Date Published: March 14, 2019
Publisher: Public Library of Science
Author(s): Khaloud Al-Zahrani, Timothy Licknack, Destiny L. Watson, Nicholas B. Anthony, Douglas D. Rhoads, Arda Yildirim.
We have extended our previous survey of the association of mitochondrial prevalence in particular tissues with ascites susceptibility in broilers. We previously reported that in breast muscle of 22 week old susceptible line male birds had significantly higher mtDNA copy number relative to nuclear copy number (mtDNA/nucDNA), compared to resistant line male birds. The higher copy number correlated with higher expression of PPARGC1A mRNA gene. Ascites is a significant metabolic disease associated with fast-growing meat-type chickens (broilers) and is a terminal result of pulmonary hypertension syndrome. We now report the mtDNA/nucDNA ratio in lung, liver, heart, thigh, and breast of both genders at 3, and 20 weeks old. At 3 weeks the mtDNA/nucDNA ratio is significantly higher in lung, breast, and thigh for susceptible line males compared to the resistant line males. Conversely, we see the opposite for lung and breast in females. At 20 weeks of age the differences between males from the two lines is lost for lung, and thigh. Although there is a significant reduction in the mtDNA/nucDNA ratio of breast from 3 weeks to 20 weeks in the susceptible line males, the susceptible males remain higher than resistant line males for this specific tissue. We assessed relative expression of five genes known to regulate mitochondrial biogenesis for lung, thigh and breast muscle from males and females of both lines with no consistent pattern to explain the marked gender and line differences for these tissues. Our results indicate clear sex differences in mitochondrial biogenesis establishing a strong association between the mtDNA quantity in a tissue-specific manner and correlated with ascites-phenotype. We propose that mtDNA/nucDNA levels could serve as a potential predictive marker in breeding programs to reduce ascites.
Ascites, Pulmonary hypertension syndrome PHS, or ‘water belly” is a cardiovascular, metabolic disease affecting fast-growing broilers. Ascites is a complex problem resulting from many interacting factors such as genetics, environment and management, but also occurs in normal conditions as a response to high metabolic rate [1–6]. The high metabolic oxygen requirement of rapid growth combined with insufficient capacity of the pulmonary capillaries appears to be the most important cause of ascites incidence in modern broilers [7–9]. Inadequate oxygen levels trigger a series of events, including peripheral vasodilation, increased cardiac output, increased pulmonary arterial pressure, right ventricular hypertrophy (RVH; elevated right ventricular to total ventricular ratios- RV: TV), and ultimitly accumulation of fluid in the abdominal cavity and pericardium [3, 8–12]. Advances in management practices, rearing programs, and improved selection techniques have decreased ascites incidence in modern broilers. However, ascites syndrome remains an economic concern throughout the world, causing estimated losses of $100 million annually in the US [9, 13] (personal communications: Rossi, 2004; Cooper, 2018). The etiology of ascites in poultry has been classified into three categories: 1) mainly pulmonary hypertension, 2) various cardiac pathologies, and 3) cellular damage caused by reactive oxygen species (ROS) . Mitochondria are the powerhouses of the eukaryotic cell and are the major contributor to oxidative stress through the generation of reactive oxygen species (ROS). Mitochondria are the primary oxygen consumer for energy production to sustain rapid growth in broilers [15–17]. Mitochondria are known to be involved in the regulation of several fundamental cellular processes, including metabolism, apoptosis, intracellular signaling, and energy production in the form of ATP via the oxidative phosphorylation. Mitochondrial biogenesis can be defined as the process of growth and division of pre-existing mitochondria to increase ATP production in response to growing demand for energy or stress conditions . During times of environmental stress (e.g., hypoxia, cold temperature, etc.), ROS levels can increase dramatically in mitochondria which may result in significant damage and long term damage to the mitochondrial DNA [19–21]. Oxidative damage to mitochondrial DNA has been shown to persist longer than nuclear DNA [21, 22]. Changes in mitochondrial copy number have been associated with aging, Alzheimer’s disease, neurodegeneration, heart disease, oxidative stress, and cancer [23–32]. In such cases, quantification of mtDNA copy number may be a good biomarker for disease progression [23, 27, 29]. Ascites can be induced at early ages by several methods such as altering the environment’s temperature [33, 34], air quality , and altitude . Researchers at the University of Arkansas established divergently selected ascites experimental lines derived from a former full pedigreed elite line beginning in the 1990s through sibling-selection based on a hypobaric challenge [9, 36, 37]. The lines are the ascites resistant (RES) line, ascites susceptible (SUS) line, and a relaxed (REL) unselected line.
Previously we evaluated the mitochondrial biogenesis and PPARGC1A mRNA gene expression in male broiler chickens at 22 weeks of age . The analyses compared two experimental lines produced through divergent selection for ascites phenotype; the ascites-susceptible (SUS) and ascites resistant (RES) broiler lines [9, 36]. The comparison was based on five males from each line and the evaluation was for right ventricle and breast. Results showed that birds from SUS had significantly higher mtDNA copy number (p = 0.038) and PPARGC1A RNA gene (p = 0.033) in breast muscle; with no difference in right ventricle. Thus, we suggested that mitochondrial biogenesis and PPARGC1A mRNA gene expression differ between male boilers from RES and SUS lines in a tissue-specific manner. The present report extends our previous analyses to additional muscles and other critical tissues at additional ages and for both genders.
Mitochondrial dysfunction is well documented in a wide array of diseases and conditions, such as Alzheimer’s disease, cancer, and aging [23, 25, 30]. Mitochondria are central to ATP synthesis, heat production, radical oxygen species (ROS) generation, fatty acid and steroid metabolism, cell proliferation, and apoptosis [18, 26, 28, 31, 46]. Alterations in mtDNA sequence or copy number may contribute to mitochondrial dysfunction [23, 26, 29]. Thus, it is likely that imbalances within the cell concerning mitochondria-centered metabolic pathways may contribute to ascites syndrome [5, 15, 16, 47]. Our observations indicate that variations in mtDNA copy number could be an important component in the pathoetiology of ascites syndrome in broilers. Using different tissues, we have demonstrated that mtDNA copy number can be an important biomarker during early developmental age for ascites syndrome susceptibility. Our results showed sizable tissue-specific, and gender differences in the mtDNA/nucDNA ratio at early ages of broilers. The possible existence of gender-specific differences in energy metabolism for particular tissues might be a consequence of interplay between maternally inherited mitochondria and sex chromosomes or differences in endocrine responses. In males, mtDNA/nucDNA ratio was significantly higher in lung, thigh, and breast tissues from SUS line males at 3 weeks of age in comparison with RES line males. Conversely, mtDNA levels were significantly lower in lung and breast tissues of SUS line females as compared to RES line females. The gender differences may impact ascites phenotype considering that males are documented to have higher ascites mortality than females [48–51]. The observed elevation in the amount of mtDNA in lung, thigh and breast muscle of SUS line males might be attributable to a compensatory response to the decline in the respiratory function of mitochondria or a response to other metabolic regulatory processes. An alternate explanation is needed for the reduced mtDNA copy number in lung and breast muscle of SUS line females. One possible explanation is that differences in mtDNA content of different sexes can be attributed to imbalances in oxidative stress due to higher female estrogen levels [24, 52]. Previous work found that oxidative damage to mtDNA is 4-fold higher in males than in females [24, 52]. The lower oxidative damage in females may be attributable to the protective effect of estrogens by upregulating the expression of antioxidant enzymes in mitochondria via intracellular signaling pathways, thus decreasing oxidative damage and increasing antioxidants defenses. Moreover, fundamental sex differences in metabolism under stressful conditions have long been observed in several organisms and may also be influenced by intrinsic differences in genomic maintenance . Absent from our analysis is any determination of whether the differences in mtDNA content is associated with functional or non-functional (defective) mitochondria. Future work could involve fluorescent detection systems for visualizing mitochondria in SUS vs RES tissues to assess relative mitochondrial abundance and enzymatic staining to assess mitochondrial functional state.