Transdifferentiation Of Cardiac Fibroblasts Research Articles

JAK/STAT3 signaling in cardiac fibrosis: a promising therapeutic target.

Cardiac fibrosis is a serious health problem because it is a common pathological change in almost all forms of cardiovascular diseases. Cardiac fibrosis is characterized by the transdifferentiation of cardiac fibroblasts (CFs) into cardiac myofibroblasts and the excessive deposition of extracellular matrix (ECM) components produced by activated myofibroblasts, which leads to fibrotic scar formation and subsequent cardiac dysfunction. However, there are currently few effective therapeutic strategies protecting against fibrogenesis. This lack is largely because the molecular mechanisms of cardiac fibrosis remain unclear despite extensive research. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling cascade is an extensively present intracellular signal transduction pathway and can regulate a wide range of biological processes, including cell proliferation, migration, differentiation, apoptosis, and immune response. Various upstream mediators such as cytokines, growth factors and hormones can initiate signal transmission via this pathway and play corresponding regulatory roles. STAT3 is a crucial player of the JAK/STAT pathway and its activation is related to inflammation, malignant tumors and autoimmune illnesses. Recently, the JAK/STAT3 signaling has been in the spotlight for its role in the occurrence and development of cardiac fibrosis and its activation can promote the proliferation and activation of CFs and the production of ECM proteins, thus leading to cardiac fibrosis. In this manuscript, we discuss the structure, transactivation and regulation of the JAK/STAT3 signaling pathway and review recent progress on the role of this pathway in cardiac fibrosis. Moreover, we summarize the current challenges and opportunities of targeting the JAK/STAT3 signaling for the treatment of fibrosis. In summary, the information presented in this article is critical for comprehending the role of the JAK/STAT3 pathway in cardiac fibrosis, and will also contribute to future research aimed at the development of effective anti-fibrotic therapeutic strategies targeting the JAK/STAT3 signaling.

Biomimetic Cardiac Fibrotic Model for Antifibrotic Drug Screening.

Cardiac fibrosis is characterized by pathological proliferation and activation of cardiac fibroblasts to myofibroblasts. Inhibition and reverse of transdifferentiation of cardiac fibroblasts to myofibroblasts is a potential strategy for cardiac fibrosis. Despite substantial progress, more effort is needed to discover effective drugs to improve and reverse cardiac fibrosis. The main reason for the slow development of antifibrotic drugs is that the traditional polystyrene culture platform does not recapitulate the microenvironment where cells reside in tissues. In this study, we propose an in vitro cardiac fibrotic model by seeding electrospun yarn scaffolds with cardiac fibroblasts. Our results show that yarn scaffolds allow three-dimensional growth of cardiac fibroblasts, promote extracellular matrix (ECM) deposition, and induce the transdifferentiation of cardiac fibroblasts to myofibroblasts. Exogenous transforming growth factor-β1 further promotes cardiac fibroblast activation and ECM deposition, which makes it a suitable fibrotic model to predict the antifibrotic potential of drugs. By using this platform, we demonstrate that both Honokiol (HKL) and Pirfenidone (PFD) show potential in antifibrosis to some extent. HKL is more efficient in antifibrosis than PFD as revealed by biochemical composition, gene, and molecular analyses as well as histological and biomechanical analysis. The electrospun yarn scaffold provides a novel platform for constructing in vitro fibrotic models to study cardiac fibrosis and to predict the antifibrotic efficacy of novel drugs.

Src tyrosine kinase promotes cardiac remodeling induced bychronic sympathetic activation.

Cardiac remodeling serves as the underlying pathological basis for numerous cardiovascular diseases and represents a pivotal stage for intervention. The excessive activation of β-adrenergic receptors (β-ARs) assumes a crucial role in cardiac remodeling. Nonetheless, theunderlyingmolecular mechanisms governing β-AR-induced cardiac remodeling remain largely unresolved. In this study, we identified Src tyrosine kinase as a key player in the cardiac remodeling triggered by excessive β-AR activation. Our findings demonstratedthat Src mediates isoproterenol (ISO)-induced cardiac hypertrophy, fibrosis, and inflammation in vivo. Furthermore, Src facilitates β-AR-mediated proliferation and transdifferentiation of cardiac fibroblasts, and hypertrophy and cardiomyocytes in vitro. Subsequent investigations have substantiated that Src mediates β-AR induced the extracellular signal-regulated protein kinase (ERK1/2) signaling pathway activated by β-AR. Our research presents compelling evidence that Src promotes β-AR-induced cardiac remodeling in both in vivo and in vitro settings. It establishes the promoting effect of the β-AR/Src/ERK signaling pathway on overall cardiac remodeling in cardiac fibroblasts and underscores the potential of Src as a therapeutic target for cardiac remodeling.

P63 silencing induces epigenetic modulation to enhance human cardiac fibroblast to cardiomyocyte-like differentiation

Direct cell reprogramming represents a promising new myocardial regeneration strategy involving in situ transdifferentiation of cardiac fibroblasts into induced cardiomyocytes. Adult human cells are relatively resistant to reprogramming, however, likely because of epigenetic restraints on reprogramming gene activation. We hypothesized that modulation of the epigenetic regulator gene p63 could improve the efficiency of human cell cardio-differentiation. qRT-PCR analysis demonstrated significantly increased expression of a panel of cardiomyocyte marker genes in neonatal rat and adult rat and human cardiac fibroblasts treated with p63 shRNA (shp63) and the cardio-differentiation factors Hand2/Myocardin (H/M) versus treatment with Gata4, Mef2c and Tbx5 (GMT) with or without shp63 (p < 0.001). FACS analysis demonstrated that shp63+ H/M treatment of human cardiac fibroblasts significantly increased the percentage of cells expressing the cardiomyocyte marker cTnT compared to GMT treatment with or without shp63 (14.8% ± 1.4% versus 4.3% ± 1.1% and 3.1% ± 0.98%, respectively; p < 0.001). We further demonstrated that overexpression of the p63—transactivation inhibitory domain (TID) interferes with the physical interaction of p63 with the epigenetic regulator HDAC1 and that human cardiac fibroblasts treated with p63-TID+ H/M demonstrate increased cardiomyocyte marker gene expression compared to cells treated with shp63+ H/M (p < 0.05). Whereas human cardiac fibroblasts treated with GMT alone failed to contract in co-culture experiments, human cardiac fibroblasts treated with shp63+ HM or p63-TID+ H/M demonstrated calcium transients upon electrical stimulation and contractility synchronous with surrounding neonatal cardiomyocytes. These findings demonstrate that p63 silencing provides enhanced rat and human cardiac fibroblast transdifferentiation into induced cardiomyocytes compared to a standard reprogramming strategy. p63-TID overexpression may be a useful reprogramming strategy for overcoming epigenetic barriers to human fibroblast cardio-differentiation.

Huoxin Pill inhibits isoproterenol-induced transdifferentiation and collagen synthesis in cardiac fibroblasts through the TGF-β/Smads pathway

Ethnopharmacological relevanceThe abnormal proliferation and differentiation of cardiac fibroblasts (CFs) are universally regarded as the key process for the progressive development of cardiac fibrosis following various cardiovascular diseases. Huoxin Pill (Concentrated pill, HXP) is a Chinese herbal formula for treating coronary heart disease. However, the cellular and molecular mechanisms of HXP in the treatment of myocardial fibrosis are still unclear. Aim of the studyTo investigate the effects of HXP on CFs transdifferentiation and collagen synthesis under isoproterenol (ISO) conditions, as well as the potential mechanism of action. Materials and methodsIn vivo, we established a rat model of cardiac fibrosis induced by ISO, and administered with low or high dose of HXP (10 mg/kg/day or 30 mg/kg/day). The level of α-SMA was detected by immunohistochemistry examination, and combined with RNA-sequencing analysis to determine the protective effect of HXP on myocardial fibrosis rats. In vitro, by culturing primary rat CFs, we examined the effects of HXP on the proliferation and transdifferentiation of CFs using CCK8, scratch wound healing and immunofluorescence assays. Western blot was used to determine protein expression. ResultsThe findings revealed that HXP protects against ISO-induced cardiac fibrosis and CFs transdifferentiation in rats. RNA-sequencing and pathway analyses demonstrated 238 or 295 differentially expressed genes (DEGs) and multiple enriched signal pathways, including transforming growth factor-beta (TGF-β) receptor signaling activates Smads, downregulation of TGF-β receptor signaling, signaling by TGF-β receptor complex, and collagen formation under treatment with low or high-dose of HXP. Moreover, HXP also markedly inhibited ISO-induced primary rat CFs proliferation, transdifferentiation, collagen synthesis and the upregulation of TGF-β1 and phosphorylated Smad2/3 protein expression. ConclusionHXP suppresses ISO-induced CFs transdifferentiation and collagen synthesis, and it may exert these effects in part by inhibiting the activation of the TGF-β/Smads pathway. This may be a new therapeutic tool for cardiac fibrosis.

MicroRNA-21-Dependent Macrophage-to-Fibroblast Signaling Determines the Cardiac Response to Pressure Overload.

Cardiac macrophages (cMPs) are increasingly recognized as important regulators of myocardial homeostasis and disease, yet the role of noncoding RNA in these cells is largely unknown. Small RNA sequencing of the entire miRNomes of the major cardiac cell fractions revealed microRNA-21 (miR-21) as the single highest expressed microRNA in cMPs, both in health and disease (25% and 43% of all microRNA reads, respectively). MiR-21 has been previously reported as a key microRNA driving tissue fibrosis. Here, we aimed to determine the function of macrophage miR-21 on myocardial homeostasis and disease-associated remodeling. Macrophage-specific ablation of miR-21 in mice driven by Cx3cr1-Cre was used to determine the function of miR-21 in this cell type. As a disease model, mice were subjected to pressure overload for 6 and 28 days. Cardiac function was assessed in vivo by echocardiography, followed by histological analyses and single-cell sequencing. Cocultures of macrophages and cardiac fibroblasts were used to study macrophage-to-fibroblast signaling. Mice with macrophage-specific genetic deletion of miR-21 were protected from interstitial fibrosis and cardiac dysfunction when subjected to pressure overload of the left ventricle. Single-cell sequencing of pressure-overloaded hearts from these mice revealed that miR-21 in macrophages is essential for their polarization toward a M1-like phenotype. Systematic quantification of intercellular communication mediated by ligand-receptor interactions across all cell types revealed that miR-21 primarily determined macrophage-fibroblast communication, promoting the transition from quiescent fibroblasts to myofibroblasts. Polarization of isolated macrophages in vitro toward a proinflammatory (M1-like) phenotype activated myofibroblast transdifferentiation of cardiac fibroblasts in a paracrine manner and was dependent on miR-21 in cMPs. Our data indicate a critical role of cMPs in pressure overload-induced cardiac fibrosis and dysfunction and reveal macrophage miR-21 as a key molecule for the profibrotic role of cMPs.

Matrine attenuates pathological cardiac fibrosis via RPS5/p38 in mice.

Pathological cardiac fibrosis is a common feature in multiple cardiovascular diseases that contributes to the occurrence of heart failure and life-threatening arrhythmias. Our previous study demonstrated that matrine could attenuate doxorubicin-induced oxidative stress and cardiomyocyte apoptosis. In this study, we investigated the effect of matrine on cardiac fibrosis. Mice received aortic banding (AB) operation or continuous injection of isoprenaline (ISO) to generate pathological cardiac fibrosis and then were exposed to matrine lavage (200 mg·kg-1·d-1) or an equal volume of vehicle as the control. We found that matrine lavage significantly attenuated AB or ISO-induced fibrotic remodeling and cardiac dysfunction. We also showed that matrine (200 μmol/L) significantly inhibited the proliferation, migration, collagen production, and phenotypic transdifferentiation of cardiac fibroblasts. Mechanistically, matrine suppressed p38 activation in vivo and in vitro, and overexpression of constitutively active p38 completely abolished the protective effects of matrine. We also demonstrated that ribosomal protein S5 (RPS5) upregulation was responsible for matrine-mediated inhibition on p38 and fibrogenesis. More importantly, matrine was capable of ameliorating preexisting cardiac fibrosis in mice. In conclusion, matrine treatment attenuates cardiac fibrosis by regulating RPS5/p38 signaling in mice, and it might be a promising therapeutic agent for treating pathological cardiac fibrosis.

Knock-out of MicroRNA 145 impairs cardiac fibroblast function and wound healing post-myocardial infarction.

Prevention of infarct scar thinning and dilatation and stimulation of scar contracture can prevent progressive heart failure. Since microRNA 145 (miR‐145) plays an important role in cardiac fibroblast response to wound healing and cardiac repair after an myocardial infarction (MI), using a miR‐145 knock‐out (KO) mouse model, we evaluated contribution of down‐regulation of miR‐145 to cardiac fibroblast and myofibroblast function during adverse cardiac remodelling. Cardiac function decreased more and the infarct size was larger in miR‐145 KO than that in WT mice after MI and this phenomenon was accompanied by a decrease in cardiac fibroblast‐to‐myofibroblast differentiation. Quantification of collagen I and α‐SMA protein levels as well as wound contraction revealed that transdifferentiation of cardiac fibroblasts into myofibroblasts was lower in KO than WT mice. In vitro restoration of miR‐145 induced more differentiation of fibroblasts to myofibroblasts and this effect involved the target genes Klf4 and myocardin. MiR‐145 contributes to infarct scar contraction in the heart and the absence of miR‐145 contributes to dysfunction of cardiac fibroblast, resulting in greater infarct thinning and dilatation. Augmentation of miR‐145 could be an attractive target to prevent adverse cardiac remodelling after MI by enhancing the phenotypic switch of cardiac fibroblasts to myofibroblasts.

Fibroblast transdifferentiation promotes conversion of M1 macrophages and replenishment of cardiac resident macrophages following cardiac injury in mice.

Resident cardiac macrophages play important roles in homeostasis, maintenance of cardiac function, and tissue repair. After cardiac injury, monocytes infiltrate the tissue, undergo phenotypic and functional changes, and are involved in inflammatory injury and functional remodelling. However, the fate of cardiac infiltrating/polarized macrophages and the relationship between these cells and resident cardiac macrophage replenishment following injury remain unclear. Our results showed that angiotensin II induces cardiac fibroblast transdifferentiation into cardiac myofibroblasts (MFBs). In cocultures with MFBs and murine macrophages, the MFBs promoted macrophage polarization to M1 phenotype, followed by selective apoptosis, which was associated with TNF/TNFR1 axis and independent of NO production. Surprisingly, after 36 h of coculture, the surviving macrophages were converted to M2 phenotype and settled in heart, which was dependent on leptin produced by MFBs or polarized macrophages via the PI3K or Akt pathway. CCR2+ CD45.2+ cells adoptively transferred into CD45.1+ mice with viral myocarditis, differentiated into CD45.2+ CCR2+ CX3CR1+ M2 cells during the resolution of inflammation and settled within the heart. Our data highlight a novel mechanism related to the renewal or replenishment of cardiac resident macrophages following cardiac injury; and suggest that transdifferentiation of cardiac fibroblasts may promote the resolution of inflammation.

Pro-fibrotic effect of oxidized LDL in cardiac myofibroblasts

Inflammatory signals associated with cardiac diseases trigger trans-differentiation of cardiac fibroblasts to cardiac myofibroblasts. Cardiac myofibroblasts are the main cell type involved in the development of cardiac fibrosis, a diffuse and disproportionate accumulation of collagen in the myocardium. Although the role of the scavenger like-lectin receptor LOX-1 was previously investigated in cardiac fibroblasts and fibrosis, the involvement of the LOX-1 ligand -oxidized low-density lipoprotein (oxLDL)- on cardiac myofibroblast function still remains unexplored. In the present work, we investigated the effect of oxLDL/LOX-1 on fibrotic markers and cardiac myofibroblast function. Our in vitro results showed that oxLDL increased cardiac myofibroblast proliferation, triggered an increase in the synthesis of collagen type I and fibronectin containing extra domain A, and stimulated collagen type I secretion. oxLDL also decreased cardiac myofibroblast migration, collagen gel contraction and cell area, without modifying α-smooth muscle actin protein levels. These effects were dependent on LOX-1, because LOX-1 knockdown abolished oxLDL effects. Collectively these data showed that oxLDL has important modulatory effects on cardiac myofibroblast function.

Inhibitory Effects of Oxymatrine on Transdifferentiation of Neonatal Rat Cardiac Fibroblasts to Myofibroblasts Induced by Aldosterone via Keap1/Nrf2 Signaling Pathways In Vitro.

BackgroundOxymatrine (OMT), a quinolizidine alkaloid derived from the traditional Chinese herb Radix Sophorae flavescentis, has widely reported pharmacological efficacy in treating cardiovascular dysfunction-related diseases. However, the underlying mechanism has been unclear. Here, we investigated the potential inhibitory effects and mechanism of OMT on transdifferentiation of cardiac fibroblast to myofibroblasts induced by aldosterone in vitro.Material/MethodsThe cardiac fibroblasts (CFBs) proliferation and migration capacity were evaluated by MTT assay, cell cycle assay, and scratch analysis, respectively. The protein expression of the Nrf2/Keap1 signal pathway, FN, Collagen I, Collagen III, α-SMA, CTGF, and mineralocorticoid receptor (MR) protein was detected by Western blot analysis. The mRNA expression of Nrf2 was detected by qRT-PCR. Immunofluorescence staining was used to observe the expression of α-SMA protein. Nrf2 siRNA was used to explore the role of Nrf2 in OMT-treated CFBs. GSH, SOD, and MDA levels and hydroxyproline content were measured by colorimetric assay with commercial kits. The DCFH-DA fluorescent probe was used to assess cellular ROS levels.ResultsOMT and Curcumin (an Nrf2 agonist) attenuated aldosterone (ALD)-induced proliferation and migration in CFBs, as well as the fibrosis-associated protein expression levels. Moreover, OMT activated Nrf2 and promoted the nucleus translocation of Nrf2. OMT alleviated the elevated levels of α-SMA, Collagen I, Collagen III, and CTGF, which were abrogated by the Nrf2 siRNA transfection. We also found that OMT decreased oxidative stress levels.ConclusionsOur results confirm that OMT alleviates transdifferentiation of cardiac fibroblasts to myofibroblasts induced by aldosterone via activating the Nrf2/Keap1 pathway in vitro.

YAP activation promotes the transdifferentiation of cardiac fibroblasts to myofibroblasts in matrix remodeling of dilated cardiomyopathy

Yes-associated protein (YAP) is an important regulator of cellular proliferation and transdifferentiation. However, little is known about the mechanisms underlying myofibroblast transdifferentiation in dilated cardiomyopathy (DCM). We investigated the role of YAP in the pathological process of cardiac matrix remodeling. A classic model of DCM was established in BALB/c mice by immunization with porcine cardiac myosin. Cardiac fibroblasts were isolated from neonatal Sprague-Dawley rats by density gradient centrifugation. The expression levels of α-smooth muscle actin (α-SMA) and collagen volume fraction (CVF) were significantly increased in DCM mice. Angiotensin II (Ang II)-mediated YAP activation promoted the proliferation and transdifferentiation of neonatal rat cardiac fibroblasts, and this effect was significantly suppressed in the shRNA YAP + Ang II group compared with the shRNA Control + Ang II group in vitro (2.98±0.34 ×105 vs 5.52±0.82 ×105, P<0.01). Inhibition of endogenous Ang II-stimulated YAP improved the cardiac function by targeting myofibroblast transdifferentiation to attenuate matrix remodeling in vivo. In the valsartan group, left ventricular ejection fraction and fractional shortening were significantly increased compared with the DCM group (52.72±5.51% vs 44.46±3.01%, P<0.05; 34.84±3.85% vs 26.65±3.12%, P<0.01). Our study demonstrated that YAP was a regulator of cardiac myofibroblast differentiation, and regulation of YAP signaling pathway contributed to improve cardiac function of DCM mice, possibly in part by decreasing myofibroblast transdifferentiation to inhibit matrix remodeling.

GW29-e0371 YAP activation promotes the transdifferentiation of cardiac fibroblasts to myofibroblasts in matrix remodeling of dilated cardiomyopathy

GW29-e0371 YAP activation promotes the transdifferentiation of cardiac fibroblasts to myofibroblasts in matrix remodeling of dilated cardiomyopathy

SIRT3 inhibits Ang II-induced transdifferentiation of cardiac fibroblasts through β-catenin/PPAR-γ signaling

AimsCardiac fibrosis is an inevitable process of numerous cardiovascular diseases in which the transdifferentiation of cardiac fibroblasts plays a pivotal role. Sirtuin3 (SIRT3) has been believed to protect against cardiac fibrosis. However, the mechanism underlying this beneficial effect has not yet been elucidated. In this study, we investigated the potential mechanism of SIRT3 on the inhibition of fibroblast-to-myoblast transdifferentiation. Main methodsCells were stimulated by angiotension II (Ang II) with SIRT3 overexpression or knockdown. Also, PPARγ agonist (Pioglitazone PIO) and inhibitor (GW9662) were used to confirm the antifibrotic effect of PPARγ. Western blot, qRT-PCR, CCK-8 and immunofluorescence staining analysis were used for investigation. Key findingsOur data demonstrated that overexpression of SIRT3 prevented the transdifferentiation of CFs while SIRT3 knockdown promoted the process. Simultaneously, SIRT3 overexpression increased total PPARγ expression and suppressed the acetylated PPARγ. Besides, pretreatment with PPARγ agonist, pioglitazone protected CFs from transdifferentiation while PPARγ inhibitor prevented the protective effect of SIRT3. In addition, we have found that SIRT3 upregulated the expression of PPARγ by degeration of β-catenin. SignificanceOur findings indicate that this newly identified SIRT3/β-catenin/PPAR-γ axis will provide novel insight into the understanding of the mechanism of transdifferentiation of CFs to myofibroblasts.

Abstract 410: Rhein Administration Prevents Cardiac Fibrosis by Interfering with the TGF-β Pathway

Fibrosis, which occurs in various heart diseases like acute myocardial ischemia and pressure overload, is triggered by the differentiation of fibroblasts into myofibroblasts. Dysregulation of this reparative mechanism results in excessive collagen accumulation leading to cardiac stiffness and impaired heart function. The aim of this study was to determine whether the rhubarb anthraquinone Rhein, a drug already used as treatment for chondroarthritis, prevents the transdifferentiation of cardiac fibroblasts. We observed that Rhein pre-treatment ameliorates the cardiac function and reduces adverse remodeling after acute myocardial infarction in mice, in vivo . In primary human cardiac fibroblasts, Rhein incubation dose-dependently inhibited the TGF-β-mediated upregulation of α-SMA, the master marker for myofibrolasts, and prevented the contraction of fibroblast-populated collagen gel lattices upon TGF-β stimulation. Further, Rhein reduced TGFβ-R1 expression in primary human cardiac fibroblast, resulting in decreased SMAD2 phosphorylation and blunting of the fibrogenic response. Furthermore, Rhein stabilized protein levels of SMAD7, a key inhibitor of TGF-β signaling. Collectively, these data show for the first time that Rhein administration prevents cardiac fibrosis in vivo and in vitro by blunting the TGF-β signaling pathway, and identify Rhein as potential therapeutic treatment to prevent excessive fibrosis and adverse remodeling in cardiac pathologies.

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

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 17995: Inhibition of Myofibroblast Differentiation by FOXO3a - Implications for Acute Myocardial Infarction and Cardiac Remodeling

Introduction: Transdifferentiation of cardiac fibroblasts into myofibroblasts regulated by TGFβ/SMAD3 signaling is a major mechanism of scar formation and adverse remodeling following myocardial infarction. Hypothesis: We hypothesized that the transcription factor FOXO3a, a key regulator of cell differentiation, cycle and size, might inhibit this process. Methods: Acute myocardial infarction was induced in FOXO3-/- and WT mice (FVB) by permanent LAD ligation and myofibroblast transdifferentiation markers were assessed. FOXO3a-/- and WT cardiac fibroblasts were investigated in transdifferentiation assays ex vivo. FOXO3a gene transfer was performed with gain of function adenoviral vectors. IP/IF and Western blotting were used to test for a direct interaction between FOXO3a and SMAD3. Results: FOXO3a-/- mice had significantly higher survival rates compared to WT mice due to reduced rates of ventricle perforation. Myocardial expression of alpha smooth muscle actin (ASMA) and Collagen1A1 (Col1A1) was significantly enhanced in FOXO3a-/- mice 3 and 14 days post infarction. Moreover, Foxo3a-/- mice showed larger fibrotic areas following MI. In line with these results, cardiac fibroblasts isolated from FOXO3a-/- mice showed significantly enhanced expression levels of Col1A1 and ASMA 24 hours following stretch or TGF-β stimulation when compared to WT cells in vitro. Furthermore supernatants of FOXO3a -/- fibroblasts showed significantly higher protein expression of Col1A1 in (p&lt;0.01) while FOXO3a gene-transfer dose-dependently downregulated Col1A1. Immunfluorescence staining for ASMA protein was significantly attenuated following FOXO3a gene transfer in cardiac fibroblasts. Mechanistically, immunoprecipitation showed direct interaction of FOXO3a with SMAD3a that was enhanced following activation of the transcription factor leading to diminished SMAD3 downstream gene expression. Conclusions: Our results identify FOXO3a as a direct inhibitor of TGF- β regulated matrix remodeling via FOXO3a-SMAD3 interaction. FOXO3a regulates fibrosis and scar formation following myocardial infarction and thus targeting the FOXO3a-SMAD3 axis might be of therapeutic interest.

Engineering cardiac muscle: new ways to refurbish old hearts?

Myocardial infarction leads to irreversible cell loss and scar formation, which result in impaired cardiac function, cardiac hypertrophy and, eventually, heart failure. As the intrinsic regenerative capacity of the human heart is very limited, organ transplantation or ventricular assist devices remain the only end-stage therapy options to date. For at least two decades, alternative therapeutic concepts of heart repair based either on direct cell injection or on the transplantation of in vitro engineered tissue constructs have been considered potential approaches to overcome the shortage of donor organs. However, the multitude of recent experimental and clinical studies applying various adult stem cells have thus far not been able to fulfil the initial high hopes vested in adult stem cell-based therapies. As summarized in a recent meta study, the vast majority of these studies resulted either in no or in only minor functional improvement [1], most likely due to paracrine effects, including accelerated revascularization, enhanced myocyte survival in the infarct border zone and the modulation of scar formation, resulting in improved mechanical properties [2]. Despite sporadic myocyte formation, a significant generation of de novo myocardium could not be demonstrated [3]. Interestingly, very recent studies have demonstrated the possibility of targeted trans-differentiation of cardiac fibroblasts into functional cardiomyocytes (CMs). Inspired by a recent approach utilizing the transcription factor MyoD to drive transdifferentiation of fibroblasts into skeletal muscle [4], a set of cardiac transcription factors was overexpressed for the targeted differentiation of mouse cardiac and tail-tip fibroblasts (Fbs) into cardiomyocytes in vitro [5]. Although trans-differentiation of cardiac Fbs in vivo could be demonstrated [6], the efficiency of such direct conversion experiments is still controversial [7, 8]. At this point, the usefulness of this kind of cell conversion remains uncertain and will critically depend not only on a much higher efficiency, but also on the functional similarity of these artificially converted cells into normal cardiomyocytes. For many years, embryonic stem cells (ESCs) were considered the only cell source suitable to supply the huge numbers of cardiomyocytes lost after myocardial infarction. However, a potential clinical use of ESCs is highly controversial, since their generation requires the destruction of human embryos and, if not produced through therapeutic cloning, no autologous ESCs are available for cellular therapies. In 2006, these limitations were overcome by the groundbreaking development of induced pluripotent stem cells (iPSCs) by S. Yamanaka [9], who was awarded the Nobel Prize in Medicine in 2012. In the meantime, the generation of human iPSCs has become a standard procedure in many laboratories and it is now clear that these cells are almost indistinguishable from ESCs with respect to their phenotype, culture characteristics and potential for proliferation and differentiation. Unfortunately, the availability of human iPSCs and CMs derived from these cells (Figure 1) has solved only one of the most severe limitations for current concepts for myocardial repair. Further critical hurdles are low cell survival and the lack of functional integration of the cellular transplants. In particular, the functional coupling and formation of well-organized myocardium appears to be difficult to achieve. The direct injection of cells to improve heart function was investigated in different animal models with a variety of mouse and human cell sources, including undifferentiated cells as well as cells that had been differentiated to cardiac progenitors or cardiomyocytes. Regardless of the cell type or application method (intramyocardial or intracoronary injection), only up to 5% of the cells remained in the heart, and neither significant integration nor long-term survival could be shown ([10], reviewed by ref. [11]). Although survival and cardiovascular in vitro differentiation was demonstrated, the injection of iPSC-derived cardiovascular progenitors did not result in the formation of structured myocardium [12]. In contrast, a very recent study was the first to report the formation of relatively large electrically coupled and well-structured muscle islands from injected human ESC-derived myocytes in a guinea pig model of myocardial infarction [13]. These data are considered to be very promising, although it was necessary to inject extremely high numbers of myocytes, (i.e. 10 cells, which correlates to 2 × 10 or approximately four times the total number of myocytes in the adult human left ventricle, when projected from the guinea pig to the human heart, which is ca. two hundred times larger). Even more remarkable is the fact that the formation of large contractile human ESC-derived muscle islands have evidently been confirmed by the same group in a

Role of miR-145 in cardiac myofibroblast differentiation

Following a myocardial infarction (MI), fibroblasts differentiate to myofibroblasts, which possess some of the characteristics of smooth muscle cells (SMCs) and contribute to wound healing. Previous studies suggested that the miR-143/-145 cluster plays a critical role in SMC differentiation. Therefore, we determined whether miR-145 promoted differentiation of cardiac fibroblasts to myofibroblasts. Following coronary occlusion in mice, myocardial miR-145 expression was downregulated at 3days but was restored at 7days. In vitro studies showed that hypoxia also downregulated miR-145 in cardiac fibroblasts. The number of α-smooth muscle actin (α-SMA) positive cells in fibroblast cultures was employed to determine their transdifferentiation to cardiac myofibroblasts and was increased by 73.5% after transient transfection with miR-145. Ultrastructural analysis of α-SMA stress fibers revealed that ~95% of the α-SMA+ cells treated with miR-145 organized their actin-filament bundles with a specific orientation compared to only 15% in the scrambled control group. This orientation of the SMA bundles and their integration with the filamentous actin fibers of the cytoskeleton permit infarct wound contraction. Structural and functional studies showed that miR-145 induced a myofibroblast phenotype, and miR-145 also potentiated the production of mature collagen by myofibroblasts. Repression of KLF5, a target of miR-145, was validated by a chimeric luciferase construct tagged with the full-length 3′-UTR of KLF5. A dramatic decrease in KLF5 and a corresponding increase in myocardin expression were observed after transfecting cultured fibroblasts with miR-145. Similar results were found in vivo: the transient decrease in miR-145 expression 3days post-MI was associated with an increase in KLF5 and a decrease in myocardin. In addition, in vivo delivery of a miR-145 antagomir 1day prior to and 2 and 6days after MI decreased myofibroblast formation and increased scar size. The antagomir also reversed the suppressed expression of KLF5 protein in the scar region at day 7 after MI. In summary, we describe a novel association between miR-145 and fibroblast differentiation toward myofibroblasts. These observations provide a new approach to promote endogenous scar healing and contracture by stimulating the transdifferentiation of cardiac fibroblasts to myofibroblasts.

Immunohistologic evidence supports apoptosis, IgG deposition, and novel macrophage/fibroblast crosstalk in the pathologic cascade leading to congenital heart block

To assess in vivo the pathologic cascade leading to fibrosis in congenital heart block (CHB). In vitro studies suggest that CHB is initiated via apoptosis, resulting in translocation of SSA/Ro and SSB/La antigens and surface binding by maternal autoantibodies. These opsonized cardiocytes are phagocytosed by macrophages, which secrete factors inducing fibrosis. Immunohistochemistry analysis was performed on formalin-fixed sections of 4 fetal hearts identified in utero as having CHB or isolated myocarditis; mothers had anti-SSA/Ro and anti-SSB/La antibodies. Apoptosis was most extensive in fetuses dying early and most pronounced in regions containing conduction tissue. Deposition of IgG was observed in hearts from fetuses with CHB/myocarditis, but not in 3 control hearts, and was colocalized with apoptotic cells. Giant cells and macrophages (frequently seen proximal to IgG and apoptotic cells) were present in septal and thickened fibrous subendocardial regions, most apparent in the youngest fetuses. Septal tissue also revealed extensive areas of fibrosis and microcalcification in which a predominant smooth muscle actin (SMA)-positive infiltrate (myofibroblast scarring phenotype) was observed. In contrast, there were no macrophages or SMA-positive cells (other than those lining blood vessels) in septal tissue from control hearts, although rare macrophages were seen in the working myocardium. In summary, findings in this unique autopsy material paralleled those in in vitro studies. These data support the notion of exaggerated apoptosis, probably due to ongoing inflammation caused by IgG binding and ingestion by macrophages. Transdifferentiation of cardiac fibroblasts to a scarring phenotype may be a pathologic process initiated by maternal antibodies, and persistence of this phenotype even after birth may relate to the progression of block seen in some infants postpartum.