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SHOX2 overexpression favors differentiation of embryonic stem cells into cardiac pacemaker cells, improving biological pacing ability.

ABSTRACT: When pluripotency factors are removed, embryonic stem cells (ESCs) undergo spontaneous differentiation, which, among other lineages, also gives rise to cardiac sublineages, including chamber cardiomyocytes and pacemaker cells. Such heterogeneity complicates the use of ESC-derived heart cells in therapeutic and diagnostic applications. We sought to direct ESCs to differentiate specifically into cardiac pacemaker cells by overexpressing a transcription factor critical for embryonic patterning of the native cardiac pacemaker (the sinoatrial node). Overexpression of SHOX2 during ESC differentiation upregulated the pacemaker gene program, resulting in enhanced automaticity in vitro and induced biological pacing upon transplantation in vivo. The accentuated automaticity is accompanied by temporally evolving changes in the effectors and regulators of Wnt signaling. Our findings provide a strategy for enriching the cardiac pacemaker cell population from ESCs.

SUBMITTER: Ionta V

PROVIDER: S-EPMC4297875 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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SHOX2 overexpression favors differentiation of embryonic stem cells into cardiac pacemaker cells, improving biological pacing ability.

Ionta Vittoria V Liang Wenbin W Kim Elizabeth H EH Rafie Reza R Giacomello Alessandro A Marbán Eduardo E Cho Hee Cheol HC

Stem cell reports 20141218 1

When pluripotency factors are removed, embryonic stem cells (ESCs) undergo spontaneous differentiation, which, among other lineages, also gives rise to cardiac sublineages, including chamber cardiomyocytes and pacemaker cells. Such heterogeneity complicates the use of ESC-derived heart cells in therapeutic and diagnostic applications. We sought to direct ESCs to differentiate specifically into cardiac pacemaker cells by overexpressing a transcription factor critical for embryonic patterning of t ...[more]

PMID: 25533636

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Project description:BackgroundEstablishment of a biological pacemaker is expected to solve the persisting problems of a mechanical pacemaker including the problems of battery life and electromagnetic interference. Enhancement of the funny current (If) flowing through hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and attenuation of the inward rectifier K+ current (IK1) flowing through inward rectifier potassium (Kir) channels are essential for generation of a biological pacemaker. Therefore, we generated HCN4-overexpressing mouse embryonic stem cells (mESCs) and induced cardiomyocytes that originally show poor IK1 currents, and we investigated whether the HCN4-overexpressing mESC-derived cardiomyocytes (mESC-CMs) function as a biological pacemaker in vitro.Methods and resultsThe rabbit Hcn4 gene was transfected into mESCs, and stable clones were selected. mESC-CMs were generated via embryoid bodies and purified under serum/glucose-free and lactate-supplemented conditions. Approximately 90% of the purified cells were troponin I-positive by immunostaining. In mESC-CMs, expression level of the Kcnj2 gene encoding Kir2.1, which is essential for generation of IK1 currents that are responsible for stabilizing the resting membrane potential, was lower than that in an adult mouse ventricle. HCN4-overexpressing mESC-CMs expressed about a 3-times higher level of the Hcn4 gene than did non-overexpressing mESC-CMs. Expression of the Cacna1h gene, which encodes T-type calcium channel and generates diastolic depolarization in the sinoatrial node, was also confirmed. Additionally, genes required for impulse conduction including Connexin40, Connexin43, and Connexin45 genes, which encode connexins forming gap junctions, and the Scn5a gene, which encodes sodium channels, are expressed in the cells. HCN4-overexpressing mESC-CMs showed significantly larger If currents and more rapid spontaneous beating than did non-overexpressing mESC-CMs. The beating rate of HCN4-overexpressing mESC-CMs responded to ivabradine, an If inhibitor, and to isoproterenol, a beta-adrenergic receptor agonist. Co-culture of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with aggregates composed of mESC-CMs resulted in synchronized contraction of the cells. The beating rate of hiPSC-CMs co-cultured with aggregates of HCN4-overexpressing mESC-CMs was significantly higher than that of non-treated hiPSC-CMs and that of hiPSC-CMs co-cultured with aggregates of non-overexpressing mESC-CMs.ConclusionsWe generated HCN4-overexpresssing mESC-CMs expressing genes required for impulse conduction, showing rapid spontaneous beating, responding to an If inhibitor and beta-adrenergic receptor agonist, and having pacing ability in an in vitro co-culture system with other excitable cells. The results indicated that these cells could be applied to a biological pacemaker.

Project description:Conjugation of small ubiquitin-like modifiers (SUMOs) to substrate proteins is a posttranslational protein modification that affects a diverse range of physiological processes. Global inhibition of SUMO conjugation in mice results in embryonic lethality, reflecting the importance of the SUMO pathways for embryonic development. Here, we demonstrated that SUMO1 overexpression was not well tolerated in murine embryonic carcinoma and embryonic stem (ES) cells and that only a few clones were recovered after transduction with vectors delivering SUMO1 expression constructs. Differentiated NIH/3T3 cells overexpress SUMO1 without deleterious effects and maintain high levels of both conjugated and free forms of SUMO1. The few embryonic cells surviving after forced overexpression retained all their SUMO1 in the form of a few high-molecular-weight conjugates and maintained undetectable levels of free SUMO1. The absence of free SUMO in embryonic cells was seen specifically upon overexpression of SUMO1, but not SUMO2. Moreover, blocking SUMO1 conjugation to endogenous substrates by C-terminal mutations of SUMO1 or by overexpression of a SUMO1 substrate "sponge" or by overexpression of the deSUMOylating enzyme SUMO-specific peptidase 1 (SENP1) dramatically restored free SUMO1 overexpression. The data suggest that overexpression of SUMO1 protein leading to an excess accumulation of critical SUMO1-conjugated substrates is not tolerated in embryonic cells. Surviving embryonic cells exhibit SUMO1 conjugation to allowed substrates but a complete absence of free SUMO1.IMPORTANCE Embryonic stem (ES) cells exhibit unusual transcriptional, proteomic, and signal response profiles, reflecting their unusual needs for rapid differentiation and replication. The work reported here demonstrated that mouse embryonic cell lines did not tolerate the overexpression of SUMO1, the small ubiquitin-like modifier protein that is covalently attached to many substrates to alter their intracellular localization and functionality. Forced SUMO1 overexpression is toxic to ES cells, and surviving cell populations adapt by dramatically reducing the levels of free SUMO1. Such a response is not seen in differentiated cells or with SUMO2 or with nonconjugatable SUMO1 mutants or in the presence of a SUMO1 "sponge" substrate that accepts the modification. The findings suggest that excess SUMO1 modification of specific substrates is not tolerated by embryonic cells and highlight a distinctive need for these cells to control the levels of SUMO1 available for conjugation.

Project description:Cardiac dyssynchrony is the proposed mechanism for pacemaker-induced cardiomyopathy, which can be prevented by biventricular pacing. Left bundle branch pacing and His bundle pacing are novel interventions that imitate the natural conduction of the heart with, theoretically, less interventricular dyssynchrony. One of the surrogate markers of interventricular synchrony is QRS duration. Our study aimed to compare the change of QRS duration before and after implantation between types of cardiac implantable electronic devices (CIEDs): left bundle branch pacing versus His bundle pacing versus biventricular pacing and conventional right ventricular pacing. A literature search for studies that reported an interval change of QRS duration after CIED implantation was conducted utilizing the MEDLINE, EMBASE, and Cochrane databases. All relevant works from database inception through November 2020 were included in this analysis. A random-effects model, Bayesian network meta-analysis was used to analyze QRS duration changes (eg, electrical cardiac synchronization) across different CIED implantations. The mean study sample size, from 14 included studies, was 185 subjects. The search found 707 articles. After exclusions, 14 articles remained with 2,054 patients. The His bundle pacing intervention resulted in the most dramatic decline in QRS duration (mean difference, - 53 ms; 95% CI - 67, - 39), followed by left bundle branch pacing (mean difference, - 46 ms; 95% CI - 60, - 33), and biventricular pacing (mean difference, - 19 ms; 95% CI - 37, - 1.8), when compared to conventional right ventricle apical pacing. When compared between LBBP and HBP, showed no statistically significant wider QRS duration in LBBP with mean different 6.5 ms. (95% CI - 6.7, 21). Our network meta-analysis found that physiologic pacing has the greatest effect on QRS duration after implantation. Thus, HBP and LBBP showed no significant difference between QRS duration after implantation. Physiologic pacing interventions result in improved electrocardiography markers of cardiac synchrony, narrower QRS duration, and might lower electromechanical dyssynchrony.

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SHOX2 overexpression favors differentiation of embryonic stem cells into cardiac pacemaker cells, improving biological pacing ability.

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SHOX2 overexpression favors differentiation of embryonic stem cells into cardiac pacemaker cells, improving biological pacing ability.

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