Lack of the myotendinous junction marker col22a1 results in posture and locomotion disabilities in zebrafish

Highlights

• Collagen XXII is the unique extracellular matrix marker of the myotendinous junction.

• CRISPR/Cas9-mediated col22a1 gene knockout in zebrafish results in loss of the myotendinous unit integrity and dysfunctions in force transmission but with phenotypic variability

• A subset of the col22a1^−/− progeny displays severe phenotype accompanied with erratic movements and postural defects and dies before metamorphosis

• The moderate col22a1^−/− phenotype is characterized by muscle fatigue and mitochondrial adaptation to exercise in adults.

• COL22A1 represents a novel candidate gene for human myopathies characterized by muscle weakness and dysfunctional force transmission.

Abstract

The myotendinous junction (MTJ) is essential for the integrity of the musculoskeletal unit. Here, we show that gene ablation of the MTJ marker col22a1 in zebrafish results in MTJ dysfunction but with variable degrees of expression and distinct phenotypic classes. While most individuals reach adulthood with no overt muscle phenotype (class 1), a subset of the progeny displays severe movement impairment and die before metamorphosis (class 2). Yet all mutants display muscle weakness due to ineffective muscle force transmission that is ultimately detrimental for class-specific locomotion-related functions. Movement impairment at the critical stage of swimming postural learning causes class 2 larval death by compromising food intake. In class 1 adults, intensive exercise is required to uncover a decline in muscle performance, accompanied by higher energy demand and mitochondrial adaptation. This study underscores COL22A1 as a candidate gene for myopathies associated with dysfunctional force transmission and anticipates a phenotypically heterogeneous disease.

Introduction

The myotendinous junction (MTJ) is a specialized anatomical region where tendon collagen fibers insert into the muscle basem6ent membrane. These muscle-tendon connection sites are appropriately located and organized to transmit muscle contractile forces to tendons and create a movement. This is mainly due to the presence of two independent trans-sarcolemmal linkage systems that structurally link the intracellular contractile elements of muscle cells to the extracellular matrix (ECM) of tendons, the dystrophin-glycoprotein complex (DGC) and the α7β1 integrin complex. Mutations in their associated genes, such as dystrophin, α-7integrin, and α-2 laminin have been associated with myopathies in patients and animal models revealing that most of the MTJ components are unconditionally required for proper muscle function and integrity [[1], [2], [3]]. The impact of these mutations in MTJ formation and/or function remains poorly documented, particularly in the context of human diseases for which biopsies at this particular location would be too prejudicial for the patients. Yet mice that lack dystrophin (mdx; Dmd, [4,5]), laminin α2 (dy; Lama2, [6]) or integrin α7 [7,8] all exhibit a striking reduction in the number of membrane folds at MTJ. Zebrafish has proven instrumental in the study of MTJ components in developing skeletal muscle. In zebrafish, mutant lines of the structural proteins enriched at the MTJ (sapje/dystrophin, caf/lama2 and patchytail/dag1) or morpholino (MO)-knockdown in zebrafish embryos of itga7, thbs4b, or col22a1 all displayed compromised muscle attachments that result in muscular dystrophies of various severities [[9], [10], [11], [12]]. However in vivo function of these components was generally limited to zebrafish larval stage while muscular dystrophies are often progressive muscle disorders.

Collagen XXII (ColXXII) is a recognized marker of the MTJ that was first described by Koch and colleagues [13]. Functional analysis in zebrafish showed that col22a1 expression concentrates at the ends of muscle fibers guiding the protein deposition at the junctional ECM. ECM proteome across muscle-tendon interface found ColXXII restricted to the muscle-tendon junction tissue [14]. Single-nucleus RNA-seq analysis in mice recently suggested that myonuclei near the muscle ends and tenocytes may both contribute this collagen [15]. While ColXXII binding partners have not been biochemically identified yet, synergistic interactions suggested that ColXXII is a constitutive protein of the transmembrane α7β1 linkage system. In addition, transmission electron microscopy (TEM) revealed that ColXXII is located at the outer surface of the MTJ suggesting a structural role linking the basement membrane to the tendinous ECM [11,13]. As such, ColXXII could be the missing link that anchors muscle basement membrane network to the tendon collagen fibers. Although recognized as an important structural protein of the MTJ, the role of ColXXII in adult muscle function and performance remains overlooked. A recent study nonetheless described that high mRNA expression of COL22A1 at the MTJ is associated with muscle injury risk in athletes [16].

Here, we generated loss-of-function mutants in zebrafish col22a1 using CRISPR/Cas9 technology to detail the function of ColXXII beyond early larval stages. Phenotype discrepancy between morphants and knock-out (KO) stable lines have been previously reported [[17], [18], [19]]. But, as a rare example of a strong correlation between morpholino-induced and mutant phenotypes [17], we further documented the requirement of ColXXII for proper development of the myotendinous system, for contractile force transmission and ultimately movements not only in larvae but in adults. By combining ultrastructural analyses, muscle performance measurements and behavioral assays, we demonstrate that the lack of ColXXII results in the systematic dysfunction of the myotendinous unit that impairs postural behavior learning and swimming performance but with variable expressivity as often observed in human disease.