Muscleblind-like 1 is required for normal heart valve development in vivo.

BACKGROUND: Development of the valves and septa of the heart depends on the formation and remodeling of the endocardial cushions in the atrioventricular canal and outflow tract. These cushions are populated by mesenchyme produced from the endocardium by epithelial-mesenchymal transition (EMT). The endocardial cushions are remodeled into the valves at post-EMT stages via differentiation of the mesenchyme and changes in the extracellular matrix (ECM). Transforming growth factor ß (TGFß) signaling has been implicated in both the induction of EMT in the endocardial cushions and the remodeling of the valves at post-EMT stages. We previously identified the RNA binding protein muscleblind-like 1 (MBNL1) as a negative regulator of TGFß signaling and EMT in chicken endocardial cushions ex vivo. Here, we investigate the role of MBNL1 in endocardial cushion development and valvulogenesis in Mbnl1(∆E3/∆E3) mice, which are null for MBNL1 protein. METHODS: Collagen gel invasion assays, histology, immunohistochemistry, real-time RT-PCR, optical coherence tomography, and echocardiography were used to evaluate EMT and TGFß signaling in the endocardial cushions, and morphogenesis, ECM composition, and function of the heart valves. RESULTS: As in chicken, the loss of MBNL1 promotes precocious TGFß signaling and EMT in the endocardial cushions. Surprisingly, this does not lead to the production of excess mesenchyme, but later valve morphogenesis is aberrant. Adult Mbnl1(∆E3/∆E3) mice exhibit valve dysmorphia with elevated TGFß signaling, changes in ECM composition, and increased pigmentation. This is accompanied by a high incidence of regurgitation across both inflow and outflow valves. Mbnl1(∆E3/∆E3) mice also have a high incidence of ostium secundum septal defects accompanied by atrial communication, but do not develop overt cardiomyopathy. CONCLUSIONS: Together, these data indicate that MBNL1 plays a conserved role in negatively regulating TGFß signaling, and is required for normal valve morphogenesis and homeostasis in vivo.

Gene Expression Analyses during Spontaneous Reversal of Cardiomyopathy in Mice with Repressed Nuclear CUG-BP, Elav-Like Family (CELF) Activity in Heart Muscle.

CUG-BP, Elav-like family (CELF) proteins regulate cell type- and developmental stage-specific alternative splicing in the heart. Repression of CELF-mediated splicing activity via expression of a nuclear dominant negative CELF protein in heart muscle was previously shown to induce dysregulation of alternative splicing, cardiac dysfunction, cardiac hypertrophy, and dilated cardiomyopathy in MHC-CELFΔ transgenic mice. A "mild" line of MHC-CELFΔ mice that expresses a lower level of the dominant negative protein exhibits cardiac dysfunction and myopathy at a young age, but spontaneously recovers normal cardiac function and heart size with age despite the persistence of splicing defects. To the best of our knowledge, this was the first example of a genetically induced cardiomyopathy that spontaneously recovers without intervention. In this study, we explored the basis for this recovery. We examined whether a transcriptional program regulated by serum response factor (SRF) that is dysregulated in juvenile MHC-CELFΔ mice is restored in the mild line with age, and evaluated global changes in gene expression by microarray analyses. We found that differences in gene expression between the mild line and wild type hearts are greatly reduced in older animals, including a partial recovery of SRF target gene expression. We did not find evidence of a new compensatory pathway being activated in the mild line with age, and propose that recovery may occur due to developmental stage-specific compatibility of CELF-dependent splice variants with the cellular environment of the cardiomyocyte.

Gene expression analyses implicate an alternative splicing program in regulating contractile gene expression and serum response factor activity in mice.

Members of the CUG-BP, Elav-like family (CELF) regulate alternative splicing in the heart. In MHC-CELFΔ transgenic mice, CELF splicing activity is inhibited postnatally in heart muscle via expression of a nuclear dominant negative CELF protein under an α-myosin heavy chain promoter. MHC-CELFΔ mice develop dilated cardiomyopathy characterized by alternative splicing defects, enlarged hearts, and severe contractile dysfunction. In this study, gene expression profiles in the hearts of wild type, high- and low-expressing lines of MHC-CELFΔ mice were compared using microarrays. Gene ontology and pathway analyses identified contraction and calcium signaling as the most affected processes. Network analysis revealed that the serum response factor (SRF) network is highly affected. Downstream targets of SRF were up-regulated in MHC-CELFΔ mice compared to the wild type, suggesting an increase in SRF activity. Although SRF levels remained unchanged, known inhibitors of SRF activity were down-regulated. Conversely, we found that these inhibitors are up-regulated and downstream SRF targets are down-regulated in the hearts of MCKCUG-BP1 mice, which mildly over-express CELF1 in heart and skeletal muscle. This suggests that changes in SRF activity are a consequence of changes in CELF-mediated regulation rather than a secondary result of compensatory pathways in heart failure. In MHC-CELFΔ males, where the phenotype is only partially penetrant, both alternative splicing changes and down-regulation of inhibitors of SRF correlate with the development of cardiomyopathy. Together, these results strongly support a role for CELF-mediated alternative splicing in the regulation of contractile gene expression, achieved in part through modulating the activity of SRF, a key cardiac transcription factor.

Loss of muscleblind-like 1 promotes invasive mesenchyme formation in endocardial cushions by stimulating autocrine TGFß3.

BACKGROUND: Valvulogenesis and septation in the developing heart depend on the formation and remodeling of endocardial cushions in the atrioventricular canal (AVC) and outflow tract (OFT). These cushions are invaded by a subpopulation of endocardial cells that undergo an epithelial-mesenchymal transition in response to paracrine and autocrine transforming growth factor ß (TGFß) signals. We previously demonstrated that the RNA binding protein muscleblind-like 1 (MBNL1) is expressed specifically in the cushion endocardium, and knockdown of MBNL1 in stage 14 embryonic chicken AVC explants enhances TGFß-dependent endocardial cell invasion. RESULTS: In this study, we demonstrate that the effect of MBNL1 knockdown on invasion remains dependent on TGFß3 after it is no longer required to induce basal levels of invasion. TGFß3, but not TGFß2, levels are elevated in medium conditioned by MBNL1-depleted AVC explants. TGFß3 is elevated even when the myocardium is removed, indicating that MBNL1 modulates autocrine TGFß3 production in the endocardium. More TGFß3-positive cells are observed in the endocardial monolayer following MBNL1 knockdown. Addition of exogenous TGFß3 to AVC explants recapitulates the effects of MBNL1 knockdown. Time course experiments demonstrate that knockdown of MBNL1 induces precocious TGFß3 secretion, and early exposure to excess TGFß3 induces precocious invasion. MBNL1 expression precedes TGFß3 in the AVC endocardium, consistent with a role in preventing precocious autocrine TGFß3 signaling. The stimulatory effects of MBNL1 knockdown on invasion are lost in stage 16 AVC explants. Knockdown of MBNL1 in OFT explants similarly enhances cell invasion, but not activation. TGFß is necessary and sufficient to mediate this effect. CONCLUSIONS: Taken together, these data support a model in which MBNL1 negatively regulates cell invasion in the endocardial cushions by restricting the magnitude and timing of endocardial-derived TGFß3 production.