STARS is essential to maintain cardiac development and function in vivo via a SRF pathway

Abstract

Background: STARS (STriated muscle Activator of Rho Signaling) is a sarcomeric protein expressed early in cardiac development that acts as an acute stress sensor for pathological remodeling. However the role of STARS in cardiac development and function is incompletely understood. Here, we investigated the role of STARS in heart development and function in the zebrafish model and in vitro.

Methodology and principal findings: Expression of zebrafish STARS (zSTARS) first occurs in the somites by the 16 somite stage [17 hours post fertilization (hpf)]. zSTARS is expressed in both chambers of the heart by 48 hpf, and also in the developing brain, jaw structures and pectoral fins. Morpholino-induced knockdown of zSTARS alters atrial and ventricular dimensions and decreases ventricular fractional shortening (measured by high-speed video microscopy), with pericardial edema and decreased or absent circulation [abnormal cardiac phenotypes in 126/164 (77%) of morpholino-injected embryos vs. 0/152 (0%) of control morpholino embryos]. Co-injection of zsrf (serum response factor) mRNA rescues the cardiac phenotype of zSTARS knockdown, resulting in improved fractional shortening and ventricular end-diastolic dimensions. Ectopic over-expression of STARS in vitro activates the STARS proximal promoter, which contains a conserved SRF site. Chromatin immunoprecipitation demonstrates that SRF binds to this site in vivo and the SRF inhibitor CCG-1423 completely blocks STARS proximal reporter activity in H9c2 cells.

Conclusions/significance: This study demonstrates for the first time that STARS deficiency severely disrupts cardiac development and function in vivo and revealed a novel STARS-SRF feed-forward autoregulatory loop that could play an essential role in STARS regulation and cardiac function.

Figure 1. Whole mount in situ expression of zSTARS in zebrafish embryos.

A. Frontal view of 48 hpf embryo showing zSTARS expression in ventricle (v) and atrium (a). B. Schematic depicting position of ventricular (v) and atrium (a) in the view from part A. C. Expression in somites at 16 somite stage (17 hpf). D. and E. Expression in somites and central nervous system at 24 hpf (D) and 48 hpf (E).

Figure 2. Developmental phenotype of morpholino-induced knockdown of zSTARS: lateral views of 56 hpf embryos.

A. Embryo injected with control morpholino (with 5 mismatches). The cardiac silhouette is demarcated by arrowheads. The heart tube is looped so that the ventricle (v) and atrium (a) are closely apposed (inset). Circulating red blood cells (rbc) are visible in a thin rim along the inferior aspect of the yolk and within the heart. B. Embryo injected with zSTARS morpholino. The heart tube is unlooped so that the ventricle (v) and atrium (a) are co-linear, with atrial dilation (inset). There is significant edema in the pericardium and over the yolk, with stasis of red blood cells (rbc) over the yolk. C. Lateral view of entire 56 hpf embryo following injection of control, mismatch morpholino. D. and E. 56 hpf embryos showing representative phenotypes of zSTARS morpholino injection.

Figure 3. Ventricular function and dimensions based on quantitative analysis of high-speed video microscopy.

A. Ventricular dimensions at end-diastole (black bars) and end-systole (white bars). Conditions are identical to those in part a. Values plotted are mean (n = 4 embryos) ± standard deviation. Asterisk (*) denotes statistically significant difference by ANOVA. B. Ventricular fractional shortening observed after injection of: control mismatched morpholino (MM MO), zSTARS morpholino + srf mRNA (MO + SRF), srf mRNA only (SRF only), or zSTARS morpholino only (MO only). Values plotted are mean (n = 4 embryos) ± standard deviation. Asterisk (*) denotes statistically significant difference by ANOVA.

Figure 4. Binding of SRF to the STARS promoter.

A. STARS expression activates reporter gene constructs containing the conserved serum response element (SRE). Luciferase activity is shown for two constructs upstream of the STARS transcription start site. B. Chromatin immunoprecipitation assays were performed with formaldehyde cross-linked chromatin isolated from feline adult cardiomyocytes. Asterisk (*) denotes PCR primer locations. Immunoprecipitations were performed without primary antibody (No Ab) as a negative control, with anti-SRF antibody. Input DNA is also shown as a positive control. Similar results were observed in four independent experiments. C. The SRF inhibitor CCG-1423 (1 µM) abolished STARS −365/+60 promoter-reporter activity in H9c2 cells (n = 3 experiments, in triplicates).