Progressive Structural Defects in Canine Centronuclear Myopathy Indicate a Role for HACD1 in Maintaining Skeletal Muscle Membrane Systems

Gemma L Walmsley,Stéphane Blot,Kerrie Venner,Caroline Sewry,Jocelyn Laporte,Jordan Blondelle,Inès Barthélémy,Marie Maurer,Nicolas Blanchard-Gutton,Fanny Pilot-Storck,Laurent Tiret,Richard J Piercy

doi:10.1016/j.ajpath.2016.10.002

Abstract

Mutations in HACD1/PTPLA cause recessive congenital myopathies in humans and dogs. Hydroxyacyl-coA dehydratases are required for elongation of very long chain fatty acids, and HACD1 has a role in early myogenesis, but the functions of this striated muscle-specific enzyme in more differentiated skeletal muscle remain unknown. Canine HACD1 deficiency is histopathologically classified as a centronuclear myopathy (CNM). We investigated the hypothesis that muscle from HACD1-deficient dogs has membrane abnormalities in common with CNMs with different genetic causes. We found progressive changes in tubuloreticular and sarcolemmal membranes and mislocalized triads and mitochondria in skeletal muscle from animals deficient in HACD1. Furthermore, comparable membranous abnormalities in cultured HACD1-deficient myotubes provide additional evidence that these defects are a primary consequence of altered HACD1 expression. Our novel findings, including T-tubule dilatation and disorganization, associated with defects in this additional CNM-associated gene provide a definitive pathophysiologic link with these disorders, confirm that dogs deficient in HACD1 are relevant models, and strengthen the evidence for a unifying pathogenesis in CNMs via defective membrane trafficking and excitation-contraction coupling in muscle. These results build on previous work by determining further functional roles of HACD1 in muscle and provide new insight into the pathology and pathogenetic mechanisms of HACD1 CNM. Consequently, alterations in membrane properties associated withHACD1 mutations should be investigated in humans with related phenotypes.

Highlights

Ed in affected dogs included the absence of a clear fiber-type distribution and the presence of necklace fibers with an internal, dense-staining ring, running parallel to the sarcolemma, lobulated fibers with dense subsarcolemmal mitochondrial deposits and many fibers with a halo or peripheral rim devoid of oxidative staining (Figure 1, E, H, K, and N)
Ultrastructural abnormalities observed in affected dogs with HACD1 centronuclear myopathy (CNM) included internalized or centralized nuclei (Figure 1, F, I, and L), mitochondrial mislocalization and clumping (Figure 1F), presence of lipid bodies (Figure 1, I and L), and myofibrillar disorganization (Figure 1, I and L)
The causative HACD1 mutation’s effect on muscle ultrastructure and subcellular membrane systems, which are implicated in CNMs with different genetic causes, has not previously been investigated

Summary

Introduction

Ed in affected dogs included the absence of a clear fiber-type distribution and the presence of necklace fibers with an internal, dense-staining ring, running parallel to the sarcolemma, lobulated fibers with dense subsarcolemmal mitochondrial deposits and many fibers with a halo or peripheral rim devoid of oxidative staining (Figure 1, E, H, K, and N). Subjective scoring (G.L.W.) revealed that most fibers from HACD1 CNM dogs of all ages had abnormal internal cytoarchitecture with histologic and oxidative stains (Table 1). Ultrastructural abnormalities observed in affected dogs with HACD1 CNM included internalized or centralized nuclei (Figure 1, F, I, and L), mitochondrial mislocalization and clumping (Figure 1F), presence of lipid bodies (Figure 1, I and L), and myofibrillar disorganization (Figure 1, I and L). These features appeared most severe in the older animals. Prominent features observed by electron microscopy were probed further by IHC on cryosections from the same biopsy samples

Methods

Results

Conclusion