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The cardioprotective effect of hypoxic and ischemic preconditioning in dogs with myocardial ischemia–reperfusion injury using a double-bypass model
The cardioprotective effect of hypoxic and ischemic preconditioning in dogs with myocardial ischemia–reperfusion injury using a double-bypass model Chun-Ling Chen, Hong Zheng, Yan Xuan, Ablikim Amat, Lin Chen, Jin Yu, Jiang Wang PII: DOI: Reference:
S0024-3205(15)30005-9 doi: 10.1016/j.lfs.2015.09.002 LFS 14489
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
20 March 2015 22 July 2015 8 September 2015
Please cite this article as: Chen Chun-Ling, Zheng Hong, Xuan Yan, Amat Ablikim, Chen Lin, Yu Jin, Wang Jiang, The cardioprotective effect of hypoxic and ischemic preconditioning in dogs with myocardial ischemia–reperfusion injury using a doublebypass model, Life Sciences (2015), doi: 10.1016/j.lfs.2015.09.002
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ACCEPTED MANUSCRIPT The cardioprotective effect of hypoxic and ischemic preconditioning in dogs with
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myocardial ischemia-reperfusion injury using a double-bypass model
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Short title: Cardioprotection and preconditioning
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Chun-Ling Chen, MD, Hong Zheng, MD*, Yan Xuan, MS, Ablikim Amat, MS,
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Lin Chen, MS, Jin Yu, MS, Jiang Wang, MD
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Department of Anesthesiology, the First Affiliated Hospital of Xinjiang Medical University,
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Urumqi, 830054, Xinjiang, China
Correspondence to: Hong Zheng
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Department of Anesthesiology, the First Affiliated Hospital of Xinjiang Medical University, No. 137, Li yu Shan Street, Urumqi, 830054, Xinjiang, China Tel: +86-0991-4366167
Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract Aims: The effects of preconditioning on cardioprotection have mainly been studied in vitro. No
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sufficient in vivo experiments have been performed to optimize ischemic preconditioning (IPC)
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or hypoxic preconditioning (HPC) for clinical applications. The purpose of this study was to establish a canine double-bypass model to examine the effect of IPC and HPC on
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cardiomyocytes and heart function.
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Materials and Methods: A double-bypass procedure to enable independent control of systemic and coronary circulation was established in dogs. The animals were divided into control, HPC,
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and IPC groups (n=6 each). Indicators of cardiac function, including cardiodynamics, hemodynamics, ATP, and cardiac troponin I (cTnI) levels; myocardium morphology; and
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myocardiocyte apoptosis were determined.
Key findings: Both IPC and HPC attenuated the reperfusion-induced decrease in left ventricular
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end systolic pressure seen in the control group. Both the HPC and IPC groups had lower serum cTnI levels, better myocardiocyte histology, and lower rates of apoptosis compared to the control group without preconditioning. HPC reduced the abnormal cardiomyocyte histology and apoptosis to a greater extent than IPC, and only HPC significantly restored the depletion of ATP. Significance: This study demonstrates the effectiveness of the double-bypass model for the optimized study of both HPC and IPC. The results suggest that HPC may provide better cardioprotection than IPC.
Keyword: Cardiopulmonary bypass, CPB; hypoxia preconditioning (heart); myocardial injury; ischemia/reperfusion injury; apoptosis
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ACCEPTED MANUSCRIPT 1. Introduction Studies have shown that both ischemic preconditioning (IPC) and hypoxic preconditioning (HPC)
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increase tolerance of cardiomyocytes to damage, i.e., a sublethal traumatic stimulus may elevate
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the tolerance of tissues or cells to additional severe injury (Minamino 2012; Murry et al. 1986; Shizukuda et al. 1992). Therefore,the mechanisms underlying HPC, IPC, and pharmacological
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preconditioning have been a focus of medical research. Preconditioning, however, has not been
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used to achieve cardioprotection in clinical practice except for the limited application of pharmacological preconditioning (Shohet and Garcia 2007; Wilcox 2010). Cardioprotection
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through IPC or HPC has been previously demonstrated through cardiomyocyte culture, in vitro heart perfusion, and in vivo systemic HPC studies (Bautista et al. 2009; Kohler et al. 2007; Mei
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et al. 1998). HPC has been shown to attenuate hypoxia/reoxygenation-induced apoptosis in mesenchymal stem cells (Wang et al. 2008), and Duan et al. (2012) showed that there was an
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early protective effect of HPC against systemic injury from hemorrhagic shock and resuscitation in a rat model. Using isolated rat hearts, Xiang et al. (2013) showed that multiple-cycle short duration HPC exerts a cardioprotective effect equivalent to that of IPC. In another rat study, Xu and Lamanna (2014) reported that short-term HPC improved survival following cardiac arrest and resuscitation. Despite the available evidence, however, it has not been conclusively confirmed whether in vivo cardioprotection with IPC and HPC exists. The current study utilized a novel double-bypass model in which the oxygenation circuits for systemic and coronary circulation are separated. The double-bypass model allows for the application of ischemia or hypoxia to the heart alone without involving the systemic circulation. This model avoids damage to endothelial cells, plaque rupture, and thromboembolism due to the
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ACCEPTED MANUSCRIPT repeated clamping of the coronary artery. Its use may provide data with respect to myocardium-specific IPC and HPC.
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Thus, the purpose of this study was to establish a canine double-bypass model to examine
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the effect of IPC and HPC on cardiomyocytes and heart function.
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2. Materials and methods
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2.1. Animals
Animals were purchased from the Experimental Animal Center of Affiliated First Hospital of
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Xinjiang Medical University, and the study was conducted in accordance with Guidelines for Animal Surgery in Research. This study was approved by the Ethics Committee of our hospital.
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with 10% potassium chloride.
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At the end of the experimental procedures, the animals were sacrificed by intravenous injection
2.2. Anesthesia
Healthy dogs of either sex weighing 21±3.5 kg were randomly assigned to the control, IPC, and HPC groups (n=6 each group). After sedation with intramuscular injection of ketamine (100 mg; Fujian Gutian Pharmacy) and atropine (1 mg; Shanghai Hefeng Pharmacy), the animals were placed on an operating table and their vital signs were monitored. General anesthesia was induced with midazolam 0.2 mg/kg (Jiangsu Enhua Pharmacy, China), vecuronium 0.15 mg/kg (Organon, Netherlands), and fentanyl 10 μg/kg (Jiangxi Yichang Renfu Pharmacy, China), and endotracheal intubation and mechanical ventilation (Ohmeda S/5 Aespire,7100) were performed. The tidal volume was adjusted according to the blood gas analysis data. Anesthesia was maintained with continuous infusion of midazolam (0.1-0.2 mg/kg/h) and fentanyl (0.02 mg
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ACCEPTED MANUSCRIPT /kg/h) and inhalation of 1-2% isoflurane (Shanghai Abbott Laboratories). Monitoring of the mean arterial pressure (MAP) and arterial blood gas analysis were performed via the femoral
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artery. The pH was maintained at 7.35-7.45, PO2 >100 mm Hg, and PCO2 35-45 mm Hg. The
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right jugular vein was punctured and cannulated (7F tube, Shenzhen Yixinda, China) for monitoring of central venous pressure (CVP), and for fluid supplementation. After endotracheal
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intubation, a 7-F tube was inserted into the left ventricle via the right carotid artery to the left
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atrium and mitral valve, and the cardiac function was monitored using a Powerlab biological signal acquisition system (ML870 AD instruments, Australia). Intravenous heparin (3 mg/kg)
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was injected for anti-coagulation to maintain an activated clotting time (ACT) of at least 480
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seconds.
2.3. Double bypass surgery and preconditioning
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2.3.1. Bypass 1: coronary circulation was bypassed and systemic circulation was connected to an oxygenator and pump
After sternotomy and exposure of the heart, a 14-F tube (Kewei, China) was inserted through the root of the aorta, an 18-F tube was inserted into the superior vena cava, and a 24-F tube (Kewei, China) was inserted into the inferior vena cava. The aorta and superior and inferior vena cava were then connected to a membrane oxygenator (Kewei, China) and a Stockert S2 heart-lung machine (Sorin, Milan, Italy) using a non-heparin-coated tube. The membrane oxygenator and tube were primed with a solution of Voluven® (6% hydroxyethyl starch 130/0.4 in 0.9% sodium chloride injection; 200 ml), Ringer's lactate solution (200 ml), and allogeneic fresh blood from a donor dog (200 ml).
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ACCEPTED MANUSCRIPT 2.3.2. Bypass 2: systemic circulation was bypassed and coronary circulation was connected to an oxygenator and pump.
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After systemic circulation was established, oxygenation and perfusion of the heart was
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achieved through a circuit that received blood from the right and left atria, which was then infused into the coronary sinus and separated from the aorta and systemic circulation by an aortic
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clamp. A standard cardioplegic cannula (9 Fr, dlp-CB20012, Aortic root cannula, Medtronic,
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USA) with a side branch for venting the left ventricle was placed in the ascending aorta and connected with a membrane oxygenator (Kewei, China) and a non-heparin-coated tube. The
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membrane oxygenator and tube were primed with a solution containing Voluven® (6% hydroxyethyl starch 130/0.4 in 0.9% sodium chloride injection; 100 ml), Ringer's lactate
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solution (100 ml), and allogeneic fresh blood from a donor dog (100 ml). A small amount of venous blood from the blood container of systemic circulation was shunted into an oxygenator
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and pump supplying blood to the coronary circulation through a standard perfusion tube placed in the coronary sinus orifice.
For HPC, the oxygenator for the coronary circulation was filled with nitrogen. For IPC, the coronary circulation was shut off by stopping the pump (Figure 1).
2.4. Ischemia/reperfusion (I/R) After IPC or HPC was completed, 10 ml/kg of St. Thomas’ II crystalloid cardioplegic solution at 4°C was perfused into the ascending aorta. Thirty minutes later, crystalloid cardioplegic solution was perfused again at 5 ml/kg. After cardiac arrest for 1 hour, the aortic clamp was released to allow reperfusion and the return of cardiac contractions for 2 hours. The nasopharyngeal temperature was maintained at 32-36°C by a heat exchanger (Sarns Dual
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ACCEPTED MANUSCRIPT Heater/Cooler, Terumo Cardiovascular Systems Co., Ann Arbor, MI, USA) during ischemia.
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MAP was maintained at 50-80 mm Hg by adjusting the arterial blood flow.
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2.5. Group protocols
The animals were divided into the 3 groups, and the protocols are shown in Figure 1. In the
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control group, after double-bypass preparation no preconditioning was performed, and 30
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minutes (a period similar to that used for pre-conditioning) was allowed to elapse before cardiopulmonary bypass was initiated. In the IPC group, myocardial IPC was performed after
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double-bypass preparation. After clamping the aorta, cannulating the coronary artery, and initiating oxygenation of the beating heart, coronary circulation was obstructed by stopping the
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pump for 5 minutes followed by reperfusion (opening the pump) for 5 minutes. This sequence was repeated three times. In the HPC group, after clamping the aorta, cannulating the coronary
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artery, and initiating oxygenation of the beating heart, nitrogen (100%) was administered for 5 minutes through the coronary circulation oxygenator to induce myocardial hypoxia, followed by administration of oxygen for 5 minutes, a sequence that was repeated three times. After the waiting time for the control group and the preconditioning time around 30 minutes, cardiopulmonary bypass was then performed for 60 minutes in the presence of depolarized cardiac arrest induced by St. Thomas’ II crystalloid cardioplegic solution, followed by reperfusion for 120 minutes. 2.6. Detection 2.6.1. Time points T1: before the thoracotomy; T2: after establishing the double-bypass; T3: after
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ACCEPTED MANUSCRIPT preconditioning; T4: 60 minutes after initiating reperfusion; T5: 120 minutes after initiating
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reperfusion.
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2.6.2. Cardiac function and hemodynamics
The hemodynamic parameters evaluated were the heart rate (HR), MAP, and CVP. Indicators of
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left ventricular function included left ventricular end systolic pressure (LVESP), left ventricular
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end diastolic pressure (LVEDP), +dp/dt max, and –dp/dtmax.
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2.6.3. Cardiac ATP concentration
Myocardial tissue from the right ventricular anterior wall was collected via a biopsy needle
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before clamping the aorta and initiating the cardiopulmonary bypass (CPB; control), immediately after releasing the clamp (ischemia), and 30 minutes after initiating reperfusion (reperfusion).
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The tissues were immediately placed in liquid nitrogen, and the intracellular concentration of ATP was measured with an ATP fluorescence kit (Sigma, USA).
2.6.4. Cardiac troponin (cTnI) levels Venous blood (3 ml) was collected from the right jugular vein at T1, T3, T4, and T5 and then centrifuged at 3000 g for 10 minutes. The serum was collected and stored at -80°C. Serum cTnI was measured by ELISA (Shanghai Xitang Biotech Co., China). The results were adjusted using Talor’s formula: dilution adjusted value = ELISA measurement × hematocrit at baseline.
2.6.5. Electron microscopy of the myocardium
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ACCEPTED MANUSCRIPT At the end of the study, myocardial tissues at the apex were collected, fixed in 2.5% glutaraldehyde for 2 hours, and cut into blocks (1 mm3) that were then fixed in glutaraldehyde
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for another 2 hours. After washing in 0.1 M PBS, the blocks were fixed in 1% osmic acid for 2
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hours. After dehydration in acetone at different concentrations and embedding in EPON812 epoxy resin, ultra-thin sections were obtained and subjected to double-staining with uranyl
2.6.6. Detection of myocardiocyte apoptosis
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acetate and lead citrate. Observations were performed with an electron microscope (JEOL1230).
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Tissues for assessing apoptosis were collected from the right ventricle wall, fixed in formalin for 24 hours, embedded in paraffin, and sectioned. TUNEL staining was performed according to the
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manufacturer’s instructions (TUNEL Apoptosis Assay Kit, Roche, USA). Representative photographs were captured at a magnification of 200×, and the number of TUNEL positive cells
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was determined. Five fields were randomly selected from each section at a magnification of 400×, and the number of apoptotic cells was determined among a total of 100 cells, followed by the calculation of the apoptosis index (AI) as follows: AI = (apoptotic cells/total cells) × 100.
2.7. Statistical analysis
Continuous data were presented as the means and standard deviations (SDs). Due to the repeated measurement of the heart function, a linear mixed model was used to investigate the effect of groups (denoted as the Group Effect) and test times (denoted as the Time Effect). When the main effects or interactions were found to have statistical significance, Bonferroni corrections were used to control type I errors during post-hoc multiple comparisons. Statistical analyses were performed with SAS software version 9.2 (SAS Institute Inc., Cary, NC, USA). A two-tailed
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ACCEPTED MANUSCRIPT p-value <0.05 indicated statistical significance. 3. Results
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3.1. Changes in the hemodynamic characteristics
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3.1.1. HR, MAP, and CVP
In the control group, CPB increased the HR, decreased MAP, and increased CVP, and the
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changes were intensified during the reperfusion period. IPC and HPC had no significant effect on
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the CPB-mediated changes in these three parameters. For HR, MAP, and CVP, a significant difference was found in the Time Effect (all, p≤0.001; Table 1), but not in the Group Effects
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(data not shown).
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3.1.2. LVESP and LVEDP
End systolic and diastolic pressure in the LV decreased in the control group during reperfusion
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after CPB. Both IPC and HPC attenuated the reperfusion-induced decrease in LVESP seen in the control group. However, the effect of IPC and HPC on LVEDP did not reach significance (Table 1).
3.1.3. +dp/dtmax and –dp/dtmax
Both + dp/dtmax and -dp/dtmax decreased in the control group during reperfusion after CPB. IPC and HPC somewhat lessened the decrease in +dp/dtmax observed in control hearts during the reperfusion period; however the effect of preconditioning did not reach statistical significance. Neither IPC or HPC had any effect on -dp/dtmax (Table 1).
3.2. Changes in ATP
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ACCEPTED MANUSCRIPT Both IPC and HPC reduced the decrease in ATP observed in control hearts during I/R. The ATP levels for all of the groups were significantly decreased compared to baseline during ischemia
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and reperfusion (p≤0.012). The level of ATP in the HPC group was significantly higher after
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reperfusion compared to that of the control group (p=0.01) (Table 2) and reached a level that was
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not significantly different from the baseline level.
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3.3. Changes in cTnI
The levels of cTnI increased significantly after CPB, and even more significantly during
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reperfusion in all of the groups. However the increase during reperfusion was significantly
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reduced by both IPC and HPC, although not to baseline levels (Table 3).
3.4. The influence of IPC and HPC on myocardial morphology
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Myocardial tissue micrographs are shown in Figure 2. In the control group (Figure 2A), the cardiomyocytes showed severe swelling, nuclei were irregular in shape, filaments were irregularly arranged or lysed, swollen mitochondria showed vacuolization, and sarcomeres were not found. In the IPC group (Figure 2B), the cardiomyocyte swelling was mild, although the nuclei were irregularly shaped and the sarcoplasmic reticulum was slightly expanded. In the HPC group (Figure 2C), the cardiomyocyte swelling was mild, the myofibrils were in order, and the mitochondria had nearly normal morphology.
3.5. AI The AI was significantly lower in the IPC and HPC groups than in the control group (13.69% and 10.02% vs. 18.49%, respectively, p<0.001) and was significantly lower in the HPC group
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ACCEPTED MANUSCRIPT compared to the IPC group (p<0.001). Representative images from each group are shown in
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Figure 3.
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4. Discussion
The results of this study showed that both IPC and HPC attenuated I/R injury-induced decreases
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in LVESP and injury induced cTn1 levels. HPC reduced abnormal cardiomyocyte histology and
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apoptosis to a greater extent than IPC, and only HPC significantly restored the CPB-induced depletion of the ATP levels. The myocardial ATP levels in the HPC group were significantly
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higher than in the control group, supporting the conclusion that injury to myocardial mitochondria was milder in the HPC than in the control group (Kohler et al. 2007).
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These findings are consistent with those of in vitro heart perfusion studies (Guyton and Hall 2000). Both IPC and HPC may initiate endogenous protective mechanisms at an early stage of
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I/R, which may then attenuate the injury to cardiomyocytes (Williams and Benjamin 2000). Cai et al. (2003) found that 5 cycles of 6-minutes of hypoxia followed by 6-minutes of re-oxygenation for HPC exerted cardioprotection that were observed at 24 hours, but not at 30 minutes. Our results are not consistent with this finding. ,The difference might be attributed to the systemic hypoxia used in Cai’s study, which was actually a process of tolerance to hypoxia, and also to the fact that Cai used an isolated heart preparation and we used an in vivo model in which systemic effects on other organs and tissues were present. In a study using isolated rat hearts, Xiang et al. (2013) reported the effects of HPC and IPC to be similar. Our finding of the superiority of HPC might be explained by the fact that in the presence of double-bypass, although coronary artery blood is hypoxic during HPC, blood flow is
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ACCEPTED MANUSCRIPT uninterrupted, and this blood flow may carry away acidic metabolites that are produced during anaerobic metabolism. In contrast, both ischemia and hypoxia occur during IPC.
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During I/R injury, changes in the myocardial structure are characterized by cardiomyocyte
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necrosis and apoptosis (Fliss and Gattinger 1996). Fischer et al. (2003) found apoptotic cardiomyocytes in the heart undergoing in vitro perfusion, and the inhibition of cardiomyocyte
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apoptosis was beneficial for the recovery of cardiac function. Schmitt et al. (2002) described the
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biochemical features of cell apoptosis in the hearts of patients with acute myocardial infarction. In the present study, both HPC and IPC attenuated cardiomyocyte apoptosis observed after
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myocardial I/R injury, but the effect of HPC was better than that of IPC. These results again support the possibility that the increased cardioprotection was because only a decrease in O2 was
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present and the blood flow in the coronary was maintained. Interestingly, Neckár et al. (2002) exposed adult male rats to chronic hypoxia in a hypobaric chamber and then to IPC and reported
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that adaptation to hypoxia decreased the efficiency of IPC, and that the cardioprotective effects of HPC and IPC are not additive.
There are limitations of the current study. Because of limited funds, only one pattern of preconditioning was used in each group. To date, there is no consensus on the duration of hypoxia or the oxygen concentration to be used for hypoxia preconditioning (Semenza 2007). In the presence of double-bypass, the coronary artery is not clamped during HPC and the blood flow of the coronary artery may carry away acidic metabolites produced during the hypoxic condition. The metabolites in the coronary blood were not assessed in this study. 5. Conclusions
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ACCEPTED MANUSCRIPT The double-bypass model is useful for the in vivo study of IPC and HPC, and their contributions to cardioprotection after I/R injury. The results of this study support the conclusion that HPC
Conflict of interest
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The authors declare that there are no conflicts of interest.
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may provide better cardioprotection than IPC.
Acknowledgments
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This work was supported by the National Natural Science Foundation of China (U1303123) and
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the Innovation Fund Designated for Graduate Students of Xinjiang Province (XJGRI2013078).
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ACCEPTED MANUSCRIPT References Bautista, L., Castro, M.J., Lopez-Barneo, J., Castellano, A., 2009. Hypoxia inducible
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factor-2alpha stabilization and maxi-K+ channel beta1-subunit gene repression by hypoxia
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in cardiac myocytes: role in preconditioning. Circ. Res. 104, 1364-1372. Cai, Z., Manalo, D.J., Wei, G., Rodriguez, E.R., Fox-Talbot, K., Lu, H., Zweier, J.L. Semenza,
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G.L., 2003. Hearts from rodents exposed to intermittent hypoxia or erythropoietin are
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protected against ischemia-reperfusion injury. Circulation. 108, 79-85. Duan, Z., Zhang, L., Liu, J., Xiang, X., Lin, H., 2012. Early protective effect of total hypoxic
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preconditioning on rats against systemic injury from hemorrhagic shock and resuscitation. J Surg. Res. 178, 842-850.
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Fischer, U.M., Klass, O., Stock, U., Easo, J., Geissler, H.J., Fischer, J.H., Bloch, W.,Mehlhorn, U., 2003. Cardioplegic arrest induces apoptosis signal-pathway in myocardial endothelial
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cells and cardiac myocytes. Eur. J. Cardiothorac Surg. 23, 984-990. Fliss, H., Gattinger, D., 1996. Apoptosis in ischemic and reperfused rat myocardium. Circ. Res. 79, 949-956.
Guyton, A.C., Hall, J.E., 2000. Muscle blood flow and cardiac output during exercise: the coronary circulation and ischemic heart disease, tenth ed. Textbook of Medical Physiology, Chapter 21. Saunders, Philadelphia, pp. 223-224. Kohler, D., Eckle, T., Faigle, M., Grenz, A., Mittelbronn, M., Laucher, S., Hart, M.L., Robson, S.C.,
Muller,
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Eltzschig,
H.K.,
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CD39/ectonucleoside
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diphosphohydrolase 1 provides myocardial protection during cardiac ischemia/reperfusion injury. Circulation. 116, 1784-1794.
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ACCEPTED MANUSCRIPT Mei, D.A., Nithipatikom, K., Lasley, R.D., Gross, G.J., 1998. Myocardial preconditioning produced by ischemia, hypoxia, and a KATP channel opener: effects on interstitial
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adenosine in dogs. J. Mol. Cell Cardiol. 30, 1225-1236.
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Minamino, T., 2012. Cardioprotection from ischemia/reperfusion injury: basic and translational research. Circ. J. 76, 1074-1082.
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Murry, C.E., Jennings, R.B., Reimer, K.A., 1986. Preconditioning with ischemia: a delay of
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lethal cell injury in ischemic myocardium. Circulation. 74, 1124-1136. Neckar, J., Papousek, F., Novakova, O., Ost'adal, B., Kolar, F., 2002. Cardioprotective effects of
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chronic hypoxia and ischaemic preconditioning are not additive. Basic Res. Cardiol. 97, 161-167.
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Schmitt, J.P., Schroder, J., Schunkert, H., Birnbaum, D.E., Aebert, H., 2002. Role of apoptosis in myocardial stunning after open heart surgery. Ann Thorac Surg. 73, 1229-1235.
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Shizukuda, Y., Mallet, R.T., Lee, S.C., Downey, H.F., 1992. Hypoxic preconditioning of ischaemic canine myocardium. Cardiovasc. Res. 26, 534-542. Semenza, G.L., 2007. Life with oxygen. Science. 318, 62-64. Shohet, R.V., Garcia, J.A., 2007. Keeping the engine primed: HIF factors as key regulators of cardiac metabolism and angiogenesis during ischemia. J. Mol. Med (Berl). 85, 1309-1315. Wang, J.A., Chen, T.L., Jiang, J., Shi, H., Gui, C., Luo, R. H., Xie, X.J., Xiang, M.X., Zhang, X., 2008. Hypoxic preconditioning attenuates hypoxia/reoxygenation-induced apoptosis in mesenchymal stem cells. Acta. Pharmacol. Sin. 29, 74-82. Wilcox, C.S., 2010. Effects of tempol and redox-cycling nitroxides in models of oxidative stress. Pharmacol. Ther. 126, 119-145.
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ACCEPTED MANUSCRIPT Williams, R.S., Benjamin, I.J., 2000. Protective responses in the ischemic myocardium. J. Clin. Invest. 106, 813-818.
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Xiang, X., Lin, H., Liu, J.,Duan, Z., 2013. Equivalent cardioprotection induced by ischemic and
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hypoxic preconditioning. Thorac. Cardiovasc. Surg. 61, 229-233.
Xu, K., Lamanna. J.C., 2014. Short-term hypoxic preconditioning improved survival following
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cardiac arrest and resuscitation in rats. Adv. Exp. Med. Biol. 812, 309-315.
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ACCEPTED MANUSCRIPT Figure Legends Figure 1. The experimental design. Treatment of groups (A), and (B) a diagram of the
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double-bypass procedure.
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Figure 2. Representative electron microscopy micrographs of myocardial tissue (400×). (A) Control group. (B) IPC group. (C) HPC group. (D) Sham negative control.
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Figure 3. Representative micrographs and a bar graph showing the apoptosis of myocytes
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identified by TUNEL staining (200×). Nuclei are indicated by arrows: nuclei of apoptotic cells are brown, and those of normal cells are blue. Letters in the images were indicated specific cell
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types: morphologies A: Normal myocyte; . B: Apoptotic myocyte.; C: Normal smooth muscle cells; . D: Apoptotic smooth muscle cells. The four images of this figure are: (A) Control group.
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(B) IPC group. (C) HPC group. (D) A bar graph of the apoptosis results.
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ACCEPTED MANUSCRIPT Table 1. Hemodynamic and cardiodynamic characteristics
IPC
117.17±15.2
114.83±15.04
T3
126.5±18.96
121.33±16.75
T4
139.33±16.42*†
130±16.48
T5
142.5±12.66*†
124.67±13.63
T2
91±10
T3
85.5±7.56
T4 T5
CVP
0.524
<0.001
0.794
113±11.17
123.5±17.52
96.83±9.39
122.17±11.48
94.33±10.8
90.33±10.76
89±8.07
82.33±12.89*
85.17±10.57
72.83±6.77*†‡
73.5±12.24*†
79±7.01*
68.5±8.6*†‡
73.67±6.86*†
80.33±9.93*
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96.33±8.29
<0.001
112.67±9
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T1
0.053
114.67±11.43
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MAP (mmHg)
0.001
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T2
Group Effect
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113.83±12.86
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112.5±15
Time Effect
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HR (beats/min) T1
p-value for
HPC
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Control
p-value for
T1
4.5±1.05
4.17±1.17
4.67±1.63
T2
6.17±1.17*
6.83±1.47*
7±1.41*
T3
7.5±1.05*
7.83±0.75*
7.5±1.38*
T4
9.33±1.86*†‡
9.5±1.87*†‡
10±2.1*†‡
T5
10.33±1.63*†‡
9.67±1.63*†‡
9.83±2.14*†‡
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Control
IPC
p-value for
p-value for
Time Effect
Group Effect
<0.001
0.030
<0.001
0.744
<0.001
0.076
<0.001
0.846
HPC
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LVESP (mm
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Hg) 99.42±6.56
100.22±11.44
100.03±10.33
T2
95.17±8.72
99.93±17.86
T3
92.73±10.04
94.82±6.33
T4
55.28±6.61*†‡
67.93±8.89*†‡§
69.45±9.59*†‡§
T5
64.07±7.56*†‡
77.72±11.64*†‡§
80.53±11.84*†‡§
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T1
97.82±12.94
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96.07±7.48
LVEDP (mm
9.04±1.26
9.08±1.72
9.29±1.32
T2
8.52±1.33
8.68±1.58
8.97±1.45
T3
8.03±1.47
8.23±1.53
8.52±1.18
T4
12.21±1.67*†‡
12.57±2.25*†‡
12.78±1.71*†‡
T5
12.05±1.34*†‡
12.27±1.98*†‡
11.95±1.83*†‡
+dp/dtmax (mm Hg/s)
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Hg)
T1
1860.00±365.08
1861.67±160.30
1861.00±268.83
T2
1898.33±286.60
1923.33±344.71
2003.33±360.54
T3
1660.83±250.97
1709.50±323.92
1754.67±361.90
T4
990.50±57.50*†‡
1288.50±259.81*†‡
1302.17±201.47*†‡
1162.83±100.24*†‡ 1476.67±204.91*†
1488.50±112.86*†
T5
- dp/dtmax (mm Hg/s)
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Control
IPC
1250.5±180.98
1295.67±200.42
T2
1276.67±146.56
1306±178.89
1351.17±239.22
T3
1125.83±159.81
1190.83±192.71
1197.83±206.26
T4
654.33±127.03*†‡
696±137.23*†‡
647.5±139.74*†‡
T5
854.83±136.54*†‡
854.5±181.53*†‡
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891.67±140.95*†‡
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‡Significant difference compared to T3.
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1293.17±169.04
†Significant difference compared to T2.
p-value for
Time Effect
Group Effect
HPC
T1
*Significant difference compared to T1.
p-value for
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§Significant difference compared to the control group.
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IPC, ischemic preconditioning; HPC, hypoxic preconditioning.
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p-value for the
Time Effect
Group Effect
<0.001
0.045
HPC
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Control
p-value for the
41.00±8.68
Ischemia
23.98±3.72*
27.72±4.44*
Reperfusion
25.77±4.08*
32.15±4.23*
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*Significant difference compared to baseline.
41.45±3.27
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40.85±8.24
30.38±4.76* 34.80±6.34†
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Baseline
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ATP (nmol/mg protein)
†Significant difference compared to the control group.
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IPC, ischemic preconditioning; HPC, hypoxic preconditioning.
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IPC
HPC
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Time Effect <0.001
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cTnI (ng/ml) 0.04±0.01
0.03±0.02
0.03±0.02
T3
1.92±0.72*
1.68±0.49*
1.63±0.50*
T4
6.03±0.84*†
4.57±0.65*†§
3.87±0.77*†§
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T1
*Significant difference compared to T1. †Significant difference compared to T3.
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T5 6.87±0.91*†‡ 5.32±0.83*†‡§ 4.92±0.76*†‡§
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‡Significant difference compared to T4.
§Significant difference compared to the control group.
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IPC, ischemic preconditioning; HPC, hypoxic preconditioning.
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Group Effect <0.001
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S0024-3205(15)30005-9 doi: 10.1016/j.lfs.2015.09.002 LFS 14489
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
20 March 2015 22 July 2015 8 September 2015
Please cite this article as: Chen Chun-Ling, Zheng Hong, Xuan Yan, Amat Ablikim, Chen Lin, Yu Jin, Wang Jiang, The cardioprotective effect of hypoxic and ischemic preconditioning in dogs with myocardial ischemia–reperfusion injury using a doublebypass model, Life Sciences (2015), doi: 10.1016/j.lfs.2015.09.002
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ACCEPTED MANUSCRIPT The cardioprotective effect of hypoxic and ischemic preconditioning in dogs with
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myocardial ischemia-reperfusion injury using a double-bypass model
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Short title: Cardioprotection and preconditioning
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Chun-Ling Chen, MD, Hong Zheng, MD*, Yan Xuan, MS, Ablikim Amat, MS,
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Lin Chen, MS, Jin Yu, MS, Jiang Wang, MD
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Department of Anesthesiology, the First Affiliated Hospital of Xinjiang Medical University,
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Urumqi, 830054, Xinjiang, China
Correspondence to: Hong Zheng
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Department of Anesthesiology, the First Affiliated Hospital of Xinjiang Medical University, No. 137, Li yu Shan Street, Urumqi, 830054, Xinjiang, China Tel: +86-0991-4366167
Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract Aims: The effects of preconditioning on cardioprotection have mainly been studied in vitro. No
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sufficient in vivo experiments have been performed to optimize ischemic preconditioning (IPC)
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or hypoxic preconditioning (HPC) for clinical applications. The purpose of this study was to establish a canine double-bypass model to examine the effect of IPC and HPC on
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cardiomyocytes and heart function.
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Materials and Methods: A double-bypass procedure to enable independent control of systemic and coronary circulation was established in dogs. The animals were divided into control, HPC,
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and IPC groups (n=6 each). Indicators of cardiac function, including cardiodynamics, hemodynamics, ATP, and cardiac troponin I (cTnI) levels; myocardium morphology; and
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myocardiocyte apoptosis were determined.
Key findings: Both IPC and HPC attenuated the reperfusion-induced decrease in left ventricular
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end systolic pressure seen in the control group. Both the HPC and IPC groups had lower serum cTnI levels, better myocardiocyte histology, and lower rates of apoptosis compared to the control group without preconditioning. HPC reduced the abnormal cardiomyocyte histology and apoptosis to a greater extent than IPC, and only HPC significantly restored the depletion of ATP. Significance: This study demonstrates the effectiveness of the double-bypass model for the optimized study of both HPC and IPC. The results suggest that HPC may provide better cardioprotection than IPC.
Keyword: Cardiopulmonary bypass, CPB; hypoxia preconditioning (heart); myocardial injury; ischemia/reperfusion injury; apoptosis
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ACCEPTED MANUSCRIPT 1. Introduction Studies have shown that both ischemic preconditioning (IPC) and hypoxic preconditioning (HPC)
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increase tolerance of cardiomyocytes to damage, i.e., a sublethal traumatic stimulus may elevate
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the tolerance of tissues or cells to additional severe injury (Minamino 2012; Murry et al. 1986; Shizukuda et al. 1992). Therefore,the mechanisms underlying HPC, IPC, and pharmacological
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preconditioning have been a focus of medical research. Preconditioning, however, has not been
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used to achieve cardioprotection in clinical practice except for the limited application of pharmacological preconditioning (Shohet and Garcia 2007; Wilcox 2010). Cardioprotection
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through IPC or HPC has been previously demonstrated through cardiomyocyte culture, in vitro heart perfusion, and in vivo systemic HPC studies (Bautista et al. 2009; Kohler et al. 2007; Mei
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et al. 1998). HPC has been shown to attenuate hypoxia/reoxygenation-induced apoptosis in mesenchymal stem cells (Wang et al. 2008), and Duan et al. (2012) showed that there was an
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early protective effect of HPC against systemic injury from hemorrhagic shock and resuscitation in a rat model. Using isolated rat hearts, Xiang et al. (2013) showed that multiple-cycle short duration HPC exerts a cardioprotective effect equivalent to that of IPC. In another rat study, Xu and Lamanna (2014) reported that short-term HPC improved survival following cardiac arrest and resuscitation. Despite the available evidence, however, it has not been conclusively confirmed whether in vivo cardioprotection with IPC and HPC exists. The current study utilized a novel double-bypass model in which the oxygenation circuits for systemic and coronary circulation are separated. The double-bypass model allows for the application of ischemia or hypoxia to the heart alone without involving the systemic circulation. This model avoids damage to endothelial cells, plaque rupture, and thromboembolism due to the
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ACCEPTED MANUSCRIPT repeated clamping of the coronary artery. Its use may provide data with respect to myocardium-specific IPC and HPC.
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Thus, the purpose of this study was to establish a canine double-bypass model to examine
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the effect of IPC and HPC on cardiomyocytes and heart function.
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2. Materials and methods
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2.1. Animals
Animals were purchased from the Experimental Animal Center of Affiliated First Hospital of
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Xinjiang Medical University, and the study was conducted in accordance with Guidelines for Animal Surgery in Research. This study was approved by the Ethics Committee of our hospital.
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with 10% potassium chloride.
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At the end of the experimental procedures, the animals were sacrificed by intravenous injection
2.2. Anesthesia
Healthy dogs of either sex weighing 21±3.5 kg were randomly assigned to the control, IPC, and HPC groups (n=6 each group). After sedation with intramuscular injection of ketamine (100 mg; Fujian Gutian Pharmacy) and atropine (1 mg; Shanghai Hefeng Pharmacy), the animals were placed on an operating table and their vital signs were monitored. General anesthesia was induced with midazolam 0.2 mg/kg (Jiangsu Enhua Pharmacy, China), vecuronium 0.15 mg/kg (Organon, Netherlands), and fentanyl 10 μg/kg (Jiangxi Yichang Renfu Pharmacy, China), and endotracheal intubation and mechanical ventilation (Ohmeda S/5 Aespire,7100) were performed. The tidal volume was adjusted according to the blood gas analysis data. Anesthesia was maintained with continuous infusion of midazolam (0.1-0.2 mg/kg/h) and fentanyl (0.02 mg
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artery. The pH was maintained at 7.35-7.45, PO2 >100 mm Hg, and PCO2 35-45 mm Hg. The
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right jugular vein was punctured and cannulated (7F tube, Shenzhen Yixinda, China) for monitoring of central venous pressure (CVP), and for fluid supplementation. After endotracheal
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intubation, a 7-F tube was inserted into the left ventricle via the right carotid artery to the left
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atrium and mitral valve, and the cardiac function was monitored using a Powerlab biological signal acquisition system (ML870 AD instruments, Australia). Intravenous heparin (3 mg/kg)
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was injected for anti-coagulation to maintain an activated clotting time (ACT) of at least 480
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seconds.
2.3. Double bypass surgery and preconditioning
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2.3.1. Bypass 1: coronary circulation was bypassed and systemic circulation was connected to an oxygenator and pump
After sternotomy and exposure of the heart, a 14-F tube (Kewei, China) was inserted through the root of the aorta, an 18-F tube was inserted into the superior vena cava, and a 24-F tube (Kewei, China) was inserted into the inferior vena cava. The aorta and superior and inferior vena cava were then connected to a membrane oxygenator (Kewei, China) and a Stockert S2 heart-lung machine (Sorin, Milan, Italy) using a non-heparin-coated tube. The membrane oxygenator and tube were primed with a solution of Voluven® (6% hydroxyethyl starch 130/0.4 in 0.9% sodium chloride injection; 200 ml), Ringer's lactate solution (200 ml), and allogeneic fresh blood from a donor dog (200 ml).
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ACCEPTED MANUSCRIPT 2.3.2. Bypass 2: systemic circulation was bypassed and coronary circulation was connected to an oxygenator and pump.
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After systemic circulation was established, oxygenation and perfusion of the heart was
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achieved through a circuit that received blood from the right and left atria, which was then infused into the coronary sinus and separated from the aorta and systemic circulation by an aortic
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clamp. A standard cardioplegic cannula (9 Fr, dlp-CB20012, Aortic root cannula, Medtronic,
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USA) with a side branch for venting the left ventricle was placed in the ascending aorta and connected with a membrane oxygenator (Kewei, China) and a non-heparin-coated tube. The
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membrane oxygenator and tube were primed with a solution containing Voluven® (6% hydroxyethyl starch 130/0.4 in 0.9% sodium chloride injection; 100 ml), Ringer's lactate
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solution (100 ml), and allogeneic fresh blood from a donor dog (100 ml). A small amount of venous blood from the blood container of systemic circulation was shunted into an oxygenator
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and pump supplying blood to the coronary circulation through a standard perfusion tube placed in the coronary sinus orifice.
For HPC, the oxygenator for the coronary circulation was filled with nitrogen. For IPC, the coronary circulation was shut off by stopping the pump (Figure 1).
2.4. Ischemia/reperfusion (I/R) After IPC or HPC was completed, 10 ml/kg of St. Thomas’ II crystalloid cardioplegic solution at 4°C was perfused into the ascending aorta. Thirty minutes later, crystalloid cardioplegic solution was perfused again at 5 ml/kg. After cardiac arrest for 1 hour, the aortic clamp was released to allow reperfusion and the return of cardiac contractions for 2 hours. The nasopharyngeal temperature was maintained at 32-36°C by a heat exchanger (Sarns Dual
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MAP was maintained at 50-80 mm Hg by adjusting the arterial blood flow.
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2.5. Group protocols
The animals were divided into the 3 groups, and the protocols are shown in Figure 1. In the
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control group, after double-bypass preparation no preconditioning was performed, and 30
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minutes (a period similar to that used for pre-conditioning) was allowed to elapse before cardiopulmonary bypass was initiated. In the IPC group, myocardial IPC was performed after
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double-bypass preparation. After clamping the aorta, cannulating the coronary artery, and initiating oxygenation of the beating heart, coronary circulation was obstructed by stopping the
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pump for 5 minutes followed by reperfusion (opening the pump) for 5 minutes. This sequence was repeated three times. In the HPC group, after clamping the aorta, cannulating the coronary
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artery, and initiating oxygenation of the beating heart, nitrogen (100%) was administered for 5 minutes through the coronary circulation oxygenator to induce myocardial hypoxia, followed by administration of oxygen for 5 minutes, a sequence that was repeated three times. After the waiting time for the control group and the preconditioning time around 30 minutes, cardiopulmonary bypass was then performed for 60 minutes in the presence of depolarized cardiac arrest induced by St. Thomas’ II crystalloid cardioplegic solution, followed by reperfusion for 120 minutes. 2.6. Detection 2.6.1. Time points T1: before the thoracotomy; T2: after establishing the double-bypass; T3: after
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reperfusion.
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2.6.2. Cardiac function and hemodynamics
The hemodynamic parameters evaluated were the heart rate (HR), MAP, and CVP. Indicators of
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left ventricular function included left ventricular end systolic pressure (LVESP), left ventricular
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end diastolic pressure (LVEDP), +dp/dt max, and –dp/dtmax.
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2.6.3. Cardiac ATP concentration
Myocardial tissue from the right ventricular anterior wall was collected via a biopsy needle
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before clamping the aorta and initiating the cardiopulmonary bypass (CPB; control), immediately after releasing the clamp (ischemia), and 30 minutes after initiating reperfusion (reperfusion).
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The tissues were immediately placed in liquid nitrogen, and the intracellular concentration of ATP was measured with an ATP fluorescence kit (Sigma, USA).
2.6.4. Cardiac troponin (cTnI) levels Venous blood (3 ml) was collected from the right jugular vein at T1, T3, T4, and T5 and then centrifuged at 3000 g for 10 minutes. The serum was collected and stored at -80°C. Serum cTnI was measured by ELISA (Shanghai Xitang Biotech Co., China). The results were adjusted using Talor’s formula: dilution adjusted value = ELISA measurement × hematocrit at baseline.
2.6.5. Electron microscopy of the myocardium
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ACCEPTED MANUSCRIPT At the end of the study, myocardial tissues at the apex were collected, fixed in 2.5% glutaraldehyde for 2 hours, and cut into blocks (1 mm3) that were then fixed in glutaraldehyde
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for another 2 hours. After washing in 0.1 M PBS, the blocks were fixed in 1% osmic acid for 2
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hours. After dehydration in acetone at different concentrations and embedding in EPON812 epoxy resin, ultra-thin sections were obtained and subjected to double-staining with uranyl
2.6.6. Detection of myocardiocyte apoptosis
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acetate and lead citrate. Observations were performed with an electron microscope (JEOL1230).
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Tissues for assessing apoptosis were collected from the right ventricle wall, fixed in formalin for 24 hours, embedded in paraffin, and sectioned. TUNEL staining was performed according to the
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manufacturer’s instructions (TUNEL Apoptosis Assay Kit, Roche, USA). Representative photographs were captured at a magnification of 200×, and the number of TUNEL positive cells
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was determined. Five fields were randomly selected from each section at a magnification of 400×, and the number of apoptotic cells was determined among a total of 100 cells, followed by the calculation of the apoptosis index (AI) as follows: AI = (apoptotic cells/total cells) × 100.
2.7. Statistical analysis
Continuous data were presented as the means and standard deviations (SDs). Due to the repeated measurement of the heart function, a linear mixed model was used to investigate the effect of groups (denoted as the Group Effect) and test times (denoted as the Time Effect). When the main effects or interactions were found to have statistical significance, Bonferroni corrections were used to control type I errors during post-hoc multiple comparisons. Statistical analyses were performed with SAS software version 9.2 (SAS Institute Inc., Cary, NC, USA). A two-tailed
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ACCEPTED MANUSCRIPT p-value <0.05 indicated statistical significance. 3. Results
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3.1. Changes in the hemodynamic characteristics
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3.1.1. HR, MAP, and CVP
In the control group, CPB increased the HR, decreased MAP, and increased CVP, and the
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changes were intensified during the reperfusion period. IPC and HPC had no significant effect on
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the CPB-mediated changes in these three parameters. For HR, MAP, and CVP, a significant difference was found in the Time Effect (all, p≤0.001; Table 1), but not in the Group Effects
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(data not shown).
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3.1.2. LVESP and LVEDP
End systolic and diastolic pressure in the LV decreased in the control group during reperfusion
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after CPB. Both IPC and HPC attenuated the reperfusion-induced decrease in LVESP seen in the control group. However, the effect of IPC and HPC on LVEDP did not reach significance (Table 1).
3.1.3. +dp/dtmax and –dp/dtmax
Both + dp/dtmax and -dp/dtmax decreased in the control group during reperfusion after CPB. IPC and HPC somewhat lessened the decrease in +dp/dtmax observed in control hearts during the reperfusion period; however the effect of preconditioning did not reach statistical significance. Neither IPC or HPC had any effect on -dp/dtmax (Table 1).
3.2. Changes in ATP
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ACCEPTED MANUSCRIPT Both IPC and HPC reduced the decrease in ATP observed in control hearts during I/R. The ATP levels for all of the groups were significantly decreased compared to baseline during ischemia
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and reperfusion (p≤0.012). The level of ATP in the HPC group was significantly higher after
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reperfusion compared to that of the control group (p=0.01) (Table 2) and reached a level that was
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not significantly different from the baseline level.
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3.3. Changes in cTnI
The levels of cTnI increased significantly after CPB, and even more significantly during
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reperfusion in all of the groups. However the increase during reperfusion was significantly
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reduced by both IPC and HPC, although not to baseline levels (Table 3).
3.4. The influence of IPC and HPC on myocardial morphology
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Myocardial tissue micrographs are shown in Figure 2. In the control group (Figure 2A), the cardiomyocytes showed severe swelling, nuclei were irregular in shape, filaments were irregularly arranged or lysed, swollen mitochondria showed vacuolization, and sarcomeres were not found. In the IPC group (Figure 2B), the cardiomyocyte swelling was mild, although the nuclei were irregularly shaped and the sarcoplasmic reticulum was slightly expanded. In the HPC group (Figure 2C), the cardiomyocyte swelling was mild, the myofibrils were in order, and the mitochondria had nearly normal morphology.
3.5. AI The AI was significantly lower in the IPC and HPC groups than in the control group (13.69% and 10.02% vs. 18.49%, respectively, p<0.001) and was significantly lower in the HPC group
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Figure 3.
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4. Discussion
The results of this study showed that both IPC and HPC attenuated I/R injury-induced decreases
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in LVESP and injury induced cTn1 levels. HPC reduced abnormal cardiomyocyte histology and
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apoptosis to a greater extent than IPC, and only HPC significantly restored the CPB-induced depletion of the ATP levels. The myocardial ATP levels in the HPC group were significantly
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higher than in the control group, supporting the conclusion that injury to myocardial mitochondria was milder in the HPC than in the control group (Kohler et al. 2007).
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These findings are consistent with those of in vitro heart perfusion studies (Guyton and Hall 2000). Both IPC and HPC may initiate endogenous protective mechanisms at an early stage of
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I/R, which may then attenuate the injury to cardiomyocytes (Williams and Benjamin 2000). Cai et al. (2003) found that 5 cycles of 6-minutes of hypoxia followed by 6-minutes of re-oxygenation for HPC exerted cardioprotection that were observed at 24 hours, but not at 30 minutes. Our results are not consistent with this finding. ,The difference might be attributed to the systemic hypoxia used in Cai’s study, which was actually a process of tolerance to hypoxia, and also to the fact that Cai used an isolated heart preparation and we used an in vivo model in which systemic effects on other organs and tissues were present. In a study using isolated rat hearts, Xiang et al. (2013) reported the effects of HPC and IPC to be similar. Our finding of the superiority of HPC might be explained by the fact that in the presence of double-bypass, although coronary artery blood is hypoxic during HPC, blood flow is
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During I/R injury, changes in the myocardial structure are characterized by cardiomyocyte
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necrosis and apoptosis (Fliss and Gattinger 1996). Fischer et al. (2003) found apoptotic cardiomyocytes in the heart undergoing in vitro perfusion, and the inhibition of cardiomyocyte
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apoptosis was beneficial for the recovery of cardiac function. Schmitt et al. (2002) described the
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biochemical features of cell apoptosis in the hearts of patients with acute myocardial infarction. In the present study, both HPC and IPC attenuated cardiomyocyte apoptosis observed after
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myocardial I/R injury, but the effect of HPC was better than that of IPC. These results again support the possibility that the increased cardioprotection was because only a decrease in O2 was
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present and the blood flow in the coronary was maintained. Interestingly, Neckár et al. (2002) exposed adult male rats to chronic hypoxia in a hypobaric chamber and then to IPC and reported
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that adaptation to hypoxia decreased the efficiency of IPC, and that the cardioprotective effects of HPC and IPC are not additive.
There are limitations of the current study. Because of limited funds, only one pattern of preconditioning was used in each group. To date, there is no consensus on the duration of hypoxia or the oxygen concentration to be used for hypoxia preconditioning (Semenza 2007). In the presence of double-bypass, the coronary artery is not clamped during HPC and the blood flow of the coronary artery may carry away acidic metabolites produced during the hypoxic condition. The metabolites in the coronary blood were not assessed in this study. 5. Conclusions
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Conflict of interest
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The authors declare that there are no conflicts of interest.
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may provide better cardioprotection than IPC.
Acknowledgments
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This work was supported by the National Natural Science Foundation of China (U1303123) and
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the Innovation Fund Designated for Graduate Students of Xinjiang Province (XJGRI2013078).
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Schmitt, J.P., Schroder, J., Schunkert, H., Birnbaum, D.E., Aebert, H., 2002. Role of apoptosis in myocardial stunning after open heart surgery. Ann Thorac Surg. 73, 1229-1235.
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Shizukuda, Y., Mallet, R.T., Lee, S.C., Downey, H.F., 1992. Hypoxic preconditioning of ischaemic canine myocardium. Cardiovasc. Res. 26, 534-542. Semenza, G.L., 2007. Life with oxygen. Science. 318, 62-64. Shohet, R.V., Garcia, J.A., 2007. Keeping the engine primed: HIF factors as key regulators of cardiac metabolism and angiogenesis during ischemia. J. Mol. Med (Berl). 85, 1309-1315. Wang, J.A., Chen, T.L., Jiang, J., Shi, H., Gui, C., Luo, R. H., Xie, X.J., Xiang, M.X., Zhang, X., 2008. Hypoxic preconditioning attenuates hypoxia/reoxygenation-induced apoptosis in mesenchymal stem cells. Acta. Pharmacol. Sin. 29, 74-82. Wilcox, C.S., 2010. Effects of tempol and redox-cycling nitroxides in models of oxidative stress. Pharmacol. Ther. 126, 119-145.
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ACCEPTED MANUSCRIPT Williams, R.S., Benjamin, I.J., 2000. Protective responses in the ischemic myocardium. J. Clin. Invest. 106, 813-818.
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Xiang, X., Lin, H., Liu, J.,Duan, Z., 2013. Equivalent cardioprotection induced by ischemic and
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hypoxic preconditioning. Thorac. Cardiovasc. Surg. 61, 229-233.
Xu, K., Lamanna. J.C., 2014. Short-term hypoxic preconditioning improved survival following
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cardiac arrest and resuscitation in rats. Adv. Exp. Med. Biol. 812, 309-315.
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ACCEPTED MANUSCRIPT Figure Legends Figure 1. The experimental design. Treatment of groups (A), and (B) a diagram of the
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double-bypass procedure.
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Figure 2. Representative electron microscopy micrographs of myocardial tissue (400×). (A) Control group. (B) IPC group. (C) HPC group. (D) Sham negative control.
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Figure 3. Representative micrographs and a bar graph showing the apoptosis of myocytes
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identified by TUNEL staining (200×). Nuclei are indicated by arrows: nuclei of apoptotic cells are brown, and those of normal cells are blue. Letters in the images were indicated specific cell
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types: morphologies A: Normal myocyte; . B: Apoptotic myocyte.; C: Normal smooth muscle cells; . D: Apoptotic smooth muscle cells. The four images of this figure are: (A) Control group.
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(B) IPC group. (C) HPC group. (D) A bar graph of the apoptosis results.
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ACCEPTED MANUSCRIPT Table 1. Hemodynamic and cardiodynamic characteristics
IPC
117.17±15.2
114.83±15.04
T3
126.5±18.96
121.33±16.75
T4
139.33±16.42*†
130±16.48
T5
142.5±12.66*†
124.67±13.63
T2
91±10
T3
85.5±7.56
T4 T5
CVP
0.524
<0.001
0.794
113±11.17
123.5±17.52
96.83±9.39
122.17±11.48
94.33±10.8
90.33±10.76
89±8.07
82.33±12.89*
85.17±10.57
72.83±6.77*†‡
73.5±12.24*†
79±7.01*
68.5±8.6*†‡
73.67±6.86*†
80.33±9.93*
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96.33±8.29
<0.001
112.67±9
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0.053
114.67±11.43
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MAP (mmHg)
0.001
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T2
Group Effect
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113.83±12.86
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112.5±15
Time Effect
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p-value for
HPC
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Control
p-value for
T1
4.5±1.05
4.17±1.17
4.67±1.63
T2
6.17±1.17*
6.83±1.47*
7±1.41*
T3
7.5±1.05*
7.83±0.75*
7.5±1.38*
T4
9.33±1.86*†‡
9.5±1.87*†‡
10±2.1*†‡
T5
10.33±1.63*†‡
9.67±1.63*†‡
9.83±2.14*†‡
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Control
IPC
p-value for
p-value for
Time Effect
Group Effect
<0.001
0.030
<0.001
0.744
<0.001
0.076
<0.001
0.846
HPC
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LVESP (mm
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Hg) 99.42±6.56
100.22±11.44
100.03±10.33
T2
95.17±8.72
99.93±17.86
T3
92.73±10.04
94.82±6.33
T4
55.28±6.61*†‡
67.93±8.89*†‡§
69.45±9.59*†‡§
T5
64.07±7.56*†‡
77.72±11.64*†‡§
80.53±11.84*†‡§
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T1
97.82±12.94
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LVEDP (mm
9.04±1.26
9.08±1.72
9.29±1.32
T2
8.52±1.33
8.68±1.58
8.97±1.45
T3
8.03±1.47
8.23±1.53
8.52±1.18
T4
12.21±1.67*†‡
12.57±2.25*†‡
12.78±1.71*†‡
T5
12.05±1.34*†‡
12.27±1.98*†‡
11.95±1.83*†‡
+dp/dtmax (mm Hg/s)
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Hg)
T1
1860.00±365.08
1861.67±160.30
1861.00±268.83
T2
1898.33±286.60
1923.33±344.71
2003.33±360.54
T3
1660.83±250.97
1709.50±323.92
1754.67±361.90
T4
990.50±57.50*†‡
1288.50±259.81*†‡
1302.17±201.47*†‡
1162.83±100.24*†‡ 1476.67±204.91*†
1488.50±112.86*†
T5
- dp/dtmax (mm Hg/s)
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Control
IPC
1250.5±180.98
1295.67±200.42
T2
1276.67±146.56
1306±178.89
1351.17±239.22
T3
1125.83±159.81
1190.83±192.71
1197.83±206.26
T4
654.33±127.03*†‡
696±137.23*†‡
647.5±139.74*†‡
T5
854.83±136.54*†‡
854.5±181.53*†‡
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891.67±140.95*†‡
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‡Significant difference compared to T3.
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1293.17±169.04
†Significant difference compared to T2.
p-value for
Time Effect
Group Effect
HPC
T1
*Significant difference compared to T1.
p-value for
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ACCEPTED MANUSCRIPT Table 2. Changes in the ATP levels
IPC
p-value for the
Time Effect
Group Effect
<0.001
0.045
HPC
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Control
p-value for the
41.00±8.68
Ischemia
23.98±3.72*
27.72±4.44*
Reperfusion
25.77±4.08*
32.15±4.23*
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*Significant difference compared to baseline.
41.45±3.27
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40.85±8.24
30.38±4.76* 34.80±6.34†
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Baseline
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ATP (nmol/mg protein)
†Significant difference compared to the control group.
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ACCEPTED MANUSCRIPT Table 3 Changes in the cTnI concentrations. p-value for the p-value for the Control
IPC
HPC
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Time Effect <0.001
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cTnI (ng/ml) 0.04±0.01
0.03±0.02
0.03±0.02
T3
1.92±0.72*
1.68±0.49*
1.63±0.50*
T4
6.03±0.84*†
4.57±0.65*†§
3.87±0.77*†§
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*Significant difference compared to T1. †Significant difference compared to T3.
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T5 6.87±0.91*†‡ 5.32±0.83*†‡§ 4.92±0.76*†‡§
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‡Significant difference compared to T4.
§Significant difference compared to the control group.
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Group Effect <0.001
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