<i>Nkx2-5</i> defines a subpopulation of pacemaker cells and is essential for the physiological function of the sinoatrial node in mice

Li, Hua; Li, Dainan; Wang, Yuzhi; Huang, Zhen; Xu, Jue; Yang, Tianfang; Wang, Linyan; Tang, Qi; Cai, Chen-Leng; Huang, Hai; Zhang, Yanding

doi:10.1242/dev.178145

Cited by 27 publications

(44 citation statements)

References 55 publications

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“…Among the cobinding peaks, two sites (termed Gja5-S1 and Gja5-S2) co-occupied by Shox2 and Nkx2-5 downstream of Gja5 raised our particular interest ( Fig. 1A), because Gja5 expression has been proven to be regulated by Shox2 and Nkx2-5 during SAN development (Li et al, 2019;Ye et al, 2015). Gja5-S1 locates at around 9-Kb downstream of the Gja5 coding region, whereas Gja5-S2 sits downstream of Gja5-S1 separated by a 12-Kb non-coding sequence.…”

Section: Identification Of Gja5-s1 and Gja5-s2 And Generation Of Gja5mentioning

confidence: 99%

“…Hearts were then isolated, and atria were separated from ventricles. Samples from about 20 pups of each genotype were pooled, homogenized, and subjected to protein extraction or RNA extraction followed by cDNA preparation, as previously described (Li et al, 2019;Ye et al, 2015). For Western Blot, the following primary antibodies were used: anti-Cx40 (Cx40-A; Alpha Diagnostic International;…”

Section: Western Blot and Real-time Quantitative Pcr (Rt-qpcr)mentioning

confidence: 99%

“…1×10 7 cells of each genotype (about 30 wild type embryos or 50 Nkx2-5 Cre/Cre embryos) were pooled for each 3C library preparation following standard procedures (Cope and Fraser, 2009). The relative interaction frequency (RIF) of each fragment of interest was measured by RT-qPCR, as described previously (Li et al, 2019). Primer details are as follows: F1 (5' -GCCAAGGCCCTCAAGGTGA-3'), F2 (5' -GGAGGGATTTGATATG AATGTAAGCACTG-3'), F3 (5' -ATTCATGTAAGAGGGTCTGATCTCCAG-3'), F4 (5' -A CAACCTTATCTCCAAACCTTTGCTC-3'), R1 (5' -CCATTCCCTTTAGGACGGTTACCTT C-3'), R2 (5' -CATGGACTGACCTCATTGGAGTG-3'), R3 (5' -GGGAGGGATTCAGGAC ATGTTG-3').…”

Section: Chromatin Conformation Capture (3c) Assaysmentioning

confidence: 99%

“…In contrast, Shox2 is essential for SAN development and represses Nkx2-5 expression in the developing SAN head domain (Espinoza-Lewis et al, 2009;Liu et al, 2014), thus ensuring the absence of Nkx2-5 in the SAN head from the surrounding Nkx2-5 + working myocardium. It was recently demonstrated the existence of a Shox2 and Nkx2-5 co-expression domain in the developing SAN, genetically dividing the SAN into a Shox2 + /Nkx2-5 -"head" domain and a Shox2 + /Nkx2-5 + "jFunction" domain (Li et al, 2019;Ye et al, 2015). In the Shox2 + /Nkx2-5 + SAN junction domain, a Shox2-Nkx2-5 antagonistic mechanism appears to direct the pacemaker cell fate (Ye et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

“…During murine cardiogenesis, Gja5 is selectively expressed in the atrial myocardium, the ventricular conduction system, and the arterial endothelial cells (Beyer et al, 2011;Christoffels and Moorman, 2009;van Weerd and Christoffels, 2016). Cardiac Gja5 expression depends on Nkx2-5 (Dupays et al, 2005;Espinoza-Lewis et al, 2011;Linhares et al, 2004) but is repressed by Shox2 (Blaschke et al, 2007;Espinoza-Lewis et al, 2009), and is controlled tightly by the Shox2-Nkx2-5 antagonism in the Nkx2-5 + /Shox2 + domains (Li et al, 2019;Ye et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

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Conjugated activation of myocardial-specific transcription ofGja5by a pair of Nkx2-5-Shox2 co-responsive elements

Yang

Huang

et al. 2020

Preprint

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AbstractThe sinoatrial node (SAN) is the primary pacemaker in the heart. During cardiogenesis, Shox2 and Nkx2-5 are co-expressed in the junction domain of the SAN and regulate pacemaker cell fate through a Shox2-Nkx2-5 antagonism. Cx40 is a marker of working myocardium and an Nkx2-5 transcriptional output antagonized by Shox2, but the underlying regulatory mechanisms remain elusive. Here we characterized a bona fide myocardial-specific Gja5 (coding gene of Cx40) distal enhancer consisting of a pair of Nkx2-5 and Shox2 co-bound elements in the regulatory region of Gja5. Transgenic reporter assays revealed that neither element alone, but the conjugation of both elements together, drives myocardial-specific transcription. Genetic analyses confirmed that the activation of this enhancer depends on Nkx2-5 but is inhibited by Shox2 in vivo, and its presence is essential for Gja5 expression in the myocardium but not the endothelial cells of the heart. Furthermore, chromatin conformation analysis showed an Nkx2-5-dependent loop formation between these two elements and the Gja5 promoter in vivo, indicating that Nkx2-5 bridges the conjugated activation of this enhancer by pairing the two elements to the Gja5 promoter.

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Section: Identification Of Gja5-s1 and Gja5-s2 And Generation Of Gja5mentioning

confidence: 99%

Section: Western Blot and Real-time Quantitative Pcr (Rt-qpcr)mentioning

confidence: 99%

Section: Chromatin Conformation Capture (3c) Assaysmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Conjugated activation of myocardial-specific transcription ofGja5by a pair of Nkx2-5-Shox2 co-responsive elements

Yang

Huang

et al. 2020

Preprint

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Development of ventricular trabeculae affects electrical conduction in the early endothermic heart

Olejníčková

Hamor

Janáček

et al. 2022

Developmental Dynamics

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BackgroundThe ventricular trabeculae play a role, among others, in the impulse spreading in ectothermic hearts. Despite the morphological similarity with the early developing hearts of endotherms, this trabecular function in mammalian and avian embryos was poorly addressed.ResultsWe simulated impulse propagation inside the looping ventricle and revealed delayed apical activation in the heart with inhibited trabecular growth. This finding was corroborated by direct imaging of the endocardial surface showing early activation within the trabeculae implying preferential spreading of depolarization along with them. Targeting two crucial pathways of trabecular formation (Neuregulin/ErbB and Nkx2.5), we showed that trabecular development is also essential for proper conduction patterning. Persistence of the slow isotropic conduction likely contributed to the pumping failure in the trabeculae‐deficient hearts.ConclusionsOur results showed the essential role of trabeculae in intraventricular impulse spreading and conduction patterning in the early endothermic heart. Lack of trabeculae leads to the failure of conduction parameters differentiation resulting in primitive ventricular activation with consequent impact on the cardiac pumping function.

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Analysis of changes in the action potential morphology of the mouse sinoatrial node true pacemaker cells during ontogenetic development in vitro and in silico

Ryvkin,

Furman,

Lebedeva

et al. 2024

Developmental Dynamics

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BackgroundMaturation of the mouse is accompanied by the increase in heart rate. However, the mechanisms underlying this process remain unclear. We performed an action potentials (APs) recordings in mouse sinoatrial node (SAN) true pacemaker cells and in silico analysis to clarify the mechanisms underlying pre–postnatal period heart rate changes.ResultsThe APs of true pacemaker cells at different stages had similar configurations and dV/dtmax values. The cycle length, action potential duration (APD90), maximal diastolic potential (MDP), and AP amplitude decreased, meanwhile the velocity of diastolic depolarization (DDR) increased from E12.5 stage to adult. Using a pharmacological approach we found that in SAN true pacemaker cells ivabradine reduces the DDR and the cycle length significantly stronger in E12.5 than in newborn and adult mice, whereas the effects of Ni2+ and nifedipine were significantly stronger in adult mice. Computer simulations further suggested that the density of the hyperpolarization–activated pacemaker сurrent (If) decreased during development, whereas transmembrane and intracellular Ca2+ flows increased.ConclusionsThe ontogenetic decrease in IK1 density from E12.5 to adult leads to depolarization of MDP to the voltage range in which calcium currents are activated, thereby shifting the balance from the “membrane‐clock” to the “calcium‐clock.”

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Nkx2-5 defines a subpopulation of pacemaker cells and is essential for the physiological function of the sinoatrial node in mice

Cited by 27 publications

References 55 publications

Conjugated activation of myocardial-specific transcription ofGja5by a pair of Nkx2-5-Shox2 co-responsive elements

Conjugated activation of myocardial-specific transcription ofGja5by a pair of Nkx2-5-Shox2 co-responsive elements

Development of ventricular trabeculae affects electrical conduction in the early endothermic heart

Analysis of changes in the action potential morphology of the mouse sinoatrial node true pacemaker cells during ontogenetic development in vitro and in silico

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