Andrew H. Williams

A signature pattern of stress-responsive microRNAs that can evoke cardiac hypertrophy and heart failure

Diverse forms of injury and stress evoke a hypertrophic growth response in adult cardiac myocytes, which is characterized by an increase in cell size, enhanced protein synthesis, assembly of sarcomeres, and reactivation of fetal genes, often culminating in heart failure and sudden death. Given the emerging roles of microRNAs (miRNAs) in modulation of cellular phenotypes, we searched for miRNAs that were regulated during cardiac hypertrophy and heart failure. We describe >12 miRNAs that are up- or down-regulated in cardiac tissue from mice in response to transverse aortic constriction or expression of activated calcineurin, stimuli that induce pathological cardiac remodeling. Many of these miRNAs were similarly regulated in failing human hearts. Forced overexpression of stress-inducible miRNAs was sufficient to induce hypertrophy in cultured cardiomyocytes. Similarly, cardiac overexpression of miR-195, which was up-regulated during cardiac hypertrophy, resulted in pathological cardiac growth and heart failure in transgenic mice. These findings reveal an important role for specific miRNAs in the control of hypertrophic growth and chamber remodeling of the heart in response to pathological signaling and point to miRNAs as potential therapeutic targets in heart disease.

microRNA-133a regulates cardiomyocyte proliferation and suppresses smooth muscle gene expression in the heart

MicroRNAs (miRNAs) modulate gene expression by inhibiting mRNA translation and promoting mRNA degradation, but little is known of their potential roles in organ formation or function. miR-133a-1 and miR-133a-2 are identical, muscle-specific miRNAs that are regulated during muscle development by the SRF transcription factor. We show that mice lacking either miR-133a-1 or miR-133a-2 are normal, whereas deletion of both miRNAs causes lethal ventricular-septal defects in approximately half of double-mutant embryos or neonates; miR-133a double-mutant mice that survive to adulthood succumb to dilated cardiomyopathy and heart failure. The absence of miR-133a expression results in ectopic expression of smooth muscle genes in the heart and aberrant cardiomyocyte proliferation. These abnormalities can be attributed, at least in part, to elevated expression of SRF and cyclin D2, which are targets for repression by miR-133a. These findings reveal essential and redundant roles for miR-133a-1 and miR-133a-2 in orchestrating cardiac development, gene expression, and function and point to these miRNAs as critical components of an SRF-dependent myogenic transcriptional circuit.

MicroRNA-206 Delays ALS Progression and Promotes Regeneration of Neuromuscular Synapses in Mice

An Innervative Small RNA Amyotrophic lateral sclerosis (ALS) is a relentless disease characterized by progressive degeneration of motor neurons that control muscle movement, leading to muscle atrophy and paralysis. Williams et al. (p. 1549; see the Perspective by Brown) show that a small noncoding RNA that is selectively expressed in skeletal muscle, miR-206, senses motor neuron injury or loss and helps ameliorate resultant muscle damage by promoting regeneration of neuromuscular synapses. Expression of miR-206 was dramatically induced in a mouse model of ALS, and when this RNA was removed from mice by genetic manipulation, the disease progressed at a faster rate. The salutary effects of miR-206 appear to be mediated through a signaling pathway in muscle cells involving histone deacetylase 4 and a fibro-blast growth factor modulator, activation of which leads to release of factors that promote nerve-muscle interactions. A small noncoding RNA promotes nerve-muscle interactions in response to motor neuron injury and slows disease progression. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by loss of motor neurons, denervation of target muscles, muscle atrophy, and paralysis. Understanding ALS pathogenesis may require a fuller understanding of the bidirectional signaling between motor neurons and skeletal muscle fibers at neuromuscular synapses. Here, we show that a key regulator of this signaling is miR-206, a skeletal muscle–specific microRNA that is dramatically induced in a mouse model of ALS. Mice that are genetically deficient in miR-206 form normal neuromuscular synapses during development, but deficiency of miR-206 in the ALS mouse model accelerates disease progression. miR-206 is required for efficient regeneration of neuromuscular synapses after acute nerve injury, which probably accounts for its salutary effects in ALS. miR-206 mediates these effects at least in part through histone deacetylase 4 and fibroblast growth factor signaling pathways. Thus, miR-206 slows ALS progression by sensing motor neuron injury and promoting the compensatory regeneration of neuromuscular synapses.

An intragenic MEF2-dependent enhancer directs muscle-specific expression of microRNAs 1 and 133

The muscle-specific microRNAs, miR-1 and miR-133, play important roles in muscle growth and differentiation. Here, we show that the MEF2 transcription factor, an essential regulator of muscle development, directly activates transcription of a bicistronic primary transcript encoding miR-1-2 and 133a-1 via an intragenic muscle-specific enhancer located between the miR-1-2 and 133a-1 coding regions. This MEF2-dependent enhancer is activated in the linear heart tube during mouse embryogenesis and thereafter controls transcription throughout the atrial and ventricular chambers of the heart. MEF2 together with MyoD also regulates the miR-1-2/-133a-1 intragenic enhancer in the somite myotomes and in all skeletal muscle fibers during embryogenesis and adulthood. A similar muscle-specific intragenic enhancer controls transcription of the miR-1-1/-133a-2 locus. These findings reveal a common architecture of regulatory elements associated with the miR-1/-133 genes and underscore the central role of MEF2 as a regulator of the transcriptional and posttranscriptional pathways that control cardiac and skeletal muscle development.

Myogenin and Class II HDACs Control Neurogenic Muscle Atrophy by Inducing E3 Ubiquitin Ligases

Maintenance of skeletal muscle structure and function requires innervation by motor neurons, such that denervation causes muscle atrophy. We show that myogenin, an essential regulator of muscle development, controls neurogenic atrophy. Myogenin is upregulated in skeletal muscle following denervation and regulates expression of the E3 ubiquitin ligases MuRF1 and atrogin-1, which promote muscle proteolysis and atrophy. Deletion of myogenin from adult mice diminishes expression of MuRF1 and atrogin-1 in denervated muscle and confers resistance to atrophy. Mice lacking histone deacetylases (HDACs) 4 and 5 in skeletal muscle fail to upregulate myogenin and also preserve muscle mass following denervation. Conversely, forced expression of myogenin in skeletal muscle of HDAC mutant mice restores muscle atrophy following denervation. Thus, myogenin plays a dual role as both a regulator of muscle development and an inducer of neurogenic atrophy. These findings reveal a specific pathway for muscle wasting and potential therapeutic targets for this disorder.

microRNA-206 promotes skeletal muscle regeneration and delays progression of Duchenne muscular dystrophy in mice

Skeletal muscle injury activates adult myogenic stem cells, known as satellite cells, to initiate proliferation and differentiation to regenerate new muscle fibers. The skeletal muscle-specific microRNA miR-206 is upregulated in satellite cells following muscle injury, but its role in muscle regeneration has not been defined. Here, we show that miR-206 promotes skeletal muscle regeneration in response to injury. Genetic deletion of miR-206 in mice substantially delayed regeneration induced by cardiotoxin injury. Furthermore, loss of miR-206 accelerated and exacerbated the dystrophic phenotype in a mouse model of Duchenne muscular dystrophy. We found that miR-206 acts to promote satellite cell differentiation and fusion into muscle fibers through suppressing a collection of negative regulators of myogenesis. Our findings reveal an essential role for miR-206 in satellite cell differentiation during skeletal muscle regeneration and indicate that miR-206 slows progression of Duchenne muscular dystrophy.

Control of mitochondrial metabolism and systemic energy homeostasis by microRNAs 378 and 378

Obesity and metabolic syndrome are associated with mitochondrial dysfunction and deranged regulation of metabolic genes. Peroxisome proliferator-activated receptor γ coactivator 1β (PGC-1β) is a transcriptional coactivator that regulates metabolism and mitochondrial biogenesis through stimulation of nuclear hormone receptors and other transcription factors. We report that the PGC-1β gene encodes two microRNAs (miRNAs), miR-378 and miR-378*, which counterbalance the metabolic actions of PGC-1β. Mice genetically lacking miR-378 and miR-378* are resistant to high-fat diet-induced obesity and exhibit enhanced mitochondrial fatty acid metabolism and elevated oxidative capacity of insulin-target tissues. Among the many targets of these miRNAs, carnitine O-acetyltransferase, a mitochondrial enzyme involved in fatty acid metabolism, and MED13, a component of the Mediator complex that controls nuclear hormone receptor activity, are repressed by miR-378 and miR-378*, respectively, and are elevated in the livers of miR-378/378* KO mice. Consistent with these targets as contributors to the metabolic actions of miR-378 and miR-378*, previous studies have implicated carnitine O-acetyltransferase and MED13 in metabolic syndrome and obesity. Our findings identify miR-378 and miR-378* as integral components of a regulatory circuit that functions under conditions of metabolic stress to control systemic energy homeostasis and the overall oxidative capacity of insulin target tissues. Thus, these miRNAs provide potential targets for pharmacologic intervention in obesity and metabolic syndrome.

Efferocytosis produces a prometastatic landscape during postpartum mammary gland involution

Breast cancers that occur in women 2-5 years postpartum are more frequently diagnosed at metastatic stages and correlate with poorer outcomes compared with breast cancers diagnosed in young, premenopausal women. The molecular mechanisms underlying the malignant severity associated with postpartum breast cancers (ppBCs) are unclear but relate to stromal wound-healing events during postpartum involution, a dynamic process characterized by widespread cell death in milk-producing mammary epithelial cells (MECs). Using both spontaneous and allografted mammary tumors in fully immune-competent mice, we discovered that postpartum involution increases mammary tumor metastasis. Cell death was widespread, not only occurring in MECs but also in tumor epithelium. Dying tumor cells were cleared through receptor tyrosine kinase MerTK-dependent efferocytosis, which robustly induced the transcription of genes encoding wound-healing cytokines, including IL-4, IL-10, IL-13, and TGF-β. Animals lacking MerTK and animals treated with a MerTK inhibitor exhibited impaired efferocytosis in postpartum tumors, a reduction of M2-like macrophages but no change in total macrophage levels, decreased TGF-β expression, and a reduction of postpartum tumor metastasis that was similar to the metastasis frequencies observed in nulliparous mice. Moreover, TGF-β blockade reduced postpartum tumor metastasis. These data suggest that widespread cell death during postpartum involution triggers efferocytosis-induced wound-healing cytokines in the tumor microenvironment that promote metastatic tumor progression.