PgmNr Z6065: Loss of SET- and MYND-domain-containing protein 1a (SMYD1a) leads to sarcomeric disorganization in zebrafish.

Authors:
S. Rudeck; W. Rottbauer; S. Just


Institutes
University Hospital, Ulm, Baden-Württemberg, DE.


Abstract:

Assembly, maintenance and renewal of sarcomeric units require highly organized and balanced folding, transport, modification and degradation of sarcomeric proteins. However, mechanisms that regulate these fundamental processes are only poorly understood, but of great clinical importance, since many cardiac and skeletal muscle diseases are associated with defective sarcomerogenesis.
The mutant flatline (fla) shows disturbed sarcomere assembly exclusively in heart and fast-twitch skeletal muscle. We identified a nonsense mutation within the SET- and MYND-domain-containing protein 1 gene (smyd1b) to be responsible for the fla phenotype. Smyd1b localizes to the sarcomeric M-line, where it associates with myosin. Moreover, the zebrafish genome contains a second highly conserved smyd1-orthologue, smyd1a, which has minor importance for myofiber organization. We found that smyd1a mRNA, similar to smyd1b, is strongly expressed in skeletal muscle cells and to a weaker extent in the embryonic heart. To characterize the in vivo function of smyd1a, we performed a morpholino-mediated knock-down of smyd1a, which leads to a cardiac defect accompanied by progressive reduction of ventricular contractility. To investigate whether the observed cardiac defects are caused by defective specification of cardiomyocytes, we performed a MF20/S46 immunostaining and found normal specification of atrial and ventricular cardiomyocytes. Furthermore, MF20 staining of smyd1a morphants revealed disorganization of skeletal muscle sarcomeres. To assess whether the observed sarcomeric disorganization is caused by impaired skeletal muscle development, we further investigated the expression of myoG and myoD by performing in situ hybridization and found no alteration compared to controls, indicating that loss of Smyd1a function does not affect early skeletal muscle development. To compare the localization of the different smyd1-orthologues, we generated transgenic lines using a heart and skeletal muscle specific unc45b-promoter followed by smyd1a- or smyd1b-gfp fusion constructs. Interestingly, both orthologues, Smyd1a and Smyd1b localize in an alternating pattern to α-actinin, indicating M-band localization, and implying that both proteins might have redundant functions. To evaluate this hypothesis, we performed rescue experiments by injection of smyd1a- or smyd1b-mRNA into fla mutant oocytes. Remarkably, smyd1b deficiency was functionally and structurally rescued by ectopic expression of either smyd1a-mRNA or smyd1b-mRNA, demonstrating redundant roles for Smyd1a and Smyd1b regarding the control of sarcomere organization in zebrafish.



ZFIN Genetics Index
1. smyd1a
2. smyd1b