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Myeloperoxidase increased cardiomyocyte protein nitration in mice subjected to nonlethal mechanical trauma
Biochemical and Biophysical Research Communications 393 (2010) 531–535
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Myeloperoxidase increased cardiomyocyte protein nitration in mice subjected to nonlethal mechanical trauma Zi Yan a,b,1, Feng Liang c,1, Li Guo b, Jin Wang a,b, Xiao-Liang Wang d, Xiao-Long Cheng e, Xin-Liang Ma d, Hui-Rong Liu f,* a
State Key Laboratory of Cellular Physiology, Shanxi Medical University, Taiyuan, Shanxi, PR China Department of Physiology, Shanxi Medical University, Taiyuan, Shanxi, PR China Cardiothoracic Surgery, Steel General Hospital of Taiyuan, Taiyuan, Shanxi, PR China d Department of Emergency Medicine, Thomas Jefferson University, 1020 Sansom Street, Philadelphia, PA 19107, Unites States e Department of Anatomy, Shanxi Medical University, Taiyuan, Shanxi, PR China f Department of Pathophysiology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, PR China b c
a r t i c l e
i n f o
Article history: Received 1 February 2010 Available online 12 February 2010 Keywords: Trauma Myocardial inflammation Apoptosis Endothelium 3-Nitrotyrosine
a b s t r a c t Nonlethal mechanical trauma causes cardiomyocyte apoptosis which contributes to posttraumatic cardiac dysfunction. Apoptosis is positively correlated with protein nitration in the traumatic heart. However, the mechanisms responsible for the cardiomyocyte protein nitration remain unclear. The present study was designed to identify whether myeloperoxidase may contribute to protein nitration in nonlethal mechanical trauma and subsequent cardiomyocyte apoptosis, and, if so, to determine the possible mechanisms responsible. We used Noble-Collip drum to make nonlethal traumatic mice models. Male adult C57B16/J mice were placed in the Noble-Collip drum and subjected to a total of 200 revolutions at a rate of 40 r/min. Then myeloperoxidase activity and release, protein nitration, cardiomyocyte apoptosis, endothelial function and intercellular adhesion molecule-1 expression were determined. Nonlethal mechanical trauma was characterized by the 100% survival rate during the first 24 h after trauma, the lack of circulatory shock and without direct heart injury. However, myeloperoxidase activity significantly increased 6 h after trauma, and reached a maximum level 12 h after trauma. Obviously, protein nitration and cardiomyocyte apoptosis increased 12 h after trauma and could be blocked by administration of R15.7, a monoclonal antibody that blocks polymorphonuclear neutrophils adhesion. Moreover, endothelial dysfunction and intercellular adhesion molecule-1 upregulation were observed in traumatic mice. Our present study demonstrated for the first time that myeloperoxidase caused protein nitration and cardiomyocyte apoptosis in nonlethal traumatic mice. Inhibition of polymorphonuclear neutrophils adhesion and antinitration treatments may be novel measures in reducing posttraumatic cardiomyocyte apoptosis and secondary heart injury. Ó 2010 Elsevier Inc. All rights reserved.
Introduction With the development of modern industry and transportation, the morbidity of mechanical trauma has gradually risen. Nowadays trauma has become the most common cause of death under the age of 45 years old [1]. Most studies confirm that mechanical trauma can cause direct heart damage, such as coronary artery dissection and cardiac contusion [2,3]. However, recently published clinical reports [4–7] have indicated that blunt chest trauma causes myocardial infarction even in the absence of direct cardiomyocyte injury. These results suggest that trauma induces not only * Corresponding author. Fax: +86 351 4135076. E-mail address: [email protected] (H.-R. Liu). 1 Zi Yan and Feng Liang contributed equally to this work. 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.02.049
primary heart injury but also secondary heart injury. Nevertheless, the mechanisms responsible for trauma-induced secondary heart injury still remain unknown. Our previous results revealed that mechanical trauma caused significant cardiomyocyte apoptosis which contributed to posttraumatic secondary cardiac dysfunction [8]. Moreover, we also proved that tyrosine nitration was positively correlated with cardiomyocyte apoptosis in nonlethal traumatic cardiomyocytes. Most studies [9,10] considered that there were mainly two mechanisms involved in tyrosine nitration, including the formation of peroxynitrite (ONOO ) and mediation via hemeperoxidase, most notably myeloperoxidase (MPO). In a recent study, we demonstrated that ONOO -mediated signaling pathway caused tyrosine nitration and subsequent cardiomyocyte apoptosis in nonlethal traumatic injury [11]. In addition, it is well known that traumatic
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Z. Yan et al. / Biochemical and Biophysical Research Communications 393 (2010) 531–535
injury elicits a systemic inflammatory response. As one of the most important inflammatory components, it remains unknown whether MPO increased and contributed to tyrosine nitration and subsequent cardiomyocyte apoptosis in nonlethal traumatic mice. Therefore, the aims of the present study were (1) to investigate whether MPO increased in nonlethal traumatic mice; and, if so, (2) to determine whether MPO may contribute to protein nitration induced by mechanical trauma; and (3) to identify the possible mechanisms. Materials and methods Animals. The experimental procedures were conducted in adherence to the ‘‘Guiding Principles in the Use and Care of Animals” published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996), the Guide for the Care and Use of Laboratory Animals protocol, published by the Ministry of the People’s Republic of China (issued at 3 June, 2004), and approved by the Institutional Animal Care and Use Committee of Shanxi Medical University. Nonlethal traumatic mice model. Nonlethal mechanical trauma was induced as previously described [8]. In brief, Male adult C57B16/J mice were anesthetized with pentobarbital sodium (40 mg/kg). Then the mice were placed in a Noble-Collip drum and subjected to a total of 200 revolutions at a rate of 40 r/min. Trauma mice were injured when the wheel was rotated, while sham trauma mice were taped on the inner wall of drum, so without traumatic injury. Immediately after trauma, a PE catheter filled with heparinized 0.9% NaCl solution was inserted into the right common carotid artery for recording mean arterial blood pressure (MABP) through a PowerLab data analysis system. Determination of myocardial MPO activity. As an index of polymorphonuclear neutrophils (PMNs) infiltration, MPO activity in cardiac tissue was assayed according to the method as described in our previous study [12]. In brief, cardiac tissue was homogenized in 50 mmol/L potassium phosphate buffer at pH 6 containing 0.5% hexadecyltrimethyl ammonium bromide (HTAB). The homogenates were centrifuged for 30 min at 12,500g at 4 °C. The supernatants were then collected and reacted with 0.167 mg/mL Odianisodine dihydrochloride and 0.0005% H2O2 in 50 mmol/L phosphate buffer at pH 6. The measurement was performed at 460 nm at 25 °C using a microplate reader (SpectraMAX Plus, Molecular Devices, Sunnyvale, CA) in duplicate. One unit of MPO activity was defined as that quantity of enzyme hydrolyzing 1 mmol of peroxide per minute at 25 °C. Quantitation of tissue nitrotyrosine content. Cardiac nitrotyrosine content, a footprint of in vivo protein nitration level, was determined using an enzyme linked immunosorbent assay (ELISA) method described in our recent publication [13]. In brief, the free wall of the left ventricle was separated and homogenized in icecold PBS. The homogenates were centrifuged for 10 min at 12,500g at 4 °C. The supernatants were collected and protein concentrations were determined by the BCA method. A nitrated protein solution was prepared for use as a standard. These standard samples, along with tissue samples from hearts, were applied to disposable sterile ELISA plates and incubated overnight with primary antibody. The secondary antibody was added, and the peroxidase reaction product was generated by using O-phenylenediamine dihydrochloride (OPD) solution. The optical density was measured at 460 nm with a microplate reader (BioTek Instruments, Inc. Winooski, VT). The amount of nitrotyrosine content in tissue samples was calculated using standard curves generated from nitrated BSA containing known amounts of nitrotyrosine and expressed as nanomoles per gram of protein.
Determination of myocardial apoptosis with the terminal deoxyneucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. To determine myocardial apoptosis in a quantitative manner, the hearts were perfused first with 0.9% NaCl for 5 min and then with 4% paraformaldehyde in PBS (pH 7.4) for 20 min. Four longitudinal sections from the free wall of the left ventricle were cut and further fixed in 4% paraformaldehyde in PBS for 24 h at room temperature. Fixed tissues were embedded in a paraffin block and two slides at 4- to 5-lm thickness were cut from each tissue block. Immunohistochemical procedures for detecting apoptotic cardiomyocytes were performed by using an apoptosis detection kit (Boehringer Mannheim, Ridgefield, CT) according to the manufacturer’s instructions. An additional staining was performed with monoclonal anti-a-sarcomeric actin. This staining enables the identification of myocyte and, therefore, a distinction between myocyte nuclei and nuclei of other cells in cardiac tissue. After being rinsed with PBS, slides were coverslipped with mounting medium containing DAPI to permit total nuclei counting. With the use of a 20 objective, the tissue slide was digitally photographed with a QICAM-Fast Digital Camera mounted onto an Olympus BX51 fluorescence microscope. Total nuclei (DAPI staining, blue) and TUNEL-positive nuclei (green) in each field were counted by IP Lab Imagine Analysis Software (Version 3.5, Scanalytics, Fairfax, VA) with a custom-made script (by Ken Anderson, Bio Vision Technologies, North Exton, PA). The index of apoptosis (number of TUNEL-positive nuclei/total number of nuclei 100%) was automatically calculated and exported to Microsoft Excel for further analysis. Results from different fields taken from the same animal were averaged and counted as one sample. Determination of endothelial function. Endothelial function was determined by comparing the vasorelaxation response to acetylcholine (ACh), an endothelium-dependent vasodilator, with that of acidified NaNO2, an endothelium-independent vasodilator, as described previously [13]. Briefly, aortic rings were mounted onto hooks, suspended in organ chambers filled with Krebs buffer and aerated with 95% O2 and 5% CO2 at 37 °C, and connected to force transducers (WPI, Sarasota, FL) to record changes via a Maclab data acquisition system. After equilibration for 60 min at a preload of 1 g, the rings were precontracted with norepinephrine (NE, 0.1 nM). Once a stable contraction was achieved, the rings were exposed to cumulative concentrations of ACh (10 9–10 5 M). After the cumulative response stabilized, the rings were washed and allowed to equilibrate to baseline. The procedure was then repeated with an endothelium-independent vasodilator (acidified NaNO2, 10 9–10 6 M) to determine smooth muscle function. Endothelial dysfunction was defined as a reduced vasorelaxation in response to ACh with a normal response to acidified NaNO2. Immunohistochemical detection of MPO and intercellular adhesion molecule-1 (ICAM-1). Immunostaining of MPO and ICAM-1 were determined using the method described in our recent publication [14]. Briefly, optimal cutting temperature compound-embedded tissues were cut into 6 mm thickness and stained with primary antibody (anti-MPO monoclonal antibody, Abcam; anti-ICAM-1 monoclonal antibody, BD Bio-sciences) and biotinylated secondary antibody. MPO was detected with alkaline phosphatase kit (VECTASTAINÒ ABC-AP kit with Vector Red as a substrate, Vector Laboratories) and ICAM-1 expression was detected with horseradishperoxidase kit (VECTASTAINÒ ABC kit, Vector Laboratories). Statistical analysis. All values in the text and figures are presented as means ± SEM. Time and group differences were determined by two-way ANOVA for repeated measures. For nonrepeated measures, data were subjected to ANOVA followed by the Scheffe’s correction for post hoc t-test comparison. Probabilities of P 6 0.05 were considered to be statistically significant.
Z. Yan et al. / Biochemical and Biophysical Research Communications 393 (2010) 531–535
Results Nonlethal traumatic mice were established Nonlethal mechanical trauma by Noble-Collip drum rotation is characterized by the lack of circulatory shock (MABP > 75 mmHg within 24 h after trauma), no direct cardiac injury (i.e., cardiac contusion and pericardial bleeding), and a 100% 24 h survival rate [8]. Nonlethal traumatic injury caused increased MPO activity and release MPO, an enzyme occurring virtually exclusively in PMNs, was an index that has been shown to correlate closely with PMNs accumulation and infiltration in the heart. As summarized in Fig. 1A, MPO activity significantly increased 6 h after trauma, and reached a maximal level at 12 h after trauma. Moreover, compared with the sham trauma, MPO immunostaining was markedly increased in cardiac tissue from mice subjected to nonlethal traumatic injury (Fig. 1B). These results indicated for the first time that nonlethal trauma caused significant PMNs accumulation and increased MPO activity and release in cardiac tissue. MPO increased protein nitration that contributed to posttraumatic myocardial apoptosis To investigate whether increased MPO resulted in protein nitration and subsequent myocardial apoptosis, we observed the effect of R15.7 (a monoclonal antibody that blocks PMNs adhesion) treatment on myocardial nitrotyrosine level and apoptosis index. As illustrated in Fig. 2A 12 h after trauma, the tissue nitrotyrosine level was significantly increased. And as summarized in Fig. 2B, there were no TUNEL-positive nuclei detected in samples isolated from mice subjected to sham trauma. However, significant cardiomyocytes were labeled TUNEL-positive in samples from mice subjected to nonlethal traumatic injury. Treatment with R15.7 significantly reduced cardiac tissue nitrotyrosine level and decreased myocardial apoptosis. These results demonstrated that PMNs and MPO are critical contributors to posttrauma protein nitration and myocardial apoptosis. Mechanisms responsible for increased myocardial MPO activity and release To determine the mechanisms responsible for increased myocardial MPO activity and release in nonlethal traumatized mice, two additional studies were performed in which endothelial function and ICAM-1 expression were determined 6 h after trauma. As illus-
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trated in Fig. 3A, the dose–response curve to ACh, an endotheliumdependent vasodilator, was significantly shifted to the right in aortic segments isolated from traumatic animals. However, the dose–response curve to acidified NaNO2, an endothelium-independent vasodilator, was unaltered in these vascular segments (Fig. 3B). Moreover, ICAM-1 expression was markedly upregulated in myocardial tissue obtained from traumatized animals (Fig. 4). These results demonstrated that traumatic injury caused significant endothelial dysfunction and upregulation of adhesion molecules which might be the reason of PMNs accumulation and MPO release. Discussion In our present study we have made several novel observations. First, we have demonstrated that nonlethal mechanical trauma resulted in significantly increased MPO activity 6 h after trauma, and MPO release also markedly increased in myocardial tissue. Second, we have provided direct evidence that MPO increased posttraumatic protein nitration and subsequent myocardial apoptosis. Third, we have demonstrated that nonlethal mechanical trauma caused endothelial dysfunction and ICAM-1 upregulation which might be responsible for trauma-induced increased MPO activity and release. It is well known that PMNs play an important role in inflammatory response. A typical inflammatory reaction can result in PMNs adhesion, aggregation and extravasation. Activated PMNs have a considerable effect on the development and turnover of inflammatory response via the release of inflammatory factor, oxygen-derived free radicals and elastase. MPO, an enzyme occurring virtually exclusively in azurophil granules of PMNs in high concentrations, was released when PMNs were activated and was used to quantify the accumulation of PMNs in the injured tissues. MPO activity and immunostaining can be used to reflect the accumulation and localization of PMNs [15]. Released MPO can oxidize chloride to generate hypochlorous acid (HOCl) [16,17] and nitrate tyrosine to form nitrotyrosine [18,19] which are both responsible for tissue injury. In the present study, the result that MPO activity and release increased demonstrated that nonlethal mechanical trauma caused MPO release which might play an important role in inflammatory response induced by trauma. Protein nitration generally occurs in an organism that contributes to regulate protein metabolism, protein function and signal pathway process [20]. Increased 3-nitrotyrosinated proteins in the aging process also demonstrate that there is protein nitration under physiological conditions [21–23]. But more importantly, protein nitration (primarily including tyrosine nitration) became an injury mediator in many inflammatory diseases. Emerging evi-
Fig. 1. The activity and release of myeloperoxidase (MPO) after trauma. (A) Time-course of myocardial MPO activity after trauma. **P < 0.01 vs. sham control, n = 8–10 mice/ group; (B) typical photographs for MPO immunostaining (20) by streptavidin-peroxidase conjugated method (SP) in myocardial tissue 12 h after trauma.
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Fig. 2. Effects of R15.7 on myocardial nitrotyrosine contents and myocardial apoptosis 12 h after trauma. (A) Myocardial nitrotyrosine contents were determined by ELISA method. (B) Myocardial apoptosis was determined by the terminal deoxyneucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Cardiac myocytes were identified by anti-a-actinin antibody (red), total nuclei were labeled with DAPI (blue) and apoptotic nuclei were detected by TUNEL staining (green). The results were expressed as an apoptosis index. **P < 0.01 vs. sham control, P < 0.01 vs. trauma, n = 8–9 mice/group. R15.7, a monoclonal antibody that blocks PMNs adhesion. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Vasorelaxation responses of isolated aortic rings 6 h after trauma to each concentration of acetylcholine (ACh, an endothelium-dependent vasodilator) or acidified NaNO2 (an endothelium-independent vasodilator). **P < 0.01 vs. sham control, n = 12–13 mice/group.
Fig. 4. Typical photographs for intercellular adhesion molecule-1(ICAM-1) immunostaining (20) by streptavidin-peroxidase conjugated method (SP) in myocardial tissue 6 h after trauma.
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dence indicates that protein nitration is not just a marker of disease, but rather is a critical post-translational modification and may play a significant pathogenic role in many cardiovascular diseases [24,25]. Many mechanisms of tyrosine nitration have been proposed, however, the two that are widely believed to exist in vivo involve the formation of ONOO or mediation via hemeperoxidase. For the former, the ONOO pathway involves the direct reaction of nitric oxide (NO) with superoxide. Under pathological conditions where iNOS is expressed, substantially high concentrations of NO and excessive generation of superoxide reacts to produce ONOO [26]. For the latter, tyrosine nitration is mediated by hemeperoxidase, most notably MPO. In the presence of hydrogen peroxide, MPO utilizes nitrite, a breakdown product of NO, to generate nitrogen dioxide, a strong nitrating molecule [27–29]. Our previous study indicated that tyrosine nitration was positively correlated with cardiomyocyte apoptosis in nonlethal traumatic mice. In the present study, treatment with R15.7 to inhibit PMNs adhesion significantly reduced myocardial nitrotyrosine level and decreased myocardial apoptosis. This result suggested that MPO increased downstream target protein nitration and then resulted in myocardial apoptosis in nonlethal trauma. Moreover, previous studies have demonstrated that endothelial dysfunction and upregulation of adhesion molecules are two major factors that facilitate PMNs accumulation. The inflammatory response is triggered by an early endothelial dysfunction characterized by a decreased release of NO from the endothelium. NO mediates a variety of cellular processes, including vasodilation, inhibition of platelet aggregation, and attenuation of PMNs adherence to the endothelium. Thus the loss of NO production promotes PMNs adherence, accumulation and activation. ICAM-1, as one of the most critical adhesion molecules, plays an important role in PMNs adherence to the endothelium. In the present study, nonlethal traumatic injury caused significant endothelial dysfunction and upregulation of adhesion molecules. This result revealed that endothelial dysfunction and upregulation of ICAM-1 might be the reason behind PMNs aggregation and increased MPO in mechanical trauma. But to confirm it in further studies we should treat mice with L-arginine and ICAM-1 inhibitor and then investigate the activity and release of MPO and protein nitration level. In summary, in the present study we demonstrated for the first time that MPO activity and release increased in nonlethal mechanical trauma, and increased MPO caused protein nitration, subsequent myocardial apoptosis and cardiac injury. Moreover, PMNs accumulation and MPO release may be relevant with endothelial dysfunction and upregulation of ICAM-1 in nonlethal trauma, but this requires further study to provide a more definitive answer. Though our study has some limitations, it strongly suggests that we can evaluate and predict the degree of mechanical trauma by detecting some valuable inflammatory factors, and we can reduce the posttrauma secondary cardiac injury by inhibition of PMNs adhesion and antinitration treatment. Acknowledgment This research is supported partially by Natural Scientific Foundation of China (30872680) to Feng Liang. References [1] R.M. Hardaway, Traumatic shock alias posttrauma critical illness, Am. Surg. 66 (2000) 284–290. [2] L.T. Wang, S.M. Cheng, L.W. Chang, M.Y. Liu, C.P. Wu, D.S. Hseih, Acute myocardial infarction caused by occult coronary intimal dissection after a heel stomp: a case report, J. Trauma 64 (2008) 824–826. [3] Y.W. Yoon, S. Park, S.H. Lee, M. Cho, B. Hong, D. Kim, H.M. Kwon, H.S. Kim, Posttraumatic myocardial infarction complicated with left ventricular aneurysm and pericardial effusion, J. Trauma 63 (2007) E73–E75.
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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Myeloperoxidase increased cardiomyocyte protein nitration in mice subjected to nonlethal mechanical trauma Zi Yan a,b,1, Feng Liang c,1, Li Guo b, Jin Wang a,b, Xiao-Liang Wang d, Xiao-Long Cheng e, Xin-Liang Ma d, Hui-Rong Liu f,* a
State Key Laboratory of Cellular Physiology, Shanxi Medical University, Taiyuan, Shanxi, PR China Department of Physiology, Shanxi Medical University, Taiyuan, Shanxi, PR China Cardiothoracic Surgery, Steel General Hospital of Taiyuan, Taiyuan, Shanxi, PR China d Department of Emergency Medicine, Thomas Jefferson University, 1020 Sansom Street, Philadelphia, PA 19107, Unites States e Department of Anatomy, Shanxi Medical University, Taiyuan, Shanxi, PR China f Department of Pathophysiology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, PR China b c
a r t i c l e
i n f o
Article history: Received 1 February 2010 Available online 12 February 2010 Keywords: Trauma Myocardial inflammation Apoptosis Endothelium 3-Nitrotyrosine
a b s t r a c t Nonlethal mechanical trauma causes cardiomyocyte apoptosis which contributes to posttraumatic cardiac dysfunction. Apoptosis is positively correlated with protein nitration in the traumatic heart. However, the mechanisms responsible for the cardiomyocyte protein nitration remain unclear. The present study was designed to identify whether myeloperoxidase may contribute to protein nitration in nonlethal mechanical trauma and subsequent cardiomyocyte apoptosis, and, if so, to determine the possible mechanisms responsible. We used Noble-Collip drum to make nonlethal traumatic mice models. Male adult C57B16/J mice were placed in the Noble-Collip drum and subjected to a total of 200 revolutions at a rate of 40 r/min. Then myeloperoxidase activity and release, protein nitration, cardiomyocyte apoptosis, endothelial function and intercellular adhesion molecule-1 expression were determined. Nonlethal mechanical trauma was characterized by the 100% survival rate during the first 24 h after trauma, the lack of circulatory shock and without direct heart injury. However, myeloperoxidase activity significantly increased 6 h after trauma, and reached a maximum level 12 h after trauma. Obviously, protein nitration and cardiomyocyte apoptosis increased 12 h after trauma and could be blocked by administration of R15.7, a monoclonal antibody that blocks polymorphonuclear neutrophils adhesion. Moreover, endothelial dysfunction and intercellular adhesion molecule-1 upregulation were observed in traumatic mice. Our present study demonstrated for the first time that myeloperoxidase caused protein nitration and cardiomyocyte apoptosis in nonlethal traumatic mice. Inhibition of polymorphonuclear neutrophils adhesion and antinitration treatments may be novel measures in reducing posttraumatic cardiomyocyte apoptosis and secondary heart injury. Ó 2010 Elsevier Inc. All rights reserved.
Introduction With the development of modern industry and transportation, the morbidity of mechanical trauma has gradually risen. Nowadays trauma has become the most common cause of death under the age of 45 years old [1]. Most studies confirm that mechanical trauma can cause direct heart damage, such as coronary artery dissection and cardiac contusion [2,3]. However, recently published clinical reports [4–7] have indicated that blunt chest trauma causes myocardial infarction even in the absence of direct cardiomyocyte injury. These results suggest that trauma induces not only * Corresponding author. Fax: +86 351 4135076. E-mail address: [email protected] (H.-R. Liu). 1 Zi Yan and Feng Liang contributed equally to this work. 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.02.049
primary heart injury but also secondary heart injury. Nevertheless, the mechanisms responsible for trauma-induced secondary heart injury still remain unknown. Our previous results revealed that mechanical trauma caused significant cardiomyocyte apoptosis which contributed to posttraumatic secondary cardiac dysfunction [8]. Moreover, we also proved that tyrosine nitration was positively correlated with cardiomyocyte apoptosis in nonlethal traumatic cardiomyocytes. Most studies [9,10] considered that there were mainly two mechanisms involved in tyrosine nitration, including the formation of peroxynitrite (ONOO ) and mediation via hemeperoxidase, most notably myeloperoxidase (MPO). In a recent study, we demonstrated that ONOO -mediated signaling pathway caused tyrosine nitration and subsequent cardiomyocyte apoptosis in nonlethal traumatic injury [11]. In addition, it is well known that traumatic
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injury elicits a systemic inflammatory response. As one of the most important inflammatory components, it remains unknown whether MPO increased and contributed to tyrosine nitration and subsequent cardiomyocyte apoptosis in nonlethal traumatic mice. Therefore, the aims of the present study were (1) to investigate whether MPO increased in nonlethal traumatic mice; and, if so, (2) to determine whether MPO may contribute to protein nitration induced by mechanical trauma; and (3) to identify the possible mechanisms. Materials and methods Animals. The experimental procedures were conducted in adherence to the ‘‘Guiding Principles in the Use and Care of Animals” published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996), the Guide for the Care and Use of Laboratory Animals protocol, published by the Ministry of the People’s Republic of China (issued at 3 June, 2004), and approved by the Institutional Animal Care and Use Committee of Shanxi Medical University. Nonlethal traumatic mice model. Nonlethal mechanical trauma was induced as previously described [8]. In brief, Male adult C57B16/J mice were anesthetized with pentobarbital sodium (40 mg/kg). Then the mice were placed in a Noble-Collip drum and subjected to a total of 200 revolutions at a rate of 40 r/min. Trauma mice were injured when the wheel was rotated, while sham trauma mice were taped on the inner wall of drum, so without traumatic injury. Immediately after trauma, a PE catheter filled with heparinized 0.9% NaCl solution was inserted into the right common carotid artery for recording mean arterial blood pressure (MABP) through a PowerLab data analysis system. Determination of myocardial MPO activity. As an index of polymorphonuclear neutrophils (PMNs) infiltration, MPO activity in cardiac tissue was assayed according to the method as described in our previous study [12]. In brief, cardiac tissue was homogenized in 50 mmol/L potassium phosphate buffer at pH 6 containing 0.5% hexadecyltrimethyl ammonium bromide (HTAB). The homogenates were centrifuged for 30 min at 12,500g at 4 °C. The supernatants were then collected and reacted with 0.167 mg/mL Odianisodine dihydrochloride and 0.0005% H2O2 in 50 mmol/L phosphate buffer at pH 6. The measurement was performed at 460 nm at 25 °C using a microplate reader (SpectraMAX Plus, Molecular Devices, Sunnyvale, CA) in duplicate. One unit of MPO activity was defined as that quantity of enzyme hydrolyzing 1 mmol of peroxide per minute at 25 °C. Quantitation of tissue nitrotyrosine content. Cardiac nitrotyrosine content, a footprint of in vivo protein nitration level, was determined using an enzyme linked immunosorbent assay (ELISA) method described in our recent publication [13]. In brief, the free wall of the left ventricle was separated and homogenized in icecold PBS. The homogenates were centrifuged for 10 min at 12,500g at 4 °C. The supernatants were collected and protein concentrations were determined by the BCA method. A nitrated protein solution was prepared for use as a standard. These standard samples, along with tissue samples from hearts, were applied to disposable sterile ELISA plates and incubated overnight with primary antibody. The secondary antibody was added, and the peroxidase reaction product was generated by using O-phenylenediamine dihydrochloride (OPD) solution. The optical density was measured at 460 nm with a microplate reader (BioTek Instruments, Inc. Winooski, VT). The amount of nitrotyrosine content in tissue samples was calculated using standard curves generated from nitrated BSA containing known amounts of nitrotyrosine and expressed as nanomoles per gram of protein.
Determination of myocardial apoptosis with the terminal deoxyneucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. To determine myocardial apoptosis in a quantitative manner, the hearts were perfused first with 0.9% NaCl for 5 min and then with 4% paraformaldehyde in PBS (pH 7.4) for 20 min. Four longitudinal sections from the free wall of the left ventricle were cut and further fixed in 4% paraformaldehyde in PBS for 24 h at room temperature. Fixed tissues were embedded in a paraffin block and two slides at 4- to 5-lm thickness were cut from each tissue block. Immunohistochemical procedures for detecting apoptotic cardiomyocytes were performed by using an apoptosis detection kit (Boehringer Mannheim, Ridgefield, CT) according to the manufacturer’s instructions. An additional staining was performed with monoclonal anti-a-sarcomeric actin. This staining enables the identification of myocyte and, therefore, a distinction between myocyte nuclei and nuclei of other cells in cardiac tissue. After being rinsed with PBS, slides were coverslipped with mounting medium containing DAPI to permit total nuclei counting. With the use of a 20 objective, the tissue slide was digitally photographed with a QICAM-Fast Digital Camera mounted onto an Olympus BX51 fluorescence microscope. Total nuclei (DAPI staining, blue) and TUNEL-positive nuclei (green) in each field were counted by IP Lab Imagine Analysis Software (Version 3.5, Scanalytics, Fairfax, VA) with a custom-made script (by Ken Anderson, Bio Vision Technologies, North Exton, PA). The index of apoptosis (number of TUNEL-positive nuclei/total number of nuclei 100%) was automatically calculated and exported to Microsoft Excel for further analysis. Results from different fields taken from the same animal were averaged and counted as one sample. Determination of endothelial function. Endothelial function was determined by comparing the vasorelaxation response to acetylcholine (ACh), an endothelium-dependent vasodilator, with that of acidified NaNO2, an endothelium-independent vasodilator, as described previously [13]. Briefly, aortic rings were mounted onto hooks, suspended in organ chambers filled with Krebs buffer and aerated with 95% O2 and 5% CO2 at 37 °C, and connected to force transducers (WPI, Sarasota, FL) to record changes via a Maclab data acquisition system. After equilibration for 60 min at a preload of 1 g, the rings were precontracted with norepinephrine (NE, 0.1 nM). Once a stable contraction was achieved, the rings were exposed to cumulative concentrations of ACh (10 9–10 5 M). After the cumulative response stabilized, the rings were washed and allowed to equilibrate to baseline. The procedure was then repeated with an endothelium-independent vasodilator (acidified NaNO2, 10 9–10 6 M) to determine smooth muscle function. Endothelial dysfunction was defined as a reduced vasorelaxation in response to ACh with a normal response to acidified NaNO2. Immunohistochemical detection of MPO and intercellular adhesion molecule-1 (ICAM-1). Immunostaining of MPO and ICAM-1 were determined using the method described in our recent publication [14]. Briefly, optimal cutting temperature compound-embedded tissues were cut into 6 mm thickness and stained with primary antibody (anti-MPO monoclonal antibody, Abcam; anti-ICAM-1 monoclonal antibody, BD Bio-sciences) and biotinylated secondary antibody. MPO was detected with alkaline phosphatase kit (VECTASTAINÒ ABC-AP kit with Vector Red as a substrate, Vector Laboratories) and ICAM-1 expression was detected with horseradishperoxidase kit (VECTASTAINÒ ABC kit, Vector Laboratories). Statistical analysis. All values in the text and figures are presented as means ± SEM. Time and group differences were determined by two-way ANOVA for repeated measures. For nonrepeated measures, data were subjected to ANOVA followed by the Scheffe’s correction for post hoc t-test comparison. Probabilities of P 6 0.05 were considered to be statistically significant.
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Results Nonlethal traumatic mice were established Nonlethal mechanical trauma by Noble-Collip drum rotation is characterized by the lack of circulatory shock (MABP > 75 mmHg within 24 h after trauma), no direct cardiac injury (i.e., cardiac contusion and pericardial bleeding), and a 100% 24 h survival rate [8]. Nonlethal traumatic injury caused increased MPO activity and release MPO, an enzyme occurring virtually exclusively in PMNs, was an index that has been shown to correlate closely with PMNs accumulation and infiltration in the heart. As summarized in Fig. 1A, MPO activity significantly increased 6 h after trauma, and reached a maximal level at 12 h after trauma. Moreover, compared with the sham trauma, MPO immunostaining was markedly increased in cardiac tissue from mice subjected to nonlethal traumatic injury (Fig. 1B). These results indicated for the first time that nonlethal trauma caused significant PMNs accumulation and increased MPO activity and release in cardiac tissue. MPO increased protein nitration that contributed to posttraumatic myocardial apoptosis To investigate whether increased MPO resulted in protein nitration and subsequent myocardial apoptosis, we observed the effect of R15.7 (a monoclonal antibody that blocks PMNs adhesion) treatment on myocardial nitrotyrosine level and apoptosis index. As illustrated in Fig. 2A 12 h after trauma, the tissue nitrotyrosine level was significantly increased. And as summarized in Fig. 2B, there were no TUNEL-positive nuclei detected in samples isolated from mice subjected to sham trauma. However, significant cardiomyocytes were labeled TUNEL-positive in samples from mice subjected to nonlethal traumatic injury. Treatment with R15.7 significantly reduced cardiac tissue nitrotyrosine level and decreased myocardial apoptosis. These results demonstrated that PMNs and MPO are critical contributors to posttrauma protein nitration and myocardial apoptosis. Mechanisms responsible for increased myocardial MPO activity and release To determine the mechanisms responsible for increased myocardial MPO activity and release in nonlethal traumatized mice, two additional studies were performed in which endothelial function and ICAM-1 expression were determined 6 h after trauma. As illus-
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trated in Fig. 3A, the dose–response curve to ACh, an endotheliumdependent vasodilator, was significantly shifted to the right in aortic segments isolated from traumatic animals. However, the dose–response curve to acidified NaNO2, an endothelium-independent vasodilator, was unaltered in these vascular segments (Fig. 3B). Moreover, ICAM-1 expression was markedly upregulated in myocardial tissue obtained from traumatized animals (Fig. 4). These results demonstrated that traumatic injury caused significant endothelial dysfunction and upregulation of adhesion molecules which might be the reason of PMNs accumulation and MPO release. Discussion In our present study we have made several novel observations. First, we have demonstrated that nonlethal mechanical trauma resulted in significantly increased MPO activity 6 h after trauma, and MPO release also markedly increased in myocardial tissue. Second, we have provided direct evidence that MPO increased posttraumatic protein nitration and subsequent myocardial apoptosis. Third, we have demonstrated that nonlethal mechanical trauma caused endothelial dysfunction and ICAM-1 upregulation which might be responsible for trauma-induced increased MPO activity and release. It is well known that PMNs play an important role in inflammatory response. A typical inflammatory reaction can result in PMNs adhesion, aggregation and extravasation. Activated PMNs have a considerable effect on the development and turnover of inflammatory response via the release of inflammatory factor, oxygen-derived free radicals and elastase. MPO, an enzyme occurring virtually exclusively in azurophil granules of PMNs in high concentrations, was released when PMNs were activated and was used to quantify the accumulation of PMNs in the injured tissues. MPO activity and immunostaining can be used to reflect the accumulation and localization of PMNs [15]. Released MPO can oxidize chloride to generate hypochlorous acid (HOCl) [16,17] and nitrate tyrosine to form nitrotyrosine [18,19] which are both responsible for tissue injury. In the present study, the result that MPO activity and release increased demonstrated that nonlethal mechanical trauma caused MPO release which might play an important role in inflammatory response induced by trauma. Protein nitration generally occurs in an organism that contributes to regulate protein metabolism, protein function and signal pathway process [20]. Increased 3-nitrotyrosinated proteins in the aging process also demonstrate that there is protein nitration under physiological conditions [21–23]. But more importantly, protein nitration (primarily including tyrosine nitration) became an injury mediator in many inflammatory diseases. Emerging evi-
Fig. 1. The activity and release of myeloperoxidase (MPO) after trauma. (A) Time-course of myocardial MPO activity after trauma. **P < 0.01 vs. sham control, n = 8–10 mice/ group; (B) typical photographs for MPO immunostaining (20) by streptavidin-peroxidase conjugated method (SP) in myocardial tissue 12 h after trauma.
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Fig. 2. Effects of R15.7 on myocardial nitrotyrosine contents and myocardial apoptosis 12 h after trauma. (A) Myocardial nitrotyrosine contents were determined by ELISA method. (B) Myocardial apoptosis was determined by the terminal deoxyneucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Cardiac myocytes were identified by anti-a-actinin antibody (red), total nuclei were labeled with DAPI (blue) and apoptotic nuclei were detected by TUNEL staining (green). The results were expressed as an apoptosis index. **P < 0.01 vs. sham control, P < 0.01 vs. trauma, n = 8–9 mice/group. R15.7, a monoclonal antibody that blocks PMNs adhesion. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Vasorelaxation responses of isolated aortic rings 6 h after trauma to each concentration of acetylcholine (ACh, an endothelium-dependent vasodilator) or acidified NaNO2 (an endothelium-independent vasodilator). **P < 0.01 vs. sham control, n = 12–13 mice/group.
Fig. 4. Typical photographs for intercellular adhesion molecule-1(ICAM-1) immunostaining (20) by streptavidin-peroxidase conjugated method (SP) in myocardial tissue 6 h after trauma.
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dence indicates that protein nitration is not just a marker of disease, but rather is a critical post-translational modification and may play a significant pathogenic role in many cardiovascular diseases [24,25]. Many mechanisms of tyrosine nitration have been proposed, however, the two that are widely believed to exist in vivo involve the formation of ONOO or mediation via hemeperoxidase. For the former, the ONOO pathway involves the direct reaction of nitric oxide (NO) with superoxide. Under pathological conditions where iNOS is expressed, substantially high concentrations of NO and excessive generation of superoxide reacts to produce ONOO [26]. For the latter, tyrosine nitration is mediated by hemeperoxidase, most notably MPO. In the presence of hydrogen peroxide, MPO utilizes nitrite, a breakdown product of NO, to generate nitrogen dioxide, a strong nitrating molecule [27–29]. Our previous study indicated that tyrosine nitration was positively correlated with cardiomyocyte apoptosis in nonlethal traumatic mice. In the present study, treatment with R15.7 to inhibit PMNs adhesion significantly reduced myocardial nitrotyrosine level and decreased myocardial apoptosis. This result suggested that MPO increased downstream target protein nitration and then resulted in myocardial apoptosis in nonlethal trauma. Moreover, previous studies have demonstrated that endothelial dysfunction and upregulation of adhesion molecules are two major factors that facilitate PMNs accumulation. The inflammatory response is triggered by an early endothelial dysfunction characterized by a decreased release of NO from the endothelium. NO mediates a variety of cellular processes, including vasodilation, inhibition of platelet aggregation, and attenuation of PMNs adherence to the endothelium. Thus the loss of NO production promotes PMNs adherence, accumulation and activation. ICAM-1, as one of the most critical adhesion molecules, plays an important role in PMNs adherence to the endothelium. In the present study, nonlethal traumatic injury caused significant endothelial dysfunction and upregulation of adhesion molecules. This result revealed that endothelial dysfunction and upregulation of ICAM-1 might be the reason behind PMNs aggregation and increased MPO in mechanical trauma. But to confirm it in further studies we should treat mice with L-arginine and ICAM-1 inhibitor and then investigate the activity and release of MPO and protein nitration level. In summary, in the present study we demonstrated for the first time that MPO activity and release increased in nonlethal mechanical trauma, and increased MPO caused protein nitration, subsequent myocardial apoptosis and cardiac injury. Moreover, PMNs accumulation and MPO release may be relevant with endothelial dysfunction and upregulation of ICAM-1 in nonlethal trauma, but this requires further study to provide a more definitive answer. Though our study has some limitations, it strongly suggests that we can evaluate and predict the degree of mechanical trauma by detecting some valuable inflammatory factors, and we can reduce the posttrauma secondary cardiac injury by inhibition of PMNs adhesion and antinitration treatment. Acknowledgment This research is supported partially by Natural Scientific Foundation of China (30872680) to Feng Liang. References [1] R.M. Hardaway, Traumatic shock alias posttrauma critical illness, Am. Surg. 66 (2000) 284–290. [2] L.T. Wang, S.M. Cheng, L.W. Chang, M.Y. Liu, C.P. Wu, D.S. Hseih, Acute myocardial infarction caused by occult coronary intimal dissection after a heel stomp: a case report, J. Trauma 64 (2008) 824–826. [3] Y.W. Yoon, S. Park, S.H. Lee, M. Cho, B. Hong, D. Kim, H.M. Kwon, H.S. Kim, Posttraumatic myocardial infarction complicated with left ventricular aneurysm and pericardial effusion, J. Trauma 63 (2007) E73–E75.
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