Estrogen receptor-α (ERα) is a nuclear receptor family member thought to substantially contribute to the metabolic regulation of skeletal muscle. However, previous mouse models utilized to assess the necessity of ERα signaling in skeletal muscle are confounded by altered developmental programming and/or influenced by secondary effects, making it difficult to assign a causal role for ERα. The objective of the study was to determine the role of skeletal muscle ERα in regulating metabolism in the absence of confounding factors of development.

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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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Background: Hypertrophic cardiomyopathy (HCM) is characterized by myocyte hypertrophy and fibrosis. Studies in 2 mouse models (R92W-TnT/R403Q-MyHC) at early HCM stage revealed upregulation of endothelin (ET1) signaling in both mutants, but TGFβ signaling only in TnT mutants. Dysregulation of miR29 expression has been implicated in cardiac fibrosis. But it is unknown whether expression of miR29a/b/c and profibrotic genes is commonly regulated in mouse and human HCM.
Methods: In order to understand mechanisms underlying fibrosis in HCM, and examine similarities/differences in expression of miR29a/b/c and several profibrotic genes in mouse and human HCM, we performed parallel studies in rat cardiac myocyte/fibroblast cultures, examined gene expression in 2 mouse models of (non-obstructive) HCM (R92W-TnT, R403Q-MyHC)/controls at early (5wks) and established (24wks) disease stage, and analyzed publicly available mRNA/miRNA expression data from obstructive-HCM patients undergoing septal myectomy/controls (unused donor hearts).
Results:
Myocyte cultures: ET1 increased superoxide/H2O2, stimulated TGFβ expression/secretion and suppressed miR29a expression in myocytes. The effect of ET1 on miR29 and TGFβ expression/secretion was antagonized by N-acetyl-cysteine, a reactive oxygen species scavenger.
Fibroblast cultures: ET1 had no effect on pro-fibrotic gene expression in fibroblasts. TGFβ1/TGFβ2 suppressed miR29a and increased collagen expression, which was abolished by miR29a overexpression.
Mouse and human HCM: Expression of miR29a/b/c was lower, and TGFB1/collagen gene expression was higher in TnT mutant-LV at 5&24 weeks; no difference was observed in expression of these genes in MyHC mutant-LV and in human myectomy tissue. TGFB2 expression was higher in LV of both mutant mice and human myectomy tissue. ACE2, a negative regulator of the renin-angiotensin-aldosterone system, was the most upregulated transcript in human myectomy tissue. Pathway analysis predicted upregulation of the anti-hypertrophic/anti-fibrotic liver X receptor/retinoid X receptor (LXR/RXR) pathway only in human myectomy tissue.
Conclusions: Our in vitro studies suggest that activation of ET1 signaling in cardiac myocytes increases reactive oxygen species and stimulates TGFβ secretion, which downregulates miR29a and increases collagen in fibroblasts, thus contributing to fibrosis. Our gene expression studies in mouse and human HCM reveal allele-specific differences in miR29 family/profibrotic gene expression in mouse HCM, and activation of anti-hypertrophic/anti-fibrotic genes and pathways in human HCM.

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There is a growing interest in materials that can dynamically change their properties in the presence of cells to study mechanobiology. Herein, we exploit the 365 nm light mediated [4+4] photodimerization of anthracene groups to develop cytocompatible PEG‐based hydrogels with tailorable initial moduli that can be further stiffened. A hydrogel formulation that can stiffen from 10 to 50 kPa, corresponding to the stiffness of a healthy and fibrotic heart, respectively, was prepared. This system was used to monitor the stiffness‐dependent localization of NFAT, a downstream target of intracellular calcium signaling using a reporter in live cardiac fibroblasts (CFbs). NFAT translocates to the nucleus of CFbs on stiffening hydrogels within 6 h, whereas it remains cytoplasmic when the CFbs are cultured on either 10 or 50 kPa static hydrogels. This finding demonstrates how dynamic changes in the mechanical properties of a material can reveal the kinetics of mechanoresponsive cell signaling pathways that may otherwise be missed in cells cultured on static substrates.

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Numerous diseases, including those of the heart, are characterized by increased stiffness due to excessive deposition of extracellular matrix proteins. Cardiomyocytes continuously adapt their morphology and function to the mechanical changes of their microenvironment. Because traditional cell culture is conducted on substrates that are many orders of magnitude stiffer than any environment encountered by a cardiomyocyte in health or disease, alternate culture systems are necessary to model these processes in vitro. Here, we employ photo-clickable thiol-ene poly(ethylene glycol) (PEG) hydrogels for three-dimensional cell culture of adult mouse cardiomyocytes. PEG hydrogels serve as versatile biocompatible scaffolds, whose stiffness can be precisely tuned to mimic physiological and pathological microenvironments. Compared to traditional culture, adult cardiomyocytes encapsulated in PEG hydrogels exhibited longer survival and preserved sarcomeric and T-tubular architecture. Culture in PEG hydrogels of varying stiffnesses regulated the subcellular localization of the mechanosensitive transcription factor, YAP, in adult cardiomyocytes, indicating PEG hydrogels offer a versatile platform to study the role of mechanical cues in cardiomyocyte biology.

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The transcatheter aortic valve replacement (TAVR) procedure has emerged as a minimally invasive treatment for patients with aortic valve stenosis (AVS). However, alterations in serum factor composition and biological activity after TAVR remain unknown. Here, we quantified the systemic inflammatory effects of the TAVR procedure and hypothesized that alterations in serum factor composition would modulate valve and cardiac fibrosis. Serum samples were obtained from patients with AVS immediately before their TAVR procedure (pre-TAVR) and about 1 month afterward (post-TAVR). Aptamer-based proteomic profiling revealed alterations in post-TAVR serum composition, and ontological analysis identified inflammatory macrophage factors implicated in myofibroblast activation and deactivation. Hydrogel biomaterials used as valve matrix mimics demonstrated that post-TAVR serum reduced myofibroblast activation of valvular interstitial cells relative to pre-TAVR serum from the same patient. Transcriptomics and curated network analysis revealed a shift in myofibroblast phenotype from pre-TAVR to post-TAVR and identified p38 MAPK signaling as one pathway involved in pre-TAVR–mediated myofibroblast activation. Post-TAVR serum deactivated valve and cardiac myofibroblasts initially exposed to pre-TAVR serum to a quiescent fibroblast phenotype. Our in vitro deactivation data correlated with patient disease severity measured via echocardiography and multimorbidity scores, and correlations were dependent on hydrogel stiffness. Sex differences in cellular responses to male and female sera were also observed and may corroborate clinical observations regarding sex-specific TAVR outcomes. Together, alterations in serum composition after TAVR may lead to an antifibrotic fibroblast phenotype, which suggests earlier interventions may be beneficial for patients with advanced AVS to prevent further disease progression.

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Hypertrophic cardiomyopathy (HCM) is a common genetic disorder characterized by left ventricular hypertrophy and cardiac hyper-contractility. Mutations in the β cardiac myosin heavy chain gene (β-MyHC) are a major cause of HCM, but the specific mechanistic changes to myosin function that lead to this disease remain incompletely understood. Predicting the severity of any β-MyHC mutation is hindered by a lack of detailed examinations at the molecular level. Moreover, since HCM can take ≥20 years to develop, the severity of the mutations must be somewhat subtle. We hypothesized that mutations that result in early onset disease would have more severe changes in function than do later onset mutations. Here, we performed steady-state and transient kinetic analyses of myosins carrying one of seven missense mutations in the motor domain. Of these seven, four were previously identified in early onset cardiomyopathy screens. We used the parameters derived from these analyses to model the ATP driven cross-bridge cycle. Contrary to our hypothesis, the results indicated no clear differences between early and late onset HCM mutations. Despite the lack of distinction between early and late onset HCM, the predicted occupancy of the force-holding actin.myosin.ADP complex at [Actin] = 3 Kapp along with the closely related duty ratio (DR; the fraction of myosin in strongly attached force-holding states) and the measured ATPases all changed in parallel (in both sign and degree of change) compared to wild type (WT) values. Six of the seven HCM mutations were clearly distinct from a set of previously characterized DCM mutations.

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Numerous diseases, including those of the heart, are characterized by increased stiffness due to excessive deposition of extracellular matrix proteins. Cardiomyocytes continuously adapt their morphology and function to the mechanical changes of their microenvironment. Because traditional cell culture is conducted on substrates that are many orders of magnitude stiffer than any environment encountered by a cardiomyocyte in health or disease, alternate culture systems are necessary to model these processes in vitro. Here, we employ photo-clickable thiol-ene poly(ethylene glycol) (PEG) hydrogels for three-dimensional cell culture of adult mouse cardiomyocytes. PEG hydrogels serve as versatile biocompatible scaffolds, whose stiffness can be precisely tuned to mimic physiological and pathological microenvironments. Compared to traditional culture, adult cardiomyocytes encapsulated in PEG hydrogels exhibited longer survival and preserved sarcomeric and T-tubular architecture. Culture in PEG hydrogels of varying stiffnesses regulated the subcellular localization of the mechanosensitive transcription factor, YAP, in adult cardiomyocytes, indicating PEG hydrogels offer a versatile platform to study the role of mechanical cues in cardiomyocyte biology.

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What can we learn from prairie voles, Burmese pythons, shortfin mollies, and naked mole rats? Researchers from across the world are studying unusual laboratory animals with astonishing traits in their quest to answer important questions in the fields of biomedicine and neuroscience.

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CU �鶹ӰԺ engineers and faculty from the  (CFReT) at the CU Anschutz Medical Campus have teamed up to develop biomaterial-based “mimics” of heart tissues to measure patients’ responses to an aortic valve replacement procedure, offering new insight into the ways that cardiac tissue re-shapes itself post-surgery.

Aortic valve stenosis (AVS), a progressive disease characterized by heart valve tissue stiffening and obstructed blood flow from the heart, is known as a “silent killer,” affecting 12.4 percent of the population over 75 years old with a mortality range of 2-5 years if left untreated. Transcatheter aortic valve replacement (TAVR) procedures, which place an artificial valve at the site of the blockage, have been widely and successfully adopted as a remedy in recent decades.

Details of the broader biological reaction to the valve replacement have remained largely unknown, but nevertheless hold significant ramifications for quantifying the quality of recovery, the risk of complications and the assessment of overall patient outcomes.

During AVS disease progression, tissue-specific cells known as fibroblasts transition into myofibroblasts, which promote tissue stiffening. The researchers were interested in understanding how and why, post TAVR, myofibroblasts revert to the more benign fibroblasts. 

“Previous studies have shown significant remodeling of cardiac tissues post-intervention,” said Dr. Brian Aguado, lead author of the study and a post-doctoral researcher in CU �鶹ӰԺ’s Department of Chemical and Biological Engineering. “Our hypothesis was that perhaps there are biochemical cues in a patient’s blood that may revert myofibroblasts back to fibroblasts.”

Modeling such a transformation in the lab is one thing, Aguado said, but the key to the new study was obtaining blood samples from real AVS patients and then using biomaterials to replicate the microenvironment of the heart. 

“The heart is not made of plastic like a petri dish is,” he said. “We needed to engineer materials that could reflect the various stiffnesses of both healthy and diseased valve and cardiac tissue.” 

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