Multiplatform modeling of atrial fibrillation identifies phospholamban as a central regulator of cardiac rhythm.

Atrial fibrillation (AF) is a common and genetically inheritable form of cardiac arrhythmia; however, it is currently not known how these genetic predispositions contribute to the initiation and/or maintenance of AF-associated phenotypes. One major barrier to progress is the lack of experimental systems to investigate the effects of gene function on rhythm parameters in models with human atrial and whole-organ relevance. Here, we assembled a multi-model platform enabling high-throughput characterization of the effects of gene function on action potential duration and rhythm parameters using human induced pluripotent stem cell-derived atrial-like cardiomyocytes and a Drosophila heart model, and validation of the findings using computational models of human adult atrial myocytes and tissue. As proof of concept, we screened 20 AF-associated genes and identified phospholamban loss of function as a top conserved hit that shortens action potential duration and increases the incidence of arrhythmia phenotypes upon stress. Mechanistically, our study reveals that phospholamban regulates rhythm homeostasis by functionally interacting with L-type Ca2+ channels and NCX. In summary, our study illustrates how a multi-model system approach paves the way for the discovery and molecular delineation of gene regulatory networks controlling atrial rhythm with application to AF.

Metabolic Maturation Media Improve Physiological Function of Human iPSC-Derived Cardiomyocytes.

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have enormous potential for the study of human cardiac disorders. However, their physiological immaturity severely limits their utility as a model system and their adoption for drug discovery. Here, we describe maturation media designed to provide oxidative substrates adapted to the metabolic needs of human iPSC (hiPSC)-CMs. Compared with conventionally cultured hiPSC-CMs, metabolically matured hiPSC-CMs contract with greater force and show an increased reliance on cardiac sodium (Na+) channels and sarcoplasmic reticulum calcium (Ca2+) cycling. The media enhance the function, long-term survival, and sarcomere structures in engineered heart tissues. Use of the maturation media made it possible to reliably model two genetic cardiac diseases: long QT syndrome type 3 due to a mutation in the cardiac Na+ channel SCN5A and dilated cardiomyopathy due to a mutation in the RNA splicing factor RBM20. The maturation media should increase the fidelity of hiPSC-CMs as disease models.

Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells.

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is a pre-requisite for cell-based cardiac therapies and disease modeling. We have recently shown that Id genes are both necessary and sufficient to specify first heart field progenitors during vertebrate development. This differentiation protocol leverages these findings and uses Id1 overexpression in combination with Activin A as potent specifying cues to produce first heart field-like (FHF-L) progenitors. Importantly, resulting progenitors efficiently differentiate (~70-90%) into ventricular-like cardiomyocytes. Here we describe a detailed method to 1) generate Id1-overexpressing hPSCs and 2) differentiate scalable quantities of cryopreservable FHF-L progenitors and ventricular-like cardiomyocytes.

Id genes are essential for early heart formation.

Deciphering the fundamental mechanisms controlling cardiac specification is critical for our understanding of how heart formation is initiated during embryonic development and for applying stem cell biology to regenerative medicine and disease modeling. Using systematic and unbiased functional screening approaches, we discovered that the Id family of helix-loop-helix proteins is both necessary and sufficient to direct cardiac mesoderm formation in frog embryos and human embryonic stem cells. Mechanistically, Id proteins specify cardiac cell fate by repressing two inhibitors of cardiogenic mesoderm formation-Tcf3 and Foxa2-and activating inducers Evx1, Grrp1, and Mesp1. Most importantly, CRISPR/Cas9-mediated ablation of the entire Id (Id1-4) family in mouse embryos leads to failure of anterior cardiac progenitor specification and the development of heartless embryos. Thus, Id proteins play a central and evolutionarily conserved role during heart formation and provide a novel means to efficiently produce cardiovascular progenitors for regenerative medicine and drug discovery applications.

High content screening for modulators of cardiac differentiation in human pluripotent stem cells.

Chemical genomics has the unique potential to expose novel mechanisms of complex cellular biology through screening of small molecules in in vitro assays of a biological phenotype of interest, followed by target identification. In the case of disease-specific assays, the cellular proteins identified might constitute novel drug targets, and the small molecules themselves might be developed as drug leads. In cardiovascular biology, a chemical genomics approach to study the formation of cardiomyocyte, vascular endothelial, and smooth muscle lineages might contribute to therapeutic regeneration. Here, we describe methods used to develop high content screening assays implementing multipotent cardiovascular progenitors derived from human pluripotent stem cells and have identified novel compounds that direct cardiac differentiation.

Coordinate Nodal and BMP inhibition directs Baf60c-dependent cardiomyocyte commitment.

A critical but molecularly uncharacterized step in heart formation and regeneration is the process that commits progenitor cells to differentiate into cardiomyocytes. Here, we show that the endoderm-derived dual Nodal/bone morphogenetic protein (BMP) antagonist Cerberus-1 (Cer1) in embryonic stem cell cultures orchestrates two signaling pathways that direct the SWI/SNF chromatin remodeling complex to cardiomyogenic loci in multipotent (KDR/Flk1+) progenitors, activating lineage-specific transcription. Transient inhibition of Nodal by Cer1 induces Brahma-associated factor 60c (Baf60c), one of three Baf60 variants (a, b, and c) that are mutually exclusively assembled into SWI/SNF. Blocking Nodal and BMP also induces lineage-specific transcription factors Gata4 and Tbx5, which interact with Baf60c. siRNA to Cer1, Baf60c, or the catalytic SWI/SNF subunit Brg1 prevented the developmental opening of chromatin surrounding the Nkx2.5 early cardiac enhancer and cardiomyocyte differentiation. Overexpression of Baf60c fully rescued these deficits, positioning Baf60c and SWI/SNF function downstream from Cer1. Thus, antagonism of Nodal and BMP coordinates induction of the myogenic Baf60c variant and interacting transcription factors to program the developmental opening of cardiomyocyte-specific loci in chromatin. This is the first demonstration that cues from the progenitor cell environment direct the subunit variant composition of SWI/SNF to remodel the transcriptional landscape for lineage-specific differentiation.

Studying arrhythmogenic right ventricular dysplasia with patient-specific iPSCs.

Cellular reprogramming of somatic cells to patient-specific induced pluripotent stem cells (iPSCs) enables in vitro modelling of human genetic disorders for pathogenic investigations and therapeutic screens. However, using iPSC-derived cardiomyocytes (iPSC-CMs) to model an adult-onset heart disease remains challenging owing to the uncertainty regarding the ability of relatively immature iPSC-CMs to fully recapitulate adult disease phenotypes. Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an inherited heart disease characterized by pathological fatty infiltration and cardiomyocyte loss predominantly in the right ventricle, which is associated with life-threatening ventricular arrhythmias. Over 50% of affected individuals have desmosome gene mutations, most commonly in PKP2, encoding plakophilin-2 (ref. 9). The median age at presentation of ARVD/C is 26 years. We used previously published methods to generate iPSC lines from fibroblasts of two patients with ARVD/C and PKP2 mutations. Mutant PKP2 iPSC-CMs demonstrate abnormal plakoglobin nuclear translocation and decreased ß-catenin activity in cardiogenic conditions; yet, these abnormal features are insufficient to reproduce the pathological phenotypes of ARVD/C in standard cardiogenic conditions. Here we show that induction of adult-like metabolic energetics from an embryonic/glycolytic state and abnormal peroxisome proliferator-activated receptor gamma (PPAR-γ) activation underlie the pathogenesis of ARVD/C. By co-activating normal PPAR-alpha-dependent metabolism and abnormal PPAR-γ pathway in beating embryoid bodies (EBs) with defined media, we established an efficient ARVD/C in vitro model within 2 months. This model manifests exaggerated lipogenesis and apoptosis in mutant PKP2 iPSC-CMs. iPSC-CMs with a homozygous PKP2 mutation also had calcium-handling deficits. Our study is the first to demonstrate that induction of adult-like metabolism has a critical role in establishing an adult-onset disease model using patient-specific iPSCs. Using this model, we revealed crucial pathogenic insights that metabolic derangement in adult-like metabolic milieu underlies ARVD/C pathologies, enabling us to propose novel disease-modifying therapeutic strategies.

Laser-Based Propagation of Human iPS and ES Cells Generates Reproducible Cultures with Enhanced Differentiation Potential.

Proper maintenance of stem cells is essential for successful utilization of ESCs/iPSCs as tools in developmental and drug discovery studies and in regenerative medicine. Standardization is critical for all future applications of stem cells and necessary to fully understand their potential. This study reports a novel approach for the efficient, consistent expansion of human ESCs and iPSCs using laser sectioning, instead of mechanical devices or enzymes, to divide cultures into defined size clumps for propagation. Laser-mediated propagation maintained the pluripotency, quality, and genetic stability of ESCs/iPSCs and led to enhanced differentiation potential. This approach removes the variability associated with ESC/iPSC propagation, significantly reduces the expertise, labor, and time associated with manual passaging techniques and provides the basis for scalable delivery of standardized ESC/iPSC lines. Adoption of standardized protocols would allow researchers to understand the role of genetics, environment, and/or procedural effects on stem cells and would ensure reproducible production of stem cell cultures for use in clinical/therapeutic applications.

Wnt inhibition correlates with human embryonic stem cell cardiomyogenesis: a structure-activity relationship study based on inhibitors for the Wnt response.

Human embryonic stem cell-based high-content screening of 550 known signal transduction modulators showed that one "lead" (1, a recently described inhibitor of the proteolytic degradation of Axin) stimulated cardiomyogenesis. Because Axin controls canonical Wnt signaling, we conducted an investigation to determine whether the cardiogenic activity of 1 is Wnt-dependent, and we developed a structure-activity relationship to optimize the cardiogenic properties of 1. We prepared analogues with a range of potencies (low nanomolar to inactive) for Wnt/ß-catenin inhibition and for cardiogenic induction. Both functional activities correlated positively (r(2) = 0.72). The optimal compounds induced cardiogenesis 1.5-fold greater than 1 at 30-fold lower concentrations. In contrast, no correlation was observed for cardiogenesis and modulation of transforming growth factor ß (TGFß)/Smad signaling that prominently influences cardiogenesis. Taken together, these data show that Wnt signaling inhibition is essential for cardiogenic activity and that the pathway can be targeted for the design of druglike cardiogenic molecules.

Small-molecule inhibitors of the Wnt pathway potently promote cardiomyocytes from human embryonic stem cell-derived mesoderm.

RATIONALE: Human embryonic stem cells can form cardiomyocytes when cultured under differentiation conditions. Although the initiating step of mesoderm formation is well characterized, the subsequent steps that promote for cardiac lineages are poorly understood and limit the yield of cardiomyocytes. OBJECTIVE: Our aim was to develop a human embryonic stem cell-based high-content screening assay to discover small molecules that drive cardiogenic differentiation after mesoderm is established to improve our understanding of the biology involved. Screening of libraries of small-molecule pathway modulators was predicted to provide insight into the cellular proteins and signaling pathways that control stem cell cardiogenesis. METHODS AND RESULTS: Approximately 550 known pathway modulators were screened in a high-content screening assay, with hits being called out by the appearance of a red fluorescent protein driven by the promoter of the cardiac-specific MYH6 gene. One potent small molecule was identified that inhibits transduction of the canonical Wnt response within the cell, which demonstrated that Wnt inhibition alone was sufficient to generate cardiomyocytes from human embryonic stem cell-derived mesoderm cells. Transcriptional profiling of inhibitor-treated compared with vehicle-treated samples further indicated that inhibition of Wnt does not induce other mesoderm lineages. Notably, several other Wnt inhibitors were very efficient in inducing cardiogenesis, including a molecule that prevents Wnts from being secreted by the cell, which confirmed that Wnt inhibition was the relevant biological activity. CONCLUSIONS: Pharmacological inhibition of Wnt signaling is sufficient to drive human mesoderm cells to form cardiomyocytes; this could yield novel tools for the benefit of pharmaceutical and clinical applications.

Non-cardiomyocytes influence the electrophysiological maturation of human embryonic stem cell-derived cardiomyocytes during differentiation.

Various types of cardiomyocytes undergo changes in automaticity and electrical properties during fetal heart development. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs), like fetal cardiomyocytes, are electrophysiologically immature and exhibit automaticity. We used hESC-CMs to investigate developmental changes in mechanisms of automaticity and to determine whether electrophysiological maturation is driven by an intrinsic developmental clock and/or is regulated by interactions with non-cardiomyocytes in embryoid bodies (EBs). We isolated pure populations of hESC-CMs from EBs by lentivirus-engineered Puromycin resistance at various stages of differentiation. Using pharmacological agents, calcium (Ca(2+)) imaging, and intracellular recording techniques, we found that intracellular Ca(2+)-cycling mechanisms developed early and contributed to dominant automaticity throughout hESC-CM differentiation. Sarcolemmal ion channels evolved later upon further differentiation within EBs and played an increasing role in controlling automaticity and electrophysiological properties of hESC-CMs. In contrast to the development of intracellular Ca(2+)-handling proteins, ion channel development and electrophysiological maturation of hESC-CMs did not occur when hESC-CMs were isolated from EBs early and maintained in culture without further interaction with non-cardiomyocytes. Adding back non-cardiomyocytes to early-isolated hESC-CMs rescued the arrest of electrophysiological maturation, indicating that non-cardiomyocytes in EBs drive electrophysiological maturation of early hESC-CMs. Non-cardiomyocytes in EBs contain most cell types present in the embryonic heart that are known to influence early cardiac development. Our study is the first to demonstrate that non-cardiomyocytes influence electrophysiological maturation of early hESC-CMs in cultures. Defining the nature of these extrinsic signals will aid in the directed maturation of immature hESC-CMs to mitigate arrhythmogenic risks of cell-based therapies.

Lentiviral vectors and protocols for creation of stable hESC lines for fluorescent tracking and drug resistance selection of cardiomyocytes.

BACKGROUND: Developmental, physiological and tissue engineering studies critical to the development of successful myocardial regeneration therapies require new ways to effectively visualize and isolate large numbers of fluorescently labeled, functional cardiomyocytes. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe methods for the clonal expansion of engineered hESCs and make available a suite of lentiviral vectors for that combine Blasticidin, Neomycin and Puromycin resistance based drug selection of pure populations of stem cells and cardiomyocytes with ubiquitous or lineage-specific promoters that direct expression of fluorescent proteins to visualize and track cardiomyocytes and their progenitors. The phospho-glycerate kinase (PGK) promoter was used to ubiquitously direct expression of histone-2B fused eGFP and mCherry proteins to the nucleus to monitor DNA content and enable tracking of cell migration and lineage. Vectors with T/Brachyury and alpha-myosin heavy chain (alphaMHC) promoters targeted fluorescent or drug-resistance proteins to early mesoderm and cardiomyocytes. The drug selection protocol yielded 96% pure cardiomyocytes that could be cultured for over 4 months. Puromycin-selected cardiomyocytes exhibited a gene expression profile similar to that of adult human cardiomyocytes and generated force and action potentials consistent with normal fetal cardiomyocytes, documenting these parameters in hESC-derived cardiomyocytes and validating that the selected cells retained normal differentiation and function. CONCLUSION/SIGNIFICANCE: The protocols, vectors and gene expression data comprise tools to enhance cardiomyocyte production for large-scale applications.