A novel method for the percutaneous induction of myocardial infarction by occlusion of small coronary arteries in the rabbit.

Arrhythmic sudden cardiac death (SCD) is an important cause of mortality following myocardial infarction (MI). The rabbit has similar cardiac electrophysiology to humans and is therefore an important small animal model to study post-MI arrhythmias. The established approach of surgical coronary ligation results in thoracic adhesions that impede epicardial electrophysiological studies. Adhesions are absent following a percutaneously induced MI, which is also associated with reduced surgical morbidity and so represents a clear refinement of the approach. Percutaneous procedures have previously been described in large rabbits (3.5-5.5 kg). Here, we describe a novel method of percutaneous MI induction in smaller rabbits (2.5-3.5 kg) that are readily available commercially. New Zealand White rabbits (n = 51 males, 3.1 ± 0.3 kg) were anesthetized using isoflurane (1.5-3%) and underwent either a percutaneous MI procedure involving microcatheter tip deployment (≤1.5 Fr, 5 mm), coronary ligation surgery, or a sham procedure. Electrocardiography (ECG) recordings were used to confirm ST-segment elevation indicating coronary occlusion. Blood samples (1 and 24 h) were taken for cardiac troponin I (cTnI) levels. Ejection fraction (EF) was measured at 6-8 wk. Rabbits were then euthanized (Euthatal) and hearts were processed for magnetic resonance imaging and histology. Mortality rates were similar in both groups. Scar volume, cTnI, and EF were similar between both MI groups and significantly different from their respective sham controls. Thus, percutaneous coronary occlusion by microcatheter tip deployment is feasible in rabbits (2.5-3.5 kg) and produces an MI with similar characteristics to surgical ligation with lower procedural trauma and without epicardial adhesions.NEW & NOTEWORTHY Surgical coronary ligation is the standard technique to induce myocardial infarction (MI) in rabbits but is associated with procedural trauma and the generation of thoracic adhesions. Percutaneous coronary occlusion avoids these shortcomings and is established in pigs but has only been applicable to large rabbits because of a mismatch between the equipment used and target vessel size. Here, we describe a new scalable approach to percutaneous MI induction that is safe and effective in 2.5-3.5-kg rabbits.

Unlocking Personalized Biomedicine and Drug Discovery with Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Fit for Purpose or Forever Elusive?

In recent decades, drug development costs have increased by approximately a hundredfold, and yet about 1 in 7 licensed drugs are withdrawn from the market, often due to cardiotoxicity. This review considers whether technologies using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could complement existing assays to improve discovery and safety while reducing socioeconomic costs and assisting with regulatory guidelines on cardiac safety assessments. We draw on lessons from our own work to suggest a panel of 12 drugs that will be useful in testing the suitability of hiPSC-CM platforms to evaluate contractility. We review issues, including maturity versus complexity, consistency, quality, and cost, while considering a potential need to incorporate auxiliary approaches to compensate for limitations in hiPSC-CM technology. We give examples on how coupling hiPSC-CM technologies with Cas9/CRISPR genome engineering is starting to be used to personalize diagnosis, stratify risk, provide mechanistic insights, and identify new pathogenic variants for cardiovascular disease.

Relationship Between Anxiety, Depression, and Susceptibility to Severe Acute Respiratory Syndrome Coronavirus 2 Infection: Proof of Concept.

BACKGROUND: Psychological factors can influence susceptibility to viral infections. We examined whether such influences are evident in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: Participants (n = 102) completed measures of anxiety, depression, positive mood, and loneliness and provided a blood sample for the measurement of antibodies to the SARS-CoV-2 spike and nucleocapsid proteins. RESULTS: SARS-CoV-2 was significantly negatively associated with anxiety and depression. The model remained significant after adjustment for age and gender, although anxiety and depression were no longer significant independent predictors. CONCLUSIONS: These findings offer early support for the hypothesis that psychological factors may influence susceptibility to SARS-CoV-2 infection.

The use of fluorescence correlation spectroscopy to monitor cell surface ß2-adrenoceptors at low expression levels in human embryonic stem cell-derived cardiomyocytes and fibroblasts.

The importance of cell phenotype in determining the molecular mechanisms underlying ß2 -adrenoceptor (ß2AR) function has been noted previously when comparing responses in primary cells and recombinant model cell lines. Here, we have generated haplotype-specific SNAP-tagged ß2AR human embryonic stem (ES) cell lines and applied fluorescence correlation spectroscopy (FCS) to study cell surface receptors in progenitor cells and in differentiated fibroblasts and cardiomyocytes. FCS was able to quantify SNAP-tagged ß2AR number and diffusion in both ES-derived cardiomyocytes and CRISPR/Cas9 genome-edited HEK293T cells, where the expression level was too low to detect using standard confocal microscopy. These studies demonstrate the power of FCS in investigating cell surface ß2ARs at the very low expression levels often seen in endogenously expressing cells. Furthermore, the use of ES cell technology in combination with FCS allowed us to demonstrate that cell surface ß2ARs internalize in response to formoterol-stimulation in ES progenitor cells but not following their differentiation into ES-derived fibroblasts. This indicates that the process of agonist-induced receptor internalization is strongly influenced by cell phenotype and this may have important implications for drug treatment with long-acting ß2AR agonists.

Isogenic models of hypertrophic cardiomyopathy unveil differential phenotypes and mechanism-driven therapeutics.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a prevalent and complex cardiovascular condition. Despite being strongly associated with genetic alterations, wide variation of disease penetrance, expressivity and hallmarks of progression complicate treatment. We aimed to characterize different human isogenic cellular models of HCM bearing patient-relevant mutations to clarify genetic causation and disease mechanisms, hence facilitating the development of effective therapeutics. METHODS: We directly compared the p.ß-MHC-R453C and p.ACTC1-E99K HCM-associated mutations in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and their healthy isogenic counterparts, generated using CRISPR/Cas9 genome editing technology. By harnessing several state-of-the-art HCM phenotyping techniques, these mutations were investigated to identify similarities and differences in disease progression and hypertrophic signaling pathways, towards establishing potential targets for pharmacological treatment. CRISPR/Cas9 knock-in of the genetically-encoded calcium indicator R-GECO1.0 to the AAVS1 locus into these disease models resulted in calcium reporter lines. RESULTS: Confocal line scan analysis identified calcium transient arrhythmias and intracellular calcium overload in both models. The use of optogenetics and 2D/3D contractility assays revealed opposing phenotypes in the two mutations. Gene expression analysis highlighted upregulation of CALM1, CASQ2 and CAMK2D, and downregulation of IRF8 in p.ß-MHC-R453C mutants, whereas the opposite changes were detected in p.ACTC1-E99K mutants. Contrasting profiles of nuclear translocation of NFATc1 and MEF2 between the two HCM models suggest differential hypertrophic signaling pathway activation. Calcium transient abnormalities were rescued with combination of dantrolene and ranolazine, whilst mavacamten reduced the hyper-contractile phenotype of p.ACTC1-E99K hiPSC-CMs. CONCLUSIONS: Our data show that hypercontractility and molecular signaling within HCM are not uniform between different gene mutations, suggesting that a 'one-size fits all' treatment underestimates the complexity of the disease. Understanding where the similarities (arrhythmogenesis, bioenergetics) and differences (contractility, molecular profile) lie will allow development of therapeutics that are directed towards common mechanisms or tailored to each disease variant, hence providing effective patient-specific therapy.

MUSCLEMOTION: A Versatile Open Software Tool to Quantify Cardiomyocyte and Cardiac Muscle Contraction In Vitro and In Vivo.

RATIONALE: There are several methods to measure cardiomyocyte and muscle contraction, but these require customized hardware, expensive apparatus, and advanced informatics or can only be used in single experimental models. Consequently, data and techniques have been difficult to reproduce across models and laboratories, analysis is time consuming, and only specialist researchers can quantify data. OBJECTIVE: Here, we describe and validate an automated, open-source software tool (MUSCLEMOTION) adaptable for use with standard laboratory and clinical imaging equipment that enables quantitative analysis of normal cardiac contraction, disease phenotypes, and pharmacological responses. METHODS AND RESULTS: MUSCLEMOTION allowed rapid and easy measurement of movement from high-speed movies in (1) 1-dimensional in vitro models, such as isolated adult and human pluripotent stem cell-derived cardiomyocytes; (2) 2-dimensional in vitro models, such as beating cardiomyocyte monolayers or small clusters of human pluripotent stem cell-derived cardiomyocytes; (3) 3-dimensional multicellular in vitro or in vivo contractile tissues, such as cardiac "organoids," engineered heart tissues, and zebrafish and human hearts. MUSCLEMOTION was effective under different recording conditions (bright-field microscopy with simultaneous patch-clamp recording, phase contrast microscopy, and traction force microscopy). Outcomes were virtually identical to the current gold standards for contraction measurement, such as optical flow, post deflection, edge-detection systems, or manual analyses. Finally, we used the algorithm to quantify contraction in in vitro and in vivo arrhythmia models and to measure pharmacological responses. CONCLUSIONS: Using a single open-source method for processing video recordings, we obtained reliable pharmacological data and measures of cardiac disease phenotype in experimental cell, animal, and human models.

Surface plasmon resonance imaging of excitable cells.

Surface plasmons (SPs) are surface charge density oscillations occuring at a metal/dieletric interface and are highly sensitive to refractive index variations adjacent to the surface. This sensitivity has been exploited successfully for chemical and biological assays. In these systems, a surface plasmon resonance (SPR)-based sensor detects temporal variations in the refractive index at a point. SPR has also been used in imaging systems where the spatial variations of refractive index in the sample provide the contrast mechanism. SPR imaging systems using high numerical aperture (NA) objective lenses have been designed to image adherent live cells with high magnification and near-diffraction limited spatial resolution. Addressing research questions in cell physiology and pharmacology often requires the development of a multimodal microscope where complementary information can be obtained. In this paper, we present the development of a multimodal microscope that combines SPR imaging with a number of additional imaging modalities including bright-field, epifluorescence, total internal reflection microscopy and SPR fluorescence microscopy. We used a high NA objective lens for SPR and TIR microscopy and the platform has been used to image live cell cultures demonstrating both fluorescent and label-free techniques. Both the SPR and TIR imaging systems feature a wide field of view (~300 µm) that allows measurements from multiple cells whilst maintaining a resolution sufficient to image fine cellular processes. The capability of the platform to perform label-free functional imaging of living cells was demonstrated by imaging the spatial variations in contractions from stem cell-derived cardiomyocytes. This technique shows promise for non-invasive imaging of cultured cells over very long periods of time during development.

Highly efficient delivery of functional cargoes by the synergistic effect of GAG binding motifs and cell-penetrating peptides.

Protein transduction domains (PTDs) are powerful nongenetic tools that allow intracellular delivery of conjugated cargoes to modify cell behavior. Their use in biomedicine has been hampered by inefficient delivery to nuclear and cytoplasmic targets. Here we overcame this deficiency by developing a series of novel fusion proteins that couple a membrane-docking peptide to heparan sulfate glycosaminoglycans (GAGs) with a PTD. We showed that this GET (GAG-binding enhanced transduction) system could deliver enzymes (Cre, neomycin phosphotransferase), transcription factors (NANOG, MYOD), antibodies, native proteins (cytochrome C), magnetic nanoparticles (MNPs), and nucleic acids [plasmid (p)DNA, modified (mod)RNA, and small inhibitory RNA] at efficiencies of up to two orders of magnitude higher than previously reported in cell types considered hard to transduce, such as mouse embryonic stem cells (mESCs), human ESCs (hESCs), and induced pluripotent stem cells (hiPSCs). This technology represents an efficient strategy for controlling cell labeling and directing cell fate or behavior that has broad applicability for basic research, disease modeling, and clinical application.

CRISPR/Cas9 editing in human pluripotent stem cell-cardiomyocytes highlights arrhythmias, hypocontractility, and energy depletion as potential therapeutic targets for hypertrophic cardiomyopathy.

Aims: Sarcomeric gene mutations frequently underlie hypertrophic cardiomyopathy (HCM), a prevalent and complex condition leading to left ventricle thickening and heart dysfunction. We evaluated isogenic genome-edited human pluripotent stem cell-cardiomyocytes (hPSC-CM) for their validity to model, and add clarity to, HCM. Methods and results: CRISPR/Cas9 editing produced 11 variants of the HCM-causing mutation c.C9123T-MYH7 [(p.R453C-ß-myosin heavy chain (MHC)] in 3 independent hPSC lines. Isogenic sets were differentiated to hPSC-CMs for high-throughput, non-subjective molecular and functional assessment using 12 approaches in 2D monolayers and/or 3D engineered heart tissues. Although immature, edited hPSC-CMs exhibited the main hallmarks of HCM (hypertrophy, multi-nucleation, hypertrophic marker expression, sarcomeric disarray). Functional evaluation supported the energy depletion model due to higher metabolic respiration activity, accompanied by abnormalities in calcium handling, arrhythmias, and contraction force. Partial phenotypic rescue was achieved with ranolazine but not omecamtiv mecarbil, while RNAseq highlighted potentially novel molecular targets. Conclusion: Our holistic and comprehensive approach showed that energy depletion affected core cardiomyocyte functionality. The engineered R453C-ßMHC-mutation triggered compensatory responses in hPSC-CMs, causing increased ATP production and αMHC to energy-efficient ßMHC switching. We showed that pharmacological rescue of arrhythmias was possible, while MHY7: MYH6 and mutant: wild-type MYH7 ratios may be diagnostic, and previously undescribed lncRNAs and gene modifiers are suggestive of new mechanisms.

Stem cell-derived models to improve mechanistic understanding and prediction of human drug-induced liver injury.

Current preclinical drug testing does not predict some forms of adverse drug reactions in humans. Efforts at improving predictability of drug-induced tissue injury in humans include using stem cell technology to generate human cells for screening for adverse effects of drugs in humans. The advent of induced pluripotent stem cells means that it may ultimately be possible to develop personalized toxicology to determine interindividual susceptibility to adverse drug reactions. However, the complexity of idiosyncratic drug-induced liver injury means that no current single-cell model, whether of primary liver tissue origin, from liver cell lines, or derived from stem cells, adequately emulates what is believed to occur during human drug-induced liver injury. Nevertheless, a single-cell model of a human hepatocyte which emulates key features of a hepatocyte is likely to be valuable in assessing potential chemical risk; furthermore, understanding how to generate a relevant hepatocyte will also be critical to efforts to build complex multicellular models of the liver. Currently, hepatocyte-like cells differentiated from stem cells still fall short of recapitulating the full mature hepatocellular phenotype. Therefore, we convened a number of experts from the areas of preclinical and clinical hepatotoxicity and safety assessment, from industry, academia, and regulatory bodies, to specifically explore the application of stem cells in hepatotoxicity safety assessment and to make recommendations for the way forward. In this short review, we particularly discuss the importance of benchmarking stem cell-derived hepatocyte-like cells to their terminally differentiated human counterparts using defined phenotyping, to make sure the cells are relevant and comparable between labs, and outline why this process is essential before the cells are introduced into chemical safety assessment. (Hepatology 2017;65:710-721).

Cardiomyocytes from human pluripotent stem cells: From laboratory curiosity to industrial biomedical platform.

Cardiomyocytes from human pluripotent stem cells (hPSCs-CMs) could revolutionise biomedicine. Global burden of heart failure will soon reach USD $90bn, while unexpected cardiotoxicity underlies 28% of drug withdrawals. Advances in hPSC isolation, Cas9/CRISPR genome engineering and hPSC-CM differentiation have improved patient care, progressed drugs to clinic and opened a new era in safety pharmacology. Nevertheless, predictive cardiotoxicity using hPSC-CMs contrasts from failure to almost total success. Since this likely relates to cell immaturity, efforts are underway to use biochemical and biophysical cues to improve many of the ~30 structural and functional properties of hPSC-CMs towards those seen in adult CMs. Other developments needed for widespread hPSC-CM utility include subtype specification, cost reduction of large scale differentiation and elimination of the phenotyping bottleneck. This review will consider these factors in the evolution of hPSC-CM technologies, as well as their integration into high content industrial platforms that assess structure, mitochondrial function, electrophysiology, calcium transients and contractility. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

Combined hydrogels that switch human pluripotent stem cells from self-renewal to differentiation.

The ability of materials to define the architecture and microenvironment experienced by cells provides new opportunities to direct the fate of human pluripotent stem cells (HPSCs) [Robinton DA, Daley GQ (2012) Nature 481(7381):295-305]. However, the conditions required for self-renewal vs. differentiation of HPSCs are different, and a single system that efficiently achieves both outcomes is not available [Giobbe GG, et al. (2012) Biotechnol Bioeng 109(12):3119-3132]. We have addressed this dual need by developing a hydrogel-based material that uses ionic de-cross-linking to remove a self-renewal permissive hydrogel (alginate) and switch to a differentiation-permissive microenvironment (collagen). Adjusting the timing of this switch can preferentially steer the HPSC differentiation to mimic lineage commitment during gastrulation to ectoderm (early switch) or mesoderm/endoderm (late switch). As an exemplar differentiated cell type, we showed that directing early lineage specification using this single system can promote cardiogenesis with increased gene expression in high-density cell populations. This work will facilitate regenerative medicine by allowing in situ HPSC expansion to be coupled with early lineage specification within defined tissue geometries.

Materials for stem cell factories of the future.

Polymeric substrates are being identified that could permit translation of human pluripotent stem cells from laboratory-based research to industrial-scale biomedicine. Well-defined materials are required to allow cell banking and to provide the raw material for reproducible differentiation into lineages for large-scale drug-screening programs and clinical use. Yet more than 1 billion cells for each patient are needed to replace losses during heart attack, multiple sclerosis and diabetes. Producing this number of cells is challenging, and a rethink of the current predominant cell-derived substrates is needed to provide technology that can be scaled to meet the needs of millions of patients a year. In this Review, we consider the role of materials discovery, an emerging area of materials chemistry that is in large part driven by the challenges posed by biologists to materials scientists.

Current status of drug screening and disease modelling in human pluripotent stem cells.

The emphasis in human pluripotent stem cell (hPSC) technologies has shifted from cell therapy to in vitro disease modelling and drug screening. This review examines why this shift has occurred, and how current technological limitations might be overcome to fully realise the potential of hPSCs. Details are provided for all disease-specific human induced pluripotent stem cell lines spanning a dozen dysfunctional organ systems. Phenotype and pharmacology have been examined in only 17 of 63 lines, primarily those that model neurological and cardiac conditions. Drug screening is most advanced in hPSC-cardiomyocytes. Responses for almost 60 agents include examples of how careful tests in hPSC-cardiomyocytes have improved on existing in vitro assays, and how these cells have been integrated into high throughput imaging and electrophysiology industrial platforms. Such successes will provide an incentive to overcome bottlenecks in hPSC technology such as improving cell maturity and industrial scalability whilst reducing cost.

Allele-specific RNA interference rescues the long-QT syndrome phenotype in human-induced pluripotency stem cell cardiomyocytes.

AIMS: Long-QT syndromes (LQTS) are mostly autosomal-dominant congenital disorders associated with a 1:1000 mutation frequency, cardiac arrest, and sudden death. We sought to use cardiomyocytes derived from human-induced pluripotency stem cells (hiPSCs) as an in vitro model to develop and evaluate gene-based therapeutics for the treatment of LQTS. METHODS AND RESULTS: We produced LQTS-type 2 (LQT2) hiPSC cardiomyocytes carrying a KCNH2 c.G1681A mutation in a IKr ion-channel pore, which caused impaired glycosylation and channel transport to cell surface. Allele-specific RNA interference (RNAi) directed towards the mutated KCNH2 mRNA caused knockdown, while leaving the wild-type mRNA unaffected. Electrophysiological analysis of patient-derived LQT2 hiPSC cardiomyocytes treated with mutation-specific siRNAs showed normalized action potential durations (APDs) and K(+) currents with the concurrent rescue of spontaneous and drug-induced arrhythmias (presented as early-afterdepolarizations). CONCLUSIONS: These findings provide in vitro evidence that allele-specific RNAi can rescue diseased phenotype in LQTS cardiomyocytes. This is a potentially novel route for the treatment of many autosomal-dominant-negative disorders, including those of the heart.

Non-invasive label-free monitoring the cardiac differentiation of human embryonic stem cells in-vitro by Raman spectroscopy.

BACKGROUND: Online label-free monitoring of in-vitro differentiation of stem cells remains a major challenge in stem cell research. In this paper we report the use of Raman micro-spectroscopy (RMS) to measure time- and spatially-resolved molecular changes in intact embryoid bodies (EBs) during in-vitro cardiogenic differentiation. METHODS: EBs formed by aggregation of human embryonic stem cells (hESCs) were cultured in defined medium to induce differentiation towards cardiac phenotype and maintained in purpose-built micro-bioreactors on the Raman microscope for 5days (between days 5 and 9 of differentiation) and spatially-resolved spectra were recorded at 24h intervals. RESULTS: The Raman spectra showed that the onset of spontaneous beating of EBs at day 7 coincided with an increase in the intensity of the Raman bands at 1340cm(-1), 1083cm(-1), 937cm(-1), 858cm(-1), 577cm(-1) and 482cm(-1). The spectral maps corresponding to these bands had a high positive correlation with the expression of the cardiac-specific α-actinin obtained by immuno-fluorescence imaging of the same EBs. The spectral markers obtained here are also in agreement with previous studies performed on individual live hESC-derived CMs. CONCLUSIONS: The intensity profile of these Raman bands can be used for label-free in-situ monitoring of EBs to estimate the efficacy of cardiogenic differentiation. GENERAL SIGNIFICANCE: As the acquisition of the time-course Raman spectra did not affect the viability or the differentiation potential of the hESCs, this study demonstrates the feasibility of using RMS for on-line non-invasive continuous monitoring of such processes inside bioreactor culture systems.

A secreted proteomic footprint for stem cell pluripotency.

With a view to developing a much-needed non-invasive method for monitoring the healthy pluripotent state of human stem cells in culture, we undertook proteomic analysis of the waste medium from cultured embryonic (Man-13) and induced (Rebl.PAT) human pluripotent stem cells (hPSCs). Cells were grown in E8 medium to maintain pluripotency, and then transferred to FGF2 and TGFß deficient E6 media for 48 hours to replicate an early, undirected dissolution of pluripotency. We identified a distinct proteomic footprint associated with early loss of pluripotency in both hPSC lines, and a strong correlation with changes in the transcriptome. We demonstrate that multiplexing of four E8- against four E6- enriched secretome biomarkers provides a robust, diagnostic metric for the pluripotent state. These biomarkers were further confirmed by Western blotting which demonstrated consistent correlation with the pluripotent state across cell lines, and in response to a recovery assay.

SnoRNAs in cardiovascular development, function, and disease.

Small nucleolar RNAs (snoRNAs) are emerging as important regulators of cardiovascular (patho)biology. Several roles of snoRNAs have recently been identified in heart development and congenital heart diseases, as well as their dynamic regulation in hypertrophic and dilated cardiomyopathies, coronary heart disease (CHD), myocardial infarction (MI), cardiac fibrosis, and heart failure. Furthermore, reports of changes in vesicular snoRNA expression and altered levels of circulating snoRNAs in response to cardiac stress suggest that snoRNAs also function in cardiac signaling and intercellular communication. In this review, we summarize and discuss key findings and outline the clinical potential of snoRNAs considering current challenges and gaps in the field of cardiovascular diseases (CVDs).

Strengthening cardiac therapy pipelines using human pluripotent stem cell-derived cardiomyocytes.

Advances in hiPSC isolation and reprogramming and hPSC-CM differentiation have prompted their therapeutic application and utilization for evaluating potential cardiovascular safety liabilities. In this perspective, we showcase key efforts toward the large-scale production of hiPSC-CMs, implementation of hiPSC-CMs in industry settings, and recent clinical applications of this technology. The key observations are a need for traceable gender and ethnically diverse hiPSC lines, approaches to reduce cost of scale-up, accessible clinical trial datasets, and transparent guidelines surrounding the safety and efficacy of hiPSC-based therapies.

Perlecan (HSPG2) promotes structural, contractile, and metabolic development of human cardiomyocytes.

Perlecan (HSPG2), a heparan sulfate proteoglycan similar to agrin, is key for extracellular matrix (ECM) maturation and stabilization. Although crucial for cardiac development, its role remains elusive. We show that perlecan expression increases as cardiomyocytes mature in vivo and during human pluripotent stem cell differentiation to cardiomyocytes (hPSC-CMs). Perlecan-haploinsuffient hPSCs (HSPG2+/-) differentiate efficiently, but late-stage CMs have structural, contractile, metabolic, and ECM gene dysregulation. In keeping with this, late-stage HSPG2+/- hPSC-CMs have immature features, including reduced ⍺-actinin expression and increased glycolytic metabolism and proliferation. Moreover, perlecan-haploinsuffient engineered heart tissues have reduced tissue thickness and force generation. Conversely, hPSC-CMs grown on a perlecan-peptide substrate are enlarged and display increased nucleation, typical of hypertrophic growth. Together, perlecan appears to play the opposite role of agrin, promoting cellular maturation rather than hyperplasia and proliferation. Perlecan signaling is likely mediated via its binding to the dystroglycan complex. Targeting perlecan-dependent signaling may help reverse the phenotypic switch common to heart failure.