Eric M. Small

Pervasive roles of microRNAs in cardiovascular biology

First recognized as regulators of development in worms and fruitflies, microRNAs are emerging as pivotal modulators of mammalian cardiovascular development and disease. Individual microRNAs modulate the expression of collections of messenger RNA targets that often have related functions, thereby governing complex biological processes. The wideranging functions of microRNAs in the cardiovascular system have provided new perspectives on disease mechanisms and have revealed intriguing therapeutic targets, as well as diagnostics, for a variety of cardiovascular disorders.

Activation of Cardiac Gene Expression by Myocardin, a Transcriptional Cofactor for Serum Response Factor

Serum response factor (SRF) regulates transcription of numerous muscle and growth factor-inducible genes. Because SRF is not muscle specific, it has been postulated to activate muscle genes by recruiting myogenic accessory factors. Using a bioinformatics-based screen for unknown cardiac-specific genes, we identified a novel and highly potent transcription factor, named myocardin, that is expressed in cardiac and smooth muscle cells. Myocardin belongs to the SAP domain family of nuclear proteins and activates cardiac muscle promoters by associating with SRF. Expression of a dominant negative mutant of myocardin in Xenopus embryos interferes with myocardial cell differentiation. Myocardin is the founding member of a class of muscle transcription factors and provides a mechanism whereby SRF can convey myogenic activity to cardiac muscle genes.

MicroRNAs miR-143 and miR-145 modulate cytoskeletal dynamics and responsiveness of smooth muscle cells to injury

Vascular injury triggers dedifferentiation and cytoskeletal remodeling of smooth muscle cells (SMCs), culminating in vessel occlusion. Serum response factor (SRF) and its coactivator, myocardin, play a central role in the control of smooth muscle phenotypes by regulating the expression of cytoskeletal genes. We show that SRF and myocardin regulate a cardiovascular-specific microRNA (miRNA) cluster encoding miR-143 and miR-145. To assess the functions of these miRNAs in vivo, we systematically deleted them singly and in combination in mice. Mice lacking both miR-143 and miR-145 are viable and do not display overt abnormalities in smooth muscle differentiation, although they show a significant reduction in blood pressure due to reduced vascular tone. Remarkably, however, neointima formation in response to vascular injury is profoundly impeded in mice lacking these miRNAs, due to disarray of actin stress fibers and diminished migratory activity of SMCs. These abnormalities reflect the regulation of a cadre of modulators of SRF activity and actin dynamics by miR-143 and miR-145. Thus, miR-143 and miR-145 act as integral components of the regulatory network whereby SRF controls cytoskeletal remodeling and phenotypic switching of SMCs during vascular disease.

MicroRNAs add a new dimension to cardiovascular disease

Cardiovascular disease is the predominant cause of human morbidity and mortality in developed countries. Thus, extraordinary effort has been devoted to determining the molecular and pathophysiological characteristics of the diseased heart and vasculature with the goal of developing novel diagnostic and therapeutic strategies to combat cardiovascular disease. The collective work of multiple research groups has uncovered a complex transcriptional and posttranscriptional regulatory circuit, the integrity of which is essential for the maintenance of cardiac homeostasis. Mutations in or aberrant expression of various transcriptional and posttranscriptional regulators have now been correlated with human cardiac disease, and pharmacological modulation of the activity of these target genes is a major focus of ongoing research. Recently, a novel class of small noncoding RNAs, called microRNAs (miRNAs), was identified as important transcriptional and posttranscriptional inhibitors of gene expression thought to “fine tune” the translational output of target messenger RNAs (mRNAs).1,2 miRNAs are implicated in the pathogenesis of various cardiovascular diseases and have become an intriguing target for therapeutic intervention. This review focuses on the basic biology and mechanism of action of miRNAs specifically pertaining to cardiovascular disorders and addresses the potential for miRNAs to be targeted therapeutically in the treatment of cardiovascular disease. miRNAs originate from longer precursor RNAs called primary miRNAs that are regulated by conventional transcription factors and transcribed by RNA polymerase II. Primary miRNAs are hundreds to thousands of nucleotides long and are processed in the nucleus into an ≈70- to 100-nucleotide hairpin-shaped precursor miRNA by the RNase III enzyme Drosha and the double-stranded RNA binding protein DGCR8. The precursor miRNA is then transported into the cytoplasm by the nuclear export factor exportin 5 and further processed into an ≈19- to 25-nucleotide double-stranded RNA by the RNaseIII enzyme Dicer. This duplex miRNA is then incorporated into the RNA-induced silencing complex. One …

Regulation of PI3-kinase/Akt signaling by muscle-enriched microRNA-486

microRNAs (miRNAs) play key roles in modulating a variety of cellular processes through repression of mRNA targets. In a screen for miRNAs regulated by myocardin-related transcription factor-A (MRTF-A), a coactivator of serum response factor (SRF), we discovered a muscle-enriched miRNA, miR-486, controlled by an alternative promoter within intron 40 of the Ankyrin-1 gene. Transcription of miR-486 is directly controlled by SRF and MRTF-A, as well as by MyoD. Among the most strongly predicted targets of miR-486 are phosphatase and tensin homolog (PTEN) and Foxo1a, which negatively affect phosphoinositide-3-kinase (PI3K)/Akt signaling. Accordingly, PTEN and Foxo1a protein levels are reduced by miR-486 overexpression, which, in turn, enhances PI3K/Akt signaling. Similarly, we show that MRTF-A promotes PI3K/Akt signaling by up-regulating miR-486 expression. Conversely, inhibition of miR-486 expression enhances the expression of PTEN and Foxo1a and dampens signaling through the PI3K/Akt-signaling pathway. Our findings implicate miR-486 as a downstream mediator of the actions of SRF/MRTF-A and MyoD in muscle cells and as a potential modulator of PI3K/Akt signaling.

Myocardin-Related Transcription Factor-A Controls Myofibroblast Activation and Fibrosis in Response to Myocardial Infarction

Rationale: Myocardial infarction (MI) results in loss of cardiac myocytes in the ischemic zone of the heart, followed by fibrosis and scar formation, which diminish cardiac contractility and impede angiogenesis and repair. Myofibroblasts, a specialized cell type that switches from a fibroblast-like state to a contractile, smooth muscle-like state, are believed to be primarily responsible for fibrosis of the injured heart and other tissues, although the transcriptional mediators of fibrosis and myofibroblast activation remain poorly defined. Myocardin-related transcription factors (MRTFs) are serum response factor (SRF) cofactors that promote a smooth muscle phenotype and are emerging as components of stress-responsive signaling. Objective: We aimed to examine the effect of MRTF-A on cardiac remodeling and fibrosis. Methods and Results: Here, we show that MRTF-A controls the expression of a fibrotic gene program that includes genes involved in extracellular matrix production and smooth muscle cell differentiation in the heart. In MRTF-A–null mice, fibrosis and scar formation following MI or angiotensin II treatment are dramatically diminished compared with wild-type littermates. This protective effect of MRTF-A deletion is associated with a reduction in expression of fibrosis-associated genes, including collagen 1a2, a direct transcriptional target of SRF/MRTF-A. Conclusions: We conclude that MRTF-A regulates myofibroblast activation and fibrosis in response to the renin–angiotensin system and post-MI remodeling.

MicroRNA-218 regulates vascular patterning by modulation of Slit-Robo signaling

Rationale: Establishment of a functional vasculature requires the interconnection and remodeling of nascent blood vessels. Precise regulation of factors that influence endothelial cell migration and function is essential for these stereotypical vascular patterning events. The secreted Slit ligands and their Robo receptors constitute a critical signaling pathway controlling the directed migration of both neurons and vascular endothelial cells during embryonic development, but the mechanisms of their regulation are incompletely understood. Objective: To identify microRNAs regulating aspects of the Slit-Robo pathway and vascular patterning. Methods and Results: Here, we provide evidence that microRNA (miR)-218, which is encoded by an intron of the Slit genes, inhibits the expression of Robo1 and Robo2 and multiple components of the heparan sulfate biosynthetic pathway. Using in vitro and in vivo approaches, we demonstrate that miR-218 directly represses the expression of Robo1, Robo2, and glucuronyl C5-epimerase (GLCE), and that an intact miR-218–Slit–Robo regulatory network is essential for normal vascularization of the retina. Knockdown of miR-218 results in aberrant regulation of this signaling axis, abnormal endothelial cell migration, and reduced complexity of the retinal vasculature. Conclusions: Our findings link Slit gene expression to the posttranscriptional regulation of Robo receptors and heparan sulfate biosynthetic enzymes, allowing for precise control over vascular guidance cues influencing the organization of blood vessels during development.

Transgenic analysis of the atrialnatriuretic factor (ANF) promoter: Nkx2-5 and GATA-4 binding sites are required for atrial specific expression of ANF

The atrial natriuretic factor (ANF) gene is initially expressed throughout the myocardial layer of the heart, but during subsequent development, expression becomes limited to the atrial chambers. Mouse knockout and mammalian cell culture studies have shown that the ANF gene is regulated by combinatorial interactions between Nkx2-5, GATA-4, Tbx5, and SRF; however, the molecular mechanisms leading to chamber-specific expression are currently unknown. We have isolated the Xenopus ANF promoter in order to examine the temporal and spatial regulation of the ANF gene in vivo using transgenic embryos. The mammalian and Xenopus ANF promoters show remarkable sequence similarity, including an Nkx2-5 binding site (NKE), two GATA sites, a T-box binding site (TBE), and two SRF binding sites (SREs). Our transgenic studies show that mutation of either SRE, the TBE or the distal GATA element, strongly reduces expression from the ANF promoter. However, mutations of the NKE, the proximal GATA, or both elements together, result in relatively minor reductions in transgene expression within the myocardium. Surprisingly, mutation of these elements results in ectopic ANF promoter activity in the kidneys, facial muscles, and aortic arch artery-associated muscles, and causes persistent expression in the ventricle and outflow tract of the heart. We propose that the NKE and proximal GATA elements serve as crucial binding sites for assembly of a repressor complex that is required for atrial-specific expression of the ANF gene.

Essential roles of the bHLH transcription factor Hrt2 in repression of atrial gene expression and maintenance of postnatal cardiac function

The basic helix–loop–helix transcriptional repressor Hairy-related transcription factor 2 (Hrt2) is expressed in ventricular, but not atrial, cardiomyocytes, and in endothelial and vascular smooth muscle cells. Mice homozygous for a null mutation of Hrt2 die perinatally from a spectrum of cardiac abnormalities, raising questions about the specific functions of this transcriptional regulator in individual cardiac cell lineages. Using a conditional Hrt2 null allele, we show that cardiomyocyte-specific deletion of Hrt2 in mice results in ectopic activation of atrial genes in ventricular myocardium with an associated impairment of cardiac contractility and a unique distortion in morphology of the right ventricular chamber. Consistent with the atrialization of ventricular gene expression in Hrt2 mutant mice, forced expression of Hrt2 in atrial cardiomyocytes is sufficient to repress atrial cardiac genes. These findings reveal a ventricular myocardial cell-autonomous function for Hrt2 in the suppression of atrial cell identity and the maintenance of postnatal cardiac function.

Myocardin is sufficient and necessary for cardiac gene expression in Xenopus

Myocardin is a cardiac- and smooth muscle-specific cofactor for the ubiquitous transcription factor serum response factor (SRF). Using gain-of-function approaches in the Xenopus embryo, we show that myocardin is sufficient to activate transcription of a wide range of cardiac and smooth muscle differentiation markers in non-muscle cell types. We also demonstrate that, for the myosin light chain 2 gene (MLC2), myocardin cooperates with the zinc-finger transcription factor Gata4 to activate expression. Inhibition of myocardin activity in Xenopus embryos using morpholino knockdown methods results in inhibition of cardiac development and the absence of expression of cardiac differentiation markers and severe disruption of cardiac morphological processes. We conclude that myocardin is an essential component of the regulatory pathway for myocardial differentiation.