Sarcomeres, the repetitive intracellular units within striated muscle fibers, are the basic contractile unit in skeletal and cardiac muscles. Sarcomeres contain highly organized arrangements of proteins and are responsible for the contractile properties of muscles. Two major types of filaments- thick and thin- can be found within these complex sarcomeric structures. In addition, sarcomeres contain other major structures, including the Z-lines and M- lines, which provide structural support to the sarcomere. Myofibrillogenesis, the process of assembling myofibrillar proteins into a highly organized sarcomere, is pivotal for normal muscle function and motility. Defective sarcomere organization often leads to malfunction and diseases of skeletal and cardiac muscles, including muscular dystrophy and cardiomyopathy .
The major protein components of the thick and thin filaments are myosin and α-actin, respectively. Recent studies indicate that molecular chaperones play an important role in myosin folding and thick filament assembly [2–4]. Heat shock protein 90α (Hsp90α) and Unc-45 are key myosin chaperones that are specifically expressed in skeletal and cardiac muscles and play crucial roles in myofibrillogenesis [5–11]. Knockdown or mutation of Hsp90α1 results in poor myofibril organization, myosin degradation, and paralysis of zebrafish embryos [7, 8]. Hsp90α associates with Unc-45, a myosin chaperone which was first characterized in the nematode Caenorhabditis elegans, to control myofibril assembly. The C. elegans Unc-45 mutants showed decreased body movement and disorganized myofilament arrays in body wall muscles .
While C. elegans and Drosophila melanogaster express one isoform of Unc-45, many vertebrates, including the zebrafish, express two isoforms [9, 13]. These two isoforms are known as Unc-45a and Unc-45b. Unc-45a, also known as general cell Unc-45, is expressed in many tissues and has roles in smooth muscle myosin maturation and development of the aortic arches [14, 15]. Unc-45b, on the other hand, has been identified as a muscle-specific isoform expressed in striated muscles . Biochemical and functional studies have revealed that Unc-45b binds to the myosin motor domain and plays key roles in myosin folding and sarcomere assembly [3, 15, 16]. Unc-45b knockdown or mutation results in cardiac defects and paralysis of zebrafish embryos [10, 11].
Previous studies in C. elegans demonstrated that the protein levels of Unc-45 are highly regulated by the ubiquitin/proteasome pathway . A new E3/E4 complex, formed by CHN-1, the C. elegans ortholog of CHIP (carboxyl terminus of Hsc70-interacting protein), and UFD-2, an enzyme known to have ubiquitin-conjugating E4 activity in yeast, is necessary and sufficient to multiubiquitylate Unc-45 in vitro and directs Unc-45 degradation . Overexpression of Unc-45 in C. elegans results in thick filament defects, decreased myosin expression, and mild paralysis . The decrease in myosin expression is attributed to increased protein degradation through the ubiquitin/proteasome pathway . A similar conserved pathway was identified in humans, which links Unc-45b degradation and myosin assembly . Remarkably, mutations in human p97, an ubiquitin-selective chaperone, cause hereditary inclusion body myopathy, abrogate Unc-45 degradation and result in severely disorganized myofibrils [18, 19].
Structurally, Unc-45b is comprised of three major domains: an N-terminal domain containing three tetratricopeptide repeats (TPR), a central domain, and a C-terminal UCS (Unc-45/CRO1/She4p) domain . The TPR domain interacts with the MMEVD motif at the C-terminus of Hsp90 . The UCS domain is required for interaction with myosin [11, 16]. Although the function of the central domain remains unknown, it contains regions of limited homology to β-catenin, a key player in the Wnt signaling pathway .
While previous studies have contributed significantly to our understanding of the role of Unc-45 in myosin folding, several questions regarding Unc-45 structure and function remain unanswered. Is Unc-45b required for organization of other sarcomeric structures in addition to the thick and thin filaments? Does overexpression of Unc-45b disrupt myofibril organization in vertebrate skeletal muscles? Which protein domain is involved in the disruptive effect of Unc-45b when overexpressed? Addressing these questions may lead to increased understanding of Unc-45b function in muscle physiology and development that promote new approaches combating chronic muscular disorders.
This study attempts to answer these questions and better ascertain the role of Unc-45b in sarcomere assembly in zebrafish embryos. Our results indicate that knockdown of Unc-45b result in disruption of sarcomere organization and decreased levels of myosin protein expression. We further showed that overexpression of Unc-45b resulted in similar disruption of myosin thick filament organization. The disruption of thick filament organization by Unc-45b depended on the C-terminal UCS domain required for interaction with myosin. Deletion of the C-terminal UCS domain abolished the disruptive effect of Unc-45b overexpression on thick filament organization. Together, these studies indicate that the expression levels of Unc-45b must be precisely regulated to ensure normal myofibril organization during muscle development. Knockdown and overexpression of Unc-45b result in defective myofibril organization.