Ronald A. Li

Modeling susceptibility to drug-induced long QT with a panel of subject-specific induced pluripotent stem cells

A large number of drugs can induce prolongation of cardiac repolarization and life-threatening cardiac arrhythmias. The prediction of this side effect is however challenging as it usually develops in some genetically predisposed individuals with normal cardiac repolarization at baseline. Here, we describe a platform based on a genetically diverse panel of induced pluripotent stem cells (iPSCs) that reproduces susceptibility to develop a cardiotoxic drug response. We generated iPSC-derived cardiomyocytes from patients presenting in vivo with extremely low or high changes in cardiac repolarization in response to a pharmacological challenge with sotalol. In vitro, the responses to sotalol were highly variable but strongly correlated to the inter-individual differences observed in vivo. Transcriptomic profiling identified dysregulation of genes (DLG2, KCNE4, PTRF, HTR2C, CAMKV) involved in downstream regulation of cardiac repolarization machinery as underlying high sensitivity to sotalol. Our findings offer novel insights for the development of iPSC-based screening assays for testing individual drug reactions. DOI: http://dx.doi.org/10.7554/eLife.19406.001

Electrophysiological Mechanisms of Brugada Syndrome: Insights from Pre-clinical and Clinical Studies

Brugada syndrome (BrS), is a primary electrical disorder predisposing affected individuals to sudden cardiac death via the development of ventricular tachycardia and fibrillation (VT/VF). Originally, BrS was linked to mutations in the SCN5A, which encodes for the cardiac Na+ channel. To date, variants in 19 genes have been implicated in this condition, with 11, 5, 3, and 1 genes affecting the Na+, K+, Ca2+, and funny currents, respectively. Diagnosis of BrS is based on ECG criteria of coved- or saddle-shaped ST segment elevation and/or T-wave inversion with or without drug challenge. Three hypotheses based on abnormal depolarization, abnormal repolarization, and current-load-mismatch have been put forward to explain the electrophysiological mechanisms responsible for BrS. Evidence from computational modeling, pre-clinical, and clinical studies illustrates that molecular abnormalities found in BrS lead to alterations in excitation wavelength (λ), which ultimately elevates arrhythmic risk. A major challenge for clinicians in managing this condition is the difficulty in predicting the subset of patients who will suffer from life-threatening VT/VF. Several repolarization risk markers have been used thus far, but these neglect the contributions of conduction abnormalities in the form of slowing and dispersion. Indices incorporating both repolarization and conduction and based on the concept of λ have recently been proposed. These may have better predictive values than the existing markers.

Nitric Oxide‐cGMP‐PKG Pathway Acts on Orai1 to Inhibit the Hypertrophy of Human Embryonic Stem Cell‐Derived Cardiomyocytes

Cardiac hypertrophy is an abnormal enlargement of heart muscle. It frequently results in congestive heart failure, which is a leading cause of human death. Previous studies demonstrated that the nitric oxide (NO), cyclic GMP (cGMP), and protein kinase G (PKG) signaling pathway can inhibit cardiac hypertrophy and thus improve cardiac function. However, the underlying mechanisms are not fully understood. Here, based on the human embryonic stem cell‐derived cardiomyocyte (hESC‐CM) model system, we showed that Orai1, the pore‐forming subunit of store‐operated Ca2+ entry (SOCE), is the downstream effector of PKG. Treatment of hESC‐CMs with an α‐adrenoceptor agonist phenylephrine (PE) caused a marked hypertrophy, which was accompanied by an upregulation of Orai1. Moreover, suppression of Orai1 expression/activity using Orai1‐siRNAs or a dominant‐negative construct Orai1G98A inhibited the hypertrophy, suggesting that Orai1‐mediated SOCE is indispensable for the PE‐induced hypertrophy of hESC‐CMs. In addition, the hypertrophy was inhibited by NO and cGMP via activating PKG. Importantly, substitution of Ala for Ser34 in Orai1 abolished the antihypertrophic effects of NO, cGMP, and PKG. Furthermore, PKG could directly phosphorylate Orai1 at Ser34 and thus prevent Orai1‐mediated SOCE. Together, we conclude that NO, cGMP, and PKG inhibit the hypertrophy of hESC‐CMs via PKG‐mediated phosphorylation on Orai1‐Ser‐34. These results provide novel mechanistic insights into the action of cGMP‐PKG‐related antihypertrophic agents, such as NO donors and sildenafil. Stem Cells 2015;33:2973–2984

Machine Learning of Human Pluripotent Stem Cell-Derived Engineered Cardiac Tissue Contractility for Automated Drug Classification

Summary Accurately predicting cardioactive effects of new molecular entities for therapeutics remains a daunting challenge. Immense research effort has been focused toward creating new screening platforms that utilize human pluripotent stem cell (hPSC)-derived cardiomyocytes and three-dimensional engineered cardiac tissue constructs to better recapitulate human heart function and drug responses. As these new platforms become increasingly sophisticated and high throughput, the drug screens result in larger multidimensional datasets. Improved automated analysis methods must therefore be developed in parallel to fully comprehend the cellular response across a multidimensional parameter space. Here, we describe the use of machine learning to comprehensively analyze 17 functional parameters derived from force readouts of hPSC-derived ventricular cardiac tissue strips (hvCTS) electrically paced at a range of frequencies and exposed to a library of compounds. A generated metric is effective for then determining the cardioactivity of a given drug. Furthermore, we demonstrate a classification model that can automatically predict the mechanistic action of an unknown cardioactive drug.

Polycystin‐2 Plays an Essential Role in Glucose Starvation‐Induced Autophagy in Human Embryonic Stem Cell‐Derived Cardiomyocytes

Autophagy is a process essential for cell survival under stress condition. The patients with autosomal dominant polycystic kidney disease, which is caused by polycystin‐1 or polycystin‐2 (PKD2) mutation, display cardiovascular abnormalities and dysregulation in autophagy. However, it is unclear whether PKD2 plays a role in autophagy. In the present study, we explored the functional role of PKD2 in autophagy and apoptosis in human embryonic stem cell‐derived cardiomyocytes. HES2 hESC line‐derived cardiomyocytes (HES2‐CMs) were transduced with adenoviral‐based PKD2‐shRNAs (Ad‐PKD2‐shRNAs), and then cultured with normal or glucose‐free medium for 3 hours. Autophagy was upregulated in HES2‐CMs under glucose starvation, as indicated by increased microtubule‐associated protein 1 light chain 3‐II level in immunoblots and increased autophagosome and autolysosome formation. Knockdown of PKD2 reduced the autophagic flux and increased apoptosis under glucose starvation. In Ca2+ measurement, Ad‐PKD2‐shRNAs reduced caffeine‐induced cytosolic Ca2+ rise. Co‐immunoprecipitation and in situ proximity ligation assay demonstrated an increased physical interaction of PKD2 with ryanodine receptor 2 (RyR2) under glucose starvation condition. Furthermore, Ad‐PKD2‐shRNAs substantially attenuated the starvation‐induced activation of AMP‐activated protein kinase (AMPK) and inactivation of mammalian target of rapamycin (mTOR). The present study for the first time demonstrates that PKD2 functions to promote autophagy under glucose starvation, thereby protects cardiomyocytes from apoptotic cell death. The mechanism may involve PKD2 interaction with RyR2 to alter Ca2+ release from sarcoplasmic reticulum, consequently modulating the activity of AMPK and mTOR, resulting in alteration of autophagy and apoptosis. Stem Cells 2018;36:501–513

A Singular Role of IK1 Promoting the Development of Cardiac Automaticity during Cardiomyocyte Differentiation by IK1–Induced Activation of Pacemaker Current

The inward rectifier potassium current (IK1) is generally thought to suppress cardiac automaticity by hyperpolarizing membrane potential (MP). We recently observed that IK1 could promote the spontaneously-firing automaticity induced by upregulation of pacemaker funny current (If) in adult ventricular cardiomyocytes (CMs). However, the intriguing ability of IK1 to activate If and thereby promote automaticity has not been explored. In this study, we combined mathematical and experimental assays and found that only IK1 and If, at a proper-ratio of densities, were sufficient to generate rhythmic MP-oscillations even in unexcitable cells (i.e. HEK293T cells and undifferentiated mouse embryonic stem cells [ESCs]). We termed this effect IK1-induced If activation. Consistent with previous findings, our electrophysiological recordings observed that around 50% of mouse (m) and human (h) ESC-differentiated CMs could spontaneously fire action potentials (APs). We found that spontaneously-firing ESC-CMs displayed more hyperpolarized maximum diastolic potential and more outward IK1 current than quiescent-yet-excitable m/hESC-CMs. Rather than classical depolarization pacing, quiescent mESC-CMs were able to fire APs spontaneously with an electrode-injected small outward-current that hyperpolarizes MP. The automaticity to spontaneously fire APs was also promoted in quiescent hESC-CMs by an IK1-specific agonist zacopride. In addition, we found that the number of spontaneously-firing m/hESC-CMs was significantly decreased when If was acutely upregulated by Ad-CGI-HCN infection. Our study reveals a novel role of IK1 promoting the development of cardiac automaticity in m/hESC-CMs through a mechanism of IK1-induced If activation and demonstrates a synergistic interaction between IK1 and If that regulates cardiac automaticity.

A Micropatterned Human Pluripotent Stem Cell‐Based Ventricular Cardiac Anisotropic Sheet for Visualizing Drug‐Induced Arrhythmogenicity

A novel cardiomimetic biohybrid material, termed as the human ventricular cardiac anisotropic sheet (hvCAS) is reported. Well-characterized human pluripotent stem-cell-derived ventricular cardiomyocytes are strategically aligned to reproduce key electrophysiological features of native human ventricle, which, along with specific selection criteria, allows for a direct visualization of arrhythmic spiral re-entry and represents a revolutionary tool to assess preclinical drug-induced arrhythmogenicity.

Human ISL1+ Ventricular Progenitors Self-Assemble into an In Vivo Functional Heart Patch and Preserve Cardiac Function Post Infarction

The generation of human pluripotent stem cell (hPSC)-derived ventricular progenitors and their assembly into a 3-dimensional in vivo functional ventricular heart patch has remained an elusive goal. Herein, we report the generation of an enriched pool of hPSC-derived ventricular progenitors (HVPs), which can expand, differentiate, self-assemble, and mature into a functional ventricular patch in vivo without the aid of any gel or matrix. We documented a specific temporal window, in which the HVPs will engraft in vivo. On day 6 of differentiation, HVPs were enriched by depleting cells positive for pluripotency marker TRA-1-60 with magnetic-activated cell sorting (MACS), and 3 million sorted cells were sub-capsularly transplanted onto kidneys of NSG mice where, after 2 months, they formed a 7 mm × 3 mm × 4 mm myocardial patch resembling the ventricular wall. The graft acquired several features of maturation: expression of ventricular marker (MLC2v), desmosomes, appearance of T-tubule-like structures, and electrophysiological action potential signature consistent with maturation, all this in a non-cardiac environment. We further demonstrated that HVPs transplanted into un-injured hearts of NSG mice remain viable for up to 8 months. Moreover, transplantation of 2 million HVPs largely preserved myocardial contractile function following myocardial infarction. Taken together, our study reaffirms the promising idea of using progenitor cells for regenerative therapy.

Gene Delivery for the Generation of Bioartificial Pacemaker

Electronic pacemakers have been used in patients with heart rhythm disorders for device-supported pacing. While effective, there are such shortcomings as limited battery life, permanent implantation of catheters, the lack of autonomic neurohumoral responses, and risks of lead dislodging. Here we describe protocols for establishing porcine models of sick sinus syndrome and complete heart block, and the generation of bioartificial pacemaker by delivering a strategically engineered form of hyperpolarization-activated cyclic nucleotide-gated pacemaker channel protein via somatic gene transfer to convert atrial or ventricular muscle cardiomyocytes into nodal-like cells that rhythmically fire action potentials.

An abnormal TRPV4-related cytosolic Ca2+ rise in response to uniaxial stretch in induced pluripotent stem cells-derived cardiomyocytes from dilated cardiomyopathy patients.

Dilated cardiomyopathy (DCM) is cardiac disease characterized by increased left ventricular chamber volume and decreased systolic function. DCM patient-specific human induced-pluripotent stem cells-derived cardiomyocytes (DCM-hiPSC-CMs) were generated. We found that uniaxial stretch elicited a cytosolic [Ca2+]i rise in hiPSC-CMs. Compared to control-hiPSC-CMs, DCM-hiPSC-CMs displayed a greater magnitude of [Ca2+]i responses to the cell stretch of 10-15% elongation in length. This stretch-induced [Ca2+]i rise was abolished by removal of extracellular Ca2+ and markedly attenuated by TRPV4 inhibitors HC-067047 and RN-1734. Application of nifedipine and tranilast also reduced the [Ca2+]i response but to a lesser degree. Moreover, the augmented [Ca2+]i was decreased by cytochalasin D treatment. Taken together, our study for the first time demonstrated an abnormal TRPV4-related mechanosensitive Ca2+ signaling in DCM-hiPSC-CMs.