3048
Chin Med J 2009;122(24):3048-3054
SUN Hai-yan, XUE Fu-shan, XU Ya-chao, LI Cheng-wen, XIONG Jun, LIAO Xu and ZHANG Yan-ming
Keywords: nitric oxide; nitric oxide synthase; myocardial ischemia-reperfusion injury; propofol; myocardial preservation
Background There are few studies to assess whether propofol attenuates myocardial ischemia-reperfusion injury via a mechanism related to nitric oxide (NO) route, so we designed this randomized blinded experiment to observe the changes of NO contents, nitric oxide synthase (NOS) activity, NOS contents in the myocardium, and cardiac function in ischemic reperfused isolated rat hearts, and to assess the relation between myocardial NO system and cardioprotection of propofol.
Methods The hearts of 30 Sprague-Dawley male rats were removed, mounted on a Langendorff apparatus, and randomly assigned to one of three groups ( n =10 each group) to be treated with the following treatments in a blinded manner: Group 1, control group, after perfusion with pure Krebs Henseleit bicarbonate (K-HBB) buffer solution for 15 minutes, hearts were subjected to 20 minutes global ischemia followed by 60 minutes reperfusion with pure K-HBB buffer;
Group 2, after perfusion with K-HBB buffer solution containing propofol (10 µg/ml) for 15 minutes, the hearts underwent
20 minutes global ischemia followed by 60 minutes reperfusion with the same K-HBB buffer solution; Group 3, after perfusion with K-HBB buffer solution containing propofol (10 µg/ml) and L-NAME (100 µmol/L) for 15 minutes, the hearts underwent 20 minutes global ischemia followed by 60 minutes reperfusion with the same K-HBB buffer solution. The cardiac function was continuously monitored throughout the experiment. The coronary flow was also measured. An
ISO-NO electrode was placed into the right atrium close to the coronary sinus to continuously measure NO concentration in the coronary effluent. The tissue samples from apex of hearts in Groups 1 and 2 were obtained to measure the NOS activity by spectrophotometry and the NOS contents by immunohistochemistry, respectively.
Results The cardiac function was significantly inhibited after ischemia and then gradually improved with reperfusion in all three groups. As compared with Group 1, the cardiac function variables and coronary flow at all the observed points were significantly improved in Group 2. The cardiac function variables and coronary flow were better in Group 3 than in
Group 1, but were inferior in Group 3 than in Group 2. Both NO contents and NOS activity in the myocardium were significantly higher in Group 2 than in Group 1. However, NOS contents in the myocardium did not significantly differ between Groups 1 and 2.
Conclusions In isolated rat hearts, propofol can improve cardiac functional recovery after ischemia-reperfusion by upregulating NOS activity in the myocardium. The NO system may play an important role in the preservation of myocardial ischemia-reperfusion injury produced by propofol.
Chin Med J 2009;122(24):3048-3054
W ith the widespread use of propofol in cardiac surgery, its cardioprotective effect has been increasingly recognized. It has been demonstrated that propofol can enhance myocardial functional recovery, reduce myocardial necrosis and retain adenosine triphosphate (ATP) in the ischemic reperfused isolated hearts.
1,2
The mechanisms of cardioprotection by propofol remain unclear, but may include oxygen free radical scavenging, reductions in Ca
2+
overloading, increases in coronary flow, resistance to the neutrophil accumulation and platelet aggregation, and inhibition of the release of inflammatory factors.
3-6
In addition, propofol has also been shown to involve in the modulation of apoptosis and preconditioning.
7-10
Nitric oxide (NO), an important cellular signaling molecular or chemical mediator, has been shown to play an important role in the myocardial preservation via its vasodilator, antioxidant, antiplatelet and antineutrophil actions.
11
It has been confirmed that NO mediates the ischemic preconditioning
12,13
and many drugs can
DOI: 10.3760/cma.j.issn.0366-6999.2009.24.026
Department of Anesthesiology, Xuanwu Hospital, Capital Medical
University, Beijing 100053, China (Sun HY and Xu YC)
Department of Anesthesiology, Plastic Surgery Hospital, Chinese
Academy of Medical Sciences and Peking Union Medical College,
Beijing 100144, China (Xue FS, Li CW, Xiong J, Liao X and
Zhang YM)
Correspondence to: Prof. XUE Fu-shan, Department of
Anesthesiology, Plastic Surgery Hospital, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing
100144, China (Email: fruitxue@yahoo.com.cn. Fax:
86-10-88772106)
SUN Hai-yan and ZHANG Yan-ming contributed equally to this work.

Chinese Medical Journal 2009;122(24):3048-3054 alleviate myocardial ischemia-reperfusion injury by promoting NO production.
14
Some studies have also found that propofol dilates blood vessel, decreases blood pressure and heart rate, and inhibits platelet aggregation by increasing NO production in the endothelial cells, myocardial cells, lymphocyte and platelet.
15-18
However, it still remains unclear whether propofol attenuates myocardial ischemia-reperfusion injury via a mechanism related to NO route. In addition, the relation between NO production of myocardium and cardioprotection of propofol has also still not been investigated as yet.
Therefore, this randomized, double-blind experiment was designed to observe the changes of the NO contents, nitric oxide synthase (NOS) activity, NOS contents in the myocardium and cardiac function in ischemic reperfused isolated rat hearts, and to assess the relation between myocardial NO system and cardioprotection of propofol.
Our main purpose was to determine the role of myocardial NO system in the mechanism of propofol attenuating ischemia-reperfusion injury.
METHODS
Animals
Healthy adult male Sprague-Dawley rats weighing
220–310 g (9 weeks old) were used in this study. Animals were provided by Peking Union Medical College animal centre and were housed in plastic cages, six to a cage, with water and food available ad libitum. They were placed in a quiet, temperature ((22±2)°C) and humidity
((60±6)%) controlled room in which a 12:12 hours light-dark cycle (light beginning at 8:00 AM), and all tests were performed at the light phase of the cycle. This study was conducted in accordance with our institutional guidelines on the use of live animals for research, and the experimental protocol was approved by the Animal Care and Use Committee of Peking Union Medical College.
Preparation of isolated hearts
Rats were firstly anesthetized with an intraperitoneal injection of sodium thiopental (65 mg/kg). Sodium thiopental was chosen for initial anesthesia because this drug has been shown not to influence preconditioning.
2
Isolated hearts were prepared using the methods previously described.
1,2
Briefly, when no response to tail clamping was obtained, heparin sodium 1000 IU was intravenously and rats were sacrificed by decapitation with a guillotine. Then hearts were excised via thoracotomy and promptly immersed in the perfusion medium (modified Krebs Henseleit bicarbonate buffer
(K-HBB) solution) at 4°C. Hearts were retrogradely perfused via the aorta in a Langendorff apparatus with
K-HBB solution that contained (in mmol/L) 118 NaCl,
27.2 NaHCO
3
, 4.8 KCl, 1.2 MgSO
4
CaCl
2
, and 10 C
6
H
12 mixture of 95% O
2
O
6
and 5% CO
2
, 1.0 KH
2
PO
4
, 1.25
. The perfusate was bubbled with a
, which produced a PO
2
range of 36–42 mmHg, range of 400–500 mmHg, a PCO
2 and a pH range of 7.35–7.45. The temperature of both perfusate and bath was maintained at (36.5±0.1)°C using
3049 a thermostatically controlled water circulating system.
Perfusion pressure was maintained at 73 mmHg by a 100 cmH
2
O fluid column with an overflow pump
(BT00-100M, Lange constant flow pump Limited
Company, Baoding, China), and it was measured at the aortic root via a pressure transducer.
Experimental protocol
All isolated hearts were initially equilibrated for 10 minutes. The exclusion criteria were a preparation time of isolated heart of >60 seconds and a left ventricular systolic pressure of <70 mmHg after initial equilibration period.
1
By computer generated random numbers, the isolated hearts were then assigned to one of the three groups ( n =10 each group) to be perfused with the following in a blinded manner: pure K-HBB solution in
Group 1, the K-HBB solution containing propofol 10
µg/ml (Diprivan; Zeneca, Cheshire, UK) in Group 2, and the K-HBB solution containing propofol 10 µg/ml and a nonspecific nitric oxide synthase inhibitor,
N
ω
-nitro-L-arginine methyl ester (L-NAME) 100 µmol/L
(Sigma Chemical Co., USA) in Group 3. An independent investigator who was not involved in data recording prepared the different perfusates according to the group assignments. The other investigators were blinded to the identity of the perfusates.
The isolated hearts were perfused with the different perfusates for 15 minutes before global ischemia, which was initiated by discontinuating perfusate flow and maintaining for 20 minutes. Then a reperfusion period of
60 minutes was begun (also at constant pressure) with the same perfusate as that given before ischemia each group.
Assessments of cardiac function and coronary flow
A thin latex balloon was inserted into the left ventricle through the left auricle incision and mitral valve, and connected to a pressure transducer (P10EZ, Gould,
Oxnard, CA, USA) for the isovolumetric measurement of left ventricular pressure (LVP). By a pressure transducer connected to a biological mechanic experiment system
(BL-420, Taimeng Sciences and Technology Limited
Company, Chengdu, China), the left ventricular peak systolic pressure, heart rate (HR), left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (LVDP), peak rates of rise in the first derivative of left ventricular pressure (+ dp/dt max
), peak rates of fall in the first derivative of left ventricular pressure (– dp/dt max
) were continuously monitored on an recorder. LVEDP was adjusted to approximately 10 mmHg before the start of the experiment by adjusting the volume of the intraventricular balloon. This balloon volume was maintained throughout the experiment.
LVDP was regarded as contractile function of the isolated rat heart. The effluent from the right ventricle was collected on time to measure the coronary flow (CF).
After an equilibration period of 10 minutes, the baseline values of HR, CF, LVEDP, LVDP, + dp/dt max
and – dp/dt max

3050
Chin Med J 2009;122(24):3048-3054 were taken. Then they were recorded every 10 minutes during the reperfusion. To exclude a bias caused by individual variation, HR, CF, LVDP, + dp/dt max
– dp/dt max
and
at every observed point during the reperfusion were expressed as their recovery rates relative to the baseline values, which were calculated by the following formula: Recovery rate (%) ＝ measured value during the reperfusion/baseline value×100%.
19
The baseline value of
LVEDP was adjusted to approximately 10 mmHg and the
LVEDP at every observed point during the reperfusion was expressed by its measurement value.
Measurements of NO concentration in the coronary effluent
NO concentration in the coronary effluent was measured using a commercially available NO meter and a ISO-NOP
ISO-NO, World Precision Instruments, Sarasota, FL). The principle of this electrode to measure NO is the oxidation of NO at a working electrode which is kept at a constant potential of 0.85 V against an Ag/AgCl reference electrode.
20,21
Selectivity to NO is maintained by a gas permeable membrane covering the electrode. NO diffuses through the gas permeable membrane of the electrode and is then oxidized at the working platinum electrode, resulting in an electrical current. The redox current is detected by a current-voltage converter circuit and continuously recorded by the DUO 18 software supplied by World Precision Instruments. This redox current is proportional to the rate of NO diffusion through the membrane, which is in turn proportional to the NO concentration outside the membrane. To reduce the noise, the generated redox current is filtered at 0.5 Hz. The NO meter, the electrode, and the sample are grounded.
Electrode calibration was performed daily before each experiment using a standard method of nitrite reduction via the quantitative potassium nitrite (KNO
2 solution containing sulfuric acid (H
2
SO
4
) added to a
) and potassium iodide (KI) at 37°C. This produces the same millimole of
NO as KNO
2
according to the following equation:
2KNO
2
+2H
2
SO
4
+2KI → 2NO+I
2
+2H
2
O+2KSO
4
.
A calibration curve was obtained by measurement of the current generated by the successive additions of 100, 200 and 400 nmol/L potassium nitrite, respectively, to the calibration solution containing sulfuric acid and potassium iodide. The relationships between the NO concentrations and output current of the electrode were always linear. The probe was then allowed to equilibrate for 60 minutes in a calibrating cup containing the K-HBB solution balanced with the mixed gases of 95% O
2
5% CO
2
and
at 37°C before being transferred to the right atrium. During the experiment, an ISO-NOP 200 electrode was inserted into the right atrium and immersed in the coronary effluent completely. NO concentration in the coronary effluent was calculated from the current measured with the electrode and the calibration curve, and automatically provided by the DUO 18 software.
After an equilibration period of 10 minutes, NO concentration in the coronary effluent was measured as the baseline value. Then it was determined and recorded at 2, 5, 10, 20, 30, 40, 50 and 60 minutes after initiation of the reperfusion.
Assay of myocardial NOS activity and NOS contents
After a reperfusion period of 60 minutes, the myocardial tissues from apex of hearts in Groups 1 and 2 were obtained. One part of the myocardial tissues was homogenized and centrifuged at 4000 r/min for 10 minutes at 4°C. The total activity of NOS in the myocardium was measured with the spectrophotometic method via the NOS detect assay kit (Nanjing Jiancheng
Biology Engineering company, Nanjing, China) according to the manufacturer’s instructions. The contents of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) in the myocardium were assessed semi-quantitatively using the SABC assay kit and the antibodies of eNOS and iNOS (Wuhan Boster
Biological Technology Ltd., Wuhan, China). The myocardial sample was fixed with 4% paraformaldehyde for 24 hours, then transferred to 20% sucrose-PBS solution and incubated for 48 hours at 4°C. This was followed by paraffin processing through increasing grades of ethanol, xylene, and paraplast.
Paraffin-embedded tissue blocks were sectioned at 5 µ m, and sections were mounted on positively charged slides.
For immunostaining, sections were deparaffinized, rehydrated, treated with target retrieval buffer, blocked with 3 % hydrogen peroxide for 10 minutes (to block endogenous peroxidase activity, washed with phosphate-buffered saline (PBS)) and blocked with 5% normal goat serum for 30 minutes. The slides were subsequently incubated with primary mouse monoclonal eNOS antibody or rabbit polyclonal iNOS antibody in
PBS containing 1% normal goat serum overnight at 4°C.
The primary antibody was rinsed off with PBS, and the sections were incubated with either goat anti-mouse eNOS or goat anti-rabbit iNOS for 30 minutes. After three washing steps with PBS were completed, sections were stained using ABC staining system for 10 minutes, washed with distilled water, and counterstained using hematoxylin. Sections were dipped in lithium carbonate, washed in running water, dehydrated in increasing grades of alcohol, and cleared in xylene before being mounted in resinous mounting medium with coverslips. According to the requirements of antibodies for iNOS, microwave restoring program for iNOS immunohistochemistry was needed to be adopted before slides were treated with 5% normal goat serum.
Five sections per heart were randomly selected to evaluate semi-quantitatively the labeling of NOS immunoreactivity under a high power microscope. The cell dyeing conditions in five scopes selected randomly from each section were evaluated and classed as follows: the blank dyeing is zero; the focal dyeing is one; the mild diffuse dyeing is two; the moderate diffuse dyeing is three; the strong diffuse dyeing is four.
22

Chinese Medical Journal 2009;122(24):3048-3054
3051
Table.
The changes of cardiac function variables and coronary flow during reperfusion in the three groups
During reperfusion (minutes) Groups
10
HR (%) LVEDP (mmHg) LVDP (%) + dp/dt max
(%) – dp/dt max
(%) CF (%)
Group 1 60±6 47±6 31±5 30±5
Group 2 78±7 * 28±6 * 46±7 * 61±8 *
30±5
55±7 *
61±3
79±5 *
Group 3 71±7 *† 38±6 *† 36±6 *† 40±6 *† 37±6 *† 69±5 *†
20
30
Group 1 66±6 40±5 40±6 46±6
Group 2 86±6 * 24±5 * 58±7 * 74±8 *
38±8
68±6 *
48±4
69±5 *
Group 3 72±4 *† 32±6 *† 49±7 *† 60±8 *† 47±8 *† 60±4 *†
Group 1 67±4 38±3
Group 2 87±4 * 23±5 *
Group 3 72±4 *† 32±6 *†
45±4
51±8 *†
51±8
61±8 *†
42±11 46±4
73±8 * 71±10 * 63±6 *
50±9 *† 54±5 *†
40
50
60
Group 1 70±4 35±7
Group 3 76±5 *† 31±6 *†
43±6
Group 2 92±5 * 21±6 * 58±6 * 70±6 *
50±6 *†
49±6
61±6 *†
43±5 42±3
68 ± 8 * 57±4 *
51±6 *† 50±6 *†
Group 1 70±4 37±6 37±5 47±6
Group 2 91±6 * 20±5 * 56±5 * 64±7 *
41±5
63±7 *
39±3
54±5 *
Group 3 77±5 *† 31±7 *† 44±6 *† 55±6 *† 46±5 *† 45±4 *†
Group 1 68±4 35±4 37±3 46±7
Group 2 85±4 * 19±4 * 51±6 * 61±8 *
38±7
59±8 *
34±5
52±3 *
Group 3 77±4 *† 30±4 *† 41±9 *† 52±8 *† 44±6 *† 44±4 *†
HR: heart rate. LVEDP: left ventricular end-diastolic pressure. LVDP: left ventricular developed pressure. + dp/dt max
: peak rates of rise in the first derivative of left ventricular pressure. – dp/dt max
, peak rates of fall in the first derivative of left ventricular pressure. CF: coronary flow. HR, CF, LVDP, + dp/dt max
and – dp/dt max
at every observed point during the reperfusion were their recovery rates relative to baseline values taken after equilibration period. Data are expressed as means ± SD. n =10 in each group. *
P <0.05, compared with Group 1. †
P <0.05, compared with Group 2.
Statistical analysis
The number of animals needed to demonstrate direct effects of propofol on cardiac function variables was calculated with the assumption that a 20% change would be significantly relevant. Based on a statistical power of
0.9, an α =0.05 and β =0.2, 10 rats per group was suggested. The statistical analysis of data was performed with SPSS (Version 11.5, SPSS Inc., Chicago, IL, USA).
The comparisons of HR, cardiac function, CF and NO concentration in the coronary effluent among three groups were done using the factorial analysis of variance
(ANOVA). Where significance was different, Fisher ′ s protected least significance difference (PLSD) post hoc test was applied. The intragroup differences of these data recorded over a period of time were analyzed using the repeated measures ANOVA. Where significance was different, the Fisher ′ s PLSD post hoc test was applied.
The differences in the total activity of NOS and contents of eNOS and iNOS in the myocardium between Groups 1 and 2 were compared using the two tailed t
-tests. All data are presented as means ± standard deviation (SD).
Statistical difference was considered significant if a P value was less than 0.05. effluent at the end of equilibration did not differ among three groups. During the reperfusion, NO concentrations in the coronary effluent in three groups decreased significantly compared with the baseline values ( P <0.05).
NO concentrations in the coronary effluent at all observing points after the reperfusion were highest in
Group 2, secondary in Group 3 and lowest in Group 1, with significant differences among three groups ( P <0.05)
(Figure 1).
RESULTS
Cardiac function and coronary flow
The cardiac function was significantly inhibited after ischemia and then gradually improved with reperfusion in all three groups. As compared with Group 1, the recovery rates of HR, LVDP, + dp/dt max
, – dp/dt max
and CF at all observed points after the reperfusion were significantly higher and LVEDP was significantly lower in Group 2 ( P
<0.05) . Although all these observed variables were significantly better in Group 3 than with in Group 1, but they were inferior in Group 3 than in Group 2 (Table).
NO concentrations in coronary effluent
The baseline values of NO concentration in the coronary
Figure 1.
NO concentrations in the coronary effluent during reperfusion in the three groups. Data are expressed as means±SD. n
=10 in each group.
1. †
*
P
<0.05, compared with Group
P
<0.05, compared with Group 2.
Immunohistochemistry assessments of NOS in myocardium
The immunoreactants for eNOS, as evidenced by the

3052
Chin Med J 2009;122(24):3048-3054
Figure 2.
The immunoreactivity of eNOS in the myocardium in the Groups 1 (
A
) and 2 (
B
) (SABC staining, original magnification
×100).
Figure 3.
The Immunohistochemistry assessments of myocardial iNOS in Group 1 (
A
) and Group 2 (
B
) (SABC staining, original magnification ×100). yellow granular precipitates, were observed in the reperfusion. This suggests that the ischemia-reperfusion myocardium and endothelial cells. The immunoreactivity of iNOS in the myocardium was less than that of eNOS in can decrease NO production in the myocardium. Our results may contribute to the following factors. First, the myocardium. The values of optical density for eNOS and iNOS immunoreactivity were 2.7±1.0 and 0.7±0.3, respectively, in Group 1, 2.7±0.8 and 0.7±0.3 in Group 2, with no significant difference between groups (
P
>0.05,
Figure 2).
Spectrophotometic assay of NOS activity in the myocardium
Total activity of NOS in the myocardium was (0.23±0.07) and (0.36±0.06) U/mg prot in Groups 1 and 2, respect- tively, with significant difference between groups (
P
<0.05).
DISCUSSION
The main purpose of this experiment was to assess the role of myocardial NO system in the mechanism of ischemia-reperfusion can induce endothelial cell injury and activate endogenous NOS inhibitors, leading to eNOS activity inhibition and NO production dysfunction.
23,24 Second, reperfusion may produce significant reactive oxygen species, such as the superoxide anion and hydroxyl radicals, which damage lipids and proteins.
3,4 NO is a strong antioxidant and can substantially be degraded by reacting directly with free radicals.
11,25
In the current study, a propofol concentration of 10 µg/ml was contained in perfusates in Groups 2 and 3. This concentration corresponds to propofol 56 µmol/L and is a maximal blood concentration of propofol frequently used in clinical intravenous anesthesia.
23 Xu et al 24 propofol attenuating ischemia-reperfusion injury in isolated rat hearts. To determine accurately the real-time changes of myocardial NO during the ischemia- reperfusion process, an electrochemical detection technique was selected in our study. It has been accepted as the most sensitive technique available that can reliably measure physiological levels of NO in vitro and in vivo .
21
The main advantages of this technique include high demonstrated that following ischemia-reperfusion, recovery of the heart mechanical function by the suppression of catecholamine release was best in a propofol concentration of 50 µmol/L compared with propofol concentration of 10 µmol/L and 100 µmol/L.
25
In ischemic reperfused isolated rat hearts, propofol at concentrations of 25 µmol/L and 50 µmol/L could also result in significant improvement in the recovery of mechanical function by the reduction of lipid peroxidation.
1 Moreover, the results of our preliminary selectivity against other potentially interfering molecules, low detection limit (<1 nmol/L), rapid real-time response
(millisecond to second), and wide dynamic concentration range (<1 nmol/L to >100 µmol/L).
Using this sensitive electrochemical NO microsensor in present study, we demonstrated that NO production in the myocardium decreased significantly after ischemia. NO production in the myocardium increased with reperfusion, but it was still significantly lower than baseline level, even in the best restoration state at 30 minutes following experiment showed that propofol at a concentration of 10
µg/ml could provide a cardioprotection against ischemia reperfusion injury.
The present study showed that the perfusion with propofol in Group 2 could significantly increase NO release in the myocardium following the reperfusion and improve cardiac function depression induced by ischemia-reperfusion. In 60 minutes following the reperfusion, the recovery rates of LVDP, + dp/dt
– dp/dt max
and max
in Group 2 increased 14%, 15% and 20%

Chinese Medical Journal 2009;122(24):3048-3054
3053 respectively, compared with Group 1 in which the isolated hearts were perfused with pure K-HBB solution.
When a nonselective NOS inhibitor L-NAME was administered in Group 3, however, the improvement of cardiac function recovery caused by propofol was decreased. These results indicate that increasing NO production in the myocardium may be a key for propofol alleviating myocardial ischemia-reperfusion injury.
The exact mechanism that propofol increases NO production in the myocardium is not clear, but may related to improvement on the myocardial NO metabolic disorders after ischemia-reperfusion. First, propofol has been shown to promote the synthesis of endogenous NO by stimulating myocardial eNOS activity.
17,18
and production and significantly decrease coronary flow.
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In conclusion, our study demonstrates that propofol can improve cardiac function after ischemia-reperfusion by upregulating NOS activity in the isolated rat hearts. The
NO system may play an important role in the preservation of myocardial ischemia-reperfusion injury produced by propofol.
29
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(Received July 20, 2009)
Edited by GUO Li-shao