Regulation of extracellular matrix composition by fibroblasts during perinatal cardiac maturation.

BACKGROUND: Cardiac fibroblasts are the main non-myocyte population responsible for extracellular matrix (ECM) production. During perinatal development, fibroblast expansion coincides with the transition from hyperplastic to hypertrophic myocardial growth. Therefore, we investigated the consequences of fibroblast loss at the time of cardiomyocyte maturation by depleting fibroblasts in the perinatal mouse. METHODS AND RESULTS: We evaluated the microenvironment of the perinatal heart in the absence of fibroblasts and the potential functional impact of fibroblast loss in regulation of cardiomyocyte cell cycle arrest and binucleation. Cre-mediated expression of diphtheria toxin A in PDGFRα expressing cells immediately after birth eliminated 70-80% of the cardiac fibroblasts. At postnatal day 5, hearts lacking fibroblasts appeared similar to controls with normal morphology and comparable numbers of endothelial and smooth muscle cells, despite a pronounced reduction in fibrillar collagen. Immunoblotting and proteomic analysis of control and fibroblast-deficient hearts identified differential abundance of several ECM proteins. In addition, fibroblast loss decreased tissue stiffness and resulted in increased cardiomyocyte mitotic index, DNA synthesis, and cytokinesis. Moreover, decellularized matrix from fibroblast-deficient hearts promoted cardiomyocyte DNA replication. While cardiac architecture was not overtly affected by fibroblast reduction, few pups survived past postnatal day 11, suggesting an overall requirement for PDGFRα expressing fibroblasts. CONCLUSIONS: These studies demonstrate the key role of fibroblasts in matrix production and cardiomyocyte cross-talk during mouse perinatal heart maturation and revealed that fibroblast-derived ECM may modulate cardiomyocyte maturation in vivo. Neonatal depletion of fibroblasts demonstrated that although hearts can tolerate reduced ECM composition, fibroblast loss eventually leads to perinatal death as the approach simultaneously reduced fibroblast populations in other organs.

Cardiac Remodeling and Repair: Recent Approaches, Advancements, and Future Perspective.

The limited ability of mammalian adult cardiomyocytes to proliferate following an injury to the heart, such as myocardial infarction, is a major factor that results in adverse fibrotic and myocardial remodeling that ultimately leads to heart failure. The continued high degree of heart failure-associated morbidity and lethality requires the special attention of researchers worldwide to develop efficient therapeutics for cardiac repair. Recently, various strategies and approaches have been developed and tested to extrinsically induce regeneration and restoration of the myocardium after cardiac injury have yielded encouraging results. Nevertheless, these interventions still lack adequate success to be used for clinical interventions. This review highlights and discusses both cell-based and cell-free therapeutic approaches as well as current advancements, major limitations, and future perspectives towards developing an efficient therapeutic method for cardiac repair.

The Tcf21 lineage constitutes the lung lipofibroblast population.

Transcription factor 21 (Tcf21) is a basic helix-loop-helix transcription factor required for mesenchymal development in several organs. Others have demonstrated that Tcf21 is expressed in embryonic lung mesenchyme and that loss of Tcf21 results in a pulmonary hypoplasia phenotype. Although recent single-cell transcriptome analysis has described multiple mesenchymal cell types in the lung, few have characterized the Tcf21 expressing population. To explore the Tcf21 mesenchymal lineage, we traced Tcf21-expressing cells during embryogenesis and in the adult. Our results showed that Tcf21 progenitor cells at embryonic day (E)11.5 generated a subpopulation of fibroblasts and lipofibroblasts and a limited number of smooth muscle cells. After E15.5, Tcf21 progenitor cells exclusively become lipofibroblasts and interstitial fibroblasts. Lipid metabolism genes were highly expressed in perinatal and adult Tcf21 lineage cells. Overexpression of Tcf21 in primary neonatal lung fibroblasts led to increases in intracellular neutral lipids, suggesting a regulatory role for Tcf21 in lipofibroblast function. Collectively, our results reveal that Tcf21 expression after E15.5 delineates the lipofibroblast and a population of interstitial fibroblasts. The Tcf21 inducible Cre mouse line provides a novel method for identifying and manipulating the lipofibroblast.

Platelet-derived growth factor receptor-α is essential for cardiac fibroblast survival.

Platelet-derived growth factor receptor α (PDGFRα), a receptor tyrosine kinase required for cardiac fibroblast development, is uniquely expressed by fibroblasts in the adult heart. Despite the consensus that PDGFRα is expressed in adult cardiac fibroblasts, we know little about its function when these cells are at rest. Here, we demonstrate that loss of PDGFRα in cardiac fibroblasts resulted in a rapid reduction of resident fibroblasts. Furthermore, we observe that phosphatidylinositol 3-kinase signaling was required for PDGFRα-dependent fibroblast maintenance. Interestingly, this reduced number of fibroblasts was maintained long-term, suggesting that there is no homeostatic mechanism to monitor fibroblast numbers and restore hearts to wild-type levels. Although we did not observe any systolic functional changes in hearts with depleted fibroblasts, the basement membrane and microvasculature of these hearts were perturbed. Through in vitro analyses, we showed that PDGFRα signaling inhibition resulted in an increase in fibroblast cell death, and PDGFRα stimulation led to increased levels of the cell survival factor activating transcription factor 3. Our data reveal a unique role for PDGFRα signaling in fibroblast maintenance and illustrate that a 50% loss in cardiac fibroblasts does not result in lethality.NEW & NOTEWORTHY Platelet-derived growth factor receptor α (PDGFRα) is required in developing cardiac fibroblasts, but a functional role in adult, quiescent fibroblasts has not been identified. Here, we demonstrate that PDGFRα signaling is essential for cardiac fibroblast maintenance and that there are no homeostatic mechanisms to regulate fibroblast numbers in the heart. PDGFR signaling is generally considered mitogenic in fibroblasts, but these data suggest that this receptor may direct different cellular processes depending on the cell's maturation and activation status.

Resident fibroblast expansion during cardiac growth and remodeling.

Cardiac fibrosis, denoted by the deposition of extracellular matrix, manifests with a variety of diseases such as hypertension, diabetes, and myocardial infarction. Underlying this pathological extracellular matrix secretion is an expansion of fibroblasts. The mouse is now a common experimental model system for the study of cardiovascular remodeling and elucidation of fibroblast responses to cardiac growth and stress is vital for understanding disease processes. Here, using diverse but fibroblast specific markers, we report murine fibroblast distribution and proliferation in early postnatal, adult, and injured hearts. We find that perinatal fibroblasts and endothelial cells proliferate at similar rates. Furthermore, regardless of the injury model, fibroblast proliferation peaks within the first week after injury, a time window similar to the period of the inflammatory phase. In addition, fibroblast densities remain high weeks after the initial insult. These results provide detailed information regarding fibroblast distribution and proliferation in experimental methods of heart injury.

Revisiting Cardiac Cellular Composition.

RATIONALE: Accurate knowledge of the cellular composition of the heart is essential to fully understand the changes that occur during pathogenesis and to devise strategies for tissue engineering and regeneration. OBJECTIVE: To examine the relative frequency of cardiac endothelial cells, hematopoietic-derived cells, and fibroblasts in the mouse and human heart. METHODS AND RESULTS: Using a combination of genetic tools and cellular markers, we examined the occurrence of the most prominent cell types in the adult mouse heart. Immunohistochemistry revealed that endothelial cells constitute >60%, hematopoietic-derived cells 5% to 10%, and fibroblasts <20% of the nonmyocytes in the heart. A refined cell isolation protocol and an improved flow cytometry approach provided an independent means of determining the relative abundance of nonmyocytes. High-dimensional analysis and unsupervised clustering of cell populations confirmed that endothelial cells are the most abundant cell population. Interestingly, fibroblast numbers are smaller than previously estimated, and 2 commonly assigned fibroblast markers, Sca-1 and CD90, under-represent fibroblast numbers. We also describe an alternative fibroblast surface marker that more accurately identifies the resident cardiac fibroblast population. CONCLUSIONS: This new perspective on the abundance of different cell types in the heart demonstrates that fibroblasts comprise a relatively minor population. By contrast, endothelial cells constitute the majority of noncardiomyocytes and are likely to play a greater role in physiological function and response to injury than previously appreciated.

Genetic tools for identifying and manipulating fibroblasts in the mouse.

The use of mouse genetic tools to track and manipulate fibroblasts has provided invaluable in vivo information regarding the activities of these cells. Recently, many new mouse strains have been described for the specific purpose of studying fibroblast behavior. Colorimetric reporter mice and lines expressing Cre are available for the study of fibroblasts in the organs prone to fibrosis, including heart, kidney, liver, lung, and skeletal muscle. In this review we summarize the current state of the models that have been used to define tissue resident fibroblast populations. While these complex genetic reagents provide unique insights into the process of fibrosis, they also require a thorough understanding of the caveats and limitations. Here, we discuss the specificity and efficiency of the available genetic models and briefly describe how they have been used to document the mechanisms of fibrosis.

Defining the Cardiac Fibroblast.

Cardiac fibrosis remains an important health concern, but the study of fibroblast biology has been hindered by a lack of effective means for identifying and tracking fibroblasts. Recent advances in fibroblast-specific lineage tags and reporters have permitted a better understanding of these cells. After injury, multiple cell types have been implicated as the source for extracellular matrix-producing cells, but emerging studies suggest that resident cardiac fibroblasts contribute substantially to the remodeling process. In this review, we discuss recent findings regarding cardiac fibroblast origin and identity. Our understanding of cardiac fibroblast biology and fibrosis is still developing and will expand profoundly in the next few years, with many of the recent findings regarding fibroblast gene expression and behavior laying down the groundwork for interpreting the purpose and utility of these cells before and after injury. (Circ J 2016; 80: 2269-2276).

Fast skeletal myosin binding protein-C expression exacerbates dysfunction in heart failure.

During heart failure, gene and protein expression profiles undergo extensive compensatory and pathological remodeling. We previously observed that fast skeletal myosin binding protein-C (fMyBP-C) is upregulated in diseased mouse hearts. While fMyBP-C shares significant homology with its cardiac paralog, cardiac myosin binding protein-C (cMyBP-C), there are key differences that may affect cardiac function. However, it is unknown if the expression of fMyBP-C expression in the heart is a pathological or compensatory response. We aim to elucidate the cardiac consequence of either increased or knockout of fMyBP-C expression. To determine the sufficiency of fMyBP-C to cause cardiac dysfunction, we generated cardiac-specific fMyBP-C over-expression mice. These mice were further crossed into a cMyBP-C null model to assess the effect of fMyBP-C in the heart in the complete absence of cMyBP-C. Finally, fMyBP-C null mice underwent transverse aortic constriction (TAC) to define the requirement of fMyBP-C during heart failure development. We confirmed the upregulation of fMyBP-C in several models of cardiac disease, including the use of lineage tracing. Low levels of fMyBP-C caused mild cardiac remodeling and sarcomere dysfunction. Exclusive expression of fMyBP-C in a heart failure model further exacerbated cardiac pathology. Following 8 weeks of TAC, fMyBP-C null mice demonstrated greater protection against heart failure development. Mechanistically, this may be due to the differential regulation of the myosin super-relaxed state. These findings suggest that the elevated expression of fMyBP-C in diseased hearts is a pathological response. Targeted therapies to prevent upregulation of fMyBP-C may prove beneficial in the treatment of heart failure. Significance Statement: Recently, the sarcomere - the machinery that controls heart and muscle contraction - has emerged as a central target for development of cardiac therapeutics. However, there remains much to understand about how the sarcomere is modified in response to disease. We recently discovered that a protein normally expressed in skeletal muscle, is present in the heart in certain settings of heart disease. How this skeletal muscle protein affects the function of the heart remained unknown. Using genetically engineered mouse models to modulate expression of this skeletal muscle protein, we determined that expression of this skeletal muscle protein in the heart negatively affects cardiac performance. Importantly, deletion of this protein from the heart could improve heart function suggesting a possible therapeutic avenue.

IL-1ß-mediated adaptive reprogramming of endogenous human cardiac fibroblasts to cells with immune features during fibrotic remodeling.

The source and roles of fibroblasts and T-cells during maladaptive remodeling and myocardial fibrosis in the setting of pulmonary arterial hypertension (PAH) have been long debated. We demonstrate, using single-cell mass cytometry, a subpopulation of endogenous human cardiac fibroblasts expressing increased levels of CD4, a helper T-cell marker, in addition to myofibroblast markers distributed in human fibrotic RV tissue, interstitial and perivascular lesions in SUGEN/Hypoxia (SuHx) rats, and fibroblasts labeled with pdgfrα CreERt2/+ in R26R-tdTomato mice. Recombinant IL-1ß increases IL-1R, CCR2 receptor expression, modifies the secretome, and differentiates cardiac fibroblasts to form CD68-positive cell clusters. IL-1ß also activates stemness markers, such as NANOG and SOX2, and genes involved in dedifferentiation, lymphoid cell function and metabolic reprogramming. IL-1ß induction of lineage traced primary mouse cardiac fibroblasts causes these cells to lose their fibroblast identity and acquire an immune phenotype. Our results identify IL-1ß induced immune-competency in human cardiac fibroblasts and suggest that fibroblast secretome modulation may constitute a therapeutic approach to PAH and other diseases typified by inflammation and fibrotic remodeling.

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes.

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression. The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Characterization of Burn Eschar Pericytes.

Pericytes are cells that reside adjacent to microvasculature and regulate vascular function. Pericytes gained great interest in the field of wound healing and regenerative medicine due to their multipotential fate and ability to enhance angiogenesis. In burn wounds, scarring and scar contractures are the major pathologic feature and cause loss of mobility. The present study investigated the influence of burn wound environment on pericytes during wound healing. Pericytes isolated from normal skin and tangentially excised burn eschar tissues were analyzed for differences in gene and protein expression using RNA-seq., immunocytochemistry, and ELISA analyses. RNA-seq identified 443 differentially expressed genes between normal- and burn eschar-derived pericytes. Whereas, comparing normal skin pericytes to normal skin fibroblasts identified 1021 distinct genes and comparing burn eschar pericytes to normal skin fibroblasts identified 2449 differential genes. Altogether, forkhead box E1 (FOXE1), a transcription factor, was identified as a unique marker for skin pericytes. Interestingly, FOXE1 levels were significantly elevated in burn eschar pericytes compared to normal. Additionally, burn wound pericytes showed increased expression of profibrotic genes periostin, fibronectin, and endosialin and a gain in contractile function, suggesting a contribution to scarring and fibrosis. Our findings suggest that the burn wound environment promotes pericytes to differentiate into a myofibroblast-like phenotype promoting scar formation and fibrosis.

Genetic lineage tracing defines myofibroblast origin and function in the injured heart.

Cardiac fibroblasts convert to myofibroblasts with injury to mediate healing after acute myocardial infarction (MI) and to mediate long-standing fibrosis with chronic disease. Myofibroblasts remain a poorly defined cell type in terms of their origins and functional effects in vivo. Here we generate Postn (periostin) gene-targeted mice containing a tamoxifen-inducible Cre for cellular lineage-tracing analysis. This Postn allele identifies essentially all myofibroblasts within the heart and multiple other tissues. Lineage tracing with four additional Cre-expressing mouse lines shows that periostin-expressing myofibroblasts in the heart derive from tissue-resident fibroblasts of the Tcf21 lineage, but not endothelial, immune/myeloid or smooth muscle cells. Deletion of periostin(+) myofibroblasts reduces collagen production and scar formation after MI. Periostin-traced myofibroblasts also revert back to a less-activated state upon injury resolution. Our results define the myofibroblast as a periostin-expressing cell type necessary for adaptive healing and fibrosis in the heart, which arises from Tcf21(+) tissue-resident fibroblasts.