Independent compartmentalization of functional, metabolic, and transcriptional maturation of hiPSC-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) recapitulate numerous disease and drug response phenotypes, but cell immaturity may limit their accuracy and fidelity as a model system. Cell culture medium modification is a common method for enhancing maturation, yet prior studies have used complex media with little understanding of individual component contribution, which may compromise long-term hiPSC-CM viability. Here, we developed high-throughput methods to measure hiPSC-CM maturation, determined factors that enhanced viability, and then systematically assessed the contribution of individual maturation medium components. We developed a medium that is compatible with extended culture. We discovered that hiPSC-CM maturation can be sub-specified into electrophysiological/EC coupling, metabolism, and gene expression and that induction of these attributes is largely independent. In this work, we establish a defined baseline for future studies of cardiomyocyte maturation. Furthermore, we provide a selection of medium formulae, optimized for distinct applications and priorities, that promote measurable attributes of maturation.

Functional Validation of Doxorubicin-Induced Cardiotoxicity-Related Genes.

Background: Genome-wide association studies and candidate gene association studies have identified more than 180 genetic variants statistically associated with anthracycline-induced cardiotoxicity (AIC). However, the lack of functional validation has hindered the clinical translation of these findings. Objectives: The aim of this study was to functionally validate all genes associated with AIC using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Methods: Through a systemic literature search, 80 genes containing variants significantly associated with AIC were identified. Additionally, 3 more genes with potential roles in AIC (GSTM1, CBR1, and ERBB2) were included. Of these, 38 genes exhibited expression in human fetal heart, adult heart, and hiPSC-CMs. Using clustered regularly interspaced short palindromic repeats/Cas9-based genome editing, each of these 38 genes was systematically knocked out in control hiPSC-CMs, and the resulting doxorubicin-induced cardiotoxicity (DIC) phenotype was assessed using hiPSC-CMs. Subsequently, functional assays were conducted for each gene knockout on the basis of hypothesized mechanistic implications in DIC. Results: Knockout of 26 genes increased the susceptibility of hiPSC-CMs to DIC. Notable genes included efflux transporters (ABCC10, ABCC2, ABCB4, ABCC5, and ABCC9), well-established DIC-associated genes (CBR1, CBR3, and RAC2), and genome-wide association study-discovered genes (RARG and CELF4). Conversely, knockout of ATP2B1, HNMT, POR, CYBA, WDR4, and COL1A2 had no significant effect on the in vitro DIC phenotype of hiPSC-CMs. Furthermore, knockout of the uptake transporters (SLC28A3, SLC22A17, and SLC28A1) demonstrated a protective effect against DIC. Conclusions: The present findings establish a comprehensive platform for the functional validation of DIC-associated genes, providing insights for future studies in DIC variant associations and potential mechanistic targets for the development of cardioprotective drugs.

Nutritional requirements of human induced pluripotent stem cells.

The nutritional requirements for human induced pluripotent stem cell (hiPSC) growth have not been extensively studied. Here, building on our prior work that established the suitable non-basal medium components for hiPSC growth, we develop a simplified basal medium consisting of just 39 components, demonstrating that many ingredients of DMEM/F12 are either not essential or are at suboptimal concentrations. This new basal medium along with the supplement, which we call BMEM, enhances the growth rate of hiPSCs over DMEM/F12-based media, supports derivation of multiple hiPSC lines, and allows differentiation to multiple lineages. hiPSCs cultured in BMEM consistently have enhanced expression of undifferentiated cell markers such as POU5F1 and NANOG, along with increased expression of markers of the primed state and reduced expression of markers of the naive state. This work describes titration of the nutritional requirements of human pluripotent cell culture and identifies that suitable nutrition enhances the pluripotent state.

A review of protocols for human iPSC culture, cardiac differentiation, subtype-specification, maturation, and direct reprogramming.

The methods for the culture and cardiomyocyte differentiation of human embryonic stem cells, and later human induced pluripotent stem cells (hiPSC), have moved from a complex and uncontrolled systems to simplified and relatively robust protocols, using the knowledge and cues gathered at each step. HiPSC-derived cardiomyocytes have proven to be a useful tool in human disease modelling, drug discovery, developmental biology, and regenerative medicine. In this protocol review, we will highlight the evolution of protocols associated with hPSC culture, cardiomyocyte differentiation, sub-type specification, and cardiomyocyte maturation. We also discuss protocols for somatic cell direct reprogramming to cardiomyocyte-like cells.

Identification of Drug Transporter Genomic Variants and Inhibitors That Protect Against Doxorubicin-Induced Cardiotoxicity.

BACKGROUND: Multiple pharmacogenomic studies have identified the synonymous genomic variant rs7853758 (G > A, L461L) and the intronic variant rs885004 in SLC28A3 (solute carrier family 28 member 3) as statistically associated with a lower incidence of anthracycline-induced cardiotoxicity. However, the true causal variant(s), the cardioprotective mechanism of this locus, the role of SLC28A3 and other solute carrier (SLC) transporters in anthracycline-induced cardiotoxicity, and the suitability of SLC transporters as targets for cardioprotective drugs has not been investigated. METHODS: Six well-phenotyped, doxorubicin-treated pediatric patients from the original association study cohort were recruited again, and human induced pluripotent stem cell-derived cardiomyocytes were generated. Patient-specific doxorubicin-induced cardiotoxicity (DIC) was then characterized using assays of cell viability, activated caspase 3/7, and doxorubicin uptake. The role of SLC28A3 in DIC was then queried using overexpression and knockout of SLC28A3 in isogenic human-induced pluripotent stem cell-derived cardiomyocytes using a CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9). Fine-mapping of the SLC28A3 locus was then completed after SLC28A3 resequencing and an extended in silico haplotype and functional analysis. Genome editing of the potential causal variant was done using cytosine base editor. SLC28A3-AS1 overexpression was done using a lentiviral plasmid-based transduction and was validated using stranded RNA-sequencing after ribosomal RNA depletion. Drug screening was done using the Prestwick Chemical Library (n = 1200), followed by in vivo validation in mice. The effect of desipramine on doxorubicin cytotoxicity was also investigated in 8 cancer cell lines. RESULTS: Here, using the most commonly used anthracycline, doxorubicin, we demonstrate that patient-derived cardiomyocytes recapitulate the cardioprotective effect of the SLC28A3 locus and that SLC28A3 expression influences the severity of DIC. Using Nanopore-based fine-mapping and base editing, we identify a novel cardioprotective single nucleotide polymorphism, rs11140490, in the SLC28A3 locus; its effect is exerted via regulation of an antisense long noncoding RNA (SLC28A3-AS1) that overlaps with SLC28A3. Using high-throughput drug screening in patient-derived cardiomyocytes and whole organism validation in mice, we identify the SLC competitive inhibitor desipramine as protective against DIC. CONCLUSIONS: This work demonstrates the power of the human induced pluripotent stem cell model to take a single nucleotide polymorphism from a statistical association through to drug discovery, providing human cell-tested data for clinical trials to attenuate DIC.

RARG variant predictive of doxorubicin-induced cardiotoxicity identifies a cardioprotective therapy.

Doxorubicin is an anthracycline chemotherapy agent effective in treating a wide range of malignancies, but its use is limited by dose-dependent cardiotoxicity. A recent genome-wide association study identified a SNP (rs2229774) in retinoic acid receptor-γ (RARG) as statistically associated with increased risk of anthracycline-induced cardiotoxicity. Here, we show that human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with rs2229774 and who suffered doxorubicin-induced cardiotoxicity (DIC) are more sensitive to doxorubicin. We determine that the mechanism of this RARG variant effect is mediated via suppression of topoisomerase 2ß (TOP2B) expression and activation of the cardioprotective extracellular regulated kinase (ERK) pathway. We use patient-specific hiPSC-CMs as a drug discovery platform, determining that the RARG agonist CD1530 attenuates DIC, and we confirm this cardioprotective effect in an established in vivo mouse model of DIC. This study provides a rationale for clinical prechemotherapy genetic screening for rs2229774 and a foundation for the clinical use of RARG agonist treatment to protect cancer patients from DIC.

An updated protocol for the cost-effective and weekend-free culture of human induced pluripotent stem cells.

The protocol provided here describes methodologies for making a highly cost-effective, chemically defined medium for culturing hiPSCs we call B8 medium. The typical cost of B8 medium is US$10 per liter, which with modifications included here is more affordable than standard media. We provide simple protocols for making B8 supplement aliquots, making the basal media DMEM/F12, Matrigel-coated plates, thawing, passaging, culturing, and cryopreserving hiPSCs. We show typical differentiation results and provide a comprehensive troubleshooting guide. For complete details on the use and execution of this protocol, please refer to Kuo et al. (2020).

Mitochondrial architecture in cardiac myocytes depends on cell shape and matrix rigidity.

Contraction of cardiac myocytes depends on energy generated by the mitochondria. During cardiac development and disease, the structure and function of the mitochondrial network in cardiac myocytes is known to remodel in concert with many other factors, including changes in nutrient availability, hemodynamic load, extracellular matrix (ECM) rigidity, cell shape, and maturation of other intracellular structures. However, the independent role of each of these factors on mitochondrial network architecture is poorly understood. In this study, we tested the hypothesis that cell aspect ratio (AR) and ECM rigidity regulate the architecture of the mitochondrial network in cardiac myocytes. To do this, we spin-coated glass coverslips with a soft, moderate, or stiff polymer. Next, we microcontact printed cell-sized rectangles of fibronectin with AR matching cardiac myocytes at various developmental or disease states onto the polymer surface. We then cultured neonatal rat ventricular myocytes on the patterned surfaces and used confocal microscopy and image processing techniques to quantify sarcomeric α-actinin volume, nucleus volume, and mitochondrial volume, surface area, and size distribution. On some substrates, α-actinin volume increased with cell AR but was not affected by ECM rigidity. Nucleus volume was mostly uniform across all conditions. In contrast, mitochondrial volume increased with cell AR on all substrates. Furthermore, mitochondrial surface area to volume ratio decreased as AR increased on all substrates. Large mitochondria were also more prevalent in cardiac myocytes with higher AR. For select AR, mitochondria were also smaller as ECM rigidity increased. Collectively, these results suggest that mitochondrial architecture in cardiac myocytes is strongly influenced by cell shape and moderately influenced by ECM rigidity. These data have important implications for understanding the factors that impact metabolic performance during heart development and disease.

Pluripotent Stem Cell Modeling of Anticancer Therapy-Induced Cardiotoxicity.

PURPOSE OF REVIEW: In this article, we review the different model systems based on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and how they have been applied to identify the cardiotoxic effects of anticancer therapies. RECENT FINDINGS: Developments on 2D and 3D culture systems enabled the use of hiPSC-CMs as screening platforms for cardiotoxic effects of anticancer therapies such as anthracyclines, monoclonal antibodies, and tyrosine kinase inhibitors. Combined with computational approaches and higher throughput screening technologies, they have also enabled mechanistic studies and the search for cardioprotective strategies. As the population ages and cancer treatments become more effective, the cardiotoxic effects of anticancer drugs become a bigger problem leading to an increased role of cardio-oncology. In the past decade, human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important platform for preclinical drug tests, elucidating mechanisms of action for drugs, and identifying cardioprotective pathways that could be further explored in the development of combined treatments. In this article, we highlight 2D and 3D model systems based on hiPSC-CMs that have been used to study the cardiotoxic effects of anticancer drugs, investigating their mechanisms of action and the potential for patient-specific prediction. We also present some of the important challenges and opportunities in the field, indicating possible future developments and how they could impact the landscape of cardio-oncology.

Generating a Cost-Effective, Weekend-Free Chemically Defined Human Induced Pluripotent Stem Cell (hiPSC) Culture Medium.

We have previously developed a cost-effective chemically defined medium formula for weekend-free culture of human induced pluripotent stem cells (hiPSCs), costing ∼3% of the price of commercial medium. This medium, which we termed B8, is specifically optimized for robust and fast growth of hiPSCs and for a weekend-free medium change regimen. We demonstrated that this medium is suitable for reprogramming of somatic cells into hiPSCs and for differentiation into a variety of lineages. Here, we provide a protocol for simple generation of the most cost-effective variant of this medium, along with a protocol for making Matrigel-coated plates and culturing, passaging, cryopreserving, and thawing hiPSCs. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of a highly optimized, robust, and cost-effective human induced pluripotent stem cell culture medium Basic Protocol 2: Weekend-free maintenance and passaging of human induced pluripotent stem cells in B8 medium.

Regulation of calcium dynamics and propagation velocity by tissue microstructure in engineered strands of cardiac tissue.

Disruptions to cardiac tissue microstructure are common in diseased or injured myocardium and are known substrates for arrhythmias. However, we have a relatively coarse understanding of the relationships between myocardial tissue microstructure, propagation velocity and calcium cycling, due largely to the limitations of conventional experimental tools. To address this, we used microcontact printing to engineer strands of cardiac tissue with eight different widths, quantified several structural and functional parameters and established correlation coefficients. As strand width increased, actin alignment, nuclei density, sarcomere index and cell aspect ratio decreased with unique trends. The propagation velocity of calcium waves decreased and the rise time of calcium transients increased with increasing strand width. The decay time constant of calcium transients decreased and then slightly increased with increasing strand width. Based on correlation coefficients, actin alignment was the strongest predictor of propagation velocity and calcium transient rise time. Sarcomere index and cell aspect ratio were also strongly correlated with propagation velocity. Actin alignment, sarcomere index and cell aspect ratio were all weak predictors of the calcium transient decay time constant. We also measured the expression of several genes relevant to propagation and calcium cycling and found higher expression of the genes that encode for connexin 43 (Cx43) and a subunit of L-type calcium channels in thin strands compared to isotropic tissues. Together, these results suggest that thinner strands have higher values of propagation velocity and calcium transient rise time due to a combination of favorable tissue microstructure and enhanced expression of genes for Cx43 and L-type calcium channels. These data are important for defining how microstructural features regulate intercellular and intracellular calcium handling, which is needed to understand mechanisms of propagation in physiological situations and arrhythmogenesis in pathological situations.

Matrix-guided control of mitochondrial function in cardiac myocytes.

In ventricular myocardium, extracellular matrix (ECM) remodeling is a hallmark of physiological and pathological growth, coincident with metabolic rewiring of cardiac myocytes. However, the direct impact of the biochemical and mechanical properties of the ECM on the metabolic function of cardiac myocytes is mostly unknown. Furthermore, understanding the impact of distinct biomaterials on cardiac myocyte metabolism is critical for engineering physiologically-relevant models of healthy and diseased myocardium. For these reasons, we systematically measured morphological and metabolic responses of neonatal rat ventricular myocytes cultured on fibronectin- or gelatin-coated polydimethylsiloxane (PDMS) of three elastic moduli and gelatin hydrogels with four elastic moduli. On all substrates, total protein content, cell morphology, and the ratio of mitochondrial DNA to nuclear DNA were preserved. Cytotoxicity was low on all substrates, although slightly higher on PDMS compared to gelatin hydrogels. We also quantified oxygen consumption rates and extracellular acidification rates using a Seahorse extracellular flux analyzer. Our data indicate that several metrics associated with baseline glycolysis and baseline and maximum mitochondrial function are enhanced when cardiac myocytes are cultured on gelatin hydrogels compared to all PDMS substrates, irrespective of substrate rigidity. These results yield new insights into how mechanical and biochemical cues provided by the ECM impact mitochondrial function in cardiac myocytes. STATEMENT OF SIGNIFICANCE: Cardiac development and disease are associated with remodeling of the extracellular matrix coincident with metabolic rewiring of cardiac myocytes. However, little is known about the direct impact of the biochemical and mechanical properties of the extracellular matrix on the metabolic function of cardiac myocytes. In this study, oxygen consumption rates were measured in neonatal rat ventricular myocytes maintained on several commonly-used biomaterial substrates to reveal new relationships between the extracellular matrix and cardiac myocyte metabolism. Several mitochondrial parameters were enhanced on gelatin hydrogels compared to synthetic PDMS substrates. These data are important for comprehensively understanding matrix-regulation of cardiac myocyte physiology. Additionally, these data should be considered when selecting scaffolds for engineering in vitro cardiac tissue models.

Engineering cardiac microphysiological systems to model pathological extracellular matrix remodeling.

Many cardiovascular diseases are associated with pathological remodeling of the extracellular matrix (ECM) in the myocardium. ECM remodeling is a complex, multifactorial process that often contributes to declines in myocardial function and progression toward heart failure. However, the direct effects of the many forms of ECM remodeling on myocardial cell and tissue function remain elusive, in part because conventional model systems used to investigate these relationships lack robust experimental control over the ECM. To address these shortcomings, microphysiological systems are now being developed and implemented to establish direct relationships between distinct features in the ECM and myocardial function with unprecedented control and resolution in vitro. In this review, we will first highlight the most prominent characteristics of ECM remodeling in cardiovascular disease and describe how these features can be mimicked with synthetic and natural biomaterials that offer independent control over multiple ECM-related parameters, such as rigidity and composition. We will then detail innovative microfabrication techniques that enable precise regulation of cellular architecture in two and three dimensions. We will also describe new approaches for quantifying multiple aspects of myocardial function in vitro, such as contractility, action potential propagation, and metabolism. Together, these collective technologies implemented as cardiac microphysiological systems will continue to uncover important relationships between pathological ECM remodeling and myocardial cell and tissue function, leading to new fundamental insights into cardiovascular disease, improved human disease models, and novel therapeutic approaches.

Microenvironmental Modulation of Calcium Wave Propagation Velocity in Engineered Cardiac Tissues.

INTRODUCTION: In the myocardium, rapid propagation of action potentials and subsequent calcium waves is critical for synchronizing the contraction of cardiac myocytes and maximizing cardiac output. In many pathological settings, diverse remodeling of the tissue microenvironment is correlated with arrhythmias and decreased cardiac output, but the precise impact of tissue remodeling on propagation is not completely understood. Our objective was to delineate how multiple features within the cardiac tissue microenvironment modulate propagation velocity. METHODS: To recapitulate diverse myocardial tissue microenvironments, we engineered substrates with tunable elasticity, patterning, composition, and topography using two formulations of polydimethylsiloxane (PDMS) micropatterned with fibronectin and gelatin hydrogels with flat or micromolded features. We cultured neonatal rat ventricular myocytes on these substrates and quantified cell density, tissue alignment, and cell shape. We used a fluorescent calcium indicator, high-speed microscopy, and newly-developed analysis software to record and quantify calcium wave propagation velocity (CPV). RESULTS: For all substrates, tissue alignment and cell aspect ratio were higher in aligned compared to isotropic tissues. Isotropic CPV and longitudinal CPV were similar across conditions, but transverse CPV was lower on micromolded gelatin hydrogels compared to micropatterned soft and stiff PDMS. In aligned tissues, the anisotropy ratio of CPV (longitudinal CPV/transverse CPV) was lower on micropatterned soft PDMS compared to micropatterned stiff PDMS and micromolded gelatin hydrogels. CONCLUSION: Propagation velocity in engineered cardiac tissues is sensitive to features in the tissue microenvironment, such as alignment, matrix elasticity, and matrix topography, which may underlie arrhythmias in conditions with pathological tissue remodeling.

Engineering micromyocardium to delineate cellular and extracellular regulation of myocardial tissue contractility.

Cardiovascular diseases are a leading cause of death, in part due to limitations of existing models of the myocardium. Myocardium consists of aligned, contractile cardiac myocytes interspersed with fibroblasts that synthesize extracellular matrix (ECM). The cellular demographics and biochemical and mechanical properties of the ECM remodel in many different cardiac diseases. However, the impact of diverse cellular and extracellular remodeling on the contractile output of the myocardium are poorly understood. To address this, we micropatterned 13 kPa and 90 kPa polyacrylamide gels with aligned squares of fibronectin (FN) or laminin (LN). We seeded gels with two concentrations of primary neonatal rat ventricular myocytes, which naturally contain fibroblasts. Cells assembled into aligned "µMyocardia" with fibroblast : myocyte ratios dependent on initial seeding concentration. Using traction force microscopy (TFM), we found that the peak systolic longitudinal cross-sectional force was similar across conditions, but the peak systolic work was significantly lower on 90 kPa gels. This indicates that ECM elasticity dominates over ECM ligand and cell demographics in regulating contractile output. Because our platform provides independent control over cell-cell and cell-matrix interactions, it has many applications for cardiac disease modeling.

Mitochondrial function in engineered cardiac tissues is regulated by extracellular matrix elasticity and tissue alignment.

Mitochondria in cardiac myocytes are critical for generating ATP to meet the high metabolic demands associated with sarcomere shortening. Distinct remodeling of mitochondrial structure and function occur in cardiac myocytes in both developmental and pathological settings. However, the factors that underlie these changes are poorly understood. Because remodeling of tissue architecture and extracellular matrix (ECM) elasticity are also hallmarks of ventricular development and disease, we hypothesize that these environmental factors regulate mitochondrial function in cardiac myocytes. To test this, we developed a new procedure to transfer tunable polydimethylsiloxane disks microcontact-printed with fibronectin into cell culture microplates. We cultured Sprague-Dawley neonatal rat ventricular myocytes within the wells, which consistently formed tissues following the printed fibronectin, and measured oxygen consumption rate using a Seahorse extracellular flux analyzer. Our data indicate that parameters associated with baseline metabolism are predominantly regulated by ECM elasticity, whereas the ability of tissues to adapt to metabolic stress is regulated by both ECM elasticity and tissue alignment. Furthermore, bioenergetic health index, which reflects both the positive and negative aspects of oxygen consumption, was highest in aligned tissues on the most rigid substrate, suggesting that overall mitochondrial function is regulated by both ECM elasticity and tissue alignment. Our results demonstrate that mitochondrial function is regulated by both ECM elasticity and myofibril architecture in cardiac myocytes. This provides novel insight into how extracellular cues impact mitochondrial function in the context of cardiac development and disease.NEW & NOTEWORTHY A new methodology has been developed to measure O2 consumption rates in engineered cardiac tissues with independent control over tissue alignment and matrix elasticity. This led to the findings that matrix elasticity regulates basal mitochondrial function, whereas both matrix elasticity and tissue alignment regulate mitochondrial stress responses.

Parallel imaging acceleration of spiral Fourier velocity encoded MRI using SPIRiT.

This paper demonstrates parallel imaging acceleration of spiral Fourier velocity encoded MRI using the iterative self-consistent parallel imaging reconstruction (SPIRiT) technique. Magnitude images and time-velocity distributions obtained with image domain SPIRiT and sum-of-squares reconstruction are compared, for 2-fold and 4-fold undersampling. We show that SPIRiT is able to reduce spatial aliasing from undersampled time-velocity distributions, with good results for 2-fold undersampling, and moderately good results for 4-fold undersampling.