The fundamental importance of chaperones for muscle integrity strongly suggests that correcting chaperone function might be a therapeutic option to tackle hereditary myopathies, however, no proven therapies exist at the moment. By monitoring myopathy-related defects in Caenorhabditis elegans and patient muscle combined with structure-function analysis of UNC45B mutations, we provide novel insights into myofibrillogenesis and regeneration mechanisms of therapeutic relevance for the treatment of muscle diseases.

Skeletal muscle integrity and function is constantly challenged by stress and strain placed upon sarcomeric and structural proteins. Therefore, pathogenic mutations in chaperones or co-chaperones often trigger inherited neuromuscular disorders collectively termed chaperonopathies. In contrast to the rather broad role of general chaperones in protein homeostasis and myofibrillar integrity, the assembly of myosin requires precise temporal and spatial control, which is governed by the highly specialized co-chaperone UNC-45. The repetitive arrangement of UNC-45 oligomers defines the periodicity of myosin organization in growing sarcomers. However, despite the conserved role in myofibrillogenesis from C. elegans to vertebrates, the relevance of muscle-specific UNC-45 dysfunction for the pathology and pathogenesis of human myopathies had remained completely unclear until now.

The CMMC-funded project took advantage of the unique opportunity to explore the patho-mechanism associated with four novel patient-derived mutations in the myosin-directed co-chaperone UNC45B. The detailed characterization of UNC45B function in the formation and maintenance of skeletal muscle is important for the molecular understanding of congenital muscle diseases and other forms of muscle weakness.

In the current funding period, we

1. characterized structure-functional properties of disease-associated UNC45B mutations;
2. explored the role of UNC45B mutations on myofibrillogenesis in C. elegans; and
3. defined myopathy-related UNC45B dysfunction in patient muscle.

We identified a common UNC45B variant that acts as a complex hypomorph splice variant. Purified UNC-45B mutants showed changes in folding and solubility. In situ localization studies further demonstrated reduced expression of mutant UNC-45B in muscle combined with abnormal localization away from the A-band towards the Z-disk of the sarcomere. The physiological relevance of these observations was investigated in C. elegans by transgenic expression of conserved UNC-45 missense variants, which showed impaired myosin binding for one and defective muscle function for three. Together, our results demonstrate that UNC-45B impairment manifests as a chaperonopathy with progressive muscle pathology, which discovers the previously unknown conserved role of UNC-45B in myofibrillar organization.

The major findings of this CMMC funded project were published in the American Journal of Human Genetics (Donkervoort, Kutzner et al., 2020).

Proper muscle development and function require a complex system of structural and motor proteins organized into contractile units called the sarcomeres. The sarcomeric repeat is a supra-molecular structure, in which actin and myosin filaments together with associated proteins are arranged in a precise order that coordinates actin-myosin cross bridges with filament gliding and muscle contraction. The folding, stability, and organization of sarcomeric proteins into muscle filaments is governed by molecular chaperones (Kim et al., 2008). 

The fundamental importance of chaperones for the development and maintenance of skeletal muscle is underscored by recent studies indicating that chaperone dysfunction is responsible for many hereditary myopathies. These so called chaperonopathies are directly caused by pathogenic mutations in genes encoding chaperones and co-chaperones. The first described entity was a myofibrillar myopathy associated with mutations in the heat shock protein (HSP) HSPB5 (CRYAB), although most later identified chaperonopathies were Charcot-Marie-Tooth-types of neuropathies caused by HSPB1, HSPB8, CCTdelta/MKKS, and DNAJB2 gene defects. 

In contrast to the rather broad regulation of myofibrillar integrity by these general chaperones, the precise temporal and spatial control of myosin assembly is governed by the Hsp90 co-chaperone UNC-45 (Kim et al., 2008). Recent work of our lab importantly contributed to the mechanistic understanding of UNC-45 function in muscle formation and maintenance. We found that UNC-45 oligomers serve as multisite-docking platforms, which support precisely defined collaboration with Hsp70 and Hsp90 to form myosin filaments (Gazda et al., 2013). Thus, UNC-45 provides substrate specificity for the partner chaperones during late stages of myofibrillogenesis. Detailed analysis of structure-related mutations indicated that the elongated part of the conserved UCS (UNC-45/CRO1/She4p) domain of UNC-45 acts as myosin-binding site (Figure 1and 4).

Therefore, the repetitive arrangement of UNC-45 oligomers serves as template that defines the periodicity of myosin organization in growing sarcomers (Figure 1 and 2).

This idea is strongly supported by point mutations in UNC-45, which trigger myosin disorganization and severe motility defects in C. elegans, Drosophila, Zebrafish, and Xenopus (Figure 3) (Kim et al., 2008).

UNC-45 homologs also exist in humans, which indicates a conserved requirement for myosin-directed co-chaperones. Our work has shown that UNC-45 levels are tightly controlled by ubiquitin-dependent proteolysis, which coordinates myosin folding and assembly both in C. elegans and human myoblasts (Hoppe et al., 2004; Janiesch et al., 2007). Given the constant use and mechanical stress exposure of the muscle sarcomere, maintenance of muscle function involves a network of protein folding and degradation pathways (Hoppe et al., 2004; Kim et al., 2008). Due to its central role in myosin assembly, UNC-45 protein localization and amount are tightly coordinated with muscle development and stress conditions. In zebrafish it has been shown that UNC-45B shuttles between the Z-disc and the myosin containing A-band of the muscle sarcomere upon eccentric exercise or induced damage to the myofiber (Figure 4B) (Etard et al., 2008). Our work has shown that UNC-45 levels are tightly controlled by ubiquitin-dependent proteolysis, which coordinates myosin folding and assembly both in C. elegans and human myoblasts (Hoppe et al., 2004; Janiesch et al., 2007). Thus, the growth and maintenance of myofilaments depend on the interplay of protein folding and degradation.

To define the myopathy-related UNC45B dysfunction in patient muscle, we collaborated with the laboratories of Carsten Bönnemann, Coen Ottenheijm, Jocelyn Laporte, and many clinical pathologists all over the world. Histological analyses of patient muscle biopsies showed internalized nuclei, cytoplasmic bodies, oxidative defects, and reduced oxidative activity with core-like regions, consistent with eccentric cores, among others. Electron microscopy unveiled large areas of myofibrillar disorganization and unstructured cores (Figure 7A-C). Single muscle fiber mechanics revealed a type I fiber dominance but otherwise no impairment of fiber contractile function. Immunofluorescence location studies suggested a reduction and mislocalization of UNC45B variant proteins compared to wild type (Figure 7D-E). In muscle RNA sequencing, a recurring UNC45B variant was identified to act as a complex hypomorph splice variant causing the inclusion of an in-frame STOP codon and subsequent degradation of the nonsense transcript via nonsense-mediated decay (Figure 7F).

Taken together, we report that UNC45B impairment manifests as a chaperonopathy with progressive muscle pathology, which discovers the previously unknown, conserved role of UNC45B in myofibrillar organization. The disease mechanism involves alterations in UNC45B mRNA splicing and UNC45B protein levels, as well as disruptions of myosin binding, folding, and localization.