The physiological targets regulated by MEF2 in striated muscle are not completely known. Several recent studies have identified novel downstream target genes and shed light on the global transcriptional network regulated by MEF2 in muscle. In our continuing effort to identify novel, downstream pathways controlled by MEF2, we have used mef2a knock-out mice to find those genes dependent on MEF2A transcriptional activity. Here, we describe the characterization of a direct, downstream target gene for the MEF2A transcription factor encoding a large, muscle-specific protein that localizes to the Z-disc/costameric region in striated muscle. This gene, called myomaxin, was identified as a gene markedly down-regulated in MEF2A knock-out hearts. Myomaxin is the mouse ortholog of a partial human cDNA of unknown function named cardiomyopathy associated gene 3 (CMYA3). Myomaxin is expressed as a single, large transcript of ϳ11 kilobases in adult heart and skeletal muscle with an open reading frame of 3,283 amino acids. The protein encoded by the myomaxin gene is related to the actin-binding protein Xin and interacts with the sarcomeric Z-disc protein, ␣-actinin-2. Our findings demonstrate that Myomaxin functions directly downstream of MEF2A at the peripheral Z-disc complex in striated muscle potentially playing a role in regulating cytoarchitectural integrity.

4 suggesting that CMYA3 is directly regulated by Ang II signaling. This gene, since named Xirp2 (also known as mXin␤ and myomaxin), is a direct target of the MEF2A transcription factor and is markedly downregulated in hearts lacking MEF2A. 5,6 Xirp2 belongs to the ancient, muscle-specific, actin-binding Xin gene family whose expression can be traced to ancestral vertebrates with a 2-chambered heart. 7-9 Xirp2 is expressed in cardiac and skeletal muscle where it interacts with filamentous actin and ␣-actinin through the novel actin-binding motif, the Xin repeat. 5,8 In striated muscle, Xirp2 localizes to the peripheral Z-disc region, or costamere, 5 and the intercalated disk. 10,11 The subcellular localization of Xirp2 is significant in that the costamere and intercalated disk harbor mechanical stress sensors that are critical for normal muscle function. [12][13][14] Antisense knockdown of Xin in developing chick embryos, the sole Xin family member in this species, results in a severe disruption of cardiac looping morphogenesis. 9 In mice, a lossof-function mutation of mXin␣, the mammalian ortholog of Xin, results in cardiomyopathy and conduction defects. 11 In the present study we sought to determine the role of Xirp2 in cardiac development and/or function. Mice harboring a hypomorphic Xirp2 allele are viable but display cardiac hypertrophy. As Xirp2 is regulated by Ang II, we also examined cardiac pathology in hypomorphic mice with long-term administration of this hormone. In contrast to wild type mice exposed to a chronic Ang II infusion, hypomorphic mice displayed diminished cardiac hypertrophy, fibrosis, and apoptosis. Furthermore, we demonstrate that regulation of Xirp2 gene expression in response to Ang II signaling is mediated by MEF2A. Our results suggest that Original

The Mef2 family of transcription factors regulates muscle differentiation, but the specific gene programs controlled by each member remain unknown. Characterization of Mef2A knockout mice has revealed severe myofibrillar defects in cardiac muscle indicating a requirement for Mef2A in cytoarchitectural integrity. Through comprehensive expression analysis of Mef2A-deficient hearts, we identified a cohort of dysregulated genes whose products localize to the peripheral Z-disc/ costamere region. Many of these genes are essential for costamere integrity and function. Here we demonstrate that these genes are directly regulated by Mef2A, establishing a mechanism by which Mef2A controls the costamere. In an independent model system, acute knockdown of Mef2A in primary neonatal cardiomyocytes resulted in profound malformations of myofibrils and focal adhesions accompanied by adhesion-dependent programmed cell death. These findings indicate a role for Mef2A in cardiomyocyte survival through regulation of costamere integrity. Finally, bioinformatics analysis identified over-represented transcription factor-binding sites in this network of costamere promoters that may provide insight into the mechanism by which costamere genes are regulated by Mef2A. The global control of costamere gene expression adds another dimension by which this essential macromolecular complex may be regulated in health and disease.

Mutations in the aldolase B gene (ALDOB) impairing enzyme activity toward fructose-1-phosphate cleavage cause hereditary fructose intolerance (HFI). Diagnosis of the disease is possible by identifying known mutant ALDOB alleles in suspected patients; however, the frequencies of mutant alleles can differ by population. Here, 153 American HFI patients with 268 independent alleles were analyzed to identify the prevalence of seven known HFI-causing alleles (A149P, A174D, N334K, Δ4E4, R59Op, A337V, and L256P) in this population. Allele-specific oligonucleotide hybridization analysis was performed on polymerase chain reaction (PCR)-amplified genomic DNA from these patients. In the American population, the missense mutations A149P and A174D are the two most common alleles, with frequencies of 44% and 9%, respectively. In addition, the nonsense mutations Δ4E4 and R59Op are the next most common alleles, with each having a frequency of 4%. Together, the frequencies of all seven alleles make up 65% of HFI-causing alleles in this population. Worldwide, these same alleles make up 82% of HFI-causing mutations. This difference indicates that screening for common HFI alleles is more difficult in the American population. Nevertheless, a genetic screen for diagnosing HFI in America can be improved by including all seven alleles studied here. Lastly, identification of HFI patients presenting with classic symptoms and who have homozygous null genotypes indicates that aldolase B is not required for proper development or metabolic maintenance.

Skeletal muscle has the ability to regenerate, in response to disease or injury, through the activation of muscle stem cells and their subsequent differentiation into myofibers. This process is controlled by a hierarchy of just a few transcription factors. The role of the MEF2 transcription factor family in skeletal muscle regeneration has not been studied. We performed RNAi‐mediated knockdown of MEF2A in C2C12 cells, an established line of proliferating myoblasts isolated from injured muscle. Unlike developmental myogenesis, MEF2A is the first MEF2 factor expressed in this model. Knockdown of MEF2A in myoblasts resulted in markedly impaired differentiation. Given the source of C2C12 myoblasts we investigated the potential function of MEF2A in regenerative myogenesis in vivo. Cardiotoxin‐induced muscle injury in MEF2A knockout mice resulted in widespread necrosis and reduced myofiber cross‐sectional area indicating defective regeneration. Mechanistically, microarray analysis revealed a downregulation of more than 40 microRNAs (miRNAs); all of which are located in a large miRNA gene cluster. These miRNAs are not related to any of the well‐characterized skeletal muscle‐specific miRNAs. We demonstrate that a single, upstream promoter is bound and activated by MEF2A, suggesting its ability to regulate the entire miRNA domain. Furthermore, miRNA target gene prediction analysis revealed that a cohort of these miRNAs targets inhibitors of Wnt signaling, a key pathway promoting skeletal muscle regeneration. Consistent with the miRNA downregulation these Wnt inhibitors are massively upregulated in injured MEF2A‐deficient muscle. Thus, miRNA‐mediated modulation of Wnt signaling by MEF2A is a requisite step for proper muscle regeneration. Supported by NIH grant HL73304 to FJN.