Developmental Changes in Cardiomyocytes Differentiated from Human Embryonic Stem Cells: A Molecular and Electrophysiological Approach

Sartiani, Laura; Bettiol, Esther; Stillitano, Francesca; Mugelli, Alessandro; Cerbai, Elisabetta; Jaconi, Marisa

doi:10.1634/stemcells.2006-0466

Cited by 332 publications

(327 citation statements)

References 45 publications

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“…In this subset of cells, the I f blockers ZD7288 or zatebradine strongly decreased the funny current [174] , as monitored under voltage clamp, and led to a bradycardia and a depolarization of the maximum diastolic potential (MDP), which is frequently associated with a cessation of the pacemaker activity [173] . Similar concentrations of zatebradine were previously reported in hESC-CMs [175] or rabbit SAN, and, interestingly, a suppression of the MDP notch could already be observed in those studies but without a clear explanation [176] . The automaticity of those cells was not affected by KB-R7943 or the peptide FRCRCFa, two structurally different inhibitors of the NCX exchanger [177,178] .…”

Section: The Calcium-activated Potassium Channel Familysupporting

confidence: 87%

Mechanisms underlying the cardiac pacemaker: the role of SK4 calcium-activated potassium channels

et al. 2016

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The proper expression and function of the cardiac pacemaker is a critical feature of heart physiology. The sinoatrial node (SAN) in human right atrium generates an electrical stimulation approximately 70 times per minute, which propagates from a conductive network to the myocardium leading to chamber contractions during the systoles. Although the SAN and other nodal conductive structures were identified more than a century ago, the mechanisms involved in the generation of cardiac automaticity remain highly debated. In this short review, we survey the current data related to the development of the human cardiac conduction system and the various mechanisms that have been proposed to underlie the pacemaker activity. We also present the human embryonic stem cellderived cardiomyocyte system, which is used as a model for studying the pacemaker. Finally, we describe our latest characterization of the previously unrecognized role of the SK4 Ca 2+ -activated K + channel conductance in pacemaker cells. By exquisitely balancing the inward currents during the diastolic depolarization, the SK4 channels appear to play a crucial role in human cardiac automaticity.

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Section: The Calcium-activated Potassium Channel Familysupporting

confidence: 87%

Mechanisms underlying the cardiac pacemaker: the role of SK4 calcium-activated potassium channels

et al. 2016

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“…S1). These data show that hERG 1b is present in human iPSC-CMs at both the mRNA and protein levels, which was expected from studies of human ventricular myocardium (17,18) and embryonic stem cell-derived cardiomyocytes (19). Furthermore, shRNA transfection predominantly targeted hERG 1b mRNA and reduced 1b mRNA and protein by roughly one-half.…”

Section: Significancesupporting

confidence: 73%

hERG 1b is critical for human cardiac repolarization

Jones¹,

Liu²,

Vaidyanathan

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

108

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The human ether-à-go-go-related gene (hERG; or KCNH2) encodes the voltage-gated potassium channel underlying I Kr , a repolarizing current in the heart. Mutations in KCNH2 or pharmacological agents that reduce I Kr slow action potential (AP) repolarization and can trigger cardiac arrhythmias associated with long QT syndrome. Two channel-forming subunits encoded by KCNH2 (hERG 1a and 1b) are expressed in cardiac tissue. In heterologous expression systems, these subunits avidly coassemble and exhibit biophysical and pharmacological properties distinct from those of homomeric hERG 1a channels. Despite these findings, adoption of hERG 1a/1b heteromeric channels as a model for cardiac I Kr has been hampered by the lack of evidence for a direct functional role for the 1b subunit in native tissue. In this study, we measured I Kr and APs at physiological temperature in cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs). We found that specific knockdown of the 1b subunit using shRNA caused reductions in 1b mRNA, 1b protein levels, and I Kr magnitude by roughly one-half. AP duration was increased and AP variability was enhanced relative to controls. Early afterdepolarizations, considered cellular substrates for arrhythmia, were also observed in cells with reduced 1b expression. Similar behavior was elicited when channels were effectively converted from heteromers to 1a homomers by expressing a fragment corresponding to the 1a-specific N-terminal Per-Arnt-Sim domain, which is omitted from hERG 1b by alternate transcription. These findings establish that hERG 1b is critical for normal repolarization and that loss of 1b is proarrhythmic in human cardiac cells.T he human ether-à-go-go-related gene (hERG; or KCNH2) encodes the voltage-gated potassium channel underlying a native cardiac current known as I Kr (1, 2). Mutations in the gene or drugs that block the channel can diminish I Kr and slow ventricular repolarization, thus prolonging the QT interval on the surface electrocardiogram and causing long QT syndrome (LQTS) (1-3). Individuals with LQTS have an increased risk for torsades de pointes arrhythmia, syncope, and sudden cardiac death. Since 2005, a cell-based assay expressing the hERG 1a isoform as a proxy for I Kr has been the cornerstone of drug safety screening for new drug development (4). Thus, whether current drug safety assays accurately represent the native channel is of paramount importance.The first studies of heterologously expressed hERG channels described biophysical (1, 2) and pharmacological (2, 5) properties uniquely characteristic of cardiac I Kr . Subsequently, alternate transcripts of KCNH2 in mouse and human heart were shown to encode two subunits: 1a (the original isolate) and 1b (6, 7). In the hERG 1b transcript, an alternate 5′ exon replaces 1a exons 1-5, resulting in a shorter, unique N terminus that lacks a Per-Arnt-Sim (PAS) domain (also known as the ether-à-go-go domain) (8, 9). In heterologous systems, hERG 1b subunits avidly associate with hERG 1a but fail as homomer...

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“…7,14,15 A higher expression of HCN4 was detected in CO-exposed rat hearts at 4 days after birth (2.73±0.54 vs 1.04±0.11) (Figure 5a); this was accompanied by a relative upregulation of the mRNA coding for MinK-related peptide 1 (MiRP-1) (2.06 ± 0.02 vs 1.03 ± 0.2 at 4 days) (Figure 5c). MiRP-1 is a b-subunit that enhances the expression and speeds up the kinetics of activation of the HCN-derived proteins; in the adult heart, MiRP-1 is expressed in the sinus node region but hardly detectable in the ventricle.…”

Section: Molecular Properties Of F-channels In Co-exposed Neonatal Ratsmentioning

confidence: 98%

“…7,8 Briefly, tissue specimens were homogenized in OMNIzol reagent (Celbio). Genomic DNA was eliminated by incubating in DNase I. RNA was quantified by spectrophotometry.…”

Section: Mrna Extractionmentioning

confidence: 99%

Prenatal exposure to carbon monoxide delays postnatal cardiac maturation

Sartiani

Stillitano

Luceri

et al. 2010

Laboratory Investigation

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Prenatal exposure to toxicants, such as maternal smoking, may impair cardiovascular autonomic maturation in infants. We recently showed that exposure of pregnant rats to a mild concentration of carbon monoxide (CO), a component of cigarette smoke, delays postnatal electrophysiological maturation of ventricular myocytes from newborns rats, likely predisposing to life-threatening arrhythmias. To get a comprehensive view of developmental molecular abnormalities induced, at cardiac level, by prenatal CO exposure, we used microarray analysis approach on the rat heart at 4, 7 and 20 days postnatal life. The relationship between molecular and functional alterations was investigated by assessing the ventricular expression of f-current, an electrophysiological marker of immature cardiac phenotype. Rats were prenatally exposed to 0 (CTR) or 150 p.p.m. CO and mRNA obtained from ventricular samples. Differential analysis and biological pathway analysis of microarray data were performed by using Newton's approach and the GENMAPP/MAPPFinder, respectively. The real-time RT-PCR reactions were performed by TaqMan probe-based chemistry. Freshly isolated patch-clamped ventricular cardiomyocytes were used to measure I f . Genes and pathways controlling cell cycle and excitation-contraction coupling were significantly modified in CO-exposed rats. The higher effect was observed in cardiomyocytes harvested from 7-day-old rats, in which mRNA expression for crucial sarcomeric proteins (myosin and actin subunits, troponin I), transporters (Ca 2 þ transporting ATPase) and enzymes (aldolase) were significantly downregulated. Accordingly, the molecular and functional expression of f-channels, which represents a marker of fetal ventricular phenotype, was transiently greater in CO-exposed rats ( þ 200%) than in control ones. In conclusion, our study provides new insights into the molecular and functional mechanisms underlying cardiac maturation and its impairment by prenatal exposure to toxic components of smoking, such as CO.

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Developmental Changes in Cardiomyocytes Differentiated from Human Embryonic Stem Cells: A Molecular and Electrophysiological Approach

Cited by 332 publications

References 45 publications

Mechanisms underlying the cardiac pacemaker: the role of SK4 calcium-activated potassium channels

Mechanisms underlying the cardiac pacemaker: the role of SK4 calcium-activated potassium channels

hERG 1b is critical for human cardiac repolarization

Prenatal exposure to carbon monoxide delays postnatal cardiac maturation

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