Fibroblast Origin

Cardiac fibroblasts are the only cell type in the heart not associated with the basement membrane. They are recognized as single-nucleated, spindle-shaped cells with multiple processes and prominent endoplasmic reticulum and Golgi apparatus. Fibroblasts reside in the self-secreted extracellular matrix arranged in sheets and strands that tightly envelop cardiomyocyte fibers.1,2 Their spatial distribution in the healthy heart is mainly uniform, with the highest density found in the sinoatrial (SA) node and annulus fibrosis, where these cells provide electrical insulation to enable successful pacemaking function and coordinated activation of atria and ventricles. Developmentally, cardiac fibroblasts are derived from mesenchymal cells of a proepicardial organ, which migrate over the heart surface to form the epicardium.^2,5 Through epithelial-to-mesenchymal transition, the epicardium gives rise to epicardium-derived cells that migrate into the heart wall and gradually attain a fibroblast or smooth muscle phenotype. This process is regulated by the spatially and temporally coordinated expression of different growth factors, including platelet-derived growth factor (PDGF), fibroblast growth factors (FGFs) and transforming growth factor (TGF) β.⁶ During prenatal growth, the number of fibroblasts in the heart steadily increases. Shortly after birth, the proliferative capacity of cardiomyocytes ceases, while the fibroblast number abruptly increases (greater than twofold) for reasons that may be related to the postnatal increase in blood pressure (and associated mechanical strains in the heart wall), oxygen tension, or both.

Phenotypic Diversity and Lack of Specific Markers

Although there is significant knowledge of the role of cardiac fibroblasts in the heart, the molecular properties of these cells remain poorly characterized, mainly because of the lack of specific and ubiquitous markers of their phenotype.⁷ The markers currently used are either expressed in only a fraction of fibroblasts or also label other cells (Table 30-1). For example, immunostaining for vimentin has been used widely to label cardiac fibroblasts; however, this intracellular protein is expressed in other mesoderm-derived cells, including endothelial and smooth muscle cells. Similarly, Thy-1 (CD90), a surface marker that labels cardiac fibroblasts and other mesenchymal cells also labels endothelial cells. Discoidin domain receptor 2 (DDR2), a membrane collagen-binding tyrosine kinase receptor, is expressed in a subset of cardiac fibroblasts, but is also found in endothelial and smooth muscle cells. Fibroblast-specific protein-1 (FSP1, S100A4) is another commonly used fibroblast marker that is only sparsely expressed in the normal heart, but is significantly upregulated in heart disease, where it may also label smooth muscle cells.⁸ Recently, Acharya et al⁹ reported the generation of transgenic mice in which transcription factor Tcf21 (Epicardin/Pod1/Capsulin) was selectively expressed in a subset of epicardially derived cardiac fibroblasts, but also in a multitude of other tissues.⁹ In general, the best existing methods to identify and isolate cardiac fibroblasts rely on the labeling with multiple positive and negative markers, such as CD31^–/CD90⁺/DDR2⁺ for live cell isolation,⁴ or von Willebrand factor^–/smooth muscle actin (SMA)^–/vimentin⁺/DDR2⁺ for immunolabeling.^1,10 Although genetic fate mapping methods for in situ labeling of cardiac fibroblasts in mice are extremely valuable, they require a careful interpretation that accounts for the potential pitfalls of this system.⁵

Identifying specific molecular markers of cardiac fibroblasts is additionally complicated by the diversity of their phenotype as contributed by their specific developmental origin (e.g., epicardial vs. endocardial), location in the heart (e.g., atria vs. ventricle, left vs. right heart, valves, SA node, atrioventricular groove), pathologic state (e.g., infarction, pressure overload), and aging. For example, a subset of fibroblastic cells positive for PDGF receptor-α and stem cell antigen 1 (Sca-1) was recently identified in perivascular interstitial regions of the mouse heart.¹¹ These epicardially derived cells, termed cardiac colony-forming-unit fibroblasts, can undergo a long-term expansion for approximately 40 passages and differentiate into various cells of mesodermal lineage. The potential role of these highly proliferative cells in cardiac repair, remodeling, and fibrotic disease remains to be explored. Phenotypic diversity was also identified between canine ventricular and atrial fibroblasts, with atrial fibroblasts being more proliferative in response to a variety of growth stimuli (e.g., fetal bovine serum, PDGF, FGF-2, TGF-β1, angiotensin II [AngII], endothelin-1 [ET-1]).¹² These differences were amplified in congestive heart failure and eliminated using a PDGF receptor blocker, AG1295. Different functional roles were also reported for fetal and adult mouse cardiac fibroblasts. Ieda et al⁴ have shown that adult fibroblasts promote cardiomyocyte hypertrophy, whereas fetal fibroblasts promote embryonic cardiomyocyte proliferation by secreting fibronectin, collagen, and heparin-binding endothelial growth factor that activate β1-integrin signaling in cardiomyocytes.⁴ This finding suggests the possibility that the observed reactivation of the cardiac fetal gene program during hypertrophic cardiomyopathy or heart failure may be contributed by analogous phenotypic changes in cardiac fibroblasts.

Detailed phenotypic and functional characterization of cardiac fibroblasts can be performed in vitro, but these studies must be interpreted with caution because of various confounding factors, including the possibility that enzymatic or outgrowth cell isolation selects for a particular pool of cardiac fibroblasts and that fibroblast phenotype is significantly altered by higher stiffness of the cell attachment substrate (several GPa for glass or plastic vs. tens of KPa for intact tissue),¹³ increased oxygen tension (21% for ambient air vs. 5% in intact tissue),¹⁴ and the lack of neurohumoral and inflammatory factors, cyclic stretch, restricted extracellular space, and vasculature. Direct culture of freshly isolated fibroblasts within a biomimetic three-dimensional environment (e.g., cyclically stretched soft hydrogel) is likely to better preserve native cell phenotype compared with the use of standard two-dimensional culture conditions.¹⁵

Cardiac Myofibroblasts

Although cardiac fibroblasts are the most dominant nonmyocyte cell type in the healthy heart, cardiac disease or myocyte loss owing to myocardial infarction, hypertension, inflammation, and other stress signals is associated with the appearance and proliferation of cardiac myofibroblasts to either replace dead myocytes with a collagenous scar (replacement fibrosis) or yield interstitial or perivascular collagen accumulation (reactive fibrosis). Myofibroblasts exhibit a contractile cell phenotype intermediate between that of fibroblasts and smooth muscle cells.16,17 Compared with fibroblasts, myofibroblasts are larger cells with increased expression of stress fibers that exhibit enhanced proliferation, migration, and secretion of ECM proteins (e.g., collagen I, collagen III, fibronectin), ECM degradation enzymes (matrix metalloproteinases [MMPs]) and their inhibitors (tissue inhibitors of metalloproteinases [TIMPs]).¹⁸ In addition, microfibroblasts show both the increased secretion of and responsiveness to various cytokines and growth factors (e.g., AngII, TGF-β, PDGF, ET-1, tumor necrosis factor [TNF] α, interleukin [IL] 1β).^17,18 Although mainly absent from the normal heart (with the exception of the heart valves), myofibroblasts are found to participate actively in the cardiac wound healing response, where they support rapid tissue remodeling and the formation of a fibrous scar. The efficient scar formation initially serves to prevent harmful dilatation of the heart; however, the long-term persistence and activity of myofibroblasts in the infarct scar or other regions of the myocardium can lead to excessive collagen accumulation, stiffening of the heart, pathologic remodeling, and eventually cardiac malfunction and failure. This persistence of the activated myofibroblasts in the heart after the mature scar is formed contrasts wound-healing processes observed in other organs where myofibroblasts undergo apoptosis and disappear upon scar formation.¹⁶

Although myofibroblasts express all the known molecular markers used to label cardiac fibroblasts (DDR2, vimentin, Thy1, FSP1, periostin), no specific myofibroblast markers have been identified (see Table 30-1).¹⁶ Compared with fibroblasts, de novo myofibroblasts also express or increase the expression of various cytoskeletal (α smooth muscle actin [α-SMA], SM22α, myosin heavy chain-B, tropomyosin) and cell adhesion (paxillin, tensin, fibronectin ED-A) proteins.^13,16 Furthermore, myofibroblasts and fibroblasts do not express the intermediate filament marker desmin, and they lack the expression of mature smooth muscle markers, such as smooth muscle myosin heavy chains. Regarding their origin in fibrotic disease, myofibroblasts are traditionally thought to be derived from the resident (interstitial and adventitial) fibroblasts that proliferate, migrate, and differentiate into a myofibroblast phenotype.⁵ This view is based on in vitro studies showing increased fibroblast proliferation, migration, and conversion to a myofibroblast phenotype in response to a variety of cytokines related to cardiac injury and disease (TGF-β, AngII, TNF-α, IL-1β, IL-6, ET-1).¹⁸ However, the need to form a collagenous scar rapidly to replace dead myocardium (thereby preventing wall rupture or dilatation after injury) would suggest that a significant portion of myofibroblasts at the injury site should be derived from nonresident or nonfibroblastic cells, rather than the activation and long-range migration of remote resident cardiac fibroblasts. In line with this reasoning, recent genetic fate mapping studies in mice have demonstrated that during acute cardiac injury, pressure overload, prolonged ischemia, or chronic AngII treatment, myofibroblasts can originate from various nonfibroblastic sources such as (1) coronary endothelium in which endothelial cells after cardiac damage undergo endothelial-to-mesenchymal transition and migrate from the microvascular bed into interstitium to become myofibroblasts,¹⁹ (2) epicardial epithelium where epicardium-derived cells formed by an epithelial-to-mesenchymal transition differentiate (potentially via Notch activation) into fibroblasts and myofibroblasts that remain to reside in the epicardium,^20,21 and (3) circulating bone marrow-derived cells, such as fibrocytes, that express both markers of hematopoietic origin (CD45, CD13, CD34) and ECM proteins (collagen I and III) or monocytes that upon recruitment to the site of injury or inflammation express both myofibroblast (FSP1, α-SMA) and monocytic (CD45, CD11b, CD14) markers.⁵ In several studies, these non–fibroblast-derived myofibroblasts are reported to comprise a significant fraction (20% to 75%) of all myofibroblasts found in the fibrotic areas.^7,19 Although some of these cells might not persist in the heart at later stages of the disease,²² their exact role in the initial and late adaptive and maladaptive fibrotic sequelae remains to be explored.

The mechanisms of fibroblast-to-myofibroblast conversion have been extensively studied in vitro despite the fact that, with time in culture, cardiac fibroblasts spontaneously attain a myofibroblast phenotype and significantly upregulate α-SMA expression.²³ This spontaneous phenotypic change does not occur in the homeostatic milieu of the healthy heart, but is rather an artifact of cell culture attributed to the high stiffness of the attachment substrate or a switch to hyperoxic (ambient air) conditions.^13,14 The time course and extent of this process strongly depend on the particulars of cell isolation and culture conditions, with some reports showing ubiquitous α-SMA expression as early as 1 to 2 days after fibroblast plating,²⁴ whereas others describe little or no phenotype switch until passage 2 to 3.¹⁰ A contributing factor to this variability might be the use of different anti–α-SMA antibodies by different groups. Figure 30-1 shows passage-1 cultured ventricular fibroblasts isolated from adult human, neonatal rat, and neonatal mouse tissues stained by two commonly used anti–α-SMA antibodies, a monoclonal mouse antibody from Sigma-Aldrich and polyclonal rabbit antibody from Abcam (MA, USA). The same antibodies were used to stain paraformaldehyde-fixed adult mouse ventricular sections. The difference in the specificity of the two antibodies is obvious and suggests nonspecific staining of fibroblasts by the antibody from Sigma-Aldrich. The extent to which cultured fibroblasts and myofibroblasts faithfully represent the phenotype and function of their in vivo counterparts from healthy or diseased hearts remains unknown and certainly warrants further study.

Fibroblast Voltage-sensitive Channels

Cardiac fibroblasts and myofibroblasts lack the required ion channels to initiate an action potential (AP) and are thus considered unexcitable cells. They exhibit a relatively depolarized resting membrane potential (RMP) of –50 to –20 mV, a cell capacitance from approximately 6 pF for fibroblasts to 60 pF for myofibroblasts, and an input resistance at rest of 1 to 10 GΩ.25–27 Figure 30-2, A1, shows typical current traces elicited in cultured human ventricular fibroblasts when membrane voltage in these cells was initially held at –40mV and increased by 10-mV increments from –80 to 30 mV. Lowering the initial holding potential to –100 mV increased the amplitude of both the steady-state and small time-dependent current components (see Figure 30-2, A2). Furthermore, the steady-state current-voltage (I-V) relationships of different types of fibroblasts and myofibroblasts exhibited similar shapes, typical of unexcitable cells, with a moderate outward rectification present at higher membrane potentials (see Figure 30-2, B). In a recent study, cultured fibroblasts isolated from infarcted rat ventricles exhibited a hyperpolarized resting potential and increased outward current density compared with fibroblasts isolated from healthy ventricles.²⁵

Cardiac fibroblasts and myofibroblasts express a variety of voltage-sensitive currents and related ion channel genes and proteins.27–29 For example, inward rectifier, Ba²⁺-sensitive K⁺ current (I_K1) controls RMP in freshly isolated adult rat ventricular fibroblasts (where it is likely mediated by Kir2.1 channels) and cultured commercially available (ScienCell Research Laboratory [CA, USA]) human ventricular fibroblasts (in which both Kir2.1 and Kir2.3 are expressed). Both rat and human adult ventricular fibroblasts express Ca²⁺-activated large conductance K⁺ channels (BK_Ca) as well as a delayed outward rectifier K⁺ current (I_K), which is mediated by Kv1.6 channels in rat cells and by Kv1.5 and Kv1.6 in human cells. Similar slow and rapid delayed rectifier currents (I_Kr and I_Ks) have been recorded in neonatal rat ventricular fibroblasts and are presumably conducted via Kv1.2, Kv1.4, Kv1.5, and Kv2.1 channels.²⁷ Cultured adult rat and mouse ventricular myofibroblasts express the gene encoding the Kir6.1 channel; in mouse cells, the expression of Kir6.1 along with subunits of the sulfonylurea receptor-2 (SUR2) channel generates a robust ATP-sensitive K⁺ current (I_KATP) potentiated by pinacidil.²⁸ Adult human and neonatal rat ventricular fibroblasts were also found to express a transient outward K⁺ current (I_to) that in human cells is conducted by Kv4.2 and Kv4.3 channels and in rat cells by Kv4.2 and Kv1.4. Beside K⁺ currents, cultured human ventricular fibroblasts express TTX-sensitive and TTX-resistant Na⁺ currents and swelling-induced Cl^– current,²⁹ whereas human atrial fibroblasts express voltage-gated H⁺ currents.²⁷ Interestingly, a recent study has demonstrated that differentiation of human atrial fibroblasts into myofibroblasts in cell culture is associated with the de novo expression of a fast voltage-gated Na⁺ current predominantly carried by the α-subunit of the cardiac Na⁺ channel (Nav1.5).³⁰ The potential relevance of this finding for atrial fibrotic disease and arrhythmogenesis is unknown. Endogenous expression of a functional Na⁺-Ca²⁺ exchanger (NCX1 or NCX3 isoform) and L-type Ca²⁺ channel α-subunit (Cav1.2) has been shown to modulate the Ca²⁺ inflow in cultured fibroblasts and potentially contribute to regulation of myofibroblast proliferation, migration, contraction, and collagen secretion.^27,31,32 Importantly, the majority of the ion currents described in the studies mentioned here were detected in only a fraction of all the fibroblasts or myofibroblasts studied, thereby confirming the large phenotypic and functional diversity of these cells.

Fibroblast Mechanosensing and Transient Receptor Potential Channels

Both fibroblasts and myofibroblasts have been considered mechanosensitive cells, whereby their patterns of gene expression, proliferation rate, contractile and electrical properties, and sensitivity to and secretion of different soluble factors and ECM proteins are directly influenced by the mechanical state of their environment.¹³ The phenomenon in which cell electrical properties are altered in response to a mechanical stimulus is called mechanoelectric feedback. In cardiac fibroblasts, mechanoelectric feedback is likely mediated via Ca²⁺-permeable, stretch-sensitive channels of unknown molecular identity that likely belong to a family of transient receptor potential (TRP) channels.^27,33,34 TRP channels are weakly sensitive to changes in membrane voltage and are regulated instead by stretch, oxidative stress, osmotic pressure, temperature, pH, or membrane receptor activation. The activity of these channels sensitizes cardiac fibroblasts to physicochemical changes in their environment. Transcripts of several TRP channels from the canonical (TRPC1,4,6), vanilloid (TRPV2,4), and melastatin (TRPM4,7) subfamilies were identified in human atrial fibroblasts, and TRMP7 (but not TRPC6 or TRPV2,4) currents were also successfully recorded in these cells using single-channel and whole-cell patch clamp. Similarly, adult rat ventricular fibroblasts were reported to express transcripts of TRPC2,3,5,6, TRPV2,4,6, and TRPM4,7 channels, and TRPV4 and TRPC6 channels were shown to mediate Ca²⁺ entry into these cells. Furthermore, adult rat ventricular (but not human atrial) fibroblasts were found to express nonselective cation currents likely carried by TRPC3 and TRPC6 channels, or their heteromers.³⁴ In response to mechanical compression, cardiac fibroblasts generate membrane potential depolarizations known as mechanically induced potentials that, through capacitive or potential electrotonic coupling with cardiomyocytes, can modulate cardiac electrical properties.³⁵ In addition to voltage-gated and TRP channels, cardiac fibroblasts and myofibroblasts express a variety of membrane-bound receptors and integrins that mediate their sensitivity to different chemical and ECM-mediated stimuli and potentially support their long-range communication and signal integration.