STARS Is Essential to Maintain Cardiac Development and Function In Vivo via a SRF Pathway

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.


Introduction
Pathological cardiac remodeling due to sustained mechanical stress, tissue injury and neurohormonal stimulus can lead to hypertrophic growth, cardiomyopathy and heart failure. In order to decipher the complexity of the multiple gene networks that converge to initiate and coordinate such response, essential upstream integral factors need to be identified and characterized. STARS (STriated muscle Activator of Rho Signaling [also known as MS1 (Myocyte Stress 1) and ABRA (Actin-Binding Rho Activating-protein)], is an evolutionarily-conserved sarcomeric actin binding protein that is acutely (and transiently) up-regulated in response to pressure overload left ventricular hypertrophy with a peak expression well before any detectable increase in left ventricular (LV) mass [1], [2]. This suggests a possible role for STARS in the initial signaling of cardiac remodeling such as the hypertrophic response. Sustained over-expression of STARS in the mouse heart have no effect on LV mass but resulted in an increased sensitivity to hemodynamic stress leading to cardiac hypertrophy and heart failure [3].
STARS expression parallels the development and regression of skeletal muscle hypertrophy in humans [4]. Moreover, STARS can stimulate serum response factor (SRF)-dependent transcription in cultured cells by inducing the nuclear accumulation of the SRF co-factors, myocardin-related transcription factors (MRTFs) through a mechanism dependent on RhoA (ras homolog gene family, member A) and actin polymerization [2], [5]. Forced overexpression of STARS in vitro results in an increase in cell size, induces expression of several MRTF/SRF target genes and provides protection against apoptosis [6], providing further evidence of its role in striated muscle pathophysiology.
STARS is expressed during embryonic cardiac development [1], [2] and has been implicated as a key factor involved in myogenic differentiation [7]. STARS could play a more widespread role in muscle pathophysiology such as fluid shear stress induced blood vessel formation (arteriogenesis) [8], insulin resistance and fat metabolism in skeletal muscle [9], [10]. To gain a better understanding on the function and mechanism of STARS in cardiac biology in vivo, we investigated the orthologue in zebrafish and report that knockdown of STARS in zebrafish results in severe contractile dysfunction that can be rescued by SRF. We also provide evidence indicating that SRF can regulate STARS transcription establishing a newly discovered autoregulatory feed-forward loop for STARS-SRF signaling.

Expression of zSTARS in the Zebrafish
The zebrafish STARS orthologue (zSTARS) contains a high degree of homology to other STARS orthologues, particularly in the actin-binding regions critical for signaling from actin to SRFmediated gene transcription ( Figure S1, Text S1). Whole mount in situ hybridization was performed in order to investigate the developmental expression pattern of zSTARS. zSTARS expression was detected in both the atrium and ventricle of the zebrafish heart beginning at 48 hpf ( Figure 1A-B). zSTARS transcript was also detected in somites by the 16 somite stage (17 hpf) ( Figure 1C-E). Other notable embryonic sites of expression include the developing brain ( Figure 1D, E), jaw structures, and pectoral fins (data not shown).

Knockdown of zSTARS in Zebrafish Leads to Structural and Functional Cardiac Abnormalities
To assess the in vivo role of zSTARS during cardiac development, we performed morpholino-induced knockdown of zSTARS during zebrafish embryonic development. Injection of a morpholino directed against the translation initiation site of zSTARS causes a striking cardiovascular phenotype by 48-56 hpf (126 out of 164 embryos, 77%). Morphologically, the heart tube is incompletely looped, causing the atrium and ventricle to retain an immature linear form (Figure 2A and B, Figure S2A and S2B). The atrium is dilated, and overall contractility of both chambers appears impaired (Movie S1, Movie S2). These cardiac abnormalities lead to significant pericardial edema, with congestion and pooling of blood in the sinus venosus. The most significantly affected embryos show complete absence of circulation despite a beating heart. zSTARS morphants also exhibited abnormal somite formation and curvature of the longitudinal axis, suggesting an important developmental role for zSTARS in the non-cardiac sites of expression ( Figure 2C-E). In contrast, injection of a control morpholino with five mismatches to the zSTARS sequence does not cause obvious developmental abnormalities (0 out of 152 embryos; x 2 = 194, p,0.001) or obstruction of the outflow tract ( Figure S2C).

Over-expression of SRF Rescues the Cardiac Phenotype Induced by zSTARS Knockdown
We quantified cardiac function and dimensions in zSTARS morphants using high-speed video microscopy [11]. In addition to the abnormal morphology of heart development, zSTARS knockdown results in a smaller ventricle. Specifically, ventricular end diastolic size is decreased compared to controls. At end systole, the morphant ventricle is unable to contract to the same degree as control ventricles resulting in a larger ventricular end diastolic size ( Figure 3A, Figure S3, Movie S1, Movie S2). Together, these changes result in impaired cardiac performance shown by a significant decrease in ventricular fractional shortening compared to control ( Figure 3B). Injection of zsrf mRNA suppresses the cardiac phenotype induced by zSTARS knockdown: fractional shortening (p,0.05) and ventricular systolic and diastolic dimensions are restored to values indistinguishable from control embryos ( Figure 3, Movie S3).

SRF Regulate STARS Transcription via an Autoregulatory Feed-forward Loop
Ectopic over-expression of STARS significantly increased the activity of the STARS proximal reporter, which contains a conserved SRF binding site (position 2305 bp). In vivo binding of SRF to this site was confirmed by ChIP ( Figure 4). This and similar SRF-chromatin preparations have been used to detect SRF binding to the sodium-calcium exchanger (NCX1) gene [12]. The SRF inhibitor CCG-1423, which works by inhibiting MRTF nuclear localization [9], completely blocked STARS proximal reporter activity in H9c2 cells. Of interest, over-expression of rat STARS also significantly increased srf mRNA and several MRTF-SRF target genes in H9c2 cells [6], [13].

Discussion
In this study, we isolated the mammalian homologue of STARS in zebrafish and examined its role in cardiac function and development. Morpholino knockdown of STARS in the zebrafish results in abnormal cardiac looping, an enlarged atrium, and severe contractile dysfunction with resulting marked impaired circulation.These phenotypes confirm an important role for STARS in cardiac development and function. Notably, the cardiac phenotype of zSTARS knockdown resembles that of a zebrafish morpholino against cardiac troponin T (tnnt2), which can be mutated in human hypertrophic cardiomyopathy [14]. Both zSTARS and zTnnt2 knockdown result in enlarged atria, decreased ventricular fractional shortening, and decreased end diastolic dimension. Thus, genes such as STARS and Tnnt2 which regulate hypertrophic response in adults may also play critical roles in cardiac embryonic development. Furthermore, these examples illustrate how studying developmental phenotypes arising from zebrafish knockdown can shed light on physiological mechanisms of cardiac stress response (such as cardiomyopathy and contractility dysfunction).
Previous studies have demonstrated that STARS can activate srf transcription activity in vitro [2], [3]. In the present study, we provide the first in vivo evidence that srf is an essential downstream target of STARS by showing that injection of zsrf mRNA rescues cardiac abnormalities caused by morpholino-mediated zSTARS knockdown. SRF is known to be involved in cardiac development, structure and function [15], [16] and knockdown of STARS was shown to reduce SRF activity [5]. In addition, it was demonstrated that cardiac-specific deletion of SRF in the embryonic heart results in cardiac defects [15] and deletion of SRF from the adult heart caused reduced contractility leading to dilated cardiomyopathy [17]. It is conceivable that knockdown of STARS decreased SRF activity, which resulted in decreased cardiac function. Consistent with this hypothesis, SRF expression restored SRF activity and rescued the cardiac abnormalities as shown here.
In addition to its effects on cardiac function, zSTARS knockdown causes phenotypes consistent with its non-cardiac sites of expression, including abnormal somite formation and curvature and shortening of the longitudinal axis. As the axial skeleton and skeletal muscle are derived from somites [18], this novel finding implies an important role for STARS in skeletal muscle development. STARS levels were up-regulated in human skeletal hypertrophy and down-regulated in skeletal muscle atrophy [4] and in aged skeletal muscle of mice [19] demonstrating a role for STARS in the maintenance of skeletal muscle. Interestingly, the same finding was observed for SRF [4], [19] and deletion of SRF in mice resulted in impaired muscle growth [20]. Collectively, these results extend the findings in cardiac tissue and suggest that The present study demonstrates for the first time that STARSdeficient animals display severe abnormalities in cardiac development and function, which were rescued by SRF overexpression. Our study further highlights the importance of sarcomeric proteins in cardiac development and biology [21], [22]. Both STARS and SRF play comparable roles in not just establishing and maintaining cardiac physiology but also in skeletal muscle and brain development and function. These findings provide support for a widespread role of the STARS-SRF signaling axis in multiple tissues in vivo. Understanding the precise mechanism of STARS expression and regulation could provide a novel avenue to dissect the initiation process of cardiac dysfunction and heart failure.

Zebrafish Experiments
All zebrafish experiments were approved by the Subcommittee on Research Animal Care at Massachusetts General Hospital. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). IACUC approval number A3596-01. Zebrafish (AB strain) were raised and maintained using standard protocols.
An antisense morpholino oligonucleotide was designed against the translation initiation site, with the sequence: GCTGTACT-CATGGTGTTTTAATTTG (Gene-Tools, Philomath, OR). Antisense and scrambled morpholinos were injected according to standard procedures [23]. For expression of zebrafish (z) SRF, the T7 phage promoter sequence was added to the 59 end of the zsrf cDNA (Open Biosystems, Huntsville, AL) by PCR; zsrf mRNA was synthesized by in vitro transcription and injected using published methods [11], [23], [24].

In situ Hybridization
Digoxigenin labeled RNA probes were generated by in vitro transcription according to manufacturer's instructions (MEGAscript SP6, Ambion). Embryos were fixed at the indicated times in 4% formaldehyde in PBS overnight at 4uC, and transferred to methanol for storage at 220uC. In situ hybridization on whole mount embryos was performed as previously described [11].

Antisense Morpholino Injection
An antisense morpholino oligonucleotide was designed against the translation initiation site, with the sequence: GCTGTACT-CATGGTGTTTTAATTTG (Gene-Tools). A control morpholino with 5 mismatches was also designed: GCTcTACT-gATGcTGTTTaAATaTG. Morpholinos were dissolved in Danieau's buffer according to manufacturer's instructions at a concentration of 300 mM, and injected into embryos at the 1 cell stage [23].

RNA Injection
The T7 phage promoter sequence was added to the 59 end of the cDNA encoding zsrf (Open Biosystems, Huntsville, AL) by PCR. This template was used to synthesize capped mRNA encoding the full-length coding sequence of srf by in vitro transcription (mMessage Machine, Ambion). mRNA was diluted in Danieau's buffer to a final concentration of 100 ng/mL and coinjected with morpholino (at 400 mM) as above. n = 4 for each condition.

Ventricular Function Measurements and M-mode Recordings
Extensive description on the measurement of cardiovascular function and the creation of m-mode images have been published elsewhere. Ventricular size and fractional shortening were measured as previously described [24].
Transfection of STARS Luciferase reporters were done as described [7]. Briefly, 0.5 ug of STARS reporter plasmids (or its empty control vector pGL3-promoter) and 10 ng of Renilla reporter was cotransfected into H9c2 cells (60% confluency) in 24well plates using the JetPei reagent (Source BioScience). For standard transfection, cell extracts were measured for activity after 48 h using a Dual-Luciferase Assay kit (Promega). For SRF inhibition experiments, CCG-1423 (1 mM; Sigma) was added 24 h post-transfection and left for a further 24 h before assayed.

Statistical Analysis
Data are presented as mean6SEM. Data were analyzed by ANOVA followed by Bonferroni post hoc testing (Graph Pad Prism, La Jolla, CA). Text S1

Supporting Information
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