Human engineered cardiac tissue model of hypertrophic cardiomyopathy recapitulates key hallmarks of the disease and the effect of chronic mavacamten treatment.

Introduction: The development of patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offers an opportunity to study genotype-phenotype correlation of hypertrophic cardiomyopathy (HCM), one of the most common inherited cardiac diseases. However, immaturity of the iPSC-CMs and the lack of a multicellular composition pose concerns over its faithfulness in disease modeling and its utility in developing mechanism-specific treatment. Methods: The Biowire platform was used to generate 3D engineered cardiac tissues (ECTs) using HCM patient-derived iPSC-CMs carrying a ß-myosin mutation (MYH7-R403Q) and its isogenic control (WT), withal ECTs contained healthy human cardiac fibroblasts. ECTs were subjected to electro-mechanical maturation for 6 weeks before being used in HCM phenotype studies. Results: Both WT and R403Q ECTs exhibited mature cardiac phenotypes, including a lack of automaticity and a ventricular-like action potential (AP) with a resting membrane potential < -75 mV. Compared to WT, R403Q ECTs demonstrated many HCM-associated pathological changes including increased tissue size and cell volume, shortened sarcomere length and disorganized sarcomere structure. In functional assays, R403Q ECTs showed increased twitch amplitude, slower contractile kinetics, a less pronounced force-frequency relationship, a smaller post-rest potentiation, prolonged AP durations, and slower Ca2+ transient decay time. Finally, we observed downregulation of calcium handling genes and upregulation of NPPB in R403Q vs. WT ECTs. In an HCM phenotype prevention experiment, ECTs were treated for 5-weeks with 250 nM mavacamten or a vehicle control. We found that chronic mavacamten treatment of R403Q ECTs: (i) shortened relaxation time, (ii) reduced APD90 prolongation, (iii) upregulated ADRB2, ATP2A2, RYR2, and CACNA1C, (iv) decreased B-type natriuretic peptide (BNP) mRNA and protein expression levels, and (v) increased sarcomere length and reduced sarcomere disarray. Discussion: Taken together, we demonstrated R403Q ECTs generated in the Biowire platform recapitulated many cardiac hypertrophy phenotypes and that chronic mavacamten treatment prevented much of the pathology. This demonstrates that the Biowire ECTs are well-suited to phenotypic-based drug discovery in a human-relevant disease model.

Activin A directly impairs human cardiomyocyte contractile function indicating a potential role in heart failure development.

Activin A has been linked to cardiac dysfunction in aging and disease, with elevated circulating levels found in patients with hypertension, atherosclerosis, and heart failure. Here, we investigated whether Activin A directly impairs cardiomyocyte (CM) contractile function and kinetics utilizing cell, tissue, and animal models. Hydrodynamic gene delivery-mediated overexpression of Activin A in wild-type mice was sufficient to impair cardiac function, and resulted in increased cardiac stress markers (N-terminal pro-atrial natriuretic peptide) and cardiac atrophy. In human-induced pluripotent stem cell-derived (hiPSC) CMs, Activin A caused increased phosphorylation of SMAD2/3 and significantly upregulated SERPINE1 and FSTL3 (markers of SMAD2/3 activation and activin signaling, respectively). Activin A signaling in hiPSC-CMs resulted in impaired contractility, prolonged relaxation kinetics, and spontaneous beating in a dose-dependent manner. To identify the cardiac cellular source of Activin A, inflammatory cytokines were applied to human cardiac fibroblasts. Interleukin -1ß induced a strong upregulation of Activin A. Mechanistically, we observed that Activin A-treated hiPSC-CMs exhibited impaired diastolic calcium handling with reduced expression of calcium regulatory genes (SERCA2, RYR2, CACNB2). Importantly, when Activin A was inhibited with an anti-Activin A antibody, maladaptive calcium handling and CM contractile dysfunction were abrogated. Therefore, inflammatory cytokines may play a key role by acting on cardiac fibroblasts, causing local upregulation of Activin A that directly acts on CMs to impair contractility. These findings demonstrate that Activin A acts directly on CMs, which may contribute to the cardiac dysfunction seen in aging populations and in patients with heart failure.

Engineering microenvironment for human cardiac tissue assembly in heart-on-a-chip platform.

Organ-on-a-chip systems have the potential to revolutionize drug screening and disease modeling through the use of human stem cell-derived cardiomyocytes. The predictive power of these tissue models critically depends on the functional assembly and maturation of human cells that are used as building blocks for organ-on-a-chip systems. To resemble a more adult-like phenotype on these heart-on-a-chip systems, the surrounding micro-environment of individual cardiomyocyte needs to be controlled. Herein, we investigated the impact of four microenvironmental cues: cell seeding density, types and percentages of non-myocyte populations, the types of hydrogels used for tissue inoculation and the electrical conditioning regimes on the structural and functional assembly of human pluripotent stem cell-derived cardiac tissues. Utilizing a novel, plastic and open-access heart-on-a-chip system that is capable of continuous non-invasive monitoring of tissue contractions, we were able to study how different micro-environmental cues affect the assembly of the cardiomyocytes into a functional cardiac tissue. We have defined conditions that resulted in tissues exhibiting hallmarks of the mature human myocardium, such as positive force-frequency relationship and post-rest potentiation.

Engineered Cardiac Tissues Generated in the Biowire II: A Platform for Human-Based Drug Discovery.

Recent advances in techniques to differentiate human induced pluripotent stem cells (hiPSCs) hold the promise of an unlimited supply of human derived cardiac cells from both healthy and disease populations. That promise has been tempered by the observation that hiPSC-derived cardiomyocytes (hiPSC-CMs) typically retain a fetal-like phenotype, raising concern about the translatability of the in vitro data obtained to drug safety, discovery, and development studies. The Biowire II platform was used to generate 3D engineered cardiac tissues (ECTs) from hiPSC-CMs and cardiac fibroblasts. Long term electrical stimulation was employed to obtain ECTs that possess a phenotype like that of adult human myocardium including a lack of spontaneous beating, the presence of a positive force-frequency response from 1 to 4 Hz and prominent postrest potentiation. Pharmacology studies were performed in the ECTs to confirm the presence and functionality of pathways that modulate cardiac contractility in humans. Canonical responses were observed for compounds that act via the ß-adrenergic/cAMP-mediated pathway, eg, isoproterenol and milrinone; the L-type calcium channel, eg, FPL64176 and nifedipine; and indirectly effect intracellular Ca2+ concentrations, eg, digoxin. Expected positive inotropic responses were observed for compounds that modulate proteins of the cardiac sarcomere, eg, omecamtiv mecarbil and levosimendan. ECTs generated in the Biowire II platform display adult-like properties and have canonical responses to cardiotherapeutic and cardiotoxic agents that affect contractility in humans via a variety of mechanisms. These data demonstrate that this human-based model can be used to assess the effects of novel compounds on contractility early in the drug discovery and development process.

A Platform for Generation of Chamber-Specific Cardiac Tissues and Disease Modeling.

Tissue engineering using cardiomyocytes derived from human pluripotent stem cells holds a promise to revolutionize drug discovery, but only if limitations related to cardiac chamber specification and platform versatility can be overcome. We describe here a scalable tissue-cultivation platform that is cell source agnostic and enables drug testing under electrical pacing. The plastic platform enabled on-line noninvasive recording of passive tension, active force, contractile dynamics, and Ca2+ transients, as well as endpoint assessments of action potentials and conduction velocity. By combining directed cell differentiation with electrical field conditioning, we engineered electrophysiologically distinct atrial and ventricular tissues with chamber-specific drug responses and gene expression. We report, for the first time, engineering of heteropolar cardiac tissues containing distinct atrial and ventricular ends, and we demonstrate their spatially confined responses to serotonin and ranolazine. Uniquely, electrical conditioning for up to 8 months enabled modeling of polygenic left ventricular hypertrophy starting from patient cells.

Strategies and Challenges to Myocardial Replacement Therapy.

UNLABELLED: Cardiovascular diseases account for the majority of deaths globally and are a significant drain on economic resources. Although heart transplants and left-ventricle assist devices are the solution for some, the best chance for many patients who suffer because of a myocardial infarction, heart failure, or a congenital heart disease may be cell-based regenerative therapies. Such therapies can be divided into two categories: the application of a cell suspension and the implantation of an in vitro engineered tissue construct to the damaged area of the heart. Both strategies have their advantages and challenges, and in this review, we discuss the current state of the art in myocardial regeneration, the challenges to success, and the future direction of the field. SIGNIFICANCE: This article outlines the advantages and limitations of the cell injection and patch approaches to cardiac regenerative therapy. If the field is to move forward, some fundamental questions require answers, including the limitations to the use of animal models for human cell-transplantation studies; the best way to measure success in terms of functional improvements, histological integration, electrical coupling, and arrhythmias; and where the cells should be applied for maximal benefit-the epicardium or the myocardium.

Maturing human pluripotent stem cell-derived cardiomyocytes in human engineered cardiac tissues.

Engineering functional human cardiac tissue that mimics the native adult morphological and functional phenotype has been a long held objective. In the last 5 years, the field of cardiac tissue engineering has transitioned from cardiac tissues derived from various animal species to the production of the first generation of human engineered cardiac tissues (hECTs), due to recent advances in human stem cell biology. Despite this progress, the hECTs generated to date remain immature relative to the native adult myocardium. In this review, we focus on the maturation challenge in the context of hECTs, the present state of the art, and future perspectives in terms of regenerative medicine, drug discovery, preclinical safety testing and pathophysiological studies.

Combined hypoxia and sodium nitrite pretreatment for cardiomyocyte protection in vitro.

Methods that increase cardiomyocyte survival upon exposure to ischemia, hypoxia and reoxygenation injuries are required to improve the efficacy of cardiac cell therapy and enhance the viability and function of engineered tissues. We investigated the effect of combined hypoxia/NaNO2 pretreatment on rat neonatal cardiomyocyte (CM), cardiac fibroblast, and human embryonic stem cell-derived CM (hESC-CM) survival upon exposure to hypoxia/reoxygenation (H/R) injury in vitro. Cells were pretreated with and without hypoxia and/or various concentrations of NaNO2 for 20 min, then incubated for 2 h under hypoxic conditions, followed by 2 h in normoxia. The control cells were maintained under normoxia for 4 h. Pretreatment with either hypoxia or NaNO2 significantly increased CM viability but had no effect on cardiac fibroblast viability. Combined hypoxia/NaNO2 pretreatment significantly increased CM viability but significantly decreased cardiac fibroblast viability. In rat neonatal CMs, cell death, as determined by lactate dehydrogenase (LDH) activity, was significantly reduced with hypoxia/NaNO2 pretreatment; and in hESC-CMs, hypoxia/NaNO2 pretreatment increased the BCL-2/BAX gene expression ratio, suggesting that hypoxia/NaNO2 pretreatment promotes cell viability by downregulating apoptosis. Additionally, we found a correlation between the prosurvival effect of hypoxia/NaNO2 pretreatment and the myoglobin content of the cells by comparing neonatal rat ventricular and atrial CMs, which express high and low myoglobin respectively. Functionally, hypoxia/NaNO2 pretreatment significantly improved the excitation threshold upon H/R injury to the level observed for uninjured cells, whereas pretreatment did not affect the maximum capture rate. Hence, hypoxia/NaNO2 pretreatment may serve as a strategy to increase CM survival in cardiac regenerative therapy applications and tissue engineering.

The role of tissue engineering and biomaterials in cardiac regenerative medicine.

In recent years, the development of 3-dimensional engineered heart tissue (EHT) has made large strides forward because of advances in stem cell biology, materials science, prevascularization strategies, and nanotechnology. As a result, the role of tissue engineering in cardiac regenerative medicine has become multifaceted as new applications become feasible. Cardiac tissue engineering has long been established to have the potential to partially or fully restore cardiac function after cardiac injury. However, EHTs may also serve as surrogate human cardiac tissue for drug-related toxicity screening. Cardiotoxicity remains a major cause of drug withdrawal in the pharmaceutical industry. Unsafe drugs reach the market because preclinical evaluation is insufficient to weed out cardiotoxic drugs in all their forms. Bioengineering methods could provide functional and mature human myocardial tissues, ie, physiologically relevant platforms, for screening the cardiotoxic effects of pharmaceutical agents and facilitate the discovery of new therapeutic agents. Finally, advances in induced pluripotent stem cells have made patient-specific EHTs possible, which opens up the possibility of personalized medicine. Herein, we give an overview of the present state of the art in cardiac tissue engineering, the challenges to the field, and future perspectives.

Inhibition of apoptosis in human induced pluripotent stem cells during expansion in a defined culture using angiopoietin-1 derived peptide QHREDGS.

Adhesion molecule signaling is critical to human pluripotent stem cell (hPSC) survival, self-renewal, and differentiation. Thus, hPSCs are grown as clumps of cells on feeder cell layers or poorly defined extracellular matrices such as Matrigel. We sought to define a small molecule that would initiate adhesion-based signaling to serve as a basis for a defined substrate for hPSC culture. Soluble angiopoeitin-1 (Ang-1)-derived peptide QHREDGS added to defined serum-free media increased hPSC colony cell number and size during long- and short-term culture when grown on feeder cell layers or Matrigel, i.e. on standard substrates, without affecting hPSC morphology, growth rate or the ability to differentiate into multiple lineages both in vitro and in vivo. Importantly, QHREDGS treatment decreased hPSC apoptosis during routine passaging and single-cell dissociation. Mechanistically, the interaction of QHREDGS with ß1-integrins increased expression of integrin-linked kinase (ILK), increased expression and activation of extracellular signal-regulated kinases 1/2 (ERK1/2), and decreased caspase-3/7 activity. QHREDGS immobilization to polyethylene glycol hydrogels significantly increased cell adhesion in a dose-dependent manner. We propose QHREDGS as a small molecule inhibitor of hPSC apoptosis and the basis of an affordable defined substrate for hPSC maintenance.

Apolipoprotein(a) acts as a chemorepellent to human vascular smooth muscle cells via integrin αVß3 and RhoA/ROCK-mediated mechanisms.

Lipoprotein(a) (Lp(a)) is an independent risk factor for the development of cardiovascular disease. Vascular smooth muscle cell (SMC) motility and plasticity, functions that are influenced by environmental cues, are vital to adaptation and remodelling in vascular physiology and pathophysiology. Lp(a) is reportedly damaging to SMC function via unknown molecular mechanisms. Apolipoprotein(a) (apo(a)), a unique glycoprotein moiety of Lp(a), has been demonstrated as its active component. The aims of this study were to determine functional effects of recombinant apo(a) on human vascular SMC motility and explore the underlying mechanism(s). Exposure of SMC to apo(a) in migration assays induced a potent, concentration-dependent chemorepulsion that was RhoA and integrin αVß3-dependent, but transforming growth factor ß-independent. SMC manipulation through RhoA gene silencing, Rho kinase inhibition, statin pre-treatment, αVß3 neutralising antibody and tyrosine kinase inhibition all markedly inhibited apo(a)-mediated SMC migration. Our data reveal unique and potent activities of apo(a) that may negatively influence SMC remodelling in cardiovascular disease. Circulating levels of Lp(a) are resistant to lipid-lowering strategies and hence a greater understanding of the mechanisms underlying its functional effects on SMC may provide alternative therapeutic targets.

Atherogenic lipids and lipoproteins trigger CD36-TLR2-dependent apoptosis in macrophages undergoing endoplasmic reticulum stress.

Macrophage apoptosis in advanced atheromata, a key process in plaque necrosis, involves the combination of ER stress with other proapoptotic stimuli. We show here that oxidized phospholipids, oxidized LDL, saturated fatty acids (SFAs), and lipoprotein(a) trigger apoptosis in ER-stressed macrophages through a mechanism requiring both CD36 and Toll-like receptor 2 (TLR2). In vivo, macrophage apoptosis was induced in SFA-fed, ER-stressed wild-type but not Cd36⁻(/)⁻ or Tlr2⁻(/)⁻ mice. For atherosclerosis, we combined TLR2 deficiency with that of TLR4, which can also promote apoptosis in ER-stressed macrophages. Advanced lesions of fat-fed Ldlr⁻(/)⁻ mice transplanted with Tlr4⁻(/)⁻Tlr2⁻(/)⁻ bone marrow were markedly protected from macrophage apoptosis and plaque necrosis compared with WT →Ldlr⁻(/)⁻ lesions. These findings provide insight into how atherogenic lipoproteins trigger macrophage apoptosis in the setting of ER stress and how TLR activation might promote macrophage apoptosis and plaque necrosis in advanced atherosclerosis.

A polymorphism in the protease-like domain of apolipoprotein(a) is associated with severe coronary artery disease.

OBJECTIVES: The purpose of this study was to identify genetic variants associated with severe coronary artery disease (CAD). METHODS AND RESULTS: We used 3 case-control studies of white subjects whose severity of CAD was assessed by angiography. The first 2 studies were used to generate hypotheses that were then tested in the third study. We tested 12,077 putative functional single nucleotide polymorphisms (SNPs) in Study 1 (781 cases, 603 controls) and identified 302 SNPs nominally associated with severe CAD. Testing these 302 SNPs in Study 2 (471 cases, 298 controls), we found 5 (in LPA, CALM1, HAP1, AP3B1, and ABCG2) were nominally associated with severe CAD and had the same risk alleles in both studies. We then tested these 5 SNPs in Study 3 (554 cases, 373 controls). We found 1 SNP that was associated with severe CAD: LPA I4399M (rs3798220). LPA encodes apolipoprotein(a), a component of lipoprotein(a). I4399M is located in the protease-like domain of apolipoprotein(a). Compared with noncarriers, carriers of the 4399M risk allele (2.7% of controls) had an adjusted odds ratio for severe CAD of 3.14 (confidence interval 1.51 to 6.56), and had 5-fold higher median plasma lipoprotein(a) levels (P=0.003). CONCLUSIONS: The LPA I4399M SNP is associated with severe CAD and plasma lipoprotein(a) levels.