Trimethylamine-N-oxide acutely increases cardiac muscle contractility.

Cardiovascular disease is a major cause of morbidity and mortality among patients with chronic kidney disease (CKD). Trimethylamine-N-oxide (TMAO), a uremic metabolite that is elevated in the setting of CKD, has been implicated as a nontraditional risk factor for cardiovascular disease. While association studies have linked elevated plasma levels of TMAO to adverse cardiovascular outcomes, its direct effect on cardiac and smooth muscle function remains to be fully elucidated. We hypothesized that pathological concentrations of TMAO would acutely increase cardiac and smooth muscle contractility. These effects may ultimately contribute to cardiac dysfunction during CKD. High levels of TMAO significantly increased paced, ex vivo human cardiac muscle biopsy contractility (P < 0.05). Similarly, TMAO augmented contractility in isolated mouse hearts (P < 0.05). Reverse perfusion of TMAO through the coronary arteries via a Langendorff apparatus also enhanced cardiac contractility (P < 0.05). In contrast, the precursor molecule, trimethylamine (TMA), did not alter contractility (P > 0.05). Multiphoton microscopy, used to capture changes in intracellular calcium in paced, adult mouse hearts ex vivo, showed that TMAO significantly increased intracellular calcium fluorescence (P < 0.05). Interestingly, acute administration of TMAO did not have a statistically significant influence on isolated aortic ring contractility (P > 0.05). We conclude that TMAO directly increases the force of cardiac contractility, which corresponds with TMAO-induced increases in intracellular calcium but does not acutely affect vascular smooth muscle or endothelial function of the aorta. It remains to be determined if this acute inotropic action on cardiac muscle is ultimately beneficial or harmful in the setting of CKD.NEW & NOTEWORTHY We demonstrate for the first time that elevated concentrations of TMAO acutely augment myocardial contractile force ex vivo in both murine and human cardiac tissue. To gain mechanistic insight into the processes that led to this potentiation in cardiac contraction, we used two-photon microscopy to evaluate intracellular calcium in ex vivo whole hearts loaded with the calcium indicator dye Fluo-4. Acute treatment with TMAO resulted in increased Fluo-4 fluorescence, indicating that augmented cytosolic calcium plays a role in the effects of TMAO on force production. Lastly, TMAO did not show an effect on aortic smooth muscle contraction or relaxation properties. Our results demonstrate novel, acute, and direct actions of TMAO on cardiac function and help lay the groundwork for future translational studies investigating the complex multiorgan interplay involved in cardiovascular pathogenesis during CKD.

Osteocyte lacunar strain determination using multiscale finite element analysis.

Osteocytes are thought to be the primary mechanosensory cells within bone, regulating both osteoclasts and osteoblasts to control load induced changes in bone resorption and formation. Osteocytes initiate intracellular responses including activating the Wnt/ß-catenin signaling pathway after experiencing mechanical forces. In response to changing mechanical loads (strain) the osteocytes signal to cells on the bone surface. However, this process of osteocyte activation appears heterogeneous since it occurs in sub-populations of osteocytes, even within regions predicted to be experiencing similar global strain magnitudes determined based on traditional finite element modeling approaches. Several studies have investigated the strain responses of osteocyte lacunae using finite element (FE) models, but many were limited by the use of idealized geometries (e.g., ellipsoids) and analysis of a single osteocyte. Finite element models by other groups included more details, such as canaliculi, but all were done on models consisting of a single osteocyte. We hypothesized that variation in size and orientation of the osteocyte lacunae within bone would give rise to micro heterogeneity in the strain fields that could better explain the observed patterns of osteocyte activation following load. The osteocytes in our microscale and nanoscale models have an idealized oval shape and some are based on confocal scans. However, all the FE models in this preliminary study consist of multiple osteocytes. The number of osteocytes in the 3D confocal scan models ranged from five to seventeen. In this study, a multi-scale computational approach was used to first create an osteocyte FE model at the microscale level to examine both the theoretical lacunar and perilacunar strain responses based on two parameters: 1) lacunar orientation and 2) lacunar size. A parametric analysis was performed by steadily increasing the perilacunar modulus (5, 10, 15, and 20 GPa). Secondly, a nanoscale FE model was built using known osteocyte dimensions to determine the predicted strains in the perilacunar matrix, fluid space, and cell body regions. Finally, 3-D lacunar models were created using confocal image stacks from mouse femurs to determine the theoretical strain in the lacunae represented by realistic geometries. Overall, lacunar strains decreased by 14% in the cell body, 15% in the fluid space region and 25% in the perilacunar space as the perilacunar modulus increased, indicating a stress shielding effect. Lacunar strains were lower for the osteocytes aligned along the loading axis compared to those aligned perpendicular to axis. Increases in lacuna size also led to increased lacunar strains. These finite element model findings suggest that orientation and lacunar size may contribute to the heterogeneous initial pattern of osteocyte strain response observed in bone following in vivo applied mechanical loads. A better understanding of how mechanical stimuli directly affect the lacunae and perilacunar tissue strains may ultimately lead to a better understanding of the process of osteocyte activation in response to mechanical loading.

Changes in the osteocyte lacunocanalicular network with aging.

Osteoporosis is an aging-related disease of reduced bone mass that is particularly prevalent in post-menopausal women, but also affects the aged male population and is associated with increased fracture risk. Osteoporosis is the result of an imbalance whereby bone formation by osteoblasts no longer keeps pace with resorption of bone by osteoclasts. Osteocytes are the most abundant cells in bone and, although previously thought to be quiescent, they are now known to be active, multifunctional cells that play a key role in the maintenance of bone mass by regulating both osteoblast and osteoclast activity. They are also thought to regulate bone mass through their role as mechanoresponsive cells in bone that coordinate adaptive responses to mechanical loading. Osteocytes form an extensive interconnected network throughout the mineralized bone matrix and receive their nutrients as well as hormones and signaling factors through the lacunocanalicular system. Several studies have shown that the extent and connectivity of the lacunocanalicular system and osteocyte networks degenerates in aged humans as well as in animal models of aging. It is also known that the bone anabolic response to loading is decreased with aging. This review summarizes recent research on the degenerative changes that occur in osteocytes and their lacunocanalicular system as a result of aging and discusses the implications for skeletal health and homeostasis as well as potential mechanisms that may underlie these degenerative changes. Since osteocytes are such key regulators of skeletal homeostasis, maintaining the health of the osteocyte network would seem critical for maintenance of bone health. Therefore, a more complete understanding of the structure and function of the osteocyte network, its lacunocanalicular system, and the degenerative changes that occur with aging should lead to advances in our understanding of age related bone loss and potentially lead to improved therapies.

Live Cell Imaging of Bone Cell and Organ Cultures.

Over the past two decades there have been unprecedented advances in the capabilities for live cell imaging using light and confocal microscopy. Together with the discovery of green fluorescent protein and its derivatives and the development of a vast array of fluorescent imaging probes and conjugates, it is now possible to image virtually any intracellular or extracellular protein or structure. Traditional static imaging of fixed bone cells and tissues takes a snapshot view of events at a specific time point, but can often miss the dynamic aspects of the events being investigated. This chapter provides an overview of the application of live cell imaging approaches for the study of bone cells and bone organ cultures. Rather than emphasizing technical aspects of the imaging equipment, which may vary in different laboratories, we focus on what we consider to be the important principles that are of most practical use for an investigator setting up these techniques in their own laboratory. We also provide detailed protocols that our laboratory has used for live imaging of bone cell and organ cultures.

Collagen Dynamics During the Process of Osteocyte Embedding and Mineralization.

Bone formation, remodeling and repair are dynamic processes, involving cell migration, ECM assembly, osteocyte embedding, and bone resorption. Using live-cell imaging, we previously showed that osteoblast assembly of the ECM proteins fibronectin and collagen is highly dynamic and is integrated with cell motility. Additionally, osteoblast-to-osteocyte transition involved arrest of cell motility, followed by dendrite extension and retraction that may regulate positioning of embedding osteocytes. To further understand how osteocytes differentiate and embed in collagen, mice were generated that co-expressed GFPtopaz-tagged collagen with a Dmp1-Cre-inducible tdTomato reporter targeted to preosteocytes/osteocytes. Dual live-cell imaging of collagen and osteocyte dynamics in mineralizing primary calvarial cell cultures showed that Dmp1-Cre/tdTomato turned on in early bone nodule forming regions, demarcated by foci of concentrated GFP-collagen bundles that appeared structurally distinct from the surrounding collagen. Dmp1-Cre/tdTomato-positive cells were post-mitotic and were continuously induced throughout the 2 week timecourse, whereas the majority of collagen was assembled by day 7. GFP-collagen fibrils showed global (tissue-level) motions, suggesting coordinated cell layer movement, and local fibril motions mediated by cell-generated forces. Condensation of collagen fibril networks occurred within bone nodules prior to mineralization. Intravital imaging confirmed a similar structural appearance of GFP-collagen in calvarial bone, with analogous global motions of mineralizing areas adjacent to sutures. In early (unmineralized) calvarial cell cultures, Dmp1-Cre/tdTomato-positive cells were motile (mean velocity 4.8 µm/h), moving freely in and around the forming bone nodule, with a small number of these cells embedded in collagen, constraining their motion. In mineralizing cultures, the average velocity of Dmp1-Cre/tdTomato-positive cells was significantly reduced (0.7 µm/h), with many immobilized in the mineralizing nodule. Three apparent mechanisms for embedding of Dmp1-Cre/tdTomato-positive cells were observed. In some cases, a previously motile Dmp1-Cre/tdTomato-positive cell became immobilized in collagen fibril networks that were newly assembled around the cell, thereby entrapping it. In other cases, a motile Dmp1-Cre/tdTomato-positive cell moved into an already formed "collagen lacuna," arrested its motility and became embedded. Alternatively, some cells switched on tdTomato expression in situ within a lacuna. These data provide new insight into the dynamic process of bone collagen assembly and suggest multiple mechanisms for osteocyte entrapment in collagen matrix.

How Boundaries Form: Linked Nonautonomous Feedback Loops Regulate Pattern Formation in Yeast Colonies.

Under conditions in which budding yeast form colonies and then undergo meiosis/sporulation, the resulting colonies are organized such that a sharply defined layer of meiotic cells overlays a layer of unsporulated cells termed "feeder cells." This differentiation pattern requires activation of both the Rlm1/cell-wall integrity pathway and the Rim101/alkaline-response pathway. In the current study, we analyzed the connection between these two signaling pathways in regulating colony development by determining expression patterns and cell-autonomy relationships. We present evidence that two parallel cell-nonautonomous positive-feedback loops are active in colony patterning, an Rlm1-Slt2 loop active in feeder cells and an Rim101-Ime1 loop active in meiotic cells. The Rlm1-Slt2 loop is expressed first and subsequently activates the Rim101-Ime1 loop through a cell-nonautonomous mechanism. Once activated, each feedback loop activates the cell fate specific to its colony region. At the same time, cell-autonomous mechanisms inhibit ectopic fates within these regions. In addition, once the second loop is active, it represses the first loop through a cell-nonautonomous mechanism. Linked cell-nonautonomous positive-feedback loops, by amplifying small differences in microenvironments, may be a general mechanism for pattern formation in yeast and other organisms.

A Novel Osteogenic Cell Line That Differentiates Into GFP-Tagged Osteocytes and Forms Mineral With a Bone-Like Lacunocanalicular Structure.

Osteocytes, the most abundant cells in bone, were once thought to be inactive, but are now known to have multifunctional roles in bone, including in mechanotransduction, regulation of osteoblast and osteoclast function and phosphate homeostasis. Because osteocytes are embedded in a mineralized matrix and are challenging to study, there is a need for new tools and cell models to understand their biology. We have generated two clonal osteogenic cell lines, OmGFP66 and OmGFP10, by immortalization of primary bone cells from mice expressing a membrane-targeted GFP driven by the Dmp1-promoter. One of these clones, OmGFP66, has unique properties compared with previous osteogenic and osteocyte cell models and forms 3-dimensional mineralized bone-like structures, containing highly dendritic GFP-positive osteocytes, embedded in clearly defined lacunae. Confocal and electron microscopy showed that structurally and morphologically, these bone-like structures resemble bone in vivo, even mimicking the lacunocanalicular ultrastructure and 3D spacing of in vivo osteocytes. In osteogenic conditions, OmGFP66 cells express alkaline phosphatase (ALP), produce a mineralized type I collagen matrix, and constitutively express the early osteocyte marker, E11/gp38. With differentiation they express osteocyte markers, Dmp1, Phex, Mepe, Fgf23, and the mature osteocyte marker, Sost. They also express RankL, Opg, and Hif1α, and show expected osteocyte responses to PTH, including downregulation of Sost, Dmp1, and Opg and upregulation of RankL and E11/gp38. Live cell imaging revealed the dynamic process by which OmGFP66 bone-like structures form, the motile properties of embedding osteocytes and the integration of osteocyte differentiation with mineralization. The OmGFP10 clone showed an osteocyte gene expression profile similar to OmGFP66, but formed less organized bone nodule-like mineral, similar to other osteogenic cell models. Not only do these cell lines provide useful new tools for mechanistic and dynamic studies of osteocyte differentiation, function, and biomineralization, but OmGFP66 cells have the unique property of modeling osteocytes in their natural bone microenvironment. © 2019 American Society for Bone and Mineral Research.

Live Imaging of Type I Collagen Assembly Dynamics in Osteoblasts Stably Expressing GFP and mCherry-Tagged Collagen Constructs.

Type I collagen is the most abundant extracellular matrix protein in bone and other connective tissues and plays key roles in normal and pathological bone formation as well as in connective tissue disorders and fibrosis. Although much is known about the collagen biosynthetic pathway and its regulatory steps, the mechanisms by which it is assembled extracellularly are less clear. We have generated GFPtpz and mCherry-tagged collagen fusion constructs for live imaging of type I collagen assembly by replacing the α2(I)-procollagen N-terminal propeptide with GFPtpz or mCherry. These novel imaging probes were stably transfected into MLO-A5 osteoblast-like cells and fibronectin-null mouse embryonic fibroblasts (FN-null-MEFs) and used for imaging type I collagen assembly dynamics and its dependence on fibronectin. Both fusion proteins co-precipitated with α1(I)-collagen and remained intracellular without ascorbate but were assembled into α1(I) collagen-containing extracellular fibrils in the presence of ascorbate. Immunogold-EM confirmed their ultrastuctural localization in banded collagen fibrils. Live cell imaging in stably transfected MLO-A5 cells revealed the highly dynamic nature of collagen assembly and showed that during assembly the fibril networks are continually stretched and contracted due to the underlying cell motion. We also observed that cell-generated forces can physically reshape the collagen fibrils. Using co-cultures of mCherry- and GFPtpz-collagen expressing cells, we show that multiple cells contribute collagen to form collagen fiber bundles. Immuno-EM further showed that individual collagen fibrils can receive contributions of collagen from more than one cell. Live cell imaging in FN-null-MEFs expressing GFPtpz-collagen showed that collagen assembly was both dependent upon and dynamically integrated with fibronectin assembly. These GFP-collagen fusion constructs provide a powerful tool for imaging collagen in living cells and have revealed novel and fundamental insights into the dynamic mechanisms for the extracellular assembly of collagen. © 2018 American Society for Bone and Mineral Research.

Osteocytes Acidify Their Microenvironment in Response to PTHrP In Vitro and in Lactating Mice In Vivo.

Osteocytes appear to mobilize calcium within minutes in response to PTH injections; we have previously shown that osteocytes remove their perilacunar matrix during lactation through activation of the PTH type 1 receptor. Mechanisms utilized by osteocytes to mobilize calcium are unknown but we hypothesized that the molecular components may be similar to those used by osteoclasts. Here we show, using IDG-SW3 cells that ATP6V0D2, an essential component of vacuolar ATPase in osteoclasts, and other genes associated with osteoclastic bone resorption, increase with osteoblast to osteocyte differentiation. Furthermore, PTHrP increases ATP6V0D2 expression and induces proton generation by primary osteocytes, which is blocked by bafilomycin, a vacuolar ATPase inhibitor. These in vitro proton measurements raised the question of osteocyte viability in an acidic environment. Interestingly, osteocytes, showed enhanced viability at pH as low as 5 compared to osteoblasts and fibroblasts in vitro. To study in vivo acidification by osteocytes, virgin and lactating CD1 mice on a low calcium diet were injected with the pH indicator dye, acridine orange, and their osteocyte lacuno-canalicular system imaged by confocal microscopy. Lower pH was observed in lactating compared to virgin animals. In addition, a novel transgenic mouse line with a topaz variant of green fluorescent protein (GFPtpz)-tagged collagen α2(I) chain was used. Instead of the expected reduction in GFP-fluorescence only in the perilacunar matrix, reduced fluorescence was observed in the entire bone matrix of lactating mice. Based on our experiments showing quenching of GFP in vitro, we propose that the observed reduction in GFP fluorescence in lactating mice is due to quenching of GFP by the acidic pH generated by osteocytes. Together these findings provide novel mechanistic insight into how osteocytes remove calcium from their perilacunar/pericanalicular matrices through active acidification of their microenvironment and show that osteocytes, like osteoclasts, are resistant to the negative effects of acid on viability. © 2017 American Society for Bone and Mineral Research.

Shrinking Daughters: Rlm1-Dependent G1/S Checkpoint Maintains Saccharomyces cerevisiae Daughter Cell Size and Viability.

The Rlm1 transcription factor is a target of the cell wall integrity pathway. We report that an rlm1Δ mutant grown on a nonfermentable carbon source at low osmolarity forms cell groups in which a mother cell is surrounded by smaller "satellite-daughter" cells. Mother cells in these groups progressed through repeated rounds of cell division with normal rates of bud growth and genetic stability; however, these cells underwent precocious START relative to wild-type mothers. Thus, once activated, Rlm1 delays the transition from G1 to S, a mechanism we term the cell wall/START (CW/START) checkpoint. The rlm1Δ satellite-cell phenotype is suppressed by deletion of either SLT2, which encodes the kinase that activates Rlm1, or SWI4, which is also activated by Slt2; suggesting that Slt2 can have opposing roles in regulating the START transition. Consistent with an Rlm1-dependent CW/START checkpoint, rlm1Δ satellite daughters were unable to grow or divide further even after transfer to rich medium, but UV irradiation in G1 could partially rescue rlm1Δ satellite daughters in the next division. Indeed, after cytokinesis, these satellite daughters shrank rapidly, displayed amorphous actin staining, and became more permeable. As a working hypothesis, we propose that duplication of an "actin-organizing center" in late G1 may be required both to progress through START and to reestablish the actin cytoskeleton in daughter cells.

Degeneration of the osteocyte network in the C57BL/6 mouse model of aging.

Age-related bone loss and associated fracture risk are major problems in musculoskeletal health. Osteocytes have emerged as key regulators of bone mass and as a therapeutic target for preventing bone loss. As aging is associated with changes in the osteocyte lacunocanalicular system, we focused on the responsible cellular mechanisms in osteocytes. Bone phenotypic analysis was performed in young-(5mo) and aged-(22mo) C57BL/6 mice and changes in bone structure/geometry correlated with alterations in osteocyte parameters determined using novel multiplexed-3D-confocal imaging techniques. Age-related bone changes analogous to those in humans were observed, including increased cortical diameter, decreased cortical thickness, reduced trabecular BV/TV and cortical porosities. This was associated with a dramatic reduction in osteocyte dendrite number and cell density, particularly in females, where osteocyte dendricity decreased linearly from 5, 12, 18 to 22mo and correlated significantly with cortical bone parameters. Reduced dendricity preceded decreased osteocyte number, suggesting dendrite loss may trigger loss of viability. Age-related degeneration of osteocyte networks may impair bone anabolic responses to loading and gender differences in osteocyte cell body and lacunar fluid volumes we observed in aged mice may lead to gender-related differences in mechanosensitivity. Therapies to preserve osteocyte dendricity and viability may be beneficial for bone health in aging.

Deletion of Rac in Mature Osteoclasts Causes Osteopetrosis, an Age-Dependent Change in Osteoclast Number, and a Reduced Number of Osteoblasts In Vivo.

Rac1 and Rac2 are thought to have important roles in osteoclasts. Therefore, mice with deletion of both Rac1 and Rac2 in mature osteoclasts (DKO) were generated by crossing Rac1(flox/flox) mice with mice expressing Cre in the cathepsin K locus and then mating these animals with Rac2(-/-) mice. DKO mice had markedly impaired tooth eruption. Bone mineral density (BMD) was increased 21% to 33% in 4- to 6-week-old DKO mice at all sites when measured by dual-energy X-ray absorptiometry (DXA) and serum cross-linked C-telopeptide (CTx) was reduced by 52%. The amount of metaphyseal trabecular bone was markedly increased in DKO mice, but the cortices were very thin. Spinal trabecular bone mass was increased. Histomorphometry revealed significant reductions in both osteoclast and osteoblast number and function in 4- to 6-week-old DKO animals. In 14- to 16-week-old animals, osteoclast number was increased, although bone density was further increased. DKO osteoclasts had severely impaired actin ring formation, an impaired ability to generate acid, and reduced resorptive activity in vitro. In addition, their life span ex vivo was reduced. DKO osteoblasts expressed normal differentiation markers except for the expression of osterix, which was reduced. The DKO osteoblasts mineralized normally in vitro, indicating that the in vivo defect in osteoblast function was not cell autonomous. Confocal imaging demonstrated focal disruption of the osteocytic dendritic network in DKO cortical bone. Despite these changes, DKO animals had a normal response to treatment with once-daily parathyroid hormone (PTH). We conclude that Rac1 and Rac2 have critical roles in skeletal metabolism.

Novel approaches for two and three dimensional multiplexed imaging of osteocytes.

Although osteocytes have historically been viewed as quiescent cells, it is now clear that they are highly active cells in bone and play key regulatory roles in diverse skeletal functions, including mechanotransduction, phosphate homeostasis and regulation of osteoblast and osteoclast activity. Three dimensional imaging of embedded osteocytes and their dendritic connections within intact bone specimens can be quite challenging and many of the currently available methods are actually imaging the lacunocanalicular network rather than the osteocytes themselves. With the explosion of interest in the field of osteocyte biology, there is an increased need for reliable ways to image these cells in live and fixed bone specimens. Here we report the development of reproducible methods for 2D and 3D imaging of osteocytes in situ using multiplexed imaging approaches in which the osteocyte cell membrane, nucleus, cytoskeleton and extracellular matrix can be imaged simultaneously in various combinations. We also present a new transgenic mouse line expressing a membrane targeted-GFP variant selectively in osteocytes as a novel tool for in situ imaging of osteocytes and their dendrites in fixed or living bone specimens. These methods have been multiplexed with a novel method for labeling of the lacunocanalicular network using fixable dextran, which enables aspects of the osteocyte cell structure and lacunocanalicular system to be simultaneously imaged. The application of these comprehensive approaches for imaging of osteocytes in situ should advance research into osteocyte biology and function in health and disease.