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Relatively little is known about the epigenetic control mechanisms that guide postnatal organ maturation. The goal of this study was to determine whether DNA methylation plays an important role in guiding transcriptional changes during the first 2 wk of mouse heart development, which is an important period for cardiomyocyte maturation, loss of proliferative capacity and loss of regenerative potential. Gene expression profiling (RNA-seq) and genome-wide sequencing of methylated DNA (MBD-seq) identified dynamic changes in the cardiac methylome during postnatal development [2545 differentially methylated regions (DMRs) from P1 to P14 in the mouse].The vast majority (~80%) of DMRs were hypermethylated between P1 and P14, and these hypermethylated regions were associated with transcriptional shut down of important developmental signaling pathways, including Hedgehog, bone morphogenetic protein, TGF-β, fibroblast growth factor, and Wnt/β-catenin signaling. Postnatal inhibition of DNA methylation with 5-aza-2'-deoxycytidine induced a marked increase (~3-fold) in cardiomyocyte proliferation and ~50% reduction in the percentage of binucleated cardiomyocytes compared with saline-treated controls. This study provides novel evidence for widespread alterations in DNA methylation during postnatal heart maturation and suggests that cardiomyocyte cell cycle arrest during the neonatal period is subject to regulation by DNA methylation. [ABSTRACT FROM AUTHOR]

The adult mammalian heart has limited potential for regeneration. Thus, after injury, cardiomyocytes are permanently lost, and contractility is diminished. In contrast, the neonatal heart can regenerate owing to sustained cardiomyocyte proliferation. Identification of critical regulators of cardiomyocyte proliferation and quiescence represents an important step toward potential regenerative therapies. Yes-associated protein (Yap), a transcriptional cofactor in the Hippo signaling pathway, promotes proliferation of embryonic cardiomyocytes by activating the insulin-like growth factor and Wnt signaling pathways. Here we report that mice bearing mutant alleles of Yap and its paralog WW domain containing transcription regulator 1 (Taz) exhibit gene dosage-dependent cardiac phenotypes, suggesting redundant roles of these Hippo pathway effectors in establishing proper myocyte number and maintaining cardiac function. Cardiac-specific deletion of Yap impedes neonatal heart regeneration, resulting in a default fibrotic response. Conversely, forced expression of a constitutively active form of Yap in the adult heart stimulates cardiac regeneration and improves contractility after myocardial infarction. The regenerative activity of Yap is correlated with its activation of embryonic and proliferative gene programs in cardiomyocytes. These findings identify Yap as an important regulator of cardiac regeneration and provide an experimental entry point to enhance this process. [ABSTRACT FROM AUTHOR]

With a high incidence of acute kidney injury among hospitalized COVID-19 patients, considerable attention has been focussed on whether SARS-CoV-2 specifically targets kidney cells to directly impact renal function, or whether renal damage is primarily an indirect outcome. To date, several studies have utilized kidney organoids to understand the pathogenesis of COVID-19, revealing the ability for SARS-CoV-2 to predominantly infect cells of the proximal tubule (PT), with reduced infectivity following administration of soluble ACE2. However, the immaturity of standard human kidney organoids represents a significant hurdle, leaving the preferred SARS-CoV-2 processing pathway, existence of alternate viral receptors, and the effect of common hypertensive medications on the expression of ACE2 in the context of SARS-CoV-2 exposure incompletely understood. Utilizing a novel kidney organoid model with enhanced PT maturity, genetic- and drug-mediated inhibition of viral entry and processing factors confirm ed the requirement for ACE2 for SARS-CoV-2 entry but showed that the virus can utilize dual viral spike protein processing pathways downstream of ACE2 receptor binding. These include TMPRSS- and CTSL/CTSB-mediated non-endosomal and endocytic pathways, with TMPRSS10 likely playing a more significant role in the non-endosomal pathway in renal cells than TMPRSS2. Finally, treatment with the antihypertensive ACE inhibitor, lisinopril, showed negligible impact on receptor expression or susceptibility of renal cells to infection. This study represents the first in-depth characterization of viral entry in stem cell-derived human kidney organoids with enhanced PTs, providing deeper insight into the renal implications of the ongoing COVID-19 pandemic. [ABSTRACT FROM AUTHOR]

Background: A strong focus of the post-genomic era is mining of the non-coding regulatory genome in order to unravel the function of regulatory elements that coordinate gene expression (Nat 489:57–74, 2012; Nat 507:462–70, 2014; Nat 507:455–61, 2014; Nat 518:317–30, 2015). Whole-genome approaches based on next-generation sequencing (NGS) have provided insight into the genomic location of regulatory elements throughout different cell types, organs and organisms. These technologies are now widespread and commonly used in laboratories from various fields of research. This highlights the need for fast and user-friendly software tools dedicated to extracting cis-regulatory information contained in these regulatory regions; for instance transcription factor binding site (TFBS) composition. Ideally, such tools should not require prior programming knowledge to ensure they are accessible for all users. Results: We present TrawlerWeb, a web-based version of the Trawler_standalone tool (Nat Methods 4:563–5, 2007; Nat Protoc 5:323–34, 2010), to allow for the identification of enriched motifs in DNA sequences obtained from next-generation sequencing experiments in order to predict their TFBS composition. TrawlerWeb is designed for online queries with standard options common to web-based motif discovery tools. In addition, TrawlerWeb provides three unique new features: 1) TrawlerWeb allows the input of BED files directly generated from NGS experiments, 2) it automatically generates an input-matched biologically relevant background, and 3) it displays resulting conservation scores for each instance of the motif found in the input sequences, which assists the researcher in prioritising the motifs to validate experimentally. Finally, to date, this web-based version of Trawler_standalone remains the fastest online de novo motif discovery tool compared to other popular web-based software, while generating predictions with high accuracy. Conclusions: TrawlerWeb provides users with a fast, simple and easy-to-use web interface for de novo motif discovery. This will assist in rapidly analysing NGS datasets that are now being routinely generated. TrawlerWeb is freely available and accessible at: http://trawler.erc.monash.edu.au. [ABSTRACT FROM AUTHOR]

Recent studies in non-human model systems have shown therapeutic potential of nucleoside-modified messenger RNA (modRNA) treatments for lysosomal storage diseases. Here, we assessed the efficacy of a modRNA treatment to restore the expression of the galactosidase alpha (GLA), which codes for α-Galactosidase A (α-GAL) enzyme, in a human cardiac model generated from induced pluripotent stem cells (iPSCs) derived from two individuals with Fabry disease. Consistent with the clinical phenotype, cardiomyocytes from iPSCs derived from Fabry-affected individuals showed accumulation of the glycosphingolipid Globotriaosylceramide (GB3), which is an α-galactosidase substrate. Furthermore, the Fabry cardiomyocytes displayed significant upregulation of lysosomal-associated proteins. Upon GLA modRNA treatment, a subset of lysosomal proteins were partially restored to wild-type levels, implying the rescue of the molecular phenotype associated with the Fabry genotype. Importantly, a significant reduction of GB3 levels was observed in GLA modRNA-treated cardiomyocytes, demonstrating that α-GAL enzymatic activity was restored. Together, our results validate the utility of iPSC-derived cardiomyocytes from affected individuals as a model to study disease processes in Fabry disease and the therapeutic potential of GLA modRNA treatment to reduce GB3 accumulation in the heart. [Display omitted] Nucleoside-modified messenger RNA (modRNA) has shown great promise as an enzyme replacement tool. Here, we show that GLA modRNA almost completely abolishes the accumulation of globotriaosylceramides observed in cardiomyocytes derived from individuals with Fabry disease. Moreover, changes in the proteome between Fabry and healthy cardiomyocytes were rescued upon modRNA treatment. [ABSTRACT FROM AUTHOR]

Understanding the spatial gene expression and regulation in the heart is key to uncovering its developmental and physiological processes, during homeostasis and disease. Numerous techniques exist to gain gene expression and regulation information in organs such as the heart, but few utilize intuitive true-to-life three-dimensional representations to analyze and visualise results. Here we combined transcriptomics with 3D-modelling to interrogate spatial gene expression in the mammalian heart. For this, we microdissected and sequenced transcriptome-wide 18 anatomical sections of the adult mouse heart. Our study has unveiled known and novel genes that display complex spatial expression in the heart sub-compartments. We have also created 3D-cardiomics, an interface for spatial transcriptome analysis and visualization that allows the easy exploration of these data in a 3D model of the heart. 3D-cardiomics is accessible from http://3d-cardiomics.erc.monash.edu/. [Display omitted] • Transcriptome-wide profiling of the murine heart reveals complex spatial patterns. • Identification of novel markers for cardiac subsections. • 3D-cardiomics allows for interrogation of gene expression in the adult heart. See https://3d-cardiomics.erc.monash.edu.au/. • Users can run a standalone 3D-cardiomics version for custom visualization. See https://github.com/Ramialison-Lab/3DCardiomics. [ABSTRACT FROM AUTHOR]

Abstract Three dimensional engineered culture systems are powerful tools to rapidly expand our knowledge of human biology and identify novel therapeutic targets for disease. Bioengineered skeletal muscle has been recently shown to recapitulate many features of native muscle biology. However, current skeletal muscle bioengineering approaches require large numbers of cells, reagents and labour, limiting their potential for high-throughput studies. Herein, we use a miniaturized 96-well micro-muscle platform to facilitate semi-automated tissue formation, culture and analysis of human skeletal micro muscles (hμMs). Utilising an iterative screening approach we define a serum-free differentiation protocol that drives rapid, directed differentiation of human myoblast to skeletal myofibres. The resulting hμMs comprised organised bundles of striated and functional myofibres, which respond appropriately to electrical stimulation. Additionally, we developed an optogenetic approach to chronically stimulate hμM to recapitulate known features of exercise training including myofibre hypertrophy and increased expression of metabolic proteins. Taken together, our miniaturized approach provides a new platform to enable high-throughput studies of human skeletal muscle biology and exercise physiology. [ABSTRACT FROM AUTHOR]

C18ORF25 was recently shown to be phosphorylated at S67 by AMP-activated protein kinase (AMPK) in the skeletal muscle, following acute exercise in humans. Phosphorylation was shown to improve the ex vivo skeletal muscle contractile function in mice, but our understanding of the molecular mechanisms is incomplete. Here, we profiled the interactome of C18ORF25 in mouse myotubes using affinity purification coupled to mass spectrometry. This analysis included an investigation of AMPK-dependent and S67-dependent protein/protein interactions. Several nucleocytoplasmic and contractile-associated proteins were identified, which revealed a subset of GTPases that associate with C18ORF25 in an AMPK- and S67 phosphorylation-dependent manner. We confirmed that C18ORF25 is localized to the nucleus and the contractile apparatus in the skeletal muscle. Mice lacking C18Orf25 display defects in calcium handling specifically in fast-twitch muscle fibers. To investigate these mechanisms, we developed an integrated single fiber physiology and single fiber proteomic platform. The approach enabled a detailed assessment of various steps in the excitation-contraction pathway including SR calcium handling and force generation, followed by paired single fiber proteomic analysis. This enabled us to identify >700 protein/phenotype associations and 36 fiber-type specific differences, following loss of C18Orf25 . Taken together, our data provide unique insights into the function of C18ORF25 and its role in skeletal muscle physiology.

The cardiac endothelium influences ventricular chamber development by coordinating trabeculation and compaction. However, the endothelial-specific molecular mechanisms mediating this coordination are not fully understood. Here, we identify the Sox7 transcription factor as a critical cue instructing cardiac endothelium identity during ventricular chamber development. Endothelial-specific loss of Sox7 function in mice results in cardiac ventricular defects similar to non-compaction cardiomyopathy, with a change in the proportions of trabecular and compact cardiomyocytes in the mutant hearts. This phenotype is paralleled by abnormal coronary artery formation. Loss of Sox7 function disrupts the transcriptional regulation of the Notch pathway and connexins 37 and 40, which govern coronary arterial specification. Upon Sox7 endothelial-specific deletion, single-nuclei transcriptomics analysis identifies the depletion of a subset of Sox9/Gpc3-positive endocardial progenitor cells and an increase in erythro-myeloid cell lineages. Fate mapping analysis reveals that a subset of Sox7-null endothelial cells transdifferentiate into hematopoietic but not cardiomyocyte lineages. Our findings determine that Sox7 maintains cardiac endothelial cell identity, which is crucial to the cellular cross-talk that drives ventricular compaction and coronary artery development., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Identifying spatially variable genes (SVGs) is a key step in the analysis of spatially resolved transcriptomics data. SVGs provide biological insights by defining transcriptomic differences within tissues, which was previously unachievable using RNA-sequencing technologies. However, the increasing number of published tools designed to define SVG sets currently lack benchmarking methods to accurately assess performance. This study compares results of 6 purpose-built packages for SVG identification across 9 public and 5 simulated datasets and highlights discrepancies between results. Additional tools for generation of simulated data and development of benchmarking methods are required to improve methods for identifying SVGs., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Here, we provide a protocol for next-generation human cardiac organoid modeling containing markers of vascularized tissues. We describe steps for cardiac differentiation, harvesting cardiac cells, and generating vascularized human cardiac organoids. We then detail downstream analysis of functional parameters and fluorescence labeling of human cardiac organoids. This protocol is useful for high throughput disease modeling, drug discovery, and providing mechanistic insight into cell-cell and cell-matrix interactions. For complete details on the use and execution of this protocol, please refer to Voges et al. 1 and Mills et al. 2 ., Competing Interests: Declaration of interests R.J.M., J.E.H., and E.R.P. are listed as co-inventors on pending patents that relate to cardiac organoid maturation and cardiac regeneration therapeutics. E.R.P. and J.E.H. are listed as co-inventors on pending patents that relate to endothelial cells in 3D cardiac products. J.E.H. is a co-inventor on licensed patents relating to engineered heart muscle. R.J.M., E.R.P., and J.E.H. are co-founders, scientific advisors, and stockholders in Dynomics., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Crosstalk between cardiac cells is critical for heart performance. Here we show that vascular cells within human cardiac organoids (hCOs) enhance their maturation, force of contraction, and utility in disease modeling. Herein we optimize our protocol to generate vascular populations in addition to epicardial, fibroblast, and cardiomyocyte cells that self-organize into in-vivo-like structures in hCOs. We identify mechanisms of communication between endothelial cells, pericytes, fibroblasts, and cardiomyocytes that ultimately contribute to cardiac organoid maturation. In particular, (1) endothelial-derived LAMA5 regulates expression of mature sarcomeric proteins and contractility, and (2) paracrine platelet-derived growth factor receptor β (PDGFRβ) signaling from vascular cells upregulates matrix deposition to augment hCO contractile force. Finally, we demonstrate that vascular cells determine the magnitude of diastolic dysfunction caused by inflammatory factors and identify a paracrine role of endothelin driving dysfunction. Together this study highlights the importance and role of vascular cells in organoid models., Competing Interests: Declaration of interests R.J.M., J.E.H., G.A.Q.-R., and E.R.P. are listed as co-inventors on pending patents that relate to cardiac organoid maturation and cardiac regeneration therapeutics. E.R.P. and J.E.H. are listed as co-inventors on pending patents that relate to endothelial cells in 3D cardiac products. J.E.H. is a co-inventor on licensed patents relating to engineered heart muscle. R.J.M., E.R.P., and J.E.H. are co-founders, scientific advisors, and stockholders in Dynomics., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Muscle contraction is driven by the molecular machinery of the sarcomere. As phosphorylation is a critical regulator of muscle function, the identification of regulatory kinases is important for understanding sarcomere biology. Pathogenic variants in alpha kinase 3 (ALPK3) cause cardiomyopathy and musculoskeletal disease, but little is known about this atypical kinase. Here we show that ALPK3 is an essential component of the M-band of the sarcomere and define the ALPK3-dependent phosphoproteome. ALPK3 deficiency impaired contractility both in human cardiac organoids and in the hearts of mice harboring a pathogenic truncating Alpk3 variant. ALPK3-dependent phosphopeptides were enriched for sarcomeric components of the M-band and the ubiquitin-binding protein sequestosome-1 (SQSTM1) (also known as p62). Analysis of the ALPK3 interactome confirmed binding to M-band proteins including SQSTM1. In human pluripotent stem cell-derived cardiomyocytes modeling cardiomyopathic ALPK3 mutations, sarcomeric organization and M-band localization of SQSTM1 were abnormal suggesting that this mechanism may underly disease pathogenesis., (© 2023. The Author(s).)

Long non-coding RNAs (lncRNAs) have been demonstrated to influence numerous biological processes, being strongly implicated in the maintenance and physiological function of various tissues including the heart. The lncRNA OIP5-AS1 ( 1700020I14Rik /C yrano ) has been studied in several settings; however its role in cardiac pathologies remains mostly uncharacterized. Using a series of in vitro and ex vivo methods, we demonstrate that OIP5-AS1 is regulated during cardiac development in rodent and human models and in disease settings in mice. Using CRISPR, we engineered a global OIP5-AS1 knockout (KO) mouse and demonstrated that female KO mice develop exacerbated heart failure following cardiac pressure overload (transverse aortic constriction [TAC]) but male mice do not. RNA-sequencing of wild-type and KO hearts suggest that OIP5-AS1 regulates pathways that impact mitochondrial function. Thus, these findings highlight OIP5-AS1 as a gene of interest in sex-specific differences in mitochondrial function and development of heart failure., Competing Interests: The authors declare they have no conflict of interest., (© 2021 The Authors.)

The mammalian heart undergoes maturation during postnatal life to meet the increased functional requirements of an adult. However, the key drivers of this process remain poorly defined. We are currently unable to recapitulate postnatal maturation in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), limiting their potential as a model system to discover regenerative therapeutics. Here, we provide a summary of our studies, where we developed a 96-well device for functional screening in human pluripotent stem cell-derived cardiac organoids (hCOs). Through interrogation of >10,000 organoids, we systematically optimize parameters, including extracellular matrix (ECM), metabolic substrate, and growth factor conditions, that enhance cardiac tissue viability, function, and maturation. Under optimized maturation conditions, functional and molecular characterization revealed that a switch to fatty acid metabolism was a central driver of cardiac maturation. Under these conditions, hPSC-CMs were refractory to mitogenic stimuli, and we found that key proliferation pathways including β-catenin and Yes-associated protein 1 (YAP1) were repressed. This proliferative barrier imposed by fatty acid metabolism in hCOs could be rescued by simultaneous activation of both β-catenin and YAP1 using genetic approaches or a small molecule activating both pathways. These studies highlight that human organoids coupled with higher-throughput screening platforms have the potential to rapidly expand our knowledge of human biology and potentially unlock therapeutic strategies., Competing Interests: Conflict of interest statement: R.J.M., D.M.T., E.R.P., and J.E.H. are listed as coinventors on a pending patent held by The University of Queensland that relates to the Heart-Dyno device and maturation medium, which are described in this paper. R.J.M., G.A.Q.-R., E.R.P., and J.E.H. are listed as coinventors on a pending patent held by The University of Queensland that relates to the reactivation of cardiomyocyte cell cycle for cardiac regeneration. L.D., A.T.P., and Q.-D.W. are employees of AstraZeneca.

Regenerative medicine aims to replace injured tissues to restore normal physiological function. One possibility for achieving this goal is to activate or enhance endogenous regenerative pathways. Therefore, human tissue regeneration models may be useful tools for the discovery and development of novel regenerative therapeutics. In this chapter, we describe methods for the generation of three-dimensional bioengineered striated muscle in vitro and a cryoinjury model that can be applied to these tissues. This technique enables mechanistic in vitro analysis of the endogenous regenerative response of human striated muscle to injury, which is not possible using other in vivo approaches.

Relatively little is known about the epigenetic control mechanisms that guide postnatal organ maturation. The goal of this study was to determine whether DNA methylation plays an important role in guiding transcriptional changes during the first 2 wk of mouse heart development, which is an important period for cardiomyocyte maturation, loss of proliferative capacity and loss of regenerative potential. Gene expression profiling (RNA-seq) and genome-wide sequencing of methylated DNA (MBD-seq) identified dynamic changes in the cardiac methylome during postnatal development [2545 differentially methylated regions (DMRs) from P1 to P14 in the mouse]. The vast majority (~80%) of DMRs were hypermethylated between P1 and P14, and these hypermethylated regions were associated with transcriptional shut down of important developmental signaling pathways, including Hedgehog, bone morphogenetic protein, TGF-β, fibroblast growth factor, and Wnt/β-catenin signaling. Postnatal inhibition of DNA methylation with 5-aza-2'-deoxycytidine induced a marked increase (~3-fold) in cardiomyocyte proliferation and ~50% reduction in the percentage of binucleated cardiomyocytes compared with saline-treated controls. This study provides novel evidence for widespread alterations in DNA methylation during postnatal heart maturation and suggests that cardiomyocyte cell cycle arrest during the neonatal period is subject to regulation by DNA methylation., (© FASEB.)

Background: Perinatal glucocorticoid treatment is associated with hypertrophic cardiomyopathy, but the cellular mechanism is controversial. An underlying interaction between glucocorticoids and the renin-angiotensin system may be important, but whether glucocorticoids modulate angiotensin II (AngII)-dependent cardiomyocyte growth responses in the neonate has not been investigated., Objectives: The major aim of this investigation was to determine whether glucocorticoids modulate the neonatal cardiomyocyte growth response to AngII. In particular we sought evidence to determine whether angiotensin II type 2 (AT(2)) receptor co-expression with angiotensin II type 1 (AT(1)) receptor is of specific importance in this modulatory function., Methods: In this study, we used AT(1) and AT(2) receptor-expressing adenoviruses (Ad-AT(1) and Ad-AT(2)) in a well-defined in vitro neonatal cardiomyocyte culture model to assess whether glucocorticoids affect cardiomyocyte growth responses (i.e. total protein content)., Results: Following addition of AngII (0.1 micromol/l) to neonatal cardiomyocytes infected with Ad-AT(1) alone, a significant growth response was measured (133.2 +/- 4.8%). Expression of Ad-AT(2) alone induced a approximately 20% increase in total cellular protein content, which was unaffected by addition of AngII. Neither corticosterone (1 micromol/l) nor dexamethasone (1 micromol/l) had any significant effect on the AT(1)- or AT(2)-mediated growth responses. In contrast, the growth response to AngII was augmented following co-expression of AT(2) and AT(1) receptors (149.2 +/- 4.2%), which was reduced by approximately 20% in the presence of either corticosterone or dexamethasone (p < 0.05)., Conclusions: The present study provides novel evidence that glucocorticoids suppress neonatal cardiomyocyte growth responsiveness when AT(2 )and AT(1) receptor subtypes are co-expressed., (Copyright 2009 S. Karger AG, Basel.)