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1. DNA methylation analysis reveals epimutation hotspots in patients with dilated cardiomyopathy-associated laminopathies

   https://doi.org/10.1186/s13148-021-01127-0  

   Morival, Julien L. ; Widyastuti, Halida P. ; Nguyen, Cecilia H. ; Zaragoza, Michael V. ; Downing, Timothy L. ( December 2021 , Clinical Epigenetics)

   Abstract Background Mutations in LMNA , encoding lamin A/C, lead to a variety of diseases known as laminopathies including dilated cardiomyopathy (DCM) and skeletal abnormalities. Though previous studies have investigated the dysregulation of gene expression in cells from patients with DCM, the role of epigenetic (gene regulatory) mechanisms, such as DNA methylation, has not been thoroughly investigated. Furthermore, the impact of family-specific LMNA mutations on DNA methylation is unknown. Here, we performed reduced representation bisulfite sequencing on ten pairs of fibroblasts and their induced pluripotent stem cell (iPSC) derivatives from two families with DCM due to distinct LMNA mutations, one of which also induces brachydactyly. Results Family-specific differentially methylated regions (DMRs) were identified by comparing the DNA methylation landscape of patient and control samples. Fibroblast DMRs were found to enrich for distal regulatory features and transcriptionally repressed chromatin and to associate with genes related to phenotypes found in tissues affected by laminopathies. These DMRs, in combination with transcriptome-wide expression data and lamina-associated domain (LAD) organization, revealed the presence of inter-family epimutation hotspots near differentially expressed genes, most of which were located outside LADs redistributed in LMNA -related DCM. Comparison of DMRs found in fibroblasts and iPSCs identified regions where epimutationsmore »were persistent across both cell types. Finally, a network of aberrantly methylated disease-associated genes revealed a potential molecular link between pathways involved in bone and heart development. Conclusions Our results identified both shared and mutation-specific laminopathy epimutation landscapes that were consistent with lamin A/C mutation-mediated epigenetic aberrancies that arose in somatic and early developmental cell stages.« less
2. Arrhythmogenic Effects of Genetic Mutations Affecting Potassium Channels in Human Atrial Fibrillation: A Simulation Study

   https://doi.org/10.3389/fphys.2021.681943  

   Belletti, Rebecca ; Romero, Lucia ; Martinez-Mateu, Laura ; Cherry, Elizabeth M. ; Fenton, Flavio H. ; Saiz, Javier ( May 2021 , Frontiers in Physiology)

   Genetic mutations in genes encoding for potassium channel protein structures have been recently associated with episodes of atrial fibrillation in asymptomatic patients. The aim of this study is to investigate the potential arrhythmogenicity of three gain-of-function mutations related to atrial fibrillation—namely, KCNH2 T895M, KCNH2 T436M, and KCNE3-V17M—using modeling and simulation of the electrophysiological activity of the heart. A genetic algorithm was used to tune the parameters’ value of the original ionic currents to reproduce the alterations experimentally observed caused by the mutations. The effects on action potentials, ionic currents, and restitution properties were analyzed using versions of the Courtemanche human atrial myocyte model in different tissues: pulmonary vein, right, and left atrium. Atrial susceptibility of the tissues to spiral wave generation was also investigated studying the temporal vulnerability. The presence of the three mutations resulted in an overall more arrhythmogenic substrate. Higher current density, action potential duration shortening, and flattening of the restitution curves were the major effects of the three mutations at the single-cell level. The genetic mutations at the tissue level induced a higher temporal vulnerability to the rotor’s initiation and progression, by sustaining spiral waves that perpetuate until the end of the simulation. The mutation with themore »highest pro-arrhythmic effects, exhibiting the widest sustained VW and the smallest meandering rotor’s tip areas, was KCNE3-V17M. Moreover, the increased susceptibility to arrhythmias and rotor’s stability was tissue-dependent. Pulmonary vein tissues were more prone to rotor’s initiation, while in left atrium tissues rotors were more easily sustained. Re-entries were also progressively more stable in pulmonary vein tissue, followed by the left atrium, and finally the right atrium. The presence of the genetic mutations increased the susceptibility to arrhythmias by promoting the rotor’s initiation and maintenance. The study provides useful insights into the mechanisms underlying fibrillatory events caused by KCNH2 T895M, KCNH2 T436M, and KCNE3-V17M and might aid the planning of patient-specific targeted therapies.« less
3. Gain-of-function cardiomyopathic mutations in RBM20 rewire splicing regulation and re-distribute ribonucleoprotein granules within processing bodies

   https://doi.org/10.1038/s41467-021-26623-y  

   Fenix, Aidan M. ; Miyaoka, Yuichiro ; Bertero, Alessandro ; Blue, Steven M. ; Spindler, Matthew J. ; Tan, Kenneth K. B. ; Perez-Bermejo, Juan A. ; Chan, Amanda H. ; Mayerl, Steven J. ; Nguyen, Trieu D. ; et al ( November 2021 , Nature Communications)

   Mutations in the cardiac splicing factor RBM20 lead to malignant dilated cardiomyopathy (DCM). To understand the mechanism of RBM20-associated DCM, we engineered isogenic iPSCs with DCM-associated missense mutations in RBM20 as well as RBM20 knockout (KO) iPSCs. iPSC-derived engineered heart tissues made from these cell lines recapitulate contractile dysfunction of RBM20-associated DCM and reveal greater dysfunction with missense mutations than KO. Analysis of RBM20 RNA binding by eCLIP reveals a gain-of-function preference of mutant RBM20 for 3′ UTR sequences that are shared with amyotrophic lateral sclerosis (ALS) and processing-body associated RNA binding proteins (FUS, DDX6). Deep RNA sequencing reveals that the RBM20 R636S mutant has unique gene, splicing, polyadenylation and circular RNA defects that differ from RBM20 KO. Super-resolution microscopy verifies that mutant RBM20 maintains very limited nuclear localization potential; rather, the mutant protein associates with cytoplasmic processing bodies (DDX6) under basal conditions, and with stress granules (G3BP1) following acute stress. Taken together, our results highlight a pathogenic mechanism in cardiac disease through splicing-dependent and -independent pathways.
4. A 3D Bioprinted Human Cardiac Cell Platform to Model the Pathophysiology of Diabetes

   Binata Joddar, Shweta AnilKumar ( January 2020 , Circulation research)

   Type-II diabetes (T2D) patients affected by underlying hyperglycemic (high glucose/blood sugar) conditions often suffer from cardiac atrophy, resulting in tissue mass reduction and debilitating cardiac health. To understand pathophysiological mechanisms during progression of cardiac atrophy, a 3D bioprinted organoid platform was developed from a mixture of hydrogels containing human cardiac cells, including cardiomyocytes (CM), fibroblasts (CF) and endothelial cells (EC), to mimic the functionality of the in-vivo tissue. The organoids were cultured using normoglycemic- or hyperglycemic-conditions. The expression of essential biomarkers in these organoids, for myocardin (Myocd), troponin-I (TRP-I), fibroblast protein-1 (FSP-1) and endothelin-1 (ET-1) was confirmed. To assess the physiological cellular connections during hyperglycemia, the presence of Connexin-43 (CX-43) was assessed in the presence of a CX-43 blocker, gap26. Epigenomic tools were used to simultaneously interrogate histone-modifications by histone 3 lysine 9 mono-methylation (H3K9me1) along with the co-regulation of inflammatory mediators, such as the high mobility group box 1 (HMGB1) and toll like receptor 4 (TLR4) in the cardiac organoids cultured using normal versus hyperglycemic conditions. Organoids exposed to high glucose showed an increased expression of H3K9me1 as well as inflammatory mediators HMGB1 and TLR4. Hyperglycemia also exhibited alterations in expression of Myocd and FSP-1 in the organoids, comparedmore »to normoglycemic conditions. Treatment with gap26 affected the CX-43 expression significantly, in organoids cultured under hyperglycemia suggesting that high glucose conditions associated with prolonged diabetes may lead to compromised CM-CF coupling, essential for maintenance of cardiac functionality. Increased levels of H3K9me1 suggest decreased expression of Myocd, which may lead to CM degeneration. Epigenetic modifications including alterations in histone methylation in regulation of the myocardial genes and gap junction proteins under hyperglycemic conditions, may lead to cardiac atrophy. We expect to establish an actual T2D patient iPSC cell derived cardiac platform, to offer new therapeutic opportunities within the field.« less
5. BIN1 is a key regulator of proinflammatory and neurodegeneration-related activation in microglia

   https://doi.org/10.1186/s13024-022-00535-x  

   Sudwarts, Ari ; Ramesha, Supriya ; Gao, Tianwen ; Ponnusamy, Moorthi ; Wang, Shuai ; Hansen, Mitchell ; Kozlova, Alena ; Bitarafan, Sara ; Kumar, Prateek ; Beaulieu-Abdelahad, David ; et al ( May 2022 , Molecular Neurodegeneration)

   TheBIN1locus contains the second-most significant genetic risk factor for late-onset Alzheimer’s disease.BIN1undergoes alternate splicing to generate tissue- and cell-type-specific BIN1 isoforms, which regulate membrane dynamics in a range of crucial cellular processes. Whilst the expression of BIN1 in the brain has been characterized in neurons and oligodendrocytes in detail, information regarding microglial BIN1 expression is mainly limited to large-scale transcriptomic and proteomic data. Notably, BIN1 protein expression and its functional roles in microglia, a cell type most relevant to Alzheimer’s disease, have not been examined in depth.

   Microglial BIN1 expression was analyzed by immunostaining mouse and human brain, as well as by immunoblot and RT-PCR assays of isolated microglia or human iPSC-derived microglial cells.Bin1expression was ablated by siRNA knockdown in primary microglial cultures in vitro and Cre-lox mediated conditional deletion in adult mouse brain microglia in vivo. Regulation of neuroinflammatory microglial signatures by BIN1 in vitro and in vivo was characterized using NanoString gene panels and flow cytometry methods. The transcriptome data was explored by in silico pathway analysis and validated by complementary molecular approaches.

   Here, we characterized microglial BIN1 expression in vitro and in vivo and ascertained microglia expressed BIN1 isoforms. By silencingBin1expression in primary microglial cultures, wemore »demonstrate that BIN1 regulates the activation of proinflammatory and disease-associated responses in microglia as measured by gene expression and cytokine production. Our transcriptomic profiling revealed key homeostatic and lipopolysaccharide (LPS)-induced inflammatory response pathways, as well as transcription factors PU.1 and IRF1 that are regulated by BIN1. Microglia-specificBin1conditional knockout in vivo revealed novel roles of BIN1 in regulating the expression of disease-associated genes while counteracting CX3CR1 signaling. The consensus from in vitro and in vivo findings showed that loss ofBin1impaired the ability of microglia to mount type 1 interferon responses to proinflammatory challenge, particularly the upregulation of a critical type 1 immune response gene,Ifitm3.

   Our convergent findings provide novel insights into microglial BIN1 function and demonstrate an essential role of microglial BIN1 in regulating brain inflammatory response and microglial phenotypic changes. Moreover, for the first time, our study shows a regulatory relationship betweenBin1andIfitm3, two Alzheimer’s disease-related genes in microglia. The requirement for BIN1 to regulateIfitm3upregulation during inflammation has important implications for inflammatory responses during the pathogenesis and progression of many neurodegenerative diseases.

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