Precise correction of Duchenne muscular dystrophy exon deletion mutations by base and prime editing.

Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by the lack of dystrophin, which maintains muscle membrane integrity. We used an adenine base editor (ABE) to modify splice donor sites of the dystrophin gene, causing skipping of a common DMD deletion mutation of exon 51 (∆Ex51) in cardiomyocytes derived from human induced pluripotent stem cells, restoring dystrophin expression. Prime editing was also capable of reframing the dystrophin open reading frame in these cardiomyocytes. Intramuscular injection of ∆Ex51 mice with adeno-associated virus serotype-9 encoding ABE components as a split-intein trans-splicing system allowed gene editing and disease correction in vivo. Our findings demonstrate the effectiveness of nucleotide editing for the correction of diverse DMD mutations with minimal modification of the genome, although improved delivery methods will be required before these strategies can be used to sufficiently edit the genome in patients with DMD.

G protein-coupled receptor (GPR)40-dependent potentiation of insulin secretion in mouse islets is mediated by protein kinase D1.

AIMS/HYPOTHESIS: Activation of the G protein-coupled receptor (GPR)40 by long-chain fatty acids potentiates glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells, and GPR40 agonists are in clinical development for type 2 diabetes therapy. GPR40 couples to the G protein subunit Gα(q/11) but the signalling cascade activated downstream is unknown. This study aimed to determine the mechanisms of GPR40-dependent potentiation of GSIS by fatty acids. METHODS: Insulin secretion in response to glucose, oleate or diacylglycerol (DAG) was assessed in dynamic perifusions and static incubations in islets from wild-type (WT) and Gpr40 (-/-) mice. Depolymerisation of filamentous actin (F-actin) was visualised by phalloidin staining and epifluorescence. Pharmacological and molecular approaches were used to ascertain the roles of protein kinase D (PKD) and protein kinase C delta in GPR40-mediated potentiation of GSIS. RESULTS: Oleate potentiates the second phase of GSIS, and this effect is largely dependent upon GPR40. Accordingly, oleate induces rapid F-actin remodelling in WT but not in Gpr40 (-/-) islets. Exogenous DAG potentiates GSIS in both WT and Gpr40 (-/-) islets. Oleate induces PKD phosphorylation at residues Ser-744/748 and Ser-916 in WT but not Gpr40 (-/-) islets. Importantly, oleate-induced F-actin depolymerisation and potentiation of GSIS are lost upon pharmacological inhibition of PKD1 or deletion of Prkd1. CONCLUSIONS/INTERPRETATION: We conclude that the signalling cascade downstream of GPR40 activation by fatty acids involves activation of PKD1, F-actin depolymerisation and potentiation of second-phase insulin secretion. These results provide important information on the mechanisms of action of GPR40, a novel drug target for type 2 diabetes.

Transcriptome analysis of subcutaneous adipose tissues in beef cattle using 3' digital gene expression-tag profiling.

The molecular mechanisms that regulate fat deposition in bovine adipose tissue have not been well studied. To elucidate the genes and gene networks involved in bovine fat development, transcriptional profiles of backfat (BF) tissues from Hereford × Aberdeen Angus (HEAN, n = 6) and Charolais × Red Angus (CHRA, n = 6) steers with high or low BF thickness were characterized by digital gene expression-tag profiling. Approximately 9.8 to 21.9 million tags were obtained for each library, and a total of 18,034 genes were identified. In total, 650 genes were found to be differentially expressed, with a greater than 1.5-fold difference between the 2 crossbreds (Benjamini-Hochberg false discovery rate ≤ 0.05). The majority of differentially expressed genes that were more highly expressed in CHRA vs. HEAN were associated with development, whereas the differentially expressed genes with greater expression in HEAN vs. CHRA were overrepresented in biological processes such as metabolism and immune response. Thirty-six and 152 differentially expressed genes were detected between animals with high (n = 3) and low (n = 3) BF thickness in HEAN and CHRA, respectively (Benjamini-Hochberg false discovery rate ≤0.05). The differentially expressed genes between high and low groups in CHRA were related to cell proliferation and development processes. In addition, lipid metabolism was 1 of the top 5 molecular and cellular functions identified in both crossbreds. Ten and 17 differentially expressed genes were found to be involved in fat metabolism in HEAN and CHRA, respectively. Genes associated with obesity, such as PTX3 (pentraxin 3, long) and SERPINE1 (serpin peptidase inhibitor, clade E, member 1), were more highly expressed (P < 0.05) in the subset of CHRA animals with greater BF thickness. Our study revealed that the expression patterns of genes in BF tissues differed depending on the genetic background of the cattle.

Cardiac hypertrophy: the good, the bad, and the ugly.

Cardiac hypertrophy is the heart's response to a variety of extrinsic and intrinsic stimuli that impose increased biomechanical stress. While hypertrophy can eventually normalize wall tension, it is associated with an unfavorable outcome and threatens affected patients with sudden death or progression to overt heart failure. Accumulating evidence from studies in human patients and animal models suggests that in most instances hypertrophy is not a compensatory response to the change in mechanical load, but rather is a maladaptive process. Accordingly, modulation of myocardial growth without adversely affecting contractile function is increasingly recognized as a potentially auspicious approach in the prevention and treatment of heart failure. In this review, we summarize recent insights into hypertrophic signaling and consider several novel antihypertrophic strategies.

Molecular mechanisms of ventricular hypoplasia.

We have established the beginnings of a road map to understand how ventricular cells become specified, differentiate, and expand into a functional cardiac chamber (Fig. 5). The transcriptional networks described here provide clear evidence that disruption of pathways affecting ventricular growth could be the underlying etiology in a subset of children born with malformation of the right or left ventricle. As we learn details of the precise mechanisms through which the critical factors function, the challenge will lie in devising innovative methods to augment or modify the effects of gene mutations on ventricular development. Because most congenital heart disease likely occurs in a setting of heterozygous, predisposing mutations of one or more genes, modulation of activity of critical pathways in a preventive fashion may be useful in averting disease in genetically susceptible individuals.

The Mef2c gene is a direct transcriptional target of myogenic bHLH and MEF2 proteins during skeletal muscle development.

Members of the MEF2 family of transcription factors are upregulated during skeletal muscle differentiation and cooperate with the MyoD family of myogenic basic helix-loop-helix (bHLH) transcription factors to control the expression of muscle-specific genes. To determine the mechanisms that regulate MEF2 gene expression during skeletal muscle development, we analyzed the mouse Mef2c gene for cis-regulatory elements that direct expression in the skeletal muscle lineage in vivo. We describe a skeletal muscle-specific control region for Mef2c that is sufficient to direct lacZ reporter gene expression in a pattern that recapitulates that of the endogenous Mef2c gene in skeletal muscle during pre- and postnatal development. This control region is a direct target for the binding of myogenic bHLH and MEF2 proteins. Mutagenesis of the Mef2c control region shows that a binding site for myogenic bHLH proteins is essential for expression at all stages of skeletal muscle development, whereas an adjacent MEF2 binding site is required for maintenance but not for initiation of Mef2c transcription. Our findings reveal the existence of a regulatory circuit between these two classes of transcription factors that induces, amplifies and maintains their expression during skeletal muscle development.

Role of Dlx6 in regulation of an endothelin-1-dependent, dHAND branchial arch enhancer.

Neural crest cells play a key role in craniofacial development. The endothelin family of secreted polypeptides regulates development of several neural crest sublineages, including the branchial arch neural crest. The basic helix-loop-helix transcription factor dHAND is also required for craniofacial development, and in endothelin-1 (ET-1) mutant embryos, dHAND expression in the branchial arches is down-regulated, implicating it as a transcriptional effector of ET-1 action. To determine the mechanism that links ET-1 signaling to dHAND transcription, we analyzed the dHAND gene for cis-regulatory elements that control transcription in the branchial arches. We describe an evolutionarily conserved dHAND enhancer that requires ET-1 signaling for activity. This enhancer contains four homeodomain binding sites that are required for branchial arch expression. By comparing protein binding to these sites in branchial arch extracts from endothelin receptor A (EdnrA) mutant and wild-type mouse embryos, we identified Dlx6, a member of the Distal-less family of homeodomain proteins, as an ET-1-dependent binding factor. Consistent with this conclusion, Dlx6 was down-regulated in branchial arches from EdnrA mutant mice. These results suggest that Dlx6 acts as an intermediary between ET-1 signaling and dHAND transcription during craniofacial morphogenesis.

Dilated cardiomyopathy and sudden death resulting from constitutive activation of protein kinase a.

beta-Adrenergic receptor (betaAR) signaling, which elevates intracellular cAMP and enhances cardiac contractility, is severely impaired in the failing heart. Protein kinase A (PKA) is activated by cAMP, but the long-term physiological effect of PKA activation on cardiac function is unclear. To investigate the consequences of chronic cardiac PKA activation in the absence of upstream events associated with betaAR signaling, we generated transgenic mice that expressed the catalytic subunit of PKA in the heart. These mice developed dilated cardiomyopathy with reduced cardiac contractility, arrhythmias, and susceptibility to sudden death. As seen in human heart failure, these abnormalities correlated with PKA-mediated hyperphosphorylation of the cardiac ryanodine receptor/Ca(2+)-release channel, which enhances Ca(2+) release from the sarcoplasmic reticulum, and phospholamban, which regulates the sarcoplasmic reticulum Ca(2+)-ATPase. These findings demonstrate a specific role for PKA in the pathogenesis of heart failure, independent of more proximal events in betaAR signaling, and support the notion that PKA activity is involved in the adverse effects of chronic betaAR signaling.

Activation of MEF2 by muscle activity is mediated through a calcineurin-dependent pathway.

Gene expression in skeletal muscles of adult vertebrates is altered profoundly by changing patterns of contractile work. Here we observed that the functional activity of MEF2 transcription factors is stimulated by sustained periods of endurance exercise or motor nerve pacing, as assessed by expression in trans genic mice of a MEF2-dependent reporter gene (desMEF2-lacZ). This response is accompanied by transformation of specialized myofiber subtypes, and is blocked either by cyclosporin A, a specific chemical inhibitor of calcineurin, or by forced expression of the endogenous calcineurin inhibitory protein, myocyte-enriched calcineurin interacting protein 1. Calcineurin removes phosphate groups from MEF2, and augments the potency of the transcriptional activation domain of MEF2 fused to a heterologous DNA binding domain. Across a broad range, the enzymatic activity of calcineurin correlates directly with expression of endogenous genes that are transcriptionally activated by muscle contractions. These results delineate a molecular pathway in which calcineurin and MEF2 participate in the adaptive mechanisms by which skeletal myofibers acquire specialized contractile and metabolic properties as a function of changing patterns of muscle contraction.

Antisocial, an intracellular adaptor protein, is required for myoblast fusion in Drosophila.

Somatic muscle formation in Drosophila requires fusion of muscle founder cells with fusion-competent myoblasts. In a genetic screen for genes that control muscle development, we identified antisocial (ants), a gene that encodes an ankyrin repeat-, TPR repeat-, and RING finger-containing protein, required for myoblast fusion. In ants mutant embryos, founder cells and fusion-competent myoblasts are properly specified and patterned, but they are unable to form myotubes. ANTS, which is expressed specifically in founder cells, interacts with the cytoplasmic domain of Dumbfounded, a founder cell transmembrane receptor, and with Myoblast city, a cytoskeletal protein, both of which are also required for myoblast fusion. These findings suggest that ANTS functions as an intracellular adaptor protein that relays signals from Dumbfounded to the cytoskeleton during myoblast fusion.

Analysis of the tendon cell fate using Scleraxis, a specific marker for tendons and ligaments.

Little is known about the genesis and patterning of tendons and other connective tissues, mostly owing to the absence of early markers. We have found that Scleraxis, a bHLH transcription factor, is a highly specific marker for all the connective tissues that mediate attachment of muscle to bone in chick and mouse, including the limb tendons, and show that early scleraxis expression marks the progenitor cell populations for these tissues. In the early limb bud, the tendon progenitor population is found in the superficial proximomedial mesenchyme. Using the scleraxis gene as a marker we show that these progenitors are induced by ectodermal signals and restricted by bone morphogenetic protein (BMP) signaling within the mesenchyme. Application of Noggin protein antagonizes this endogenous BMP activity and induces ectopic scleraxis expression. However, the presence of excess tendon progenitors does not lead to the production of additional or longer tendons, indicating that additional signals are required for the final formation of a tendon. Finally, we show that the endogenous expression of noggin within the condensing digit cartilage contributes to the induction of distal tendons.

Control of muscle development by dueling HATs and HDACs.

Skeletal muscle cells have provided an especially auspicious system in which to dissect the roles of chromatin structure in the control of cell growth, differentiation, and development. The MyoD and MEF2 families of transcription factors act cooperatively to regulate the expression of skeletal muscle-specific genes. Recent studies have shown that these two classes of transcription factors associate with histone acetyltransferases and histone deacetylases to control the activation and repression, respectively, of the muscle differentiation program. Signaling systems that regulate the growth and differentiation of muscle cells act, at least in part, by regulating the intracellular localization and associations of these chromatin remodeling enzymes with myogenic transcription factors. We describe the molecules and mechanisms involved in chromatin remodeling during skeletal muscle development.

Identification of a signal-responsive nuclear export sequence in class II histone deacetylases.

Activation of muscle-specific genes by the MEF2 transcription factor is inhibited by class II histone deacetylases (HDACs) 4 and 5, which contain carboxy-terminal deacetylase domains and amino-terminal extensions required for association with MEF2. The inhibitory action of HDACs is overcome by myogenic signals which disrupt MEF2-HDAC interactions and stimulate nuclear export of these transcriptional repressors. Nucleocytoplasmic trafficking of HDAC5 is mediated by binding of the chaperone protein 14-3-3 to two phosphoserine residues (Ser-259 and Ser-498) in its amino-terminal extension. Here we show that HDAC4 and -5 each contain a signal-responsive nuclear export sequence (NES) at their extreme carboxy termini. The NES is conserved in another class II HDAC, HDAC7, but is absent in class I HDACs and the HDAC-related corepressor, MEF2-interacting transcription repressor. Our results suggest that this conserved NES is inactive in unphosphorylated HDAC5, which is localized to the nucleus, and that calcium-calmodulin-dependent protein kinase (CaMK)-dependent binding of 14-3-3 to phosphoserines 259 and 498 activates the NES, with consequent export of the transcriptional repressor to the cytoplasm. A single amino acid substitution in this NES is sufficient to retain HDAC5 in the nucleus in the face of CaMK signaling. These findings provide molecular insight into the mechanism by which extracellular cues alter chromatin structure to promote muscle differentiation and other MEF2-regulated processes.

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.

CHAMP, a novel cardiac-specific helicase regulated by MEF2C.

MEF2C is a MADS-box transcription factor required for cardiac myogenesis and morphogenesis. In MEF2C mutant mouse embryos, heart development arrests at the looping stage (embryonic day 9.0), the future right ventricular chamber fails to form, and cardiomyocyte differentiation is disrupted. To identify genes regulated by MEF2C in the developing heart, we performed differential array analysis coupled with subtractive cloning using RNA from heart tubes of wild-type and MEF2C-null embryos. Here, we describe a novel MEF2C-dependent gene that encodes a cardiac-restricted protein, called CHAMP (cardiac helicase activated by MEF2 protein), that contains seven conserved motifs characteristic of helicases involved in RNA processing, DNA replication, and transcription. During mouse embryogenesis, CHAMP expression commences in the linear heart tube at embryonic day 8.0, shortly after initiation of MEF2C expression in the cardiogenic region. Thereafter, CHAMP is expressed specifically in embryonic and postnatal cardiomyocytes. At the trabeculation stage of heart development, CHAMP expression is highest in the trabecular region in which cardiomyocytes have exited the cell cycle and is lowest in the proliferative compact zone. These findings suggest that CHAMP acts downstream of MEF2C in a cardiac-specific regulatory pathway for RNA processing and/or transcriptional control.

The transcriptional corepressor MITR is a signal-responsive inhibitor of myogenesis.

Activation of muscle-specific genes by members of the myocyte enhancer factor 2 (MEF2) and MyoD families of transcription factors is coupled to histone acetylation and is inhibited by class II histone deacetylases (HDACs) 4 and 5, which interact with MEF2. The ability of HDAC4 and -5 to inhibit MEF2 is blocked by phosphorylation of these HDACs at two conserved serine residues, which creates docking sites for the intracellular chaperone protein 14-3-3. When bound to 14-3-3, HDACs are released from MEF2 and transported to the cytoplasm, thereby allowing MEF2 to stimulate muscle-specific gene expression. MEF2-interacting transcription repressor (MITR) shares homology with the amino-terminal regions of HDAC4 and -5, but lacks an HDAC catalytic domain. Despite the absence of intrinsic HDAC activity, MITR acts as a potent inhibitor of MEF2-dependent transcription. Paradoxically, however, MITR has minimal inhibitory effects on the skeletal muscle differentiation program. We show that a substitution mutant of MITR containing alanine in place of two serine residues, Ser-218 and Ser-448, acts as a potent repressor of myogenesis. Our findings indicate that promyogenic signals antagonize the inhibitory action of MITR by targeting these serines for phosphorylation. Phosphorylation of Ser-218 and Ser-448 stimulates binding of 14-3-3 to MITR, disrupts MEF2:MITR interactions, and alters the nuclear distribution of MITR. These results reveal a role for MITR as a signal-dependent regulator of muscle differentiation.

Activated MEK5 induces serial assembly of sarcomeres and eccentric cardiac hypertrophy.

Mitogen-activated protein kinase (MAPK) pathways couple intrinsic and extrinsic signals to hypertrophic growth of cardiomyocytes. The MAPK kinase MEK5 activates the MAPK ERK5. To investigate the potential involvement of MEK5-ERK5 in cardiac hypertrophy, we expressed constitutively active and dominant-negative forms of MEK5 in cardiomyocytes in vitro. MEK5 induced a form of hypertrophy in which cardiomyocytes acquired an elongated morphology and sarcomeres were assembled in a serial manner. The cytokine leukemia inhibitory factor (LIF), which stimulates MEK5 activity, evoked a similar response. Moreover, a dominant-negative MEK5 mutant specifically blocked LIF-induced elongation of cardiomyocytes and reduced expression of fetal cardiac genes without blocking other aspects of LIF-induced hypertrophy. Consistent with the ability of MEK5 to induce serial assembly of sarcomeres in vitro, cardiac-specific expression of activated MEK5 in transgenic mice resulted in eccentric cardiac hypertrophy that progressed to dilated cardiomyopathy and sudden death. These findings reveal a specific role for MEK5-ERK5 in the induction of eccentric cardiac hypertrophy and in transduction of cytokine signals that regulate serial sarcomere assembly.