Smooth muscle caldesmon modulates peristalsis in the wild type and non-innervated zebrafish intestine.
BACKGROUND
The high molecular weight isoform of the actin-binding protein Caldesmon (h-CaD) regulates smooth muscle contractile function by modulating cross-bridge cycling of myosin heads. The normal inhibitory activity of h-CaD is regulated by the enteric nervous system; however, the role of h-CaD during intestinal peristalsis has never been studied.


METHODS
We identified a zebrafish paralog of the human CALD1 gene that encodes an h-CaD isoform expressed in intestinal smooth muscle. We examined the role of h-CaD during intestinal peristalsis in zebrafish larvae by knocking down the h-CaD protein using an antisense morpholino oligonucleotide. We also developed transgenic zebrafish that express inhibitory peptides derived from the h-CaD myosin and actin-binding domains, and examined their effect on peristalsis in wild-type zebrafish larvae and sox10 (colourless) mutant larvae that lack enteric nerves.


RESULTS
Genomic analyses identified two zebrafish Caldesmon paralogs. The cald1a ortholog encoded a high molecular weight isoform generated by alternative splicing whose intestinal expression was restricted to smooth muscle. Propulsive intestinal peristalsis was increased in wild-type zebrafish larvae by h-CaD knockdown and by expression of transgenes encoding inhibitory myosin and actin-binding domain peptides. Peristalsis in the non-innervated intestine of sox10 (colourless) larvae was partially restored by h-CaD knockdown and expression of the myosin-binding peptide.


CONCLUSIONS
Disruption of the normal inhibitory function of h-CaD enhances intestinal peristalsis in both wild-type zebrafish larvae and mutant larvae that lack enteric nerves, thus confirming a physiologic role for regulation of smooth muscle contraction at the actin filament.
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.
J
J
Abrams
G
G
Davuluri
C
C
Seiler
M
M
Pack
D000199:Actins / Q000378:metabolism; D017398:Alternative Splicing; D000818:Animals; D030801:Animals, Genetically Modified; D002148:Calmodulin-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007422:Intestines / Q000294:innervation* / Q000502:physiology*; D009119:Muscle Contraction / Q000502:physiology; D009130:Muscle, Smooth / Q000294:innervation / Q000502:physiology*; D009218:Myosins / Q000378:metabolism; D010528:Peristalsis / Q000502:physiology*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D015027:Zebrafish / Q000033:anatomy & histology* / Q000502:physiology*
D000199:Actins; D002148:Calmodulin-Binding Proteins; D020033:Protein Isoforms; D009218:Myosins