Micro-Engineered Heart Tissues On-Chip with Heterotypic Cell Composition Display Self-Organization and Improved Cardiac Function.

Advanced in vitro models that recapitulate the structural organization and function of the human heart are highly needed for accurate disease modeling, more predictable drug screening, and safety pharmacology. Conventional 3D Engineered Heart Tissues (EHTs) lack heterotypic cell complexity and culture under flow, whereas microfluidic Heart-on-Chip (HoC) models in general lack the 3D configuration and accurate contractile readouts. In this study, an innovative and user-friendly HoC model is developed to overcome these limitations, by culturing human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs), endothelial (ECs)- and smooth muscle cells (SMCs), together with human cardiac fibroblasts (FBs), underflow, leading to self-organized miniaturized micro-EHTs (µEHTs) with a CM-EC interface reminiscent of the physiological capillary lining. µEHTs cultured under flow display enhanced contractile performance and conduction velocity. In addition, the presence of the EC layer altered drug responses in µEHT contraction. This observation suggests a potential barrier-like function of ECs, which may affect the availability of drugs to the CMs. These cardiac models with increased physiological complexity, will pave the way to screen for therapeutic targets and predict drug efficacy.

A safety screening platform for individualized cardiotoxicity assessment.

Cardiotoxicity remains a major cause of drug withdrawal, partially due to lacking predictability of animal models. Additionally, risk of cardiotoxicity following treatment of cancer patients is treatment limiting. It is unclear which patients will develop heart failure following therapy. Human pluripotent stem cell (hPSC)-derived cardiomyocytes present an unlimited cell source and may offer individualized solutions to this problem. We developed a platform to predict molecular and functional aspects of cardiotoxicity. Our platform can discriminate between the different cardiotoxic mechanisms of existing and novel anthracyclines Doxorubicin, Aclarubicin, and Amrubicin. Doxorubicin and Aclarubicin unlike Amrubicin substantially affected the transcriptome, mitochondrial membrane integrity, contractile force and transcription factor availability. Cardiomyocytes recovered fully within two or three weeks, corresponding to the intermittent clinical treatment regimen. Our system permits the study of hPSC-cardiomyocyte recovery and the effects of accumulated dose after multiple dosing, allowing individualized cardiotoxicity evaluation, which effects millions of cancer patients treated annually.

Automated assessment of human engineered heart tissues using deep learning and template matching for segmentation and tracking.

The high rate of drug withdrawal from the market due to cardiovascular toxicity or lack of efficacy, the economic burden, and extremely long time before a compound reaches the market, have increased the relevance of human in vitro models like human (patient-derived) pluripotent stem cell (hPSC)-derived engineered heart tissues (EHTs) for the evaluation of the efficacy and toxicity of compounds at the early phase in the drug development pipeline. Consequently, the EHT contractile properties are highly relevant parameters for the analysis of cardiotoxicity, disease phenotype, and longitudinal measurements of cardiac function over time. In this study, we developed and validated the software HAARTA (Highly Accurate, Automatic and Robust Tracking Algorithm), which automatically analyzes contractile properties of EHTs by segmenting and tracking brightfield videos, using deep learning and template matching with sub-pixel precision. We demonstrate the robustness, accuracy, and computational efficiency of the software by comparing it to the state-of-the-art method (MUSCLEMOTION), and by testing it with a data set of EHTs from three different hPSC lines. HAARTA will facilitate standardized analysis of contractile properties of EHTs, which will be beneficial for in vitro drug screening and longitudinal measurements of cardiac function.

Ivabradine treatment lowers blood pressure and promotes cardiac and renal protection in spontaneously hypertensive rats.

Hypertension is linked to hyperpolarization-activated cyclic nucleotide-gated (HCN) function, expressed in excitable and non-excitable cells. Considering that the reduction in heart rate (HR) improves coronary perfusion and cardiac performance, ivabradine (IVA) emerged as an important drug for the treatment of cardiovascular diseases. AIM: Evaluate if IVA chronic treatment effect can mitigate hypertension and reverse the cardiac and renal damage in SHR. MAIN METHODS: Rats were divided into 4 groups treated for 14 days with PBS (1 ml/kg; i.p) or IVA (1 mg/kg; i.p): 1) WKY PBS; 2) SHR PBS; 3) WKY IVA; and 4) SHR IVA. The systolic blood pressure (SBP) was measured, indirectly, before and during the treatment period with IVA (day 0, 1, 7 and 11). Rats were subjected to artery cannulation for direct blood pressure (BP) measurement. Morphofunctional and gene expression were evaluated in the heart and kidneys. KEY FINDINGS: IVA reduced SBP only in SHR on the 7th day. Direct blood pressure measurement showed that IVA chronic treatment reduced HR in the SHR. Interestingly, mean arterial pressure (MAP) was reduced in SHR IVA when compared to SHR PBS. Serum and urinary biochemical data were not altered by IVA. Moreover, IVA reduced the renal inflammatory infiltrates and increased glomerular density, besides preventing the cardiac inflammatory and hypertrophic responses. SIGNIFICANCE: IVA treatment lowered blood pressure, improved cardiac remodeling and inflammation, as well as decreasing renal damage in SHR. Further, IVA increased renal HCN2 mRNA and reduced cardiac HCN4 mRNA.

Contractility analysis of human engineered 3D heart tissues by an automatic tracking technique using a standalone application.

The use of Engineered Heart Tissues (EHT) as in vitro model for disease modeling and drug screening has increased, as they provide important insight into the genetic mechanisms, cardiac toxicity or drug responses. Consequently, this has highlighted the need for a standardized, unbiased, robust and automatic way to analyze hallmark physiological features of EHTs. In this study we described and validated a standalone application to analyze physiological features of EHTs in an automatic, robust, and unbiased way, using low computational time. The standalone application "EHT Analysis" contains two analysis modes (automatic and manual) to analyzes the contractile properties and the contraction kinetics of EHTs from high speed bright field videos. As output data, the graphs of displacement, contraction force and contraction kinetics per file will be generated together with the raw data. Additionally, it also generates a summary file containing all the data from the analyzed files, which facilitates and speeds up the post analysis. From our study we highlight the importance of analyzing the axial stress which is the force per surface area (µN/mm2). This allows to have a readout overtime of tissue compaction, axial stress and leave the option to calculate at the end point of an experiment the physiological cross-section area (PSCA). We demonstrated the utility of this tool by analyzing contractile properties and compaction over time of EHTs made out of a double reporter human pluripotent stem cell (hPSC) line (NKX2.5EGFP/+-COUP-TFIImCherry/+) and different ratios of human adult cardiac fibroblasts (HCF). Our standalone application "EHT Analysis" can be applied for different studies where the physiological features of EHTs needs to be analyzed under the effect of a drug compound or in a disease model.

A New Versatile Platform for Assessment of Improved Cardiac Performance in Human-Engineered Heart Tissues.

Cardiomyocytes derived from human pluripotent stem cells (hPSC-CMs) hold a great potential as human in vitro models for studying heart disease and for drug safety screening. Nevertheless, their associated immaturity relative to the adult myocardium limits their utility in cardiac research. In this study, we describe the development of a platform for generating three-dimensional engineered heart tissues (EHTs) from hPSC-CMs for the measurement of force while under mechanical and electrical stimulation. The modular and versatile EHT platform presented here allows for the formation of three tissues per well in a 12-well plate format, resulting in 36 tissues per plate. We compared the functional performance of EHTs and their histology in three different media and demonstrated that tissues cultured and maintained in maturation medium, containing triiodothyronine (T3), dexamethasone, and insulin-like growth factor-1 (TDI), resulted in a higher force of contraction, sarcomeric organization and alignment, and a higher and lower inotropic response to isoproterenol and nifedipine, respectively. Moreover, in this study, we highlight the importance of integrating a serum-free maturation medium in the EHT platform, making it a suitable tool for cardiovascular research, disease modeling, and preclinical drug testing.

A cardiomyocyte show of force: A fluorescent alpha-actinin reporter line sheds light on human cardiomyocyte contractility versus substrate stiffness.

Cardiovascular disease is often associated with cardiac remodeling, including cardiac fibrosis, which may lead to increased stiffness of the heart wall. This stiffness in turn may cause subsequent failure of cardiac myocytes, however the response of these cells to increased substrate stiffness is largely unknown. To investigate the contractile response of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) to increased substrate stiffness, we generated a stable transgenic human pluripotent stem cell line expressing a fusion protein of α-Actinin and fluorescent mRubyII in a previously characterized NKX2.5-GFP reporter line. Cardiomyocytes differentiated from this line were subjected to a substrate with stiffness ranging from 4 kPa to 101 kPa, while contraction of sarcomeres and bead displacement in the substrate were measured for each single cardiomyocyte. We found that sarcomere dynamics in hPSC-CMs on polyacrylamide gels of increasing stiffness are not affected above physiological levels (21 kPa), but that contractile force increases up to a stiffness of 90 kPa, at which cell shortening, deducted from bead displacement, is significantly reduced compared to physiological stiffness. We therefore hypothesize that this discrepancy may be the cause of intracellular stress that leads to hypertrophy and consequent heart failure in vivo.

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.

Human Pluripotent Stem Cell Differentiation into Functional Epicardial Progenitor Cells.

Human pluripotent stem cells (hPSCs) are widely used to study cardiovascular cell differentiation and function. Here, we induced differentiation of hPSCs (both embryonic and induced) to proepicardial/epicardial progenitor cells that cover the heart during development. Addition of retinoic acid (RA) and bone morphogenetic protein 4 (BMP4) promoted expression of the mesodermal marker PDGFRα, upregulated characteristic (pro)epicardial progenitor cell genes, and downregulated transcription of myocardial genes. We confirmed the (pro)epicardial-like properties of these cells using in vitro co-culture assays and in ovo grafting of hPSC-epicardial cells into chick embryos. Our data show that RA + BMP4-treated hPSCs differentiate into (pro)epicardial-like cells displaying functional properties (adhesion and spreading over the myocardium) of their in vivo counterpart. The results extend evidence that hPSCs are an excellent model to study (pro)epicardial differentiation into cardiovascular cells in human development and evaluate their potential for cardiac regeneration.

Contractile Defect Caused by Mutation in MYBPC3 Revealed under Conditions Optimized for Human PSC-Cardiomyocyte Function.

Maximizing baseline function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is essential for their effective application in models of cardiac toxicity and disease. Here, we aimed to identify factors that would promote an adequate level of function to permit robust single-cell contractility measurements in a human induced pluripotent stem cell (hiPSC) model of hypertrophic cardiomyopathy (HCM). A simple screen revealed the collaborative effects of thyroid hormone, IGF-1 and the glucocorticoid analog dexamethasone on the electrophysiology, bioenergetics, and contractile force generation of hPSC-CMs. In this optimized condition, hiPSC-CMs with mutations in MYBPC3, a gene encoding myosin-binding protein C, which, when mutated, causes HCM, showed significantly lower contractile force generation than controls. This was recapitulated by direct knockdown of MYBPC3 in control hPSC-CMs, supporting a mechanism of haploinsufficiency. Modeling this disease in vitro using human cells is an important step toward identifying therapeutic interventions for HCM.

Altered calcium handling and increased contraction force in human embryonic stem cell derived cardiomyocytes following short term dexamethasone exposure.

One limitation in using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) for disease modeling and cardiac safety pharmacology is their immature functional phenotype compared with adult cardiomyocytes. Here, we report that treatment of human embryonic stem cell derived cardiomyocytes (hESC-CMs) with dexamethasone, a synthetic glucocorticoid, activated glucocorticoid signaling which in turn improved their calcium handling properties and contractility. L-type calcium current and action potential properties were not affected by dexamethasone but significantly faster calcium decay, increased forces of contraction and sarcomeric lengths, were observed in hESC-CMs after dexamethasone exposure. Activating the glucocorticoid pathway can thus contribute to mediating hPSC-CMs maturation.

Expansion and patterning of cardiovascular progenitors derived from human pluripotent stem cells.

The inability of multipotent cardiovascular progenitor cells (CPCs) to undergo multiple divisions in culture has precluded stable expansion of precursors of cardiomyocytes and vascular cells. This contrasts with neural progenitors, which can be expanded robustly and are a renewable source of their derivatives. Here we use human pluripotent stem cells bearing a cardiac lineage reporter to show that regulated MYC expression enables robust expansion of CPCs with insulin-like growth factor-1 (IGF-1) and a hedgehog pathway agonist. The CPCs can be patterned with morphogens, recreating features of heart field assignment, and controllably differentiated to relatively pure populations of pacemaker-like or ventricular-like cardiomyocytes. The cells are clonogenic and can be expanded for >40 population doublings while retaining the ability to differentiate into cardiomyocytes and vascular cells. Access to CPCs will allow precise recreation of elements of heart development in vitro and facilitate investigation of the molecular basis of cardiac fate determination. This technology is applicable for cardiac disease modeling, toxicology studies and tissue engineering.

Functional maturation of human pluripotent stem cell derived cardiomyocytes in vitro--correlation between contraction force and electrophysiology.

Cardiomyocytes from human pluripotent stem cells (hPSC-CM) have many potential applications in disease modelling and drug target discovery but their phenotypic similarity to early fetal stages of cardiac development limits their applicability. In this study we compared contraction stresses of hPSC-CM to 2nd trimester human fetal derived cardiomyocytes (hFetal-CM) by imaging displacement of fluorescent beads by single contracting hPSC-CM, aligned by microcontact-printing on polyacrylamide gels. hPSC-CM showed distinctly lower contraction stress than cardiomyocytes isolated from hFetal-CM. To improve maturation of hPSC-CM in vitro we made use of commercial media optimized for cardiomyocyte maturation, which promoted significantly higher contraction stress in hPSC-compared with hFetal-CM. Accordingly, other features of cardiomyocyte maturation were observed, most strikingly increased upstroke velocities and action potential amplitudes, lower resting membrane potentials, improved sarcomeric organization and alterations in cardiac-specific gene expression. Performing contraction force and electrophysiology measurements on individual cardiomyocytes revealed strong correlations between an increase in contraction force and a rise of the upstroke velocity and action potential amplitude and with a decrease in the resting membrane potential. We showed that under standard differentiation conditions hPSC-CM display lower contractile force than primary hFetal-CM and identified conditions under which a commercially available culture medium could induce molecular, morphological and functional maturation of hPSC-CM in vitro. These results are an important contribution for full implementation of hPSC-CM in cardiac disease modelling and drug discovery.

A positive feedback between p53 and miR-34 miRNAs mediates tumor suppression.

As bona fide p53 transcriptional targets, miR-34 microRNAs (miRNAs) exhibit frequent alterations in many human tumor types and elicit multiple p53 downstream effects upon overexpression. Unexpectedly, miR-34 deletion alone fails to impair multiple p53-mediated tumor suppressor effects in mice, possibly due to the considerable redundancy in the p53 pathway. Here, we demonstrate that miR-34a represses HDM4, a potent negative regulator of p53, creating a positive feedback loop acting on p53. In a Kras-induced mouse lung cancer model, miR-34a deficiency alone does not exhibit a strong oncogenic effect. However, miR-34a deficiency strongly promotes tumorigenesis when p53 is haploinsufficient, suggesting that the defective p53-miR-34 feedback loop can enhance oncogenesis in a specific context. The importance of the p53/miR-34/HDM4 feedback loop is further confirmed by an inverse correlation between miR-34 and full-length HDM4 in human lung adenocarcinomas. In addition, human lung adenocarcinomas generate an elevated level of a short HDM4 isoform through alternative polyadenylation. This short HDM4 isoform lacks miR-34-binding sites in the 3' untranslated region (UTR), thereby evading miR-34 regulation to disable the p53-miR-34 positive feedback. Taken together, our results elucidated the intricate cross-talk between p53 and miR-34 miRNAs and revealed an important tumor suppressor effect generated by this positive feedback loop.