Differentiation Of Cardiac Cells Research Articles

Differentiation, Metabolism, and Cardioprotective Secretory Functions of Human Cardiac Stromal Cells from Ischemic and Endocarditis Patients.

This study investigates the characteristics of cardiac mesenchymal stem cell-like cells (CMSCLCs) isolated from the right atrial appendage of human donors with ischemia and a young patient with endocarditis (NE-CMSCLCs). Typical CMSCLCs from ischemic heart patients were derived from coronary artery bypass grafting procedures and compared against bone marrow mesenchymal stromal cells (BM-MSCs). NE-CMSCLCs had a normal immunophenotype, but exhibited enhanced osteogenic differentiation potential, rapid proliferation, reduced senescence, reduced glycolysis, and lower reactive oxygen species generation after oxidative stress compared with typical ischemic CMSCLCs. These differences suggest a unique functional status of NE-CMSCLCs, influenced by the donor health condition. Despite large variances in their paracrine secretome, NE-CMSCLCs retained therapeutic potential, as indicated by their ability to protect hypoxia/reoxygenation-injured human cardiomyocytes, albeit less effectively than typical CMSCLCs. This research describes a unique cell phenotype and underscores the importance of donor health status in the therapeutic efficacy of autologous cardiac cell therapy.

Dynamic links between the cardiac intermediate filaments of the cytoskeleton and nucleoskeleton for proper cardiomyocyte differentiation, maturation and reprogramming

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): 1) ESPA 09SYN-21-966 2) ELIDEK 1594 Accumulating evidence suggests that mechanical forces are integral to the commitment and differentiation of cardiac stem cells. Studies have proposed that the cardiac cytoskeleton and specifically the desmin intermediate filament (IF) network and its association with nuclear lamins through the LINC complex, link the contractile apparatus to both the extracellular and nuclear matrix, thus regulating trafficking processes and mechanotransduction during development and adulthood, governing myocyte homeostasis and survival(1-3). Desmin, in addition to being one of the earliest myogenic markers both in heart and somites(4,5), regulates nuclear shape and positioning(6,7), as well as the expression of myogenic (MyoD, Myogenin)(8) and cardiogenic (MEF2, Nkx2.5) transcription regulators, during embryonic stem cell differentiation(4,5). Consistent with the above findings, desmin deficiency has negative impact in the proper regeneration of skeletal muscle cells by hematopoietic stem cells(9). Despite these overwhelming data linking desmin to the establishment and maintenance of myogenesis and cardiogenesis, the precise underlying mechanisms remain elusive. To dissect the exact mechanism of cardiogenic regulation we took advantage of the fibroblast-to-cardiomyocyte direct reprogramming, where fibroblasts trans-differentiate into functional induced cardiomyocytes (iCM) through ectopic expression of three transcription factors, GATA4, Mef2C and Tbx5 (GMT)(10). We hypothesized that desmin could enhance the reprogramming of fibroblasts into cardiomyocytes. Indeed, we have found that expression of desmin together with the GMT factors increases reprogramming by 40-60%. Involvement of a cytoskeletal protein such as desmin in cell differentiation and potentially regeneration, raises intriguing questions and exciting possibilities. To unravel the mechanism of this enhancement we focused on desmin serving as direct mechanotransducer (through the LINC complex) to chromatin. Toward this goal, we investigate the changes of the genetic profile induced by the GMT cocktail during reprogramming in the presence or absence of desmin. RNA sequence analysis has shown that the Notch developmental pathway is regulated by desmin. With the use of Chromosome Conformation Capture, Hi-C and Chip-sequence analysis, we demonstrated the importance of desmin cytoskeleton on the genome 3D organization. In addition, we show that desmin is expressed in most of the cardiac progenitor cells population declaring its role in proper cardiac formation. Moreover, immunofluorescence, calcium influx and electron microscopy studies establish desmin as a major player for cardiac maturity, a critical subject in cardiac regenerative medicine, since its absence substantial reduces expression and proper localization of cardiac specific proteins, delays myofibril formation and impairs function of the induced cardiomyocytes driving most of them to senescence. All the above data will be presented.

Proteomic and cardiac dysregulation by representative perfluoroalkyl acids of different chemical speciation during early embryogenesis of zebrafish

Perfluoroalkyl acids (PFAAs) of different chemical speciation were previously found to cause diverse toxicity. However, the toxicological mechanisms depending on chemical speciation are still largely unknown. In this follow-up study, zebrafish embryos were acutely exposed to only one concentration at 4.67 μM of the acid and salt of representative PFAAs, including perfluorooctanoic acid (PFOA), perfluorobutane carboxylic acid (PFBA), and perfluorobutanesulfonic acid (PFBS), till 96 h post-fertilization (hpf), aiming to gain more mechanistic insights. High-throughput proteomics found that PFAA acid and salt exerted discriminative effects on protein expression pattern. Bioinformatic analyses based on differentially expressed proteins underlined the developmental cardiotoxicity of PFOA acid with regard to cardiac muscle contraction, vascular smooth muscle contraction, adrenergic signaling in cardiomyocytes, and multiple terms related to myocardial contraction. PFOA salt and PFBS acid merely disrupted the cardiac muscle contraction pathway, while cardiac muscle cell differentiation was significantly enriched in PFBA acid-exposed zebrafish larvae. Consistently, under PFAA exposure, especially PFOA and PFBS acid forms, transcriptional levels of key genes for cardiogenesis and the concentrations of troponin and epinephrine associated with myocardial contraction were significantly dysregulated. Moreover, a transgenic line Tg (my17: GFP) expressing green fluorescent protein in myocardial cells was employed to visualize the histopathology of developing heart. PFOA acid concurrently caused multiple deficits in heart morphogenesis and function, which were characterized by the significant increase in sinus venosus and bulbus arteriosus distance (SV-BA distance), the induction of pericardial edema, and the decrease in heart rate, further confirming the stronger toxicity of PFOA acid than the salt counterpart on heart development. Overall, this study highlighted the developmental cardiotoxicity of PFAAs, with potency ranking PFOA > PFBS > PFBA. The acid forms of PFAAs induced stronger cardiac toxicity than their salt counterparts, providing an additional insight into the structure-toxicity relationship.

Single-cell RNA sequencing reveals the transcriptional heterogeneity of Tbx18-positive cardiac cells during heart development.

The T-box family transcription factor 18 (Tbx18) has been found to play a critical role in regulating the development of the mammalian heart during the primary stages of embryonic development while the cellular heterogeneity and landscape of Tbx18-positive (Tbx18+) cardiac cells remain incompletely characterized. Here, we analyzed prior published single-cell RNA sequencing (scRNA-seq) mouse heart data to explore the heterogeneity of Tbx18+ cardiac cell subpopulations and provide a comprehensive transcriptional landscape of Tbx18+ cardiac cells during their development. Bioinformatic analysis methods were utilized to identify the heterogeneity between cell groups. Based on the gene expression characteristics, Tbx18+ cardiac cells can be classified into a minimum of two distinct cell populations, namely fibroblast-like cells and cardiomyocytes. In terms of temporal heterogeneity, these cells exhibit three developmental stages, namely the MEM stage, ML_P0 stage, and P stage Tbx18+ cardiac cells. Furthermore, Tbx18+ cardiac cells encompass several cell types, including cardiac progenitor-like cells, cardiomyocytes, and epicardial/stromal cells, as determined by specific transcriptional regulatory networks. The scRNA-seq results revealed the involvement of extracellular matrix (ECM) signals and epicardial epithelial-to-mesenchymal transition (EMT) in the development of Tbx18+ cardiac cells. The utilization of a lineage-tracing model served to validate the crucial function of Tbx18 in the differentiation of cardiac cells. Consequently, these findings offer a comprehensive depiction of the cellular heterogeneity within Tbx18+ cardiac cells.

Design-Driven Translational Tools to Manage Cardiac Regeneration as Applicable to Clinical Practice

Cardiovascular disease is the leading cause of death, with 80% of cases occurring in developing countries. Innovative therapies are required to reduce mortality and limit or abolish the necessity for cardiac transplantation. Over the last decade, stem cells have been a promise for the cure of several diseases not only due to their plasticity but also to their capacity to act in a paracrine manner and influence the affected tissue. Human SC-based therapy derivatives are extremely attractive for therapeutic development because they have direct pharmacologic utility in clinical applications, unlike any other adult cells. Moreover, stem cell-derived paracrine factors have been shown to suppress inflammation and apoptosis, stimulate angiogenesis, and amplify the proliferation and differentiation of resident cardiac stem cells (CSCs). And SC therapies are thus viable alternatives to conventional treatments with substantial therapeutic potential; market opportunities are huge, as multiple product candidates are expected to be approved over the coming decade.

The benign nature and rare occurrence of cardiac myxoma as a possible consequence of the limited cardiac proliferative/ regenerative potential: a systematic review

BackgroundCardiac Myxoma is a primary tumor of heart. Its origins, rarity of the occurrence of primary cardiac tumors and how it may be related to limited cardiac regenerative potential, are not yet entirely known. This study investigates the key cardiac genes/ transcription factors (TFs) and signaling pathways to understand these important questions.MethodsDatabases including PubMed, MEDLINE, and Google Scholar were searched for published articles without any date restrictions, involving cardiac myxoma, cardiac genes/TFs/signaling pathways and their roles in cardiogenesis, proliferation, differentiation, key interactions and tumorigenesis, with focus on cardiomyocytes.ResultsThe cardiac genetic landscape is governed by a very tight control between proliferation and differentiation-related genes/TFs/pathways. Cardiac myxoma originates possibly as a consequence of dysregulations in the gene expression of differentiation regulators including Tbx5, GATA4, HAND1/2, MYOCD, HOPX, BMPs. Such dysregulations switch the expression of cardiomyocytes into progenitor-like state in cardiac myxoma development by dysregulating Isl1, Baf60 complex, Wnt, FGF, Notch, Mef2c and others.The Nkx2–5 and MSX2 contribute predominantly to both proliferation and differentiation of Cardiac Progenitor Cells (CPCs), may possibly serve roles based on the microenvironment and the direction of cell circuitry in cardiac tumorigenesis. The Nkx2–5 in cardiac myxoma may serve to limit progression of tumorigenesis as it has massive control over the proliferation of CPCs. The cardiac cell type-specific genetic programming plays governing role in controlling the tumorigenesis and regenerative potential.ConclusionThe cardiomyocytes have very limited proliferative and regenerative potential. They survive for long periods of time and tightly maintain the gene expression of differentiation genes such as Tbx5, GATA4 that interact with tumor suppressors (TS) and exert TS like effect. The total effect such gene expression exerts is responsible for the rare occurrence and benign nature of primary cardiac tumors. This prevents the progression of tumorigenesis. But this also limits the regenerative and proliferative potential of cardiomyocytes. Cardiac Myxoma develops as a consequence of dysregulations in these key genes which revert the cells towards progenitor-like state, hallmark of CM. The CM development in carney complex also signifies the role of TS in cardiac cells.

PCSK9 inhibition ameliorates experimental autoimmune myocarditis by reducing Th17 cell differentiation through LDLR/STAT-3/ROR-γt pathway

Proprotein convertase subtilisin kexin type 9 (PCSK9) was characterized as a protein regulating circulating cholesterol metabolism; however, recent studies demonstrated a role for PCSK9 in inflammatory and autoimmune diseases unrelated to cholesterol alterations. The implication of PCSK9 in myocarditis is unclear and we aim at investigating the roles and mechanisms of PCSK9 in myocarditis. Male BALB/c mice received subcutaneous immunization with MyHC-α peptide on days 0 and 7 to establish the experimental autoimmune myocarditis (EAM) model. PCSK9 inhibitor, evolocumab, was administered subcutaneously once a week starting on day 0 and all mice were euthanized on day 21. Our results showed that PCSK9 inhibition ameliorated the cardiac inflammation of EAM mice. PCSK9 inhibition reduced both the levels of cardiac and peripheral blood PCSK9. We found that CD4+ T cells, CD8+ T cells, macrophages, and cardiomyocytes in the heart of EAM mice could express PCSK9. PCSK9 inhibition decreased the differentiation of cardiac Th17 cells by lowering ROR-γt levels but had no effects on Th1, Th2, and Treg cell differentiation. In vitro experiments of CD4+ T cells, we found that PCSK9 directly promoted Th17 cell differentiation through LDLR/STAT3/ROR-γt pathway. Collectively, we demonstrated that PCSK9 inhibition ameliorated the severity of EAM mice by reducing Th17 cell differentiation. PCSK9 is a promising target for treating myocarditis.

Wnt9 directs zebrafish heart tube assembly via a combination of canonical and non-canonical pathway signaling.

During zebrafish heart formation, cardiac progenitor cells converge at the embryonic midline where they form the cardiac cone. Subsequently, this structure transforms into a heart tube. Only little is known about the molecular mechanisms that control these morphogenetic processes. Here, we use light sheet microscopy and combine genetic, molecular biological and pharmacological tools to show that the paralogous genes wnt9a/b are required for the assembly of the nascent heart tube. In wnt9a/b double mutants, cardiomyocyte progenitor cells are delayed in their convergence towards the embryonic midline, the formation of the heart cone is impaired and the transformation into an elongated heart tube fails. The same cardiac phenotype occurs when both canonical and non-canonical Wnt signaling pathways are simultaneously blocked by pharmacological inhibition. This demonstrates that Wnt9a/b and canonical and non-canonical Wnt signaling regulate the migration of cardiomyocyte progenitor cells and control the formation of the cardiac tube. This can be partly attributed to their regulation of the timing of cardiac progenitor cell differentiation. Our study demonstrates how these morphogens activate a combination of downstream pathways to direct cardiac morphogenesis.

Abstract GS.102: Cd206 + Il-4rα + Macrophages Are Drivers Of Adverse Cardiac Remodeling In Ischemic Cardiomyopathy

Background: Cardiac macrophages are significantly expanded in chronic heart failure (HF); however, their role is unclear. We tested the hypothesis that CD206 + macrophages expressing interleukin (IL)-4Rα in the failing heart are key drivers of adverse left ventricular (LV) remodeling and ischemic cardiomyopathy. Methods and Results: Adult male C57BL/6 (WT) mice underwent non-reperfused myocardial infarction (MI) to induce HF, or sham operation, and cardiac macrophages were profiled using flow cytometry. As compared to sham hearts, both the frequency and absolute number of CD206 + macrophages steadily increased in post-MI hearts during the progression of LV remodeling and HF, such that at 8 w post-MI they comprised ~85% of macrophages. These macrophages were exclusively proliferative and predominantly C-C motif chemokine receptor (CCR2) - with nearly half expressing IL-4Rα, and correlated with LV dysfunction and fibrosis. IL-4-polarized bone marrow derived CD206 + macrophages exhibited marked upregulation of found in inflammatory zone (FIZZ)1 and induced FIZZ1-dependent myofibroblast differentiation of cardiac mesenchymal stem cells (cMSCs), in part related to DLL-4/Jagged1-Notch1 signaling. Intramyocardial adoptive transfer of M[IL-4], but not M[IL-10], macrophages induced progressive LV dilation and dysfunction and augmented cardiac fibrosis over 4 weeks in naïve mice. In vivo IL-4Rα gene silencing in mice with established HF effectively depleted cardiac CD206 + IL-4Rα + macrophages and induced reverse LV remodeling, improving fibrosis, neovascularization, and dysfunction, and suppressing local and systemic inflammation. Lastly, alternatively activated CD206 + and CD163 + macrophages were significantly expanded in human failing hearts and correlated with cardiac fibrosis, with the majority of CD163 + macrophages expressing IL-4Rα and FIZZ3, the human homolog of FIZZ1. Conclusion: CD206 + IL-4Rα + macrophages proliferate and expand in the failing heart and are key mediators of pathological remodeling and fibrosis, in part through the secretion of FIZZ1. Inhibition of CD206 + macrophage IL-4Rα signaling may be a fruitful therapeutic approach for alleviating LV remodeling in HF.

Keratose Hydrogel Drives Differentiation of Cardiac Vascular Smooth Muscle Progenitor Cells: Implications in Ischemic Treatment.

Peripheral artery disease (PAD) is a common vascular disorder in the extremity of limbs with limited clinical treatments. Stem cells hold great promise for thetreatment of PAD, but their therapeutic efficiency is limited due to multiple factors, such as poor engraftment and non-optimal selection of cell type. To date, stem cells from a variety of tissue sources have been tested, but little information is available regarding vascular smooth muscle cells (VSMCs) for PAD therapy. The present study examines the effects of keratose (KOS) hydrogels on c-kit+/CD31- cardiac vascular smooth muscle progenitor cell (cVSMPC) differentiation and the therapeutic potential of the resultant VSMCs in a mouse hindlimb ischemic model of PAD. The results demonstrated that KOS but not collagen hydrogel was able to drive the majority of cVSMPCs into functional VSMCs in a defined Knockout serum replacement (SR) medium in the absence of differentiation inducers. This effect could be inhibited by TGF-β1 antagonists. Further, KOS hydrogel increased expression of TGF-β1-associated proteins and modulated the level of free TGF-β1 during differentiation. Finally, transplantation of KOS-driven VSMCs significantly increased blood flow and vascular densities of ischemic hindlimbs. These findings indicate that TGF-β1 signaling is involved in KOS hydrogel-preferred VSMC differentiation and that enhanced blood flow are likely resulted from angiogenesis and/or arteriogenesis induced by transplanted VSMCs.

Tissue Engineering and Targeted Drug Delivery in Cardiovascular Disease: The Role of Polymer Nanocarrier for Statin Therapy

Atherosclerosis-related coronary artery disease (CAD) is the leading cause of mortality and morbidity worldwide. This requires effective primary and secondary prevention in reducing the complications related to CAD; the regression or stabilization of the pathology remains the mainstay of treatment. Statins have proved to be the most effective treatment in reducing adverse effects, but there are limitations related to the administration and achievement of effective doses as well as side effects due to the lack of target-related molecular specificity. The implemented technological steps are polymers and nanoparticles for the administration of statins, as it has been seen how the conjugation of drug delivery systems (DDSs) with statins increases bioavailability by circumventing the hepatic-renal filter and increases the related target specificity, enhancing their action and decreasing side effects. Reduction of endothelial dysfunction, reduced intimal hyperplasia, reduced ischemia-reperfusion injury, cardiac regeneration, positive remodeling in the extracellular matrix, reduced neointimal growth, and increased reendothelialization are all drug-related effects of statins enhanced by binding with DDSs. Recent preclinical studies demonstrate how the effect of statins stimulates the differentiation of endogenous cardiac stem cells. Poly-lactic-co-glycolic acid (PLGA) seems to be the most promising DDS as it succeeds more than the others in enhancing the effect of the bound drug. This review intends to summarize the current evidence on polymers and nanoparticles for statin delivery in the field of cardiovascular disease, trying to shed light on this topic and identify new avenues for future studies.

The Calcineurin-Drp1-Mediated Mitochondrial Fragmentation Is Aligned with the Differentiation of c-Kit Cardiac Progenitor Cells.

The heart contains a pool of c-kit+ progenitor cells which is believed to be able to regenerate. The differentiation of these progenitor cells is reliant on different physiological cues. Unraveling the underlying signals to direct differentiation of progenitor cells will be beneficial in controlling progenitor cell fate. In this regard, the role of the mitochondria in mediating cardiac progenitor cell fate remains unclear. Specifically, the association between changes in mitochondrial morphology with the differentiation status of c-kit+ CPCs remains elusive. In this study, we investigated the relationship between mitochondrial morphology and the differentiation status of c-kit+ progenitor cells. c-kit+ CPCs were isolated from 2-month-old male wild-type FVB mice. To activate differentiation, CPCs were incubated in α-minimal essential medium containing 10 nM dexamethasone for up to 7 days. To inhibit Drp1-mediated mitochondrial fragmentation, either 10 μM or 50 μM mdivi-1 was administered once at Day 0 and again at Day 2 of differentiation. To inhibit calcineurin, either 1 μM or 5 μM ciclosporin-A (CsA) was administered once at Day 0 and again at Day 2 of differentiation. Dexamethasone-induced differentiation of c-kit+ progenitor cells is aligned with fragmentation of the mitochondria via a calcineurin-Drp1 pathway. Pharmacologically inhibiting mitochondrial fragmentation retains the undifferentiated state of the c-kit+ progenitor cells. The findings from this study provide an alternative view of the role of mitochondrial fusion-fission in the differentiation of cardiac progenitor cells and the potential of pharmacologically manipulating the mitochondria to direct progenitor cell fate.

Extracellular Matrix-Based Approaches in Cardiac Regeneration: Challenges and Opportunities.

Cardiac development is characterized by the active proliferation of different cardiac cell types, in particular cardiomyocytes and endothelial cells, that eventually build the beating heart. In mammals, these cells lose their regenerative potential early after birth, representing a major obstacle to our current capacity to restore the myocardial structure and function after an injury. Increasing evidence indicates that the cardiac extracellular matrix (ECM) actively regulates and orchestrates the proliferation, differentiation, and migration of cardiac cells within the heart, and that any change in either the composition of the ECM or its mechanical properties ultimately affect the behavior of these cells throughout one's life. Thus, understanding the role of ECMs' proteins and related signaling pathways on cardiac cell proliferation is essential to develop effective strategies fostering the regeneration of a damaged heart. This review provides an overview of the components of the ECM and its mechanical properties, whose function in cardiac regeneration has been elucidated, with a major focus on the strengths and weaknesses of the experimental models so far exploited to demonstrate the actual pro-regenerative capacity of the components of the ECM and to translate this knowledge into new therapies.

Molecular perspectives in hypertrophic heart disease: An epigenetic approach from chromatin modification.

Epigenetic changes induced by environmental factors are increasingly relevant in cardiovascular diseases. The most frequent molecular component in cardiac hypertrophy is the reactivation of fetal genes caused by various pathologies, including obesity, arterial hypertension, aortic valve stenosis, and congenital causes. Despite the multiple investigations performed to achieve information about the molecular components of this pathology, its influence on therapeutic strategies is relatively scarce. Recently, new information has been taken about the proteins that modify the expression of fetal genes reactivated in cardiac hypertrophy. These proteins modify the DNA covalently and induce changes in the structure of chromatin. The relationship between histones and DNA has a recognized control in the expression of genes conditioned by the environment and induces epigenetic variations. The epigenetic modifications that regulate pathological cardiac hypertrophy are performed through changes in genomic stability, chromatin architecture, and gene expression. Histone 3 trimethylation at lysine 4, 9, or 27 (H3-K4; -K9; -K27me3) and histone demethylation at lysine 9 and 79 (H3-K9; -K79) are mediators of reprogramming in pathologic hypertrophy. Within the chromatin architecture modifiers, histone demethylases are a group of proteins that have been shown to play an essential role in cardiac cell differentiation and may also be components in the development of cardiac hypertrophy. In the present work, we review the current knowledge about the influence of epigenetic modifications in the expression of genes involved in cardiac hypertrophy and its possible therapeutic approach.

Abstract 14259: Novel Long Noncoding RNA, Ppp1r1b-LncRNA , Epigenetically Promotes Myogenic Differentiation Program in Cardiac and Skeletal Myocytes

Introduction: Emerged as critical regulators of the transcriptome, the lncRNAs play important roles in cardiac development and might be targeted to treat human cardiomyocyte dysfunction in congenital heart disease. In our work, we identified a novel lncRNA, Ppp1r1b-lncRNA , as an essential, functionally conserved, positive regulator of myogenic differentiation. Hypothesis: Ppp1r1b-lncRNA promotes myogenic differentiation program of cardiac and skeletal myocytes via epigenic mechanisms. Methods: Antisense oligonucleotides (GapmeR) were used to suppress Ppp1r1b-lncRNA . Chromatin immunoprecipitation (CHIP) and chromatin isolation by RNA purification (CHIRP) were used to analyze gene specific histone modification level and lncRNA: chromatin interaction, respectively. RNA pull-down and RNA immunoprecipitation (RIP) were used to identify Ppp1r1b-lncRNA : protein interactome. Results: By silencing Ppp1r1b-lncRNA, skeletal myoblasts and human-induced pluripotent stem cell derived cardiomyocytes (hiPSCs-CMs) failed to develop fully differentiated myotubes. Analysis of GapmeR injected neonatal mouse heart further confirmed the role of Ppp1r1b-lncRNA in normal myogenesis. Members of muscle specific transcription factors, including MyoD1 and TBX5, were not induced during myogenic differentiation in the Ppp1r1b-lncRNA depleted cardiac and skeletal muscle precursor cells. Histone modification analysis revealed enrichment of H3K27 tri-methylation at MyoD1 and TBX5 promoters in GapmeR treated skeletal myoblasts. Furthermore, Ppp1r1b-lncRNA was found to bind to Ezh2, and ChIRP analysis revealed enriched interaction of Ppp1r1b-lncRNA with Myod1 and Tbx5 promoters, suggesting that Ppp1r1b-lncRNA induces transcription of myogenic regulatory factors by interacting with the polycomb repressive complex 2 (PRC2) at the chromatin interface. Correspondingly, the silencing of Ppp1r1b-lncRNA increased EZH2 binding at promoter regions of myogenic transcription factors. Conclusions: Ppp1r1b-lncRNA promotes myogenic differentiation by inhibiting histone methylation on the promotors of myogenic master regulators via competing for PRC2 binding with chromatin during heart and skeletal muscle development.

Polycomb repressive complex 2 controls cardiac cell fate decision via interacting with RNA: Promiscuously or well-ordered.

The epigenetic landscape determines cell fate during heart development. Polycomb repressive complex 2 (PRC2) mediates histone methyltransferase activity during cardiac cell differentiation. The PRC2 complex contains the proteins embryonic ectoderm development (EED), suppressor of zeste (SUZ12), the chromatin assembly factor 1 (CAF1) histone-binding proteins RBBP4 and RBBP7, and the histone methyltransferase called enhancer of zeste (EZH2 or EZH1), which incorporates the Su(var)3-9, Enhancer-of-zeste, Trithorax (SET) domain. Cardiac PRC2-deficient mice display lethal congenital heart malformations. The dynamic process of cardiac cell fate decisions is controlled by PRC2 and the PRC2-mediated epigenetic landscape. Although specific individual long noncoding RNAs (lncRNAs) including Braveheart were widely reported to regulate the recruitments of PRC2 to their specific targets, a promiscuous RNA binding profile by PRC2 was also identified to play an essential role in cardiac cell fate decision. In this review, we focus on RNA-mediated PRC2 recruitment machinery in the process of cardiac cell fate decisions. The roles of individual lncRNAs which recruit PRC2, as well as promiscuous RNA binding by PRC2 in heart development are summarized. Since the binding priority of RNAs with different primary and secondary structures differs in its affinity to PRC2, the competitive relationship between individual lncRNAs binding and promiscuous RNA binding by PRC2 may be important for understanding the machinery by which biding of individual lncRNA and promiscuous RNA by PRC2 coordinately control the well-ordered dynamic cardiac cell lineage differentiation process.

Pioglitazone Mediates Cardiac Progenitor Formation through Increasing ROS Levels.

In order to achieve a sufficient population of cardiac-committed progenitor cells, it is crucial to know the mechanisms of cardiac progenitor formation. Previous studies suggested ROS effect on cardiac commitment events to play a key role in the cell signaling and activate cardiac differentiation of pluripotent stem cells. We previously reported that PPARγ activity is essential for cardiac progenitor cell commitment. Although several studies have conducted the involvement of PPARγ-related signaling pathways in cardiac differentiation, so far, the regulatory mechanisms of these signaling pathways have not been discussed and cleared. In this study, we focus on the role of PPARγ agonist in ROS generation and its further effects on the differentiation of cardiac cells from mESCs. The results of this study show that the presence of ROS is necessary for heart differentiation in the precursor stage of cardiac cells, and the coenzyme Q10 antioxidant precludes proper cardiac differentiation. In addition, this antioxidant prevents the action of pioglitazone in increasing oxygen radicals as well as beating cardiomyocyte differentiation properties. In this case, it can be concluded that PPARγ is required to modulate ROS levels during cardiac differentiation.

Abstract P1070: Role Of Vezf1 In Cardiomyocyte Development

Cardiovascular diseases (CVDs) are the leading cause of high morbidity rates and death worldwide. The heart is the first organ that is formed during mammalian development. Progenitor cells that form the cardiovascular system (heart and blood vessels) originate from the mesoderm post gastrulation. To date, there remain considerable gaps in the molecular mechanisms that govern the differentiation of cardiac progenitor cells into a functional heart, knowledge of which will provide advancements in cardiac regenerative medicine. Vascular endothelial zinc finger 1 (Vezf1) is a nuclear protein expressed predominantly in the mesoderm of a developing mouse embryo. A recent study showed that diseased human myocardia had reduced expression of Vezf1 when compared to healthy hearts. However, the function of Vezf1 in heart development is largely uncharacterized and understudied. Using murine embryonic stem cells (mESCs), we sought to investigate the early role(s) of Vezf1 in cardiovascular development. We show that genetic ablation or conditional knockdown of Vezf1 in mESCs impairs cardiomyocyte differentiation, as determined by the absence of contractile activity in these mutants. The absence of cardiomyocytes from Vezf1 mutant cell lines was confirmed using gene expression analysis and immunofluorescence. We observed impaired mesoderm induction in Vezf1 mutants despite external stimulation of Wnt signaling using small molecule inhibitors, suggesting a potential role of Vezf1 in the modulation of genes in the signaling pathway. Indeed, our gene expression analysis and ChIP-qPCR indicate that Vezf1 potentially associates with the promoters of Wnt signaling genes to activate their expression. These studies point towards a model where Vezf1 regulates the formation of cardiac progenitor cells by modulating the Wnt signaling pathway. Our observations suggest that Vezf1 may sit at the center of determining the fate of mesodermal cells into cardiovascular progenitors, the absence of which impairs cardiomyocyte differentiation. This study is the first to delineate the functional role of Vezf1 in the early development of cardiomyocytes and may form the foundation for future cardiac regenerative medicine.

Reduction of Cardiac Fibrosis by Interference With YAP-Dependent Transactivation.

Conversion of cardiac stromal cells into myofibroblasts is typically associated with hypoxia conditions, metabolic insults, and/or inflammation, all of which are predisposing factors to cardiac fibrosis and heart failure. We hypothesized that this conversion could be also mediated by response of these cells to mechanical cues through activation of the Hippo transcriptional pathway. The objective of the present study was to assess the role of cellular/nuclear straining forces acting in myofibroblast differentiation of cardiac stromal cells under the control of YAP (yes-associated protein) transcription factor and to validate this finding using a pharmacological agent that interferes with the interactions of the YAP/TAZ (transcriptional coactivator with PDZ-binding motif) complex with their cognate transcription factors TEADs (TEA domain transcription factors), under high-strain and profibrotic stimulation. We employed high content imaging, 2-dimensional/3-dimensional culture, atomic force microscopy mapping, and molecular methods to prove the role of cell/nuclear straining in YAP-dependent fibrotic programming in a mouse model of ischemia-dependent cardiac fibrosis and in human-derived primitive cardiac stromal cells. We also tested treatment of cells with Verteporfin, a drug known to prevent the association of the YAP/TAZ complex with their cognate transcription factors TEADs. Our experiments suggested that pharmacologically targeting the YAP-dependent pathway overrides the profibrotic activation of cardiac stromal cells by mechanical cues in vitro, and that this occurs even in the presence of profibrotic signaling mediated by TGF-β1 (transforming growth factor beta-1). In vivo administration of Verteporfin in mice with permanent cardiac ischemia reduced significantly fibrosis and morphometric remodeling but did not improve cardiac performance. Our study indicates that preventing molecular translation of mechanical cues in cardiac stromal cells reduces the impact of cardiac maladaptive remodeling with a positive effect on fibrosis.

Receptor tyrosine kinase inhibitors negatively impact on pro-reparative characteristics of human cardiac progenitor cells

Receptor tyrosine kinase inhibitors improve cancer survival but their cardiotoxicity requires investigation. We investigated these inhibitors’ effects on human cardiac progenitor cells in vitro and rat heart in vivo. We applied imatinib, sunitinib or sorafenib to human cardiac progenitor cells, assessing cell viability, proliferation, stemness, differentiation, growth factor production and second messengers. Alongside, sunitinib effects were assessed in vivo. Inhibitors decreased (p < 0.05) cell viability, at levels equivalent to ‘peak’ (24 h; imatinib: 91.5 ± 0.9%; sunitinib: 83.9 ± 1.8%; sorafenib: 75.0 ± 1.6%) and ‘trough’ (7 days; imatinib: 62.3 ± 6.2%; sunitinib: 86.2 ± 3.5%) clinical plasma levels, compared to control (100% viability). Reduced (p < 0.05) cell cycle activity was seen with imatinib (29.3 ± 4.3% cells in S/G2/M-phases; 50.3 ± 5.1% in control). Expression of PECAM-1, Nkx2.5, Wnt2, linked with cell differentiation, were decreased (p < 0.05) 2, 2 and 6-fold, respectively. Expression of HGF, p38 and Akt1 in cells was reduced (p < 0.05) by sunitinib. Second messenger (p38 and Akt1) blockade affected progenitor cell phenotype, reducing c-kit and growth factor (HGF, EGF) expression. Sunitinib for 9 days (40 mg/kg, i.p.) in adult rats reduced (p < 0.05) cardiac ejection fraction (68 ± 2% vs. baseline (83 ± 1%) and control (84 ± 4%)) and reduced progenitor cell numbers. Receptor tyrosine kinase inhibitors reduce cardiac progenitor cell survival, proliferation, differentiation and reparative growth factor expression.