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Fibroblasts Inform the Heart: Control of Cardiomyocyte Cycling and Size by Age-Dependent Paracrine Signals
Abstract
The heart is the earliest organ to form in the incipient embryo. Its complex three-dimensional patterning is essential to survival even in midgestation, and derangements of cardiac morphogenesis comprise the commonest birth defects of the newborn (Olson and Schneider, 2003). Highly ordered spatial programs of proliferative growth underlie the expansion of early cardiac progenitor cells, elongation of the linear heart tube, and thickening of the ventricular walls to ensure appropriate mechanical pump function. By contrast, most growth of the heart after birth occurs by cell enlargement. Whereas much is known about developmentally regulated transitions in cell cycle machinery that execute this shift from hyperplasia to hypertrophy as the predominant mode of organ-level growth, such changes cannot by themselves explain the intricate spatial patterning of growth that ultimately fashions the working heart.
... What is little appreciated is the fact that, even in this tight structural straightjacket, 50% of cardiomyocytes are renewed during the lifespan of individual humans, showing that the basic principles of regeneration per se persist in adult hearts 25, 26 . It is very likely that paracrine signals originating from the "non-cardiomyocytes" and the extracellular matrix might be the "controller" of coordinated repair [27][28][29] . Even arrested developmental pathways, such as the Hippo signalling controlling organ size in animals through the regulation of cell proliferation and apoptosis or the fact that cardiomyocyte proliferation is by and large aborted in newborn rats, illustrate that "myocardial growth signals" are present in mammals, though actively suppressed for the greater good of organ homeostasis 30 . ...
Clinical heart failure prevention and contemporary therapy often involve breaking the vicious cycle of global haemodynamic consequences of myocardial decay. The lack of effective regenerative therapies results in a primary focus on preventing further deterioration of cardiac performance. The cellular transplantation hypothesis has been evaluated in many different preclinical models and a handful of important clinical trials. The primary expectation that cellular transplants will be embedded into failing myocardium and fuse with existing functioning cells appears unlikely. A multitude of cellular formulas, access routes and clinical surrogate endpoints for evaluation add to the complexity of cellular therapies. Several recent large clinical trials have provided insights into both the regenerative potential and clinical improvement from non-regenerative mechanisms. Initiating endogenous repair seems to be another meaningful alternative to recover structural integrity in myocardial injury. This option may be achieved using a transcoronary sinus catheter intervention, implying the understanding of basic principles in biology. With intermittent reduction of outflow in cardiac veins (PICSO), vascular cells appear to be activated and restart a programme similar to pathways in the developing heart. Structural regeneration may be possible without requiring exogenous agents, or a combination of both approaches may become clinical reality in the next decade.
... Fibroblasts are the major cells involved in extracellular matrix remodeling and the repair processes following injury through cytokine-dependent transformation into myofibroblasts [1][2][3][4]. Differentiation of fibroblasts into myofibroblasts for active repair of damaged tissue is accompanied by major changes in cell phenotype with conversion of non-excitable precursors into excitable myofibroblasts, cells with increased contractility and higher synthetic and secretory capabilities [5][6][7][8][9], processes that increase cellular energy demands [10]. Although phenotypic changes with fibroblast differentiation are well characterized, little information is available about mitochondrial remodeling associated with fibroblast differentiation. ...
Objectivs:
Cytokine-dependent activation of fibroblasts to myofibroblasts, a key event in fibrosis, is accompanied by phenotypic changes with increased secretory and contractile properties dependent on increased energy utilization, yet changes in the energetic profile of these cells are not fully described. We hypothesize that the TGF-β1-mediated transformation of myofibroblasts is associated with an increase in mitochondrial content and function when compared to naive fibroblasts.
Methods:
Cultured NIH/3T3 mouse fibroblasts treated with TGF-β1, a profibrotic cytokine, or vehicle were assessed for transformation to myofibroblasts (appearance of α-smooth muscle actin [α-SMA] stress fibers) and associated changes in mitochondrial content and functions using laser confocal microscopy, Seahorse respirometry, multi-well plate reader and biochemical protocols. Expression of mitochondrial-specific proteins was determined using western blotting, and the mitochondrial DNA quantified using Mitochondrial DNA isolation kit.
Results:
Treatment with TGF-β1 (5 ng/mL) induced transformation of naive fibroblasts into myofibroblasts with a threefold increase in the expression of α-SMA (6.85 ± 0.27 RU) compared to cells not treated with TGF-β1 (2.52 ± 0.11 RU). TGF-β1 exposure increased the number of mitochondria in the cells, as monitored by membrane potential sensitive dye tetramethylrhodamine, and expression of mitochondria-specific proteins; voltage-dependent anion channels (0.54 ± 0.05 vs. 0.23 ± 0.05 RU) and adenine nucleotide transporter (0.61 ± 0.11 vs. 0.22 ± 0.05 RU), as well as mitochondrial DNA content (530 ± 12 μg DNA/106 cells vs. 307 ± 9 μg DNA/106 cells in control). TGF-β1 treatment was associated with an increase in mitochondrial function with a twofold increase in baseline oxygen consumption rate (2.25 ± 0.03 vs. 1.13 ± 0.1 nmol O2/min/106 cells) and FCCP-induced mitochondrial respiration (2.87 ± 0.03 vs. 1.46 ± 0.15 nmol O2/min/106 cells).
Conclusions:
TGF-β1 induced differentiation of fibroblasts is accompanied by energetic remodeling of myofibroblasts with an increase in mitochondrial respiration and mitochondrial content.
... What is little appreciated is the fact that even in this tight structural straightjacket, 50% of cardiomyocytes are renewed during a lifespan of individual humans showing that the basic principles of regeneration per se persist in adult hearts [2]. It may very likely be, that paracrine signals originating from the -non cardiomyocytes‖ and extracellular matrix might be the -controller‖ of coordinated repair [1][2][3]. Even arrested developmental pathways as -Hippo signaling‖ or the fact that cardiomyocyte proliferation is by and large aborted as shown in newborn rats, illustrates that -myocardial growth signals‖ are present in mammals however actively suppressed for a better good of organ homeostasis [4]. ...
Cardiac regeneration remains a clinical target regardless of numerous therapeutic concepts. We formulated a hypothesis claiming that periodic coronary venous pressure elevation (PICSO; Pressure controlled Intermittent Coronary Sinus Occlusion) initiates embedded, but dormant developmental processes in adult jeopardized hearts. Hemodynamics in the primitive beating heart tube is sensed transducing “mechanical” epigenetic information during normal cardiac development. In analogy mechanotransduction via shear stress and pulsatile stretch induced by periodic elevation of blood pressure in cardiac veins reconnects this dormant developmental signal, setting regenerative impulses in the adult heart. Significant increase of hemeoxygenase-1 gene expression (p < 0.001) and vascular endothelial growth factor (VEGF) (p < 0.002) as well as production of VEGRF2 in experimental infarction underscores the resurgence of developmental stimuli by PICSO. Molecular findings correspond with risk reduction (p < 0.0001) in patients with acute coronary syndromes as well as observations in heart failure patients showing substantial risk reduction up to 5 years endorsing our hypothesis and preclinical experience that PICSO via hemodynamic power activates regenerative processes also in adult human hearts. These results emphasize that our proposed hypothesis “embryonic recall” claiming revival of an imbedded albeit dormant “epigenetic” process is able not only to sculpture myocardium in the embryo, but also to redesign structure in the adult and failing heart.
... Their cell surface lacks a basal lamina and often forms grooves containing thin bundles of collagen microfibrils, indicating that these cells can preside over the spatial orientation of the newly formed ECM macromolecules (Fig. 1). Besides being primarily responsible for ECM production and remodelling, cardiac fibroblasts can also regulate cardiomyocyte proliferation and growth during development through paracrine and juxtacrine signals [35][36][37]. Thus, they are currently viewed as a dynamic, multifunctional lineage crucial for both developmental and post-natal repair pathways. ...
The term stromal cells is referred to cells of direct or indirect (hematopoietic) mesenchymal origin, and encompasses different cell populations residing in the connective tissue, which share the ability to produce the macromolecular components of the extracellular matrix and to organize them in the correct spatial assembly. In physiological conditions, stromal cells are provided with the unique ability to shape a proper three-dimensional scaffold and stimulate the growth and differentiation of parenchymal precursors to give rise to tissues and organs. Thus, stromal cells have an essential function in the regulation of organ morphogenesis and regeneration. In pathological conditions, under the influence of local pro-inflammatory mediators, stromal cells can be prompted to differentiate into myofibroblasts, which rather express a fibrogenic phenotype required for prompt deposition of reparatory scar tissue. Indeed, scarring may be interpreted as an emergency healing response to injury typical of evolved animals, like mammals, conceivably directed to preserve survival at the expense of function. However, under appropriate conditions, the original ability of stromal cells to orchestrate organ regeneration, which is typical of some lower vertebrates and mammalian embryos, can be resumed. These concepts underline the importance of expanding the knowledge on the biological properties of stromal cells and their role as key regulators of the three-dimensional architecture of the organs in view of the refinement of the therapeutic protocols of regenerative medicine.
... The age of the CF donor tissue can be especially critical given that it is known that the growth factor expression of CFs changes significantly with age. 23 Although there is a trend that the CFs aid in longer EC sprout formation, culture with MSCs resulted in significantly more multicellular sprouts in the ratio study (Fig. 7d), which is more indicative of the in vivo process. 29 MSCs have been previously demonstrated to aid in vessel stability during in vitro co-culture with ECs. 10 Moreover, MSCs are known to produce TGFb which can lead to decreased EC migration/proliferation and enhanced vessel maturity via Alk1 and Alk5 receptors. ...
A primary impediment to cardiac tissue engineering lies in the inability to adequately vascularize the constructs to optimize survival upon implantation. During normal angiogenesis, endothelial cells (ECs) require a support cell to form mature patent lumens and it has been demonstrated that pericytes, vascular smooth muscle cells and mesenchymal stem cells (MSCs) are all able to support the formation of mature vessels. In the heart, cardiac fibroblasts (CFs) provide important electrical and mechanical functions, but to date have not been sufficiently studied for their role in angiogenesis. To study CFs role in angiogenesis, we co-cultured different concentrations of various cell types in fibrin hemispheres with appropriate combinations of their specific media, to determine the optimal conditions for EC growth and sprout formation through DNA analysis, flow cytometry and immunohistology. ECs proliferated best when co-cultured with CFs and analysis of immunohistological images demonstrated that ECs formed the longest and most numerous sprouts with CFs as compared to MSCs. However, ECs were able to produce more multicellular sprouts when in culture with the MSCs. Moreover, these effects were dependent on the ratio of support cell to EC in co-culture. Overall, CFs provide a good support system for EC proliferation and sprout formation; however, MSCs allow for more multicellular sprouts, which is more indicative of the in vivo process.
... With regard to the function in cardiac fibroblasts, miR-29b may attenuate scar barriers to progenitor cell infiltration thereby facilitating iPSC NCX1+ penetration from the Tri-P into the infarcted area. Cardiac fibroblasts, the most numerous non-cardiomyocyte cell populations in heart tissue, generate essential autocrine/paracrine factors to maintain the functional integrity of the myocardium [25,26,27,28]. Since miRNAs target not only single genes but also functionally related gene networks, the paracrine effect of miR-29b overexpressing fibroblasts may contribute to increased iPSC NCX1+ migration and survival. ...
The purpose of this study was to assess the effect of collagen composition on engraftment of progenitor cells within infarcted myocardium.
We previously reported that intramyocardial penetration of stem/progenitor cells in epicardial patches was enhanced when collagen was reduced in hearts overexpressing adenylyl cyclase-6 (AC6). In this study we hypothesized an alternative strategy wherein overexpression of microRNA-29b (miR-29b), inhibiting mRNAs that encode cardiac fibroblast proteins involved in fibrosis, would similarly facilitate progenitor cell migration into infarcted rat myocardium.
In vitro: A tri-cell patch (Tri-P) consisting of cardiac sodium-calcium exchanger-1 (NCX1) positive iPSC (iPSC(NCX1+)), endothelial cells (EC), and mouse embryonic fibroblasts (MEF) was created, co-cultured, and seeded on isolated peritoneum. The expression of fibrosis-related genes was analyzed in cardiac fibroblasts (CFb) by qPCR and Western blot. In vivo: Nude rat hearts were administered mimic miRNA-29b (miR-29b), miRNA-29b inhibitor (Anti-29b), or negative mimic (Ctrl) before creation of an ischemically induced regional myocardial infarction (MI). The Tri-P was placed over the infarcted region 7 days later. Angiomyogenesis was analyzed by micro-CT imaging and immunofluorescent staining. Echocardiography was performed weekly.
The number of green fluorescent protein positive (GFP(+)) cells, capillary density, and heart function were significantly increased in hearts overexpressing miR-29b as compared with Ctrl and Anti-29b groups. Conversely, down-regulation of miR-29b with anti-29b in vitro and in vivo induced interstitial fibrosis and cardiac remodeling.
Overexpression of miR-29b significantly reduced scar formation after MI and facilitated iPSC(NCX1+) penetration from the cell patch into the infarcted area, resulting in restoration of heart function after MI.
... Although research has identified a key role for CFs in regulating heart function through both the release of and response to paracrine signals, further investigation should elucidate how soluble factor signaling by CFs is affected by matrix composition under both normal and pathophysiological conditions. For a more extensive overview of CF's role in paracrine signaling in the myocardium through development and disease, see the works of Tian and Morrisey [49], Noseda and Schneider [50], and Rosenkranz [51]. ...
The extracellular matrix is no longer considered a static support structure for cells, but a dynamic signaling network with the power to influence cell, tissue and whole organ physiology. In the myocardium, cardiac fibroblasts are the primary cell type responsible for the synthesis, deposition and degradation of matrix proteins and they therefore play a critical role in the development and maintenance of functional heart tissue. This review will summarize the extensive research conducted in vivo and in vitro demonstrating the influence of both physical and chemical stimuli on cardiac fibroblasts and how these interactions impact both cardiomyocytes and the extracellular matrix. This work is of considerable significance given that cardiovascular diseases are marked by extensive remodeling of the extracellular matrix, which ultimately impairs the functional capacity of the heart. We seek to summarize the unique role of cardiac fibroblasts in normal cardiac development and the most prevalent cardiac pathologies including congenital heart defects, hypertension, hypertrophy, and the remodeled heart following myocardial infarction. We will conclude by identifying existing holes in the research that, if answered, have the potential to dramatically improve current therapeutic strategies for the repair and regeneration of damaged myocardium via mechanotransductive signaling.
... Functionally, the notion of cardiac fibroblasts has transitioned over the past several years from the classification of fibroblasts as a cell lineage primarily responsible for contributing to the extracellular matrix (ECM) to our current understanding of cardiac fibroblasts as a dynamic, multi-functional lineage critical for both developmental and postnatal repair pathways. Cardiac fibroblasts not only produce and remodel the ECM in response to different physiological cues (reviewed in [1,[20][21][22][23]), but also regulate cardiomyocyte proliferation and growth during development [24,25], directly connect to cardiomyocytes via connexins [20,[26][27][28][29], electrically isolate various portions of the conduction system in the heart [1,23,30], secrete factors to regulate signaling of cardiomyocytes in a paracrine fashion (reviewed in [1,9,[21][22][23]31]), as well as induce cardiomyocyte hypertrophic and fibrotic responses to injury in the adult heart (reviewed in [19,23]). Within the impressive breadth of cardiac fibroblast functions, this review focuses on the roles of cardiac fibroblasts in the context of development and injury, in order to compare and contrast these two physiologic processes and emphasize the dynamic nature of cardiac fibroblasts. ...
Cardiac fibroblasts are the most abundant cell in the mammalian heart. While they have been historically overlooked in terms of functional contributions to development and physiology, cardiac fibroblasts are now front and center. They are currently recognized as key protagonists during both normal development and cardiomyopathy disease, and work together with cardiomyocytes through paracrine, structural, and potentially electrical interactions. However, the lack of specific biomarkers and fibroblast heterogeneous nature currently convolutes the study of this dynamic cell lineage; though, efforts to advance marker analysis and lineage mapping technologies are ongoing. These tools will help elucidate the functional significance of fibroblast-cardiomyocyte interactions in vivo and delineate the dynamic nature of normal and pathological cardiac fibroblasts. Since therapeutic promise lies in understanding the interface between developmental biology and the postnatal injury response, future studies to understand the divergent roles played by cardiac fibroblasts both in utero and following cardiac insult are essential.
... Cardiac fibroblasts organized in a myocardial threedimensional network [5] became recently a fashionable research topic because of their heterogeneity, activation capacity, and immunophenotypic switch possibilities, with contractile or secretory properties acquirement [6][7][8]. The immunohistochemical characterization of myocardial fibroblasts is difficult; these cells do not express markers with absolute specificity. ...
The cellular immunoprofile of cardiac dysfunctions and lesions of ischemic etiology are insufficiently studied to date, especially regarding the contribution of non-cardiomyocytic structures. Aiming to explore this immunoprofile, we used immunohistochemistry applied on embryonic, fetal and adult normal or ischemic myocardium. We observed a decrease of smooth muscle alpha-actin expression in fetal vs. embryonic cardiomyocytes, its absence in normal adult myocardium and its intense expression in the fibrotic scars of ischemic myocardium. DDR2 and vimentin, which are present in the interstitial cells and cardiomyocytes of the embryo, fetus and normal adult heart, are absent in the fibrotic scar tissue and cicatricial infarction, the latter expressing smooth muscle alpha-actin and CD34. This suggested that myofibroblasts and not local fibroblasts that participate in ischemic remodeling. An EGFR-positive vascular network was better represented in the ischemic heart than in the adult normal one, a fact possibly related to EGFR implication in cardiac ischemic pre- and post-conditioning. Therefore, cardiomyocytes and non-cardiomyocytic cells have an undulating immunoprofile according to the intrauterine life stage or age after birth, and a variable contribution in cardiac lesions, mostly in ischemic ones.
... This scaffold distributes mechanical forces throughout the myocardium and integrates the contractile activity of individual cells to coordinate the pump function of the heart [107]. Cardiac fibroblasts secrete autocrine and paracrine factors that control cardiomyocyte growth [108]. In addition to its primary structural role, the ECM can also act as a repository of growth factors. ...
The field of stem cell research was revolutionized with the advent of induced pluripotent stem cells. By reprogramming somatic cells to pluripotent stem cells, most ethical concerns associated with the use of embryonic stem cells are overcome, such that many hopes from the stem cell field now seem a step closer to reality. Several methods and cell sources have been described to create induced pluripotent stem cells and we discuss their characteristics in terms of feasibility and efficiency. From these cells, cardiac progenitors and cardiomyocytes can be derived by several protocols and most recent advances as well as remaining limitations are being discussed. However, in the short time period this technology has been around, evidence emerges that induced pluripotent stem cells may be more prone to genetic defects and maintain an epigenetic memory and thus may not be entirely the same as embryonic stem cells. Despite the lack of a complete fundamental understanding of stem cell biology, and even more of ways how to coax them into defined cell types, the technology is quickly adopted by industry. This paper gives an overview of the current applications of induced pluripotent stem cells in cardiovascular drug development and highlights active areas of research towards functional repair of the damaged heart. Adult stem cells have already been taken to clinical trials and we discuss these results in light of potential and hurdles to be taken to move induced pluripotent stem cells to the clinic.
... Based on the above data and considerations, it can be postulated that MSCs may behave as cardiac supporting stromal cells, whose role in regulating proliferation of cardiac progenitor cells in the embryonic and adult heart has been recently underscored [18,23,32,35,44,47]. On the other hand, our findings do not support the concept of a cardiomyogenic potential of MSCs, as we never observed MSCs expressing myocardial-specific markers in the co-cultures (not shown). ...
The possibility to induce myocardial regeneration by the activation of resident cardiac stem cells (CSCs) has raised great interest. However, to propose endogenous CSCs as therapeutic options, a better understanding of the complex mechanisms controlling heart morphogenesis is needed, including the cellular and molecular interactions that cardiomyocyte precursors establish with cells of the stromal compartment. In the present study, we co-cultured immature cardiomyocytes from neonatal mouse hearts with mouse bone marrow-derived mesenchymal stromal cells (MSCs) to investigate whether these cells could influence cardiomyocyte growth in vitro. We found that cardiomyocyte proliferation was enhanced by direct co-culture with MSCs compared with the single cultures. We also showed that the proliferative response of the neonatal cardiomyocytes involved the activation of Notch-1 receptor by its ligand Jagged-1 expressed by the adjacent MSCs. In fact, the cardiomyocytes in contact with MSCs revealed a stronger immunoreactivity for the activated Notch-intracellular domain (Notch-ICD) as compared with those cultured alone and this response was significantly attenuated when MSCs were silenced for Jagged-1. The presence of various cardiotropic cytokines and growth factors in the conditioned medium of MSCs underscored the contribution of paracrine mechanisms to Notch-1 up-regulation by the cardiomyocytes. In conclusions these findings unveil a previously unrecognized function of MSCs in regulating cardiomyocyte proliferation through Notch-1/Jagged-1 pathway and suggest that stromal-myocardial cell juxtacrine and paracrine interactions may contribute to the development of new and more efficient cell-based myocardial repair strategies.
... 36 Interestingly, recent data demonstrate that embryonic fibroblasts can induce cycling of cardiac myocytes. 37 Therefore, the phenotypic and functional characterization of the bone marrow-derived newly generated fibroblasts deserves further detailed investigation. ...
Cell therapy is a promising option to improve functional recovery after ischemia. Several subsets of bone marrow-derived cells were shown to reduce infarct size and increase ejection fraction in experimental models of ischemia. The mechanisms underlying the functional improvement are diverse and have been shown to include paracrine effects of the injected cells, as well as a variable degree of differentiation to endothelial cells, pericytes, smooth muscle, and cardiac muscle.
To elucidate the true nature of such plasticity and contribution to recovery, we engineered vectors that encoded inducible suicide genes under the control of endothelium (endothelial nitric oxide synthase)-, smooth muscle (SM22alpha)-, and cardiomyocyte (alpha-MHC)-specific promoters, thereby allowing selective depletion of the individual cell lineage acquired by the transplanted undifferentiated bone marrow-derived cells. Lentivirally delivered thymidine kinase, which converts the prodrug ganciclovir into a cytotoxic agent, was used to selectively eliminate cells 2 weeks after transplantation of bone marrow mononuclear cells in an acute myocardial infarction model. We demonstrate that elimination of transplanted endothelium-committed or SM22alpha-expressing cells, but not cardiac-committed cells, induced a significant deterioration of ejection fraction. Moreover, elimination of endothelial nitric oxide synthase-expressing cells 2 weeks after injection reduced capillary and arteriole density.
This study demonstrates that elimination of bone marrow mononuclear cells reexpressing endothelial nitric oxide synthase particularly induced a deterioration of cardiac function, which indicates a functional contribution of the vascular cell fate decision of human bone marrow-derived mononuclear cells in vivo.
Unlike mature cardiomyocytes, human pluripotent stem cell‐derived cardiomyocytes exhibit higher proliferative capacity; however, the underlying mechanisms involved are yet to be elucidated. Here, we revealed that the Yes‐associated protein (YAP) plays a critical role in regulating cell proliferation in association with epidermal growth factor receptor (EGFR) in human embryonic stem cell‐derived cardiomyocytes (hESC‐CMs). Our results show that low‐density culture significantly promotes the proliferation of hESC‐CMs via YAP. Interestingly, the low‐density culture‐induced YAP expression further induced EGFR expression, without any alterations in the activity of EGFR and its two major downstream kinases, ERK, and AKT. However, treatment of a low‐density‐culture of hESC‐CMs with epidermal growth factor (EGF) increased proliferation via phosphorylation of EGFR, ERK, and AKT, and the EGF‐induced phosphorylation of EGFR, ERK, and AKT was significantly higher in low‐density hESC‐CMs than in high‐density hESC‐CMs. Furthermore, the EGF‐induced activation of EGFR, ERK, and AKT increased YAP expression and subsequently proliferation. In conclusion, YAP mediates both low‐density culture‐induced and EGF‐induced proliferation of hESC‐CMs in low‐density culture conditions. (i) Low‐density culture promotes proliferation of hESC‐derived cardiomyocytes via YAP. (ii) EGF receptor expression is enhanced by low density culture in hESC‐derived cardiomyocytes. (iiI) YAP mediates EGF‐induced proliferation of hESC‐derived cardiomyocytes.
Mouse cardiac fibroblasts have been widely used as an in vitro model for studying fundamental biological processes and mechanisms underlying cardiac pathologies, as well as identifying potential therapeutic targets. Cardiac FBs are relatively easy to culture in a dish and can be manipulated using molecular and pharmacological tools. Because FBs rapidly decrease cell cycle division and proliferative rate after birth, they are prone to phenotypic changes and senescence in cell culture soon after a few passages. Therefore, primary cultures of differentiated fibroblasts from embryos are more desirable. Below we will describe a method that provides good cell yield and viability of E16 CD-1 mouse embryonic cardiac fibroblasts in primary cultures.
Introduction: Cardiac injury results in the death of cardiac myocytes and subsequent scar formation through extracellular matrix (ECM) deposition by fibroblasts (FB) and myofibroblasts (myoFB). Excessive fibrosis results in pathological scarring that predisposes to arrhythmogenesis and heart failure, particularly in the elderly. Strategies to limit adverse ECM remodeling are urgently needed to curtail the growing epidemic of atrial fibrillation and heart failure in the aging population. Persistence of myoFB and resistance to apoptotic cell death has been proposed to underlie the mechanism of excessive fibrosis, yet is not fully characterized.
Methods: Cultured NIH/3T3 cells (control and TGF-β1 treated) have been challenged with activators of extrinsic (FAS-Ligand, 1 μg/mL) or intrinsic (Thapsigargin 10 μM and Staurosporine 5 μM) apoptotic pathways and Caspase-3 activity was measured in cellular lysate.
Results: FAS-L exposure induced ~40-fold suppression of Caspase-3 activity in TGF-β1 treated cells as compared with control (17±12 vs 686±5 nmol AMC/min/106 cells, respectively). Similarly, Staurosporine activated Caspase-3 in TGF-β1 treated cells ~3-fold (171±38 vs 536±29 nmol AMC/min/106 cells), and Thapsigargin ~10-fold (73±33 vs 742±8 nmol AMC/min/106 cells).
Conclusion: TGF-β1 treatment increased the sensitivity of NIH/3T3 cells toward extrinsic and intrinsic apoptotic stimuli. Although, TGF-β1 treatment increased overall resistance of NIH/3T3 cells to apoptosis, the responsiveness of cells to extrinsic vs intrinsic pathways was differentially affected. This data support the hypothesis that persistence of myoFB results in pathological scarring.
The heart is a paradigm of organ provided with unique three-dimensional tissue architecture that is molded during complex organogenesis processes and is required for the heart’s physiological function. The cardiac stroma plays a critical role in the formation and maintenance of the normal heart architecture, as well as of its changes occurring in cardiac diseases. Recent studies have shown that the cardiac stroma, including the epicardium, myocardial interstitium, and endocardium, contains typical telocytes: these cells establish complex spatial relationships with cardiomyocytes and cardiac stem cells suggestive for a regulatory role over three-dimensional organization of heart tissues. Telocytes appear early during prenatal heart development and represent a major stromal cell population in the adult heart. Numerous studies have highlighted that telocytes, through juxtacrine and paracrine mechanisms, can behave as nursing cells for cardiac muscle stem cells modulating their growth and differentiation. On these grounds, a possible role of telocytes in cardiac regeneration can be postulated: this hypothesis is supported by recent experimental findings that reduction of cardiac telocytes due to hypoxia may concur to explain the negligible regenerative ability of the post-infarcted heart, while grafting of telocytes in the injured myocardium improves adverse heart remodeling. The increasing knowledge on the properties of cardiac telocytes is orienting the research toward their role as key regulators of the three-dimensional architecture of the heart and new promising targets for cardiac regenerative medicine.
Purpose: Excessive fibrosis has been suggested to result from persistence of fibroblasts in injured tissue due to impaired apoptosis, but signaling pathways are not fully defined. Methods: Suppression of apoptotic cell death following transforming growth factor-β1 (TGF-β1) exposure was studied using the culture of NIH/3T3 mouse embryonic fibroblasts. Caspase-3 activity, propidium iodide staining and annexin V binding induced by Fas-ligand (FasL) in NIH/3T3 fibroblasts in the absence and presence of TGF-β1 was determined, and relative contribution of signaling through Smad2/3 and noncanonical Erk1/2 and Akt pathways was dissected by assessing phosphorylation status of these kinases and caspase activity in the absence and presence of specific inhibitors (SB431542, PD0325901 and LY294002), respectively. Results: TGF-β1 treatment suppressed FasL-mediated fibroblasts apoptosis with a greater than threefold reduction of caspase-3 activity (from 894 ± 186 to 195 ± 56 nmol AFC/min/106 cells at 250 ng/mL of FasL) and reductions in cleaved caspase-8 and caspase-3 by 3.2-fold and 4.3-fold, respectively. The reduction in caspase activation was accompanied by a decrease in annexin V-positive cells by ~80%. TGF-β1 treatment phosphorylated Smad2/3, Erk1/2 and Akt, which were reduced by their selective inhibitors. Inhibition of Smad2/3 and Erk1/2 alone partially reduced the protective effect of TGF-β1 on caspase-3 activation, whereas inhibition of the Akt pathway had no significant effect. Concomitant inhibition of Smad2/3 and Erk1/2 completely reversed the protection by TGF-β1. Conclusions: TGF-β1-mediated suppression of apoptosis in fibroblasts involves both Smad2/3 and Erk1/2 pathways, but not the Akt pathway. A combined approach inhibiting Smad2/3 and Erk1/2 pathways can completely reverse the protective effect of TGF-β1 on apoptosis. These findings are proof of concept to help define strategies to reduce progression of fibrosis and resultant morbidities associated with conditions causing excessive fibrosis, including but not limited to keloid formation, transplant fibrosis and aging-associated fibrosis of the heart.
Cardiac progenitor cells (CPCs) in the primary and secondary heart fields contribute to the formation of all major cell types in the mammalian heart. While some CPCs remain undifferentiated in mid-gestation and postnatal hearts, very little is known about their proliferation and differentiation potential. In this study, using an Nkx2.5 cell lineage restricted reporter mouse model, we provide evidence that Nkx2.5(+) CPCs and cardiomyocytes can be readily distinguished from nonmyocyte population using a combination of Nkx2.5 and sarcomeric myosin staining of dispersed ventricular cell preparations. Assessment of cell number and G1/S transit rates during ventricular development indicates that the proliferative capacity of Nkx2.5(+) cell lineage gradually decreases despite a progressive increase in Nkx2.5+ cell number. Notably, mid-gestation ventricles (E11.5) contain a larger number of CPCs (~ 2 fold) compared to E14.5 ventricles and the embryonic CPCs retain cardiomyogenic differentiation potential. The proliferation rates are consistently higher in embryonic CPCs compared to myocyte population in both E11.5 and E14.5 ventricles. Results from two independent cell transplantation models revealed that E11.5 ventricular cells with a higher percentage of proliferating CPCs can form larger grafts compared to E14.5 ventricular cells. Furthermore, transplantation of embryonic ventricular cells did not cause any undesirable side effects such as arrhythmias. These data underscore the benefits of donor cell developmental staging in myocardial repair.
Copyright © 2014, American Journal of Physiology - Cell Physiology.
Cardiac muscle engineering is evolving rapidly, aiming at the provision of innovative models for drug development and therapeutic myocardium. The progress in this field will depend crucially on the proper exploitation of stem cell technologies. Understanding the processes governing stem cell differentiation towards a desired phenotype and subsequent maturation in an organotypic manner will be key to ultimately providing realistic tissue models or therapeutics. Cardiogenesis is controlled by milieu factors that collectively constitute a so-called cardiogenic niche. The components of the cardiogenic niche are not yet fully defined but include paracrine factors and instructive extracellular matrix. Both are provided by supportive stromal cells under strict spatial and temporal control. Detailed knowledge on the exact composition and functionality of the dynamic cardiogenic niche during development will likely be instrumental to further advance cardiac muscle engineering. This review will discuss the concept of myocardial tissue engineering from the stem cell/developmental biology perspective and put forward the hypothesis of the cardiogenic niche as a fundamental building block of tissue-engineered myocardium.
The definitive treatment for end-stage heart failure, organ transplant, is limited by the supply of donor organs. Successful allograft recipients suffer significant adverse effects from chronic antirejection medications. Positive clinical treatment of injured myocardium with stem/progenitor cells has led to hope that one day autologous stem-cell-derived whole or partial donor organs can be generated. Advances in the ability to isolate (or generate) stem or progenitor cells that can give rise to beating cardiocyte-like cells and vascular components, and the advent of human iPS cell technology when combined with recent advances in the generation of perfusable complex tissue scaffolds has moved the field closer to creation of a transplantable heart. As cardiac tissue engineering matures, several other simpler cardiac tissues, such as patches for focal use and human cell test beds for drug screening and drug discovery, are emerging.
Cardiac fibroblasts are the most populous nonmyocyte cell type within the mature heart and are required for extracellular matrix synthesis and deposition, generation of the cardiac skeleton, and to electrically insulate the atria from the ventricles. Significantly, cardiac fibroblasts have also been shown to play an important role in cardiomyocyte growth and expansion of the ventricular chambers during heart development. Although there are currently no cardiac fibroblast-restricted molecular markers, it is generally envisaged that the majority of the cardiac fibroblasts are derived from the proepicardium via epithelial-to-mesenchymal transformation. However, still relatively little is known about when and where the cardiac fibroblasts cells are generated, the lineage of each cell, and how cardiac fibroblasts move to reside in their final position throughout all four cardiac chambers. In this review, we summarize the present understanding regarding the function of Periostin, a useful marker of the noncardiomyocyte lineages, and its role during cardiac morphogenesis. Characterization of the cardiac fibroblast lineage and identification of the signals that maintain, expand and regulate their differentiation will be required to improve our understanding of cardiac function in both normal and pathophysiological states.
G-protein-coupled receptor (GPCR) agonists are well-known inducers of cardiac hypertrophy. We found that the shedding of heparin-binding epidermal growth factor (HB-EGF) resulting from metalloproteinase activation and subsequent transactivation of the epidermal growth factor receptor occurred when cardiomyocytes were stimulated by GPCR agonists, leading to cardiac hypertrophy. A new inhibitor of HB-EGF shedding, KB-R7785, blocked this signaling. We cloned a disintegrin and metalloprotease 12 (ADAM12) as a specific enzyme to shed HB-EGF in the heart and found that dominant-negative expression of ADAM12 abrogated this signaling. KB-R7785 bound directly to ADAM12, suggesting that inhibition of ADAM12 blocked the shedding of HB-EGF. In mice with cardiac hypertrophy, KB-R7785 inhibited the shedding of HB-EGF and attenuated hypertrophic changes. These data suggest that shedding of HB-EGF by ADAM12 plays an important role in cardiac hypertrophy, and that inhibition of HB-EGF shedding could be a potent therapeutic strategy for cardiac hypertrophy.
Following gastrulation and establishment of the three embryonic germ layers, the first definitive organ to form in the embryo is the heart, whose morphogenesis, growth, and integrated function are essential to embry- onic survival, even by midgestation. Abnormalities in heart development result in congenital heart disease, the most frequent form of birth defects in humans. At the opposite end of the temporal spectrum, adult cardiac dis- ease is the most common cause of death in the industri- alized world, with congestive heart failure and inad- equate pump function the end result of diverse disorders intrinsic to cardiac muscle cells, cardiac valves, systemic blood pressure, and the coronary blood supply. Despite recent therapeutic advances and mechanical devices to sustain cardiac function, only a minority of heart failure patients lives longer than 5yr. Death from heart disease therefore comprises an epidemic more prevalent than all cancers combined (Ries et al. 2003). Recent studies have begun to reveal the cellular cir- cuitry that controls cardiac growth during development and disease. Intriguingly, many of the molecules and mechanisms that regulate growth of the embryonic heart are redeployed in the adult heart in response to stress signals that provoke cardiac enlargement and heart fail- ure. Thus, understanding the mechanisms involved in heart development promises to provide insights into the molecular basis for pathogenesis of the adult heart, as well as to reveal novel therapeutic targets. In this review, we consider three aspects of cardiac development with significant implications for adult heart disease: (1) nor- mal growth during organogenesis, (2) a "fetal" cardiac gene program reactivated in hypertrophy, and (3) restor- ative growth by undifferentiated progenitor cells that have cardiogenic potential. Each of these aspects of car- diac growth could be, itself, the subject of an in-depth review. Our goal, however, is not to comprehensively review these areas, but to identify common themes in developmental biology that are reiterated in settings of abnormal growth and dysfunction of the adult heart. Al- though we focus on development and disease of the myo- cardium, many of the same principles of allometric growth and homeostasis apply to other organs whose structure and function are influenced by physiological and pathological signaling.
During cardiogenesis, perturbation of a key transition at mid-gestation from cardiac patterning to cardiac growth and chamber maturation often leads to diverse types of congenital heart disease, such as ventricular septal defect (VSD), myocardium noncompaction, and ventricular hypertrabeculation. This transition, which occurs at embryonic day (E) 9.0-9.5 in murine embryos and E24-28 in human embryos, is crucial for the developing heart to maintain normal cardiac growth and function in response to an increasing hemodynamic load. Although, ventricular trabeculation and compaction are key morphogenetic events associated with this transition, the molecular and cellular mechanisms are currently unclear. Initially, cardiac restricted cytokine bone morphogenetic protein 10 (BMP10) was identified as being upregulated in hypertrabeculated hearts from mutant embryos deficient in FK506 binding protein 12 (FKBP12). To determine the biological function of BMP10 during cardiac development, we generated BMP10-deficient mice. Here we describe an essential role of BMP10 in regulating cardiac growth and chamber maturation. BMP10 null mice display ectopic and elevated expression of p57(kip2) and a dramatic reduction in proliferative activity in cardiomyocytes at E9.0-E9.5. BMP10 is also required for maintaining normal expression levels of several key cardiogenic factors (e.g. NKX2.5 and MEF2C) in the developing myocardium at mid-gestation. Furthermore, BMP10-conditioned medium is able to rescue BMP10-deficient hearts in culture. Our data suggest an important pathway that involves a genetic interaction between BMP10, cell cycle regulatory proteins and several major cardiac transcription factors in orchestrating this transition in cardiogenesis at mid-gestation. This may provide an underlying mechanism for understanding the pathogenesis of both structural and functional congenital heart defects.
Isl1(+) cardiovascular progenitors and their downstream progeny play a pivotal role in cardiogenesis and lineage diversification of the heart. The mechanisms that control their renewal and differentiation are largely unknown. Herein, we show that the Wnt/beta-catenin pathway is a major component by which cardiac mesenchymal cells modulate the prespecification, renewal, and differentiation of isl1(+) cardiovascular progenitors. This microenvironment can be reconstituted by a Wnt3a-secreting feeder layer with ES cell-derived, embryonic, and postnatal isl1(+) cardiovascular progenitors. In vivo activation of beta-catenin signaling in isl1(+) progenitors of the secondary heart field leads to their massive accumulation, inhibition of differentiation, and outflow tract (OFT) morphogenic defects. In addition, the mitosis rate in OFT myocytes is significantly reduced following beta-catenin deletion in isl1(+) precursors. Agents that manipulate Wnt signals can markedly expand isl1(+) progenitors from human neonatal hearts, a key advance toward the cloning of human isl1(+) heart progenitors.
Growth and expansion of ventricular chambers is essential during heart development and is achieved by proliferation of cardiac progenitors. Adult cardiomyocytes, by contrast, achieve growth through hypertrophy rather than hyperplasia. Although epicardial-derived signals may contribute to the proliferative process in myocytes, the factors and cell types responsible for development of the ventricular myocardial thickness are unclear. Using a coculture system, we found that embryonic cardiac fibroblasts induced proliferation of cardiomyocytes, in contrast to adult cardiac fibroblasts that promoted myocyte hypertrophy. We identified fibronectin, collagen, and heparin-binding EGF-like growth factor as embryonic cardiac fibroblast-specific signals that collaboratively promoted cardiomyocyte proliferation in a paracrine fashion. Myocardial beta1-integrin was required for this proliferative response, and ventricular cardiomyocyte-specific deletion of beta1-integrin in mice resulted in reduced myocardial proliferation and impaired ventricular compaction. These findings reveal a previously unrecognized paracrine function of embryonic cardiac fibroblasts in regulating cardiomyocyte proliferation.
The epicardium regulates growth and survival of the underlying myocardium. This activity depends on intrinsic retinoic acid (RA) and erythropoietin signals. However, these signals do not act directly on the myocardium and instead are proposed to regulate the production of an unidentified soluble epicardial derived mitogen. Here, we show that Fgf9, Fgf16, and Fgf20 are expressed in the endocardium and epicardium and that RA can induce epicardial expression of Fgf9. Using knockout mice and an embryonic heart organ culture system, we show that endocardial and epicardial derived FGF signals regulate myocardial proliferation during midgestation heart development. We further show that this FGF signal is received by both FGF receptors 1 and 2 acting redundantly in the cardiomyoblast. In the absence of this signal, premature differentiation results in cellular hypertrophy and newborn mice develop a dilated cardiomyopathy. FGFs thus constitute all or part of the epicardial signal regulating myocardial growth and differentiation.
The epidermal growth factor (EGF)-ErbB signaling network is composed of multiple ligands of the EGF family and four tyrosine kinase receptors of the ErbB family. In higher vertebrates, these four receptors bind a multitude of ligands. Ligand binding induces the formation of various homo- and heterodimers of ErbB, potentially providing for a high degree of signal diversity. ErbB receptors and their ligands are expressed in a variety of tissues throughout development. Recent advances in gene targeting strategies in mice have revealed that the EGF-ErbB signaling network has fundamental roles in development, proliferation, differentiation, and homeostasis in mammals. The heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that binds to and activates the EGF receptor (EGFR/ErbB1) and ErbB4. Recent studies using several mutant mice lacking HB-EGF expression have revealed that HB-EGF has a critical role in normal heart function and in normal cardiac valve formation in conjunction with ErbB receptors. HB-EGF signaling through ErbB2 is essential for the maintenance of homeostasis in the adult heart, whereas HB-EGF signaling through EGFR is required during cardiac valve development. In this review, we introduce and discuss the role of ErbB receptors in heart function and development, focusing on the physiological function of HB-EGF in these processes.
Amplification of the ErbB2 locus, which encodes a receptor tyrosine kinase, is common in aggressive breast tumors and correlates with poor prognosis. The mechanisms underlying ErbB2-mediated breast carcinoma progression remain incompletely defined. To examine the role of the signaling and cell-adhesion receptor beta 4 integrin during ErbB2-mediated tumorigenesis, we introduced a targeted deletion of the beta 4 signaling domain into a mouse model of ErbB2-induced mammary carcinoma. Loss of beta 4 signaling suppresses mammary tumor onset and invasive growth. Ex vivo studies indicate that beta 4 forms a complex with ErbB2 and enhances activation of the transcription factors STAT3 and c-Jun. STAT3 contributes to disruption of epithelial adhesion and polarity, while c-Jun is required for hyperproliferation. Finally, deletion of the beta 4 signaling domain enhances the efficacy of ErbB2-targeted therapy. These results indicate that beta 4 integrin promotes tumor progression by amplifying ErbB2 signaling and identify beta 4 as a potential target for molecular therapy of breast cancer.
Ventricular chamber morphogenesis, first manifested by trabeculae formation, is crucial for cardiac function and embryonic viability and depends on cellular interactions between the endocardium and myocardium. We show that ventricular Notch1 activity is highest at presumptive trabecular endocardium. RBPJk and Notch1 mutants show impaired trabeculation and marker expression, attenuated EphrinB2, NRG1, and BMP10 expression and signaling, and decreased myocardial proliferation. Functional and molecular analyses show that Notch inhibition prevents EphrinB2 expression, and that EphrinB2 is a direct Notch target acting upstream of NRG1 in the ventricles. However, BMP10 levels are found to be independent of both EphrinB2 and NRG1 during trabeculation. Accordingly, exogenous BMP10 rescues the myocardial proliferative defect of in vitro-cultured RBPJk mutants, while exogenous NRG1 rescues differentiation in parallel. We suggest that during trabeculation Notch independently regulates cardiomyocyte proliferation and differentiation, two exquisitely balanced processes whose perturbation may result in congenital heart disease.
In the developing heart, reciprocal interactions between the epicardium and myocardium drive further sublineage specification and ventricular chamber morphogenesis. Several observations suggest that the epicardium is a source of secreted factors that influence cardiomyocyte proliferation, and these factors may have other roles as well. However, the identity of these epicardial factors remains mostly unknown. We have identified platelet-derived growth factor-A (PDGF-A) as one of several mitogens expressed by the rat EMC epicardial cell line (epicardial mesothelial cells), by embryonic epicardium and myocardium during mouse heart development, and by adult epicardium. Expression of the cognate receptor gene Pdgfra was detected in the epicardium, although a low level of expression in myocardium could not be ruled out. To address the potential role of PDGF signaling in heart development, we mutated both PDGF receptor genes in the myocardial and mesodermal compartments of the heart; however, this did not result in an observable cardiac phenotype. This finding suggests that mesodermal PDGF signaling is not essential in heart development, although its role may be redundant with other signaling pathways. Indeed, our results demonstrate the presence of additional mitogens that may have such an overlapping role.
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