The new era of cardiovascular research: revolutionizing cardiovascular research with 3D models in a dish.

Cardiovascular research has heavily relied on studies using patient samples and animal models. However, patient studies often miss the data from the crucial early stage of cardiovascular diseases, as obtaining primary tissues at this stage is impracticable. Transgenic animal models can offer some insights into disease mechanisms, although they usually do not fully recapitulate the phenotype of cardiovascular diseases and their progression. In recent years, a promising breakthrough has emerged in the form of in vitro three-dimensional (3D) cardiovascular models utilizing human pluripotent stem cells. These innovative models recreate the intricate 3D structure of the human heart and vessels within a controlled environment. This advancement is pivotal as it addresses the existing gaps in cardiovascular research, allowing scientists to study different stages of cardiovascular diseases and specific drug responses using human-origin models. In this review, we first outline various approaches employed to generate these models. We then comprehensively discuss their applications in studying cardiovascular diseases by providing insights into molecular and cellular changes associated with cardiovascular conditions. Moreover, we highlight the potential of these 3D models serving as a platform for drug testing to assess drug efficacy and safety. Despite their immense potential, challenges persist, particularly in maintaining the complex structure of 3D heart and vessel models and ensuring their function is comparable to real organs. However, overcoming these challenges could revolutionize cardiovascular research. It has the potential to offer comprehensive mechanistic insights into human-specific disease processes, ultimately expediting the development of personalized therapies.

Sphingolipid metabolism controls mammalian heart regeneration.

Utilization of lipids as energy substrates after birth causes cardiomyocyte (CM) cell-cycle arrest and loss of regenerative capacity in mammalian hearts. Beyond energy provision, proper management of lipid composition is crucial for cellular and organismal health, but its role in heart regeneration remains unclear. Here, we demonstrate widespread sphingolipid metabolism remodeling in neonatal hearts after injury and find that SphK1 and SphK2, isoenzymes producing the same sphingolipid metabolite sphingosine-1-phosphate (S1P), differently regulate cardiac regeneration. SphK2 is downregulated during heart development and determines CM proliferation via nuclear S1P-dependent modulation of histone acetylation. Reactivation of SphK2 induces adult CM cell-cycle re-entry and cytokinesis, thereby enhancing regeneration. Conversely, SphK1 is upregulated during development and promotes fibrosis through an S1P autocrine mechanism in cardiac fibroblasts. By fine-tuning the activity of each SphK isoform, we develop a therapy that simultaneously promotes myocardial repair and restricts fibrotic scarring to regenerate the infarcted adult hearts.

Recent advances in regulating the proliferation or maturation of human-induced pluripotent stem cell-derived cardiomyocytes.

In the last decade, human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM)-based cell therapy has drawn broad attention as a potential therapy for treating injured hearts. However, mass production of hiPSC-CMs remains challenging, limiting their translational potential in regenerative medicine. Therefore, multiple strategies including cell cycle regulators, small molecules, co-culture systems, and epigenetic modifiers have been used to improve the proliferation of hiPSC-CMs. On the other hand, the immaturity of these proliferative hiPSC-CMs could lead to lethal arrhythmias due to their limited ability to functionally couple with resident cardiomyocytes. To achieve functional maturity, numerous methods such as prolonged culture, biochemical or biophysical stimulation, in vivo transplantation, and 3D culture approaches have been employed. In this review, we summarize recent approaches used to promote hiPSC-CM proliferation, and thoroughly review recent advances in promoting hiPSC-CM maturation, which will serve as the foundation for large-scale production of mature hiPSC-CMs for future clinical applications.

TRF2 rescues telomere attrition and prolongs cell survival in Duchenne muscular dystrophy cardiomyocytes derived from human iPSCs.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease caused by the lack of dystrophin. Heart failure, driven by cardiomyocyte death, fibrosis, and the development of dilated cardiomyopathy, is the leading cause of death in DMD patients. Current treatments decrease the mechanical load on the heart but do not address the root cause of dilated cardiomyopathy: cardiomyocyte death. Previously, we showed that telomere shortening is a hallmark of DMD cardiomyocytes. Here, we test whether prevention of telomere attrition is possible in cardiomyocytes differentiated from patient-derived induced pluripotent stem cells (iPSC-CMs) and if preventing telomere shortening impacts cardiomyocyte function. We observe reduced cell size, nuclear size, and sarcomere density in DMD iPSC-CMs compared with healthy isogenic controls. We find that expression of just one telomere-binding protein, telomeric repeat-binding factor 2 (TRF2), a core component of the shelterin complex, prevents telomere attrition and rescues deficiencies in cell size as well as sarcomere density. We employ a bioengineered platform to micropattern cardiomyocytes for calcium imaging and perform Southern blots of telomere restriction fragments, the gold standard for telomere length assessments. Importantly, preservation of telomere lengths in DMD cardiomyocytes improves their viability. These data provide evidence that preventing telomere attrition ameliorates deficits in cell morphology, activation of the DNA damage response, and premature cell death, suggesting that TRF2 is a key player in DMD-associated cardiac failure.

Impaired Human Cardiac Cell Development due to NOTCH1 Deficiency.

BACKGROUND: NOTCH1 pathogenic variants are implicated in multiple types of congenital heart defects including hypoplastic left heart syndrome, where the left ventricle is underdeveloped. It is unknown how NOTCH1 regulates human cardiac cell lineage determination and cardiomyocyte proliferation. In addition, mechanisms by which NOTCH1 pathogenic variants lead to ventricular hypoplasia in hypoplastic left heart syndrome remain elusive. METHODS: CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 genome editing was utilized to delete NOTCH1 in human induced pluripotent stem cells. Cardiac differentiation was carried out by sequential modulation of WNT signaling, and NOTCH1 knockout and wild-type differentiating cells were collected at day 0, 2, 5, 10, 14, and 30 for single-cell RNA-seq. RESULTS: Human NOTCH1 knockout induced pluripotent stem cells are able to generate functional cardiomyocytes and endothelial cells, suggesting that NOTCH1 is not required for mesoderm differentiation and cardiovascular development in vitro. However, disruption of NOTCH1 blocks human ventricular-like cardiomyocyte differentiation but promotes atrial-like cardiomyocyte generation through shortening the action potential duration. NOTCH1 deficiency leads to defective proliferation of early human cardiomyocytes, and transcriptomic analysis indicates that pathways involved in cell cycle progression and mitosis are downregulated in NOTCH1 knockout cardiomyocytes. Single-cell transcriptomic analysis reveals abnormal cell lineage determination of cardiac mesoderm, which is manifested by the biased differentiation toward epicardial and second heart field progenitors at the expense of first heart field progenitors in NOTCH1 knockout cell populations. CONCLUSIONS: NOTCH1 is essential for human ventricular-like cardiomyocyte differentiation and proliferation through balancing cell fate determination of cardiac mesoderm and modulating cell cycle progression. Because first heart field progenitors primarily contribute to the left ventricle, we speculate that pathogenic NOTCH1 variants lead to biased differentiation of first heart field progenitors, blocked ventricular-like cardiomyocyte differentiation, and defective cardiomyocyte proliferation, which collaboratively contribute to left ventricular hypoplasia in hypoplastic left heart syndrome.

Protocol to measure contraction, calcium, and action potential in human-induced pluripotent stem cell-derived cardiomyocytes.

Multiple strategies have been developed to efficiently differentiate human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Here, we describe a protocol for measuring three key functional parameters of hiPSC-CMs, including contractile function, calcium (Ca2+) handling, and action potential. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2021).

DUSP26 induces aortic valve calcification by antagonizing MDM2-mediated ubiquitination of DPP4 in human valvular interstitial cells.

AIMS: The morbidity and mortality rates of calcific aortic valve disease (CAVD) remain high while treatment options are limited. Here, we evaluated the role and therapeutic value of dual-specificity phosphatase 26 (DUSP26) in CAVD. METHODS AND RESULTS: Microarray profiling of human calcific aortic valves and normal controls demonstrated that DUSP26 was significantly up-regulated in calcific aortic valves. ApoE-/- mice fed a normal diet or a high cholesterol diet (HCD) were infected with adeno-associated virus serotype 2 carrying DUSP26 short-hairpin RNA to examine the effects of DUSP26 silencing on aortic valve calcification. DUSP26 silencing ameliorated aortic valve calcification in HCD-treated ApoE-/- mice, as evidenced by reduced thickness and calcium deposition in the aortic valve leaflets, improved echocardiographic parameters (decreased peak transvalvular jet velocity and mean transvalvular pressure gradient, as well as increased aortic valve area), and decreased levels of osteogenic markers (Runx2, osterix, and osteocalcin) in the aortic valves. These results were confirmed in osteogenic medium-induced human valvular interstitial cells. Immunoprecipitation, liquid chromatography-tandem mass spectrometry, and functional assays revealed that dipeptidyl peptidase-4 (DPP4) interacted with DUSP26 to mediate the procalcific effects of DUSP26. High N6-methyladenosine levels up-regulated DUSP26 in CAVD; in turn, DUSP26 activated DPP4 by antagonizing mouse double minute 2-mediated ubiquitination and degradation of DPP4, thereby promoting CAVD progression. CONCLUSION: DUSP26 promotes aortic valve calcification by inhibiting DPP4 degradation. Our findings identify a previously unrecognized mechanism of DPP4 up-regulation in CAVD, suggesting that DUSP26 silencing or inhibition is a viable therapeutic strategy to impede CAVD progression.

Identification of loss-of-function RyR2 mutations associated with idiopathic ventricular fibrillation and sudden death.

Mutations in cardiac ryanodine receptor (RyR2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). Most CPVT RyR2 mutations characterized are gain-of-function (GOF), indicating enhanced RyR2 function as a major cause of CPVT. Loss-of-function (LOF) RyR2 mutations have also been identified and are linked to a distinct entity of cardiac arrhythmia termed RyR2 Ca2+ release deficiency syndrome (CRDS). Exercise stress testing (EST) is routinely used to diagnose CPVT, but it is ineffective for CRDS. There is currently no effective diagnostic tool for CRDS in humans. An alternative strategy to assess the risk for CRDS is to directly determine the functional impact of the associated RyR2 mutations. To this end, we have functionally screened 18 RyR2 mutations that are associated with idiopathic ventricular fibrillation (IVF) or sudden death. We found two additional RyR2 LOF mutations E4146K and G4935R. The E4146K mutation markedly suppressed caffeine activation of RyR2 and abolished store overload induced Ca2+ release (SOICR) in human embryonic kidney 293 (HEK293) cells. E4146K also severely reduced cytosolic Ca2+ activation and abolished luminal Ca2+ activation of single RyR2 channels. The G4935R mutation completely abolished caffeine activation of and [3H]ryanodine binding to RyR2. Co-expression studies showed that the G4935R mutation exerted dominant negative impact on the RyR2 wildtype (WT) channel. Interestingly, the RyR2-G4935R mutant carrier had a negative EST, and the E4146K carrier had a family history of sudden death during sleep, which are different from phenotypes of typical CPVT. Thus, our data further support the link between RyR2 LOF and a new entity of cardiac arrhythmias distinct from CPVT.

Method for selective ablation of undifferentiated human pluripotent stem cell populations for cell-based therapies.

Human pluripotent stem cells (PSCs), which are composed of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide an opportunity to advance cardiac cell therapy-based clinical trials. However, an important hurdle that must be overcome is the risk of teratoma formation after cell transplantation due to the proliferative capacity of residual undifferentiated PSCs in differentiation batches. To tackle this problem, we propose the use of a minimal noncardiotoxic doxorubicin dose as a purifying agent to selectively target rapidly proliferating stem cells for cell death, which will provide a purer population of terminally differentiated cardiomyocytes before cell transplantation. In this study, we determined an appropriate in vitro doxorubicin dose that (a) eliminates residual undifferentiated stem cells before cell injection to prevent teratoma formation after cell transplantation and (b) does not cause cardiotoxicity in ESC-derived cardiomyocytes (CMs) as demonstrated through contractility analysis, electrophysiology, topoisomerase activity assay, and quantification of reactive oxygen species generation. This study establishes a potentially novel method for tumorigenic-free cell therapy studies aimed at clinical applications of cardiac cell transplantation.

Effects of Cryopreservation on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Assessing Drug Safety Response Profiles.

Burgeoning applications of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in disease modeling, regenerative medicine, and drug screening have broadened the usage of hiPSC-CMs and entailed their long-term storage. Cryopreservation is the most common approach to store hiPSC-CMs. However, the effects of cryopreservation and recovery on hiPSC-CMs remain poorly understood. Here, we characterized the transcriptome, electro-mechanical function, and drug response of fresh hiPSC-CMs without cryopreservation and recovered hiPSC-CMs from cryopreservation. We found that recovered hiPSC-CMs showed upregulation of cell cycle genes, similar or reduced contractility, Ca2+ transients, and field potential duration. When subjected to treatment of drugs that affect electrophysiological properties, recovered hiPSC-CMs showed an altered drug response and enhanced propensity for drug-induced cardiac arrhythmic events. In conclusion, fresh and recovered hiPSC-CMs do not always show comparable molecular and physiological properties. When cryopreserved hiPSC-CMs are used for assessing drug-induced cardiac liabilities, the altered drug sensitivity needs to be considered.

Clinical trial in a dish using iPSCs shows lovastatin improves endothelial dysfunction and cellular cross-talk in LMNA cardiomyopathy.

Mutations in LMNA, the gene that encodes lamin A and C, causes LMNA-related dilated cardiomyopathy (DCM) or cardiolaminopathy. LMNA is expressed in endothelial cells (ECs); however, little is known about the EC-specific phenotype of LMNA-related DCM. Here, we studied a family affected by DCM due to a frameshift variant in LMNA Human induced pluripotent stem cell (iPSC)-derived ECs were generated from patients with LMNA-related DCM and phenotypically characterized. Patients with LMNA-related DCM exhibited clinical endothelial dysfunction, and their iPSC-ECs showed decreased functionality as seen by impaired angiogenesis and nitric oxide (NO) production. Moreover, genome-edited isogenic iPSC lines recapitulated the EC disease phenotype in which LMNA-corrected iPSC-ECs showed restoration of EC function. Simultaneous profiling of chromatin accessibility and gene expression dynamics by combining assay for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA sequencing (RNA-seq) as well as loss-of-function studies identified Krüppel-like factor 2 (KLF2) as a potential transcription factor responsible for the EC dysfunction. Gain-of-function studies showed that treatment of LMNA iPSC-ECs with KLF2 agonists, including lovastatin, rescued the EC dysfunction. Patients with LMNA-related DCM treated with lovastatin showed improvements in clinical endothelial dysfunction as indicated by increased reactive hyperemia index. Furthermore, iPSC-derived cardiomyocytes (iPSC-CMs) from patients exhibiting the DCM phenotype showed improvement in CM function when cocultured with iPSC-ECs and lovastatin. These results suggest that impaired cross-talk between ECs and CMs can contribute to the pathogenesis of LMNA-related DCM, and statin may be an effective therapy for vascular dysfunction in patients with cardiolaminopathy.

Activation of PDGF pathway links LMNA mutation to dilated cardiomyopathy.

Lamin A/C (LMNA) is one of the most frequently mutated genes associated with dilated cardiomyopathy (DCM). DCM related to mutations in LMNA is a common inherited cardiomyopathy that is associated with systolic dysfunction and cardiac arrhythmias. Here we modelled the LMNA-related DCM in vitro using patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Electrophysiological studies showed that the mutant iPSC-CMs displayed aberrant calcium homeostasis that led to arrhythmias at the single-cell level. Mechanistically, we show that the platelet-derived growth factor (PDGF) signalling pathway is activated in mutant iPSC-CMs compared to isogenic control iPSC-CMs. Conversely, pharmacological and molecular inhibition of the PDGF signalling pathway ameliorated the arrhythmic phenotypes of mutant iPSC-CMs in vitro. Taken together, our findings suggest that the activation of the PDGF pathway contributes to the pathogenesis of LMNA-related DCM and point to PDGF receptor-ß (PDGFRB) as a potential therapeutic target.

Modelling diastolic dysfunction in induced pluripotent stem cell-derived cardiomyocytes from hypertrophic cardiomyopathy patients.

AIMS: Diastolic dysfunction (DD) is common among hypertrophic cardiomyopathy (HCM) patients, causing major morbidity and mortality. However, its cellular mechanisms are not fully understood, and presently there is no effective treatment. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) hold great potential for investigating the mechanisms underlying DD in HCM and as a platform for drug discovery. METHODS AND RESULTS: In the present study, beating iPSC-CMs were generated from healthy controls and HCM patients with DD. Micropatterned iPSC-CMs from HCM patients showed impaired diastolic function, as evidenced by prolonged relaxation time, decreased relaxation rate, and shortened diastolic sarcomere length. Ratiometric Ca2+ imaging indicated elevated diastolic [Ca2+]i and abnormal Ca2+ handling in HCM iPSC-CMs, which were exacerbated by ß-adrenergic challenge. Combining Ca2+ imaging and traction force microscopy, we observed enhanced myofilament Ca2+ sensitivity (measured as dF/Δ[Ca2+]i) in HCM iPSC-CMs. These results were confirmed with genome-edited isogenic iPSC lines that carry HCM mutations, indicating that cytosolic diastolic Ca2+ overload, slowed [Ca2+]i recycling, and increased myofilament Ca2+ sensitivity, collectively impairing the relaxation of HCM iPSC-CMs. Treatment with partial blockade of Ca2+ or late Na+ current reset diastolic Ca2+ homeostasis, restored diastolic function, and improved long-term survival, suggesting that disturbed Ca2+ signalling is an important cellular pathological mechanism of DD. Further investigation showed increased expression of L-type Ca2+channel (LTCC) and transient receptor potential cation channels (TRPC) in HCM iPSC-CMs compared with control iPSC-CMs, which likely contributed to diastolic [Ca2+]i overload. CONCLUSION: In summary, this study recapitulated DD in HCM at the single-cell level, and revealed novel cellular mechanisms and potential therapeutic targets of DD using iPSC-CMs.

Human-Induced Pluripotent Stem Cell Model of Trastuzumab-Induced Cardiac Dysfunction in Patients With Breast Cancer.

BACKGROUND: Molecular targeted chemotherapies have been shown to significantly improve the outcomes of patients who have cancer, but they often cause cardiovascular side effects that limit their use and impair patients' quality of life. Cardiac dysfunction induced by these therapies, especially trastuzumab, shows a distinct cardiotoxic clinical phenotype in comparison to the cardiotoxicity induced by conventional chemotherapies. METHODS: We used the human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) platform to determine the underlying cellular mechanisms in trastuzumab-induced cardiac dysfunction. We assessed the effects of trastuzumab on structural and functional properties in iPSC-CMs from healthy individuals and performed RNA-sequencing to further examine the effect of trastuzumab on iPSC-CMs. We also generated human induced pluripotent stem cells from patients receiving trastuzumab and examined whether patients' phenotype could be recapitulated in vitro by using patient-specific iPSC-CMs. RESULTS: We found that clinically relevant doses of trastuzumab significantly impaired the contractile and calcium-handling properties of iPSC-CMs without inducing cardiomyocyte death or sarcomeric disorganization. RNA-sequencing and subsequent functional analysis revealed mitochondrial dysfunction and altered the cardiac energy metabolism pathway as primary causes of trastuzumab-induced cardiotoxic phenotype. Human iPSC-CMs generated from patients who received trastuzumab and experienced severe cardiac dysfunction were more vulnerable to trastuzumab treatment than iPSC-CMs generated from patients who did not experience cardiac dysfunction following trastuzumab therapy. It is important to note that metabolic modulation with AMP-activated protein kinase activators could avert the adverse effects induced by trastuzumab. CONCLUSIONS: Our results indicate that alterations in cellular metabolic pathways in cardiomyocytes could be a key mechanism underlying the development of cardiac dysfunction following trastuzumab therapy; therefore, targeting the altered metabolism may be a promising therapeutic approach for trastuzumab-induced cardiac dysfunction.

Single-Cell RNA Sequencing of Human Embryonic Stem Cell Differentiation Delineates Adverse Effects of Nicotine on Embryonic Development.

Nicotine, the main chemical constituent of tobacco, is highly detrimental to the developing fetus by increasing the risk of gestational complications and organ disorders. The effects of nicotine on human embryonic development and related mechanisms, however, remain poorly understood. Here, we performed single-cell RNA sequencing (scRNA-seq) of human embryonic stem cell (hESC)-derived embryoid body (EB) in the presence or absence of nicotine. Nicotine-induced lineage-specific responses and dysregulated cell-to-cell communication in EBs, shedding light on the adverse effects of nicotine on human embryonic development. In addition, nicotine reduced cell viability, increased reactive oxygen species (ROS), and altered cell cycling in EBs. Abnormal Ca2+ signaling was found in muscle cells upon nicotine exposure, as verified in hESC-derived cardiomyocytes. Consequently, our scRNA-seq data suggest direct adverse effects of nicotine on hESC differentiation at the single-cell level and offer a new method for evaluating drug and environmental toxicity on human embryonic development in utero.

A Human iPSC Double-Reporter System Enables Purification of Cardiac Lineage Subpopulations with Distinct Function and Drug Response Profiles.

The diversity of cardiac lineages contributes to the heterogeneity of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs). Here, we report the generation of a hiPSC TBX5Clover2 and NKX2-5TagRFP double reporter to delineate cardiac lineages and isolate lineage-specific subpopulations. Molecular analyses reveal that four different subpopulations can be isolated based on the differential expression of TBX5 and NKX2-5, TBX5+NKX2-5+, TBX5+NKX2-5-, TBX5-NKX2-5+, and TBX5-NKX2-5-, mimicking the first heart field, epicardial, second heart field, and endothelial lineages, respectively. Genetic and functional characterization indicates that each subpopulation differentiates into specific cardiac cells. We further identify CORIN as a cell-surface marker for isolating the TBX5+NKX2-5+ subpopulation and demonstrate the use of lineage-specific CMs for precise drug testing. We anticipate that this tool will facilitate the investigation of cardiac lineage specification and isolation of specific cardiac subpopulations for drug screening, tissue engineering, and disease modeling.

Disruption of mesoderm formation during cardiac differentiation due to developmental exposure to 13-cis-retinoic acid.

13-cis-retinoic acid (isotretinoin, INN) is an oral pharmaceutical drug used for the treatment of skin acne, and is also a known teratogen. In this study, the molecular mechanisms underlying INN-induced developmental toxicity during early cardiac differentiation were investigated using both human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs). Pre-exposure of hiPSCs and hESCs to a sublethal concentration of INN did not influence cell proliferation and pluripotency. However, mesodermal differentiation was disrupted when INN was included in the medium during differentiation. Transcriptomic profiling by RNA-seq revealed that INN exposure leads to aberrant expression of genes involved in several signaling pathways that control early mesoderm differentiation, such as TGF-beta signaling. In addition, genome-wide chromatin accessibility profiling by ATAC-seq suggested that INN-exposure leads to enhanced DNA-binding of specific transcription factors (TFs), including HNF1B, SOX10 and NFIC, often in close spatial proximity to genes that are dysregulated in response to INN treatment. Altogether, these results identify potential molecular mechanisms underlying INN-induced perturbation during mesodermal differentiation in the context of cardiac development. This study further highlights the utility of human stem cells as an alternative system for investigating congenital diseases of newborns that arise as a result of maternal drug exposure during pregnancy.

Determining the Pathogenicity of a Genomic Variant of Uncertain Significance Using CRISPR/Cas9 and Human-Induced Pluripotent Stem Cells.

BACKGROUND: The progression toward low-cost and rapid next-generation sequencing has uncovered a multitude of variants of uncertain significance (VUS) in both patients and asymptomatic "healthy" individuals. A VUS is a rare or novel variant for which disease pathogenicity has not been conclusively demonstrated or excluded, and thus cannot be definitively annotated. VUS, therefore, pose critical clinical interpretation and risk-assessment challenges, and new methods are urgently needed to better characterize their pathogenicity. METHODS: To address this challenge and showcase the uncertainty surrounding genomic variant interpretation, we recruited a "healthy" asymptomatic individual, lacking cardiac-disease clinical history, carrying a hypertrophic cardiomyopathy (HCM)-associated genetic variant (NM_000258.2:c.170C>A, NP_000249.1:p.Ala57Asp) in the sarcomeric gene MYL3, reported by the ClinVar database to be "likely pathogenic." Human-induced pluripotent stem cells (iPSCs) were derived from the heterozygous VUS MYL3(170C>A) carrier, and their genome was edited using CRISPR/Cas9 to generate 4 isogenic iPSC lines: (1) corrected "healthy" control; (2) homozygous VUS MYL3(170C>A); (3) heterozygous frameshift mutation MYL3(170C>A/fs); and (4) known heterozygous MYL3 pathogenic mutation (NM_000258.2:c.170C>G), at the same nucleotide position as VUS MYL3(170C>A), lines. Extensive assays including measurements of gene expression, sarcomere structure, cell size, contractility, action potentials, and calcium handling were performed on the isogenic iPSC-derived cardiomyocytes (iPSC-CMs). RESULTS: The heterozygous VUS MYL3(170C>A)-iPSC-CMs did not show an HCM phenotype at the gene expression, morphology, or functional levels. Furthermore, genome-edited homozygous VUS MYL3(170C>A)- and frameshift mutation MYL3(170C>A/fs)-iPSC-CMs lines were also asymptomatic, supporting a benign assessment for this particular MYL3 variant. Further assessment of the pathogenic nature of a genome-edited isogenic line carrying a known pathogenic MYL3 mutation, MYL3(170C>G), and a carrier-specific iPSC-CMs line, carrying a MYBPC3(961G>A) HCM variant, demonstrated the ability of this combined platform to provide both pathogenic and benign assessments. CONCLUSIONS: Our study illustrates the ability of clustered regularly interspaced short palindromic repeats/Cas9 genome-editing of carrier-specific iPSCs to elucidate both benign and pathogenic HCM functional phenotypes in a carrier-specific manner in a dish. As such, this platform represents a promising VUS risk-assessment tool that can be used for assessing HCM-associated VUS specifically, and VUS in general, and thus significantly contribute to the arsenal of precision medicine tools available in this emerging field.