Overexpression of Lifeact-GFP Disrupts F-Actin Organization in Cardiomyocytes and Impairs Cardiac Function

Abstract

Lifeact-GFP is a frequently used molecular probe to study F-actin structure and dynamic assembly in living cells. In this study, we generated transgenic zebrafish models expressing Lifeact-GFP specifically in cardiac muscles to investigate the effect of Lifeact-GFP on heart development and its application to study cardiomyopathy. The data showed that transgenic zebrafish with low to moderate levels of Lifeact-GFP expression could be used as a good model to study contractile dynamics of actin filaments in cardiac muscles in vivo. Using this model, we demonstrated that loss of Smyd1b, a lysine methyltransferase, disrupted F-actin filament organization in cardiomyocytes of zebrafish embryos. Our studies, however, also demonstrated that strong Lifeact-GFP expression in cardiomyocytes was detrimental to actin filament organization in cardiomyocytes that led to pericardial edema and early embryonic lethality of zebrafish embryos. Collectively, these data suggest that although Lifeact-GFP is a good probe for visualizing F-actin dynamics, transgenic models need to be carefully evaluated to avoid artifacts induced by Lifeact-GFP overexpression.

Keywords: F-actin filament; Lifeact-GFP; Smyd1; cardiomyocyte; sarcomere.

FIGURE 1

Analysis of congenital cardiomyopathy in smyd1b^sa15678 mutant using Tg(myl7:Lifeact-GFP) transgenic zebrafish model. (A–D) Morphology and Lifeact-GFP expression in the heart region of WT Tg(myl7:Lifeact-GFP)^mb21 (A,B), and smyd1b^sa15678; Tg(myl7:Lifeact-GFP)^mb21 mutant (C,D) transgenic larvae at 72 hpf. Pericardial edema is indicated by arrow in smyd1b^sa15678; Tg(myl7:Lifeact-GFP)^mb21 transgenic mutant larvae (C). Scale bars: 250 μm. (E–J) Actin think filaments in cardiomyocytes revealed by Lifeact-GFP (E,H) and phalloidin staining (F,I) in WT Tg(myl7:Lifeact-GFP)^mb21 (E–G), and smyd1b^sa15678; Tg(myl7:Lifeact-GFP)^mb21 transgenic mutant (H–J) embryos at 72 hpf. (G,J) are merged pictures of (E,F,H,I), respectively. Scale bars: 50 μm.

FIGURE 2

Development of pericardial edema in Tg(myl7:Lifeact-GFP)^mb22 and Tg(myl7:Lifeact-GFP)^mb23 transgenic embryos. (A–D) Morphology of representative transgenic embryos from Tg(myl7:Lifeact-GFP)^mb21 line with no edema (A,B), and Tg(myl7:Lifeact-GFP)^mb22 (C), and Tg(myl7:Lifeact-GFP)^mb23 (D) transgenic lines with edema phenotypes at 3 (C) and 14 dpf (D), respectively. Pericardial edema is indicated by arrows in Tg(myl7:Lifeact-GFP)^mb22 (C), and Tg(myl7:Lifeact-GFP)^mb23 (D) transgenic larvae. Scale bars: 500 μm. (E–G) Lifeact-GFP expression in the hearts of Tg(myl7:Lifeact-GFP)^mb21 (E), Tg(myl7:Lifeact-GFP)^mb22 (F), and Tg(myl7:Lifeact-GFP)^mb23 (G) transgenic embryos at 3 dpf. Scale bars: 100 μm.

FIGURE 3

The effect of Lifeact-GFP expression on actin filament and sarcomere organization in cardiomyocytes of Tg(myl7:Lifeact-GFP)^mb21 and Tg(myl7:Lifeact-GFP)^mb22 transgenic embryos. (A–F) Actin think filaments revealed by Lifeact-GFP (A,D) and phalloidin staining (B,E) in Tg(myl7:Lifeact-GFP)^mb21 (A–C), and Tg(myl7:Lifeact-GFP)^mb22 (D–F) transgenic embryos at 72 hpf. (C,F) are merged pictures of A-B and D-E, respectively. (G–L) The sarcomeric organization of actin think filament and Z-lines revealed by Lifeact-GFP (G,J) anti-α-actinin antibody staining (H,K) in Tg(myl7:Lifeact-GFP)^mb21 (G–I), and Tg(myl7:Lifeact-GFP)^mb22 (J–L) transgenic embryos at 72 hpf. (I,L) are merged pictures of (G,H,J,K), respectively. Scale bars: 25 μm.

FIGURE 4

Identification of transgene integration sites in Tg(myl7:Lifeact-GFP)^mb22 and Tg(myl7:Lifeact-GFP)^mb23 transgenic embryos and the rescue of heart defect by knockdown of Lifeact-GFP expression. (A,B) Mapping the transgene integration sites in Tg(myl7:Lifeact-GFP)^mb22 and Tg(myl7:Lifeact-GFP)^mb23 transgenic embryos at cadherin2 and traf4a genes, respectively. The Lifeact-GFP-MO is indicated by the red line. (C–F) Morphology of heart region of Tg(myl7:Lifeact-GFP)^mb22 transgenic embryos at 72 hpf injected with control-MO (C,D) and Lifeact-GFP-MO (E,F), respectively. Pericardial edema is indicated by the arrow in control-MO injected Tg(myl7:Lifeact-GFP)^mb22 (D) transgenic mutant larvae. H, heart; Yolk shows autofluorescence. Scale bars: 250 μm. (G–L) Lifeact-GFP expression (G,J) and phalloidin staining (H,K) showing actin think filaments in cardiomyocytes of Tg(myl7:Lifeact-GFP)^mb22 transgenic embryos injected with control-MO (G–I) or Lifeact-GFP-MO (J–L) at 72 hpf transgenic embryos. (I,L) are merged pictures of (G,H,J,K), respectively. Scale bars: 50 μm.