Musculocontractural Ehlers–Danlos Syndome (mcEDS) is a type of EDS caused by biallelic pathogenic variants in the gene for carbohydrate sulfotransferase 14/dermatan 4-O-sulfotransferase 1 (CHST14/D4ST1, mcEDS-CHST14), or in the gene for dermatan sulfate epimerase (DSE, mcEDS-DSE). Thus far, 41 patients from 28 families with mcEDS-CHST14 and five patients from four families with mcEDS-DSE have been described in the literature. Clinical features comprise multisystem congenital malformations and progressive connective tissue fragility-related manifestations. This review outlines recent advances in understanding the pathophysiology of mcEDS. Pathogenic variants in CHST14 or DSE lead to reduced activities of relevant enzymes, resulting in a negligible amount of dermatan sulfate (DS) and an excessive amount of chondroitin sulfate. Connective tissue fragility is presumably attributable to a compositional change in the glycosaminoglycan chains of decorin, a major DS-proteoglycan in the skin that contributes to collagen fibril assembly. Collagen fibrils in affected skin are dispersed in the papillary to reticular dermis, whereas those in normal skin are regularly and tightly assembled. Glycosaminoglycan chains are linear in affected skin, stretching from the outer surface of collagen fibrils to adjacent fibrils; glycosaminoglycan chains are curved in normal skin, maintaining close contact with attached collagen fibrils. Homozygous (Chst14^−/−) mice have been shown perinatal lethality, shorter fetal length and vessel-related placental abnormalities. Milder phenotypes in mcEDS-DSE might be related to a smaller fraction of decorin DS, potentially through residual DSE activity or compensation by DSE2 activity. These findings suggest critical roles of DS and DS-proteoglycans in the multisystem development and maintenance of connective tissues, and provide fundamental evidence to support future etiology-based therapies.

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.

Loss-of-function variants in CHST14 cause a dermatan 4-O-sulfotransferase deficiency named musculocontractural Ehlers–Danlos syndrome-CHST14 (mcEDS-CHST14), resulting in complete depletion of the dermatan sulfate moiety of decorin glycosaminoglycan (GAG) chains, which is replaced by chondroitin sulfate. Recently, we uncovered structural alteration of GAG chains in the skin of patients with mcEDS-CHST14. Here, we conducted the first systematic investigation of Chst14 gene-deleted homozygote (Chst14^−/−) mice. We used skin samples of wild-type (Chst14^+/+) and Chst14^−/− mice. Mechanical fragility of the skin was measured with a tensile test. Pathology was observed using light microscopy, decorin immunohistochemistry and electron microscopy (EM) including cupromeronic blue (CB) staining. Quantification of chondroitin sulfate and dermatan sulfate was performed using enzymatic digestion followed by anion-exchange HPLC. In Chst14^−/− mice, skin tensile strength was significantly decreased compared with that in Chst14^+/+ mice. EM showed that collagen fibrils were oriented in various directions to form disorganized collagen fibers in the reticular layer. Through EM-based CB staining, rod-shaped linear GAG chains were found to be attached at one end to collagen fibrils and protruded outside of the fibrils, in contrast to them being round and wrapping the collagen fibrils in Chst14^+/+ mice. A very low level of dermatan sulfate disaccharides was detected in the skin of Chst14^−/− mice by anion-exchange chromatography. Chst14^−/− mice, exhibiting similar abnormalities in the GAG structure of decorin and collagen networks in the skin, could be a reasonable model for skin fragility of patients with mcEDS-CHST14, shedding light on the role of dermatan sulfate in maintaining skin strength.

Typical skeletal muscles are composed of mixed muscle fiber types, which are classified as slow-twitch (type I) and fast-twitch (type II) fibers, whereas pectoralis major muscles (PMs) in broiler chickens are 100% composed of type IIb fast-twitch fibers. Since metabolic properties differ among muscle fiber types, the combination of muscle fiber types is involved in physiological functions and pathological conditions in skeletal muscles. In this study, using serial block-face scanning electron microscopy, we compared three-dimensional (3D) mitochondrial properties in type IIb fibers in broiler PMs and those in type I fibers of broiler gastrocnemius muscles (GMs) heterogeneously composed of slow- and fast-twitch muscle fibers. In type I fibers in the GMs, elongated mitochondria with numerous interconnections to form a substantial network among myofibrils were observed. Along with lipid droplets sandwiched by mitochondria, these features are an adaptation to effective oxidative respiration and constant oxidative damage in slow-twitch muscle fibers. In contrast, type IIb fibers in the PMs showed small and ellipsoid-shaped mitochondria with few interconnections and no lipid droplets, forming a sparse network. The mitochondrial spatial network comprises of active mitochondrial dynamics to reduce mitochondrial damage; therefore, type IIb fibers possess physiologically low capacity to maintain mitochondrial wellness due to static mitochondrial dynamics. Based on 3D mitochondrial properties, we discussed the contrasting physiological functions between type I and IIb fibers and proposed a high contractile power and low stress resistance as unique physiological properties of broiler PMs.

In skeletal muscle, myoblast differentiation results in the formation of multinucleated myofibers. Although recent studies have shown that unfolded protein responses (UPRs) play an important role in intracellular remodeling and contribute to skeletal muscle differentiation, the involvement of IRE1–XBP1 signaling, a major UPR signaling pathway, remains unclear. This study aimed to investigate the effect of the IRE1–XBP1 pathway on skeletal muscle differentiation. In C2C12 cells, knockdown of IRE1 and XBP1 in cells remarkably suppressed differentiation. In addition, apoptosis and autophagy were dramatically enhanced in the XBP1-knockdown cells, highlighting the participation of IRE1–XBP1 in cell survival maintenance with differentiation stimuli during skeletal muscle differentiation. In myogenic cells, we demonstrated that the expression of CDK5 (cyclin-dependent kinase 5) is regulated by XBP1s, and we propose that XBP1 regulates the expression of MyoD family genes via the induction of CDK5. In conclusion, this study revealed that IRE1–XBP1 signaling plays critical roles in cell viability and the expression of differentiation-related genes in predifferentiated myoblasts and during the early differentiation phase.