells were performed the same H/R treatment as described above. Scrambled
163
RNAi was used as control. Knockdown of gene expression was identified by Western blot analysis 48 h after
164
transfection.
165
2.9 Western blotting
166
Samples were lysed with lysis buffer. After sonication, the lysates were centrifuged, proteins were separated by
167
electrophoresis on SDS-PAGE and then transferred onto PVDF (polyvinylidenedifluoride)-Plus membrane
serum-free
culture
medium
and
transferred
9
into
a
Modular
Incubator
Chamber
168
(Micron Separations, Inc., Westborough, MA). After being blocked with 5% milk, the immunoblots were probed
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with anti-GRP78, CHOP, PKG1α, Akt, pAkt (Ser 473), ERK1/2, pERK1/2 (Thr202/Tyr204) and β-actin (Cell
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Signaling, Beverly, MA) overnight at 4°C followed by incubation with the corresponding secondary antibodies
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at room temperature for 1 h. The blots were visualized with ECL-plus reagent.
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2.10 Statistical analysis
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All values are presented as mean ± SEM. Differences were compared by ANOVA followed by Bonferroni
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correction for post hoc t test where appropriate; P
