The myosin-interacting protein SMYD1 is essential for sarcomere organization

Abstract

SummaryAssembly, maintenance and renewal of sarcomeres require highly organized and balanced folding, transport, modification and degradation of sarcomeric proteins. However, the molecules that mediate these processes are largely unknown. Here, we isolated the zebrafish mutant flatline (fla), which shows disturbed sarcomere assembly exclusively in heart and fast-twitch skeletal muscle. By positional cloning we identified a nonsense mutation within the SET-and MYND-domain-containing protein 1 gene (smyd1) to be responsible for the fla phenotype. We found SMYD1 expression to be restricted to the heart and fast-twitch skeletal muscle cells. Within these cell types, SMYD1 localizes to both the sarcomeric M-line, where it physically associates with myosin, and the nucleus, where it supposedly represses transcription through its SET and MYND domains. However, although we found transcript levels of thick filament chaperones, such as Hsp90a1 and UNC-45b, to be severely upregulated in fla, its histone methyltransferase activity -mainly responsible for the nuclear function of SMYD1 -is dispensable for sarcomerogenesis. Accordingly, sarcomere assembly in fla mutant embryos can be reconstituted by ectopically expressing histone methyltransferase-deficient SMYD1. By contrast, ectopic expression of myosinbinding-deficient SMYD1 does not rescue fla mutants, implicating an essential role for the SMYD1-myosin interaction in cardiac and fast-twitch skeletal muscle thick filament assembly.

A compact unc45b‐promoter drives muscle‐specific expression in zebrafish and mouse

“…Next, to assess whether the identified minimal unc45b promoter is able to direct sufficient wild‐type gene expression to rescue muscle mutant embryos, we used the smyd1b ‐deficient flatline mutants, which display severe cardiac and skeletal muscle dysfunction due to impaired myofibrillogenesis (Just et al ., 2011). We first generated a transgenic zebrafish line expressing a Smyd1b‐GFP fusion protein under the control of the compact 195 bp unc45b min promoter ( Tg(unc45b min :smyd1b‐gfp) ).…”

“…2C). This finding suggests that similar to the subcellular localization of endogenous Smyd1b, transgenic Smyd1b‐GFP properly localizes to the sarcomeric M‐band (Just et al ., 2011). Furthermore, similar to the situation in Tg(unc45b min :gfp) zebrafish embryos, 1 h heat shock significantly increased GFP fluorescence intensity in Tg(unc45b min :smyd1b‐gfp) embryos, indicating that the 195 bp unc45b min promoter fragment can be utilized to titrate transgene expression levels in vivo (Fig.…”

“…smyd1b ‐deficient homozygous fla mutant embryos ( fla −/− ) are characterized by severe cardiac and skeletal muscle dysfunction caused by defective myofibrillogenesis in cardiomyocytes and skeletal muscle cells as depicted by an α‐actinin‐specific immunostaining and birefringence analyses (Fig. 3A–D) (Just et al ., 2011). After genotyping and transgene detection, we incrossed Tg(unc45b min :smyd1b‐gfp)/(fla +/− ) fish and evaluated their offspring.…”

“…After genotyping and transgene detection, we incrossed Tg(unc45b min :smyd1b‐gfp)/(fla +/− ) fish and evaluated their offspring. Usually, homozygous flatline mutant embryos ( fla −/− ) can be detected already at 24 hpf by the lack of cardiac contractions and complete paralysis (Just et al ., 2011). None of the investigated embryos exhibited the typical fla heart and skeletal muscle phenotype as demonstrated by measuring cardiac and skeletal muscle functionality (Fig.…”

“…SMYD1 is exclusively expressed in cardiac and skeletal muscle cells from zebrafish and mice (Just et al ., 2011; Nagandla et al ., 2016) where it locates to the cell nucleus, as expected for a histone methyltransferase (HMT), and to the sarcomeric M‐band (Just et al ., 2011). In zebrafish, Smyd1b was shown to be crucial to orchestrate thick filament assembly in cardiomyocytes and fast‐twitch skeletal muscle cells and when defective leads to severe cardiac and skeletal muscle dysfunction due to impaired myofibrillogenesis (Just et al ., 2011; Li et al ., 2013; Prill et al ., 2015; Tan et al ., 2006). Consistently, nullizygous SMYD1 mice die at embryonic day E10.5 due to severe heart malformations and cardiac dysfunction (Gottlieb et al ., 2002).…”