Changes of HCN gene expression and If currents in Nkx2.5-positive cardiomyocytes derived from murine embryonic stem cells during differentiation

Yano, Shuichi; Miake, Junichiro; Mizuta, Einosuke; Manabe, Kasumi; Bahrudin, Udin; Morikawa, Kumi; Arakawa, Keita; Sasaki, Norihito; Igawa, Osamu; Shigemasa, Chiaki; Yamamoto, Yorihiro; Morisaki, Takayuki; Hidaka, Kumi; Kurata, Yasutaka; Yoshïda, Akio; Shiota, Goshi; Higaki, Kazutaka; Ninomiya, Haruaki; Lee, Jong-Kook; Shirayoshi, Yasuaki; Hisatome, Ichiro

doi:10.2220/biomedres.29.195

Cited by 16 publications

(5 citation statements)

References 27 publications

(33 reference statements)

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“…Both HCN1 and HCN2 are predominantly N-glycosylated in brain tissue (31,39,58), but whether these channels are N-glycosylated in cardiac tissue is unknown. HCN1 and HCN2 are expressed in conduction tissue of the adult heart (28 -30, 43) and are found in various regions of embryonic and neonatal hearts (45,56) and in cardiac embryonic stem cells (36,40,41,55). Studies from our lab (36,53) and others (16,24,26,45,56) have shown that HCN2 contributes to the generation and modulation of heart rate in the embryonic stage, and knockout of HCN2 leads to significant reductions in hyperpolarizationactivated current (I h ) amplitudes recorded from individual embryonic cardiac myocytes (26).…”

Section: Hcn2 Channels Undergo N-glycosylation In Native Cardiacmentioning

confidence: 99%

Evolutionary emergence of N-glycosylation as a variable promoter of HCN channel surface expression

Hegle

Nazzari

Roth

et al. 2010

American Journal of Physiology-Cell Physiology

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Hegle AP, Nazzari H, Roth A, Angoli D, Accili EA. Evolutionary emergence of N-glycosylation as a variable promoter of HCN channel surface expression. Am J Physiol Cell Physiol 298: C1066 -C1076, 2010. First published February 3, 2010 doi:10.1152/ajpcell.00389.2009.-All four mammalian hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel isoforms have been shown to undergo N-linked glycosylation in the brain. With the mouse HCN2 isoform as a prototype, HCN channels have further been suggested to require N-glycosylation for function, a provocative finding that would make them unique in the voltage-gated potassium channel superfamily. Here, we show that both the HCN1 and HCN2 isoforms are also predominantly N-glycosylated in the embryonic heart, where they are found in significant amounts and where HCN-mediated currents are known to regulate beating frequency. Surprisingly, we find that N-glycosylation is not required for HCN2 function, although its cell surface expression is highly dependent on the presence of N-glycans. Comparatively, disruption of N-glycosylation only modestly impacts cell surface expression of HCN1 and leaves permeation and gating functions almost unchanged. This difference between HCN1 and HCN2 is consistent with evolutionary trajectories that diverged in an isoform-specific manner after gene duplication from a common HCN ancestor that lacked N-glycosylation and was able to localize efficiently to the cell surface. ion channels; hyperpolarization-activated cyclic nucleotide-modulated channel-mediated current; N-linked glycosylation; embryonic heart; molecular evolution AN OUTSTANDING QUESTION in hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel biology, and indeed in the biology of most intrinsic proteins of the plasma membrane, is how expression at the cell surface is regulated to affect functional heterogeneity in health and pathological states. The number of HCN channels on the cell surface is a critical determinant of beating frequency in cardiac conduction tissue. HCN isoforms and HCN-mediated currents (I h ) are upregulated in the embryonic and neonatal ventricle (24,45,53,56), in the neonatal sinoatrial node (1), and in beating embryonic stem cells during development in culture (27,36,40,41). However, the factors that determine HCN channel supply at the cell surface during cardiac development have been studied in only a limited fashion.An important determinant of plasma membrane expression of intrinsic membrane proteins is N-linked glycosylation, which promotes proper folding, stability, and oligomeric assembly in the endoplasmic reticulum (ER) and facilitates transport and targeting to the plasma membrane (14, 15). HCN1 and HCN2 are extensively N-glycosylated in mouse brain (31,39,58) and contain only one Asn-X-Ser/Thr consensus sequon for N-glycosylation in the S5 linker, close to the channel pore (Fig.

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Section: Hcn2 Channels Undergo N-glycosylation In Native Cardiacmentioning

confidence: 99%

Evolutionary emergence of N-glycosylation as a variable promoter of HCN channel surface expression

Hegle

Nazzari

Roth

et al. 2010

American Journal of Physiology-Cell Physiology

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“…Nkx2.5, which are some of the first cardiomyocyte marker genes to be upregulated upon initiation of cardiomyocyte differentiation (Boheler et al, 2002). Nkx2.5 is the major homeobox transcription factor that regulates expression of genes in the cardiomyocyte lineage and a marker of cardiac progenitor cells (Yano et al, 2008). Nkx2.5 expression is repressed by the homeodomain transcription factor encoded by the Shox2 homeobox gene (Espinoza-Lewis et al, 2009), and as shown in Figures 2 and 3 and Table 1, one of the major effects of TCDD treatment was a 50-fold induction of Shox2, suggesting the possibility that through inhibition of Nkx2.5 expression and of Nkx2.5-regulated cardiovascular markers, TCDD treatment might block attainment of the cardiomyocyte phenotype.…”

Section: Tcdd Inhibits Cardiomyocyte Differentiationmentioning

confidence: 99%

Dioxin Exposure Disrupts the Differentiation of Mouse Embryonic Stem Cells into Cardiomyocytes

Wang¹,

Fan²,

Puga³

2009

Toxicological Sciences

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Experimental exposure of fish, birds, and rodents to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) causes multiple Ah receptor-mediated developmental abnormalities, an observation consistent with compelling evidence in human populations that TCDD exposure is responsible for a significant incidence of birth defects. To characterize molecular mechanisms that might explain the developmental effects of dioxin, we have studied the consequences of TCDD exposure on the differentiation of mouse embryonic stem (ES) cells in culture and on the expression of genes, including those coding for homeodomain containing transcription factors, with a role in progression of tissue differentiation and embryonic identity during development. We find that TCDD treatment causes expression changes in a number of homeobox genes concomitant with Ah receptor recruitment to the promoters of many of these genes, whether under naïve or dioxin-activated conditions. TCDD exposure also derails temporal expression trajectories of developmentally regulated genes in a wide diversity of differentiation pathways, including genes with functions in neural and cardiovascular development, self-renewal, hematopoiesis and mesenchymal lineage specification, and Notch and Wnt pathways. Among these, we find that TCDD represses the expression of the cardiac development-specific Nkx2.5 homeobox transcription factor, of cardiac troponin-T and of alpha- and beta-myosin heavy chains, inhibiting the formation of beating cardiomyocytes, a characteristic phenotype of differentiating mouse ES cells in culture. These data identify potential pathways for dioxin to act as a developmental teratogen, possibly critical to cardiovascular development and disease, and provide molecular targets that may help us understand the molecular basis of Ah receptor-mediated developmental toxicity.

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“…Embryonic stem cells (ESCs) are a feasible starting material, as they are pluripotent and can be differentiated to any cell lineage. Several ESC reporter lines have been engineered and used to isolate CCS cells, including (1) HCN4p-EGFP + cells that express transcripts for CCS markers Hcn4 , Cav3.2 , and Cx40 and display a hyperpolarization-activated depolarizing current (termed the funny current; Morikawa et al., 2010); (2) an ESC reporter line containing a Mink:lacz transgene and a reporter regulated by a chicken Gata6 ( cGata6 ) enhancer; the double-positive cells display pacemaker-like morphology and show pacemaking action potential waveform as well as a funny current (White and Claycomb, 2005); (3) a Shox2 promoter with a Cx30.2 enhancer to generate ESC-derived SHOX2 + and Cx30.2 + cardiomyocytes that express additional CCS markers ( Hcn4 , Cx45 , Cx30.2 , Tbx2 , and Tbx3 ) and exhibit pacemaker-nodal phenotype (Hashem and Claycomb, 2013); (4) ESC-derived Nkx2.5:GFP + cells expressing Hcn1 and Hcn4 that display the funny current (Yano et al., 2008); and (5) an ESC reporter line derived from Contactin2:EGFP BAC transgenic mice ( Cntn2:egfp ) was recently used to isolate rare cells that display transcriptional signatures and functional properties comparable to endogenous cardiac Purkinje cells (PCs) (Maass et al., 2015). Finally, CD166 has been identified as a CCS cell-surface marker (Scavone et al., 2013), although it is not specific to CCS constituents.…”

Section: Introductionmentioning

confidence: 99%

Efficient Generation of Cardiac Purkinje Cells from ESCs by Activating cAMP Signaling

et al. 2015

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SummaryDysfunction of the specialized cardiac conduction system (CCS) is associated with life-threatening arrhythmias. Strategies to derive CCS cells, including rare Purkinje cells (PCs), would facilitate models for mechanistic studies and drug discovery and also provide new cellular materials for regenerative therapies. A high-throughput chemical screen using CCS:lacz and Contactin2:egfp (Cntn2:egfp) reporter embryonic stem cell (ESC) lines was used to discover a small molecule, sodium nitroprusside (SN), that efficiently promotes the generation of cardiac cells that express gene profiles and generate action potentials of PC-like cells. Imaging and mechanistic studies suggest that SN promotes the generation of PCs from cardiac progenitors initially expressing cardiac myosin heavy chain and that it does so by activating cyclic AMP signaling. These findings provide a strategy to derive scalable PCs, along with insight into the ontogeny of CCS development.

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Changes of HCN gene expression and If currents in Nkx2.5-positive cardiomyocytes derived from murine embryonic stem cells during differentiation

Cited by 16 publications

References 27 publications

Evolutionary emergence of N-glycosylation as a variable promoter of HCN channel surface expression

Evolutionary emergence of N-glycosylation as a variable promoter of HCN channel surface expression

Dioxin Exposure Disrupts the Differentiation of Mouse Embryonic Stem Cells into Cardiomyocytes

Efficient Generation of Cardiac Purkinje Cells from ESCs by Activating cAMP Signaling

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