Abstract 370: Hedgehog Signaling Regulates Cardiac Regeneration in vivo

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Bhairab N Singh ◽  
Wuming Gong ◽  
Mary G Garry ◽  
Naoko Koyano-Nakagawa ◽  
Daniel J Garry
Keyword(s):  
Hedgehog Signaling ◽  
Cardiac Regeneration ◽  
Lineage Tracing ◽  
Heart Regeneration ◽  
Cardiomyocyte Proliferation ◽  
Post Injury ◽  
Time Period ◽  
Hh Signaling ◽  
Regenerative Therapies

The adult mammalian heart has a limited regenerative capacity due primarily to reduced cardiomyocyte (CM) proliferation. Here, we demonstrated hedgehog (HH) signaling pathway as an essential regulator of heart regeneration and CM proliferation. We undertook genome-wide screening using a novel algorithm, bootstrap, which showed an induction of HH signals in the regenerating newt heart. Blockade of HH signaling in the resected newt heart resulted in complete ablation of cardiac regeneration and scar formation. EdU-labeling revealed that inhibition of the HH pathway significantly reduced CM proliferation by 3-fold (n=4 at each time period post-injury). In mammals, cardiac specific loss- and gain-of-function of HH signals demonstrated its role in CM proliferation and regeneration in the postnatal heart. Genetic deletion of floxed-Smoothened ( Smo L/L ) allele at postnatal day 2 (P2) inhibited neonatal heart regeneration with impaired cardiac function and scarring following injury. Conversely, induction of constitutively active Smoothened (SmoM2) at P7 stimulated CM proliferation by 2.5-fold (n=3) and regeneration after myocardial infarction during the non-regenerative window. Lineage-tracing experiments showed that activation of Smo contributed to heart regeneration by promoting proliferation of the pre-existing cardiomyocytes. Activation of HH signals in the cultured CM at P1 and P7 showed an increased proliferative response by 2- and 3-fold (n=4; 1900 cells evaluated for each condition), respectively. Mechanistically, ChIP-seq analysis revealed that HH signals promoted the proliferative program by directly regulating the expression of cyclin-dependent kinases including cyclinD2, cyclinE1 and Cdc7. Finally, activation of HH signaling in the terminally differentiated hiPSC-derived CM resulted in an increase in the number of α-Actinin + /EdU + and α-Actinin + /Ki67 + cells by 2.5-fold (n=3; 645 cells assessed for each condition) and 3-fold (n=3; 685 cells assessed for each condition), respectively. These studies defined an evolutionarily conserved function of HH signaling from newt to mouse to human, as a key regulator of cardiomyocyte proliferation and regeneration that may serve as a platform for regenerative therapies.

Abstract 20451: Hedgehog Signaling and Cardiomyocyte Proliferation

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Bhairab N Singh ◽  
Stefan M Kren ◽  
Wuming Gong ◽  
Cyprian Weaver ◽  
Kathy Bowlin ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Hedgehog Signaling ◽  
Cardiac Regeneration ◽  
Pcr Analysis ◽  
Gene Clustering ◽  
Cardiomyocyte Proliferation ◽  
Post Injury ◽  
Qrt Pcr ◽  
Hh Signaling

Background: In contrast to adult mammalian heart, lower vertebrates such as newt exhibit a dramatic capacity for regeneration in response to injury. Understanding the underlying mechanisms and signaling pathways in newt could form the basis for regenerative therapies in mammals. In the present study, we explored the role of hedgehog (HH) signaling during cardiac regeneration. Methods and Results: To investigate cardiac regeneration (CR) in vivo, we performed ventricular resection studies in adult newt heart. Whole mount and histochemical studies revealed CR within 30 d of resection injury. Functional assessment by echocardiographic analysis showed improved ejection fraction from 22 ± 5% at 4 d to 42 ± 3% at 30 d of regeneration relative to control (60 ± 3%). Gene clustering and qRT-PCR analysis at 7d post injury indicated enrichment of hedgehog (HH) signaling factors including Shh and ptch1 by 3- and 2-fold respectively, suggesting a critical requirement of HH signaling at early stages of CR. We determined HH signaling is essential as pharmacological inhibition of the HH pathway resulted in complete ablation of CR. Using EdU-labeling and immunohistochemical analyses, we showed that HH signaling regulates proliferation of both epicardial and myocardial cells, as inhibition of the HH pathway reduced EdU-positive nuclei from 15 ± 2% to 5 ± 3%. In contrast, qRT-PCR analysis from murine hearts postnatal day (P) 2 to P14, showed reduced levels of HH signaling factors and cell-cycle associated mRNAs by 3.5-fold with concomitant increase of p21 by 2-fold. This implies HH signals are required for the proliferative process. Consistent with the newt studies, activation of HH in murine neonatal ventricular cardiomyocytes (NVCM) promoted cardiomyocyte proliferation, increasing positive nuclei from 10 ± 2% to 25 ± 3%, while inhibition of HH signaling reduced positivity to 6 ± 3%. qRT-PCR analysis established that activation of HH signaling in NVCM resulted in up regulation of transcripts associated with cardiomyocyte proliferation. Conclusion: These data indicated that HH signaling pathways modulate cardiomyocyte proliferation providing potential therapeutic targets for achieving mammalian cardiac regeneration.

scholarly journals DUSP5 expression in left ventricular cardiomyocytes of young hearts regulates thyroid hormone (T3)-induced proliferative ERK1/2 signaling

2020 ◽  
Author(s):  
Nikolay Bogush ◽  
Lin Tan ◽  
Hussain Naib ◽  
Ebrahim Faizullabhoy ◽  
John W. Calvert ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Enzymatic Digestion ◽  
Left Ventricular ◽  
Lineage Tracing ◽  
Cardiomyocyte Proliferation ◽  
Dual Specificity ◽  
M Phase ◽  
Immunohistochemical Studies ◽  
Regenerative Therapies

Abstract A developmental surge in thyroid hormone (T3) after postnatal day-10 (P10), in mice, results in a burst of cardiomyocyte proliferation. This finding remains controversial, which could arise from perceived homogeneity of myocardial sampling for immunoblotting and immunohistochemical studies, or from differences in enzymatic digestion efficiency for cardiomyocyte isolation used to determine total ventricular cardiomyocyte numbers. Using a highly efficient (>97%) method for cardiomyocyte isolation, we show that exogenous T3 administered in vivo to post-neonatal (after postnatal P6) mice increased cardiomyocyte numbers. Using S- and M-phase markers, and lineage-tracing tools to assay for cell cycle activation and cytokinesis, respectively, we show that the T3-mediated increase in cardiomyocytes is confined to cells of the left ventricular (LV) apex; such spatial heterogeneity also being observed during normal development, which might confound substantiation of developmental increases in cardiomyocyte proliferation. In these apical cardiomyocytes, T3 stimulates proliferative ERK1/2 signaling, whereas those in the LV base are tonically inhibited by expression of the nuclear phospho-ERK1/2-specific dual-specificity phosphatase, DUSP5. In P21 Dusp5–/– mice, cardiomyocyte endowment and LV mass are increased relative to age-matched wild-type controls, suggesting that DUSP5 regulates early heart growth. Identification of mechanisms regulating cardiomyocyte proliferation may allow development of cardiac regenerative therapies.

scholarly journals Ephrin-B1 blocks adult cardiomyocyte proliferation and heart regeneration

2019 ◽  
Author(s):  
Marie Cauquil ◽  
Céline Mias ◽  
Céline Guilbeau-Frugier ◽  
Clément Karsenty ◽  
Marie-Hélène Seguelas ◽  
...  
Keyword(s):  
Resting State ◽  
Cardiac Tissue ◽  
Hippo Pathway ◽  
Cardiac Regeneration ◽  
Mouse Heart ◽  
Heart Regeneration ◽  
Cardiomyocyte Proliferation ◽  
Postnatal Maturation

AbstractAimsDeciphering the innate mechanisms governing the blockade of proliferation in adult cardiomyocytes (CMs) is challenging for mammalian heart regeneration. Despite the exit of CMs from the cell cycle during the postnatal maturation period coincides with their morphological switch to a typical adult rod-shape, whether these two processes are connected is unknown. Here, we examined the role of ephrin-B1, a CM rod-shape stabilizer, in adult CM proliferation and cardiac regeneration.Methods and resultsTransgenic- or AAV9-based ephrin-B1 repression in adult mouse heart led to substantial proliferation of resident CMs and tissue regeneration to compensate for apex resection, myocardial infarction (MI) and senescence. Interestingly, in the resting state, CMs lacking ephrin-B1 did not constitutively proliferate, indicative of no major cardiac defects. However, they exhibited proliferation-competent signature, as indicated by higher mononucleated state and a dramatic decrease of miR-195 mitotic blocker, which can be mobilized under neuregulin-1 stimulation in vitro and in vivo. Mechanistically, the post-mitotic state of the adult CM relies on ephrin-B1 sequestering of inactive phospho-Yap1, the effector of the Hippo-pathway, at the lateral membrane. Hence, ephrin-B1 repression leads to phospho-Yap1 release in the cytosol but CM quiescence at resting state. Upon cardiac stresses (apectomy, MI, senescence), Yap1 could be activated and translocated to the nucleus to induce proliferation-gene expression and consequent CM proliferationConclusionsOur results identified ephrin-B1 as a new natural locker of adult CM proliferation and emphasize that targeting ephrin-B1 may prove a future promising approach in cardiac regenerative medicine for HF treatment.SignificanceThe mammalian adult heart is unable to regenerate due to the inability of cardiomyocytes (CMs) to proliferate and replace cardiac tissue lost. Exploiting CM-specific transgenic mice or AAV9-based gene therapy, this works identifies ephrin-B1, a specific rod-shape stabilizer of the adult CM, as a natural padlock of adult CM proliferation for compensatory adaptation to different cardiac stresses (apectomy, MI, senescence), thus emphasizing a new link between the adult CM morphology and their proliferation potential. Moreover, the study demonstrates proof-of-concept that targeting ephrin-B1 may be an innovative therapeutic approach for ischemic heart failure.

scholarly journals Prrx1b restricts fibrosis and promotes Nrg1-dependent cardiomyocyte proliferation during zebrafish heart regeneration

Development ◽  
2021 ◽  
Author(s):  
Dennis E.M. de Bakker ◽  
Mara Bouwman ◽  
Esther Dronkers ◽  
Filipa C. Simões ◽  
Paul R. Riley ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cardiac Injury ◽  
Lineage Tracing ◽  
Heart Regeneration ◽  
Cardiomyocyte Proliferation ◽  
Fibrotic Scar ◽  
Ligand Expression ◽  
Zebrafish Heart

Fibroblasts are activated to repair the heart following injury. Fibroblast activation in the mammalian heart leads to a permanent fibrotic scar that impairs cardiac function. In other organisms, like zebrafish, cardiac injury is followed by transient fibrosis and scar-free regeneration. The mechanisms that drive scarring versus scar-free regeneration are not well understood. Here we show that the homeo-box containing transcription factor Prrx1b is required for scar-free regeneration of the zebrafish heart as the loss of Prrx1b results in excessive fibrosis and impaired cardiomyocyte proliferation. Through lineage tracing and single-cell RNA-sequencing we find that Prrx1b is activated in epicardial-derived cells (EPDCs) where it restricts TGF-β ligand expression and collagen production. Furthermore, through combined in vitro experiments in human fetal EPDCs and in vivo rescue experiments in zebrafish, we conclude that Prrx1 stimulates Nrg1 expression and promotes cardiomyocyte proliferation. Collectively, these results indicate that Prrx1 is a key transcription factor that balances fibrosis and regeneration in the injured zebrafish heart.

scholarly journals Zebrafish cardiac regeneration—looking beyond cardiomyocytes to a complex microenvironment

2020 ◽  
Author(s):  
Rebecca Ryan ◽  
Bethany R. Moyse ◽  
Rebecca J. Richardson
Keyword(s):  
Cardiac Injury ◽  
Cell Communication ◽  
Cardiac Regeneration ◽  
Cell Types ◽  
Regenerative Process ◽  
Heart Regeneration ◽  
Cardiomyocyte Proliferation ◽  
Underlying Mechanisms ◽  
Different Cell Types

Abstract The study of heart repair post-myocardial infarction has historically focused on the importance of cardiomyocyte proliferation as the major factor limiting adult mammalian heart regeneration. However, there is mounting evidence that a narrow focus on this one cell type discounts the importance of a complex cascade of cell–cell communication involving a whole host of different cell types. A major difficulty in the study of heart regeneration is the rarity of this process in adult animals, meaning a mammalian template for how this can be achieved is lacking. Here, we review the adult zebrafish as an ideal and unique model in which to study the underlying mechanisms and cell types required to attain complete heart regeneration following cardiac injury. We provide an introduction to the role of the cardiac microenvironment in the complex regenerative process and discuss some of the key advances using this in vivo vertebrate model that have recently increased our understanding of the vital roles of multiple different cell types. Due to the sheer number of exciting studies describing new and unexpected roles for inflammatory cell populations in cardiac regeneration, this review will pay particular attention to these important microenvironment participants.

scholarly journals BITC induces cardiomyocyte proliferation and heart regeneration

2021 ◽  
Author(s):  
Akane Sakaguchi ◽  
Miwa Kawasaki ◽  
Kozue Murata ◽  
Hidetoshi Masumoto ◽  
Wataru Kimura
Keyword(s):  
Cell Cycle ◽  
Cardiac Regeneration ◽  
Heart Regeneration ◽  
Cardiomyocyte Proliferation ◽  
Hypoxia Exposure ◽  
Cell Cycle Reentry ◽  
Regeneration Period ◽  
Mild Hypoxia

AbstractMammalian cardiomyocytes have the ability to proliferate from the embryonic stage until early neonatal stage, with most of them being arrested from the cell cycle shortly after birth. Therefore, adult mammalian heart cannot regenerate myocardial injury. Despite much attention, pharmacological approaches for the induction of cardiomyocyte proliferation and heart regeneration have yet to be successful. To induce cardiomyocyte proliferation by drug administration, we focused on benzyl isothiocyanate (BITC). Firstly, we showed that BITC induces cardiomyocyte proliferation both in vitro and in vivo through the activation of the cyclin-dependent kinase (CDK) pathway. In addition, we demonstrated that BITC treatment induces heart regeneration in the infarcted neonatal heart even after the regeneration period. Furthermore, we administered BITC to adult mice in parallel with mild hypoxia (10% O2) treatment and showed that a combination of BITC administration and mild hypoxia exposure induces cell cycle reentry in the adult heart. The present study suggests that pharmacological activation of the CDK pathway with BITC concurrently with the activation of hypoxia-related signaling pathways may enable researchers to establish a novel strategy to induce cardiac regeneration in patients with heart disease.

scholarly journals Osteocalcin expressing cells from tendon sheaths in mice contribute to tendon repair by activating Hedgehog signaling

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Yi Wang ◽  
Xu Zhang ◽  
Huihui Huang ◽  
Yin Xia ◽  
YiFei Yao ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Hedgehog Signaling ◽  
Progenitor Cell ◽  
Tendon Repair ◽  
Tendon Sheath ◽  
Lineage Tracing ◽  
Specific Marker ◽  
Molecular Function ◽  
Hh Signaling ◽  
Necessary And Sufficient

Both extrinsic and intrinsic tissues contribute to tendon repair, but the origin and molecular functions of extrinsic tissues in tendon repair are not fully understood. Here we show that tendon sheath cells harbor stem/progenitor cell properties and contribute to tendon repair by activating Hedgehog signaling. We found that Osteocalcin (Bglap) can be used as an adult tendon-sheath-specific marker in mice. Lineage tracing experiments show that Bglap-expressing cells in adult sheath tissues possess clonogenic and multipotent properties comparable to those of stem/progenitor cells isolated from tendon fibers. Transplantation of sheath tissues improves tendon repair. Mechanistically, Hh signaling in sheath tissues is necessary and sufficient to promote the proliferation of Mkx-expressing cells in sheath tissues, and its action is mediated through TGFβ/Smad3 signaling. Furthermore, co-localization of GLI1+ and MKX+ cells is also found in human tendinopathy specimens. Our work reveals the molecular function of Hh signaling in extrinsic sheath tissues for tendon repair.

Abstract 20134: Transcriptional Profiling Identifies Candidate Regulators of Neonatal Heart Regeneration

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Caitlin O’Meara ◽  
Joseph Wamstad ◽  
Laurie Boyer ◽  
Richard T Lee
Keyword(s):  
Cell Cycle ◽  
Transcriptional Profiling ◽  
Cardiac Regeneration ◽  
Heart Regeneration ◽  
Transcriptional Signature ◽  
Adult Mice ◽  
Neonatal Heart ◽  
Sirna Knockdown

Some higher organisms, such as zebrafish and neonatal mice, are capable of complete and sufficient regeneration of the myocardium following injury, which is thought to occur primarily by proliferation of pre-existing cardiomyocytes. Although adult humans and adult mice lack this cardiac regeneration potential, there is great interest in understanding how regeneration can occur in the heart so that we can activate this process in humans suffering from heart failure. The aim of our study was to identify mechanisms by which mature, post-mitotic adult cardiomyocytes can re-enter the cell cycle to ultimately facilitate heart regeneration following injury. We derived a core transcriptional signature of injury-induced cardiomyocyte regeneration in mouse by comparing global transcriptional programs in a dynamic model of in vitro and in vivo cardiomyocyte differentiation and in an in vitro cardiomyocyte explant model, as well as a neonatal heart resection model. We identified a panel of transcription factors, growth factors, and cytokines, whose expression significantly correlated with the differentiated state of the cell in all datasets examined, suggesting that these factors play a role in regulating cardiomyocyte cell state. Furthermore, potential upstream regulators of core differentially expressed networks were identified using Ingenuity Pathway Analysis and we found that one predicted regulator, interleukin-13 (IL13), significantly induced cardiomyocyte cell cycle activity and STAT6/STAT3 signaling in vitro. siRNA knockdown experiments demonstrated that STAT3/periostin and STAT6 signaling are critical for cardiomyocyte cell cycle activity in response to IL13. These data reveal novel insights into the transcriptional regulation of mammalian heart regeneration and provide the founding circuitry for identifying potential regulators for stimulating cardiomyocyte cell cycle activity.

scholarly journals Mechanistic basis of neonatal heart regeneration revealed by transcriptome and histone modification profiling

2019 ◽  
Vol 116 (37) ◽  
pp. 18455-18465 ◽  
Author(s):  
Zhaoning Wang ◽  
Miao Cui ◽  
Akansha M. Shah ◽  
Wenduo Ye ◽  
Wei Tan ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Rna Binding ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cardiac Regeneration ◽  
Later Life ◽  
Heart Regeneration ◽  
Cardiomyocyte Proliferation ◽  
Neonatal Heart ◽  
Short Period

The adult mammalian heart has limited capacity for regeneration following injury, whereas the neonatal heart can readily regenerate within a short period after birth. To uncover the molecular mechanisms underlying neonatal heart regeneration, we compared the transcriptomes and epigenomes of regenerative and nonregenerative mouse hearts over a 7-d time period following myocardial infarction injury. By integrating gene expression profiles with histone marks associated with active or repressed chromatin, we identified transcriptional programs underlying neonatal heart regeneration, and the blockade to regeneration in later life. Our results reveal a unique immune response in regenerative hearts and a retained embryonic cardiogenic gene program that is active during neonatal heart regeneration. Among the unique immune factors and embryonic genes associated with cardiac regeneration, we identified Ccl24, which encodes a cytokine, and Igf2bp3, which encodes an RNA-binding protein, as previously unrecognized regulators of cardiomyocyte proliferation. Our data provide insights into the molecular basis of neonatal heart regeneration and identify genes that can be modulated to promote heart regeneration.

Cardiomyocyte proliferation in cardiac development and regeneration: a guide to methodologies and interpretations

2015 ◽  
Vol 309 (8) ◽  
pp. H1237-H1250 ◽  
Author(s):  
Marina Leone ◽  
Ajit Magadum ◽  
Felix B. Engel
Keyword(s):  
Cardiac Function ◽  
Molecular Mechanisms ◽  
Cardiac Development ◽  
Cardiac Regeneration ◽  
Experimental Medicine ◽  
Heart Regeneration ◽  
Cardiomyocyte Proliferation ◽  
Naturally Occurring ◽  
Ability To Regenerate ◽  
Zebrafish Heart

The newt and the zebrafish have the ability to regenerate many of their tissues and organs including the heart. Thus, a major goal in experimental medicine is to elucidate the molecular mechanisms underlying the regenerative capacity of these species. A wide variety of experiments have demonstrated that naturally occurring heart regeneration relies on cardiomyocyte proliferation. Thus, major efforts have been invested to induce proliferation of mammalian cardiomyocytes in order to improve cardiac function after injury or to protect the heart from further functional deterioration. In this review, we describe and analyze methods currently used to evaluate cardiomyocyte proliferation. In addition, we summarize the literature on naturally occurring heart regeneration. Our analysis highlights that newt and zebrafish heart regeneration relies on factors that are also utilized in cardiomyocyte proliferation during mammalian fetal development. Most of these factors have, however, failed to induce adult mammalian cardiomyocyte proliferation. Finally, our analysis of mammalian neonatal heart regeneration indicates experiments that could resolve conflicting results in the literature, such as binucleation assays and clonal analysis. Collectively, cardiac regeneration based on cardiomyocyte proliferation is a promising approach for improving adult human cardiac function after injury, but it is important to elucidate the mechanisms arresting mammalian cardiomyocyte proliferation after birth and to utilize better assays to determine formation of new muscle mass.

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