Physiological and Pathological Mitochondrial Clearance Is Related to Pectoralis Major Muscle Pathogenesis in Broilers With Wooden Breast Syndrome

Abstract

Wooden breast syndrome (WB) constitutes an emerging myopathy in the pectoralis major muscle (PM) of broiler chickens, characterized by myofiber hypertrophy and degeneration along with severe fibrosis. WB pathogenesis has been considered to involve hypoxia induced by rapid growth of the PM. In this study, we focused on mitochondrial morphology and dynamics in the myofibers, as these organelles are sensitive to damage by hypoxia, and examined the effects on WB pathogenesis. Specifically, the PMs of a flock of 35 broilers at 50 days of age were evaluated. First, the severity of disease in each bird was determined by measuring histopathological indices including the fibrotic area (FA) in the muscle and circularity of myofibers (CM). These values were 29.4 ± 9.6% and 0.70 ± 0.042, respectively, showing variety among the flock. Myofiber vacuolization was observed in all birds including numerous small- or large-rimmed vacuoles, with the former consisting of ultrastructurally autophagosome-like vacuoles engulfing degenerated mitochondria. The large-rimmed vacuoles frequently occurred in the PMs with more severe FA and CM, indicating a relationship between altered autophagy/mitophagy and WB severity. Next, the expression levels of hypoxia-adaptive and mitochondrial dynamics-related genes were analyzed, and their correlations with the histopathological indices were examined. The histopathological indices were negatively correlated with the expression of vascular endothelial growth factor A (VEGFA), indicating that less angiogenesis owing to weakened hypoxia-inducible factor signaling induces more severe WB pathology. In addition, the observed negative correlation with mitochondrial dynamics-related genes implied that WB pathology deteriorates concomitant with reduced mitochondrial dynamics. Furthermore, the expression of mitochondrial dynamics-related genes showed strong positive correlation with that of VEGFA and autophagy-/mitophagy-related genes. These results revealed that the PMs of broilers possess the mechanism of physiological clearance of mitochondria damaged by the hypoxia resulting from the continuous mitochondrial dynamics and autophagy/mitophagy accompanying rapid PM growth. In turn, the altered mitochondrial clearance induced by chronic hypoxia and the accumulation of damaged mitochondria likely underly the severe pathological features of WB.

Keywords: autophagy; hypoxia; mitochondria; mitophagy; pathology; wooden breast syndrome.

FIGURE 1

Histology of the pectoralis major muscle of broilers stained with Azan and hematoxylin–eosin. (A–E) Histological observations of the pectoralis major muscle of (A) sample number 1 (stage I; FA, 7.24% and CM, 0.70), (B) 2 (stage I; FA, 9.06% and CM, 0.69), (C) 3 (stage I; FA, 13.25% and CM, 0.68), (D) 33 (stage VI; FA, 40.90% and CM, 0.70), and (E) 34 (stage VI; FA, 40.97% and CM, 0.76). The squares indicated by dashed lines are magnified on the right. White bar = 200 mm, black bar = 100 mm. SV, myofiber with numerous small vacuoles; RV, myofiber with large-rimmed vacuoles; H, hypertrophy of myofiber; SC, myofiber with small caliber; SF, split fiber; D, myofiber degeneration; FA, fibrotic area in the muscle; CM, circularity of myofibers.

FIGURE 2

Ultrastructure of the myofibers in the pectoralis major muscle of broilers. Ultrastructure of the pectoralis major muscle of (A) sample number 2 (stage I; FA, 9.06% and CM, 0.69) and (B,C) 15 (stage III; FA, 26.28% and CM, 0.74). FA, fibrotic area in the muscle; CM, circularity of myofibers. (A) Distribution of small and clear mitochondria (arrowheads) and a swelled mitochondrion (arrow) within myofibrils (asterisks) in the normal myofiber. The square indicated by black line is magnified on the right. (B) Numerous small vacuoles (daggers) are distributed within myofibrils (asterisks) in the myofibers. The vacuole (surrounded by a dashed line) envelops the swelled and degenerated mitochondria (arrows). The square indicated by black line is magnified on the right. Arrowheads: small and clear mitochondria. (C) Multiple myoblasts accumulated in the pectoralis major muscle. The autophagosome with a double membrane (between arrows) in the myoblast envelops organelles and the small and clear mitochondria (arrowheads). The squares indicated by black line are magnified on the right. N, nuclei in the myoblast; asterisks, myofibrils.

FIGURE 3

Relationship of gene expression levels in the various stages to fibrotic area in the muscle. (A) Hypoxia-adaptive gene expression including hypoxia inducible factor 1 subunit alpha (HIF1A) and vascular endothelial growth factor A (VEGFA). (B) Mitochondrial dynamics-related gene expression including mitofusion 1, 2 (MFN1, 2), OPA1, mitochondrial dynamin-like GTPase (OPA1), and dynamin-related protein 1 (DRP1). (C) Autophagy-/mitophagy-related gene expression including microtubule-associated protein 1 light chain 3 alpha, beta (MAP1LC3A, B) and parkin RBR E3 ubiquitin protein ligase (PARK2). Data represent the means ± SE [stage I (n = 6), stage II (n = 5), stage III (n = 6), stage IV (n = 5), stage V (n = 9), and stage VI (n = 4), Tukey test (P < 0.05)].

FIGURE 4

Scheme of wooden breast syndrome (WB) pathogenesis focusing on mitochondrial clearance and myofiber pathology. In pectoralis major muscles (PM) with no or mild WB, the adaptation to hypoxia by HIF1 signaling induces physiological mitochondrial clearance mediated by mitochondrial fusion, fission, and autophagy/mitophagy, which corresponds with the appearance of myofibers with small vacuoles. At this stage, PM repair appears to occur through the differentiation of myoblasts into myofibers. Alternatively, in the muscles exhibiting severe WB, the acquired resistance to hypoxia alters the regulation of mitochondrial clearance. The accumulated damage in mitochondria exacerbates the various pathological characteristics such as myofibers with rimmed vacuoles, along with their hypertrophy and degeneration. Under severe hypoxia, the regeneration of myofibers fails, which corresponds with the frequent appearance of split fibers and myofibers with small caliber. HIF1, hypoxia-inducible factor 1.