scholarly journals Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

10.3791/52465 ◽  
2015 ◽  
Author(s):  
Christian Rimmbach ◽  
Julia J. Jung ◽  
Robert David
Keyword(s):  
Cardiac Pacemaker ◽  
Cell Aggregates ◽  
Pacemaker Cell ◽  
Es Cell

scholarly journals Mechanisms of Beat-to-Beat Regulation of Cardiac Pacemaker Cell Function by Ca2+ Cycling Dynamics

2013 ◽  
Vol 105 (7) ◽  
pp. 1551-1561 ◽  
Author(s):  
Yael Yaniv ◽  
Michael D. Stern ◽  
Edward G. Lakatta ◽  
Victor A. Maltsev
Keyword(s):  
Cell Function ◽  
Cardiac Pacemaker ◽  
Pacemaker Cell

scholarly journals Synergism of coupled subsarcolemmal Ca2+ clocks and sarcolemmal voltage clocks confers robust and flexible pacemaker function in a novel pacemaker cell model

2009 ◽  
Vol 296 (3) ◽  
pp. H594-H615 ◽  
Author(s):  
Victor A. Maltsev ◽  
Edward G. Lakatta
Keyword(s):  
Cell Model ◽  
Numerical Models ◽  
Experimental Studies ◽  
Cardiac Pacemaker ◽  
Critical Dimension ◽  
Pacemaker Cell ◽  
Pumping Rate ◽  
Rate Modulation ◽  
Wide Scale ◽  
Fail Safe

Recent experimental studies have demonstrated that sinoatrial node cells (SANC) generate spontaneous, rhythmic, local subsarcolemmal Ca2+ releases (Ca2+ clock), which occur during late diastolic depolarization (DD) and interact with the classic sarcolemmal voltage oscillator (membrane clock) by activating Na+-Ca2+ exchanger current ( INCX). This and other interactions between clocks, however, are not captured by existing essentially membrane-delimited cardiac pacemaker cell numerical models. Using wide-scale parametric analysis of classic formulations of membrane clock and Ca2+ cycling, we have constructed and initially explored a prototype rabbit SANC model featuring both clocks. Our coupled oscillator system exhibits greater robustness and flexibility than membrane clock operating alone. Rhythmic spontaneous Ca2+ releases of sarcoplasmic reticulum (SR)-based Ca2+ clock ignite rhythmic action potentials via late DD INCX over much broader ranges of membrane clock parameters [e.g., L-type Ca2+ current ( ICaL) and/or hyperpolarization-activated (“funny”) current ( If) conductances]. The system Ca2+ clock includes SR and sarcolemmal Ca2+ fluxes, which optimize cell Ca2+ balance to increase amplitudes of both SR Ca2+ release and late DD INCX as SR Ca2+ pumping rate increases, resulting in a broad pacemaker rate modulation (1.8–4.6 Hz). In contrast, the rate modulation range via membrane clock parameters is substantially smaller when Ca2+ clock is unchanged or lacking. When Ca2+ clock is disabled, the system parametric space for fail-safe SANC operation considerably shrinks: without rhythmic late DD INCX ignition signals membrane clock substantially slows, becomes dysrhythmic, or halts. In conclusion, the Ca2+ clock is a new critical dimension in SANC function. A synergism of the coupled function of Ca2+ and membrane clocks confers fail-safe SANC operation at greatly varying rates.

scholarly journals Dynamics of PKA phosphorylation and gain of function in cardiac pacemaker cells: a computational model analysis

2016 ◽  
Vol 310 (9) ◽  
pp. H1259-H1266 ◽  
Author(s):  
Joachim Behar ◽  
Yael Yaniv
Keyword(s):  
Cycle Length ◽  
Cell Function ◽  
Membrane Surface ◽  
Cardiac Pacemaker ◽  
Neonatal Rat ◽  
Hek293 Cells ◽  
Pacemaker Cell ◽  
Pacemaker Cells ◽  
Clock System ◽  
Dominant Component

Cardiac pacemaker cell function is regulated by a coupled-clock system that integrates molecular cues on the cell-membrane surface (i.e., membrane clock) and on the sarcoplasmic reticulum (SR) (i.e., Ca2+ clock). A recent study has shown that cotransfection of spontaneous beating cells (HEK293 cells and neonatal rat myocytes) with R524Q-mutant human hyperpolarization-activated cyclic nucleotide-gated molecules (the dominant component of funny channels) increases the funny channel's sensitivity to cAMP and leads to a decrease in spontaneous action potential (AP) cycle length (i.e., tachycardia). We hypothesize that in rabbit pacemaker cells, the same behavior is expected, and because of the coupled-clock system, the resultant steady-state decrease in AP cycle length will embody contributions from both clocks: the initial decrease in the spontaneous AP beating interval, arising from increased sensitivity of the f-channel to cAMP, will be accompanied by an increase in the adenylyl cyclase (AC)-cAMP-PKA-dependent phosphorylation activity, which will further decrease this interval. To test our hypothesis, we used the recently developed Yaniv-Lakatta pacemaker cell numerical model. This model predicts the cAMP signaling dynamics, as well as the kinetics and magnitude of protein phosphorylation in both normal and mutant pacemaker cells. We found that R524Q-mutant pacemaker cells have a shorter AP firing rate than that of wild-type cells and that gain in pacemaker function is the net effect of the R514Q mutation on the functioning of the coupled-clock system. Specifically, our results directly support the hypothesis that changes in Ca2+-activated AC-cAMP-PKA signaling are involved in the development of tachycardia in R524Q-mutant pacemaker cells.

scholarly journals Cardiac Pacemaker Cell Function at a Super-Resolution Scale of SIM: Distribution of RyRs, Calcium Dynamics, and Numerical Modeling

2016 ◽  
Vol 110 (3) ◽  
pp. 267a
Author(s):  
Victor A. Maltsev ◽  
Alexander V. Maltsev ◽  
Magdalena Juhaszova ◽  
Syevda Sirenko ◽  
Oliver Monfredi ◽  
...  
Keyword(s):  
Numerical Modeling ◽  
Cell Function ◽  
Cardiac Pacemaker ◽  
Super Resolution ◽  
Calcium Dynamics ◽  
Pacemaker Cell

scholarly journals The Grb2/Mek Pathway Represses Nanog in Murine Embryonic Stem Cells

2006 ◽  
Vol 26 (20) ◽  
pp. 7539-7549 ◽  
Author(s):  
Takashi Hamazaki ◽  
Sarah M. Kehoe ◽  
Toru Nakano ◽  
Naohiro Terada
Keyword(s):  
Es Cells ◽  
Tyrosine Phosphatase ◽  
Embryonic Stem ◽  
Homeobox Gene ◽  
Cell Aggregation ◽  
Cell Aggregates ◽  
Es Cell ◽  
Sodium Vanadate ◽  
Primitive Endoderm ◽  
Endoderm Differentiation

ABSTRACT The homeobox gene Nanog is a key intrinsic determinant of self renewal in embryonic stem (ES) cells, and its repression leads ES cells to selectively differentiate into primitive endoderm. Although Nanog repression occurs at the outermost layer of ES cell aggregates independent of the leukemia inhibitory factor (LIF)/STAT3 pathway, it is largely undetermined what external cues and intracellular signals cause the event. Of interest, addition of the tyrosine phosphatase inhibitor, sodium vanadate, selectively repressed Nanog transcription without any detectable changes in upstream transcriptional regulators Oct3/4 and Sox2. Furthermore, sodium vanadate induced primitive endoderm differentiation, even in the inner cells of ES cell aggregates. Expression of Gata6 and Zfp42, two putative downstream Nanog effectors, was also increased and decreased by the addition of sodium vanadate, respectively, but these changes were eliminated by exogenous Nanog expression. The effects of sodium vanadate were abrogated by Grb2 deficiency or by the addition of the Mek inhibitor, PD98059. Indeed, PD98059 prevented Nanog repression induced by ES cell aggregation as well. Furthermore, transfection of a constitutive active Mek mutant into ES cells induced Nanog repression and primitive endoderm differentiation. These data indicate that the Grb2/Mek pathway primarily mediates Nanog gene repression upon ES cell differentiation into primitive endoderm.

Abstract 196: Self-Organized Formation of Cardiac Pacemaker Tissues in Embryoid Bodies

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Sherin I Hashem ◽  
William C Claycomb
Keyword(s):  
Cell Line ◽  
Es Cells ◽  
Three Dimensional ◽  
Cardiac Pacemaker ◽  
Model System ◽  
Embryoid Bodies ◽  
Fluorescent Imaging ◽  
Es Cell ◽  
Altered Expression ◽  
Self Organized

The pacemaker tissues of the heart are a complex set of specialized cells that initiate the rhythmic heart beat. The sinoatrial node (SAN) serves as the primary pacemaker, whereas the atrioventricular node (AVN) serves as a subsidiary pacemaker under conditions of SAN failure. The elucidation of genetic networks regulating the development of these tissues is crucial for understanding the mechanisms underlying arrhythmias and for the design of biological pacemakers. At present, there is no in vitro model system in which these specialized cells are defined or targeted. Here we report efficient self-organized formation of the two pacemaker nodes in three-dimensional aggregate cultures of mouse embryonic stem (ES) cells known as embryoid bodies (EBs), as well as the isolation of ES cell-derived AVN cells. A Shox2-lacZ knockin ES cell line carrying a Cx30.2 enhancer-RFP construct was used to generate EBs. The Shox2 gene, a determinant of the SAN genetic cascade, was used to delineate the SAN in EBs. The Cx30.2 enhancer was used to direct RFP expression to the AVN in EBs. Using live fluorescent imaging and immunohistochemistry, we demonstrate that these genetic markers reproducibly delineate cell clusters which express nodal proteins. These clusters are functionally connected with, and consistently located adjacent to the contracting region of the EB in an organized manner. We demonstrate that EBs generated using Shox2 knockout ES cells exhibit a hypoplastic SAN phenotype that includes reduction in spontaneous contraction rates and altered expression of Shox2 downstream targets. We isolated an ES cell-derived AVN cell line using a genetic selection technique and demonstrated that these cells display nodal characteristics such as calcium dependent spontaneous depolarizations and an AVN gene expression profile. When these cells were grown as three-dimensional aggregates they induced synchronous contraction of surrounding cardiac myocytes in co-culture experiments. Using molecular markers, we have generated a reproducible model system and have isolated an AVN cell line that will be invaluable tools for studying the molecular pathways regulating the development of the cardiac pacemaker tissues and molecular composition of these specialized cells.

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