Formation of human cardiomyocytes is impaired in a fibrotic environment: unravelling human cardiac regeneration

Abstract Loss of myocardial tissue remains a leading cause of disease and death, as the adult heart has insufficient regenerative potential. The (pre-)clinical effects of inducing cardiac regeneration by cardiac cell therapy have been disappointing. This lack of success may result from the fact that it remains largely unclear how the receiving pathological microenvironment affects this process of cardiomyogenic differentiation of implanted cells, and thereby may (negatively) influence the therapeutic outcome. However, the tools to address this lacuna in a proper manner are lacking, as this requires tightly controllable and quantitative models of cardiomyogenic differentiation. Therefore, we have recently generated lines of conditionally immortalized human CMCs (ciCMCs) through doxycycline-dependent expression of SV40 large T antigen after genetic modification and subsequent clonal expansion. In these cells, proliferation and differentiation can be tightly controlled, allowing cardiomyogenic differentiation to be i) induced on cue, ii) precisely monitored and quantified, and iii) manipulated. The aim of this study is to improve our understanding of cardiomyogenic differentiation of guest (transplanted) cells in the context of the host (receiving) microenvironment. To create pathological microenvironments and study the effects on cardiac differentiation, co-culture experiments with human ciCMCs and cardiac fibroblasts were conducted in different ratios (10%, 30%, 60% and 90% ciCMCs). Cardiomyogenic differentiation was determined by immunostaining for cardiac specific markers and electrophysiological analysis by optical voltage mapping. After 12 days of co-culture with cardiac fibroblasts, the amount of ciCMCs that expressed the sarcomeric protein cardiac troponin T was significantly and increasingly reduced (P<0.01) in the groups with higher amounts of cardiac fibroblasts (39.4±3.9, 33±3.4, 25±1.9, 20.3±2.6, 5±1.7 for 100%, 90%, 60%, 30% and 10% ciCMCs respectively (%, mean±SD). Electrophysiological analysis showed a significantly reduced (P<0.01) conduction velocity in the co-cultures compared to the pure cultures of ciCMCs (19.1±2.1 vs 16.0±0.5, 15.8±0.9, 8.6±0.6, 5±1.67 for 100% vs 90%, 60%, 30% and 10% ciCMCs respectively (cm/s, mean±SD). However, no significant difference in conduction velocity was present between the groups with 10% and 30% ciCMCs and 30% and 60% ciCMCs present. In conclusion, a fibrotic environment has a negative effect on the formation of human cardiomyocytes as revealed by the use of ciCMCs. This not only emphasizes the need to consider the interaction between the guest (transplanted) cells and the host (receiving) microenvironment in cardiac regenerative medicine, but also offers new leads to increase the therapeutic potential of this strategy. FUNDunding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Leiden University Fund

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Modelling of atrial fibrillation at physiologically relevant scales enabled by massive expansion of native human atrial cardiomyocytes

EP Europace ◽

10.1093/europace/euab116.553 ◽

2021 ◽

Vol 23 (Supplement_3) ◽

Author(s):

N Harlaar ◽

SO Dekker ◽

J Zhang ◽

MJ Schalij ◽

RJM Klautz ◽

...

Keyword(s):

Atrial Fibrillation ◽

Conduction Velocity ◽

Lentiviral Vector ◽

Troponin T ◽

Simian Virus 40 ◽

T Antigen ◽

Atrial Cardiomyocytes ◽

Cardiomyogenic Differentiation ◽

Electrical Pacing ◽

Human Atrial Fibrillation

Abstract Funding Acknowledgements Type of funding sources: Public hospital(s). Main funding source(s): LUMC Background Current in vitro models of atrial fibrillation have limited translational potential due to a lack of relevant human physiology or the inability to reach the high activation frequencies present in human atrial fibrillation. Absence of relevant models is the result of a general deficit of readily available and standardized sources of well-differentiated human atrial cardiomyocytes. Therefore, we aimed to immortalize native human atrial cardiomyocytes to produce natural and standardized lines of these cells. Methods Human fetal atrial cardiomyocytes were transduced with a lentiviral vector directing myocyte-specific and doxycycline-inducible expression of simian virus 40 large T antigen. Addition of doxycycline to the culture medium pushed cardiomyocytes towards a highly proliferative phenotype (proliferation up to 10^12 cells). These cells were labelled hiAMs (human immortalised Atrial Myocytes). After differentiation upon doxycycline removal, hiAM cells were characterized using various molecular, biological and electrophysiological assays. Results Following cardiomyogenic differentiation, hiAMs no longer expressed the proliferation marker Ki67, revealed striated α-actinin and troponin T staining patterns and displayed synchronous contractions. Optical voltage mapping of hiAM monolayers revealed excitable cells showing homogeneous spreading of action potentials at 22.5 ± 3.1 cm/s with a mean APD80 of 139 ± 22 ms. Addition of flecainide (10 µM) to hiAM monolayers decreased the conduction velocity by 35% and increased the APD80 by 107%. Dofetilide (10 nM) addition had no effect on the conduction velocity, but did increase the APD80 by 81%. Due to their scalability, monolayers of hiAMs as big as 10 cm2 showing homogenous action potential propagation could easily be created. Following high-frequency electrical pacing, rotors could be induced with an average activation frequency of 7.5 ± 0.9 Hz. Infusion of flecainide during arrhythmic activity resulted in termination of the rotor in 18 of 24 attempts (75%), whereas addition of 0.1% DMSO (vehicle control) did not result in termination in any of the attempts. Dofetilide infusion did not result in termination. However, it did lower the average activation frequency to 2.1 ± 0.7 Hz. Conclusion We have generated first-of-a-kind lines of human atrial cardiomyocytes, allowing massive cell expansion under proliferation conditions and robust formation of cross-striated, contractile and excitable cardiomyocytes after differentiation. These characteristics allow, for the first time, the modelling, at a large-scale, of human atrial arrhythmias with frequencies similar to human atrial fibrillation. With the generation of hiAMs, a user-friendly, clinically-relevant and much-anticipated human atrial research model has been produced. Abstract Figure. hiAM AF Model

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Atrial fibrillation modelling at physiologically relevant scales enabled by massive expansion of native human atrial cardiomyocytes through immortogenetics

European Heart Journal ◽

10.1093/ehjci/ehaa946.3591 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

N Harlaar ◽

S.O Dekker ◽

J Zhang ◽

M.J Schalij ◽

R.J.M Klautz ◽

...

Keyword(s):

Atrial Fibrillation ◽

Conduction Velocity ◽

Large Scale ◽

Troponin T ◽

Simian Virus 40 ◽

T Antigen ◽

Atrial Cardiomyocytes ◽

Cardiomyogenic Differentiation ◽

Electrical Pacing ◽

Human Atrial Fibrillation

Abstract Background Current in vitro models of atrial fibrillation have limited translational potential due to a lack of relevant human physiology or the inability to reach the high activation frequencies present in human atrial fibrillation. Absence of relevant models is the result of a general deficit of readily available and standardized sources of well-differentiated human atrial cardiomyocytes. Therefore, we aimed to immortalize native human atrial cardiomyocytes to produce natural and standardized lines of these cells. Methods Human fetal atrial cardiomyocytes were transduced with a lentiviral vector directing myocyte-specific and doxycycline-inducible expression of simian virus 40 large T antigen (here defined as immortogenetics). Addition of doxycycline to the culture medium pushed cardiomyocytes towards a highly proliferative phenotype (proliferation up to 1012 cells). These cells were labelled hiAMs (human immortalised Atrial Myocytes). After differentiation upon doxycycline removal, hiAM cells were characterized using various molecular, biological and electrophysiological assays. Results Following cardiomyogenic differentiation, hiAMs no longer expressed the proliferation marker Ki67, revealed striated α-actinin and troponin T staining patterns and displayed synchronous contractions. Optical voltage mapping of hiAM monolayers revealed excitable cells showing homogeneous spreading of action potentials at 22.5±3.1 cm/s with a mean APD80 of 139±22 ms. Addition of flecainide (10 μM) to hiAM monolayers decreased the conduction velocity by 35% and increased the APD80 by 107%. Dofetilide (10 nM) addition had no effect on the conduction velocity, but did increase the APD80 by 81%. Due to their scalability, monolayers of hiAMs as big as 10 cm2 showing homogenous action potential propagation could easily be created. Following high-frequency electrical pacing, rotors could be induced with an average activation frequency of 7.5±0.9 Hz. Infusion of flecainide during arrhythmic activity resulted in termination of the rotor in 18 of 24 attempts (75%), whereas addition of 0.1% DMSO (vehicle control) did not result in termination in any of the attempts. Dofetilide infusion did not result in termination. However, it did lower the average activation frequency to 2.1±0.7 Hz. Conclusion We have generated first-of-a-kind lines of human atrial cardiomyocytes, allowing massive cell expansion under proliferation conditions and robust formation of cross-striated, contractile and excitable cardiomyocytes after differentiation. These characteristics allow, for the first time, the modelling, at a large-scale, of human atrial arrhythmias with frequencies similar to human atrial fibrillation. With the generation of hiAMs, a user-friendly, clinically-relevant and much-anticipated human atrial research model has been produced. Large-scale AF model using hiAMs Funding Acknowledgement Type of funding source: None

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Phenotypic Screen with the Human Secretome Identifies FGF16 as Inducing Proliferation of iPSC-Derived Cardiac Progenitor Cells

International Journal of Molecular Sciences ◽

10.3390/ijms20236037 ◽

2019 ◽

Vol 20 (23) ◽

pp. 6037 ◽

Cited By ~ 3

Author(s):

Karin Jennbacken ◽

Fredrik Wågberg ◽

Ulla Karlsson ◽

Jerry Eriksson ◽

Lisa Magnusson ◽

...

Keyword(s):

Progenitor Cells ◽

Cardiac Fibroblasts ◽

Cardiac Regeneration ◽

Cardiac Cells ◽

Mouse Heart ◽

Cardiac Progenitor Cells ◽

High Sequence Identity ◽

Paracrine Factors ◽

Human Cardiomyocytes ◽

Phenotypic Screen

Paracrine factors can induce cardiac regeneration and repair post myocardial infarction by stimulating proliferation of cardiac cells and inducing the anti-fibrotic, antiapoptotic, and immunomodulatory effects of angiogenesis. Here, we screened a human secretome library, consisting of 923 growth factors, cytokines, and proteins with unknown function, in a phenotypic screen with human cardiac progenitor cells. The primary readout in the screen was proliferation measured by nuclear count. From this screen, we identified FGF1, FGF4, FGF9, FGF16, FGF18, and seven additional proteins that induce proliferation of cardiac progenitor cells. FGF9 and FGF16 belong to the same FGF subfamily, share high sequence identity, and are described to have similar receptor preferences. Interestingly, FGF16 was shown to be specific for proliferation of cardiac progenitor cells, whereas FGF9 also proliferated human cardiac fibroblasts. Biosensor analysis of receptor preferences and quantification of receptor abundances suggested that FGF16 and FGF9 bind to different FGF receptors on the cardiac progenitor cells and cardiac fibroblasts. FGF16 also proliferated naïve cardiac progenitor cells isolated from mouse heart and human cardiomyocytes derived from induced pluripotent cells. Taken together, the data suggest that FGF16 could be a suitable paracrine factor to induce cardiac regeneration and repair.

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P1229Massive expansion of native human atrial cardiomyocytes through immortogenetics: generation of the hiAM cell lines

European Heart Journal ◽

10.1093/eurheartj/ehz748.0187 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Cited By ~ 1

Author(s):

N Harlaar ◽

J Liu ◽

L Volkers ◽

A A Ramkisoensing ◽

M J Schalij ◽

...

Keyword(s):

Lentiviral Vector ◽

Troponin T ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Resting Membrane Potential ◽

Cardiac Muscle Cells ◽

Atrial Cardiomyocytes ◽

Cellular Models ◽

Cardiomyogenic Differentiation

Abstract Background Preclinical cardiac research greatly depends on animal-derived cellular models, thereby hampering clinical translation. While upcoming human pluripotent stem cell technology seems to decrease this gap between bench and bedside, its complex/multi-step protocol to produce cardiac muscle cells, its required expertise, and its trouble to produce large numbers of phenotypically homogeneous cardiomyocytes so far has limited broad application. Purpose We aimed to conditionally immortalize native human atrial cardiomyocytes to produce natural and standardized lines of these cells by gaining full control over their proliferation and differentiation. Methods Human fetal atria (gestational age 18 weeks) were dissociated and transduced with a lentiviral vector directing myocyte-specific and doxycycline-inducible expression of simian virus 40 large T antigen (here defined as immortogenetics). Addition of doxycycline to the culture medium pushed cardiomyocytes towards a proliferative phenotype. In total, 125 proliferating monoclones were isolated, expanded and screened for their cardiomyogenic differentiation capacity upon doxycycline removal. Selected clones were characterised using various molecular biological and electrophysiological assays. Results Upon doxycycline removal (i.e. under differentiation conditions), cells spontaneously reacquired a cardiomyocyte-like appearance as judged by phase-contrast microscopy and were observed contracting. Simultaneously, these cells stopped proliferating, which was accompanied by a drop in large T level, loss of Ki67 expression and the development of sarcomeres with striated α-actinin and troponin T staining patterns. These cells were tagged conditionally immortalized human atrial cardiomyocytes (hereinafter called hiAMs). Optical voltage mapping of hiAM monolayers revealed excitable cells showing homogeneous spreading of action potentials at 22,5±3,1 cm/s following 1-Hz point stimulation, with a mean APD80 of 139±22 ms. Monolayers of hiAMs could easily be created as big as 10cm2 while continuing to display homogenous conduction throughout the culture. Single-cell patch clamp recordings of a hiAM clone in current-clamp mode confirmed excitability with a resting membrane potential of −62,2±4,3 mV, peak potential of 39,4±3,9 mV and APD80 of 339±9 ms. Excitable monolayer of hiAMs Conclusion We have generated first-of-a-kind lines of natural human atrial cardiomyocytes through immortogenetics, allowing massive cell expansion under proliferation conditions and robust formation of cross-striated, contractile and excitable cardiomyocytes after differentiation. Thereby, a user-friendly, clinically-relevant and much-anticipated research model has been produced, which application could range from multi-scale electrophysiological studies and drug response studies to disease modelling and myocardial regeneration.

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The underestimated issue of non-reproducible cardiac troponin I and T results: case series and systematic review of the literature

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2020-1564 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Julien Favresse ◽

Jean-Louis Bayart ◽

Damien Gruson ◽

Sergio Bernardini ◽

Aldo Clerico ◽

...

Keyword(s):

Systematic Review ◽

Clinical Judgment ◽

Troponin I ◽

Troponin T ◽

Case Reports ◽

False Negative ◽

Case Series ◽

Elevated Alkaline Phosphatase ◽

Negative Results ◽

Significant Difference

Abstract Cardiac troponins (cTn) are the preferred biomarkers for the evaluation of myocardial injury and play a key role in the diagnosis of acute myocardial infarction (MI). Pre-analytical or analytical issues and interferences affecting troponin T and I assays are therefore of major concern given the risk of misdiagnosis. False positive troponin results have been related to various interferences including anti-troponin antibodies, heterophilic antibodies, or elevated alkaline phosphatase level. On the other hand, false negative results have been reported in the case of a large biotin intake. These interferences are characterized with erroneous but reproducible troponin results. Of interest, non-reproducible results have also been reported in the literature. In other words, if the sample is reanalyzed a second time, a significant difference in troponin results will be observed. These interferences have been named “fliers” or “outliers”. Compared to the biotin interference that received major attention in the literature, troponin outliers are also able to induce harmful clinical consequences for the patient. Moreover, the prevalence of outliers in recent studies was found to be higher (0.28–0.57%) compared to the biotin interference. The aim of this systematic review is to warn clinicians about these non-reproducible results that may alter their clinical judgment. Four case reports that occurred in the Clinique of Saint-Luc Bouge are presented to attest this point. Moreover, we aimed at identifying the nature of these non-reproducible troponin results, determining their occurrence, and describing the best way for their identification.

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Cardiac regeneration: epicardial mediated repair

Proceedings of The Royal Society B Biological Sciences ◽

10.1098/rspb.2015.2147 ◽

2015 ◽

Vol 282 (1821) ◽

pp. 20152147 ◽

Cited By ~ 17

Author(s):

Teresa Kennedy-Lydon ◽

Nadia Rosenthal

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Cardiac Fibroblasts ◽

Cardiac Regeneration ◽

Repair Process ◽

Regenerative Process ◽

Cardiac Stem Cell ◽

Cardiomyocyte Proliferation ◽

Lower Vertebrates

The hearts of lower vertebrates such as fish and salamanders display scarless regeneration following injury, although this feature is lost in adult mammals. The remarkable capacity of the neonatal mammalian heart to regenerate suggests that the underlying machinery required for the regenerative process is evolutionarily retained. Recent studies highlight the epicardial covering of the heart as an important source of the signalling factors required for the repair process. The developing epicardium is also a major source of cardiac fibroblasts, smooth muscle, endothelial cells and stem cells. Here, we examine animal models that are capable of scarless regeneration, the role of the epicardium as a source of cells, signalling mechanisms implicated in the regenerative process and how these mechanisms influence cardiomyocyte proliferation. We also discuss recent advances in cardiac stem cell research and potential therapeutic targets arising from these studies.

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Efficacy of Intravitreal Triamcinolone after or Concomitant with Laser Photocoagulation in Nonproliferative Diabetic Retinopathy with Macular Edema

European Journal of Ophthalmology ◽

10.1177/112067210901900418 ◽

2009 ◽

Vol 19 (4) ◽

pp. 630-637 ◽

Cited By ~ 2

Author(s):

Erdinc Aydin ◽

Helin Deniz Demir ◽

Huseyin Yardim ◽

Unal Erkorkmaz

Keyword(s):

Diabetic Retinopathy ◽

Macular Edema ◽

Laser Photocoagulation ◽

Control Group ◽

Clinical Effects ◽

Intravitreal Triamcinolone ◽

Nonproliferative Diabetic Retinopathy ◽

Significant Difference ◽

Group 3 ◽

The Mean

Purpose To investigate the clinical effects and outcomes of intravitreal injection of 4 mg of triamcinolone acetonide (IVTA) after or concomitant with macular laser photocoagulation (MP) for clinically significant macular edema (CSME). Methods Forty-nine eyes of 49 patients with nonproliferative diabetic retinopathy and CSME were randomized into three groups. The eyes in the laser group (n=17), group 1, were subjected to MP 3 weeks after IVTA; the eyes in the IVTA group (n=13), group 2, were subjected to MP, concomitant with IVTA; the eyes in the control group (n=19), group 3, underwent only IVTA application. Visual acuity (VA), fundus fluorescein angiography, and photography were performed in each group. Results In the first group, the mean VA improved from 0.17±0.09 at baseline to 0.28±0.15 (p=0.114) and in the second group, deteriorated from 0.19±0.08 at baseline to 0.14±0.08 at the sixth month (p=0.141), respectively. In Group 3, the mean VA improved from 0.16±0.08 at baseline to 0.28±0.18 (p=0.118) at the end of the follow-up. When VA was compared between the control and study groups, significant difference was detected at the sixth month (p=0.038). Conclusions MP after IVTA improved VA, rather than MP concomitant with IVTA, and only IVTA application for CSME. MP after IVTA may reduce the recurrence of CSME and needs further investigations in a longer period.

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Myocyte-specific enhancer factor 2c triggers transdifferentiation of adipose tissue-derived stromal cells into spontaneously beating cardiomyocyte-like cells

Scientific Reports ◽

10.1038/s41598-020-80848-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Shinichiro Takashima ◽

Soichiro Usui ◽

Oto Inoue ◽

Chiaki Goten ◽

Kosei Yamaguchi ◽

...

Keyword(s):

Adipose Tissue ◽

Lentiviral Vector ◽

Troponin T ◽

Stromal Vascular Fraction ◽

Cardiac Regeneration ◽

Cardiac Lineage ◽

Cell Transcriptome ◽

Single Cell Transcriptome ◽

Culture Protocol ◽

Enhancer Factor

AbstractCardiomyocyte regeneration is limited in adults. The adipose tissue-derived stromal vascular fraction (Ad-SVF) contains pluripotent stem cells that rarely transdifferentiate into spontaneously beating cardiomyocyte-like cells (beating CMs). However, the characteristics of beating CMs and the factors that regulate the differentiation of Ad-SVF toward the cardiac lineage are unknown. We developed a simple culture protocol under which the adult murine inguinal Ad-SVF reproducibly transdifferentiates into beating CMs without induction. The beating CMs showed the striated ventricular phenotype of cardiomyocytes and synchronised oscillation of the intracellular calcium concentration among cells on day 28 of Ad-SVF primary culture. We also identified beating CM-fated progenitors (CFPs) and performed single-cell transcriptome analysis of these CFPs. Among 491 transcription factors that were differentially expressed (≥ 1.75-fold) in CFPs and the beating CMs, myocyte-specific enhancer 2c (Mef2c) was key. Transduction of Ad-SVF cells with Mef2c using a lentiviral vector yielded CFPs and beating CMs with ~ tenfold higher cardiac troponin T expression, which was abolished by silencing of Mef2c. Thus, we identified the master gene required for transdifferentiation of Ad-SVF into beating CMs. These findings will facilitate the development of novel cardiac regeneration therapies based on gene-modified, cardiac lineage-directed Ad-SVF cells.

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Abstract 255: A Fibroblast Population Shares A Developmental Origin With Cardiovascular Lineages

Circulation Research ◽

10.1161/res.115.suppl_1.255 ◽

2014 ◽

Vol 115 (suppl_1) ◽

Author(s):

Sara Ranjbarvaziri ◽

Shah Ali ◽

Mahmood Talkhabi ◽

Peng Zhao ◽

Young-Jae Nam ◽

...

Keyword(s):

Heart Development ◽

Cardiac Fibroblasts ◽

Immunohistochemical Analysis ◽

Cell Types ◽

Mouse Heart ◽

Developmental Potential ◽

Reporter Mice ◽

Significant Difference ◽

Definition Of

Rationale: The traditional definition of “cardiovascular” lineages describes the eponymous cell types - cardiomyoctes, endothelial cells, and smooth muscle cells - that arise from a common mesodermal progenitor cell during heart development. Fibroblasts are an abundant mesenchymal population in the mammalian heart which may have multiple, discrete developmental origins. Mesp1 represents the earliest marker of cardiovascular progenitors, contributing to the majority of cardiac lineages. To date no link between Mesp1 and fibroblast generation has been reported. Objective: We hypothesized progenitor cells expressing Mesp1 can also give rise to cardiac fibroblasts during heart development. Methods and Results: We generated Mesp1cre/+;R26RmTmG reporter mice where Cre-mediated recombination results in GFP activation in all Mesp1 expressing cells and their progeny. To explore their developmental potential, we isolated GFP+ cells from E7.5 Mesp1cre/+;R26RmTmG mouse. In vitro culture and transplantation studies into SCID mouse kidney capsule as wells as chick embryos showed fibroblastic adoption. Results showed that at E9.5 Mesp1+ and Mesp1- progenitors contributed to the proepicardium organ and later at E11.5 they formed epicardium. Analysis of adult hearts demonstrated that the majority of cardiac fibroblasts are derived from Mesp1 expressing cells. Immunohistochemical analysis of heart sections demonstrated expression of fibroblast markers (including DDR2, PDGFRα and Col1) in cells derived from both Mesp1+ and Mesp1- progenitors. Additionally, we investigated whether the two distinct fibroblast populations have different potency towards reprogramming to cardiomyocytes. Results showed no significant difference between Mesp1 and non-Mesp1 isolated fibroblasts to convert to cardiomyocyte fate. Conclusions: Our data demonstrates that cardiovascular progenitors expressing Mesp1 contribute to the proepicardium. These cells, as cardiovascular progenitors, also give rise to the highest portion of cardiac fibroblasts in the mouse heart.

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Abstract 313: Twist Contributes to Cardiomyocyte Renewal and is Required for Maintenance of Adult Cardiac Function

Circulation Research ◽

10.1161/res.117.suppl_1.313 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Yi-Li Min ◽

Svetlana Bezprozvannaya ◽

Drazen Šošic ◽

Young-Jae Nam ◽

Hesham Sadek ◽

...

Keyword(s):

Cardiac Function ◽

Cardiac Fibroblasts ◽

Cell Lineage ◽

Cardiac Regeneration ◽

Cell Types ◽

Bhlh Transcription Factor ◽

Adult Heart ◽

Twist 1 ◽

Essential Contribution

Cardiomyocyte renewal occurs very slowly in adult mammals, and little is known of the genetic basis of cardiac regeneration. Twist is a highly conserved bHLH transcription factor responsible for Drosophila mesoderm formation during embryogenesis. Recent studies have shown that Twist protein is essential for muscle regeneration in adult Drosophila, but the potential role of Twist in the mammalian heart has not been explored. There are two Twist genes in vertebrates, Twist-1 and -2. We show that Twist-1 and -2 are expressed in epicardium and interstitial cells but not in differentiated cardiomyocytes in mice. To understand the potential function of Twist-dependent lineages in the adult heart, we generated inducible Twist2CreERT2; ROSA26-tdTomato reporter mice. By treating these mice with tamoxifen at 8 weeks of age, we observed progressive labeling of various cell types, such as epithelial cells, cardiac fibroblasts, and cardiomyocytes in the heart. We isolated Tomato-positive nonmyocytes from these mice and found that these cells can differentiate into cardiomyocytes and other cell types in vitro. Furthermore, cardiac-specific deletion of both Twist1 and Twist2 resulted in an age-dependent lethal cardiomyopathy. These findings reveal an essential contribution of Twist to long-term maintenance of cardiac function and support the concept of slow, lifelong renewal of cardiomyocytes from a Twist-dependent cell lineage in the adult heart.

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