Polygenic Risk Score Associates With Atherosclerotic Plaque Characteristics at Autopsy.

BACKGROUND: Polygenic risk scores (PRSs) for coronary artery disease (CAD) potentially improve cardiovascular risk prediction. However, their relationship with histopathologic features of CAD has never been examined systematically. METHODS: From 4327 subjects referred to CVPath by the State of Maryland Office Chief Medical Examiner for sudden death between 1994 and 2015, 2455 cases were randomly selected for genotyping. We generated PRS from 291 known CAD risk loci. Detailed histopathologic examination of the coronary arteries was performed in all subjects. The primary study outcome measurements were histopathologic plaque features determining severity of atherosclerosis, including %stenosis, calcification, thin-cap fibroatheromas, and thrombotic CAD. RESULTS: After exclusion of cases with insufficient DNA sample quality or with missing data, 954 cases (mean age, 48.8±14.7 years; 75.7% men) remained in the final study cohort. Subjects in the highest PRS quintile exhibited more severe atherosclerosis compared with subjects in the lowest quintile, with greater %stenosis (80.3%±27.0% versus 50.4%±38.7%; adjusted P<0.001) and a higher frequency of calcification (69.6% versus 35.8%; adjusted P=0.004) and thin-cap fibroatheroma (26.7% versus 9.5%; adjusted P=0.007). Even after adjustment for traditional CAD risk factors, subjects within the highest PRS quintile had higher odds of severe atherosclerosis (ie, ≥75% stenosis; adjusted odds ratio, 3.77 [95% CI, 2.10-6.78]; P<0.001) and plaque rupture (adjusted odds ratio, 4.05 [95% CI, 2.26-7.24]; P<0.001). Moreover, subjects within the highest quintile had higher odds of CAD-associated cause of death, especially among those aged ≤50 years (adjusted odds ratio, 4.08 [95% CI, 2.01-8.30]; P<0.001). No statistically significant associations were observed with plaque erosion after adjusting for covariates. CONCLUSIONS: This is the first autopsy study investigating associations between PRS and atherosclerosis severity at the histopathologic level in subjects with sudden death. Our pathological analysis suggests PRS correlates with plaque burden and features of advanced atherosclerosis and may be useful as a method for CAD risk stratification, especially in younger subjects.

Transcriptional Dysregulation Underlies Both Monogenic Arrhythmia Syndrome and Common Modifiers of Cardiac Repolarization.

BACKGROUND: Brugada syndrome (BrS) is an inherited arrhythmia syndrome caused by loss-of-function variants in the cardiac sodium channel gene SCN5A (sodium voltage-gated channel alpha subunit 5) in ≈20% of subjects. We identified a family with 4 individuals diagnosed with BrS harboring the rare G145R missense variant in the cardiac transcription factor TBX5 (T-box transcription factor 5) and no SCN5A variant. METHODS: We generated induced pluripotent stem cells (iPSCs) from 2 members of a family carrying TBX5-G145R and diagnosed with Brugada syndrome. After differentiation to iPSC-derived cardiomyocytes (iPSC-CMs), electrophysiologic characteristics were assessed by voltage- and current-clamp experiments (n=9 to 21 cells per group) and transcriptional differences by RNA sequencing (n=3 samples per group), and compared with iPSC-CMs in which G145R was corrected by CRISPR/Cas9 approaches. The role of platelet-derived growth factor (PDGF)/phosphoinositide 3-kinase (PI3K) pathway was elucidated by small molecule perturbation. The rate-corrected QT (QTc) interval association with serum PDGF was tested in the Framingham Heart Study cohort (n=1893 individuals). RESULTS: TBX5-G145R reduced transcriptional activity and caused multiple electrophysiologic abnormalities, including decreased peak and enhanced "late" cardiac sodium current (INa), which were entirely corrected by editing G145R to wild-type. Transcriptional profiling and functional assays in genome-unedited and -edited iPSC-CMs showed direct SCN5A down-regulation caused decreased peak INa, and that reduced PDGF receptor (PDGFRA [platelet-derived growth factor receptor α]) expression and blunted signal transduction to PI3K was implicated in enhanced late INa. Tbx5 regulation of the PDGF axis increased arrhythmia risk due to disruption of PDGF signaling and was conserved in murine model systems. PDGF receptor blockade markedly prolonged normal iPSC-CM action potentials and plasma levels of PDGF in the Framingham Heart Study were inversely correlated with the QTc interval (P<0.001). CONCLUSIONS: These results not only establish decreased SCN5A transcription by the TBX5 variant as a cause of BrS, but also reveal a new general transcriptional mechanism of arrhythmogenesis of enhanced late sodium current caused by reduced PDGF receptor-mediated PI3K signaling.

Impaired Reorganization of Centrosome Structure Underlies Human Infantile Dilated Cardiomyopathy.

BACKGROUND: During cardiomyocyte maturation, the centrosome, which functions as a microtubule organizing center in cardiomyocytes, undergoes dramatic structural reorganization where its components reorganize from being localized at the centriole to the nuclear envelope. This developmentally programmed process, referred to as centrosome reduction, has been previously associated with cell cycle exit. However, understanding of how this process influences cardiomyocyte cell biology, and whether its disruption results in human cardiac disease, remains unknown. We studied this phenomenon in an infant with a rare case of infantile dilated cardiomyopathy (iDCM) who presented with left ventricular ejection fraction of 18% and disrupted sarcomere and mitochondria structure. METHODS: We performed an analysis beginning with an infant who presented with a rare case of iDCM. We derived induced pluripotent stem cells from the patient to model iDCM in vitro. We performed whole exome sequencing on the patient and his parents for causal gene analysis. CRISPR/Cas9-mediated gene knockout and correction in vitro were used to confirm whole exome sequencing results. Zebrafish and Drosophila models were used for in vivo validation of the causal gene. Matrigel mattress technology and single-cell RNA sequencing were used to characterize iDCM cardiomyocytes further. RESULTS: Whole exome sequencing and CRISPR/Cas9 gene knockout/correction identified RTTN, the gene encoding the centrosomal protein RTTN (rotatin), as the causal gene underlying the patient's condition, representing the first time a centrosome defect has been implicated in a nonsyndromic dilated cardiomyopathy. Genetic knockdowns in zebrafish and Drosophila confirmed an evolutionarily conserved requirement of RTTN for cardiac structure and function. Single-cell RNA sequencing of iDCM cardiomyocytes showed impaired maturation of iDCM cardiomyocytes, which underlie the observed cardiomyocyte structural and functional deficits. We also observed persistent localization of the centrosome at the centriole, contrasting with expected programmed perinuclear reorganization, which led to subsequent global microtubule network defects. In addition, we identified a small molecule that restored centrosome reorganization and improved the structure and contractility of iDCM cardiomyocytes. CONCLUSIONS: This study is the first to demonstrate a case of human disease caused by a defect in centrosome reduction. We also uncovered a novel role for RTTN in perinatal cardiac development and identified a potential therapeutic strategy for centrosome-related iDCM. Future study aimed at identifying variants in centrosome components may uncover additional contributors to human cardiac disease.

Mutagenesis of human genomes by endogenous mobile elements on a population scale.

Several large-scale Illumina whole-genome sequencing (WGS) and whole-exome sequencing (WES) projects have emerged recently that have provided exceptional opportunities to discover mobile element insertions (MEIs) and study the impact of these MEIs on human genomes. However, these projects also have presented major challenges with respect to the scalability and computational costs associated with performing MEI discovery on tens or even hundreds of thousands of samples. To meet these challenges, we have developed a more efficient and scalable version of our mobile element locator tool (MELT) called CloudMELT. We then used MELT and CloudMELT to perform MEI discovery in 57,919 human genomes and exomes, leading to the discovery of 104,350 nonredundant MEIs. We leveraged this collection (1) to examine potentially active L1 source elements that drive the mobilization of new Alu, L1, and SVA MEIs in humans; (2) to examine the population distributions and subfamilies of these MEIs; and (3) to examine the mutagenesis of GENCODE genes, ENCODE-annotated features, and disease genes by these MEIs. Our study provides new insights on the L1 source elements that drive MEI mutagenesis and brings forth a better understanding of how this mutagenesis impacts human genomes.

The Casein kinase 1α agonist pyrvinium attenuates Wnt-mediated CK1α degradation via interaction with the E3 ubiquitin ligase component Cereblon.

The Cullin-RING ligase 4 E3 ubiquitin ligase component Cereblon (CRBN) is a well-established target for a class of small molecules termed immunomodulatory drugs (IMiDs). These drugs drive CRBN to modulate the degradation of a number of neosubstrates required for the growth of multiple cancers. Whereas the mechanism underlying the activation of CRBN by IMiDs is well described, the normal physiological regulation of CRBN is poorly understood. We recently showed that CRBN is activated following exposure to Wnt ligands and subsequently mediates the degradation of a subset of physiological substrates. Among the Wnt-dependent substrates of CRBN is Casein kinase 1α (CK1α), a known negative regulator of Wnt signaling. Wnt-mediated degradation of CK1α occurs via its association with CRBN at a known IMiD binding pocket. Herein, we demonstrate that a small-molecule CK1α agonist, pyrvinium, directly prevents the Wnt-dependent interaction of CRBN with CK1α, attenuating the consequent CK1α degradation. We further show that pyrvinium disrupts the ability of CRBN to interact with CK1α at the IMiD binding pocket within the CRBN-CK1α complex. Of note, this function of pyrvinium is independent of its previously reported ability to enhance CK1α kinase activity. Furthermore, we also demonstrate that pyrvinium attenuates CRBN-induced Wnt pathway activation in vivo. Collectively, these results reveal a novel dual mechanism through which pyrvinium inhibits Wnt signaling by both attenuating the CRBN-mediated destabilization of CK1α and activating CK1α kinase activity.

A Novel COVID-19 Severity Score is Associated With Survival in Patients Undergoing Percutaneous Dilational Tracheostomy.

INTRODUCTION: Tracheostomy in patients with COVID-19 is a controversial and difficult clinical decision. We hypothesized that a recently validated COVID-19 Severity Score (CSS) would be associated with survival in patients considered for tracheostomy. METHODS: We reviewed 77 mechanically ventilated COVID-19 patients evaluated for decision for percutaneous dilational tracheostomy (PDT) from March to June 2020 at a public tertiary care center. Decision for PDT was based on clinical judgment of the screening surgeons. The CSS was retrospectively calculated using mean biomarker values from admission to time of PDT consult. Our primary outcome was survival to discharge, and all patient charts were reviewed through August 31, 2021. ROC curve and Youden index were used to estimate an optimal cut-point for survival. RESULTS: The mean CSS for 42 survivors significantly differed from that of 35 nonsurvivors (CSS 52 versus 66, P = 0.003). The Youden index returned an optimal CSS of 55 (95% confidence interval 43-72), which was associated with a sensitivity of 0.8 and a specificity of 0.6. The median CSS was 40 (interquartile range 27, 49) in the lower CSS (<55) group and 72 (interquartile range 66, 93) in the high CSS (≥55 group). Eighty-seven percent of lower CSS patients underwent PDT, with 74% survival, whereas 61% of high CSS patients underwent PDT, with only 41% surviving. Patients with high CSS had 77% lower odds of survival (odds ratio = 0.2, 95% confidence interval 0.1-0.7). CONCLUSIONS: Higher CSS was associated with decreased survival in patients evaluated for PDT, with a score ≥55 predictive of mortality. The novel CSS may be a useful adjunct in determining which COVID-19 patients will benefit from tracheostomy. Further prospective validation of this tool is warranted.

Antibiotic Therapy for Culture-Proven Bacterial Overgrowth in Children With Intestinal Failure Results in Improved Symptoms and Growth.

OBJECTIVES: To evaluate symptoms, enteral tolerance, growth, and antibiotic regimens in pediatric intestinal failure (IF) patients after treated with antibiotic therapy for small bowel bacterial overgrowth (SBBO). METHODS: Single-center retrospective review of children 0-18 years with IF with endoscopic cultures demonstrating >10 5 CFU/mL from 2010 to 2017. Symptoms, enteral tolerance, growth, and antibiotic regimens were evaluated at the time of endoscopy and 6 months later. RESULTS: Of 505 patients followed in our intestinal rehabilitation program, 104 underwent upper gastrointestinal endoscopy and 78 had positive duodenal cultures. Clinical data pre- and post-endoscopy were available for 56 patients. Compared to baseline, in the 6 months following targeted antibiotic treatment, children showed significant improvement in emesis or feeding intolerance (58.9% vs 23.2%, P < 0.001), abdominal pain (16.1% vs 7.1%, P = 0.02), high stool output (42.9% vs 19.6%, P = 0.002), and gross GI bleeding (19.6% vs 3.6%, P = 0.003). Mean BMI-for-age z scores increased significantly (-0.03 ± 0.94 vs 0.27 ± 0.82, P = 0.03); however, height-for-age z scores, weight-for-age z scores, and percent of calories from enteral intake were not significantly different after therapy. Antibiotic regimens remained highly variable. CONCLUSIONS: Children with IF and culture-positive SBBO showed significant improvement in symptoms and BMI-for-age z scores after duodenal culture with subsequent targeted antibiotic therapy. Longer follow-up may be needed to detect improvements in linear growth and percent of calories from enteral feeds. Antibiotic regimens remain highly variable. Long-term consequences of chronic antimicrobial therapy, including antimicrobial resistance, remain unknown. Prospective studies focused on standardizing duodenal sampling technique, correlating culture and pathology data, and evaluating antibiotic resistance patterns are needed.

TET3 controls the expression of the H3K27me3 demethylase Kdm6b during neural commitment.

The acquisition of cell identity is associated with developmentally regulated changes in the cellular histone methylation signatures. For instance, commitment to neural differentiation relies on the tightly controlled gain or loss of H3K27me3, a hallmark of polycomb-mediated transcriptional gene silencing, at specific gene sets. The KDM6B demethylase, which removes H3K27me3 marks at defined promoters and enhancers, is a key factor in neurogenesis. Therefore, to better understand the epigenetic regulation of neural fate acquisition, it is important to determine how Kdm6b expression is regulated. Here, we investigated the molecular mechanisms involved in the induction of Kdm6b expression upon neural commitment of mouse embryonic stem cells. We found that the increase in Kdm6b expression is linked to a rearrangement between two 3D configurations defined by the promoter contact with two different regions in the Kdm6b locus. This is associated with changes in 5-hydroxymethylcytosine (5hmC) levels at these two regions, and requires a functional ten-eleven-translocation (TET) 3 protein. Altogether, our data support a model whereby Kdm6b induction upon neural commitment relies on an intronic enhancer the activity of which is defined by its TET3-mediated 5-hmC level. This original observation reveals an unexpected interplay between the 5-hmC and H3K27me3 pathways during neural lineage commitment in mammals. It also questions to which extent KDM6B-mediated changes in H3K27me3 level account for the TET-mediated effects on gene expression.

Use of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in Preclinical Cancer Drug Cardiotoxicity Testing: A Scientific Statement From the American Heart Association.

It is now well recognized that many lifesaving oncology drugs may adversely affect the heart and cardiovascular system, including causing irreversible cardiac injury that can result in reduced quality of life. These effects, which may manifest in the short term or long term, are mechanistically not well understood. Research is hampered by the reliance on whole-animal models of cardiotoxicity that may fail to reflect the fundamental biology or cardiotoxic responses of the human myocardium. The emergence of human induced pluripotent stem cell-derived cardiomyocytes as an in vitro research tool holds great promise for understanding drug-induced cardiotoxicity of oncological drugs that may manifest as contractile and electrophysiological dysfunction, as well as structural abnormalities, making it possible to deliver novel drugs free from cardiac liabilities and guide personalized therapy. This article briefly reviews the challenges of cardio-oncology, the strengths and limitations of using human induced pluripotent stem cell-derived cardiomyocytes to represent clinical findings in the nonclinical research space, and future directions for their further use.

Generic and Disease-specific Health-related Quality of Life in Pediatric Intestinal Failure.

OBJECTIVE: The aim of the study was to assess overall and disease-specific health-related quality of life (hrQOL) in patients with pediatric intestinal failure (PIF) and caregivers and elucidate differences from healthy and chronic gastrointestinal (GI) illness cohorts. METHODS: Cross-sectional study of patients with PIF and their caregivers managed at a multidisciplinary intestinal rehabilitation program using the PedsQL Generic Core and the Gastrointestinal Symptoms Module to assess generic and disease-specific hrQOL, respectively. These data were compared to established healthy and chronic GI disease controls. RESULTS: A total of 53 patients (mean age 6.2 ±â€Š3.9 years) and their caregivers were studied. Patients reported lower generic hrQOL than healthy children (73.0 vs 83.84, P < 0.001), but no difference from patients with chronic GI disease (73.0 vs 77.79). In contrast, PIF caregivers perceived similar generic hrQOL compared to a healthy cohort (78.9 vs 82.70), but higher when compared to the GI disease cohort (78.9 vs 72.74, P < 0.01). Patients with PIF and caregivers reported lower psychosocial health scores than healthy controls. Patients and caregivers reported similar disease-specific hrQOL to a cohort with chronic GI disease but significantly lower disease-specific hrQOL than a healthy cohort (P < 0.001 both groups). CONCLUSIONS: Patients with PIF and their caregivers have disparate perceptions of generic hrQOL when compared to healthy and chronic GI disease controls. Both patients and caregivers, however, had significantly lower scores in psychosocial health than healthy controls. In addition, disease-specific hrQOL was substantially lower than healthy controls for PIF patients and caregivers. Further investigation to expand on these findings and identify modifiable variables to improve the psychosocial health score and disease-specific factors would be of high value.

Real-time visualization of titin dynamics reveals extensive reversible photobleaching in human induced pluripotent stem cell-derived cardiomyocytes.

Fluorescence recovery after photobleaching (FRAP) has been useful in delineating cardiac myofilament biology, and innovations in fluorophore chemistry have expanded the array of microscopic assays used. However, one assumption in FRAP is the irreversible photobleaching of fluorescent proteins after laser excitation. Here we demonstrate reversible photobleaching regarding the photoconvertible fluorescent protein mEos3.2. We used CRISPR/Cas9 genome editing in human induced pluripotent stem cells (hiPSCs) to knock-in mEos3.2 into the COOH terminus of titin to visualize sarcomeric titin incorporation and turnover. Upon cardiac induction, the titin-mEos3.2 fusion protein is expressed and integrated in the sarcomeres of hiPSC-derived cardiomyocytes (CMs). STORM imaging shows M-band clustered regions of bound titin-mEos3.2 with few soluble titin-mEos3.2 molecules. FRAP revealed a baseline titin-mEos3.2 fluorescence recovery of 68% and half-life of ~1.2 h, suggesting a rapid exchange of sarcomeric titin with soluble titin. However, paraformaldehyde-fixed and permeabilized titin-mEos3.2 hiPSC-CMs surprisingly revealed a 55% fluorescence recovery. Whole cell FRAP analysis in paraformaldehyde-fixed, cycloheximide-treated, and untreated titin-mEos3.2 hiPSC-CMs displayed no significant differences in fluorescence recovery. FRAP in fixed HEK 293T expressing cytosolic mEos3.2 demonstrates a 58% fluorescence recovery. These data suggest that titin-mEos3.2 is subject to reversible photobleaching following FRAP. Using a mouse titin-eGFP model, we demonstrate that no reversible photobleaching occurs. Our results reveal that reversible photobleaching accounts for the majority of titin recovery in the titin-mEos3.2 hiPSC-CM model and should warrant as a caution in the extrapolation of reliable FRAP data from specific fluorescent proteins in long-term cell imaging.

Discovery, synthesis and characterization of a series of 7-aryl-imidazo[1,2-a]pyridine-3-ylquinolines as activin-like kinase (ALK) inhibitors.

The activin-like kinases are a family of kinases that play important roles in a variety of disease states. Of this class of kinases, ALK2, has been shown by a gain-of-function to be the primary driver of the childhood skeletal disease fibrodysplasia ossificans progressiva (FOP) and more recently the pediatric cancer diffuse intrinsic pontine glioma (DIPG). Herein, we report our efforts to identify a novel imidazo[1,2-a]pyridine scaffold as potent inhibitors of ALK2 with good in vivo pharmacokinetic properties suitable for future animal studies.

Methods for the reliable induction of heterotopic ossification in the conditional Alk2Q207D mouse.

OBJECTIVES: Conditional Alk2Q207D-floxed (caALK2fl) mice have previously been used as a model of heterotopic ossification (HO). However, HO formation in this model can be highly variable, and it is unclear which methods reliably induce HO. Hence, these studies report validated methods for reproducibly inducing HO in caALK2fl mice. METHODS: Varying doses of Adex-cre and cardiotoxin (CTX) were injected into the calf muscles of 9, 14, or 28-day-old caALK2fl/- or caALK2fl/fl mice. HO was measured by planar radiography or microCT at 14-28 days post-injury. RESULTS: In 9-day-old caALK2fl/- or caALK2fl/fl mice, single injections of 109 PFU Adex-cre and 0.3 µg of CTX were sufficient to induce extensive HO within 14 days post-injury. In 28-day-old mice, the doses were increased to 5 x 109 PFU Adex-cre and 3.0 µg of CTX to achieve similar consistency, but at a slower rate versus younger mice. Using a crush injury, instead of CTX, also provided consistent induction of HO. Finally, the Type 1 BMPR inhibitor, DMH1, significantly reduced HO formation in 28-day-old caALK2fl/fl mice. CONCLUSIONS: These data illustrate multiple methods for reliable induction of localized HO in the caALK2flmouse that can serve as a starting point for new laboratories utilizing this model.

BMP signaling and cellular dynamics during regeneration of airway epithelium from basal progenitors.

The pseudostratified epithelium of the lung contains ciliated and secretory luminal cells and basal stem/progenitor cells. To identify signals controlling basal cell behavior we screened factors that alter their self-renewal and differentiation in a clonal organoid (tracheosphere) assay. This revealed that inhibitors of the canonical BMP signaling pathway promote proliferation but do not affect lineage choice, whereas exogenous Bmp4 inhibits proliferation and differentiation. We therefore followed changes in BMP pathway components in vivo in the mouse trachea during epithelial regeneration from basal cells after injury. The findings suggest that BMP signaling normally constrains proliferation at steady state and this brake is released transiently during repair by the upregulation of endogenous BMP antagonists. Early in repair, the packing of epithelial cells along the basal lamina increases, but density is later restored by active extrusion of apoptotic cells. Systemic administration of the BMP antagonist LDN-193189 during repair initially increases epithelial cell number but, following the shedding phase, normal density is restored. Taken together, these results reveal crucial roles for both BMP signaling and cell shedding in homeostasis of the respiratory epithelium.

Investigating pediatric disorders with induced pluripotent stem cells.

The study of disease pathophysiology has long relied on model systems, including animal models and cultured cells. In 2006, Shinya Yamanaka achieved a breakthrough by reprogramming somatic cells into induced pluripotent stem cells (iPSCs). This revolutionary discovery provided new opportunities for disease modeling and therapeutic intervention. With established protocols, investigators can generate iPSC lines from patient blood, urine, and tissue samples. These iPSCs retain ability to differentiate into every human cell type. Advances in differentiation and organogenesis move cellular in vitro modeling to a multicellular model capable of recapitulating physiology and disease. Here, we discuss limitations of traditional animal and tissue culture models, as well as the application of iPSC models. We highlight various techniques, including reprogramming strategies, directed differentiation, tissue engineering, organoid developments, and genome editing. We extensively summarize current established iPSC disease models that utilize these techniques. Confluence of these technologies will advance our understanding of pediatric diseases and help usher in new personalized therapies for patients.

Genome Editing and Induced Pluripotent Stem Cell Technologies for Personalized Study of Cardiovascular Diseases.

PURPOSE OF REVIEW: The goal of this review is to highlight the potential of induced pluripotent stem cell (iPSC)-based modeling as a tool for studying human cardiovascular diseases. We present some of the current cardiovascular disease models utilizing genome editing and patient-derived iPSCs. RECENT FINDINGS: The incorporation of genome-editing and iPSC technologies provides an innovative research platform, providing novel insight into human cardiovascular disease at molecular, cellular, and functional level. In addition, genome editing in diseased iPSC lines holds potential for personalized regenerative therapies. The study of human cardiovascular disease has been revolutionized by cellular reprogramming and genome editing discoveries. These exceptional technologies provide an opportunity to generate human cell cardiovascular disease models and enable therapeutic strategy development in a dish. We anticipate these technologies to improve our understanding of cardiovascular disease pathophysiology leading to optimal treatment for heart diseases in the future.

Matrigel Mattress: A Method for the Generation of Single Contracting Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

RATIONALE: The lack of measurable single-cell contractility of human-induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) currently limits the utility of hiPSC-CMs for evaluating contractile performance for both basic research and drug discovery. OBJECTIVE: To develop a culture method that rapidly generates contracting single hiPSC-CMs and allows quantification of cell shortening with standard equipment used for studying adult CMs. METHODS AND RESULTS: Single hiPSC-CMs were cultured for 5 to 7 days on a 0.4- to 0.8-mm thick mattress of undiluted Matrigel (mattress hiPSC-CMs) and compared with hiPSC-CMs maintained on a control substrate (<0.1-mm thick 1:60 diluted Matrigel, control hiPSC-CMs). Compared with control hiPSC-CMs, mattress hiPSC-CMs had more rod-shape morphology and significantly increased sarcomere length. Contractile parameters of mattress hiPSC-CMs measured with video-based edge detection were comparable with those of freshly isolated adult rabbit ventricular CMs. Morphological and contractile properties of mattress hiPSC-CMs were consistent across cryopreserved hiPSC-CMs generated independently at another institution. Unlike control hiPSC-CMs, mattress hiPSC-CMs display robust contractile responses to positive inotropic agents, such as myofilament calcium sensitizers. Mattress hiPSC-CMs exhibit molecular changes that include increased expression of the maturation marker cardiac troponin I and significantly increased action potential upstroke velocity because of a 2-fold increase in sodium current (INa). CONCLUSIONS: The Matrigel mattress method enables the rapid generation of robustly contracting hiPSC-CMs and enhances maturation. This new method allows quantification of contractile performance at the single-cell level, which should be valuable to disease modeling, drug discovery, and preclinical cardiotoxicity testing.