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Experimental models and measurements to study cardiovascular physiology
Published in Neil Herring, David J. Paterson, Levick's Introduction to Cardiovascular Physiology, 2018
Neil Herring, David J. Paterson
Fluorescence imaging as described in Section 19.3 with regard to single cells, can also be used in multicellular preparations using optical mapping techniques as pioneered by the Salama group. However, several limitations are particularly relevant to this approach in multicellular tissue. The first arises from the method of tissue perfusion, which may cause uneven loading of the dye throughout the tissue. The second arises in the mechanical restraint required to focus on a large area of curved tissue that may be spontaneously beating. This can be done via mechanical restraint for example, compressing a glass slide over a beating heart, or pharmacologically with a mechanical uncoupling agent such as blebbistatin, a small molecule inhibitor of myosin II. Blebbistatin will remove any mechano-electric feedback from the heart and may also directly interfere with cardiac electrophysiolog y, although this is controversial. Tissues, such as the epicardial surface of the heart, can also be loaded with more than one dye so that several parameters (membrane voltage, cytoplasmic calcium or SR Ca2+) can be monitored with high temporal and spatial resolution across the tissue. This is particularly useful for the study of cardiac arrhythmias where intracellular Ca2+ handling events can be related to the initiation and propagation of electrical waves of excitation.
Red Blood Cell and Platelet Mechanics
Published in Michel R. Labrosse, Cardiovascular Mechanics, 2018
Clot retraction (contraction, or shrinking of a blood clot) is necessary to promote wound closure, secure hemostasis, and prevent thrombotic occlusion of a vessel (Figure 8.12b). It was reported that clot retraction (duration in an in vitro assay: 20–120 min) is a multistep process that can be divided into three phases: initiation of contraction, linear contraction, and mechanical stabilization (Tutwiler et al. 2016). In Phase 1, the initiation of clot contraction is mediated by platelet activation (~2 min), fibrin network formation, platelet–fibrin binding, and start of platelet contraction. Phase 2 consists of the continuation of the latter, along with fibrin network remodeling. Finally, fibrin crosslinking via coagulation factor XIIIa causes mechanical stabilization (Phase 3). Taken together, thrombin, high platelet counts, platelet–fibrin binding, fibrin crosslinking, and platelet contraction support clot retraction, whereas high fibrinogen concentration, high hematocrit, and increased RBC mechanical interference limit clot retraction (Tutwiler et al. 2016). The importance of platelets in clot retraction is corroborated by a study in which the elasticities of platelet-rich clots and platelet-free clots were determined to be 600 Pa and 70 Pa, respectively (Jen and McIntire 1982, Carr 2003). Single platelets can generate high contraction forces, ranging from 1.5 to 79 nN, form adhesions stronger than 70 nN, have an elasticity of 10 kPa after contraction, and show an extensibility mean of about 1.57 before rupture from a fibrinogen-coated surface (Lam et al. 2011). However, it has to be noted that different adhesion force values can be obtained, depending on the coated matrix (Nguyen et al. 2016). Platelets can generate higher stall forces when exposed to a stiffer microenvironment. Thus, platelets can stiffen fibrin fibers, which contribute to the stiffening of the whole clot (Figure 8.13) (Lam et al. 2011). Moreover, considerable evidence has accumulated, suggesting that the actin–myosin complex is a crucial component for clot compaction. Platelets treated with the cell-permeable and selective drug Y-27632 to inhibit the Rho-associated, coiled-coil-containing protein kinase (ROCK), ML-7 to inhibit the myosin light-chain kinase (MLCK), and blebbistatin to inhibit the myosin ATPase activity generated strongly reduced platelet forces, as quantified by deflection of microposts (Feghhi et al. 2016). Similar observations were made using platelets from patients with the bleeding disorders Wiskott–Aldrich syndrome (WAS) and MYH9-related disease, in which the platelet cytoskeletal machinery is affected. Here, significantly lower contraction forces on soft and stiff environments were measured for platelets from patients than for platelets from healthy volunteers. Interestingly, a larger subpopulation of platelets from these patients showed almost no contractile force on the stiff environment (Myers et al. 2017). These data suggest that defects in mechanical properties of platelets can translate into increased bleeding risk.
Cell-cell junctions in developing and adult tendons
Published in Tissue Barriers, 2020
Sophia K. Theodossiou, Jett B. Murray, Nathan R. Schiele
The organization of embryonic tendon cells appears unique, with highly ordered cells tightly packed and aligned to the long-axis of the tendon, as observed in E13 chick metatarsal tendon, and E15.5 mouse tail tendon.37 A different study found that cells in chick calcaneal tendon from HH34 to HH37 possessed a highly aligned and well-organized actin cytoskeleton network, with actin filaments that appeared continuous between cells (Figure 1a).13 When the actin cytoskeleton of the embryonic tendon cells was disrupted with blebbistatin, a small molecule inhibitor of non-muscle myosin contraction, the elastic modulus of the tendon decreased significantly, suggesting that the cells contribute to the embryonic tendon mechanical properties.13 Overall, embryonic tendon is highly cellular, with a well-organized and apparently interconnected network of cells. While inherent differences between avian and mammalian tendon development may exist, embryonic tendons across species as diverse as chick, mouse, and horse appear to share the characteristics of high cellularity, alignment, and direct cell-cell contact.37,38 Given this high cellularity and cell alignment, cell-cell junction proteins are likely candidates as regulators of embryonic tendon cell organization. Therefore, we discuss the cell-cell junction proteins (mainly cadherins and connexins) that have been identified in developing tendons and explore their potential roles in tendon development.
The platelet shape change: biophysical basis and physiological consequences
Published in Platelets, 2019
Alexander E. Moskalensky, Alena L. Litvinenko
The left-hand side of Eq. (1) can be increased by the rise of the cortical tension σ. Platelet activation results in the increase in phosphorylation of myosin light chains (MLCs), which enhance actin-myosin interaction [26]. This is achieved by both calcium-dependent and calcium-independent pathways[27]. The latter is mediated by Rho-kinase, which inhibits MLC-phosphatase. On the other hand, the rise of [Ca2+]i results in the activation of MLC-kinase through calmodulin. This drive an isotropic contraction around the platelet [28], resulting in a uniform increase of the cortical tension and the centralization of granules [29]. Using blebbistatin, Johnson et al. [30] showed that the shape change could occur if myosin activity is blocked, while granule centralization is inhibited in this case. It implies that the increase of the cortical tension is not always sufficient for disk-to-sphere transformation. The contact angle α also influences the left-hand side of Eq.(1). The projection of cortical tension force which compress microtubules ring to the center is maximal if α = 0, i.e., cell shape is discoid. Platelets might control the contact angle by tuning cell volume, but there is no reliable data on this topic [31]. White and co-workers [14] reported a two- to four-fold increase of surface area, but they studied spread or dendritic forms. During cell sphering the surface area may decrease, storing the membrane in wrinkles for the subsequent spreading or the pseudopodia formation.
Rho GTPases and their downstream effectors in megakaryocyte biology
Published in Platelets, 2019
Irina Pleines, Deya Cherpokova, Markus Bender
The role of RhoA signaling in PPF has been extensively investigated. Studies using CD34+-derived human MKs demonstrated increased PPF upon overexpression of dominant-negative RhoA, whereas overexpression of an active RhoA resulted in decreased PPF (53,54). In line with RhoA as a negative regulator of PPF, unspecific inhibition of its downstream effectors ROCK1/2 (by Y27362) or myosin II (by blebbistatin) enhanced PPF in vitro (53,54). Consistently, Pf4-Cre-induced deficiency of Myh9 which encodes the non-muscle myosin IIA heavy chain (NMM-IIa) led to increased PPF of cultured MKs in vitro (54,55); however, NMM-IIa-deficient MKs exhibited reduced PPF in situ (56) or when grown in a 3D-culture system (57). Besides the multiple intrinsic MK defects (55,58), it is thus likely that a PPF defect contributed to the pronounced macrothrombocytopenia in mice with MK-specific MYH9 deficiency. Dominant macrothrombocytopenia is also characteristic of patients with MYH9-related disorders (MYH9-RD) typically caused by autosomal-dominant mutations in the MYH9 gene. However, these mutations result, at least in part, in a gain of function phenotype, and also CD34+ cell-derived MKs isolated from MYH9-RD patients exhibited reduced PPF (59). Together, these findings emphasize that the tight control of NMM-IIa expression and activity is critical for proper platelet production.