Agent-based modeling reveals benefits of heterogeneous and stochastic cell populations during cGAS-mediated IFNß production.

MOTIVATION: The cGAS pathway is a component of the innate immune system responsible for the detection of pathogenic DNA and upregulation of interferon beta (IFNß). Experimental evidence shows that IFNß signaling occurs in highly heterogeneous cells and is stochastic in nature; however, the benefits of these attributes remain unclear. To investigate how stochasticity and heterogeneity affect IFNß production, an agent-based model is developed to simulate both DNA transfection and viral infection. RESULTS: We show that heterogeneity can enhance IFNß responses during infection. Furthermore, by varying the degree of IFNß stochasticity, we find that only a percentage of cells (20-30%) need to respond during infection. Going beyond this range provides no additional protection against cell death or reduction of viral load. Overall, these simulations suggest that heterogeneity and stochasticity are important for moderating immune potency while minimizing cell death during infection. AVAILABILITY AND IMPLEMENTATION: Model repository is available at: https://github.com/ImmuSystems-Lab/AgentBasedModel-cGASPathway. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

SNRK regulates TGFß levels in atria to control cardiac fibrosis.

Atrial fibrosis is central to the pathology of heart failure (HF) and atrial fibrillation (AF). Identifying precise mechanisms underlying atrial fibrosis will provide effective strategies for clinical intervention. This study investigates a metabolic serine threonine kinase gene, sucrose non-fermenting related kinase (SNRK), that we previously reported to control cardiac metabolism and function. Conditional knockout of Snrk in mouse cardiomyocytes ( Snrk cmcKO) leads to atrial fibrosis and subsequently HF. The precise mechanism underlying cardiomyocyte SNRK-driven repression of fibrosis is not known. Here, using mouse, rat, and human tissues, we demonstrate that SNRK expression is high in atria, especially in atrial cardiomyocytes. SNRK expression correlates with lower levels of pro-fibrotic protein transforming growth factor-beta 1 (TGFß1) in the atrial cardiomyocytes. In HL-1 adult immortalized mouse atrial cells, using siRNA approaches, we show that Snrk knockdown cells show more TGFß1 secretion, which was also observed in heart lysates from Snrk cardiac-specific knockout mice in vivo. These effects were exacerbated upon infusion of Angiotensin II. Results from Snrk knockdown cardiomyocytes co-cultured with cardiac fibroblasts suggest that SNRK represses TGFß1 signaling (Smad 2/3) in atrial CMs and prevents paracrine cardiac fibroblast activation (α-SMA marker). In conclusion, high SNRK expression in atria regulates cardiac homeostasis, by preventing the release of TGFß1 secretion to block cardiac fibrosis. These studies will assist in developing heart chamber-specific fibrosis therapy for non-ischemic HF and AF.

Identifying molecular and functional similarities and differences between human primary cardiac valve interstitial cells and ventricular fibroblasts.

Introduction: Fibroblasts are mesenchymal cells that predominantly produce and maintain the extracellular matrix (ECM) and are critical mediators of injury response. In the heart, valve interstitial cells (VICs) are a population of fibroblasts responsible for maintaining the structure and function of heart valves. These cells are regionally distinct from myocardial fibroblasts, including left ventricular cardiac fibroblasts (LVCFBs), which are located in the myocardium in close vicinity to cardiomyocytes. Here, we hypothesize these subpopulations of fibroblasts are transcriptionally and functionally distinct. Methods: To compare these fibroblast subtypes, we collected patient-matched samples of human primary VICs and LVCFBs and performed bulk RNA sequencing, extracellular matrix profiling, and functional contraction and calcification assays. Results: Here, we identified combined expression of SUSD2 on a protein-level, and MEOX2, EBF2 and RHOU at a transcript-level to be differentially expressed in VICs compared to LVCFBs and demonstrated that expression of these genes can be used to distinguish between the two subpopulations. We found both VICs and LVCFBs expressed similar activation and contraction potential in vitro, but VICs showed an increase in ALP activity when activated and higher expression in matricellular proteins, including cartilage oligomeric protein and alpha 2-Heremans-Schmid glycoprotein, both of which are reported to be linked to calcification, compared to LVCFBs. Conclusion: These comparative transcriptomic, proteomic, and functional studies shed novel insight into the similarities and differences between valve interstitial cells and left ventricular cardiac fibroblasts and will aid in understanding region-specific cardiac pathologies, distinguishing between primary subpopulations of fibroblasts, and generating region-specific stem-cell derived cardiac fibroblasts.

Directed differentiation of human pluripotent stem cells to epicardial-derived fibroblasts.

Cardiac fibroblasts (CFBs) are a key therapeutic target due to their supportive roles during heart development and response to injury and disease. Here, we describe a robust protocol to differentiate human pluripotent stem cells (hPSCs) into CFBs through an epicardial intermediate. We discuss in detail the characterization of the resulting epicardial-derived fibroblasts (EpiC-FBs) using immunofluorescence microscopy, flow cytometry, and qPCR. We anticipate that these EpiC-FBs can be applied to drug testing, disease modeling, and tissue engineering. For complete details on the use and execution of this protocol, please refer to Bao et al. (2016), Floy et al. (2021), and Lian et al. (2015).

Advances in Manufacturing Cardiomyocytes from Human Pluripotent Stem Cells.

The emergence of human pluripotent stem cell (hPSC) technology over the past two decades has provided a source of normal and diseased human cells for a wide variety of in vitro and in vivo applications. Notably, hPSC-derived cardiomyocytes (hPSC-CMs) are widely used to model human heart development and disease and are in clinical trials for treating heart disease. The success of hPSC-CMs in these applications requires robust, scalable approaches to manufacture large numbers of safe and potent cells. Although significant advances have been made over the past decade in improving the purity and yield of hPSC-CMs and scaling the differentiation process from 2D to 3D, efforts to induce maturation phenotypes during manufacturing have been slow. Process monitoring and closed-loop manufacturing strategies are just being developed. We discuss recent advances in hPSC-CM manufacturing, including differentiation process development and scaling and downstream processes as well as separation and stabilization.

Adaptation and validation of Common Object Token (COT) test into the Sinhalese language.

OBJECTIVES: This manuscript presents a translation and adaptation of the Common Object Token (COT) test, which assesses speech perception, into the Sinhalese language and an attempt to validate it for use on children with normal hearing (NH) and children with a cochlear implant (CI). METHODS: Ninety-five children (70 with NH, 25 with a CI) participated in the study. The COT test was translated, back-translated, and evaluated by a team of experts until the Sinhalese translation was deemed acceptable. Data of Sinhalese children with NH and values of children with a CI were analysed. Internal reliability and consistency of the COT total score were determined. Lastly, a quick version of the COT test was created. RESULTS: The total mean scores and subtest mean scores improved with age for children with NH. For children with a CI, a strong relationship between the COT total score and device experience, i.e. hearing age, was found. A Quick Sinhalese COT test version, suitable for children with a CI, could be created from Subtests 2, 3, and 4. CONCLUSION: The Sinhalese COT test is valid for assessing the age-related development of speech perception and identification skills of children with NH. Results suggest that the COT is valid for use in children with a CI.