Project description:CUG-BP, Elav-like family member 1 (CELF1) is a highly conserved RNA binding protein that regulates pre-mRNA alternative splicing, polyadenylation, mRNA stability, and translation. In the heart, CELF1 is expressed in the myocardium, where its levels are tightly regulated during development. CELF1 levels peak in the heart during embryogenesis, and aberrant up-regulation of CELF1 in the adult heart has been implicated in cardiac pathogenesis in myotonic dystrophy type 1, as well as in diabetic cardiomyopathy. Either inhibition of CELF activity or over-expression of CELF1 in heart muscle causes cardiomyopathy in transgenic mice. Nonetheless, many of the cardiac targets of CELF1 regulation remain unknown. In this study, to identify cardiac targets of CELF1 we performed cross-linking immunoprecipitation (CLIP) for CELF1 from embryonic day 8 chicken hearts. We identified a previously unannotated exon in MYH7B as a novel target of CELF1-mediated regulation. We demonstrated that knockdown of CELF1 in primary chicken embryonic cardiomyocytes leads to increased inclusion of this exon and decreased MYH7B levels. We also investigated global changes in the transcriptome of primary embryonic cardiomyocytes following CELF1 knockdown in a published RNA-seq dataset. Pathway and network analyses identified strong associations between CELF1 and regulation of cell cycle and translation. Important regulatory proteins, including both RNA binding proteins and a cardiac transcription factor, were affected by loss of CELF1. Together, these data suggest that CELF1 is a key regulator of cardiomyocyte gene expression.
Project description:CUG-BP, Elav-like family member 1 (CELF1) is a multi-functional RNA binding protein that regulates pre-mRNA alternative splicing in the nucleus, as well as polyadenylation status, mRNA stability, and translation in the cytoplasm . Dysregulation of CELF1 has been implicated in cardiomyopathies in myotonic dystrophy type 1 and diabetes [2-5], but the targets of CELF1 regulation in the heart have not been systematically investigated. We previously demonstrated that in the developing heart CELF1 expression is restricted to the myocardium and peaks during embryogenesis [6-8]. To identify transcripts regulated by CELF1 in the embryonic myocardium, RNA-seq was used to compare the transcriptome of primary embryonic cardiomyocytes following siRNA-mediated knockdown of CELF1 to that of controls. Raw data files of the RNA-seq reads have been deposited in NCBI's Gene Expression Omnibus  under the GEO Series accession number GSE67360. These data can be used to identify transcripts whose levels or alternative processing (i.e., alternative splicing or polyadenylation site usage) are regulated by CELF1, and should provide insight into the pathways and processes modulated by this important RNA binding protein during normal heart development and during cardiac pathogenesis.
Project description:Normal blood flow is essential for proper heart formation during embryonic development, as abnormal hemodynamic load (blood pressure and shear stress) results in cardiac defects seen in congenital heart disease (CHD). However, the detrimental remodeling processes that relate altered blood flow to cardiac malformation and defects remain unclear. Heart development is a finely orchestrated process with rapid transformations that occur at the tissue, cell, and subcellular levels. Myocardial cells play an essential role in cardiac tissue maturation by aligning in the direction of stretch and increasing the number of contractile units as hemodynamic load increases throughout development. This study elucidates the early effects of altered blood flow on myofibril and mitochondrial configuration in the outflow tract myocardium in vivo. Outflow tract banding was used to increase hemodynamic load in the chicken embryo heart between Hamburger and Hamilton stages 18 and 24 (~24 h during tubular heart stages). 3D focused ion beam scanning electron microscopy analysis determined that increased hemodynamic load induced changes in the developing myocardium, characterized by thicker myofibril bundles that were more disbursed in circumferential orientation, and mitochondria that organized in large clusters around the nucleus. Proteomic mass-spectrometry analysis quantified altered protein composition after banding that is consistent with altered myofibril thin filament assembly and function, and mitochondrial maintenance and organization. Additionally, pathway analysis of the proteomics data identified possible activation of signaling pathways in response to banding, including the renin-angiotensin system (RAS). Imaging and proteomic data combined indicate that myofibril and mitochondrial arrangement in early embryonic stages is a critical developmental process that when disturbed by altered blood flow may contribute to cardiac malformation and defects.
Project description:In vitro studies of cardiac physiology and drug response have traditionally been performed on individual isolated cardiomyocytes or isotropic monolayers of cells that may not mimic desired physiological traits of the laminar adult myocardium. Recent studies have reported a number of advances to Heart-on-a-Chip platforms for the fabrication of more sophisticated engineered myocardium, but cardiomyocyte immaturity remains a challenge. In the anisotropic musculature of the heart, interactions between cardiac myocytes, the extracellular matrix (ECM), and neighboring cells give rise to changes in cell shape and tissue architecture that have been implicated in both development and disease. We hypothesized that engineered myocardium fabricated from cardiac myocytes cultured in vitro could mimic the physiological characteristics and gene expression profile of adult heart muscle. To test this hypothesis, we fabricated engineered myocardium comprised of neonatal rat ventricular myocytes with laminar architectures reminiscent of that observed in the mature heart and compared their sarcomere organization, contractile performance characteristics, and cardiac gene expression profile to that of isolated adult rat ventricular muscle strips. We found that anisotropic engineered myocardium demonstrated a similar degree of global sarcomere alignment, contractile stress output, and inotropic concentration-response to the ?-adrenergic agonist isoproterenol. Moreover, the anisotropic engineered myocardium exhibited comparable myofibril related gene expression to muscle strips isolated from adult rat ventricular tissue. These results suggest that tissue architecture serves an important developmental cue for building in vitro model systems of the myocardium that could potentially recapitulate the physiological characteristics of the adult heart. Impact statement With the recent focus on developing in vitro Organ-on-Chip platforms that recapitulate tissue and organ-level physiology using immature cells derived from stem cell sources, there is a strong need to assess the ability of these engineered tissues to adopt a mature phenotype. In the present study, we compared and contrasted engineered tissues fabricated from neonatal rat ventricular myocytes in a Heart-on-a-Chip platform to ventricular muscle strips isolated from adult rats. The results of this study support the notion that engineered tissues fabricated from immature cells have the potential to mimic mature tissues in an Organ-on-Chip platform.
Project description:Cardiac development relies on proper cardiomyocyte differentiation, including expression and assembly of cell-type-specific actomyosin subunits into a functional cardiac sarcomere. Control of this process involves not only promoting expression of cardiac sarcomere subunits but also repressing expression of noncardiac myofibril paralogs. This level of transcriptional control requires broadly expressed multiprotein machines that modify and remodel the chromatin landscape to restrict transcription machinery access. Prominent among these is the nucleosome remodeling and deacetylase (NuRD) complex, which includes the catalytic core subunit CHD4. Here, we demonstrate that direct CHD4-mediated repression of skeletal and smooth muscle myofibril isoforms is required for normal cardiac sarcomere formation, function, and embryonic survival early in gestation. Through transcriptomic and genome-wide analyses of CHD4 localization, we identified unique CHD4 binding sites in smooth muscle myosin heavy chain, fast skeletal ?-actin, and the fast skeletal troponin complex genes. We further demonstrate that in the absence of CHD4, cardiomyocytes in the developing heart form a hybrid muscle cell that contains cardiac, skeletal, and smooth muscle myofibril components. These misexpressed paralogs intercalate into the nascent cardiac sarcomere to disrupt sarcomere formation and cause impaired cardiac function in utero. These results demonstrate the genomic and physiological requirements for CHD4 in mammalian cardiac development.
Project description:A homozygous disruption or genetic mutation of the bag3 gene, a member of the Bcl-2-associated athanogene (BAG) family proteins, causes cardiomyopathy and myofibrillar myopathy that is characterized by myofibril and Z-disc disruption. However, the detailed disease mechanism is not yet fully understood.bag3(-/-) mice exhibit differences in the extent of muscle degeneration between muscle groups with muscles experiencing the most usage degenerating at an accelerated rate. Usage-dependent muscle degeneration suggests a role for BAG3 in supporting cytoskeletal connections between the Z-disc and myofibrils under mechanical stress. The mechanism by which myofibrillar structure is maintained under mechanical stress remains unclear. The purpose of the study is to clarify the detailed molecular mechanism of BAG3-mediated muscle maintenance under mechanical stress.To address the question of whether bag3 gene knockdown induces myofibrillar disorganization caused by mechanical stress, in vitro mechanical stretch experiments using rat neonatal cardiomyocytes and a short hairpin RNA-mediated gene knockdown system of the bag3 gene were performed. As expected, mechanical stretch rapidly disrupts myofibril structures in bag3 knockdown cardiomyocytes. BAG3 regulates the structural stability of F-actin through the actin capping protein, CapZ?1, by promoting association between Hsc70 and CapZ?1. BAG3 facilitates the distribution of CapZ?1 to the proper location, and dysfunction of BAG3 induces CapZ ubiquitin-proteasome-mediated degradation. Inhibition of CapZ?1 function by overexpressing CapZ?2 increased myofibril vulnerability and fragmentation under mechanical stress. On the other hand, overexpression of CapZ?1 inhibits myofibrillar disruption in bag3 knockdown cells under mechanical stress. As a result, heart muscle isolated from bag3(-/-) mice exhibited myofibrillar degeneration and lost contractile activity after caffeine contraction.These results suggest novel roles for BAG3 and Hsc70 in stabilizing myofibril structure and inhibiting myofibrillar degeneration in response to mechanical stress. These proteins are possible targets for further research to identify therapies for myofibrillar myopathy or other degenerative diseases.
Project description:The organization and maturation of ventricular cardiomyocytes from the embryonic to the adult form is crucial for normal cardiac function. We have shown that a polarity protein, Scrib, may be involved in regulating the early stages of this process. Our goal was to establish whether Scrib plays a cell autonomous role in the ventricular myocardium, and whether this involves well-known polarity pathways.Deletion of Scrib in cardiac precursors utilizing Scrib(flox) mice together with the Nkx2.5-Cre driver resulted in disruption of the cytoarchitecture of the forming trabeculae and ventricular septal defects. Although the majority of mice lacking Scrib in the myocardium survived to adulthood, they developed marked cardiac fibrosis. Scrib did not physically interact with the planar cell polarity (PCP) protein, Vangl2, in early cardiomyocytes as it does in other tissues, suggesting that the anomalies did not result from disruption of PCP signalling. However, Scrib interacted with Rac1 physically in embryonic cardiomyocytes and genetically to result in ventricular abnormalities, suggesting that this interaction is crucial for the development of the early myocardium.The Scrib-Rac1 interaction plays a crucial role in the organization of developing cardiomyocytes and formation of the ventricular myocardium. Thus, we have identified a novel signalling pathway in the early, functioning, heart muscle. These data also show that the foetus can recover from relatively severe abnormalities in prenatal ventricular development, although cardiac fibrosis can be a long-term consequence.
Project description:We are developing a novel treatment for heart failure by increasing myocardial 2 deoxy-ATP (dATP). Our studies in rodent models have shown that substitution of dATP for adenosine triphosphate (ATP) as the energy substrate in vitro or elevation of dATP in vivo increases myocardial contraction and that small increases in the native dATP pool of heart muscle are sufficient to improve cardiac function. Here we report, for the first time, the effect of dATP on human adult cardiac muscle contraction. We measured the contractile properties of chemically-demembranated multicellular ventricular wall preparations and isolated myofibrils from human subjects with end-stage heart failure. Isometric force was increased at both saturating and physiologic Ca(2+) concentrations with dATP compared to ATP. This resulted in an increase in the Ca(2+) sensitivity of force (pCa50) by 0.06 pCa units. The rate of force redevelopment (ktr) in demembranated wall muscle was also increased, as was the rate of contractile activation (kACT) in isolated myofibrils, indicating increased cross-bridge binding and cycling compared with ATP in failing human myocardium. These data suggest that dATP could increase dP/dT and end systolic pressure in failing human myocardium. Importantly, even though the magnitude and rate of force development were increased, there was no increase in the time to 50% and 90% myofibril relaxation. These data, along with our previous studies in rodent models, show the promise of elevating myocardial dATP to enhance contraction and restore cardiac pump function. These data also support further pre-clinical evaluation of this new approach for treating heart failure.
Project description:The small heat shock protein Hsp27 has been shown to be involved in a diverse array of cellular processes, including cellular stress response, protein chaperone activity, regulation of cellular glutathione levels, apoptotic signaling, and regulation of actin polymerization and stability. Furthermore, mutation within Hsp27 has been associated with the human congenital neuropathy Charcot-Marie Tooth (CMT) disease. Hsp27 is known to be expressed in developing embryonic tissues; however, little has been done to determine the endogenous requirement for Hsp27 in developing embryos. In this study, we show that depletion of XHSP27 protein results in a failure of cardiac progenitor fusion resulting in cardia bifida. Furthermore, we demonstrate a concomitant disorganization of actin filament organization and defects in myofibril assembly. Moreover, these defects are not associated with alterations in specification or differentiation. We have thus demonstrated a critical requirement for XHSP27 in developing cardiac and skeletal muscle tissues.
Project description:Valosin-containing protein (VCP) is a highly conserved mechanoenzyme that helps maintain protein homeostasis in all cells and serves specialized functions in distinct cell types. In skeletal muscle, it is critical for myofibrillogenesis and atrophy. However, little is known about VCP's role(s) in the heart. Its functional diversity is determined by differential binding of distinct cofactors/adapters, which is likely disrupted during disease. VCP mutations cause multisystem proteinopathy (MSP), a pleiotropic degenerative disorder that involves inclusion body myopathy. MSP patients display progressive muscle weakness. They also exhibit cardiomyopathy and die from cardiac and respiratory failure, which are consistent with critical myocardial roles for the enzyme. Nonetheless, efficient models to interrogate VCP in cardiac muscle remain underdeveloped and poorly studied. Here, we investigated the significance of VCP and mutant VCP in the Drosophila heart. Cardiac-restricted RNAi-mediated knockdown of TER94, the Drosophila VCP homolog, severely perturbed myofibrillar organization and heart function in adult flies. Furthermore, expression of MSP disease-causing alleles engendered cardiomyopathy in adults and structural defects in embryonic hearts. Drosophila may therefore serve as a valuable model for examining role(s) of VCP in cardiogenesis and for identifying novel heart-specific VCP interactions, which when disrupted via mutation, contribute to or elicit cardiac pathology.