Unc45b is essential for early myofibrillogenesis and costamere formation in zebrafish

Abstract

Despite the prevalence of developmental myopathies resulting from muscle fiber defects, the earliest stages of myogenesis remain poorly understood. Unc45b is a molecular chaperone that mediates the folding of thick-filament myosin during sarcomere formation; however, Unc45b may also mediate specific functions of non-muscle myosins (NMMs). unc45b Mutants have specific defects in striated muscle development, which include myocyte detachment indicative of dysfunctional adhesion complex formation. Given the necessity for non-muscle myosin function in the formation of adhesion complexes and premyofibril templates, we tested the hypothesis that the unc45b mutant phenotype is not mediated solely by interaction with muscle myosin heavy chain (mMHC). We used the advantages of a transparent zebrafish embryo to determine the temporal and spatial patterns of expression for unc45b, non-muscle myosins and mMHC in developing somites. We also examined the formation of myocyte attachment complexes (costameres) in wild-type and unc45b mutant embryos. Our results demonstrate co-expression and co-regulation of Unc45b and NMM in myogenic tissue several hours before any muscle myosin heavy chain is expressed. We also note deficiencies in the localization of costamere components and NMM in unc45b mutants that is consistent with an NMM-mediated role for Unc45b during early myogenesis. This represents a novel role for Unc45b in the earliest stages of muscle development that is independent of muscle mMHC folding.

Loss of function of myosin chaperones triggers Hsf1-mediated transcriptional response in skeletal muscle cells

“…Thus, the response to unc45b deficiency may not entirely be mediated by Hsf1 and most likely includes a further amplification of the transcriptional response by these downstream TFs. Although the skeletal myosins are the most abundant myosins in the myofiber, we cannot exclude that misfolding of the non-muscle myosins, such as the myosins implicated in the formation of costameres mediating fibril attachment [ 21 , 23 ], co-determines the types of genes induced in the unc45b mutant. Interestingly, a significant number of genes involved in visual perception (such as Alpha crystallin A [ 67 ], CNGA3 [ 68 ], and RLBP1 [ 69 ]) are down-regulated in the unc45b mutant.…”

“…Unc45a has been shown to cooperate with Hsp90 in chaperoning mammalian progesterone receptor [ 18 ] and plays a role in pharyngeal and aortic development in zebrafish [ 19 ]. Unc45b was proposed to be required for the folding of myosins in general, including those myosins that are not part of the myofibril [ 20 , 21 ]. Missense mutations in unc45b have been associated with juvenile cataract in humans, a phenotype that is also evident in the unc45b zebrafish mutant [ 22 ].…”

The UNC-45 Myosin Chaperone

“…In addition to thick and thin filament defects, UNC-45b morphants displayed disrupted M-lines and Z-disks in sarcomeres of skeletal muscles, with a significant decrease in myosin protein levels (Bernick et al, 2010). Very recently, Myhre et al demonstrated that UNC-45b co-localizes with non-muscle myosins in zebrafish myogenic tissue prior to the expression of muscle myosin and that an unc-45b mutation disrupts costamere formation and attachment of muscle fibers to the tendon-like myosepta (Myhre et al, 2014). Thus UNC-45b may be important for non-muscle myosin folding at very early stages of muscle development (Myhre et al, 2014), a role that might have been attributed to UNC-45a due its widespread expression and its necessity for myoblast proliferation (Price et al, 2002).…”

“…Knockdown of either form of Apobec2 results in reduced motility, impaired cardiac function, skeletal myofibril disorganization and disrupted muscle fibers and myosepta (tendon equivalents). Apobec2 was shown to co-localize with UNC-45b to the myoseptal boundary and the Z-disks (Etard et al, 2010; Myhre et al, 2014). Although the localization of UNC-45b was not dependent upon the presence of Apobec2, the localization of Apobec2 does depend upon UNC-45b, implicating UNC-45b in muscle fiber attachment to the myoseptum (Etard et al, 2010).…”

“…While the cataract-causing mutation (Arg805Trp) affects a UCS domain residue that is well conserved in vertebrate UCS proteins, no effects upon muscle or heart function were reported by patients (Hansen et al, 2014). To investigate the effects upon eye development, UNC-45b expression in the developing zebrafish lens was assessed; it was found to localize at cell cortices along with non-muscle myosin II (Hansen et al, 2014; Myhre et al, 2014). In support of the importance of unc-45b expression in the eye, the zebrafish unc-45b steif mutant shows a reduced lens size and lens cells contain ectopic actin fibers and swollen, anteriorly-located nuclei that are absent in the wild-type organism (Hansen et al, 2014).…”