Differential expression of mitochondrial electron transport chain ...

Differential Expression of Mitochondrial Electron Transport Chain Proteins in Cardiac Tissues of Broilers from Pulmonary Hypertension SyndromeResistant and -Susceptible Lines1 C. R. Cisar,* J. M. Balog,*,2 N. B. Anthony,† M. Iqbal,† W. G. Bottje,† and A. M. Donoghue* *Poultry Production and Product Safety Research Unit, Agricultural Research Service, USDA, Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, Arkansas, 72701; and †Department of Poultry Science, Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, Arkansas 72701 ABSTRACT Pulmonary hypertension syndrome (PHS) is a metabolic disease associated with the rapid growth rate of modern broilers. Broilers susceptible to PHS experience sustained elevation of pulmonary arterial pressure leading to right ventricular hypertrophy and ultimately heart failure. Previous studies have shown that mitochondrial function is defective in broilers with PHS; they use oxygen less efficiently than broilers without PHS. In this study mitochondrial electron transport chain (ETC) protein levels were compared in cardiac tissues from PHS resistant and susceptible line broilers using quantitative immunoblots. Seven of 9 anti-mammalian mitochondrial ETC protein antibodies tested exhibited cross-species reactivity. Six ETC proteins were differentially expressed in the right ventricles of broilers raised under simulated high altitude conditions (2,900 m above sea level). Four

ETC proteins were present at higher levels in resistant line birds without PHS than in resistant line birds with PHS or in susceptible line birds with or without PHS. One ETC protein was present at higher levels in broilers without PHS than in broilers with PHS in both lines, and one ETC protein was present at lower levels in susceptible line birds without PHS than in susceptible line birds with PHS or in resistant line birds with or without PHS. Interestingly, differential expression of mitochondrial ETC proteins was not observed in the right ventricles of broilers raised at local altitude (390 m above sea level) nor was it observed in the left ventricles of broilers exposed to simulated high altitude. These results suggest that higher levels of mitochondrial ETC proteins in right ventricle cardiac muscle may be correlated with resistance to PHS in broilers.

(Key words: ascites syndrome, cardiac muscle, mitochondrial electron transport chain, protein expression, pulmonary hypertension syndrome) 2004 Poultry Science 83:1420–1426

INTRODUCTION Pulmonary hypertension syndrome (PHS), also known as ascites syndrome, is a metabolic disease associated with the enormous oxygen demands of rapidly growing tissues in modern poultry (Julian, 1993, 2000; Wideman, 2001). It is characterized by chronically elevated pulmonary blood pressure, right ventricular hypertrophy, systemic hypoxia, and, ultimately, congestive heart failure. Several studies have shown that mitochondrial function is affected in broilers with PHS (Cawthon et al., 1999, 2001; Iqbal et al., 2001a,b; Tang et al., 2002). In aerobic

2004 Poultry Science Association, Inc. Received for publication March 2, 2004. Accepted for publication April 29, 2004. 1 Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable. 2 To whom correspondence should be addressed: [email protected]

organisms adenosine triphosphate (ATP) is synthesized in the mitochondrion in a process known as oxidative phosphorylation. During oxidative phosphorylation electrons are transferred through a series of electron carriers in the electron transport chain (ETC) to oxygen. The ETC consists of 5 multiple subunit, membrane-bound protein complexes: NADH-ubiquinone oxidoreductase (complex I), succinate-ubiquinone oxidoreductase (complex II), ubiquinone-cytochrome c (complex III), cytochrome c oxidase (complex IV), and ATP synthase (complex V). Broilers with PHS exhibit site-specific defects at complexes I and III in lung, heart, and breast muscle mitochondria (Iqbal et al., 2001a; Tang et al., 2002) and at complex II in liver mitochondria (Cawthon et al., 2001). Furthermore, there is immunohistochemical evidence

Abbreviation Key: ETC = electron transport chain; NO = nitric oxide; PHS = pulmonary hypertension syndrome; ROS = reactive oxygen species; RV:TV = right ventricle to total ventricle weight ratio.

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MITOCHONDRIAL PROTEIN EXPRESSION

that cytochrome c oxidase subunit II levels are elevated in breast muscle of broilers with PHS (Iqbal et al., 2002). It has been reported previously that heritability for susceptibility to PHS is moderate to high in broiler breeder males (Lubritz et al., 1995). Several PHS-resistant and -susceptible broiler lines have been created through divergent genetic selection (Shlosberg et al., 1996; Wideman and French, 1999, 2000; Anthony et al., 2001; Anthony and Balog, 2003; Balog, 2003; Balog et al., 2003). Response to selection has been rapid in these experiments with significant differences observed between PHS-resistant and -susceptible lines usually after only 1 or 2 generations. These data indicate that a small number of major genes are probably involved in resistance to PHS in broilers. The objective of the present study was to examine the levels of mitochondrial ETC proteins in PHS-resistant and -susceptible line broilers and determine if mitochondrial protein expression is different in the genetically selected lines.

MATERIALS AND METHODS Broiler Lines, Induction of PHS, and Collection of Tissues The PHS-resistant and -susceptible broiler lines have been described previously (Anthony et al., 2001; Anthony and Balog, 2003; Balog, 2003; Balog et al., 2003). Briefly, sire-family selection for resistance and susceptibility to PHS was applied to a commercial elite line. For each generation, 2 hatches of progeny were reared for 6 wk in a hypobaric chamber under simulated high altitude conditions (2,900 m above sea level) to induce PHS (Odom et al., 1992; Balog et al., 2000). Ascites mortality data were then used to select the most resistant and susceptible sire families for reproduction of the lines. Broilers used in these experiments were from the sixth and seventh generations. The PHS-resistant line exhibited 29.0 ± 5.3% ascites mortality, and the PHS-susceptible line exhibited 80.6 ± 8.9% ascites mortality under simulated high altitude conditions (2,900 m above sea level). For this study, chicks from the PHS-resistant and -susceptible broiler lines were placed at 1 d of age either in a hypobaric chamber at a simulated altitude of 2,900 m above sea level or in a matching chamber at the local altitude of 390 m above sea level (Balog et al., 2000). The broilers were housed in stainless-steel battery units equipped with nipple waterers and fed ad libitum. Heart tissues were harvested from birds between 32 and 42 d of age that exhibited symptoms of PHS (enlarged abdomen due to accumulation of ascitic fluid, cyanosis, and

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Pierce, Rockford, IL. Sigma, St. Louis, MO. BioRad, Hercules, CA. 6 Molecular Probes, Inc., Eugene, OR. 7 Santa Cruz Biotechnology, Inc., Santa Cruz, CA. 8 Fuji Photo Film Co., Ltd., Tokyo, Japan. 9 Invitrogen, Carlsbad, CA. 4 5

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listlessness) and from the remaining birds at the end of 6 wk. Hearts were divided into right ventricle and left ventricle plus septum as they were removed. All tissues were flash frozen in liquid nitrogen and stored at −80°C until use. Right ventricle weight to total ventricle weight ratios (RV:TV) are indicative of the severity of pulmonary hypertension (Burton et al., 1968) and were used in conjunction with physical symptoms to classify birds as with or without PHS. Broilers with PHS had RV:TV ≥ 0.429 and exhibited symptoms of PHS (ascitic fluid, cyanosis, and listlessness). Broilers without PHS had RV:TV ≤ 0.321 and were asymptomatic. Each group of birds compared was matched for sex and, as closely as possible, for age. Descriptions of the PHS-resistant and -susceptible line birds and tissues used in this study are given in Table 1.

Preparation of Protein Samples and Immunoblot Analysis Total protein was extracted from cardiac muscle using T-Per Tissue Protein Extraction Reagent3 with protease inhibitors (P8340)4 following the manufacturers’ instructions. Protein levels in the samples were determined using the Coomassie Plus Protein Assay Reagent Kit.3 Protein samples were separated on 10 to 20% gradient polyacrylamide gels (Ready Gel Tris-HCl)5 and transferred to PVDF membranes5 following the manufacturer’s recommended conditions. Immunoblot hybridizations were performed using standard methods (Ausubel et al., 1999). Primary antibodies were diluted to 0.5 or 1.0 µg/ mL, and secondary antibody was diluted to 0.06 ng/mL in 3% nonfat dry milk, 20mM Tris-HCl, pH 7.5, 500 mM NaCl, and 0.05% Tween-20 buffer. Anti-mammalian ETC protein antibodies were purchased from Molecular Probes, Inc.6 (Table 2) and HRP-conjugated anti-mouse IgG antibody was purchased from Santa Cruz Biotechnology, Inc. (sc-2314).7 Antibodies were detected by chemiluminescence (SuperSignal West Dura Extended Duration Substrate)3 using an LAS-1000plus Luminescent Image Analyzer.8 Blots contained a maximum of 12 protein samples. Initially, we had planned to use a constitutively expressed protein such as glyceraldehyde phosphate dehydrogenase or actin as an internal control on the immunoblots. However, analysis of the data revealed that glyceraldehyde phosphate dehydrogenase levels were 2fold higher in the right ventricles of broilers with PHS than in those without PHS, and the anti-mammalian actin antibody bound poorly to the chicken protein samples (data not shown). Therefore, protein levels on immunoblots were normalized using MagicMark Western protein standards.9 Occasionally, the protein standards on a blot failed, and in those instances protein levels were compared between samples on the same blot only (i.e., mitochondrial ETC protein levels in right ventricle at local altitude and in left ventricle tissues). Data between blots could not be normalized and compared for these 2 groups.

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CISAR ET AL. TABLE 1. Treatments, lines, tissues, and disease states of broilers used in this study Altitude1 390 m 2,900 m

Line2

Birds (n)

Resistant Susceptible Resistant Resistant Susceptible Susceptible

6 6 6 6 6 6

Tissue3 Right Right Right Right Right Right

ventricle ventricle and left ventricle6 and left ventricle6 ventricle ventricle

RV:TV4

PHS5

± ± ± ± ± ±

− − − + − +

0.222 0.264 0.220 0.478 0.246 0.490

0.024 0.033 0.008 0.041 0.012 0.036

1 Birds were raised at local altitude (390 m above sea level) or in a hypobaric chamber simulating an altitude of 2,900 m above sea level. 2 Broiler lines were pulmonary hypertension syndrome (PHS)-resistant and PHS-susceptible. 3 Two types of cardiac muscle tissue were used in this study: right ventricle and left ventricle plus septum. 4 RV:TV = right ventricle to total ventricle weight ratio. Data are means ± SE. 5 Birds were classified as normal (−) if they were asymptomatic and had an RV:TV ≤ 0.321. Birds were classified as having PHS (+) if they exhibited symptoms and had an RV:TV ≥ 0.429. 6 Expression patterns of 3 mitochondrial electron transport chain proteins (20-kDa subunit of complex I and subunits I and Vb of complex IV) in right ventricle and left ventricle plus septum tissues were compared in PHS-resistant line broilers with and without PHS.

The ETC proteins detected on the membranes were quantitated using the computer program Image Gauge.8 Data are presented as means ± SE. Data obtained from the local altitude and left ventricle studies were analyzed by one-way ANOVA and by using Student’s t-test. Experimental data obtained from the hypobaric chamber experiments were analyzed by two-way ANOVA and using Student’s t-test and LSD.10 A probability level of P ≤ 0.05 was considered statistically significant.

RESULTS Nine anti-mammalian mitochondrial ETC protein antibodies were tested for cross-species reactivity with chicken cardiac tissue homogenates. Seven of the antibodies bound to chicken proteins of the appropriate molecular weight (Table 2). In initial experiments, ETC protein levels in right ventricle cardiac muscle of broilers from PHS-resistant and PHS-susceptible lines raised at local altitude were compared. These birds did not exhibit symptoms of PHS, and their RV:TV were ≤ 0.321 (Table 1). No differences in mitochondrial ETC protein expression were observed between the asymptomatic resistant and susceptible line broilers (data not shown). In subsequent experiments, mitochondrial ETC protein expression was compared in right ventricle cardiac tissues from PHS-resistant and -susceptible line broilers with and without PHS. While approximately 15% of PHS-susceptible line broilers developed PHS at local altitude (390 m above sea level), fewer than 2% of PHS-resistant line birds developed PHS under the same conditions (data not shown). Therefore, a hypobaric chamber was used in subsequent experiments to provide the required number of ascitic birds for analysis. One mitochondrial ETC protein, the 70-kDa subunit of complex II, was present at the same level in the right ventricles of all 4 groups: resistant line broilers with and

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SAS Institute Inc., Cary, NC.

without PHS and susceptible line broilers with and without PHS (Figure 1). Expression of the 30-kDa subunit of complex II, however, was higher in broilers without PHS than in broilers with PHS in both lines (Figure 1). Furthermore, 4 mitochondrial ETC proteins (20-kDa subunit of complex I and subunits I, Vb, and VIb of complex IV) were present at higher levels in resistant line broilers without PHS compared to resistant line broilers with PHS and susceptible line broilers with or without PHS (Figure 1). In contrast, subunit IV of complex IV was present at lower levels in susceptible line broilers without PHS than in susceptible line broilers with PHS or resistant line broilers with or without PHS (Figure 1). Finally, the expression patterns of the 20-kDa subunit of complex I and subunits I and Vb of complex IV were examined in left ventricle plus septum cardiac tissues. Left ventricle tissues were obtained from the same resistant line broilers with and without PHS that were used in the examination of ETC protein expression in right ventricle tissues (Table 1). All 3 proteins were present at the same levels in the left ventricle tissues of resistant line broilers with or without PHS (data not shown). However, in right ventricle tissues, all 3 proteins were present at higher levels in PHS resistant line broilers without PHS (Figure 1). These results show that mitochondrial protein expression patterns in right and left ventricle cardiac tissues were dissimilar.

DISCUSSION Previous studies have shown that mitochondrial function is defective in a variety of tissues in broilers with PHS (Cawthon et al., 1999, 2001; Iqbal et al., 2001a,b; Tang et al., 2002). Furthermore, it has been shown by immunohistochemistry that complex IV subunit II levels are increased in breast muscle of broilers with PHS (Iqbal et al., 2002). The objective of the present study was to compare mitochondrial ETC protein levels in cardiac tissues of birds from PHS-resistant and -susceptible broiler lines.


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FIGURE 1. Expression of mitochondrial electron transport chain proteins, 20-kDa subunit of complex I (CI, 20 kDa), 30- and 70-kDa subunits of complex II (CII, 30 kDa and CII, 70 kDa), subunits I, IV, Vb, and VIb of complex IV (CIV, I; CIV, IV; CIV, Vb; CIV, VIb, respectively), in right ventricle cardiac muscle of broilers from pulmonary hypertension syndrome (PHS)-resistant and -susceptible lines. Broilers were exposed to hypobaric conditions for 6 wk. Four groups of broilers were compared: PHS-resistant line broilers with and without PHS (R +PHS and R −PHS, respectively) and PHS-susceptible line broilers with and without PHS (S +PHS and S −PHS, respectively). Data were obtained from immunoblots. Chemiluminescent signal was detected using a CCD camera and quantitated using the software Image Gauge. Signal intensity is expressed in arbitrary units (AU). Data are means ± SE (n = 6). a–cFor each protein, values with different letters differ significantly (P ≤ 0.05).

Mitochondrial ETC protein expression was first compared in asymptomatic, low RV:TV resistant and susceptible line broilers raised under conditions that do not induce PHS (i.e., local altitude). Under these conditions mitochondrial ETC protein levels in right ventricle cardiac muscle were not significantly different between the 2 lines. This finding indicated that genetic resistance to PHS was not correlated with ETC protein expression under conditions that do not induce PHS. In subsequent experiments broilers were raised in a hypobaric chamber to induce PHS. These experiments revealed differences in ETC protein levels related to genetic background and disease state. Four mitochondrial ETC proteins (complex I, 20-kDa subunit and complex IV, subunits I, Vb, and VIb) were present at higher levels in right ventricle cardiac muscle of resistant line birds without PHS. Furthermore, the 30-kDa subunit of complex II was present at higher levels in broilers without PHS in both lines. The 70-kDa subunit of complex II was present at a similar level in all broilers examined, and subunit IV of complex IV was present at lower levels in PHS-susceptible line broilers without PHS. These data clearly show that the levels of several mitochondrial ETC proteins (5 of 7 proteins examined) were elevated in right ventricle cardiac muscle of PHS-resistant line broilers that did not develop PHS under conditions known to induce PHS.

Interestingly, the 20-kDa subunit of complex I and subunits I and Vb of complex IV showed different patterns of expression in left ventricle plus septum and right ventricle cardiac tissues. There were no differences in the levels of these proteins in the left ventricles of resistant line broilers with or without PHS. However, these same proteins were shown to be significantly elevated in the right ventricles of resistant line birds without PHS. In a study on development of cardiac hypertrophy in rats, Wiesner et al. (1994) showed that cytochrome c oxidase (complex IV) activity and the mRNA levels of several complex IV subunit genes remained unchanged in left ventricle cardiac muscle over several days. Furthermore, cardiac tissue subgroup-specific gene expression (right ventricle vs. left ventricle vs. atria) has also been observed at the mRNA level in rats placed in a hypobaric chamber (Deindl et al., 2003). Our study confirms cardiac tissue subgroup-specific expression (mitochondrial ETC proteins in right ventricle vs. left ventricle) at the protein level in broilers under hypoxic conditions. In studies by Wiesner et al. (1994), Vijayasarathy et al. (2003), and Lehman et al. (2000) it has been shown that expression of nuclear-encoded and mitochondrial-encoded subunits of complex IV are coordinately regulated. In complex IV subunits I to III are encoded in the mitochondrial genome, and all other subunits, including IV, Vb, and VIb, are encoded in the nuclear genome (Capaldi,

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CISAR ET AL. TABLE 2. Cross-species reactivity of anti-mammalian mitochondrial electron transport chain protein antibodies with chicken proteins and mitochondrial protein gene locations Complex I II II III IV IV IV IV IV

Complex name

Subunit name

Gene encoded in1

Catalog number2

Cross-species reactivity3

NADH-ubiquinone oxidoreductase Succinate-ubiquinone oxidoreductase Succinate-ubiquinone oxidoreductase Ubiquinone-cytochrome c Cytochrome c oxidase Cytochrome c oxidase Cytochrome c oxidase Cytochrome c oxidase Cytochrome c oxidase

20 kDa 70 kDa 30 kDa Core 2 I II IV Vb VIb

nDNA nDNA nDNA nDNA mtDNA mtDNA nDNA nDNA nDNA

A-21360 A-11142 A-21345 A-11143 A-6403 A-6404 A-21348 A-21349 A-21366

+ + + − + − + + +

1 Mitochondrial protein genes are encoded in the nuclear genome (nDNA) or in the mitochondrial genome (mtDNA). 2 All antibodies were monoclonal and purchased from Molecular Probes, Inc., Eugene, OR. 3 A + indicates that the anti-mammalian antibody cross-reacts with the chicken protein on an immunoblot. A − indicates no cross-species reactivity.

1990). Coordinated regulation of gene expression between the nucleus and the mitochondrion was also observed in these experiments. The genes for the 20-kDa subunit of complex I and subunits I, Vb, and VIb of complex IV are located in the nuclear and mitochondrial genomes (Table 2); all 4 of these proteins were present at higher levels in PHS-resistant line broilers without PHS. The pattern of expression described in this study for subunit I of complex IV differs from the immunohistochemistry study performed by Iqbal et al. (2002). In that study no correlation was found between subunit I levels and disease state. The difference in results between these 2 studies may be due to the different tissues used in the analyses. Iqbal et al. used breast skeletal muscle in their study, whereas our data were obtained from right ventricle cardiac tissue. There are many examples of tissue specific expression of mitochondrial proteins (Kunz, 2003) including our own data comparing ETC protein expression in right and left ventricle cardiac tissues. Alternatively, differences in the abilities of antibodies to detect native (immunohistochemistry) and denatured (immunoblot) proteins may be significant. For example, the antimammalian complex IV subunit II antibody readily bound to the chicken protein in the immunohistochemistry study by Iqbal et al. (2002) but did not bind the chicken protein on an immunoblot under several different sets of conditions in this study. Previous studies have indicated that a small number of genes contribute significantly to resistance to PHS in broilers (Shlosberg et al., 1996; Wideman and French, 1999, 2000; Anthony et al., 2001; Anthony and Balog, 2003; Balog, 2003; Balog et al., 2003). One or more of these genes may be responsible for the correlation between elevated levels of several mitochondrial ETC proteins in right ventricle cardiac muscle under hypobaric conditions and resistance to PHS. Broilers in the susceptible line that fail to develop PHS under hypobaric conditions do not show elevated levels of ETC proteins. It might be argued that this runs contrary to the proposed connection between resistance and elevated ETC levels. However, hypobaric conditions do not induce PHS in all broilers, rather the

incidence of PHS is increased. Hence the broilers in the susceptible line may fail to develop PHS not because of the residual presence of resistance genes in the line but rather because of as yet unidentified environmental or genetic factors that affect initiation or progression of the disease. For example, the autosomal dominant mutations associated with the analogous human disease, familial primary pulmonary hypertension, are well known for incomplete penetrance within families (Loyd and Newman, 1997). If the correlation between elevated ETC protein levels and lack of development of PHS in broilers from the resistant line represents a basis for resistance (rather than fixation of alleles in the selected lines) then this would suggest that a gene involved in upstream regulation of mitochondrial protein expression may be at least partly responsible for resistance to PHS. Recent findings on the role of nitric oxide (NO) in regulation of mitochondrial protein expression and in PHS and on the role of reactive oxygen species (ROS) in PHS and in other model systems lead us to suggest that the upstream gene associated with resistance to PHS in broilers may be related to NO signal transduction pathways, ROS activity, or possibly both. The NO and ROS pathways may be linked as ROS affect NO levels in tissues (Linke et al., 2003). It has been suggested previously that ROS may be linked to susceptibility to PHS in broilers (Cawthon et al., 2001; Iqbal et al., 2001b; Tang et al., 2002). There is no doubt that mitochondrial function is defective in broilers with PHS and that these defects lead to increased production of ROS. It is not clear, however, if the increased levels of ROS are a secondary effect of development of PHS or are associated with genetic susceptibility. There is also substantial evidence that NO, a vasodilator and cell signaling molecule, is an important factor in PHS in broilers (Wideman et al., 1995; Martinez-Lemus et al., 1999; Wang et al., 2002; de Sandino and Hernandez, 2003). Interestingly, it was recently shown that elevated levels of NO stimulate mitochondrial protein synthesis and mitochondrial biogenesis in a variety of cell types including cardiac muscle by inducing expression of mitochondrial and nuclear transcription factors that control mitochon-


drial protein gene expression (Nisoli et al., 2003). Thus, the correlation between resistance to PHS and mitochondrial protein levels that we observed in the present study may be associated with a gene in a NO signal transduction pathway or a gene associated with ROS production or activity. Additional studies will be required to determine the effects of NO and ROS on mitochondrial ETC protein levels and their possible involvement in resistance to PHS in broilers.

ACKNOWLEDGMENTS The authors thank H. Sonia Tsai, Linda Stamps, Hilary Pavlidis, M. Wally McDonner, A. Scott Zornes, Dana Bassi, and David Horlick for their technical assistance. The authors also thank John de Banzie, Robert Wideman, Jr., and Neil Pumford for critical reading of the manuscript. This work has been supported in part by funds from the Poultry and Egg Association (Project #285), Cobb-Vantress, Inc., and USDA-NRI Animal Health (grant #1999-02123; W. Bottje).

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