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Immunohistochemical evidence of cytochrome C oxidase subunit II involvement in pulmonary hypertension syndrome (PHS) in broilers
Research Note Immunohistochemical Evidence of Cytochrome C Oxidase Subunit II Involvement in Pulmonary Hypertension Syndrome (PHS) in Broilers1 M. Iqbal,2 J. D. Freiburger, G. F. Erf, and W. G. Bottje Department of Poultry Science, Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, Arkansas 72701 I and II. Relative areas of multiple microscope viewing fields (400×) per tissue section of COX I and II were quantified by counting immunopositive pixels on the digital images. Whereas the number of immunopositive pixels for COX II was higher in PHS birds compared to controls, there were no difference for COX I. The amount of COX II was positively correlated with the right to total ventricular weight ratio (RV:TV), suggesting that there may be increased expression of COX II associated with severity of pulmonary arterial hypertension. Thus, it is possible that COX II expression in PHS broiler may be involved in the pathogenesis of PHS.
(Key words: mitochondria, cytochrome c oxidase subunits (COX I and COX II), pulmonary hypertension syndrome, breast muscle, broiler) 2002 Poultry Science 81:1231–1235
INTRODUCTION The mitochondrial electron transport chain (ETC) consists of a series of complexes (I to V), made up of multiple protein subunits encoded by nuclear and mitochondrial (mt)DNA (Sue and Schon, 2000). Broilers with pulmonary hypertension syndrome (PHS), a costly metabolic disease, exhibit site-specific defects in the ETC that are associated with leakage of electrons at Complexes I and III in lung, heart, and breast muscle mitochondria (Iqbal et al., 2001a; Tang et al., 2002) and Complex II in liver mitochondria (Cawthon et al., 2001). These site-specific defects in the ETC observed in PHS mitochondria might be due to mutations in DNA, post-translational modification of proteins, oxidation of proteins (from increased reactive oxygen species; ROS) or a combination of one or more of these mechanisms. At the cellular level, 85 to 90% of oxygen is consumed by mitochondria while carrying out a vital role of adenosine triphosphate (ATP) production for the cell (Shigen-
2002 Poultry Science Association, Inc. Received for publication January 25, 2002. Accepted for publication April 10, 2002. 1 This research is published with support by the Director of the Agriculture Research Experiment Station, University of Arkansas, Fayetteville, AR 72701. 2 To whom correspondence should be addressed: [email protected].
aga et al., 1994). The mitochondrial respiratory chain uses electron flow from various energy substrates to create a proton gradient (proton motive force) that is used to drive ATP synthesis (Lehninger et al., 1992). Oxygen is the terminal electron acceptor in the chain of oxidation-reduction reactions that occurs in the inner mitochondrial membrane. Proton movement through the f1f0 ATP synthase (Complex V) effectively couples the energy releasing reactions of oxidation to the energy-storing reaction of phosphorylation (Lehninger et al., 1992). Complex IV (cytochrome c oxidase; COX) plays an important role in generating energy, because, as a terminal complex of the ETC, it catalyzes the oxidation of reduced cytochrome c by oxygen and conserves energy as an electrochemical proton gradient across the inner mitochondrial membrane (Dennis and Ferguson-Miller 1995; Johns 1996). Recent in vitro studies indicate that the activity of COX can be directly regulated by the presence of molecular oxygen (Chandel et al., 1996). Increased electron leakage from the Complex I, III, or II in PHS birds (Cawthon et al., 2001; Iqbal et al., 2001a; Tang et al., 2002) causes subsequent
Abbreviation Key: C = control; COX = cytochrome c oxidase; COX I = cytochrome c oxidase subunit I; COX II = cytochrome c oxidase subunit II; ETC = electron transport chain; mt = mitochondrial; PHS = pulmonary hypertension syndrome; ROS = reactive oxygen species; RV:TV = right to total ventricular weight ratio.
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ABSTRACT Defects and variation in the relative amount of protein subunits in the mitochondrial electron transport chain (ETC) have been hypothesized to be involved, in part, in the pathogenesis of pulmonary hypertension syndrome (PHS), a costly metabolic disease. Thus, the major objective of this study was to determine whether differences in relative amounts of cytochrome c oxidase subunit I and II (COX I and II) can be detected by immunohistochemistry and digital image analysis in muscle tissue of broilers with PHS compared to control broilers. Cross sections of the breast muscle (pectoralis major) were stained with monoclonal antibodies for COX
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reduction in proton motive force and, hence, less ATP synthesis because of functional damage to mitochondrial oxidative phosphorylation due to production of more ROS (Nakahara et al., 1998). It is believed that PHS-induced hypoxaemia further exacerbates the energy supply to tissues. The response of COX activity and its protein subunits to the oxidative insult is not fully understood, and a better understanding of the role COX in the pathogenesis of PHS is warranted. It was hypothesized that differences in expression of one or more COX protein subunits might be responsible, in part, for defective energy production in PHS broilers. Thus, the major objective of this study was to determine whether differences in the quantity of COX subunits I and II (COX I and II) can be detected by immunohistochemistry in muscle tissue of broilers with PHS compared to control broilers.
Birds and Management Male broiler chicks (Cobb 500)3 obtained from a local hatchery4 were placed in an environmental chamber (8 m2 floor space) on wood shaving litter. Birds were provided ad libitum access to water and to a diet (23.7% protein, 3,200 kcal ME). Chamber temperatures were 32 and 30 C, respectively, during Weeks 1 and 2, lowered to 15 C during Week 3, and maintained between 10 and 15 C for the rest of the study. Initially, 120 chicks were placed in the chamber, but this number was reduced to 100 by 21 d due to removal of those with deformed legs, those that failed to thrive, those lost to early chick mortality. The cool temperatures combined with high protein feed to support rapid growth have been shown to induce a high incidence of PHS (Wideman et al., 1995).
Sampling Procedure Birds were randomly selected based on overt PHS symptoms (e.g., systemic cyanosis of the comb, wattle, and skin or abdominal fluid accumulation) or clinically healthy appearance (i.e., no cyanosis) as previously described (Cawthon et al., 1999). The breast muscle for immunohistochemistry was collected from the birds used in a companion study on mitochondrial membrane potential and function. Only one bird was sampled per day due to the length of time required for mitochondrial isolation and other functional studies. The birds were killed with an overdose of sodium pentobarbital by i.v. injection into the wing vein. The right to total ventricular weight ratio
3
Cobb Vantress, Siloam Springs, AR. Randall Road, Tyson, Springdale AR. 5 VWR Scientific Products Corp., West Chester, PA. 6 Micron Labs, Walldorf, Germany. 7 Molecular Probes Inc., Eugene, OR. 8 Vectors Lab Inc., 30 Ingold Rd., Burlingame, CA. 9 Sigma Chemicals Co., St. Louis, MO. 10 Diagnostic Instrument Inc., Sterling Heights, MI. 4
Immunohistochemistry Pieces of cross sections of right (lower third) superficial breast muscle (pectoralis major) were embedded in freezing compound (OCT, Tissue Tek),5 snap-frozen in liquid nitrogen, and stored at −80 C until sectioning. Muscle cross sections were processed for immunohistochemistry using methods described by Taanman et al. (1996) with modifications (Erf et al., 1995). Serial cross sections (7 µm) of frozen muscle (−23 C) were cut in a cryostat (Micron, HM505E)6 mounted on poly-L-lysine coated slides, airdried, and fixed for 5 min in acetone. Sections were preincubated overnight in PBS (pH 7.2, 0.01 M) with 20% heat-inactivated horse serum (blocking buffer). Sections were incubated with primary monoclonal antibodies (0.38 µg/75 µL/section) for COX I (A-6403, IgG2a,κ) and COX II (A-6404, IgG2a,κ)7 containing PBS with 20% heat-inactivated horse serum for 2 h. Primary monoclonal antibodies were used at dilutions optimized in preliminary studies to provide consistent immunostaining. After 1 h of incubation with biotinylated horse antimouse immunoglobulins (secondary antibodies)8 diluted in PBS with 20% horse serum (1:100) and incubation with avidin-biotinylated horseradish peroxidase complex (1:100) (Vectastain ABC kit)8 for 30 min, sections were incubated with 3,3-diaminobenzidine (DAB)9 in PBS (1:100) for 20 min. Appropriate controls were included to determine the nonspecific binding of the primary and secondary antibodies. Mouse antibody (IgG2a,κ) with irrelevant specificity was used to test for nonspecific binding of primary antibody (isotype control). To determine the degree of nonspecific binding for secondary antibodies, muscle cross sections were incubated with blocking buffer instead of primary antibodies followed by incubation with biotinylated antibodies and Vectastain ABC reagents (Erf et al., 1995). All sections were counterstained with methyl-green stain for 30 min, and slides were mounted for subsequent examination and permanent record.
Evaluation of Tissue Sections Individual sections were examined by light microscopy (400×) to quantitate the immunopositive reaction. The relative numbers of immunohistochemically stained COX I or COX II protein subunits were determined by taking digital images of each section using Spot Advance Software (version 3.0.4).10 Briefly, for each muscle tissue section, five digital images (fields, each 8 × 10 cm) were obtained and subdivided into five subfields (10 × 12 mm or 120 mm2 each). These subfields was then expanded on a large-screen monitor to give a maximum pixel size of 5 mm2. A grid having squares equal to the size of pixels was applied on the image, which facilitated counting of
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MATERIALS AND METHODS
(RV:TV) was calculated from the right (RV) and total (TV) ventricular weights (Burton et al., 1968). Birds with an RV:TV ≥ 0.30 were classified as having PHS, whereas those with an RV:TV ratio ≤ 0.27 that did not have abdominal or pericardial fluid were classified as control birds.
RESEARCH NOTE
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immunopositive reaction considerably. Pixels corresponding to the positive reactions were counted in 25 subfields (3000 mm2) per section and expressed as total number of immunopositive pixels. The same person made all quantitative evaluations randomly and without knowledge of treatment group.
of pixels between the groups was analyzed using a ttest. The relationship between RV:TV and COX protein subunits was determined using regression analysis. Software JMP IN was used in the statistical analyses.11 A probability level of P ≤ 0.05 was considered statistically significant.
Statistical Analyses Data were presented as the means (total number of immunopositive pixels/section) ± SEM. The total number
11
JMP Start Statistics, SAS Institute Inc., Cary, NC.
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FIGURE 1. Immunohistochemistry of the superficial breast muscle (pectoralis major) of broilers (400×). Representative cross sections (7 µm) shown (A to F) are stained with isotype antibodies with irrelevant specificity (A, D, isotype control) to test the nonspecific binding of primary monoclonal antibodies for cytochrome c oxidase subunits I and II (COX I, II) or stained with antibodies for COX I (B, control; C, PHS) and COX II (E, control; F, PHS) in broilers. The binding of primary antibodies was detected using biotinylated anti-mouse immunoglobulins, avidinbiotinylated horseradish proxidase, and 3,3-diaminobenzidine (DAB). Tissues were counterstaind with methyl-green stain. Immunopositive reactions are scattered throughout the cross sections of muscle (speckles), and arrowheads indicate examples of representative reactions. PHS = pulmonary hypertension syndrome.
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RESULTS AND DISCUSSION
FIGURE 2. Total number of immunopositive pixels in cross section of breast muscle (pectoralis major) of broilers. Tissue section (7 µm) stained with monoclonal antibodies for cytochrome c oxidase subunits I (COX I) and II (COX II) in control (C, non-PHS) broilers and broilers with pulmonary hypertension syndrome (PHS). The binding of primary antibodies was detected using biotinylated anti-mouse immunoglobulins, avidin-biotinylated horseradish proxidase, and 3,3-diaminobenzidine (DAB). Tissues were counterstaind with methyl-green stain. All values are means (n = 5 to 6) ± SEM. a,bMeans without common letters differ significantly (P < 0.05).
there may be increased expression of COX II associated with severity of pulmonary arterial hypertension (Figure 3). This finding would suggest that there might be a differential expression or upregulation of proteins that occurs in response to the onset of PHS symptoms. Although a genetic component to the site-specific defects in the ETC and mitochondrial function has been established for broilers susceptible to PHS (Iqbal et al., 2001a,b), it is not clear whether the differential expression of COX subunits is primary (genetic) or secondary (PHS-induced hypoxemia). Further studies are needed to delineate the exact mechanism involved in the modulation of the COX subunits in PHS. In conclusion, COX II appears to be better correlated with RV:TV than that of COX I in PHS birds. Thus, it is
FIGURE 3. Relationship between cytochrome c oxidase subunit II (COX II) and right to total weight ventricular weight ratio (RV:TV) in control broiler and broiler with pulmonary hypertension syndrome (PHS). Control birds (n = 6) are represented by solid triangles (▲) and PHS birds (n = 5) by open triangles (䉭). The regression equation shown was significant (P < 0.05).
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PHS in broilers is a basic problem of oxygen supply and demand that develops in response to cardiopulmonary insufficiency (Wideman and Bottje, 1993; Wideman and Kirby, 1995a,b; Wideman et al., 1997). A body of evidence now indicates that less efficient energy production in PHS birds may be due to defects in the ETC in broilers with PHS (Cawthon et al., 1999, 2001; Iqbal et al., 2001a,b; Tang et al., 2002). Complexes I, III, IV, and V have one or more protein subunits encoded by mtDNA, with the remaining subunits encoded by nuclear DNA (Sue and Schon, 2000). There are 13 subunits in mammalian Complex IV, three of which are encoded by mtDNA (COX I, II, III) (Keightley et al., 1996). Although both proteins (mt and nuclear encoded) are susceptible to oxidative damage because of their proximity to the ETC, encoding of protein by mtDNA may be more susceptible to such damage (Fraga et al., 1990; Merriwether et al., 1991). Unlike nuclear DNA, mtDNA are not protected by histones or DNA-binding proteins and, hence, make them an easy target for mutational or damaging effects of the high-steady state concentrations of the ROS in the matrix of the mitochondria (Wei, 1998). Defects or variation in relative amounts of protein subunits in Complex IV have been hypothesized to be involved, in part, in the pathogenesis of PHS (Iqbal et al., 2001a; Tang et al., 2002). This study was carried out in an attempt to determine whether indeed there were differences in the detectable quantity of some of the mtDNA encoded protein subunits of the Complex IV using immunohistochemical techniques. One of the underlying intents of using breast muscle was to devise a simple and noninvasive assay for the selection of broilers susceptible to PHS. We developed a novel method (see details in materials and methods) whereby Complex IV protein subunits were quantified using digital imaging and counting of immunopositive pixels. There were no differences in body weights between control (2.45 ± 0.4 kg, n = 6) and PHS (2.16 ± 0.18 kg, n = 5) broilers. The RV:TV, a sensitive indicator of prior exposure of the heart to increased pulmonary arterial pressures (Burton et al., 1968), was higher in the PHS (0.36 ± 0.01) group compared to the control (0.19 ± 0.02) group, confirming the absence or presence of prolonged pulmonary arterial hypertension in the control and PHS groups, respectively. Selected photomicrographs of cross sections of breast muscle stained immunohistochemically with monoclonal antibodies for COX I and II in control and PHS broilers are given in Figure 1. Small dark speckles within the cross section of each myofibril represent the immunopositive reactions of COX I (Figure 1B,C) and II (Figure 1E,F), and no such reactions were observed in the isotype controls with irrelevant specificity (Figure 1A,D). The number of immunopositive pixels for COX II was higher in PHS birds compared to controls, but there were no differences in the numbers of immunopositive pixels for COX I (Figure 2). Cytochrome c oxidase subunit II (COX II) was positively correlated (P < 0.05) with RV:TV, suggesting that
RESEARCH NOTE
possible that COX II expression in PHS birds may be involved in the pathogenesis of the PHS. Future studies will be focused on other mitochondrial proteins and will exploit the methods used in this study in conjunction with other techniques (e.g., Western blots) to provide additional information on the molecular basis of PHS.
ACKNOWLEDGMENT The authors thank T. Bersi for technical assistance and H. Brandenburger for editing of the manuscript. This research was funded by a USDA-NRI grant (No. 99-2123) to W. Bottje.
REFERENCES
Johns, D. R. 1996. The other human genome; mitochondrial DNA and disease. Nature Med. 2:1065–1068. Keightley, J. A., D. C. Hoffbuhr, M. D. Burton, V. M. Salas, W. S. Johnston, A. M. Penn, N. R. M., Buist, and N. G. Kennaway. 1996. A microdeletion in cytochrome c oxidase (COX) subunit III and associated with COX deficiency and recurrent myoglobinuria. Nat. Genet. 12:410–416. Lehninger, A. L., D. L. Nelson, and M. M. Cox. 1992. Oxidative phosphorylation and photophosphorylation. Pages 542–592 in Principles of Biochemistry, 2nd ed., Worth Pub., New York. Merriwether, A. A., A. G. Clark, S. W. Ballinger, T. G. Schyrr, H. Soodyall, T. Jendkins, T. T. Sheny, and D. C. Wallace. 1991. The structure of human mitochondrial DNA variation. J. Mol. Evol. 33:543–555. Nakahara, H., T. Kanno, Y. Inal, K. Utsumi, M. Hiramatsu, A. Mori, and L. Packer. 1998. Mitochondrial dysfunction in the senescence accelerated mouse (SAM). Free Rad. Biol. Med. 24: 85–92. Shigenaga, M. K., T. M. Hagen, and B. N. Ames. 1994. Oxidative damage and mitochondrial decay in aging. PNAS. USA 91:10771–10778. Sue, C. M., and E. A. Schon. 2000. Mitochondrial chain diseases and mutations in nuclear DNA: A promising start. Brain Pathol. 10:442–450. Taanman, J. W., M. D. Burton, M. F. Marusich, N. G. Kennaway, and R. A. Capaldi. 1996. Subunit specific monoclonal antibodies show different steady-state levels of various cytochrome-c oxidase subunits in chronic progressive external ophthalmoplegia. Biochimica et Biophysica Acta 1315:199– 207. Tang, Z., M. Iqbal, D. Cawthon, and W. G. Bottje. 2002. Heart and muscle mitochondrial dysfunction in pulmonary hypertension syndrome in broilers (Gallus domesticus) Comp. Biochem. Physiol. (In press). Wei, Y. H. 1998. Oxidative stress and mitochondrial DNA mutations in human aging. Exp. Biol. & Med. 127:53–63. Wideman, R. F., Jr., and W. Bottje. 1993. Current understanding of the ascites syndrome and future directions. Pages 2–20 in Proceedings of the Nutritional and Technical Symposium. Novus International, St. Louis MO. Wideman, R. F. Jr., Y. K. Kirby, M. Ismail, W. G. Bottje, R. W. Moore, and R. C., Vardeman. 1995. Supplemental L-arginine attenuates pulmonary hypertension syndrome (ascites) in broilers. Poult. Sci. 74:323–330. Wideman, R. F., Jr., and Y. K. Kirby. 1995a. Evidence of a ventilation-perfusion mismatch during acute unilateral pulmonary artery occlusion in broilers. Poult. Sci. 74:1209–1217. Wideman, R. F., Jr., and Y. K. Kirby. 1995b. A pulmonary artery clamp model for inducing pulmonary hypertension syndrome (ascites) in broilers. Poult. Sci. 74:805–812. Wideman, R. F., Jr., Y. K. Kirby, R. L. Owen, and H. French. 1997. Chronic unilateral occlusion of an extrapulmonary primary bronchus induces pulmonary hypertension syndrome (ascites) in male and female broilers. Poult. Sci. 76:400–404.
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Burton, R. R., E. L. Besch, and A. H. Smith. 1968. Effect of chronic hypoxia on the pulmonary arterial blood pressure of the chicken. Am. J. Physiol. 214:1438–1442. Cawthon, D., K. Beers, and W. Bottje. 2001. Electron transport chain defect and inefficient respiration may both underlie pulmonary hypertension syndrome (PHS)-associated mitochondrial dysfunction in broilers. Poult. Sci. 80:474–484. Cawthon, D., R. McNew, K. Beers, and W. Bottje. 1999. Evidence of mitochondrial dysfunction in broilers with pulmonary hypertension syndrome (ascites): Effect of t-butyl hydroperoxide on hepatic mitochondrial function, glutathione, and related thiols. Poult. Sci. 78:114–124. Chandel, N. S., G. R. S. Budinger, and P. T. Schumacker. 1996. Molecular oxygen modulates cytochrome c oxidase function. J. Biol. Chem. 271:18672–18677. Dennis, R., and S. Ferguson-Miller. 1995. Structure of cytochrome c oxidase, energy generator of aerobic life. Science 269:1063–1064. Erf, G. F., A. V. Trejo-Skalli, and J. R. Smyth, Jr. 1995. T cell in regenerating feathers of Smyth line chickens with vitiligo. Clinical Immunol and Immunopathol. 76:120–126. Fraga, C. G., M. K., Shigenaga, J. W. Park, P. Deagan, and B. N. Ames. 1990. Oxidative damage to DNA during aging; 8hydroxyguanosine in rat organ DNA and urine. Proc. Natl. Acad. Sci. 87:55–63. Iqbal, M., D. Cawthon, R. F. Wideman, Jr., and W. G. Bottje. 2001a. Lung mitochondrial dysfunction in pulmonary hypertension syndrome. I. Site specific defects in electron transport chain. Poult. Sci. 80:485–495. Iqbal, M., D. Cawthon, R. F. Wideman, Jr., K. W. Beers, and W. G. Bottje. 2001b. Lung mitochondrial dysfunction in pulmonary hypertension syndrome. II. Oxidative stress and inability to improve function with repeated additions of ADP. Poult. Sci. 80:656–665.
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(Key words: mitochondria, cytochrome c oxidase subunits (COX I and COX II), pulmonary hypertension syndrome, breast muscle, broiler) 2002 Poultry Science 81:1231–1235
INTRODUCTION The mitochondrial electron transport chain (ETC) consists of a series of complexes (I to V), made up of multiple protein subunits encoded by nuclear and mitochondrial (mt)DNA (Sue and Schon, 2000). Broilers with pulmonary hypertension syndrome (PHS), a costly metabolic disease, exhibit site-specific defects in the ETC that are associated with leakage of electrons at Complexes I and III in lung, heart, and breast muscle mitochondria (Iqbal et al., 2001a; Tang et al., 2002) and Complex II in liver mitochondria (Cawthon et al., 2001). These site-specific defects in the ETC observed in PHS mitochondria might be due to mutations in DNA, post-translational modification of proteins, oxidation of proteins (from increased reactive oxygen species; ROS) or a combination of one or more of these mechanisms. At the cellular level, 85 to 90% of oxygen is consumed by mitochondria while carrying out a vital role of adenosine triphosphate (ATP) production for the cell (Shigen-
2002 Poultry Science Association, Inc. Received for publication January 25, 2002. Accepted for publication April 10, 2002. 1 This research is published with support by the Director of the Agriculture Research Experiment Station, University of Arkansas, Fayetteville, AR 72701. 2 To whom correspondence should be addressed: [email protected].
aga et al., 1994). The mitochondrial respiratory chain uses electron flow from various energy substrates to create a proton gradient (proton motive force) that is used to drive ATP synthesis (Lehninger et al., 1992). Oxygen is the terminal electron acceptor in the chain of oxidation-reduction reactions that occurs in the inner mitochondrial membrane. Proton movement through the f1f0 ATP synthase (Complex V) effectively couples the energy releasing reactions of oxidation to the energy-storing reaction of phosphorylation (Lehninger et al., 1992). Complex IV (cytochrome c oxidase; COX) plays an important role in generating energy, because, as a terminal complex of the ETC, it catalyzes the oxidation of reduced cytochrome c by oxygen and conserves energy as an electrochemical proton gradient across the inner mitochondrial membrane (Dennis and Ferguson-Miller 1995; Johns 1996). Recent in vitro studies indicate that the activity of COX can be directly regulated by the presence of molecular oxygen (Chandel et al., 1996). Increased electron leakage from the Complex I, III, or II in PHS birds (Cawthon et al., 2001; Iqbal et al., 2001a; Tang et al., 2002) causes subsequent
Abbreviation Key: C = control; COX = cytochrome c oxidase; COX I = cytochrome c oxidase subunit I; COX II = cytochrome c oxidase subunit II; ETC = electron transport chain; mt = mitochondrial; PHS = pulmonary hypertension syndrome; ROS = reactive oxygen species; RV:TV = right to total ventricular weight ratio.
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ABSTRACT Defects and variation in the relative amount of protein subunits in the mitochondrial electron transport chain (ETC) have been hypothesized to be involved, in part, in the pathogenesis of pulmonary hypertension syndrome (PHS), a costly metabolic disease. Thus, the major objective of this study was to determine whether differences in relative amounts of cytochrome c oxidase subunit I and II (COX I and II) can be detected by immunohistochemistry and digital image analysis in muscle tissue of broilers with PHS compared to control broilers. Cross sections of the breast muscle (pectoralis major) were stained with monoclonal antibodies for COX
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reduction in proton motive force and, hence, less ATP synthesis because of functional damage to mitochondrial oxidative phosphorylation due to production of more ROS (Nakahara et al., 1998). It is believed that PHS-induced hypoxaemia further exacerbates the energy supply to tissues. The response of COX activity and its protein subunits to the oxidative insult is not fully understood, and a better understanding of the role COX in the pathogenesis of PHS is warranted. It was hypothesized that differences in expression of one or more COX protein subunits might be responsible, in part, for defective energy production in PHS broilers. Thus, the major objective of this study was to determine whether differences in the quantity of COX subunits I and II (COX I and II) can be detected by immunohistochemistry in muscle tissue of broilers with PHS compared to control broilers.
Birds and Management Male broiler chicks (Cobb 500)3 obtained from a local hatchery4 were placed in an environmental chamber (8 m2 floor space) on wood shaving litter. Birds were provided ad libitum access to water and to a diet (23.7% protein, 3,200 kcal ME). Chamber temperatures were 32 and 30 C, respectively, during Weeks 1 and 2, lowered to 15 C during Week 3, and maintained between 10 and 15 C for the rest of the study. Initially, 120 chicks were placed in the chamber, but this number was reduced to 100 by 21 d due to removal of those with deformed legs, those that failed to thrive, those lost to early chick mortality. The cool temperatures combined with high protein feed to support rapid growth have been shown to induce a high incidence of PHS (Wideman et al., 1995).
Sampling Procedure Birds were randomly selected based on overt PHS symptoms (e.g., systemic cyanosis of the comb, wattle, and skin or abdominal fluid accumulation) or clinically healthy appearance (i.e., no cyanosis) as previously described (Cawthon et al., 1999). The breast muscle for immunohistochemistry was collected from the birds used in a companion study on mitochondrial membrane potential and function. Only one bird was sampled per day due to the length of time required for mitochondrial isolation and other functional studies. The birds were killed with an overdose of sodium pentobarbital by i.v. injection into the wing vein. The right to total ventricular weight ratio
3
Cobb Vantress, Siloam Springs, AR. Randall Road, Tyson, Springdale AR. 5 VWR Scientific Products Corp., West Chester, PA. 6 Micron Labs, Walldorf, Germany. 7 Molecular Probes Inc., Eugene, OR. 8 Vectors Lab Inc., 30 Ingold Rd., Burlingame, CA. 9 Sigma Chemicals Co., St. Louis, MO. 10 Diagnostic Instrument Inc., Sterling Heights, MI. 4
Immunohistochemistry Pieces of cross sections of right (lower third) superficial breast muscle (pectoralis major) were embedded in freezing compound (OCT, Tissue Tek),5 snap-frozen in liquid nitrogen, and stored at −80 C until sectioning. Muscle cross sections were processed for immunohistochemistry using methods described by Taanman et al. (1996) with modifications (Erf et al., 1995). Serial cross sections (7 µm) of frozen muscle (−23 C) were cut in a cryostat (Micron, HM505E)6 mounted on poly-L-lysine coated slides, airdried, and fixed for 5 min in acetone. Sections were preincubated overnight in PBS (pH 7.2, 0.01 M) with 20% heat-inactivated horse serum (blocking buffer). Sections were incubated with primary monoclonal antibodies (0.38 µg/75 µL/section) for COX I (A-6403, IgG2a,κ) and COX II (A-6404, IgG2a,κ)7 containing PBS with 20% heat-inactivated horse serum for 2 h. Primary monoclonal antibodies were used at dilutions optimized in preliminary studies to provide consistent immunostaining. After 1 h of incubation with biotinylated horse antimouse immunoglobulins (secondary antibodies)8 diluted in PBS with 20% horse serum (1:100) and incubation with avidin-biotinylated horseradish peroxidase complex (1:100) (Vectastain ABC kit)8 for 30 min, sections were incubated with 3,3-diaminobenzidine (DAB)9 in PBS (1:100) for 20 min. Appropriate controls were included to determine the nonspecific binding of the primary and secondary antibodies. Mouse antibody (IgG2a,κ) with irrelevant specificity was used to test for nonspecific binding of primary antibody (isotype control). To determine the degree of nonspecific binding for secondary antibodies, muscle cross sections were incubated with blocking buffer instead of primary antibodies followed by incubation with biotinylated antibodies and Vectastain ABC reagents (Erf et al., 1995). All sections were counterstained with methyl-green stain for 30 min, and slides were mounted for subsequent examination and permanent record.
Evaluation of Tissue Sections Individual sections were examined by light microscopy (400×) to quantitate the immunopositive reaction. The relative numbers of immunohistochemically stained COX I or COX II protein subunits were determined by taking digital images of each section using Spot Advance Software (version 3.0.4).10 Briefly, for each muscle tissue section, five digital images (fields, each 8 × 10 cm) were obtained and subdivided into five subfields (10 × 12 mm or 120 mm2 each). These subfields was then expanded on a large-screen monitor to give a maximum pixel size of 5 mm2. A grid having squares equal to the size of pixels was applied on the image, which facilitated counting of
Downloaded from http://ps.oxfordjournals.org/ at Ryerson University on June 17, 2015
MATERIALS AND METHODS
(RV:TV) was calculated from the right (RV) and total (TV) ventricular weights (Burton et al., 1968). Birds with an RV:TV ≥ 0.30 were classified as having PHS, whereas those with an RV:TV ratio ≤ 0.27 that did not have abdominal or pericardial fluid were classified as control birds.
RESEARCH NOTE
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immunopositive reaction considerably. Pixels corresponding to the positive reactions were counted in 25 subfields (3000 mm2) per section and expressed as total number of immunopositive pixels. The same person made all quantitative evaluations randomly and without knowledge of treatment group.
of pixels between the groups was analyzed using a ttest. The relationship between RV:TV and COX protein subunits was determined using regression analysis. Software JMP IN was used in the statistical analyses.11 A probability level of P ≤ 0.05 was considered statistically significant.
Statistical Analyses Data were presented as the means (total number of immunopositive pixels/section) ± SEM. The total number
11
JMP Start Statistics, SAS Institute Inc., Cary, NC.
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FIGURE 1. Immunohistochemistry of the superficial breast muscle (pectoralis major) of broilers (400×). Representative cross sections (7 µm) shown (A to F) are stained with isotype antibodies with irrelevant specificity (A, D, isotype control) to test the nonspecific binding of primary monoclonal antibodies for cytochrome c oxidase subunits I and II (COX I, II) or stained with antibodies for COX I (B, control; C, PHS) and COX II (E, control; F, PHS) in broilers. The binding of primary antibodies was detected using biotinylated anti-mouse immunoglobulins, avidinbiotinylated horseradish proxidase, and 3,3-diaminobenzidine (DAB). Tissues were counterstaind with methyl-green stain. Immunopositive reactions are scattered throughout the cross sections of muscle (speckles), and arrowheads indicate examples of representative reactions. PHS = pulmonary hypertension syndrome.
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IQBAL ET AL.
RESULTS AND DISCUSSION
FIGURE 2. Total number of immunopositive pixels in cross section of breast muscle (pectoralis major) of broilers. Tissue section (7 µm) stained with monoclonal antibodies for cytochrome c oxidase subunits I (COX I) and II (COX II) in control (C, non-PHS) broilers and broilers with pulmonary hypertension syndrome (PHS). The binding of primary antibodies was detected using biotinylated anti-mouse immunoglobulins, avidin-biotinylated horseradish proxidase, and 3,3-diaminobenzidine (DAB). Tissues were counterstaind with methyl-green stain. All values are means (n = 5 to 6) ± SEM. a,bMeans without common letters differ significantly (P < 0.05).
there may be increased expression of COX II associated with severity of pulmonary arterial hypertension (Figure 3). This finding would suggest that there might be a differential expression or upregulation of proteins that occurs in response to the onset of PHS symptoms. Although a genetic component to the site-specific defects in the ETC and mitochondrial function has been established for broilers susceptible to PHS (Iqbal et al., 2001a,b), it is not clear whether the differential expression of COX subunits is primary (genetic) or secondary (PHS-induced hypoxemia). Further studies are needed to delineate the exact mechanism involved in the modulation of the COX subunits in PHS. In conclusion, COX II appears to be better correlated with RV:TV than that of COX I in PHS birds. Thus, it is
FIGURE 3. Relationship between cytochrome c oxidase subunit II (COX II) and right to total weight ventricular weight ratio (RV:TV) in control broiler and broiler with pulmonary hypertension syndrome (PHS). Control birds (n = 6) are represented by solid triangles (▲) and PHS birds (n = 5) by open triangles (䉭). The regression equation shown was significant (P < 0.05).
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PHS in broilers is a basic problem of oxygen supply and demand that develops in response to cardiopulmonary insufficiency (Wideman and Bottje, 1993; Wideman and Kirby, 1995a,b; Wideman et al., 1997). A body of evidence now indicates that less efficient energy production in PHS birds may be due to defects in the ETC in broilers with PHS (Cawthon et al., 1999, 2001; Iqbal et al., 2001a,b; Tang et al., 2002). Complexes I, III, IV, and V have one or more protein subunits encoded by mtDNA, with the remaining subunits encoded by nuclear DNA (Sue and Schon, 2000). There are 13 subunits in mammalian Complex IV, three of which are encoded by mtDNA (COX I, II, III) (Keightley et al., 1996). Although both proteins (mt and nuclear encoded) are susceptible to oxidative damage because of their proximity to the ETC, encoding of protein by mtDNA may be more susceptible to such damage (Fraga et al., 1990; Merriwether et al., 1991). Unlike nuclear DNA, mtDNA are not protected by histones or DNA-binding proteins and, hence, make them an easy target for mutational or damaging effects of the high-steady state concentrations of the ROS in the matrix of the mitochondria (Wei, 1998). Defects or variation in relative amounts of protein subunits in Complex IV have been hypothesized to be involved, in part, in the pathogenesis of PHS (Iqbal et al., 2001a; Tang et al., 2002). This study was carried out in an attempt to determine whether indeed there were differences in the detectable quantity of some of the mtDNA encoded protein subunits of the Complex IV using immunohistochemical techniques. One of the underlying intents of using breast muscle was to devise a simple and noninvasive assay for the selection of broilers susceptible to PHS. We developed a novel method (see details in materials and methods) whereby Complex IV protein subunits were quantified using digital imaging and counting of immunopositive pixels. There were no differences in body weights between control (2.45 ± 0.4 kg, n = 6) and PHS (2.16 ± 0.18 kg, n = 5) broilers. The RV:TV, a sensitive indicator of prior exposure of the heart to increased pulmonary arterial pressures (Burton et al., 1968), was higher in the PHS (0.36 ± 0.01) group compared to the control (0.19 ± 0.02) group, confirming the absence or presence of prolonged pulmonary arterial hypertension in the control and PHS groups, respectively. Selected photomicrographs of cross sections of breast muscle stained immunohistochemically with monoclonal antibodies for COX I and II in control and PHS broilers are given in Figure 1. Small dark speckles within the cross section of each myofibril represent the immunopositive reactions of COX I (Figure 1B,C) and II (Figure 1E,F), and no such reactions were observed in the isotype controls with irrelevant specificity (Figure 1A,D). The number of immunopositive pixels for COX II was higher in PHS birds compared to controls, but there were no differences in the numbers of immunopositive pixels for COX I (Figure 2). Cytochrome c oxidase subunit II (COX II) was positively correlated (P < 0.05) with RV:TV, suggesting that
RESEARCH NOTE
possible that COX II expression in PHS birds may be involved in the pathogenesis of the PHS. Future studies will be focused on other mitochondrial proteins and will exploit the methods used in this study in conjunction with other techniques (e.g., Western blots) to provide additional information on the molecular basis of PHS.
ACKNOWLEDGMENT The authors thank T. Bersi for technical assistance and H. Brandenburger for editing of the manuscript. This research was funded by a USDA-NRI grant (No. 99-2123) to W. Bottje.
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