scholarly journals Hippo Pathway Effector Tead1 Induces Cardiac Fibroblast to Cardiomyocyte Reprogramming

2021 ◽  
Author(s):  
Vivek P. Singh ◽  
Jaya P. Pinnamaneni ◽  
Aarthi Pugazenthi ◽  
Deepthi Sanagasetti ◽  
Megumi Mathison ◽  
...  
Keyword(s):  
Troponin T ◽  
Cardiac Fibroblasts ◽  
Hippo Pathway ◽  
Promoter Regions ◽  
Cell Contractility ◽  
Adult Rat ◽  
H3k4me3 Mark ◽  
Rat Fibroblasts ◽  
Regulator Genes ◽  
Human Cardiac Fibroblasts

Background The conversion of fibroblasts into induced cardiomyocytes may regenerate myocardial tissue from cardiac scar through in situ cell transdifferentiation. The efficiency transdifferentiation is low, especially for human cells. We explored the leveraging of Hippo pathway intermediates to enhance induced cardiomyocyte generation. Methods and Results We screened Hippo effectors Yap (yes‐associated protein), Taz (transcriptional activator binding domain), and Tead1 (TEA domain transcription factor 1; Td) for their reprogramming efficacy with cardio‐differentiating factors Gata4, Mef2C, and Tbx5 (GMT). Td induced nearly 3‐fold increased expression of cardiomyocyte marker cTnT (cardiac troponin T) by mouse embryonic and adult rat fibroblasts versus GMT administration alone ( P <0.0001), while Yap and Taz failed to enhance cTnT expression. Serial substitution demonstrated that Td replacement of TBX5 induced the greatest cTnT expression enhancement and sarcomere organization in rat fibroblasts treated with all GMT substitutions (GMTd versus GMT: 17±1.2% versus 5.4±0.3%, P <0.0001). Cell contractility (beating) was seen in 6% of GMTd‐treated cells by 4 weeks after treatment, whereas no beating GMT‐treated cells were observed. Human cardiac fibroblasts likewise demonstrated increased cTnT expression with GMTd versus GMT treatment (7.5±0.3% versus 3.0±0.3%, P <0.01). Mechanistically, GMTd administration increased expression of the trimethylated lysine 4 of histone 3 (H3K4me3) mark at the promoter regions of cardio‐differentiation genes and mitochondrial biogenesis regulator genes in rat and human fibroblast, compared with GMT. Conclusions These data suggest that the Hippo pathway intermediate Tead1 is an important regulator of cardiac reprogramming that increases the efficiency of maturate induced cardiomyocytes generation and may be a vital component of human cardiodifferentiation strategies.

Abstract 37: Mir-590 Promotes Transdifferentiation of Porcine and Human Fibroblasts Towards a Cardiomyocyte-like Fate by Directly Repressing Specificity Protein 1 (Sp1)

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Vivek P Singh ◽  
Megumi Mathison ◽  
Vivekkumar Patel ◽  
Deepthi Sanagasetti ◽  
Lina Yang ◽  
...  
Keyword(s):  
Porcine Model ◽  
Troponin T ◽  
Cardiac Fibroblasts ◽  
Human Fibroblasts ◽  
Human Cells ◽  
Cellular Reprogramming ◽  
Collagen Iii ◽  
Human Cardiac Fibroblasts ◽  
Spontaneous Contractions ◽  
Specificity Protein

Objective: Transdifferentiation of cardiac fibroblasts into cardiomyocyte-like cells (iCMs) represents a promising strategy in treating human heart disease. However, despite encouraging reprogramming data obtained from rodent models, the transgenes used in those models are not sufficient to reprogram human cells. We therefore sought to optimize a cocktail of factors which can efficiently transdifferentiate human cells. A porcine model was included to test its potential as a surrogate for human reprogramming studies. Methods: Lentivirus expressing Gata4 (G), Mef2c (M), Tbx5 (T), Hand2 (H), Myocardin (My), or two microRNAs miR-590, miR-199, were transduced into cultured porcine cardiac fibroblasts (PCFs) and human cardiac fibroblasts (HCFs) in different combinations. Two weeks after transduction, qRT-PCR, immunofluorescence (IF), and FACS assays were performed, and the efficiency of iCM production was evaluated by the expression of cardiomyocyte markers cardiac troponin T (cTnT) and α-sarcomeric actinin. Results: Combined G, M, T treatment alone was insufficient to transdifferentiate PCFs or HCFs into iCMs, despite the capability of GMT transduction to transdifferentiate up to 7% of treated rat cardiac fibroblasts, as assessed by FACS analysis for cTNT. However, the addition of H, My and miR-590 to GMT resulted in the transdifferentiation of approximately 5% of HCFs and PCFs, as measured by cTnT expression. IF analysis likewise demonstrated high expression of cTNT and α-actinin in these cells. Importantly, the transdifferentiated PCFs exhibited spontaneous contractions when co-cultured with murine cardiomyocytes. qPCR showed that administration of GMT plus either miR-590 or HMy upregulated cardiac genes MYH6 and TNNT2, and downregulated the fibroblast genes Collagen I and Collagen III. Mir-590 also directly suppressed Sp1, a fibrosis inducer and putative inhibitor of cellular reprogramming in the heart. Conclusions: Our data suggest that the porcine model can serve as an appropriate surrogate for human fibroblasts reprogramming studies. Enhanced transdifferentiation associated with miR-590-mediated repression of Sp1 suggests a novel pathway that may be targetable to enhance cardiac cellular reprogramming clinically.

Abstract 16488: Exploring the Functional Role of Exosomes in Stem Cell-mediated Cardiac Repair

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Jennifer K Lang ◽  
Rebecca F Young ◽  
Hashmat Ashraf ◽  
John M Canty
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Information Transfer ◽  
Troponin T ◽  
Cardiac Fibroblasts ◽  
Cardiac Repair ◽  
In Vitro Assays ◽  
Collagen Iii ◽  
Human Cardiac Fibroblasts

Numerous experimental and clinical studies have shown a beneficial effect of cardiosphere-derived cell (CDC) therapy on regeneration of injured myocardium. Paracrine signaling by CDC secreted exosomes is hypothesized to be the principal mediator of improved function, however the mechanism of exosome uptake, cell specificity, and information transfer has yet to be elucidated. Our aim was to define the role of physiologically secreted human CDC-derived exosomes on cardiac fibroblasts, endothelial cells and cardiomyocytes by employing a knockdown (KD) strategy against nSMase2 and Rab27a, two genes crucial in exosome secretion. Methods: Human CDCs (hCDCs) were grown from atrial tissue biopsy explant culture and characterized by flow cytometry and immunocytochemistry. CDC-derived exosomes were isolated and characterized by particle size, immunoblot analysis and electron microscopy. Protein concentration of exosome preparations was quantified by Bradford microassay. nSMase2 and Rab27a shRNAi lentivirus was generated and used to create stable CDC knockdown lines. Co-culture transwell in vitro assays were designed to assess target cell proliferation, migration, angiogenesis, and fibrotic gene expression. Results: hCDCs expressed CD105, CD90, GATA4 and Nkx2.5, and were CD45 and cardiac troponin T negative. hCDC-derived exosomes were immunoreactive for CD63 and HSP90 and negative for Cyctochrome c and EEA1. shRNAi KD of nSMase2 and Rab27a resulted in successful blockade of exosome release from hCDCs. HUVECs co-cultured with scrambled shRNAi CDCs versus shRNAi nSMase2 KD CDCs showed increased angiogenesis and migration without affecting proliferation. While TGF-β stimulation of human cardiac fibroblasts significantly increased collagen I (COLI) and collagen III (COLIII) gene expression, there was no significant decrease in COLI or COLIII after treatment with exosomes. Conclusions: Secretion of hCDC-derived exosomes was effectively inhibited by nSMase2 and Rab27a lentiviral KD. Exosome release contributed to the angiogenic and pro-migratory effects of hCDCs but did not play a role in the fibrotic gene expression of human cardiac fibroblasts, suggesting a role for physiological exosome secretion in stem cell-mediated cardiac repair.

Decreased Smad 7 expression contributes to cardiac fibrosis in the infarcted rat heart

2002 ◽  
Vol 282 (5) ◽  
pp. H1685-H1696 ◽  
Author(s):  
Baiqiu Wang ◽  
Jianming Hao ◽  
Stephen C. Jones ◽  
May-Sann Yee ◽  
Julie C. Roth ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Cardiac Fibrosis ◽  
Transforming Growth Factor ◽  
Cardiac Fibroblasts ◽  
Scar Tissue ◽  
Collagen Type I ◽  
Type I ◽  
Adult Rat ◽  
Rat Hearts ◽  
Rat Fibroblasts

We examined the role of the transforming growth factor (TGF)-β1 signaling inhibitor Smad 7 in cardiac fibrosis. TGF-β1 (10 ng/ml) was found to increase cytosolic Smad 7 expression in primary adult rat fibroblasts and induce rapid nuclear export of exogenous Smad 7 in COS-7 cells. Furthermore, overexpression of Smad 7 in primary adult fibroblasts was associated with suppressed collagen type I and III expression. We detected Smad 7, phosphorylated Smad 2, TGF-β type I receptor (TβRI), and TGF-β1 proteins in postmyocardial infarct (MI) rat hearts. In 2 and 4 wk post-MI hearts, Smad 7 and TβRI expression were decreased in scar tissue, whereas TGF-β1 expression was increased in scar and viable tissue. In the 8 wk post-MI heart, Smad 7 expression was decreased in both scar tissue and myocardium remote to the infarct scar. Finally, we confirmed that these changes are paralleled by decreased expression of cytosolic phosphorylated receptor-regulated Smad 2 in 4-wk viable myocardium and in 2- and 4-wk infarct scar tissues. Taken together, our data imply that decreased inhibitory Smad 7 signal in cardiac fibroblasts may play a role in the pathogenesis of cardiac fibrosis in the post-MI heart.

Abstract 428: Cortical Bone Stem Cell-derived Exosomes Reduce Myofibroblast Activation in Rat and Human Cardiac Fibroblasts

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Giana Schena ◽  
Hajime KUBO ◽  
Eric Feldsott ◽  
Alaina Headrick ◽  
Keith Koch ◽  
...  
Keyword(s):  
Heart Failure ◽  
Wound Healing ◽  
Stem Cell ◽  
Cortical Bone ◽  
Cardiac Fibroblasts ◽  
The United States ◽  
Adult Rat ◽  
Dose Dependent Decrease ◽  
Human Cardiac Fibroblasts

Rationale: Heart failure is the one of the leading causes of death in the United States. Post-myocardial infarction is followed by cardiac remodeling that involves extensive fibrosis and ultimately progresses into heart failure. We have seen improvements in scar size and cardiac function as a result of administration of cortical bone stem cell-derived (CBSC) exosomes. Objectives: We investigated the mechanism through which CBSC-derived exosomes altered wound healing and reduced scar formation through in vitro experimentation. We continue to broaden our understanding of how CBSCs exert their anti-fibrotic effects. Methods and Results: We cultured adult rat ventricular fibroblasts and adult human cardiac fibroblasts and treated them +/- TGF β with mouse and human CBSC-derived exosomes, respectively. We saw a dose-dependent decrease in myofibroblast activation with increasing concentrations of mCBSC and hCBSC exosomes, with 100 fold decrease compared to baseline fibroblast activation in hCBSC treated cardiac fibroblasts (9.5 x 10 5 of 1.6 x 10 7 intensity), and by 40% in mCBSC treated cardiac fibroblasts (2.4 x 10 5 of 1.0 x 10 6 intensity). In the presence of TGFβ and 2.0 x 10 6 exosome particles/cell treated, the αSMA levels were still reduced by nearly 50% vs TGFβ alone (1.2 x 10 6 of 2.2 x 10 7 intensity). We performed RNA sequencing on both the rat and human cardiac fibroblasts in order to discover which fibrosis-related genes were being altered by CBSC exosomes treatment. Conclusions: Our findings show that the wound healing induced by CBSC exosome treatment post-MI involves the reduction of myofibroblast activation and decreasing the production of pro-fibrotic mRNA in cardiac fibroblasts and cardiac endothelial cells.

scholarly journals A protocol for transdifferentiation of human cardiac fibroblasts into endothelial cells via activation of innate immunity

2021 ◽  
Vol 2 (2) ◽  
pp. 100556
Author(s):  
Chun Liu ◽  
Pedro Medina ◽  
Dilip Thomas ◽  
Ian Y. Chen ◽  
Karim Sallam ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Endothelial Cells ◽  
Cardiac Fibroblasts ◽  
Human Cardiac Fibroblasts

scholarly journals HIRA stabilizes skeletal muscle lineage identity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Joana Esteves de Lima ◽  
Reem Bou Akar ◽  
Léo Machado ◽  
Yuefeng Li ◽  
Bernadette Drayton-Libotte ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cell Lineage ◽  
Promoter Regions ◽  
Cell Fates ◽  
Regulatory Regions ◽  
Muscle Stem Cells ◽  
Adult Mice ◽  
H3k4me3 Mark ◽  
Muscle Genes ◽  
Muscle Gene

AbstractThe epigenetic mechanisms coordinating the maintenance of adult cellular lineages and the inhibition of alternative cell fates remain poorly understood. Here we show that targeted ablation of the histone chaperone HIRA in myogenic cells leads to extensive transcriptional modifications, consistent with a role in maintaining skeletal muscle cellular identity. We demonstrate that conditional ablation of HIRA in muscle stem cells of adult mice compromises their capacity to regenerate and self-renew, leading to tissue repair failure. Chromatin analysis of Hira-deficient cells show a significant reduction of histone variant H3.3 deposition and H3K27ac modification at regulatory regions of muscle genes. Additionally, we find that genes from alternative lineages are ectopically expressed in Hira-mutant cells via MLL1/MLL2-mediated increase of H3K4me3 mark at silent promoter regions. Therefore, we conclude that HIRA sustains the chromatin landscape governing muscle cell lineage identity via incorporation of H3.3 at muscle gene regulatory regions, while preventing the expression of alternative lineage genes.

Effects of methylglyoxal on human cardiac fibroblast: roles of transient receptor potential ankyrin 1 (TRPA1) channels

2014 ◽  
Vol 307 (9) ◽  
pp. H1339-H1352 ◽  
Author(s):  
Gaku Oguri ◽  
Toshiaki Nakajima ◽  
Yumiko Yamamoto ◽  
Nami Takano ◽  
Tomofumi Tanaka ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Diabetic Cardiomyopathy ◽  
Cell Cycle Progression ◽  
Transient Receptor Potential ◽  
Cardiac Fibroblasts ◽  
Receptor Potential ◽  
Rt Pcr ◽  
Cycle Progression ◽  
Transient Receptor ◽  
Human Cardiac Fibroblasts

Cardiac fibroblasts contribute to the pathogenesis of cardiac remodeling. Methylglyoxal (MG) is an endogenous carbonyl compound produced under hyperglycemic conditions, which may play a role in the development of pathophysiological conditions including diabetic cardiomyopathy. However, the mechanism by which this occurs and the molecular targets of MG are unclear. We investigated the effects of MG on Ca2+ signals, its underlying mechanism, and cell cycle progression/cell differentiation in human cardiac fibroblasts. The conventional and quantitative real-time RT-PCR, Western blot, immunocytochemical analysis, and intracellular Ca2+ concentration [Ca2+]i measurement were applied. Cell cycle progression was assessed using the fluorescence activated cell sorting. MG induced Ca2+ entry concentration dependently. Ruthenium red (RR), a general cation channel blocker, and HC030031 , a selective transient receptor potential ankyrin 1 (TRPA1) antagonist, inhibited MG-induced Ca2+ entry. Treatment with aminoguanidine, a MG scavenger, also inhibited it. Allyl isothiocyanate, a selective TRPA1 agonist, increased Ca2+ entry. The use of small interfering RNA to knock down TRPA1 reduced the MG-induced Ca2+ entry as well as TRPA1 mRNA expression. The quantitative real-time RT-PCR analysis showed the prominent existence of TRPA1 mRNA. Expression of TRPA1 protein was confirmed by Western blotting and immunocytochemical analyses. MG promoted cell cycle progression from G0/G1 to S/G2/M, which was suppressed by HC030031 or RR. MG also enhanced α-smooth muscle actin expression. The present results suggest that methylglyoxal activates TRPA1 and promotes cell cycle progression and differentiation in human cardiac fibroblasts. MG might participate the development of pathophysiological conditions including diabetic cardiomyopathy via activation of TRPA1.

scholarly journals AAV-mediated YAP expression in cardiac fibroblasts promotes inflammation and increases fibrosis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jamie Francisco ◽  
Yu Zhang ◽  
Yasuki Nakada ◽  
Jae Im Jeong ◽  
Chun-Yang Huang ◽  
...  
Keyword(s):  
Cardiac Dysfunction ◽  
Inflammatory Responses ◽  
Cardiac Fibroblasts ◽  
Hippo Pathway ◽  
Additional Stress ◽  
Myocardial Inflammation ◽  
Adeno Associated Virus ◽  
Ccl2 Expression ◽  
Underlying Mechanisms ◽  
The Hippo Pathway

AbstractFibrosis is a hallmark of heart disease independent of etiology and is thought to contribute to impaired cardiac dysfunction and development of heart failure. However, the underlying mechanisms that regulate the differentiation of fibroblasts to myofibroblasts and fibrotic responses remain incompletely defined. As a result, effective treatments to mitigate excessive fibrosis are lacking. We recently demonstrated that the Hippo pathway effector Yes-associated protein (YAP) is an important mediator of myofibroblast differentiation and fibrosis in the infarcted heart. Yet, whether YAP activation in cardiac fibroblasts is sufficient to drive fibrosis, and how fibroblast YAP affects myocardial inflammation, a significant component of adverse cardiac remodeling, are largely unknown. In this study, we leveraged adeno-associated virus (AAV) to target cardiac fibroblasts and demonstrate that chronic YAP expression upregulated indices of fibrosis and inflammation in the absence of additional stress. YAP occupied the Ccl2 gene and promoted Ccl2 expression, which was associated with increased macrophage infiltration, pro-inflammatory cytokine expression, collagen deposition, and cardiac dysfunction in mice with cardiac fibroblast-targeted YAP overexpression. These results are consistent with other recent reports and extend our understanding of YAP function in modulating fibrotic and inflammatory responses in the heart.

scholarly journals An analysis of the effects of stretch on IGF-I secretion from rat ventricular fibroblasts

2007 ◽  
Vol 293 (1) ◽  
pp. H677-H683 ◽  
Author(s):  
Betty S. Hu ◽  
Lee K. Landeen ◽  
Nakon Aroonsakool ◽  
Wayne R. Giles
Keyword(s):  
Cardiac Fibroblasts ◽  
Cyclic Stretch ◽  
Dependent Manner ◽  
Paracrine Factor ◽  
Static Stretch ◽  
Paracrine Factors ◽  
Adult Rat ◽  
Igf I ◽  
Intracellular Ca ◽  
Altered Gene

Mechanical force can induce a number of fundamental short- and long-term responses in myocardium. These include alterations in ECM, activation of cell-signaling pathways, altered gene regulation, changes in cell proliferation and growth, and secretion of a number of peptides and growth factors. It is now known that a number of these autocrine/paracrine factors are secreted from both cardiomyocytes and ventricular cardiac fibroblasts (CFb) in response to stretch. One such substance is IGF-I. IGF-I is an important autocrine/paracrine factor that can regulate physiological or pathophysiological responses, such as hypertrophy. In this study, we addressed the possible effects of mechanical perturbation, biaxial strain, on IGF-I secretion from adult rat CFb. CFb were subjected to either static stretch (3–10%) or cyclic stretch (10%; 0.1–1 Hz) over a 24-h period. IGF-1 secretion from CFb in response to selected stretch paradigms was examined using ELISA to measure IGF-I concentrations in conditioned media. Static stretch did not result in any measurable modulation of IGF-I secretion from CFb. However, cyclic stretch significantly increased IGF-I secretion from CFb in a frequency- and time-dependent manner compared with nonstretched controls. This stretch-induced increase in secretion was relatively insensitive to changes in extracellular [Ca2+] or to block of L-type Ca2+ channels. In contrast, thapsigargin, an inhibitor of sarco(endo)plasmic reticulum Ca2+ ATPase, remarkably decreased stretch-induced IGF-I secretion from CFb. We further show that IGF-I can upregulate mRNA expression of atrial natriuretic peptide in myocytes. In summary, cyclic stretch can significantly increase IGF-I secretion from CFb, and this effect is dependent on a thapsigargin-sensitive pool of intracellular [Ca2+].

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