Bjorkman, K. K. | bioRxiv Science

bioRxiv2019

miR-1/206 down-regulates splicing factor Srsf9 to promote myogenesis

BackgroundMyogenesis 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.\n\nMethodsWe 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.\n\nResultsGene 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.\n\nConclusionsOur 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.

cell biology↗

bioRxiv2019

Cardiac myocytes respond differentially and synergistically to matrix stiffness and topography

During cardiac disease progression, myocytes undergo molecular, functional and structural changes, including increases in cell size and shape, decreased myocyte alignment and contractility. The heart often increases extracellular matrix production and stiffness, which affect myocytes. The order and hierarchy of these events remain unclear as available in vitro cell culture systems do not adequately model both physiologic and pathologic environments. Traditional cell culture substrates are 5-6 orders of magnitude stiffer than even diseased native cardiac tissue. Studies that do account for substrate stiffness often do not consider intercellular alignment and vice versa. We developed a cardiac myocyte culture platform that better recapitulates native tissue stiffness while simultaneously introducing topographical cues that promote cellular alignment. We show that stiffness and topography impact myocyte molecular and functional properties. We used a spatiotemporally-tunable, photolabile hydrogel platform to generate a range of stiffness and micron-scale topographical patterns to guide neonatal rat ventricular myocyte morphology. Importantly, these substrate patterns were of subcellular dimensions to test whether cells would spontaneously respond to topographical cues rather than an imposed geometry. Cellular contractility was highest and the gene expression profile was most physiologic on gels with healthy cardiac tissue stiffness. Surprisingly, while elongated patterns in stiff gels yielded the greatest cellular alignment, the cells actually had more pathologic functional and molecular profiles. These results highlight that morphological measurements alone are not a surrogate for overall cellular health as many studies assume. In general, substrate stiffness and micropatterning synergistically affect cardiac myocyte phenotype to recreate physiologic and pathologic microenvironments.\n\nSignificance StatementHeart disease is accompanied by organ- and cellular-level remodeling, and deconvoluting their interplay is complex. Cellular-level change is best studied in vitro due to greater control and uniformity of cell types compared to animals. One common metric is degree of cellular alignment as misalignment of myocytes is a hallmark of disease. However, most studies utilize featureless culture surfaces that are orders of magnitude stiffer than, and do not mimic the scaffolding of, the heart. We developed a hydrogel platform with tunable stiffness and patterns providing topographical alignment cues. We cultured heart cells on and characterized multifactorial responses to these dynamic surfaces. Interestingly, conditions that yielded greatest alignment did not yield the healthiest functional and molecular state. Thus, morphology alone is not an indicator of overall cellular health.

cell biology↗