Differential effects of thin and thick filament disruption on zebrafish smooth muscle regulatory proteins

Abstract

Background: The smooth muscle actin binding proteins Caldesmon and Tropomyosin (Tm) promote thin filament assembly by stabilizing actin polymerization, however, whether filament assembly affects either the stability or activation of these and other smooth muscle regulatory proteins is not known.

Methods: Measurement of smooth muscle regulatory protein levels in wild type zebrafish larvae following antisense knockdown of smooth muscle actin (Acta2) and myosin heavy chain (Myh11) proteins, and in colourless mutants that lack enteric nerves. Comparison of intestinal peristalsis in wild type and colourless larvae.

Key results: Knockdown of Acta2 led to reduced levels of phospho-Caldesmon and Tm. Total Caldesmon and phospho-myosin light chain (p-Mlc) levels were unaffected. Knockdown of Myh11 had no effect on the levels of either of these proteins. Phospho-Caldesmon and p-Mlc levels were markedly reduced in colourless mutants that have intestinal motility comparable with wild type larvae.

Conclusions & inferences: These in vivo findings provide new information regarding the activation and stability of smooth muscle regulatory proteins in zebrafish larvae and their role in intestinal peristalsis in this model organism.

Figure 1

Thin and thick filament disruption in zebrafish larvae. (A, B) Western blot showing knockdown of smooth muscle actin (Acta2) and myosin heavy chain (Myh11) proteins in the intestine of 3 dpf morpholino injected larvae. (C–J) Lateral fluorescent images of live 6 dpf wild type and acta2 morpholino (C–F) and myh11 morpholino (G–J) injected larvae 2 and 18 h after ingestion of fluorescent beads. In these images, the red asterisk (*) identifies the intestinal lumen; the red arrow points to fluorescent beads adherent to the fin bud. MO, morpholino; WT, wild type control. Larvae oriented with anterior – left; posterior – right.

Figure 2

Smooth muscle regulatory proteins in smooth muscle actin (Acta2) deficient larvae. Western blot of intestinal protein derived from 3 to 5 dpf wild type and Acta2 deficient larvae. Age designations are indicated at the bottom of each lane. (A) This blot shows successful knockdown of Acta2 protein in acta2 morpholino injected larvae, along with reduced levels of Tropomyosin (Tm). beta-Actin (Actb) serves as loading control. (B) This blot shows reduced p-Caldesmon (pCad) in 4 and 5 dpf Acta2 deficient larvae but stable levels of total Caldesmon (Cad). The upper bands in these blots corresponds to the high molecular weight Caldesmon isoform. (C) This blot shows stable phosphomyosin light chain (p-Mlc) levels in Acta2 deficient larvae. WT, wild type; Act-MO, acta2 morpholino injected.

Figure 3

Caldesmon binding in smooth muscle actin (Acta2) deficient larvae. (A) Western blots of intestinal protein from wild type and Acta2 deficient larvae following immuno-precipitation using total Caldesmon antibody followed by blotting with anti-beta-Actin (Actb) and anti-myosin heavy chain (Myh11) antibodies. Caldesmon binds Acta2 and Myh11 in wild type intestine. Caldesmon remains bound to Myh11 in Acta2 deficient larvae. Total refers to intestinal protein prior to Caldesmon immunoprecipitation. (B) Western blots of intestinal protein from wild type and Acta2 deficient larvae assayed for levels of phospho-ERK (pERK) at the indicated developmental stages. Beta-actin (Actb) serves as loading control.

Figure 4

Smooth mucle regulatory protein levels are unchanged in myosin heavy chain (Myh11) deficient larvae. (A–D) Western blots of intestinal proteins derived from 3 to 5 dpf wild type and Myh11 morpholino (MO) injected larvae. (A) Knockdown of Myh11 protein in morpholino injected larvae. Acat2 levels are unchanged by the knockdown and serve as a loading control. (B) p-Caldesmon levels are unchanged with respect to total Caldesmon levels in Myh11 deficient larvae. (C, D) Tropomyosin (Tm) and phospho-myosin light chain (p-Mlc) levels are unchanged with respect to beta-actin (Actb) levels in Myh11 deficient larvae.

Figure 5

Reduced levels of activated smooth muscle regulatory proteins in colourless (cls) larvae. Western blots of phospho-myosin light chain (p-Mlc), p-Caldesmon and total Calesmond in intestinal extracts from 3 to 6 dpf wild type (WT) and cls larvae. Phospho-myosin light chain (p-Mlc) and p-Cad levels are markedly reduced in cls.

Figure 6

Peristalsis is preserved in colourless (cls). (A, B) Lateral confocal projections of live 6 dpf wild type [WT: sox10^+/+; Tg(NBT:dsRed)] and cls (sox10^m148/m148; Tg(NBT:dsRed) larvae. Red arrows point to cell bodies of enteric neurons in wild type larvae that are absent in cls. Inset shows high magnification image of intestine with enteric neuron cell bodies (red arrows), axon vesicles containing dsRed protein (green arrows) and overlying skin neurons (yellow arrows). (C–F) Lateral fluorescent images of live 6 dpf wild type and cls larvae 10 and 22 h after ingestion of fluorescent beads. At 22 h (E, F), there is low level residual fluorescence from bile in WT and cls. A small number of residual beads are present in this cls larvae (blue arrow). Blue asterisk (**) denotes spinal cord in A, B. Orientation of larvae in all panels is anterior – right; posterior – left.