8 research outputs found
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Remembering Jeff Robbins: The Father of Cardiac Transgenesis
Jeff graduated from the University of Connecticut with his PhD in 1976 and became an assistant professor in 1978 at the University of Missouri-Columbia. Jeff then moved to Ohio in 1985 to the University of Cincinnati as an associate professor where he stayed until moving his scientific research program across the street to the Cincinnati Children’s Hospital in 1993, where he served as a professor in Pediatrics of the University of Cincinnati and as the founding director of the Division of Molecular Cardiovascular Biology. Jeff retired from Cincinnati Children’s Hospital and the University of Cincinnati in 2019.
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Nonproductive Splicing Prevents Expression of MYH7b Protein in the Mammalian Heart
Background
Although the roles of alphaâmyosin heavy chain (αâMyHC) and betaâmyosin heavy chain (βâMyHC) proteins in cardiac contractility have long been appreciated, the biological contribution of another closely related sarcomeric myosin family member, MYH7b (myosin heavy chain 7b), has become a matter of debate. In mammals, MYH7b mRNA is transcribed but undergoes nonâproductive alternative splicing that prevents protein expression in a tissueâspecific manner, including in the heart. However, several studies have recently linked MYH7b variants to different cardiomyopathies or have reported MYH7b protein expression in mammalian hearts.
Methods and Results
By analyzing mammalian cardiac transcriptome and proteome data, we show that the vast majority of MYH7b RNA is subject to exon skipping and cannot be translated into a functional myosin molecule. Notably, we discovered a lag in the removal of introns flanking the alternatively spliced exon, which could retain the nonâcoding RNA in the nucleus. This process could play a significant role in controlling MYH7b expression as well as the activity of other cardiac genes. Consistent with the negligible level of fullâlength protein coding mRNA, no MYH7b protein expression was detected in adult mouse, rat, and human hearts by Western blot analysis. Furthermore, proteome surveys including quantitative mass spectrometry analyses revealed only traces of cardiac MYH7b protein and even then, only in a subset of individual samples.
Conclusions
The comprehensive analysis presented here suggests that previous studies showing cardiac MYH7b protein expression were likely attributable to antibody crossâreactivity. More importantly, our data predict that the MYH7b diseaseâassociated variants may operate through the alternately spliced RNA itself.
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miR-1/206 downregulates splicing factor Srsf9 to promote C2C12 differentiation
Background
Myogenesis is driven by specific changes in the transcriptome that occur during the different stages of muscle differentiation. In addition to controlled transcriptional transitions, several other post-transcriptional mechanisms direct muscle differentiation. Both alternative splicing and miRNA activity regulate gene expression and production of specialized protein isoforms. Importantly, disruption of either process often results in severe phenotypes as reported for several muscle diseases. Thus, broadening our understanding of the post-transcriptional pathways that operate in muscles will lay the foundation for future therapeutic interventions.
Methods
We employed bioinformatics analysis in concert with the well-established C2C12 cell system for predicting and validating novel miR-1 and miR-206 targets engaged in muscle differentiation. We used reporter gene assays to test direct miRNA targeting and studied C2C12 cells stably expressing one of the cDNA candidates fused to a heterologous, miRNA-resistant 3′ UTR. We monitored effects on differentiation by measuring fusion index, myotube area, and myogenic gene expression during time course differentiation experiments.
Results
Gene ontology analysis revealed a strongly enriched set of putative miR-1 and miR-206 targets associated with RNA metabolism. Notably, the expression levels of several candidates decreased during C2C12 differentiation. We discovered that the splicing factor Srsf9 is a direct target of both miRNAs during myogenesis. Persistent Srsf9 expression during differentiation impaired myotube formation and blunted induction of the early pro-differentiation factor myogenin as well as the late differentiation marker sarcomeric myosin, Myh8.
Conclusions
Our data uncover novel miR-1 and miR-206 cellular targets and establish a functional link between the splicing factor Srsf9 and myoblast differentiation. The finding that miRNA-mediated clearance of Srsf9 is a key myogenic event illustrates the coordinated and sophisticated interplay between the diverse components of the gene regulatory network.
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Expression of Normally Repressed Myosin Heavy Chain 7b in the Mammalian Heart Induces Dilated Cardiomyopathy
In mammals, muscle contraction is controlled by a family of 10 sarcomeric myosin motors. The expression of one of its members, MYH7b, is regulated by alternative splicing, and while the protein is restricted to specialized muscles such as extraocular muscles or muscle spindles, RNA that cannot encode protein is expressed in most skeletal muscles and in the heart. Remarkably, birds and snakes express MYH7b protein in both heart and skeletal muscles. This observation suggests that in the mammalian heart, the motor activity of MYH7b may only be needed during development since its expression is prevented in adult tissue, possibly because it could promote disease by unbalancing myocardial contractility. Methods and Results We have analyzed MYH7b null mice to determine the potential role of MYH7b during cardiac development and also generated transgenic mice with cardiac myocyte expression of MYH7b protein to measure its impact on cardiomyocyte function and contractility. We found that MYH7b null mice are born at expected Mendelian ratios and do not have a baseline cardiac phenotype as adults. In contrast, transgenic cardiac MYH7b protein expression induced early cardiac dilation in males with significantly increased left ventricular mass in both sexes. Cardiac dilation is progressive, leading to early cardiac dysfunction in males, but later dysfunction in females. Conclusions The data presented show that the expression of MYH7b protein in the mammalian heart has been inhibited during the evolution of mammals most likely to prevent the development of a severe cardiomyopathy that is sexually dimorphic.</p
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Cover Image Defining the Cardiac Fibroblast Secretome in a Fibrotic Microenvironment
Background Cardiac fibroblasts (CFs) have the ability to sense stiffness changes and respond to biochemical cues to modulate their states as either quiescent or activated myofibroblasts. Given the potential for secretion of bioactive molecules to modulate the cardiac microenvironment, we sought to determine how the CF secretome changes with matrix stiffness and biochemical cues and how this affects cardiac myocytes via paracrine signaling. Methods and Results Myofibroblast activation was modulated in vitro by combining stiffness cues with TGFβ1 (transforming growth factor β 1) treatment using engineered poly (ethylene glycol) hydrogels, and in vivo with isoproterenol treatment. Stiffness, TGFβ1, and isoproterenol treatment increased AKT (protein kinase B) phosphorylation, indicating that this pathway may be central to myofibroblast activation regardless of the treatment. Although activation of AKT was shared, different activating cues had distinct effects on downstream cytokine secretion, indicating that not all activated myofibroblasts share the same secretome. To test the effect of cytokines present in the CF secretome on paracrine signaling, neonatal rat ventricular cardiomyocytes were treated with CF conditioned media. Conditioned media from myofibroblasts cultured on stiff substrates and activated by TGFβ1 caused hypertrophy, and one of the cytokines in that media was insulin growth factor 1, which is a known mediator of cardiac myocyte hypertrophy. Conclusions Culturing CFs on stiff substrates, treating with TGFβ1, and in vivo treatment with isoproterenol all caused myofibroblast activation. Each cue had distinct effects on the secretome or genes encoding the secretome, but only the secretome of activated myofibroblasts on stiff substrates treated with TGFβ1 caused myocyte hypertrophy, most likely through insulin growth factor 1.</p
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Cardiac Fibroblasts Mediate a Sexually Dimorphic Fibrotic Response to beta-Adrenergic Stimulation
Background
Biological sex is an important modifier of cardiovascular disease and women generally have better outcomes compared with men. However, the contribution of cardiac fibroblasts (CFs) to this sexual dimorphism is relatively unexplored.
Methods and Results
Isoproterenol (ISO) was administered to rats as a model for chronic βâadrenergic receptor (βâAR)âmediated cardiovascular disease. ISOâtreated males had higher mortality than females and also developed fibrosis whereas females did not. Gonadectomy did not abrogate this sex difference. To determine the cellular contribution to this phenotype, CFs were studied. CFs from both sexes had increased proliferation in vivo in response to ISO, but CFs from female hearts proliferated more than male cells. In addition, male CFs were significantly more activated to myofibroblasts by ISO. To investigate potential regulatory mechanisms for the sexually dimorphic fibrotic response, βâAR mRNA and PKA (protein kinase A) activity were measured. In response to ISO treatment, male CFs increased expression of β1â and β2âARs, whereas expression of both receptors decreased in female CFs. Moreover, ISOâtreated male CFs had higher PKA activity relative to vehicle controls, whereas ISO did not activate PKA in female CFs.
Conclusions
Chronic in vivo βâAR stimulation causes fibrosis in male but not female rat hearts. Male CFs are more activated than female CFs, consistent with elevated fibrosis in male rat hearts and may be caused by higher βâAR expression and PKA activation in male CFs. Taken together, our data suggest that CFs play a substantial role in mediating sex differences observed after cardiac injury.
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Dietary phytoestrogens present in soy dramatically increase cardiotoxicity in male mice receiving a chemotherapeutic tyrosine kinase inhibitor
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Two novel MYH7 proline substitutions cause Laing Distal Myopathy-like phenotypes with variable expressivity and neck extensor contracture
BACKGROUND: Human skeletal muscles express three major myosin heavy chain (MyHC) isoforms: MyHCIIx (MYH1) in fast type 2B muscle fibers, MyHCIIa (MYH2) in fast type 2A fibers and MyHCI/β-cardiac MyHC (MYH7) in slow type I skeletal fibers and cardiac ventricles. In line with its expression pattern, MYH7 mutations have been reported in association with hypertrophic or dilated cardiomyopathy, skeletal myopathies or a combination of both. We analyzed the clinical and molecular phenotype of two unrelated families of Jewish Moroccan ancestry that presented with apparently autosomal dominant inheritance of progressive Laing-like distal myopathy with non-specific myopathic changes, but uncommon marked contractures and wasting of the neck extensors. METHODS: Clinical phenotyping, whole exome sequencing and restriction analysis, generation of mutants followed by cell culture transfection and imaging. RESULTS: Using whole exome sequencing we identified in both families two novel heterozygous proline substitutions located in exon 31 of MYH7 within its rod domain: c.4309G>C (p.Ala1437Pro) and c.4301G>C (p.Arg1434Pro). Here we show that the phenotype caused by these mutations includes marked cervical muscle contracture, and report that the severity of the phenotype varies significantly, to the extent of non-penetrance in one of the families. Finally, we provide evidence that both proline substitutions impair myosin self-assembly in non-muscle cells transfected with β-myosin constructs carrying the mutations, but do not prevent incorporation of the mutant molecules into the sarcomere. CONCLUSIONS: This study expands our clinical and molecular knowledge of MYH7 rod mutations causing skeletal myopathies, and underscores the importance of discussing disease penetrance during genetic counseling