BackgroundMicroRNAs (miRs) negatively regulate transcription and are important determinants of normal heart development and heart failure pathogenesis. Despite the significant knowledge gained in mouse studies, their functional roles in human (h) heart remain elusive.Methods and ResultsWe hypothesized that miRs that figure prominently in cardiac differentiation are differentially expressed in differentiating, developing, and terminally mature human cardiomyocytes (CMs). As a first step, we mapped the miR profiles of human (h) embryonic stem cells (ESCs), hESC-derived (hE), fetal (hF) and adult (hA) ventricular (V) CMs. 63 miRs were differentially expressed between hESCs and hE-VCMs. Of these, 29, including the miR-302 and -371/372/373 clusters, were associated with pluripotency and uniquely expressed in hESCs. Of the remaining miRs differentially expressed in hE-VCMs, 23 continued to express highly in hF- and hA-VCMs, with miR-1, -133, and -499 displaying the largest fold differences; others such as miR-let-7a, -let-7b, -26b, -125a and -143 were non-cardiac specific. Functionally, LV-miR-499 transduction of hESC-derived cardiovascular progenitors significantly increased the yield of hE-VCMs (to 72% from 48% of control; p<0.05) and contractile protein expression without affecting their electrophysiological properties (p>0.05). By contrast, LV-miR-1 transduction did not bias the yield (p>0.05) but decreased APD and hyperpolarized RMP/MDP in hE-VCMs due to increased Ito, IKs and IKr, and decreased If (p<0.05) as signs of functional maturation. Also, LV-miR-1 but not -499 augmented the immature Ca2+ transient amplitude and kinetics. Molecular pathway analyses were performed for further insights.ConclusionWe conclude that miR-1 and -499 play differential roles in cardiac differentiation of hESCs in a context-dependent fashion. While miR-499 promotes ventricular specification of hESCs, miR-1 serves to facilitate electrophysiological maturation.

A number of genetic mutations is associated with cardiomyopathies. A mutation in the coding region of the phospholamban (PLN) gene (R14del) is identified in families with hereditary heart failure. Heterozygous patients exhibit left ventricular dilation and ventricular arrhythmias. Here we generate induced pluripotent stem cells (iPSCs) from a patient harbouring the PLN R14del mutation and differentiate them into cardiomyocytes (iPSC-CMs). We find that the PLN R14del mutation induces Ca2+ handling abnormalities, electrical instability, abnormal cytoplasmic distribution of PLN protein and increases expression of molecular markers of cardiac hypertrophy in iPSC-CMs. Gene correction using transcription activator-like effector nucleases (TALENs) ameliorates the R14del-associated disease phenotypes in iPSC-CMs. In addition, we show that knocking down the endogenous PLN and simultaneously expressing a codon-optimized PLN gene reverses the disease phenotype in vitro. Our findings offer novel strategies for targeting the pathogenic mutations associated with cardiomyopathies.

In adult cardiomyocytes (CMs), the Na + /Ca 2+ exchanger (NCX) is a well-defi ned determinant of Ca 2+ homeostasis. Developmentally, global NCX knockout in mice leads to abnormal myofi brillar organization, electrical defects, and early embryonic death. Little is known about the expression and function of NCX in human heart development. Self-renewable, pluripotent human embryonic stem cells (hESCs) can serve as an excellent experimental model. However, hESC-derived CMs are highly heterogeneous. A stably lentivirus-transduced hESC line (MLC2v-dsRed) was generated to express dsRed under the transcriptional control of the ventricular-restricted myosin light chain-2v (MLC2v) promoter. Electrophysiologically, dsRed+ cells differentiated from MLC2v-dsRed hESCs displayed ventricular action potentials (AP), exclusively. Neither atrial nor pacemaker APs were observed. While I Ca-L , I f , and I Kr were robustly expressed, I Ks and I K1 were absent in dsRed+ ventricular hESCCMs. Upon differentiation (7+40 to +90 days), the basal [Ca 2+ ] i , Ca 2+ transient amplitude, maximum upstroke, and decay velocities signifi cantly increased (P < 0.05). The I Ca-L antagonizer nifedipine (1 μM) decreased the Ca 2+ transient amplitude (to ~30%) and slowed the kinetics (by ~2-fold), but Ca 2+ transients could still be elicited even after complete I Ca-L blockade, suggesting the presence of additional Ca 2+ infl ux(es). Indeed, Ni 2+-sensitive I NCX could be recorded in 7+40-and +90-day dsRed+ hESC-CMs, and its densities increased from −1.2 ± 0.6 pA/pF at −120 mV and 3.6 ± 1.0 pA/pF at 60 mV by 6-and 2-folds, respectively. With higher [Ca 2+ ] i , 7+90-day ventricular hESC-CMs spontaneously but irregularly fi red transients upon a single stimulus under an external Na + -free condition; however, without extracellular Na + , nifedipine could completely inhibit Ca 2+ transients. We conclude that I NCX is functionally expressed in developing ventricular hESC-CMs and contributes to their excitationcontraction coupling.

Human (h) embryonic stem cells (ESC) represent an unlimited source of cardiomyocytes (CMs); however, these differentiated cells are immature. Thus far, gene profiling studies have been performed with non-purified or non-chamber specific CMs. Here we took a combinatorial approach of using systems biology to guide functional discoveries of novel biological properties of purified hESC-derived ventricular (V) CMs. We profiled the transcriptomes of hESCs, hESC-, fetal (hF) and adult (hA) VCMs, and showed that hESC-VCMs displayed a unique transcriptomic signature. Not only did a detailed comparison between hESC-VCMs and hF-VCMs confirm known expression changes in metabolic and contractile genes, it further revealed novel differences in genes associated with reactive oxygen species (ROS) metabolism, migration and cell cycle, as well as potassium and calcium ion transport. Following these guides, we functionally confirmed that hESC-VCMs expressed IKATP with immature properties, and were accordingly vulnerable to hypoxia/reoxygenation-induced apoptosis. For mechanistic insights, our coexpression and promoter analyses uncovered a novel transcriptional hierarchy involving select transcription factors (GATA4, HAND1, NKX2.5, PPARGC1A and TCF8), and genes involved in contraction, calcium homeostasis and metabolism. These data highlight novel expression and functional differences between hESC-VCMs and their fetal counterparts, and offer insights into the underlying cell developmental state. These findings may lead to mechanism-based methods for in vitro driven maturation.

Human embryonic stem cells (hESCs) can self-renew while maintaining their pluripotency. Direct reprogramming of adult somatic cells to induced pluripotent stem cells (iPSCs) has been reported. Although hESCs and human iPSCs have been shown to share a number of similarities, such basic properties as the electrophysiology of iPSCs have not been explored. Previously, we reported that several specialized ion channels are functionally expressed in hESCs. Using transcriptomic analyses as a guide, we observed tetraethylammonium (TEA)-sensitive (IC50 = 3.3 ± 2.7 mM) delayed rectifier K+ currents ( IKDR) in 105 of 110 single iPSCs (15.4 ± 0.9 pF). IKDR in iPSCs displayed a current density of 7.6 ± 3.8 pA/pF at +40 mV. The voltage for 50% activation ( V1/2) was −7.9 ± 2.0 mV, slope factor k = 9.1 ± 1.5. However, Ca2+-activated K+ current ( IKCa), hyperpolarization-activated pacemaker current ( If), and voltage-gated sodium channel (NaV) and voltage-gated calcium channel (CaV) currents could not be measured. TEA inhibited iPSC proliferation (EC50 = 7.8 ± 1.2 mM) and viability (EC50 = 5.5 ± 1.0 mM). By contrast, 4-aminopyridine (4-AP) inhibited viability (EC50 = 4.5 ± 0.5 mM) but had less effect on proliferation (EC50 = 0.9 ± 0.5 mM). Cell cycle analysis further revealed that K+ channel blockers inhibited proliferation primarily by arresting the mitotic phase. TEA and 4-AP had no effect on iPSC differentiation as gauged by ability to form embryoid bodies and expression of germ layer markers after induction of differentiation. Neither iberiotoxin nor apamin had any function effects, consistent with the lack of IKCa in iPSCs. Our results reveal further differences and similarities between human iPSCs and hESCs. A better understanding of the basic biology of iPSCs may facilitate their ultimate clinical application.

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Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells can self-renew while maintaining their pluripotency to differentiate into virtually all cell types. In addition to their potential for regenerative medicine, hESCs and iPSCs can also serve as excellent in vitro models for the study of human organogenesis and disease models, as well as drug toxicity screening. MicroRNAs (miRNAs) are nonencoding RNAs of ∼22 nucleotides that function as negative transcriptional regulators via degradation or inhibition by RNA interference (RNAi). MiRNAs play essential roles in developmental pathways. This chapter provides a description of how miRNAs can be introduced into hESCs/iPSCs or their derivatives for experiments via lentivirus-mediated gene transfer.