Overexpression of Map3k7 activates sinoatrial node-like differentiation in mouse ES-derived cardiomyocytes

Brown, Kathy; Legros, Stephanie; Ortega, Francis A.; Dai, Yunkai; Doss, Michael Xavier; Christini, David J.; Robinson, Richard B.; Foley, Ann

doi:10.1371/journal.pone.0189818

Cited by 11 publications

(34 citation statements)

References 56 publications

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“…Similarly, beat rate can be determined by hand counting the number of beats per unit of time and converting this into beats per minute (bpm). This method is time consuming and prone to human error, particularly for fast beating cardiac cells, such as SAN cells [15]. In addition, cardiac cells may pause during imaging, which could be triggered by changes in temperature, changes in light intensity, interference from other beating areas in the dish, or experimental manipulation.…”

Section: Resultsmentioning

confidence: 99%

“…Given the recent success with the implantation of ventricular cardiomyocytes, it now seems feasible to generate SAN cells in vitro for use as biological pacemakers; however, again, this will require protocols for the efficient differentiation of pure pacemakers from pluripotent sources. Early work in this field suggests that this can be accomplished by the addition of small molecules and growth factors as cells differentiate in vitro [12][13][14][15], however much work remains to be done. One tool that is currently lacking is the ability to rapidly distinguish cardiac subtypes.…”

Section: Introductionmentioning

confidence: 99%

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Quantification of Cardiomyocyte Beating Frequency Using Fourier Transform Analysis

et al. 2018

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Pacemaker cardiomyocytes of the sinoatrial node (SAN) beat more rapidly than cells of the working myocardium. Beating in SAN cells responds to β-adrenergic and cholinergic signaling by speeding up or slowing, respectively. Beat rate has traditionally been assessed using voltage or calcium sensitive dyes, however these may not reflect the true rate of beating because they sequester calcium. Finally, in vitro differentiated cardiomyocytes sometimes briefly pause during imaging giving inaccurate beat rates. We have developed a MATLAB automation to calculate cardiac beat rates directly from video clips based on changes in pixel density at the edges of beating areas. These data are normalized to minimize the effects of secondary movement and are converted to frequency data using a fast Fourier transform (FFT). We find that this gives accurate beat rates even when there are brief pauses in beating. This technique can be used to rapidly assess beating of cardiomyocytes in organoid culture. This technique could also be combined with field scanning techniques to automatically and accurately assess beating within a complex cardiac organoid.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Quantification of Cardiomyocyte Beating Frequency Using Fourier Transform Analysis

et al. 2018

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“…Map3k7-overexpressing cell lines were transduced with the overexpression vector, hPGK::Map3k7-IRES-GFP. Clonal lines were established and checked for constitutive overexpression of Map3k7 by qRT-PCR both in undifferentiated ES cells and in differentiating EBs [17]. All lines were maintained in ES cell growth medium (10% ES-qualified FBS, 2 mM glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 10 −4 mM β-mercaptoethanol, 1× pen/strep, and 1000 units LIF).…”

Section: Cell Culturementioning

confidence: 99%

“…Mice with cardiac-specific expression of a dominant interfering form of Map3k7 die shortly after birth due to conduction system abnormalities [16]. In addition, we recently showed that myocardial differentiation within Map3k7-overexpressing EBs is directed toward the sinoatrial node (SAN) lineage [17]. Taken together, these data suggest a role for Map3k7 in both the establishment and patterning of heart tissue, but its mechanism of action is unclear.…”

Section: Introductionmentioning

confidence: 99%

“…We hypothesized that Map3k7 may function in the endoderm to mediate its heart-inducing action, either by expanding the AVE, or by increasing signaling from the AVE. To examine this, a lentivirus in which Map3k7 and green fluorescent protein (GFP) are driven under the control of the ubiquitous human promoter, hPGK (Sin18 hPGK::Map3k7-IRES2-GFP), was stably transduced into mouse ES cells [17].…”

Section: Introductionmentioning

confidence: 99%

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TAK1/Map3k7 enhances differentiation of cardiogenic endoderm from mouse embryonic stem cells

Hunter

Dai

Brown

et al. 2019

Journal of Molecular and Cellular Cardiology

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Specification of the primary heart field in mouse embryos requires signaling from the anterior visceral endoderm (AVE). The nature of these signals is not known. We hypothesized that the TGFβ-activated kinase (TAK1/Map3k7) may act as a cardiogenic factor, based on its expression in heart-inducing endoderm and its requirement for cardiac differentiation of p19 cells. To test this, mouse embryonic stem (ES) cells overexpressing Map3k7 were isolated and differentiated as embryoid bodies (EBs). Map3k7-overexpressing EBs showed increased expression of AVE markers but interestingly, showed little effect on mesoderm formation and had no impact on overall cardiomyocyte formation. To test whether the pronounced expansion of endoderm masks an expansion of cardiac lineages, chimeric EBs were made consisting of Map3k7-overexpressing ES and wild type ES cells harboring a cardiac reporter transgene, MHCα::GFP, allowing cardiac differentiation to be assessed specifically in wild type ES cells. Wild type ES cells co-cultured with Map3k7-overexpressing cells had a 4-fold increase in expression of the cardiac reporter, supporting the hypothesis that Map3k7 increases the formation of cardiogenic endoderm. To further examine the role of Map3k7 in early lineage specification, other endodermal markers were examined. Interestingly, markers that are expressed in both the VE and later in gut development were expanded, whereas transcripts that specifically mark the early definitive (streak-derived) endoderm (DE) were not. To determine if Map3k7 is necessary for endoderm differentiation, EBs were grown in the presence of the Map3k7 specific inhibitor 5Z-7-oxozeaenol. Endoderm differentiation was dramatically decreased in these cells. Western blot analysis showed that known downstream targets of Map3k7 (Jnk, Nemo-like kinase (NLK) and p38 MAPK) were all inhibited. By contrast, transcripts for another TGFβ target, Sonic Hedgehog (Shh) were markedly upregulated, as were transcripts for Gli2 (but not Gli1 and Gli3). Together these data support the hypothesis that Map3k7 governs the formation, or proliferation of cardiogenic endoderm.

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Subtype-specific cardiomyocytes for precision medicine: Where are we now?

2020

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Patient-derived pluripotent stem cells (PSCs) have greatly transformed the current understanding of human heart development and cardiovascular disease. Cardiomyocytes derived from personalized PSCs are powerful tools for modeling heart disease and performing patient-based cardiac toxicity testing. However, these PSC-derived cardiomyocytes (PSC-CMs) are a mixed population of atrial-, ventricular-, and pacemaker-like cells in the dish, hindering the future of precision cardiovascular medicine. Recent insights gleaned from the developing heart have paved new avenues to refine subtype-specific cardiomyocytes from patients with known pathogenic genetic variants and clinical phenotypes. Here, we discuss the recent progress on generating subtype-specific (atrial, ventricular, and nodal) cardiomyocytes from the perspective of embryonic heart development and how human pluripotent stem cells will expand our current knowledge on molecular mechanisms of cardiovascular disease and the future of precision medicine. K E Y W O R D S atrial cardiomyocytes, human pluripotent stem cells, nodal cardiomyocytes, subtype-specific cardiomyocytes, ventricular cardiomyocytes 1 | INTRODUCTION Human pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have greatly revolutionized modern cardiovascular medicine due to their ability to generate any cell type in the cardiovascular system. Human ESCs are derived from the inner cell mass of developing embryos (blastocysts), and thus they cannot be patient-specific [1]. Patient-specific PSCs can be generated by two reprograming mechanisms: somatic cell nuclear transfer (SCNT) [2] and iPSC reprograming [3]. SCNT is the process of cellular reprogramming of somatic cells by injecting the nucleus of a somatic cell into an enucleated oocyte that is both technically and ethically challenging. In contrast, human iPSCs can be derived by transient overexpression of four transcription factors (OCT4/SOX2/ CMYC/KLF4), thus circumventing the ethical barriers for translational medicine. Although differentiated cells from isogenic SCNT-ESCs and iPSCs are equivalent in the aspects of molecular and functional properties [4], patient-specific iPSCs have become prevalent in current biomedical research. Large banks of disease-specific iPSCs have been created and distributed around the world, which are transforming our understanding of cardiovascular science and health. Human iPSCs have been extensively employed to model prevalent heart disease, such as long QT syndrome [5-7], Brugada syndrome [8], arrhythmogenic right ventricular dysplasia [9], dilated cardiomyopathy (DCM) [10,11], and hypertrophic cardiomyopathy (HCM) [12]. In addition, human iPSC-derived cardiomyocytes (iPSC-CMs) are considered powerful platforms for novel drug screening and cardiac toxicity testing [13]. Recent CiPA (Comprehensive in vitro Proarrhythmia Assay) studies, initiated by the US Food and Drug Administration (FDA), have highlighted the importance of using human iPSC-CMs to evalua...

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Overexpression of Map3k7 activates sinoatrial node-like differentiation in mouse ES-derived cardiomyocytes

Cited by 11 publications

References 56 publications

Quantification of Cardiomyocyte Beating Frequency Using Fourier Transform Analysis

Quantification of Cardiomyocyte Beating Frequency Using Fourier Transform Analysis

TAK1/Map3k7 enhances differentiation of cardiogenic endoderm from mouse embryonic stem cells

Subtype-specific cardiomyocytes for precision medicine: Where are we now?

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