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Ischemic heart disease is the leading cause of morbidity, mortality, and healthcare expenditure worldwide due to an inability of the heart to regenerate following injury. Thus, novel heart failure therapies aimed at promoting cardiomyocyte regeneration are desperately needed. In recent years, direct reprogramming of resident cardiac fibroblasts to induced cardiac-like myocytes (iCMs) has emerged as a promising therapeutic strategy to repurpose the fibrotic response of the injured heart toward a functional myocardium. Direct cardiac reprogramming was initially achieved through the overexpression of the transcription factors (TFs) Gata4, Mef2c, and Tbx5 (GMT). However, this combination of TFs and other subsequent cocktails demonstrated limited success in reprogramming adult human and mouse fibroblasts, constraining the clinical translation of this therapy. Over the past decade, significant effort has been dedicated to optimizing reprogramming cocktails comprised of cardiac TFs, epigenetic factors, microRNAs, or small molecules to yield efficient cardiac cell fate conversion. However, efficient reprogramming of adult human fibroblasts remains a significant challenge, and underlying mechanisms driving efficient conversion have been focused on achieving epigenetic remodeling at cardiac regulatory regions. Further studies to achieve a refined understanding and directed means of overcoming epigenetic barriers is merited to more rapidly translate these promising therapies to the clinic.

Associations between human genetic variation and clinical phenotypes have become a foundation of biomedical research. Most repositories of these data seek to be disease-agnostic and therefore lack disease-focused views. The Type 2 Diabetes Knowledge Portal (T2DKP) is a public resource of genetic datasets and genomic annotations dedicated to type 2 diabetes (T2D) and related traits. Here, we seek to make the T2DKP more accessible to prospective users and more useful to existing users. First, we evaluate the T2DKP's comprehensiveness by comparing its datasets with those of other repositories. Second, we describe how researchers unfamiliar with human genetic data can begin using and correctly interpreting them via the T2DKP. Third, we describe how existing users can extend their current workflows to use the full suite of tools offered by the T2DKP. We finally discuss the lessons offered by the T2DKP toward the goal of democratizing access to complex disease genetic results.

Duchenne muscular dystrophy (DMD), caused by mutations in the X-linked dystrophin gene, is a lethal neuromuscular disease. Correction of DMD mutations in animal models has been achieved by CRISPR/Cas9 genome editing using Streptococcus pyogenes Cas9 (SpCas9) delivered by adeno-associated virus (AAV). However, due to the limited viral packaging capacity of AAV, two AAV vectors are required to deliver the SpCas9 nuclease and its single guide RNA (sgRNA), impeding its therapeutic application. We devised an efficient single-cut gene-editing method using a compact Staphylococcus aureus Cas9 (SaCas9) to restore the open reading frame of exon 51, the most commonly affected out-of-frame exon in DMD. Editing of exon 51 in cardiomyocytes derived from human induced pluripotent stem cells revealed a strong preference for exon reframing via a two-nucleotide deletion. We adapted this system to express SaCas9 and sgRNA from a single AAV9 vector. Systemic delivery of this All-In-One AAV9 system restored dystrophin expression and improved muscle contractility in a mouse model of DMD with exon 50 deletion. These findings demonstrate the effectiveness of CRISPR/SaCas9 delivered by a consolidated AAV delivery system in the correction of DMD in vivo, representing a promising therapeutic approach to correct the genetic causes of DMD., Graphical abstract, CRISPR/Cas-mediated gene editing is being used for therapeutic purposes. In this study, Zhang et al. developed a consolidated AAV delivery system to package both SaCas9 nuclease and sgRNA in a single AAV vector and correct Duchenne muscular dystrophy mutations in human cardiomyocytes and mice.

Direct cardiac reprogramming of fibroblasts to cardiomyocytes presents an attractive therapeutic strategy to restore cardiac function following injury. Cardiac reprogramming was initially achieved through overexpression of the transcription factors Gata4, Mef2c and Tbx5; later, Hand2 and Akt1 were found to further enhance this process1–5. Yet, staunch epigenetic barriers severely limit the ability of these cocktails to reprogramme adult fibroblasts6,7. We undertook a screen of mammalian gene regulatory factors to discover novel regulators of cardiac reprogramming in adult fibroblasts and identified the histone reader PHF7 as the most potent activating factor8. Mechanistically, PHF7 localizes to cardiac super enhancers in fibroblasts, and through cooperation with the SWI/SNF complex, it increases chromatin accessibility and transcription factor binding at these sites. Furthermore, PHF7 recruits cardiac transcription factors to activate a positive transcriptional autoregulatory circuit in reprogramming. Importantly, PHF7 achieves efficient reprogramming in the absence of Gata4. Here, we highlight the underexplored necessity of cardiac epigenetic readers, such as PHF7, in harnessing chromatin remodelling and transcriptional complexes to overcome critical barriers to direct cardiac reprogramming. Garry et al. report that the histone reader PHF7 cooperates with the SWI/SNF complex at cardiac super enhancers to increase accessibility to core cardiac transcription factors, thus facilitating direct cardiac reprogramming.

Evidence suggests that n-3 polyunsaturated fatty acids may act as activators of the Nrf2 antioxidant pathway. The antioxidant response, in turn, promotes neuronal differentiation and neurite outgrowth. Nrf2 has recently been suggested to be a cell intrinsic mediator of docosohexanoic acid (DHA) signaling. In the current study, we assessed whether DHA-mediated axodendritic development was dependent on activation of the Nrf2 pathway and whether Nrf2 protected from agrochemical-induced neuritic retraction. Expression profiling of the DHA-enriched Fat-1 mouse brain relative to wild type showed a significant enrichment of genes associated with neuronal development and neuronal projection and genes associated with the Nrf2-transcriptional pathway. Moreover, we found that primary cortical neurons treated with DHA showed a dose-dependent increase in Nrf2 transcriptional activity and Nrf2-target gene expression. DHA-mediated activation of Nrf2 promoted neurite outgrowth and inhibited oxidative stress-induced neuritic retraction evoked by exposure to agrochemicals. Finally, we provide evidence that this effect is largely dependent on induction of the Nrf2-target gene NAD(P)H: (quinone acceptor) oxidoreductase 1 (NQO1), and that silencing of either Nrf2 or Nqo1 blocks the effects of DHA on the axodendritic compartment. Collectively, these data support a role for the Nrf2-NQO1 pathway in DHA-mediated axodendritic development and protection from agrochemical exposure.

Duchenne muscular dystrophy (DMD) is a fatal muscle disorder characterized by cycles of degeneration and regeneration of multinucleated myofibers and pathological activation of a variety of other muscle-associated cell types. The extent to which different nuclei within the shared cytoplasm of a myofiber may display transcriptional diversity and whether individual nuclei within a multinucleated myofiber might respond differentially to DMD pathogenesis is unknown. Similarly, the potential transcriptional diversity among nonmuscle cell types within dystrophic muscle has not been explored. Here, we describe the creation of a mouse model of DMD caused by deletion of exon 51 of the dystrophin gene, which represents a prevalent disease-causing mutation in humans. To understand the transcriptional abnormalities and heterogeneity associated with myofiber nuclei, as well as other mononucleated cell types that contribute to the muscle pathology associated with DMD, we performed single-nucleus transcriptomics of skeletal muscle of mice with dystrophin exon 51 deletion. Our results reveal distinctive and previously unrecognized myonuclear subtypes within dystrophic myofibers and uncover degenerative and regenerative transcriptional pathways underlying DMD pathogenesis. Our findings provide insights into the molecular underpinnings of DMD, controlled by the transcriptional activity of different types of muscle and nonmuscle nuclei.

Stomata optimize land plants’ photosynthetic requirements and limit water vapor loss. So far, all of the molecular and electrical components identified as regulating stomatal aperture are produced, and operate, directly within the guard cells. However, a completely autonomous function of guard cells is inconsistent with anatomical and biophysical observations hinting at mechanical contributions of epidermal origins. Here, potassium (K+) assays, membrane potential measurements, microindentation, and plasmolysis experiments provide evidence that disruption of the Arabidopsis thaliana K+ channel subunit gene AtKC1 reduces pavement cell turgor, due to decreased K+ accumulation, without affecting guard cell turgor. This results in an impaired back pressure of pavement cells onto guard cells, leading to larger stomatal apertures. Poorly rectifying membrane conductances to K+ were consistently observed in pavement cells. This plasmalemma property is likely to play an essential role in K+ shuttling within the epidermis. Functional complementation reveals that restoration of the wild-type stomatal functioning requires the expression of the transgenic AtKC1 at least in the pavement cells and trichomes. Altogether, the data suggest that AtKC1 activity contributes to the building of the back pressure that pavement cells exert onto guard cells by tuning K+ distribution throughout the leaf epidermis.

Noncompaction cardiomyopathy is a common congenital cardiac disorder associated with abnormal ventricular cardiomyocyte trabeculation and impaired pump function. The genetic basis and underlying mechanisms of this disorder remain elusive. We show that the genetic deletion of RNA-binding protein with multiple splicing (Rbpms), an uncharacterized RNA-binding factor, causes perinatal lethality in mice due to congenital cardiovascular defects. The loss of Rbpms causes premature onset of cardiomyocyte binucleation and cell cycle arrest during development. Human iPSC-derived cardiomyocytes with RBPMS gene deletion have a similar blockade to cytokinesis. Sequencing analysis revealed that RBPMS plays a role in RNA splicing and influences RNAs involved in cytoskeletal signaling pathways. We found that RBPMS mediates the isoform switching of the heart-enriched LIM domain protein Pdlim5. The loss of Rbpms leads to an abnormal accumulation of Pdlim5-short isoforms, disrupting cardiomyocyte cytokinesis. Our findings connect premature cardiomyocyte binucleation to noncompaction cardiomyopathy and highlight the role of RBPMS in this process.

Cardiomyocyte loss is the underlying basis for a majority of heart diseases. Preventing cardiomyocytes from death (cardioprotection) and replenishing the lost myocardium (regeneration) are the central goals for heart repair. Although cardioprotection and heart regeneration have been traditionally thought to involve separate mechanisms, protection of cardiomyocytes from injury or disease stimuli is a prerequisite to any meaningful regenerative response. In our study, we sought to understand how neonatal cardiomyocytes cope with injury-induced stress to regenerate damaged myocardium and whether the underlying mechanisms could be leveraged to promote heart regeneration and repair in adults. Using spatial transcriptomic profiling, we visualized regenerative cardiomyocytes reconstituting damaged myocardium after ischemia, and found that they are marked by expression of Nrf1, an ER-bound stress responsive transcription factor. Single-nucleus RNA sequencing revealed that genetic deletion of Nrf1 prevented neonatal cardiomyocytes from activating a transcriptional program required for heart regeneration. Conversely, overexpression of Nrf1 protected the adult mouse heart from ischemia/reperfusion injury. Nrf1 also protected human induced pluripotent stem cell-derived cardiomyocytes from cardiotoxicity induced by the chemotherapeutic drug doxorubicin. The cardioprotective function of Nrf1 is mediated by a dual stress response mechanism involving activation of the proteasome and maintenance of redox balance. Taken together, our study uncovers a unique adaptive mechanism activated in response to injury that maintains the tissue homeostatic balance required for heart regeneration. Reactivating these mechanisms in the adult heart represents a potential therapeutic approach for cardiac repair.

Ischemic heart disease is the leading cause of death worldwide. Direct reprogramming of resident cardiac fibroblasts (CFs) to induced cardiomyocytes (iCLMs) has emerged as a potential therapeutic approach to treat heart failure and ischemic disease. Cardiac reprogramming was first achieved through forced expression of the transcription factors Gata4, Mef2c, and Tbx5 (GMT); our laboratory found that Hand2 (GHMT) and Akt1 (AGHMT) markedly enhanced reprogramming efficiency in embryonic and postnatal cell types. However, adult mouse and human fibroblasts are resistant to reprogramming due to staunch epigenetic barriers. We undertook a screen of mammalian gene regulatory factors to discover novel regulators of cardiac reprogramming in adult fibroblasts and identified the epigenetic reader PHF7 as the most potent activating factor. We validated the findings of this screen and found that PHF7 augmented reprogramming of adult fibroblasts ten-fold. Mechanistically, PHF7 localized to cardiac super enhancers in fibroblasts by reading H3K4me2 marks, and through cooperation with the SWI/SNF complex, increased chromatin accessibility and transcription factor binding at these multivalent enhancers. Further, PHF7 recruited cardiac transcription factors to activate a positive transcriptional autoregulatory circuit in reprogramming. Importantly, PHF7 achieved efficient reprogramming through these mechanisms in the absence of Gata4. Collectively, these studies highlight the underexplored necessity of cardiac epigenetic readers, such as PHF7, in harnessing chromatin remodeling and transcriptional complexes to overcome critical barriers to direct cardiac reprogramming.

Following injury, cells in regenerative tissues have the ability to regrow. The mechanisms whereby regenerating cells adapt to injury-induced stress conditions and activate the regenerative program remain to be defined. Here, using the mammalian neonatal heart regeneration model, we show that Nrf1, a stress-responsive transcription factor encoded by the Nuclear Factor Erythroid 2 Like 1 (Nfe2l1) gene, is activated in regenerating cardiomyocytes. Genetic deletion of Nrf1 prevented regenerating cardiomyocytes from activating a transcriptional program required for heart regeneration. Conversely, Nrf1 overexpression protected the adult mouse heart from ischemia/reperfusion (I/R) injury. Nrf1 also protected human induced pluripotent stem cell-derived cardiomyocytes from doxorubicin-induced cardiotoxicity and other cardiotoxins. The protective function of Nrf1 is mediated by a dual stress response mechanism involving activation of the proteasome and redox balance. Our findings reveal that the adaptive stress response mechanism mediated by Nrf1 is required for neonatal heart regeneration and confers cardioprotection in the adult heart., Following injury, cells in regenerative tissues can regrow, but how they adapt to injury conditions to regenerate the tissue is unclear. Here the authors identify a stress-responsive and cardioprotective factor Nrf1 that is critical for neonatal heart regeneration.

International audience; Duchenne muscular dystrophy (DMD) is a devastating genetic disease leading to degeneration of skeletal muscles and premature death. How dystrophin absence leads to muscle wasting remains unclear. Here, we describe an optimized protocol to differentiate human induced pluripotent stem cells (iPSC) to a late myogenic stage. This allows us to recapitulate classical DMD phenotypes (mislocalization of proteins of the dystrophin-associated glycoprotein complex, increased fusion, myofiber branching, force contraction defects, and calcium hyperactivation) in isogenic DMD-mutant iPSC lines in vitro. Treatment of the myogenic cultures with prednisolone (the standard of care for DMD) can dramatically rescue force contraction, fusion, and branching defects in DMD iPSC lines. This argues that prednisolone acts directly on myofibers, challenging the largely prevalent view that its beneficial effects are caused by antiinflammatory properties. Our work introduces a human in vitro model to study the onset of DMD pathology and test novel therapeutic approaches.

Muscular dystrophies are a heterogeneous group of monogenic neuromuscular disorders which lead to progressive muscle loss and degeneration of the musculoskeletal system. The genetic causes of muscular dystrophies are well characterized, but no effective treatments have been developed so far. The discovery and application of the CRISPR/Cas system for genome editing offers a new path for disease treatment with the potential to permanently correct genetic mutations. The post-mitotic and multinucleated features of skeletal muscle provide an ideal target for CRISPR/Cas therapeutic genome editing because correction of a subpopulation of nuclei can provide benefit to the whole myofiber. In this review, we provide an overview of the CRISPR/Cas system and its derivatives in genome editing, proposing potential CRISPR/Cas-based therapies to correct diverse muscular dystrophies, and we discuss challenges for translating CRISPR/Cas genome editing to a viable therapy for permanent correction of muscular dystrophies.

Homeothermic vertebrates produce heat in cold environments through thermogenesis, in which brown adipose tissue (BAT) increases mitochondrial oxidation along with uncoupling of the electron transport chain and activation of uncoupling protein 1 (UCP1). Although the transcription factors regulating the expression of UCP1 and nutrient oxidation genes have been extensively studied, only a few other proteins essential for BAT function have been identified. We describe the discovery of FAM195A, a BAT-enriched RNA binding protein, which is required for cold-dependent thermogenesis in mice. FAM195A knockout (KO) mice display whitening of BAT and an inability to thermoregulate. In BAT of FAM195A KO mice, enzymes involved in branched-chain amino acid (BCAA) metabolism are down-regulated, impairing their response to cold. Knockdown of FAM195A in brown adipocytes in vitro also impairs expression of leucine oxidation enzymes, revealing FAM195A to be a regulator of BCAA metabolism and a potential target for metabolic disorders.

Myocardin, a potent coactivator of serum response factor (SRF), competes with ternary complex factor (TCF) proteins for SRF binding to balance opposing mitogenic and myogenic gene programs in cardiac and smooth muscle. Here we identify a cardiac lncRNA transcribed adjacent to myocardin, named CARDINAL, which antagonizes SRF-dependent mitogenic gene transcription in the heart. CARDINAL-deficient mice show ectopic TCF/SRF-dependent mitogenic gene expression and decreased cardiac contractility in response to age and ischemic stress. CARDINAL forms a nuclear complex with SRF and inhibits TCF-mediated transactivation of the promitogenic gene c-fos, suggesting CARDINAL functions as an RNA cofactor for SRF in the heart.

SummaryNoncompaction cardiomyopathy is a common congenital cardiac disorder associated with abnormal ventricular cardiomyocyte trabeculation and impaired pump function. The genetic basis and underlying mechanisms of this disorder remain elusive. We show that genetic deletion of R NA b inding p rotein with m ultiple s plicing ( Rbpms ), an uncharacterized RNA binding and splicing factor, causes perinatal lethality in mice due to congenital cardiovascular defects. Loss of Rbpms causes premature onset of cardiomyocyte binucleation and cell cycle arrest during development. Human iPSC-derived cardiomyocytes with RBPMS gene deletion have a similar blockade to cytokinesis. Sequencing analysis revealed that RBPMS plays a role in RNA splicing and influences RNAs involved in cytoskeletal signaling pathways. We found that RBPMS mediates isoform switching of the heart-specific LIM domain protein Pdlim5 . Loss of Rbpms leads to abnormal accumulation of Pdlim5 -short isoforms, disrupting cardiomyocyte cytokinesis. Our findings connect premature cardiomyocyte binucleation to noncompaction cardiomyopathy and highlight the role of Rbpms in this process.

Direct cardiac reprogramming of fibroblasts to cardiomyocytes presents an attractive therapeutic strategy to restore cardiac function following injury. Cardiac reprogramming was initially achieved through the overexpression of the transcription factors Gata4, Mef2c, and Tbx5 (GMT), and later, Hand2 (GHMT) and Akt1 (AGHMT) were found to further enhance this process. Yet, staunch epigenetic barriers severely limit the ability of these cocktails to reprogram adult fibroblasts. We undertook a screen of mammalian gene regulatory factors to discover novel regulators of cardiac reprogramming in adult fibroblasts and identified the histone reader PHF7 as the most potent activating factor. Mechanistically, PHF7 localizes to cardiac super enhancers in fibroblasts, and through cooperation with the SWI/SNF complex, increases chromatin accessibility and transcription factor binding at these sites. Further, PHF7 recruits cardiac transcription factors to activate a core regulatory circuit in reprogramming. Importantly, PHF7 is the first epigenetic factor shown to achieve efficient reprogramming in the absence of Gata4. Here, we highlight the underexplored necessity of cardiac epigenetic modifiers, such as PHF7, in harnessing chromatin remodeling and transcriptional complexes to overcome critical barriers to direct cardiac reprogramming.

Duchenne Muscular Dystrophy (DMD) is a devastating genetic disease leading to degeneration of skeletal muscles and premature death. How dystrophin absence leads to muscle wasting remains unclear. Here, we describe an optimized protocol to differentiate human induced Pluripotent Stem Cells (iPSC) to a late myogenic stage. This allows to recapitulate classical DMD phenotypes (mislocalization of proteins of the Dystrophin-glycoprotein associated complex (DGC), increased fusion, myofiber branching, force contraction defects and calcium hyperactivation) in isogenic DMD-mutant iPSC lines in vitro. Treatment of the myogenic cultures with prednisolone (the standard of care for DMD) can dramatically rescue force contraction, fusion and branching defects in DMD iPSC lines. This argues that prednisolone acts directly on myofibers, challenging the largely prevalent view that its beneficial effects are due to anti-inflammatory properties. Our work introduces a new human in vitro model to study the onset of DMD pathology and test novel therapeutic approaches.

The adult mammalian heart is incapable of regeneration following injury. In contrast, the neonatal mouse heart can efficiently regenerate during the first week of life. The molecular mechanisms that mediate the regenerative response and its blockade in later life are not understood. Here, by single-nucleus RNA sequencing, we map the dynamic transcriptional landscape of five distinct cardiomyocyte populations in healthy, injured, and regenerating mouse hearts. We identify immature cardiomyocytes that enter the cell cycle following injury and disappear as the heart loses the ability to regenerate. These proliferative neonatal cardiomyocytes display a unique transcriptional program dependent on nuclear transcription factor Y subunit alpha (NFYa) and nuclear factor erythroid 2-like 1 (NFE2L1) transcription factors, which exert proliferative and protective functions, respectively. Cardiac overexpression of these two factors conferred protection against ischemic injury in mature mouse hearts that were otherwise non-regenerative. These findings advance our understanding of the cellular basis of neonatal heart regeneration and reveal a transcriptional landscape for heart repair following injury.

Lamins and transmembrane proteins within the nuclear envelope regulate nuclear structure and chromatin organization. Nuclear envelope transmembrane protein 39 (Net39) is a muscle nuclear envelope protein whose functions in vivo have not been explored. We show that mice lacking Net39 succumb to severe myopathy and juvenile lethality, with concomitant disruption in nuclear integrity, chromatin accessibility, gene expression, and metabolism. These abnormalities resemble those of Emery–Dreifuss muscular dystrophy (EDMD), caused by mutations in A-type lamins (LMNA) and other genes, like Emerin (EMD). We observe that Net39 is downregulated in EDMD patients, implicating Net39 in the pathogenesis of this disorder. Our findings highlight the role of Net39 at the nuclear envelope in maintaining muscle chromatin organization, gene expression and function, and its potential contribution to the molecular etiology of EDMD., The nuclear envelope tethers chromatin to the nuclear periphery to control genome architecture. Here, the authors show that Net39 preserves the integrity and gene expression of muscle nuclei in mice, and it may contribute to the pathogenesis of Emery–Dreifuss muscular dystrophy.

Regeneration of skeletal muscle in response to injury occurs through fusion of a population of stem cells, known as satellite cells, with injured myofibers. Myomixer, a muscle-specific membrane micropeptide, cooperates with the transmembrane protein Myomaker to regulate embryonic myoblast fusion and muscle formation. To investigate the role of Myomixer in muscle regeneration, we used CRISPR/Cas9-mediated genome editing to generate conditional knockout Myomixer alleles in mice. We show that genetic deletion of Myomixer in satellite cells using a tamoxifen-regulated Cre recombinase transgene under control of the Pax7 promoter abolishes satellite cell fusion and prevents muscle regeneration, resulting in severe muscle degeneration after injury. Satellite cells devoid of Myomixer maintain expression of Myomaker, demonstrating that Myomaker alone is insufficient to drive myoblast fusion. These findings, together with prior studies demonstrating the essentiality of Myomaker for muscle regeneration, highlight the obligatory partnership of Myomixer and Myomaker for myofiber formation throughout embryogenesis and adulthood.

Skeletal muscle formation requires fusion of mononucleated myoblasts to form multinucleated myofibers. The muscle-specific membrane proteins myomaker and myomixer cooperate to drive mammalian myoblast fusion. Whereas myomaker is highly conserved across diverse vertebrate species, myomixer is a micropeptide that shows relatively weak cross-species conservation. To explore the functional conservation of myomixer, we investigated the expression and function of the zebrafish myomixer ortholog. Here we show that myomixer expression during zebrafish embryogenesis coincides with myoblast fusion, and genetic deletion of myomixer using CRISPR/Cas9 mutagenesis abolishes myoblast fusion in vivo. We also identify myomixer orthologs in other species of fish and reptiles, which can cooperate with myomaker and substitute for the fusogenic activity of mammalian myomixer. Sequence comparison of these diverse myomixer orthologs reveals key amino acid residues and a minimal fusogenic peptide motif that is necessary for promoting cell-cell fusion with myomaker. Our findings highlight the evolutionary conservation of the myomaker-myomixer partnership and provide insights into the molecular basis of myoblast fusion.

Maintenance of muscle structure and function depends on the precise organization of contractile proteins into sarcomeres and coupling of the contractile apparatus to the sarcoplasmic reticulum (SR), which serves as the reservoir for calcium required for contraction. Several members of the Kelch superfamily of proteins, which modulate protein stability as substrate-specific adaptors for ubiquitination, have been implicated in sarcomere formation. The Kelch protein Klhl31 is expressed in a muscle-specific manner under control of the transcription factor MEF2. To explore its functions in vivo, we created a mouse model of Klhl31 loss of function using the CRISPR-Cas9 system. Mice lacking Klhl31 exhibited stunted postnatal skeletal muscle growth, centronuclear myopathy, central cores, Z-disc streaming, and SR dilation. We used proteomics to identify several candidate Klhl31 substrates, including Filamin-C (FlnC). In the Klhl31-knockout mice, FlnC protein levels were highly upregulated with no change in transcription, and we further demonstrated that Klhl31 targets FlnC for ubiquitination and degradation. These findings highlight a role for Klhl31 in the maintenance of skeletal muscle structure and provide insight into the mechanisms underlying congenital myopathies.

Micromanaging muscle cell fusion Adult skeletal muscles are characterized by long, multinucleated cells called myofibers. Myofibers form when muscle precursor cells, or myoblasts, differentiate and fuse together during embryogenesis. The fusion process is not fully understood. Studying cell culture and mouse models, Bi et al. identified an 84–amino acid peptide that promotes myoblast fusion. This small peptide, called Myomixer, physically interacts with and stimulates the activity of a fusogenic membrane protein called Myomaker. Notably, the Myomaker-Myomixer pair can also promote the fusion of nonmuscle cells, such as fibroblasts. Science , this issue p. 323

Summary Conversion of fibroblasts into functional cardiomyocytes represents a potential means of restoring cardiac function after myocardial infarction, but so far this process remains inefficient and little is known about its molecular mechanisms. Here we show that DAPT, a classical Notch inhibitor, enhances the conversion of mouse fibroblasts into induced cardiac-like myocytes by the transcription factors GATA4, HAND2, MEF2C, and TBX5. DAPT cooperates with AKT kinase to further augment this process, resulting in up to 70% conversion efficiency. Moreover, DAPT promotes the acquisition of specific cardiomyocyte features, substantially increasing calcium flux, sarcomere structure, and the number of spontaneously beating cells. Transcriptome analysis shows that DAPT induces genetic programs related to muscle development, differentiation, and excitation-contraction coupling. Mechanistically, DAPT increases binding of the transcription factor MEF2C to the promoter regions of cardiac structural genes. These findings provide mechanistic insights into the reprogramming process and may have important implications for cardiac regeneration therapies., Graphical Abstract, Highlights • Notch activation is a barrier for GHMT-induced cardiac cell reprogramming • Notch blockade by DAPT improves GHMT-induced cardiac reprogramming • DAPT increases sarcomere organization, calcium flux, and beating in GHMT reprogramming • DAPT enhances transcriptional activity of MEF2C in GHMT reprogramming, In this article, Olson and colleagues show that Notch signaling activation is a critical barrier for cardiac cell reprogramming. Notch signaling blockade by DAPT, a γ-secretase inhibitor, enhances fibroblast conversion to cardiomyocytes by increasing calcium flux, sarcomere structure, and beating. Mechanistically, Notch inhibition enhances the transcriptional activity of the cardiogenic transcription factor MEF2C, thereby enhancing expression of cardiac genes.

Introduction Cardiogenic transcription factors (TFs), Gata4, Mef2c, and Tbx5, can directly reprogram fibroblasts to a cardiac fate and their cardiogenic activity is enhanced by the Hand2 TF and the Akt1 kinase. Although these cardiac reprogramming factors are key regulators of heart development, their expression and biological functions are not limited to the heart, and the mechanism by which these cardiac factors orchestrate reprogramming remains unclear. Purpose We sought to study the molecular mechanisms by which cardiac reprogramming factors contribute to cell fate conversion using a genome-wide approach. Methods Here, we used chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) to explore the genomic binding sites of reprogramming TFs and the landscape of active enhancers, annotated by H3K27ac histone modification, during cardiac reprogramming. Results We found that reprogramming TFs rapidly silence fibroblast enhancers and synergistically activate cardiac enhancers which were predominantly enriched with Mef2 motifs. Addition of Hand2 and Akt1 to GMT expands TF co-occupancy and thereby activates additional cardiac enhancers, which further augments cardiac gene expression. Moreover, additional cardiac enhancers were sequentially activated during the reprogramming process, in accordance with the temporal acquisition of functional phenotypes in induced cardiac-like myocytes. We discovered that subsets of reprogramming enhancers are conserved among other cardiogenic processes, and a collection of reprogramming enhancers displayed unique spatial expression patterns in the developing heart in transgenic mouse embryos. Conclusion Our study describes the epigenomic dynamics that underlie cardiac reprogramming which is cooperatively orchestrated by reprogramming factors to convert fibroblasts toward a cardiac lineage. Acknowledgement/Funding NIH (AR-067294, HL-130253, HL-138426, HD-087351), Fondation Leducq Transatlantic Networks of Excellence in Cardiovascular Research

Duchenne muscular dystrophy (DMD) is a fatal genetic disorder caused by mutations in the dystrophin gene. To enable the non-invasive analysis of DMD gene correction strategies in vivo, we introduced a luciferase reporter in-frame with the C-terminus of the dystrophin gene in mice. Expression of this reporter mimics endogenous dystrophin expression and DMD mutations that disrupt the dystrophin open reading frame extinguish luciferase expression. We evaluated the correction of the dystrophin reading frame coupled to luciferase in mice lacking exon 50, a common mutational hotspot, after delivery of CRISPR/Cas9 gene editing machinery with adeno-associated virus. Bioluminescence monitoring revealed efficient and rapid restoration of dystrophin protein expression in affected skeletal muscles and the heart. Our results provide a sensitive non-invasive means of monitoring dystrophin correction in mouse models of DMD and offer a platform for testing different strategies for amelioration of DMD pathogenesis., Dystrophin-deficient mice are used to test corrective strategies for Duchenne muscular dystrophy, but evaluation of dystrophin expression requires collection of tissue samples from specific muscles and time points. Here, the authors generate mice in which dystrophin expression is coupled to luciferase, and show that bioluminescence allows non-invasive monitoring of dystrophin expression following genome editing.

The adult mammalian heart has limited capacity for regeneration following injury, whereas the neonatal heart can readily regenerate within a short period after birth. To uncover the molecular mechanisms underlying neonatal heart regeneration, we compared the transcriptomes and epigenomes of regenerative and nonregenerative mouse hearts over a 7-d time period following myocardial infarction injury. By integrating gene expression profiles with histone marks associated with active or repressed chromatin, we identified transcriptional programs underlying neonatal heart regeneration, and the blockade to regeneration in later life. Our results reveal a unique immune response in regenerative hearts and a retained embryonic cardiogenic gene program that is active during neonatal heart regeneration. Among the unique immune factors and embryonic genes associated with cardiac regeneration, we identified Ccl24, which encodes a cytokine, and Igf2bp3, which encodes an RNA-binding protein, as previously unrecognized regulators of cardiomyocyte proliferation. Our data provide insights into the molecular basis of neonatal heart regeneration and identify genes that can be modulated to promote heart regeneration.

Cardiac remodelling is accompanied by silencing of genes necessary for cardiac homeostasis including Krüppel-like factor 15 ( Klf15 ), an anti-hypertrophic factor. Due to a lack of tools for gene activation, we aim to adapt a CRISPR-based approach for gene expression control for mammalian cardiomyocytes. We generated a transgenic mouse model (TG) expressing an enzymatically inactive, gRNA-programmable Cas9 protein under Myh6 promoter control fused to transcriptional activators (VPR) to drive gene expression. As a proof of concept, we systemically injected mice at postnatal day P4 with AAV9 carrying a triple gRNA expression cassette containing gRNAs targeted to the Klf15 promoter region. Significant Klf15 activation was observed at P10 up to 10-fold compared to controls in three independent TG lines. This Klf15 activation was sufficient to drive target gene expression of Aldh2 and Adhfe1 (controls = WT + saline, WT + AAV9, TG + saline, n ≥ 3 per group, ANOVA and Bonferroni correction). Importantly, at the neonatal stage, the Klf15 promoter is characterized by low H3K27ac presence indicating that endogenous gene activation can enhance transcription from an epigenetically inactive locus. We demonstrated that CRISPR/Cas9 mediated loss of KLF15 expression results in impaired contractile function in a 3D model of engineering heart muscle, similar to the Klf15 -/- mouse heart phenotype. Encouraged by the evolutionary conserved KLF15 -mediated mechanisms, we aimed to generate a human induced pluripotent stem cells, which constitutively express dCas9VPR (hIPSC-dCas9VPR) for gene activation in hIPSC-derived cardiomyocytes to complement the in vivo findings upon disease. We targeted the transgene integration to the AAVS1 site by CRISPR gene editing, confirmed correct integration by PCR followed by sequencing and hIPSC-dCas9VPR were successfully differentiated into beating cardiomyocytes. To this end, we have tested gRNAs targeted to the KLF15 promoter region in HEK293 cells and observed significant KLF15 activation of up to 5.5-fold (n = 3, ANOVA and Bonferroni correction). In summary, we report transgenic mammalian tools for endogenous gene (re-) activation purposes to identify potential therapeutic targets for preventing heart failure progression.

The adult mammalian heart has limited capacity for regeneration following injury, whereas the neonatal heart can readily regenerate within a short period after birth. Deciphering the molecular underpinnings of neonatal heart regeneration and the blockade to regeneration in later life may provide novel insights for heart repair. Neonatal heart regeneration is orchestrated by multiple cell types including cardiac resident cells and immune cells that infiltrate the heart after injury. To systematically elucidate the transcriptional response after injury at cellular level during heart regeneration, we performed single cell RNA-sequencing on hearts at various time points post P1 or P8 myocardial infarction injury, and coupled that with bulk tissue RNA-sequencing data collected at the same time points. This integrated approach provides detailed transcriptome landscape and dynamics during heart regeneration at single cell resolution. We further depict a ligand-receptor interaction network that potentially reveal cellular signaling and cell-cell cross-talks during heart regeneration in cell autonomous and non-autonomous manners. Furthermore, to uncover the transcriptional dynamics in a single cardiomyocyte (CM), which was technically challenging due to incompatibility of CM diameter with Drop-Seq platforms, we performed single nucleus RNA-sequencing for CM population, and identified a subset of CMs in the regenerative hearts with unique molecular properties. Taken together, our data provide , and suggest numerous inroads that might be therapeutically manipulated to enhance cardiac function in response to injury.

Direct cardiac reprogramming of fibroblasts to cardiomyocytes is an attractive therapeutic strategy to restore cardiac function following injury. The cardiac transcription factors Gata4, Mef2c, and Tbx5 are sufficient to directly reprogram fibroblasts to a cardiac fate and their cardiogenic activity is enhanced by the addition of Hand2 and Akt1. However, the mechanisms by which these transcription factors orchestrate this cell fate conversion remains elusive. To understand the mechanistic basis of cardiac reprogramming, we performed a genome-wide analysis of cardiogenic transcription factor binding sites and enhancer activation throughout cardiac reprogramming. We found that cardiogenic transcription factors act cooperatively by co-occupying regulatory elements enriched with Mef2 binding sites during cardiac reprogramming. Importantly, these transcription factors when overexpressed in isolation are incapable of activating reprogramming enhancer elements. Further, we discovered that the early reprogramming enhancer landscape most closely resembles that of the neonatal heart. Acquisition of enhancers associated with cardiac maturation occurred at later stages in reprogramming, with addition of Hand2 and Akt1 augmenting activation of this more mature enhancer landscape. Additionally, we constructed a cardiac reprogramming gene regulatory network which demonstrated downregulation of the EGF receptor signaling pathway. We found that inhibition of EGF receptor signaling augments reprogramming toward the cardiac phenotype. Our findings demonstrate that cardiac reprogramming is coordinated by synergistic transcriptional activation across a broad landscape of cardiac enhancers and define the epigenetic landscape of the cardiac phenotype.

The cardiogenic transcription factors (TFs) Mef2c, Gata4, and Tbx5 can directly reprogram fibroblasts to induced cardiac-like myocytes (iCLMs), presenting a potential source of cells for cardiac repair. While activity of these TFs is enhanced by Hand2 and Akt1, their genomic targets and interactions during reprogramming are not well studied. We performed genome-wide analyses of cardiogenic TF binding and enhancer profiling during cardiac reprogramming. We found that these TFs synergistically activate enhancers highlighted by Mef2c binding sitesand that Hand2 and Akt1 coordinately recruit other TFs to enhancer elements. Intriguingly, these enhancer landscapes collectively resemble patterns of enhancer activation during embryonic cardiogenesis. We further constructed a cardiac reprogramming gene regulatory network and found repression of EGFR signaling pathway genes. Consistently, chemical inhibition of EGFR signaling augmented reprogramming. Thus, by defining epigenetic landscapes these findings reveal synergistic transcriptional activation across a broad landscape of cardiac enhancers and key signaling pathways that govern iCLM reprogramming.

We previously identified a unique population of interstitial muscle progenitors, marked by expression of the Twist2 transcription factor, which fuses specifically to type IIb/x fast-twitch myofibers. Tw2+ progenitors are distinct from satellite cells, a muscle progenitor that expresses Pax7 and contributes to all myofiber types. Through RNA sequencing and immunofluorescence, we identify the membrane receptor, Nrp1, as a marker of Tw2+ cells but not Pax7+ cells. We also found that Sema3a, a chemorepellent ligand for Nrp1, is expressed by type I and IIa myofibers but not IIb myofibers. Using stripe migration assays, chimeric cell-cell fusion assays, and a Sema3a transgenic mouse model, we identify Sema3a-Nrp1 signaling as a major mechanism for Tw2+ cell fiber-type specificity. Our findings reveal an extracellular signaling mechanism whereby a cell-surface receptor for a chemorepellent confers specificity of intercellular fusion of a specific muscle progenitor with its target tissue.

Rhabdomyosarcoma (RMS) is an aggressive pediatric cancer composed of myoblast-like cells. Recently, we discovered a unique muscle progenitor marked by the expression of the Twist2 transcription factor. Genomic analyses of 258 RMS patient tumors uncovered prevalent copy number amplification events and increased expression of TWIST2 in fusion-negative RMS. Knockdown of TWIST2 in RMS cells results in up-regulation of MYOGENIN and a decrease in proliferation, implicating TWIST2 as an oncogene in RMS. Through an inducible Twist2 expression system, we identified Twist2 as a reversible inhibitor of myogenic differentiation with the remarkable ability to promote myotube dedifferentiation in vitro. Integrated analysis of genome-wide ChIP-seq and RNA-seq data revealed the first dynamic chromatin and transcriptional landscape of Twist2 binding during myogenic differentiation. During differentiation, Twist2 competes with MyoD at shared DNA motifs to direct global gene transcription and repression of the myogenic program. Additionally, Twist2 shapes the epigenetic landscape to drive chromatin opening at oncogenic loci and chromatin closing at myogenic loci. These epigenetic changes redirect MyoD binding from myogenic genes toward oncogenic, metabolic, and growth genes. Our study reveals the dynamic interplay between two opposing transcriptional regulators that control the fate of RMS and provides insight into the molecular etiology of this aggressive form of cancer.

SUMMARY Micropeptide regulator of β-oxidation (MOXI) is a conserved muscle-enriched protein encoded by an RNA transcript misannotated as non-coding. MOXI localizes to the inner mitochondrial membrane where it associates with the mitochondrial trifunctional protein, an enzyme complex that plays a critical role in fatty acid β-oxidation. Isolated heart and skeletal muscle mitochondria from MOXI knockout mice exhibit a diminished ability to metabolize fatty acids, while transgenic MOXI overexpression leads to enhanced β-oxidation. Additionally, hearts from MOXI knockout mice preferentially oxidize carbohydrates over fatty acids in an isolated perfused heart system compared to wild-type (WT) animals. MOXI knockout mice also exhibit a profound reduction in exercise capacity, highlighting the role of MOXI in metabolic control. The functional characterization of MOXI provides insight into the regulation of mitochondrial metabolism and energy homeostasis and underscores the regulatory potential of additional micropeptides that have yet to be identified., In Brief Micropeptide regulator of b-oxidation (MOXI) is encoded by a muscle-enriched RNA transcript misannotated as noncoding. MOXI localizes to the inner mitochondrial membrane where it interacts with the trifunctional protein to modulate fatty acid b-oxidation and exercise capacity., Graphical Abstract

SUMMARY The adult mammalian heart has limited capacity for regeneration following injury, whereas the neonatal heart can readily regenerate within a short period after birth. Neonatal heart regeneration is orchestrated by multiple cell types intrinsic to the heart, as well as immune cells that infiltrate the heart after injury. To elucidate the transcriptional responses of the different cellular components of the mouse heart following injury, we perform single-cell RNA sequencing on neonatal hearts at various time points following myocardial infarction and couple the results with bulk tissue RNA-sequencing data collected at the same time points. Concomitant single-cell ATAC sequencing exposes underlying dynamics of open chromatin landscapes and regenerative gene regulatory networks of diverse cardiac cell types and reveals extracellular mediators of cardiomyocyte proliferation, angiogenesis, and fibroblast activation. Together, our data provide a transcriptional basis for neonatal heart regeneration at single-cell resolution and suggest strategies for enhancing cardiac function after injury., Graphical Abstract, In Brief Using single-cell sequencing technologies, Wang et al. present single-cell databases of gene expression and open chromatin landscapes of heart cells during murine neonatal heart regeneration. Comparing the injury responses of regenerative and non-regenerative hearts reveals gene regulatory networks, cellular crosstalk, and secreted factors involved in the regeneration process.

During skeletal muscle development, myoblasts fuse to form multinucleated myofibers. Myomaker [Transmembrane protein 8c (TMEM8c)] is a muscle-specific protein that is essential for myoblast fusion and sufficient to promote fusion of fibroblasts with muscle cells; however, the structure and biochemical properties of this membrane protein have not been explored. Here, we used CRISPR/Cas9 mutagenesis to disrupt myomaker expression in the C2C12 muscle cell line, which resulted in complete blockade to fusion. To define the functional domains of myomaker required to direct fusion, we established a heterologous cell-cell fusion system, in which fibroblasts expressing mutant versions of myomaker were mixed with WT myoblasts. Our data indicate that the majority of myomaker is embedded in the plasma membrane with seven membrane-spanning regions and a required intracellular C-terminal tail. We show that myomaker function is conserved in other mammalian orthologs; however, related family members (TMEM8a and TMEM8b) do not exhibit fusogenic activity. These findings represent an important step toward deciphering the cellular components and mechanisms that control myoblast fusion and muscle formation.

Editing can help build stronger muscles Much of the controversy surrounding the gene-editing technology called CRISPR/Cas9 centers on the ethics of germline editing of human embryos to correct disease-causing mutations. For certain disorders such as muscular dystrophy, it may be possible to achieve therapeutic benefit by editing the faulty gene in somatic cells. In proof-of-concept studies, Long et al. , Nelson et al. , and Tabebordbar et al. used adeno-associated virus-9 to deliver the CRISPR/Cas9 gene-editing system to young mice with a mutation in the gene coding for dystrophin, a muscle protein deficient in patients with Duchenne muscular dystrophy. Gene editing partially restored dystrophin protein expression in skeletal and cardiac muscle and improved skeletal muscle function. Science , this issue p. 400 , p. 403 , p. 407

Another micropeptide flexes its muscle Genome annotation is a complex but imperfect art. Attesting to its limitations is the growing evidence that certain transcripts annotated as long noncoding RNAs (lncRNAs) in fact code for small peptides with biologically important functions. One such lncRNA-derived micropeptide in mammals is myoregulin, which reduces muscle performance by inhibiting the activity of a key calcium pump. Nelson et al. describe the opposite activity in a second lncRNA-derived micropeptide in mammalian muscle, called DWORF (see the Perspective by Payre and Desplan). This peptide enhances muscle performance by activating the same calcium pump. DWORF may prove to be useful in improving the cardiac muscle function of mammals with heart disease. Science , this issue p. 271 ; see also p. 226

Calcium (Ca2+) dysregulation is a hallmark of heart failure and is characterized by impaired Ca2+ sequestration into the sarcoplasmic reticulum (SR) by the SR-Ca2+-ATPase (SERCA). We recently discovered a micropeptide named DWORF (DWarf Open Reading Frame) that enhances SERCA activity by displacing phospholamban (PLN), a potent SERCA inhibitor. Here we show that DWORF has a higher apparent binding affinity for SERCA than PLN and that DWORF overexpression mitigates the contractile dysfunction associated with PLN overexpression, substantiating its role as a potent activator of SERCA. Additionally, using a well-characterized mouse model of dilated cardiomyopathy (DCM) due to genetic deletion of the muscle-specific LIM domain protein (MLP), we show that DWORF overexpression restores cardiac function and prevents the pathological remodeling and Ca2+ dysregulation classically exhibited by MLP knockout mice. Our results establish DWORF as a potent activator of SERCA within the heart and as an attractive candidate for a heart failure therapeutic., eLife digest The heart is a muscular organ that contracts regularly to pump blood around the body, ensuring that nutrients and oxygen are carried to the cells and organs. Heart failure is a disease where the heart muscle becomes weakened, does not beat as strongly, and cannot pump blood as well as it should. Eventually, the heart can no longer deliver enough blood to meet the body’s needs. Although heart failure is a widespread disease, we still do not fully understand its underlying causes and the molecular machinery driving its progression. However, one common feature in many cases of heart failure is a problem with the supply of calcium to the heart muscle. Calcium is the molecule responsible for the process of muscle contraction; the strength of contraction depends on the amount of calcium available. Movement of calcium within heart cells is in turn controlled by an enzyme pump called SERCA. In 2016, researchers identified a small protein, DWORF, which increased the activity of SERCA. Makarewich et al. – including many of the researchers involved in the 2016 study – therefore wanted to find out more about how DWORF and SERCA worked together. They also wanted to test if DWORF could be used to boost the heart’s ability to pump blood efficiently, and if so, whether it could treat heart failure. Genetically modified mice that produced larger than normal amounts of DWORF had more available calcium in the heart muscle, which made it contract more strongly. This was true even when the same mice were treated with an excessive amount of a specific protein (phospholamban) that can lower the activity of SERCA, suggesting that DWORF might have a protective effect on the heart. Experiments using mice engineered to show symptoms of heart disease confirmed that DWORF treatment did indeed help their hearts beat normally, and, crucially, prevented them from developing heart failure. This work has shown for the first time that DWORF can restore the heart’s ability to pump normally in an experimental model of heart disease. In the future, Makarewich et al. hope that DWORF could be a useful target for new, more effective drugs to treat heart failure.

Twist transcription factors function as ancestral regulators of mesodermal cell fates in organisms ranging from Drosophila to mammals. Through lineage tracing of Twist2 (Tw2)-expressing cells with tamoxifen-inducible Tw2-CreERT2 and tdTomato (tdTO) reporter mice, we discovered a unique cell population that progressively contributes to cardiomyocytes (CMs), endothelial cells, and fibroblasts in the adult heart. Clonal analysis confirmed the ability of Tw2-derived tdTO(+) (Tw2-tdTO(+)) cells to form CMs in vitro. Within the adult heart, Tw2-tdTO(+) CMs accounted for ∼13% of total CMs, the majority of which resulted from fusion of Tw2-tdTO(+) cells with existing CMs. Tw2-tdTO(+) cells also contribute to cardiac remodeling after injury. We conclude that Tw2-tdTO(+) cells participate in lifelong maintenance of cardiac function, at least in part through de novo formation of CMs and fusion with preexisting CMs, as well as in the genesis of other cellular components of the adult heart.

The adult mammalian heart has very limited capacity to regenerate upon injury. Following myocardial infarction, cardiomyocytes are extensively lost and fibroblasts are activated to form a scar, resulting in compromised cardiac function. In contrast, the heart of a neonatal mouse has full capacity to regenerate after myocardial infarction, although such regenerative ability is lost by postnatal (P) day 7. The molecular properties of neonatal hearts that enable regeneration remain unknown. To uncover the mechanisms of neonatal heart regeneration, we induced myocardial infarction (MI) in P1 and P8 hearts, and profiled transcriptomic and epigenomic dynamics by RNA-Seq, H3K27ac ChIP-Seq and H3K27me3 ChIP-Seq at various time points following the surgery. Analysis of differentially expressed genes and groups of enhancers that acquire active status post-MI revealed distinct post-infarction responses between P1 and P8 hearts. Dramatic differences in immune response and developmental gene programs were identified in regenerative P1 versus non-regenerative P8 hearts. Furthermore, we analyzed transcription factors involved in the injury response post MI and constructed gene regulatory networks during neonatal cardiac injury. This work provides insights into the molecular basis of neonatal heart regeneration, and identifies potential genes and cis-regulatory elements that can be modulated to facilitate heart regeneration.

Control of protein homeostasis is an essential cellular process that, when perturbed, can result in the deregulation or toxic accumulation of proteins. Owing to constant mechanical stress, striated muscle proteins are particularly prone to wear and tear and require several protein quality–control mechanisms to coordinate protein turnover and removal of damaged proteins. Kelch-like proteins, substrate adapters for the Cullin-3 (Cul3)-RING ligase (CRL3) complex, are emerging as critical regulators of striated muscle development and function, highlighting the importance of Cul3-mediated proteostasis in muscle function. To explore the role of Cul3-mediated proteostasis in striated muscle, here we deleted Cul3 specifically in either skeletal muscle (SkM-Cul3 KO) or cardiomyocytes (CM-Cul3 KO) of mice. The loss of Cul3 caused neonatal lethality and dramatic alterations in the proteome, which were unique to each striated muscle type. Many of the proteins whose expression was significantly changed in the SkM-Cul3 KO were components of the extracellular matrix and sarcomere, whereas proteins altered in the CM-Cul3 KO were involved in metabolism. These findings highlight the requirement for striated muscle–specific CRL3 activity and indicate how the CRL3 complex can control different nodes of protein interaction networks in different types of striated muscle. Further identification of Cul3 substrates, and how these substrates are targeted, may reveal therapeutic targets and treatment regimens for striated muscle diseases.

Myocardin-Related Transcription Factors A and B (MRTF-A and MRTF-B) are highly homologous proteins that function as powerful coactivators of serum response factor (SRF), a ubiquitously expressed transcription factor essential for cardiac development. The SRF/MRTF complex binds to CArG boxes found in the control regions of genes that regulate cytoskeletal dynamics and muscle contraction, among other processes. While SRF is required for heart development and function, the role of MRTFs in the developing or adult heart has not been explored. Through cardiac-specific deletion of MRTF alleles in mice, we show that either MRTF-A or MRTF-B is dispensable for cardiac development and function, whereas deletion of both MRTF-A and MRTF-B causes a spectrum of structural and functional cardiac abnormalities. Defects observed in MRTF-A/B null mice ranged from reduced cardiac contractility and adult onset heart failure to neonatal lethality accompanied by sarcomere disarray. RNA-seq analysis on neonatal hearts identified the most altered pathways in MRTF double knockout hearts as being involved in cytoskeletal organization. Together, these findings demonstrate redundant but essential roles of the MRTFs in maintenance of cardiac structure and function and as indispensible links in cardiac cytoskeletal gene regulatory networks.

Conversion of fibroblasts to functional cardiomyocytes represents a potential approach for restoring cardiac function after myocardial injury, but the technique thus far has been slow and inefficient. To improve the efficiency of reprogramming fibroblasts to cardiac-like myocytes (iCMs) by cardiac transcription factors [Gata4, Hand2, Mef2c, and Tbx5 (GHMT)], we screened 192 protein kinases and discovered that Akt/protein kinase B dramatically accelerates and amplifies this process in three different types of fibroblasts (mouse embryo, adult cardiac, and tail tip). Approximately 50% of reprogrammed mouse embryo fibroblasts displayed spontaneous beating after 3 wk of induction by Akt plus GHMT. Furthermore, addition of Akt1 to GHMT evoked a more mature cardiac phenotype for iCMs, as seen by enhanced polynucleation, cellular hypertrophy, gene expression, and metabolic reprogramming. Insulin-like growth factor 1 (IGF1) and phosphoinositol 3-kinase (PI3K) acted upstream of Akt whereas the mitochondrial target of rapamycin complex 1 (mTORC1) and forkhead box o3 (Foxo3a) acted downstream of Akt to influence fibroblast-to-cardiomyocyte reprogramming. These findings provide insights into the molecular basis of cardiac reprogramming and represent an important step toward further application of this technique.

Background The Muscle-specific RING-finger (MuRF) protein family of E3 ubiquitin ligases is important for maintenance of muscular structure and function. MuRF proteins mediate adaptation of striated muscles to stress. MuRF2 and MuRF3 bind to microtubules and are implicated in sarcomere formation with noticeable functional redundancy. However, if this redundancy is important for muscle function in vivo is unknown. Our objective was to investigate cooperative function of MuRF2 and MuRF3 in the skeletal muscle and the heart in vivo. Methods MuRF2 and MuRF3 double knockout mice (DKO) were generated and phenotypically characterized. Skeletal muscle and the heart were investigated by morphological measurements, histological analyses, electron microscopy, immunoblotting, and real-time PCR. Isolated muscles were subjected to in vitro force measurements. Cardiac function was determined by echocardiography and working heart preparations. Function of cardiomyocytes was measured in vitro. Cell culture experiments and mass-spectrometry were used for mechanistic analyses. Results DKO mice showed a protein aggregate myopathy in skeletal muscle. Maximal force development was reduced in DKO soleus and extensor digitorum longus. Additionally, a fibre type shift towards slow/type I fibres occurred in DKO soleus and extensor digitorum longus. MuRF2 and MuRF3-deficient hearts showed decreased systolic and diastolic function. Further analyses revealed an increased expression of the myosin heavy chain isoform beta/slow and disturbed calcium handling as potential causes for the phenotype in DKO hearts. Conclusions The redundant function of MuRF2 and MuRF3 is important for maintenance of skeletal muscle and cardiac structure and function in vivo.

Direct reprogramming of fibroblasts to cardiomyocytes represents a potential means of restoring cardiac function following myocardial injury. AKT1 in the presence of four cardiogenic transcription factors, GATA4, HAND2, MEF2C, and TBX5 (AGHMT), efficiently induces the cardiac gene program in mouse embryonic fibroblasts but not adult fibroblasts. To identify additional regulators of adult cardiac reprogramming, we performed an unbiased screen of transcription factors and cytokines for those that might enhance or suppress the cardiogenic activity of AGHMT in adult mouse fibroblasts. Among a collection of inducers and repressors of cardiac reprogramming, we discovered that the zinc finger transcription factor 281 (ZNF281) potently stimulates cardiac reprogramming by genome-wide association with GATA4 on cardiac enhancers. Concomitantly, ZNF281 suppresses expression of genes associated with inflammatory signaling, suggesting the antagonistic convergence of cardiac and inflammatory transcriptional programs. Consistent with an inhibitory influence of inflammatory pathways on cardiac reprogramming, blockade of these pathways with anti-inflammatory drugs or components of the nucleosome remodeling deacetylase (NuRD) complex, which associate with ZNF281, stimulates cardiac gene expression. We conclude that ZNF281 acts at a nexus of cardiac and inflammatory gene programs, which exert opposing influences on fibroblast to cardiac reprogramming.

Maintenance of muscle function requires assembly of contractile proteins into highly organized sarcomeres. Mutations in Kelch-like protein 41 (KLHL41) cause nemaline myopathy, a fatal muscle disorder associated with sarcomere disarray. We generated KLHL41 mutant mice, which display lethal disruption of sarcomeres and aberrant expression of muscle structural and contractile proteins, mimicking the hallmarks of the human disease. We show that KLHL41 is poly-ubiquitinated and acts, at least in part, by preventing aggregation and degradation of Nebulin, an essential component of the sarcomere. Furthermore, inhibition of KLHL41 poly-ubiquitination prevents its stabilization of nebulin, suggesting a unique role for ubiquitination in protein stabilization. These findings provide new insights into the molecular etiology of nemaline myopathy and reveal a mechanism whereby KLHL41 stabilizes sarcomeres and maintains muscle function by acting as a molecular chaperone. Similar mechanisms for protein stabilization likely contribute to the actions of other Kelch proteins., eLife digest Together with the tendon and joints, muscles move our bones by contracting and relaxing. Muscles are formed of bundles of lengthy cells, which are made up of small units called sarcomeres. To contract, the proteins in the sarcomere need to be able to slide past each other. In healthy muscle cells, the proteins in the sarcomeres are evenly distributed in an organized pattern to make sure that the muscle can contract. However, when some of the proteins in the sarcomere become faulty, it can lead to diseases that affect the muscles and movement. For example, in a genetic muscle disease called nemaline myopathy, the proteins in the sarcomere are no longer organized, which leads to a build-up of proteins in the muscle fiber. These protein masses form rod-like structures that are lodged in between sarcomeres, which makes it more difficult for the muscles to contract. This can cause muscle weakness, difficulties eating or breathing, and eventually death. A common cause of nemaline myopathy is mutations in the gene that encodes the nebulin protein, which serves as a scaffold for the sarcomere assembly. Other proteins, including proteins named KLHL40 and KLHL41, have also been linked to the disease. These two proteins are both ‘Kelch’ proteins, most of which help to degrade specific proteins. However, a recent study has shown that KLHL40 actually stabilizes nebulin. As the two Kelch proteins KLHL40 and 41 are very similar in structure, scientists wanted to find out if KLHL41 plays a similar role to KLHL40. Now, Ramirez-Martinez et al. have created genetically modified mice that lacked KLHL41. These mice showed symptoms similar to people with nemaline myopathy, in which the sarcomeres were disorganized and could not form properly. Further experiments with cells grown in the laboratory showed that KLHL41 stabilized nebulin by using a specific chemical process that usually helps to degrade proteins. These results suggest that Kelch proteins have additional roles beyond degrading proteins and that some proteins linked to nemaline myopathy may actively prevent others from accumulating. A next step will be to find drugs that can compensate for the lack of KLHL41. A better understanding of the causes of this fatal disease will contribute towards developing better treatments.