Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology
Heart and breast muscle mitochondrial dysfunction in pulmonary hypertension syndrome in broilers (Gallus domesticus)
Introduction
Pulmonary hypertension syndrome (PHS) or ascites syndrome, is a metabolic disease in poultry whose pathophysiology has been broadly classified as either: (1) pulmonary hypertension; (2) miscellaneous cardiac pathologies; and/or (3) cellular damage caused by reactive oxygen species (Currie, 1999). Associated with each of these categories in birds that develop pulmonary hypertension, is impaired uptake and delivery of oxygen or impaired cellular oxygen utilization, problems magnified by the high demand for oxygen required to support rapid growth rates in commercial meat chickens (broilers). In this regard, a marginal cardio-pulmonary capacity was elegantly demonstrated (Wideman and Kirby, 1995a, Wideman and Kirby, 1995b, Wideman et al., 1997) that is clearly due to a genetic predisposition (Wideman, 2000, Wideman and French, 2000).
Key to the oxygen demand in the body are mitochondria, that account for roughly 90% of cellular oxygen consumption (Lehninger et al., 1992). The mitochondrial respiratory chain uses electron flow to create proton motive force that is used to drive adenosine triphosphate (ATP) synthesis (Lehninger et al., 1992; Fig. 1). Oxygen serves as the terminal electron acceptor for the respiratory chain that consist of a series of multi-protein complexes that reside on the inner mitochondrial membrane. Proton movement through the f0f1 ATP synthase effectively couples the energy releasing reaction of oxidation to the energy-storing reactions of phosphorylation. Lower respiratory control ratio (RCR) and ADP/O ratio, indices of respiratory chain coupling and oxidative phosphorylation, respectively (Estabrook, 1967), have been observed in mitochondria of birds with PHS (Cawthon et al., 1999). Furthermore, liver and lung mitochondrial dysfunction in PHS was magnified by repeated additions of ADP (Cawthon et al., 2001, Iqbal et al., 2001b).
Mitochondria are not only the major site of oxygen consumption in the body, but are also responsible for contributing to cellular oxidative stress due to the generation of oxygen radicals (reactive oxygen species). Rather than being completely reduced to water, it has been shown in vitro that 1–4% of the oxygen consumed by mitochondria is incompletely reduced to superoxide and hydrogen peroxide (H2O2) by univalent reduction by electrons that leak from the respiratory chain (Chance et al., 1979).
Increased mitochondrial reactive oxygen species production has been linked to metabolic diseases such as cystic fibrosis, diabetes and aging (Fiegal and Shapiro, 1979, Kristal et al., 1997, Hagen et al., 1997, Herrero and Barja, 1998, Allen et al., 1997, Lass et al., 1997), and more recently to PHS (Maxwell et al., 1996, Cawthon et al., 2001, Iqbal et al., 2001a). Herrero and Barja, 1997, Herrero and Barja, 1998 have indicated that birds generate lower amounts mitochondrial reactive oxygen species than do other species (e.g. rat, mouse) and have speculated that this might play a role in longer life spans characteristically observed in many avian species.
Mitochondrial reactive oxygen species production in broilers with PHS (Maxwell et al., 1996, Cawthon et al., 2001, Iqbal et al., 2001a) could also be responsible for the oxidative stress associated with this metabolic disease (Enkvetchakul et al., 1993, Bottje et al., 1995, Bottje and Wideman, 1995, Diaz-Cruz et al., 1996). Whereas mitochondrial radical production is associated mainly with defects in electron transport within Complex I or III (Turrens and Boveris, 1980, Turrens et al., 1985, Nohl et al., 1996, Hansford et al., 1997, Kristal et al., 1997, Herrero and Barja, 1998), Kwong and Sohal (1998) demonstrated that sites of mitochondrial H2O2 production in the electron transport chain are tissue-dependent. Similarly, Cawthon et al. (2001) indicated that electron leak occurred in Complex II in PHS liver mitochondria and contrasted with defects at Complex I and III in PHS lung mitochondria (Iqbal et al., 2001a). As electrons that leak from the electron transport chain cannot be used to support ATP synthesis, the lower ADP/O observed in PHS liver mitochondria (Cawthon et al., 1999, Cawthon et al., 2001, Iqbal et al., 2001b) could also be due to site-specific leakage of electrons from the respiratory chain as well as functional damage to mitochondrial oxidative phosphorylation (Nakahara et al., 1998).
Mitochondrial dysfunction has been observed in mitochondria obtained from the liver and lung of broilers with PHS (Cawthon et al., 1999, Cawthon et al., 2001, Iqbal et al., 2001b). If findings of similar inefficient oxygen use occur in muscle mitochondria, this could have a dramatic effect on oxygen usage in broilers with PHS as muscle tissue accounts for as much as 45% of oxygen utilization in the entire animal (Field et al., 1939, Brand et al., 1994). Thus, the objectives of the present study were to determine function and to assess if site-specific defects exist in electron transport of breast and heart muscle mitochondria obtained from broilers with PHS.
Section snippets
Birds and management
In experiment 1, male broiler chicks (Cobb 500, Gallus domesticus) obtained from a local hatchery (Randall Road, Tyson Inc., Springdale, AR 72762) at 1 day of age, were placed in an environmental chamber (8 m2 floor space per 100 chicks) on wood shaving litter. Birds were provided ad libitum access to water and to a high protein and energy diet (23.7% protein, 3200 kcal ME). Temperature in the chamber was 32 and 30 °C during week 1 and 2, lowered to 15 °C during week 3, and maintained between
Determination of mitochondrial H2O2 production
Mitochondrial H2O2 generation was determined using 2′, 7′-dichlorofluorescin diacetate (DCFH-DA, Molecular Probes Inc., Eugene, OR 97402) according to Bass et al. (1983) with recent modifications (Iqbal et al., 2001b). Briefly, H2O2 was measured in 96-well microplates with a photofluorometric detector (Cytofluor 2350, Millipore Corporation, Bedford, MA 01730) set at a sensitivity of 3 and excitation/emission wavelength at 480/530 nm, respectively. To each well, ∼0.1 mg of mitochondrial protein
Results
Broilers with PHS exhibited marginally lower body weights (P=0.10) and an elevated RV/TV (P<0.001) compared to controls (Table 1). Mitochondrial respiration and function are shown in which NADH-linked substrates (Table 2) and succinate, an FADH-linked substrate (Table 3) were provided. Respiration rates were generally higher in mitochondria provided succinate, but functional values (ACR, RCR and ADP/O) were higher in mitochondria provided NADH-linked energy substrates. In breast muscle
Mitochondrial function in PHS
The major indices of mitochondrial function, the RCR and ADP/O ratios, are measures of electron transport chain coupling and efficiency of oxidative phosphorylation, respectively (Estabrook, 1967). The RCR values in both breast and heart muscle were lower in PHS mitochondria metabolizing either NADH- or FADH-linked substrates (Table 2, Table 3), indicating less control on respiratory chain coupling in PHS as in controls. The lower RCR in PHS mitochondria was similar to that observed previously
Acknowledgments
The authors would like to thank N. Rath and D. Horlick (USDA-ARS, University of Arkansas) for assistance in the use of the Cytofluor 2350 used in the detection of DCF fluorescence, and R. McNew (Agriculture Statistics Lab, University of Arkansas) for statistical design and consultation. Parts of this study were reported at the 7th Annual Oxygen Society meeting in San Diego (Nov. 18–22, 2000). This study was supported in part by funds from the US Poultry and Egg Association (1999, #456) and
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