Tissue inhibitor of metalloproteinase-4 deletion in mice impacts maternal cardiac function during pregnancy and postpartum.

Reversible physiological cardiac hypertrophy of the maternal heart occurs during pregnancy and involves extracellular matrix (ECM) remodeling. Previous mouse studies revealed that changes in ECM molecules accompany functional changes in the left ventricle (LV) during late pregnancy and postpartum. We evaluated the effect of global Timp4 deletion in female mice on LV functional parameters and ECM molecules during pregnancy and the postpartum period. Heart weights normalized to tibia lengths were increased in Timp4 knockout (Timp4 KO) virgin, pregnant, and postpartum day 2 mice compared with wild types. Serial echocardiography performed on pregnancy days 10, 12, and 18 and postpartum days (ppds) 2, 7, 14, 21, and 28 revealed that both wild-type and Timp4 KO mice increased end systolic and end diastolic volumes (ESV, EDV) by mid to late pregnancy compared with virgins, with EDV changes persisting through the postpartum period. When compared with wild types, Timp4 KO mice exhibited higher ejection fractions in virgins, at pregnancy days 10 and 18 and ppd2 and ppd14. High-molecular weight forms of COL1A1 and COL3A1 proteins in LV were greater in Timp4 KO virgins, and COL1A1 was higher in late pregnancy and on ppd2 compared with wild types. With exceptions, Timp4 KO mice during late pregnancy and the early postpartum period were able to maintain stroke volume similar to wild-type mice through increased ejection fraction. Although TIMP4 deletion in females exhibited altered ECM molecules, it did not adversely affect cardiac function during first pregnancies and lactation.NEW & NOTEWORTHY Pregnancy and lactation increase volume load on the heart. Defects in cardiac remodeling during pregnancy and postpartum can result in peripartum cardiomyopathy. TIMPs participate in cardiac remodeling. The present study reports the cardiac function in Timp4 knockout adult female mice during pregnancy and lactation. Timp4 knockout females at many time points have higher ejection fraction to maintain stroke volume. Global deletion of Timp4 was not detrimental to maternal heart function during first pregnancies and lactation.

The ubiquitin ligase WWP1 contributes to shifts in matrix proteolytic profiles and a myocardial aging phenotype with diastolic heart.

Ubiquitylation is a key event that regulates protein turnover, and induction of the ubiquitin ligase E3 WWP1 has been associated with age. Left ventricular hypertrophy (LVH) commonly occurs as a function of age and can cause heart failure (HF) with a preserved ejection fraction (EF; HFpEF). We hypothesized that overexpression (O/E) of WWP1 in the heart would cause LVH as well as functional and structural changes consistent with the aging HFpEF phenotype. Global WWP1 O/E was achieved in mice (n = 11) and echocardiography (40 MHz) performed to measure LV mass, EF, Doppler velocities (early E, late/atrial A), myocardial relaxation (E'), and isovolumetric relaxation time (IVRT) at 4, 6, and 8 wk. Age-matched wild-type animals (n = 15) were included as referent controls. LV EF was identical (60 ± 1 vs. 60 ± 1%, P > 0.90) with no difference in LV mass (67 ± 3 vs. 75 ± 5, P > 0.25) at 4 wk. However, at 8 wk of age, LV mass increased over twofold, E/A fell (impaired passive filling), and E/E' was lower and IVRT prolonged (impaired LV relaxation) - all P < 0.05. Collagen percent area increased by over twofold and fibrillar collagen expression (RT-PCR) over 1.5-fold (P < 0.05) with WWP1 O/E. WWP1 with an anti-WWP1 antibody could be identified in isolated cardiac fibroblasts, with WWP1 increased over twofold in O/E fibroblasts (P < 0.05). Inducing WWP1 expression caused LVH and preserved systolic function but impaired diastolic dysfunction, consistent with the HFpEF phenotype. Targeting the WWP1 pathway may be a novel therapeutic target for this intractable form of HF associated with aging.NEW & NOTEWORTHY Heart failure (HF) with a preserved ejection fraction (HFpEF) is a growing cause of HF and commonly afflicts the elderly. Milestones for HFpEF include diastolic dysfunction and an abnormal extracelluar matrix (ECM). The ubiquitin ligases, such as WWP1, change with aging and regulate critical protein turnover/stability processes, such as the ECM. The present study demonstrated that induction of WWP1 in mice induced LV hypertrophy, diastolic dysfunction, and ECM accumulation, consistent with the HFpEF phenotype, and thus may identify a new therapeutic pathway.

Repeated eccentric contractions positively regulate muscle oxidative metabolism and protein synthesis during cancer cachexia in mice.

Cancer-induced wasting is accompanied by disruptions to muscle oxidative metabolism and protein turnover that have been associated with systemic inflammation, whereas exercise and stimulated muscle contractions can positively regulate muscle protein synthesis and mitochondrial homeostasis. In preclinical cancer cachexia models, a single bout of eccentric contractions (ECCs) can induce protein synthesis and repeated ECC bouts prevent myofiber atrophy. The cellular mechanisms providing this protection from atrophy have not been resolved. Therefore, the purpose of this study was to determine whether repeated stimulated ECC bouts affect basal muscle oxidative metabolism and protein synthesis during cancer cachexia, and if these changes were associated with plasma IL-6 levels. Male ApcMin/+ (MIN; n = 10) mice initiating cachexia and healthy C57BL/6 (B6; n = 11) control mice performed repeated ECC bouts over 2 wk. MIN mice exhibited body weight loss and elevated plasma IL-6 before and during repeated ECC bouts. Control MIN muscle demonstrated disrupted signaling related to inflammation, oxidative capacity, and protein synthesis regulation, which were all improved by repeated ECC bouts. With cachexia, plasma IL-6 levels were negatively correlated with myofiber cross-sectional area, oxidative capacity, and protein synthesis. Interestingly, ECC improvements in these outcomes were positively correlated with plasma IL-6 levels in MIN mice. There was also a positive relationship between muscle oxidative capacity and protein synthesis after repeated ECC bouts in MIN mice. Collectively, repeated ECC bouts altered the cachectic muscle phenotype independent of systemic wasting, and there was a strong association between muscle oxidative capacity and protein synthesis in this adaptive response.NEW & NOTEWORTHY Cancer-induced muscle wasting is accompanied by disruptions to muscle oxidative metabolism and protein turnover regulation, whereas exercise is a potent stimulator of muscle protein synthesis and mitochondrial homeostasis. In a preclinical model of cancer cachexia, we report that cachectic muscle retains anabolic and metabolic plasticity to repeated eccentric contraction bouts despite an overall systemic wasting environment. The attenuation of muscle atrophy is linked to improved oxidative capacity and protein synthesis during cancer cachexia progression.

Systemic IL-6 regulation of eccentric contraction-induced muscle protein synthesis.

Systemic cytokines and contractile activity are established regulators of muscle protein turnover. Paradoxically, the IL-6 cytokine family, which shares the ubiquitously expressed membrane gp130 receptor, has been implicated in skeletal muscle's response to both contractions and cancer-induced wasting. Although we have reported that tumor-derived cachectic factors could suppress stretch-induced protein synthesis in cultured myotubes, the ability of systemic cytokines to disrupt in vivo eccentric contraction-induced protein synthesis has not been established. Therefore, we examined whether systemic IL-6 regulates basal and eccentric contraction-induced protein synthesis through muscle gp130 signaling. Systemic IL-6 overexpression was performed for 2 wk, and we then examined basal and eccentric contraction-induced protein synthesis and mammalian target of rapamycin complex 1 (mTORC1) signaling in tibialis anterior muscle of male wild-type, muscle-specific gp130 receptor knockout, and tumor-bearing ApcMin/+ mice. Systemic IL-6 overexpression suppressed basal protein synthesis and mTORC1 signaling independently of IL-6 level, which was rescued by muscle gp130 loss. Interestingly, only high systemic IL-6 levels suppressed eccentric contraction-induced protein synthesis. Systemic IL-6 overexpression in precachectic tumor-bearing ApcMin/+ mice accelerated cachexia development, which coincided with suppressed basal and eccentric contraction-induced muscle protein synthesis. The suppression of eccentric contraction-induced protein synthesis by IL-6 occurred independently of mTORC1 activation. Collectively, these findings demonstrate that basal protein synthesis suppression was more sensitive to circulating IL-6 compared with the induction of protein synthesis by eccentric contraction. However, systemic IL-6 can interact with the cancer environment to suppress eccentric contraction-induced protein synthesis independently of mTORC1 activation.

Adsorption of cellular proteins to polyelectrolyte-functionalized gold nanorods: a mechanism for nanoparticle regulation of cell phenotype?

Cell behavior in the presence of nanomaterials is typically explored through simple viability assays, but there is mounting evidence that nanomaterials can have more subtle effects on a variety of cellular functions. Previously our lab demonstrated that gold nanorods functionalized with polyelectrolyte multi-layers inhibited rat cardiac fibroblast-mediated remodeling of type I collagen scaffolds by altering fibroblast phenotype and the mechanical properties of the collagen network. In this work, we examine a possible mechanism for these effects: adsorption of cellular proteins by the nanorods. Mass spectrometric and gel electrophoresis of media collected from cultured cells suggests that a number of proteins, some of which mediate cell-cell and cell-matrix interactions, adsorb onto the surface of these nanoparticles in vitro. Polyethylene glycol coating of the nanorods largely mitigates protein adsorption and fibroblast-mediated collagen remodeling. These results suggest that adsorption of proteins by nanorods could have a significant effect on cell functions, including fibroblast-mediated matrix remodeling.

Myocardial fibroblast-matrix interactions and potential therapeutic targets.

The cardiac extracellular matrix (ECM) is a dynamic structure, adapting to physiological and pathological stresses placed on the myocardium. Deposition and organization of the matrix fall under the purview of cardiac fibroblasts. While often overlooked compared to myocytes, fibroblasts play a critical role in maintaining ECM homeostasis under normal conditions and in response to pathological stimuli assume an activated, myofibroblast phenotype associated with excessive collagen accumulation contributing to impaired cardiac function. Complete appreciation of fibroblast function is hampered by the lack of fibroblast-specific reagents and the heterogeneity of fibroblast precursors. This is further complicated by our ability to dissect the role of myofibroblasts versus fibroblasts in myocardial in remodeling. This review highlights critical points in the regulation of collagen deposition by fibroblasts, the current panel of molecular tools used to identify fibroblasts and the role of fibroblast-matrix interactions in fibroblast function and differentiation into the myofibroblast phenotype. The clinical potential of exploiting differences between fibroblasts and myofibroblasts and using them to target specific fibroblast populations is also discussed. This article is part of a Special Issue entitled "Myocyte-Fibroblast Signalling in Myocardium."

Regulation of tissue fibrosis by the biomechanical environment.

The biomechanical environment plays a fundamental role in embryonic development, tissue maintenance, and pathogenesis. Mechanical forces play particularly important roles in the regulation of connective tissues including not only bone and cartilage but also the interstitial tissues of most organs. In vivo studies have correlated changes in mechanical load to modulation of the extracellular matrix and have indicated that increased mechanical force contributes to the enhanced expression and deposition of extracellular matrix components or fibrosis. Pathological fibrosis contributes to dysfunction of many organ systems. A variety of in vitro models have been utilized to evaluate the effects of mechanical force on extracellular matrix-producing cells. In general, application of mechanical stretch, fluid flow, and compression results in increased expression of extracellular matrix components. More recent studies have indicated that tissue rigidity also provides profibrotic signals to cells. The mechanisms whereby cells detect mechanical signals and transduce them into biochemical responses have received considerable attention. Cell surface receptors for extracellular matrix components and intracellular signaling pathways are instrumental in the mechanotransduction process. Understanding how mechanical signals are transmitted from the microenvironment will identify novel therapeutic targets for fibrosis and other pathological conditions.

Localized targeting of biomaterials following myocardial infarction: a foundation to build on.

Acute coronary syndromes can give rise to myocardial injury infarction (MI), which in turn promulgates a series of cellular and extracellular events that result in left ventricular (LV) dilation and dysfunction. Localized strategies focused upon interrupting this inexorable process include delivery of bioactive molecules and stem cell derivatives. These localized treatment strategies are often delivered in a biomaterial complex in order to facilitate elution of the bioactive molecules or stem cell engraftment. However, these biomaterials can impart significant and independent effects upon the MI remodeling process. In addition, significant changes in local cell and interstitial biology within the targeted MI region can occur following injection of certain biomaterials, which may hold important considerations when using these materials as matrices for adjuvant drug/cell therapies.

High-aspect-ratio gold nanorods: their synthesis and application to image cell-induced strain fields in collagen films.

Gold nanoparticles are receiving considerable attention due to their novel properties and the potential variety of their uses. Long gold nanorods with dimensions of approximately 20 × 400 nm exhibit strong light scattering and can be easily observed under dark-field microscopy. Here we describe the use of this light-scattering property to track micrometer scale strains in collagen gels and thick films, which result from cell traction forces applied by neonatal heart fibroblasts. The use of such collagen constructs to model cell behavior in the extracellular matrix is common, and describing local mechanical environments on such a small scale is necessary to understand the complex factors associated with the remodeling of the collagen network. Unlike other particles used for tracking purposes, gold nanorods do not photobleach, allowing their optical signal to be tracked for longer periods of time, and they can be easily synthesized and coated with various charged or neutral shells, potentially reducing the effect of their presence on the cell system or allowing selective placement. Techniques described here are ultimately applicable for investigations with a wide variety of cells and cell environments.

Type II diabetes promotes a myofibroblast phenotype in cardiac fibroblasts.

AIMS: Cardiovascular disease is the leading cause of death for individuals diagnosed with type II diabetes mellitus (DM). Changes in cardiac function, left ventricular wall thickness and fibrosis have all been described in patients and animal models of diabetes; however, the factors mediating increased matrix deposition remain unclear. The goal of this study was to evaluate whether cardiac fibroblast function is altered in a rat model of type II DM. MAIN METHODS: Cardiac fibroblasts were isolated from 14 week old Zucker diabetic and lean control (LC) adult male rat hearts. Fibroblasts were examined for their ability to remodel 3-dimensional collagen matrices, their adhesion, migration and proliferation on collagen and changes in gene expression associated with collagen remodeling. KEY FINDINGS: Cardiac fibroblasts from diabetic animals demonstrated significantly greater ability to contract 3-dimensional collagen matrices compared to cardiac fibroblasts from LC animals. The enhanced contractile behavior was associated with an increase in diabetic fibroblast proliferation and elevated expression of α-smooth muscle actin and type I collagen, suggesting the transformation of diabetic fibroblasts into a myofibroblast phenotype. SIGNIFICANCE: Cardiac fibrosis is a common complication in diabetic cardiomyopathy which may contribute to the observed cardiac dysfunction associated with this disease. Identifying and understanding the changes in fibroblast behavior which contribute to the increased deposition of collagen and other matrix proteins may provide novel therapeutic targets for reducing the devastating effects of diabetes on the heart.

Cellular mechanisms of tissue fibrosis. 2. Contributory pathways leading to myocardial fibrosis: moving beyond collagen expression.

While the term "fibrosis" can be misleading in terms of the complex patterns and processes of myocardial extracellular matrix (ECM) remodeling, fibrillar collagen accumulation is a common consequence of relevant pathophysiological stimuli, such as pressure overload (PO) and myocardial infarction (MI). Fibrillar collagen accumulation in both PO and MI is predicated on a number of diverse cellular and extracellular events, which include changes in fibroblast phenotype (transdifferentiation), posttranslational processing and assembly, and finally, degradation. The expansion of a population of transformed fibroblasts/myofibroblasts is a significant cellular event with respect to ECM remodeling in both PO and MI. The concept that this cellular expansion within the myocardial ECM may be due, at least in part, to endothelial-mesenchymal transformation and thereby not dissimilar to events observed in cancer progression holds intriguing future possibilities. Studies regarding determinants of procollagen processing, such as procollagen C-endopeptidase enhancer (PCOLCE), and collagen assembly, such as the secreted protein acidic and rich in cysteine (SPARC), have identified potential new targets for modifying the fibrotic response in both PO and MI. Finally, the transmembrane matrix metalloproteinases, such as MMP-14, underscore the diversity and complexity of this ECM proteolytic family as this protease can degrade the ECM as well as induce a profibrotic response. The growing recognition that the myocardial ECM is a dynamic entity containing a diversity of matricellular and nonstructural proteins as well as proteases and that the fibrillar collagens can change in structure and content in a rapid temporal fashion has opened up new avenues for modulating what was once considered an irreversible event--myocardial fibrosis.

Mechanical loading promotes mast cell degranulation via RGD-integrin dependent pathways.

Mast cells are known to respond to a number of stimuli, such as IgE antibody-antigen complexes, pathogens, chemical compounds, and physical stimulation, resulting in the activation of these cells and subsequent release of cytokines, inflammatory mediators and granules which can influence the pathophysiology of neighboring cells. Although different forms of physical stimulation (i.e. shear stress and acupuncture) have been investigated, the effect of cyclic tensile loading on mast cell activation has not. To characterize the response of mast cells to tensile loading, RBL-2H3 cells were embedded in a 3-dimensional fibrin construct and subjected to 24h of cyclic loading at 0%, 5% or 10% peak tensile strain. Mechanical loading significantly increased RBL-2H3 cell secretion of ß-hexosaminidase (2.1- to 2.3-fold, respectively) in a load- and time-dependent manner when compared to the controls. Furthermore, no evidence of load-induced cell death or alterations in cell proliferation was observed. To determine if RGD-dependent integrins mediated the degranulation of mast cells during mechanical loading, cell-matrix interactions were inhibited by treating the cells with echistatin, a disintegrin that binds RGD-dependent integrins. Treatment with echistatin significantly attenuated load-induced degranulation without compromising cell viability. These results suggest a novel mechanism through which mechanical loading induces mast cell activation via RGD binding integrins.

Diabetes-induced alterations in the extracellular matrix and their impact on myocardial function.

Diabetes is an increasing public health problem that is expected to escalate in the future due to the growing incidence of obesity in the western world. While this disease is well known for its devastating effects on the kidneys and vascular system, diabetic individuals can develop cardiac dysfunction, termed diabetic cardiomyopathy, in the absence of other cardiovascular risk factors such as hypertension or atherosclerosis. While much effort has gone into understanding the effects of elevated glucose or altered insulin sensitivity on cellular components within the heart, significant changes in the cardiac extracellular matrix (ECM) have also been noted. In this review article we highlight what is currently known regarding the effects diabetes has on both the expression and chemical modification of proteins within the ECM and how the fibrotic response often observed as a consequence of this disease can contribute to reduced cardiac function.

Age-dependent expression of collagen receptors and deformation of type I collagen substrates by rat cardiac fibroblasts.

Little is known about how age influences the ways in which cardiac fibroblasts interact with the extracellular matrix. We investigated the deformation of collagen substrates by neonatal and adult rat cardiac fibroblasts in monolayer and three-dimensional (3D) cultures, and quantified the expression of three collagen receptors [discoidin domain receptor (DDR)1, DDR2, and ß1 integrin] and the contractile protein alpha smooth muscle actin (α-SMA) in these cells. We report that adult fibroblasts contracted 3D collagen substrates significantly less than their neonate counterparts, whereas no differences were observed in monolayer cultures. Adult cells had lower expression of ß1 integrin and α-SMA than neonate cultures, and we detected significant correlations between the expression of α-SMA and each of the collagen receptors in neonate cells but not in adult cells. Consistent with recent work demonstrating age-dependent interactions with myocytes, our results indicate that interactions between cardiac fibroblasts and the extracellular matrix change with age.

Temporal alterations in cardiac fibroblast function following induction of pressure overload.

Increases in cardiovascular load (pressure overload) are known to elicit ventricular remodeling including cardiomyocyte hypertrophy and interstitial fibrosis. While numerous studies have focused on the mechanisms of myocyte hypertrophy, comparatively little is known regarding the response of the interstitial fibroblasts to increased cardiovascular load. Fibroblasts are the most numerous cell type in the mammalian myocardium and have long been recognized as producing the majority of the myocardial extracellular matrix. It is only now becoming appreciated that other aspects of fibroblast behavior are important to overall cardiac function. The present studies were performed to examine the temporal alterations in fibroblast activity in response to increased cardiovascular load. Rat myocardial fibroblasts were isolated at specific time-points (3, 7, 14, and 28 days) after induction of pressure overload by abdominal aortic constriction. Bioassays were performed to measure specific parameters of fibroblast function including remodeling and contraction of 3-dimensional collagen gels, migration, and proliferation. In addition, the expression of extracellular matrix receptors of the integrin family was examined. Myocardial hypertrophy and fibrosis were evident within 7 days after constriction of the abdominal aorta. Collagen gel contraction, migration, and proliferation were enhanced in fibroblasts from pressure-overloaded animals compared to fibroblasts from sham animals. Differences in fibroblast function and protein expression were evident within 7 days of aortic constriction, concurrent with the onset of hypertrophy and fibrosis of the intact myocardium. These data provide further support for the idea that rapid and dynamic changes in fibroblast phenotype accompany and contribute to the progression of cardiovascular disease.

The collagen receptor DDR2 is expressed during early cardiac development.

Discoidin Domain Receptor 2 (DDR2) is a receptor tyrosine kinase which has been shown to regulate cell migration upon binding its ligand, collagen. Expression studies determined that DDR2 mRNA and protein are present in the atrioventricular canal during epithelial-mesenchymal transformation (EMT) and the receptor is expressed in both activated endothelial and migrating mesenchymal cells in vivo.

Polyelectrolyte-coated gold nanorods and their interactions with type I collagen.

Gold nanorods (AuNRs) have unique optical properties for numerous biomedical applications, but the interactions between AuNRs and proteins, particularly those of the extracellular matrix (ECM), are poorly understood. Here the effects of AuNRs on the self-assembly, mechanics, and remodeling of type I collagen gels were examined in vitro. AuNRs were modified with polyelectrolyte multilayers (PEMs) to minimize cytotoxicity, and AuNRs with different terminal polymer chemistries were examined for their interactions with collagen by turbidity assays, rheological tests, and microscopy. Gel contraction assays were used to examine the effects of the PEM-coated AuNRs on cell-mediated collagen remodeling. Polyanion-terminated AuNRs significantly reduced the lag (nucleation) phase of collagen self-assembly and significantly increased the dynamic shear modulus of the polymerized gels, whereas polycation-terminated AuNRs had no effect on the mechanical properties of the collagen. Both polyanion- and polycation-terminated AuNRs significantly inhibited collagen gel contraction by cardiac fibroblasts, and the nanoparticles were localized in intra-, peri-, and extracellular compartments, suggesting that PEM-coated AuNRs influence cell behavior via multiple mechanisms. These results demonstrate the significance of nanoparticle-ECM interactions in determining the bioactivity of nanoparticles.

The effect of gold nanorods on cell-mediated collagen remodeling.

Cardiac fibroblasts, the noncontractile cells of the heart, contribute to myocardial maintenance through the deposition, degradation, and organization of collagen. Adding polyelectrolyte-coated gold nanorods to three-dimensional constructs composed of collagen and cardiac fibroblasts reduced contraction and altered the expression of mRNAs encoding beta-actin, alpha-smooth muscle actin, and collagen type I. These data show that nanomaterials can modulate cell-mediated matrix remodeling and suggest that the targeted delivery of nanomaterials can be applied for antifibrotic therapies.

Gold nanoparticles in biology: beyond toxicity to cellular imaging.

Gold, enigmatically represented by the target-like design of its ancient alchemical symbol, has been considered a mystical material of great value for centuries. Nanoscale particles of gold now command a great deal of attention for biomedical applications. Depending on their size, shape, degree of aggregation, and local environment, gold nanoparticles can appear red, blue, or other colors. These visible colors reflect the underlying coherent oscillations of conduction-band electrons ("plasmons") upon irradiation with light of appropriate wavelengths. These plasmons underlie the intense absorption and elastic scattering of light, which in turn forms the basis for many biological sensing and imaging applications of gold nanoparticles. The brilliant elastic light-scattering properties of gold nanoparticles are sufficient to detect individual nanoparticles in a visible light microscope with approximately 10(2) nm spatial resolution. Despite the great excitement about the potential uses of gold nanoparticles for medical diagnostics, as tracers, and for other biological applications, researchers are increasingly aware that potential nanoparticle toxicity must be investigated before any in vivo applications of gold nanoparticles can move forward. In this Account, we illustrate the importance of surface chemistry and cell type for interpretation of nanoparticle cytotoxicity studies. We also describe a relatively unusual live cell application with gold nanorods. The light-scattering properties of gold nanoparticles, as imaged in dark-field optical microscopy, can be used to infer their positions in a living cell construct. Using this positional information, we can quantitatively measure the deformational mechanical fields associated with living cells as they push and pull on their local environment. The local mechanical environment experienced by cells is part of a complex feedback loop that influences cell metabolism, gene expression, and migration.

Adaptive changes in cardiac fibroblast morphology and collagen organization as a result of mechanical environment.

There is a growing body of work in the literature that demonstrates the significant differences between 2D versus 3D environments in cell morphologies, spatial organization, cell-ECM interactions, and cell signaling. The 3D environments are generally considered more realistic tissue models both because they offer cells a surrounding environment rather than just a planar surface with which to interact, and because they provide the potential for more diverse mechanical environments. Many studies have examined cellular-mediated contraction of 3D matrices; however, because the 3D environment is much more complex and the scale more difficult to study, little is known regarding how mechanical environment, cell and collagen architecture, and collagen remodeling are linked. In the current work, we examine the spatial arrangement of neonatal cardiac fibroblasts and the associated collagen organization in constrained and unconstrained collagen gels over a 24 h period. Collagen gels that are constrained by their physical attachment to a mold and similar gels, which have been detached (unconstrained) from the mold and subsequently contract, offer two simple mechanical models by which the mechanisms of tissue homeostasis and wound repair might be examined. Our observations suggest the presence of two mechanical regimes in the unconstrained gels: an outer ring where cells orient circumferentially and local collagen aligns with the elongated cells; and a central region where unaligned stellate/bipolar cells are radially surrounded by collagen, similar to that seen throughout constrained gels. The evolving organization of cell alignment and surrounding collagen organization suggests that cellular response may be due to the cellular perception of the apparent stiffness of local physical environment.