Differentiation of Pluripotent Stem Cells for Disease Modeling: Learning from Heart Development.

Heart disease is a pressing public health problem and the leading cause of death worldwide. The heart is the first organ to gain function during embryogenesis in mammals. Heart development involves cell determination, expansion, migration, and crosstalk, which are orchestrated by numerous signaling pathways, such as the Wnt, TGF-ß, IGF, and Retinoic acid signaling pathways. Human-induced pluripotent stem cell-based platforms are emerging as promising approaches for modeling heart disease in vitro. Understanding the signaling pathways that are essential for cardiac development has shed light on the molecular mechanisms of congenital heart defects and postnatal heart diseases, significantly advancing stem cell-based platforms to model heart diseases. This review summarizes signaling pathways that are crucial for heart development and discusses how these findings improve the strategies for modeling human heart disease in vitro.

Advances in the Generation of Constructed Cardiac Tissue Derived from Induced Pluripotent Stem Cells for Disease Modeling and Therapeutic Discovery.

The human heart lacks significant regenerative capacity; thus, the solution to heart failure (HF) remains organ donation, requiring surgery and immunosuppression. The demand for constructed cardiac tissues (CCTs) to model and treat disease continues to grow. Recent advances in induced pluripotent stem cell (iPSC) manipulation, CRISPR gene editing, and 3D tissue culture have enabled a boom in iPSC-derived CCTs (iPSC-CCTs) with diverse cell types and architecture. Compared with 2D-cultured cells, iPSC-CCTs better recapitulate heart biology, demonstrating the potential to advance organ modeling, drug discovery, and regenerative medicine, though iPSC-CCTs could benefit from better methods to faithfully mimic heart physiology and electrophysiology. Here, we summarize advances in iPSC-CCTs and future developments in the vascularization, immunization, and maturation of iPSC-CCTs for study and therapy.

Circadian Control of Histone Turnover During Cardiac Development and Growth.

Rationale: During postnatal cardiac hypertrophy, cardiomyocytes undergo mitotic exit, relying on DNA replication-independent mechanisms of histone turnover to maintain chromatin organization and gene transcription. In other tissues, circadian oscillations in nucleosome occupancy influence clock-controlled gene expression, suggesting an unrecognized role for the circadian clock in temporal control of histone turnover and coordinate cardiomyocyte gene expression. Objective: To elucidate roles for the master circadian transcription factor, Bmal1, in histone turnover, chromatin organization, and myocyte-specific gene expression and cell growth in the neonatal period. Methods and Results: Bmal1 knockdown in neonatal rat ventricular myocytes (NRVM) decreased myocyte size, total cellular protein, and transcription of the fetal hypertrophic gene Nppb following treatment with increasing serum concentrations or the α-adrenergic agonist phenylephrine (PE). Bmal1 knockdown decreased expression of clock-controlled genes Per2 and Tcap, and salt-inducible kinase 1 (Sik1) which was identified via gene ontology analysis of Bmal1 targets upregulated in adult versus embryonic hearts. Epigenomic analyses revealed co-localized chromatin accessibility and Bmal1 localization in the Sik1 promoter. Bmal1 knockdown impaired Per2 and Sik1 promoter accessibility as measured by MNase-qPCR and impaired histone turnover indicated by metabolic labeling of acid-soluble chromatin fractions and immunoblots of total and chromatin-associated core histones. Sik1 knockdown basally increased myocyte size, while simultaneously impairing and driving Nppb and Per2 transcription, respectively. Conclusions: Bmal1 is required for neonatal myocyte growth, replication-independent histone turnover, and chromatin organization at the Sik1 promoter. Sik1 represents a novel clock-controlled gene that coordinates myocyte growth with hypertrophic and clock-controlled gene transcription.

Triplication of the interferon receptor locus contributes to hallmarks of Down syndrome in a mouse model.

Down syndrome (DS), the genetic condition caused by trisomy 21, is characterized by variable cognitive impairment, immune dysregulation, dysmorphogenesis and increased prevalence of diverse co-occurring conditions. The mechanisms by which trisomy 21 causes these effects remain largely unknown. We demonstrate that triplication of the interferon receptor (IFNR) gene cluster on chromosome 21 is necessary for multiple phenotypes in a mouse model of DS. Whole-blood transcriptome analysis demonstrated that IFNR overexpression associates with chronic interferon hyperactivity and inflammation in people with DS. To define the contribution of this locus to DS phenotypes, we used genome editing to correct its copy number in a mouse model of DS, which normalized antiviral responses, prevented heart malformations, ameliorated developmental delays, improved cognition and attenuated craniofacial anomalies. Triplication of the Ifnr locus modulates hallmarks of DS in mice, suggesting that trisomy 21 elicits an interferonopathy potentially amenable to therapeutic intervention.

Interferon hyperactivity impairs cardiogenesis in Down syndrome via downregulation of canonical Wnt signaling.

Congenital heart defects (CHDs) are frequent in children with Down syndrome (DS), caused by trisomy of chromosome 21. However, the underlying mechanisms are poorly understood. Here, using a human-induced pluripotent stem cell (iPSC)-based model and the Dp(16)1Yey/+ (Dp16) mouse model of DS, we identified downregulation of canonical Wnt signaling downstream of increased dosage of interferon (IFN) receptors (IFNRs) genes on chromosome 21 as a causative factor of cardiogenic dysregulation in DS. We differentiated human iPSCs derived from individuals with DS and CHDs, and healthy euploid controls into cardiac cells. We observed that T21 upregulates IFN signaling, downregulates the canonical WNT pathway, and impairs cardiac differentiation. Furthermore, genetic and pharmacological normalization of IFN signaling restored canonical WNT signaling and rescued defects in cardiogenesis in DS in vitro and in vivo. Our findings provide insights into mechanisms underlying abnormal cardiogenesis in DS, ultimately aiding the development of therapeutic strategies.

Cellular reprogramming of fibroblasts in heart regeneration.

Myocardial infarction causes the loss of cardiomyocytes and the formation of cardiac fibrosis due to the activation of cardiac fibroblasts, leading to cardiac dysfunction and heart failure. Unfortunately, current therapeutic interventions can only slow the disease progression. Furthermore, they cannot fully restore cardiac function, likely because the adult human heart lacks sufficient capacity to regenerate cardiomyocytes. Therefore, intensive efforts have focused on developing therapeutics to regenerate the damaged heart. Several strategies have been intensively investigated, including stimulation of cardiomyocyte proliferation, transplantation of stem cell-derived cardiomyocytes, and conversion of fibroblasts into cardiac cells. Resident cardiac fibroblasts are critical in the maintenance of the structure and contractility of the heart. Fibroblast plasticity makes this type of cells be reprogrammed into many cell types, including but not limited to induced pluripotent stem cells, induced cardiac progenitor cells, and induced cardiomyocytes. Fibroblasts have become a therapeutic target due to their critical roles in cardiac pathogenesis. This review summarizes the reprogramming of fibroblasts into induced pluripotent stem cell-derived cardiomyocytes, induced cardiac progenitor cells, and induced cardiomyocytes to repair a damaged heart, outlines recent findings in utilizing fibroblast-derived cells for heart regeneration, and discusses the limitations and challenges.

The molecular mechanism of sialic acid transport mediated by Sialin.

Malfunction of the sialic acid transporter caused by various genetic mutations in the SLC17A5 gene encoding Sialin leads to a spectrum of neurodegenerative conditions called free sialic acid storage disorders. Unfortunately, how Sialin transports sialic acid/proton (H+) and how pathogenic mutations impair its function are poorly defined. Here, we present the structure of human Sialin in an inward-facing partially open conformation determined by cryo-electron microscopy, representing the first high-resolution structure of any human SLC17 member. Our analysis reveals two unique features in Sialin: (i) The H+ coupling/sensing requires two highly conserved Glu residues (E171 and E175) instead of one (E175) as implied in previous studies; and (ii) the normal function of Sialin requires the stabilization of a cytosolic helix, which has not been noticed in the literature. By mapping known pathogenic mutations, we provide mechanistic explanations for corresponding functional defects. We propose a structure-based mechanism for sialic acid transport mediated by Sialin.

Cell differentiation modifies the p53 transcriptional program through a combination of gene silencing and constitutive transactivation.

The p53 transcription factor is a master regulator of cellular responses to stress that is commonly inactivated in diverse cancer types. Despite decades of research, the mechanisms by which p53 impedes tumorigenesis across vastly different cellular contexts requires further investigation. The bulk of research has been completed using in vitro studies of cancer cell lines or in vivo studies in mouse models, but much less is known about p53 action in diverse non-transformed human tissues. Here, we investigated how different cellular states modify the p53 transcriptional program in human cells through a combination of computational analyses of publicly available large-scale datasets and in vitro studies using an isogenic system consisting of induced pluripotent stem cells (iPSCs) and two derived lineages. Analysis of publicly available mRNA expression and genetic dependency data demonstrated wide variation in terms of expression and function of a core p53 transcriptional program across various tissues and lineages. To monitor the impact of cell differentiation on the p53 transcriptome within an isogenic cell culture system, we activated p53 by pharmacological inhibition of its negative regulator MDM2. Using cell phenotyping assays and genome wide transcriptome analyses, we demonstrated that cell differentiation confines and modifies the p53 transcriptional network in a lineage-specific fashion. Although hundreds of p53 target genes are transactivated in iPSCs, only a small fraction is transactivated in each of the differentiated lineages. Mechanistic studies using small molecule inhibitors and genetic knockdowns revealed the presence of two major regulatory mechanisms contributing to this massive heterogeneity across cellular states: gene silencing by epigenetic regulatory complexes and constitutive transactivation by lineage-specific transcription factors. Altogether, these results illuminate the impact of cell differentiation on the p53 program, thus advancing our understanding of how this tumor suppressor functions in different contexts.

Cardiomyocyte-Specific Long Noncoding RNA Regulates Alternative Splicing of the Triadin Gene in the Heart.

BACKGROUND: Abnormalities in Ca2+ homeostasis are associated with cardiac arrhythmias and heart failure. Triadin plays an important role in Ca2+ homeostasis in cardiomyocytes. Alternative splicing of a single triadin gene produces multiple triadin isoforms. The cardiac-predominant isoform, mouse MT-1 or human Trisk32, is encoded by triadin exons 1 to 8. In humans, mutations in the triadin gene that lead to a reduction in Trisk32 levels in the heart can cause cardiac dysfunction and arrhythmias. Decreased levels of Trisk32 in the heart are also common in patients with heart failure. However, mechanisms that maintain triadin isoform composition in the heart remain elusive. METHODS: We analyzed triadin expression in heart explants from patients with heart failure and cardiac arrhythmias and in hearts from mice carrying a knockout allele for Trdn-as, a cardiomyocyte-specific long noncoding RNA encoded by the antisense strand of the triadin gene, between exons 9 and 11. Catecholamine challenge with isoproterenol was performed on Trdn-as knockout mice to assess the role of Trdn-as in cardiac arrhythmogenesis, as assessed by ECG. Ca2+ transients in adult mouse cardiomyocytes were measured with the IonOptix platform or the GCaMP system. Biochemistry assays, single-molecule fluorescence in situ hybridization, subcellular localization imaging, RNA sequencing, and molecular rescue assays were used to investigate the mechanisms by which Trdn-as regulates cardiac function and triadin levels in the heart. RESULTS: We report that Trdn-as maintains cardiac function, at least in part, by regulating alternative splicing of the triadin gene. Knockout of Trdn-as in mice downregulates cardiac triadin, impairs Ca2+ handling, and causes premature death. Trdn-as knockout mice are susceptible to cardiac arrhythmias in response to catecholamine challenge. Normalization of cardiac triadin levels in Trdn-as knockout cardiomyocytes is sufficient to restore Ca2+ handling. Last, Trdn-as colocalizes and interacts with serine/arginine splicing factors in cardiomyocyte nuclei and is essential for efficient recruitment of splicing factors to triadin precursor mRNA. CONCLUSIONS: These findings reveal regulation of alternative splicing as a novel mechanism by which a long noncoding RNA controls cardiac function. This study indicates potential therapeutics for heart disease by targeting the long noncoding RNA or pathways regulating alternative splicing.

A simple protocol to produce mature human-induced pluripotent stem cell-derived cardiomyocytes.

When cultured under typical conditions, human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are structurally and functionally immature. We have previously demonstrated that culture of hiPSC-CMs in maturation medium containing fatty acids, in combination with culture on micropatterned surfaces, produces cells that demonstrate a more mature phenotype compared to standard approaches. Here, we show in detail the steps needed to produce mature hiPSC-CMs. Compared with many approaches, our protocol is relatively simple and can be easily adapted to new laboratories. For complete details on the use and execution of this protocol, please refer to Knight et al. (2021).

Maturation of Pluripotent Stem Cell-Derived Cardiomyocytes Enables Modeling of Human Hypertrophic Cardiomyopathy.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a powerful platform for biomedical research. However, they are immature, which is a barrier to modeling adult-onset cardiovascular disease. Here, we sought to develop a simple method that could drive cultured hiPSC-CMs toward maturity across a number of phenotypes, with the aim of utilizing mature hiPSC-CMs to model human cardiovascular disease. hiPSC-CMs were cultured in fatty acid-based medium and plated on micropatterned surfaces. These cells display many characteristics of adult human cardiomyocytes, including elongated cell morphology, sarcomeric maturity, and increased myofibril contractile force. In addition, mature hiPSC-CMs develop pathological hypertrophy, with associated myofibril relaxation defects, in response to either a pro-hypertrophic agent or genetic mutations. The more mature hiPSC-CMs produced by these methods could serve as a useful in vitro platform for characterizing cardiovascular disease.

Suppression of canonical TGF-ß signaling enables GATA4 to interact with H3K27me3 demethylase JMJD3 to promote cardiomyogenesis.

Direct reprogramming of fibroblasts into cardiomyocytes (CMs) represents a promising strategy to regenerate CMs lost after ischemic heart injury. Overexpression of GATA4, HAND2, MEF2C, TBX5, miR-1, and miR-133 (GHMT2m) along with transforming growth factor beta (TGF-ß) inhibition efficiently promote reprogramming. However, the mechanisms by which TGF-ß blockade promotes cardiac reprogramming remain unknown. Here, we identify interactions between the histone H3 lysine 27 trimethylation (H3K27me3) demethylase JMJD3, the SWI/SNF remodeling complex subunit BRG1, and cardiac transcription factors. Furthermore, canonical TGF-ß signaling regulates the interaction between GATA4 and JMJD3. TGF-ß activation impairs the ability of GATA4 to bind target genes and prevents demethylation of H3K27 at cardiac gene promoters during cardiac reprogramming. Finally, a mutation in GATA4 (V267M) that is associated with congenital heart disease exhibits reduced binding to JMJD3 and impairs cardiomyogenesis. Thus, we have identified an epigenetic mechanism wherein canonical TGF-ß pathway activation impairs cardiac gene programming, in part by interfering with GATA4-JMJD3 interactions.

Danon Disease-Associated LAMP-2 Deficiency Drives Metabolic Signature Indicative of Mitochondrial Aging and Fibrosis in Cardiac Tissue and hiPSC-Derived Cardiomyocytes.

Danon disease is a severe X-linked disorder caused by deficiency of the lysosome-associated membrane protein-2 (LAMP-2). Clinical manifestations are phenotypically diverse and consist of hypertrophic and dilated cardiomyopathies, skeletal myopathy, retinopathy, and intellectual dysfunction. Here, we investigated the metabolic landscape of Danon disease by applying a multi-omics approach and combined structural and functional readouts provided by Raman and atomic force microscopy. Using these tools, Danon patient-derived cardiac tissue, primary fibroblasts, and human induced pluripotent stem cells differentiated into cardiomyocytes (hiPSC-CMs) were analyzed. Metabolic profiling indicated LAMP-2 deficiency promoted a switch toward glycolysis accompanied by rerouting of tryptophan metabolism. Cardiomyocytes' energetic balance and NAD+/NADH ratio appeared to be maintained despite mitochondrial aging. In turn, metabolic adaption was accompanied by a senescence-associated signature. Similarly, Danon fibroblasts appeared more stress prone and less biomechanically compliant. Overall, shaping of both morphology and metabolism contributed to the loss of cardiac biomechanical competence that characterizes the clinical progression of Danon disease.

Lysosomal Abnormalities in Cardiovascular Disease.

The lysosome, a key organelle for cellular clearance, is associated with a wide variety of pathological conditions in humans. Lysosome function and its related pathways are particularly important for maintaining the health of the cardiovascular system. In this review, we highlighted studies that have improved our understanding of the connection between lysosome function and cardiovascular diseases with an emphasis on a recent breakthrough that characterized a unique autophagosome-lysosome fusion mechanism employed by cardiomyocytes through a lysosomal membrane protein LAMP-2B. This finding may impact the development of future therapeutic applications.

Cardiac Regeneration: New Insights Into the Frontier of Ischemic Heart Failure Therapy.

Ischemic heart disease is the leading cause of morbidity and mortality in the world. While pharmacological and surgical interventions developed in the late twentieth century drastically improved patient outcomes, mortality rates over the last two decades have begun to plateau. Following ischemic injury, pathological remodeling leads to cardiomyocyte loss and fibrosis leading to impaired heart function. Cardiomyocyte turnover rate in the adult heart is limited, and no clinical therapies currently exist to regenerate cardiomyocytes lost following ischemic injury. In this review, we summarize the progress of therapeutic strategies including revascularization and cell-based interventions to regenerate the heart: transiently inducing cardiomyocyte proliferation and direct reprogramming of fibroblasts into cardiomyocytes. Moreover, we highlight recent mechanistic insights governing these strategies to promote heart regeneration and identify current challenges in translating these approaches to human patients.

Dynamic Chromatin Targeting of BRD4 Stimulates Cardiac Fibroblast Activation.

RATIONALE: Small molecule inhibitors of the acetyl-histone binding protein BRD4 have been shown to block cardiac fibrosis in preclinical models of heart failure (HF). However, since the inhibitors target BRD4 ubiquitously, it is unclear whether this chromatin reader protein functions in cell type-specific manner to control pathological myocardial fibrosis. Furthermore, the molecular mechanisms by which BRD4 stimulates the transcriptional program for cardiac fibrosis remain unknown. OBJECTIVE: We sought to test the hypothesis that BRD4 functions in a cell-autonomous and signal-responsive manner to control activation of cardiac fibroblasts, which are the major extracellular matrix-producing cells of the heart. METHODS AND RESULTS: RNA-sequencing, mass spectrometry, and cell-based assays employing primary adult rat ventricular fibroblasts demonstrated that BRD4 functions as an effector of TGF-ß (transforming growth factor-ß) signaling to stimulate conversion of quiescent cardiac fibroblasts into Periostin (Postn)-positive cells that express high levels of extracellular matrix. These findings were confirmed in vivo through whole-transcriptome analysis of cardiac fibroblasts from mice subjected to transverse aortic constriction and treated with the small molecule BRD4 inhibitor, JQ1. Chromatin immunoprecipitation-sequencing revealed that BRD4 undergoes stimulus-dependent, genome-wide redistribution in cardiac fibroblasts, becoming enriched on a subset of enhancers and super-enhancers, and leading to RNA polymerase II activation and expression of downstream target genes. Employing the Sertad4 (SERTA domain-containing protein 4) locus as a prototype, we demonstrate that dynamic chromatin targeting of BRD4 is controlled, in part, by p38 MAPK (mitogen-activated protein kinase) and provide evidence of a critical function for Sertad4 in TGF-ß-mediated cardiac fibroblast activation. CONCLUSIONS: These findings define BRD4 as a central regulator of the pro-fibrotic cardiac fibroblast phenotype, establish a p38-dependent signaling circuit for epigenetic reprogramming in heart failure, and uncover a novel role for Sertad4. The work provides a mechanistic foundation for the development of BRD4 inhibitors as targeted anti-fibrotic therapies for the heart.

Deubiquitylase USP9X maintains centriolar satellite integrity by stabilizing pericentriolar material 1 protein.

Centriolar satellites are small cytoplasmic granules that play important roles in regulating the formation of centrosomes and primary cilia. Ubiquitylation of satellite proteins, including the core satellite scaffold protein pericentriolar material 1 (PCM1), regulates centriolar satellite integrity. Currently, deubiquitylases that control centriolar satellite integrity have not been identified. In this study, we find that the deubiquitylase USP9X binds PCM1, and antagonizes PCM1 ubiquitylation to protect it from proteasomal degradation. Knockdown of USP9X in human cell lines reduces PCM1 protein levels, disrupts centriolar satellite particles and causes localization of satellite proteins, such as CEP290, to centrosomes. Interestingly, knockdown of mindbomb 1 (MIB1), a ubiquitin ligase that promotes PCM1 ubiquitylation and degradation, in USP9X-depleted cells largely restores PCM1 protein levels and corrects defects caused by the loss of USP9X. Overall, our study reveals that USP9X is a constituent of centriolar satellites and functions to maintain centriolar satellite integrity by stabilizing PCM1.

LAMP-2B regulates human cardiomyocyte function by mediating autophagosome-lysosome fusion.

Mutations in lysosomal-associated membrane protein 2 (LAMP-2) gene are associated with Danon disease, which often leads to cardiomyopathy/heart failure through poorly defined mechanisms. Here, we identify the LAMP-2 isoform B (LAMP-2B) as required for autophagosome-lysosome fusion in human cardiomyocytes (CMs). Remarkably, LAMP-2B functions independently of syntaxin 17 (STX17), a protein that is essential for autophagosome-lysosome fusion in non-CMs. Instead, LAMP-2B interacts with autophagy related 14 (ATG14) and vesicle-associated membrane protein 8 (VAMP8) through its C-terminal coiled coil domain (CCD) to promote autophagic fusion. CMs derived from induced pluripotent stem cells (hiPSC-CMs) from Danon patients exhibit decreased colocalization between ATG14 and VAMP8, profound defects in autophagic fusion, as well as mitochondrial and contractile abnormalities. This phenotype was recapitulated by LAMP-2B knockout in non-Danon hiPSC-CMs. Finally, gene correction of LAMP-2 mutation rescues the Danon phenotype. These findings reveal a STX17-independent autophagic fusion mechanism in human CMs, providing an explanation for cardiomyopathy in Danon patients and a foundation for targeting defective LAMP-2B-mediated autophagy to treat this patient population.

HDAC11 suppresses the thermogenic program of adipose tissue via BRD2.

Little is known about the biological function of histone deacetylase 11 (HDAC11), which is the lone class IV HDAC. Here, we demonstrate that deletion of HDAC11 in mice stimulates brown adipose tissue (BAT) formation and beiging of white adipose tissue (WAT). Consequently, HDAC11-deficient mice exhibit enhanced thermogenic potential and, in response to high-fat feeding, attenuated obesity, improved insulin sensitivity, and reduced hepatic steatosis. Ex vivo and cell-based assays revealed that HDAC11 catalytic activity suppresses the BAT transcriptional program, in both the basal state and in response to ß-adrenergic receptor signaling, through a mechanism that is dependent on physical association with BRD2, a bromodomain and extraterminal (BET) acetyl-histone-binding protein. These findings define an epigenetic pathway for the regulation of energy homeostasis and suggest the potential for HDAC11-selective inhibitors for the treatment of obesity and diabetes.