<i>Still Heart</i> Encodes a Structural HMT, SMYD1b, with Chaperone-Like Function during Fast Muscle Sarcomere Assembly

<div><p>The vertebrate sarcomere is a complex and highly organized contractile structure whose assembly and function requires the coordination of hundreds of proteins. Proteins require proper folding and incorporation into the sarcomere by assembly factors, and they must also be maintained and replaced due to the constant physical stress of muscle contraction. Zebrafish mutants affecting muscle assembly and maintenance have proven to be an ideal tool for identification and analysis of factors necessary for these processes. The <i>still heart</i> mutant was identified due to motility defects and a nonfunctional heart. The cognate gene for the mutant was shown to be <i>smyd1b</i> and the <i>still heart</i> mutation results in an early nonsense codon. SMYD1 mutants show a lack of heart looping and chamber definition due to a lack of expression of heart morphogenesis factors <i>gata4</i>, <i>gata5</i> and <i>hand2</i>. On a cellular level, fast muscle fibers in homozygous mutants do not form mature sarcomeres due to the lack of fast muscle myosin incorporation by SMYD1b when sarcomeres are first being assembled (19hpf), supporting SMYD1b as an assembly protein during sarcomere formation.</p></div

SMYD1b is co-regulated with other myosin chaperones HSP90a1 and UNC45b.

<p>In-situ hybridization staining of wild type and <i>still heart</i> embryos shows that <i>hsp90a1</i> expression is normal in <i>still heart</i> mutants, when compared to wild type embryos at 19hpf (A&B). However, the expression of <i>hsp90a1</i> and <i>unc45b</i> dramatically increases in <i>still heart</i> mutants at 24hpf (C-F & K) and 48hpf throughout the somites (G-J & M). Additionally, <i>smyd1b</i> expression increases significantly when UNC45b is absent in <i>steif</i> mutants (N), supporting co-regulation of these three genes. (O) A time course of <i>smyd1b</i> expression during muscle formation reveals that <i>smyd1b</i> is expressed early at 10hpf when <i>unc45b</i> is expressed and increases in its expression as muscle development progresses. Due to the rapid development of <i>unc45b</i> staining in the somites of the embryo in panel J, the head while present, has no background staining and is not clear in this focal plane. (qPCR: n = 3, 30 embryos each time/phenotype. Error bars are standard deviation.).</p

SMYD1b is required for fast myosin incorporation during sarcomere assembly.

<p>At 48hpf, fast myosin (F310—green) and actin (phalloidin—red) staining is visible in the premyofibrils in wt zebrafish tails (A&B), while absent from the premyofibrils in <i>sth</i> fast muscle tissue (C&D). Slow muscle (F59) develops normally in both wild type and <i>still heart</i> zebrafish at 48hpf (E&F, G&H). At 19hpf, in wild type embryos, fast myosin (F310) is beginning to be incorporated into the maturing myofibril (I) and overlaps (white arrowheads) with the developing actin (phalloidin) (J) fibers in trunk muscle (K, merge, K’ inset, white arrowhead). Fast myosin is not incorporated into the maturing premyofibril (L), although actin fibers are still present (M&N, N’ inset).</p

<i>Still heart</i>, a <i>smyd1b</i> mutant, has defects in heart and fast skeletal muscle tissue.

<p>A lateral view of 48hpf wild type (A) and <i>still heart</i> (<i>sth</i>) mutants (B), which have pericardial edema, small eyes, malformed head and reduced motility. Black arrowheads highlight the pericardial edema in sth mutants and the absence of edema in wild type. White arrowheads indicate blood pooling in the mutant and the absence of pooling in wild type. (C&D) <i>Sth</i> mutant hearts are underdeveloped and do not beat. (E-G) Examination of lateral myofibers at 5dpf under DIC microscopy revealed striations, indicative of fully formed sarcomeres, are visible in the myofibers of wild type muscle (E, black arrowheads), while absent in the fast muscle of <i>still heart</i> mutants (F); striations are present in <i>sth</i> slow muscle (G, black arrowhead) but are disturbed by nuclei and fluid-filled spaces (G, white arrowhead). Sequencing of <i>smyd1b</i> cDNA from wild type embryos (H) and <i>sth</i> mutant embryos (I) revealed a 9 nucleotide insertion between exon 1 and 2 in the <i>smyd1b</i> mRNA, creating an in-frame stop codon (I, underlined sequence). The insertion is the first 9 nucleotides of intron one as sequenced from wild type smyd1b genomic sequence (J). This is a result of a transition mutation in the splice donor site of intron 1 (I, green letter in sequence, J, outlined letter in sequence). This results in a premature truncation of the SMYD1b protein after exon 1 (K).</p