Cellular Contraction Research Articles

Modulation by thyroid hormone of myosin light chain phosphorylation and aquaporin 5 protein expression in intact lung.

Myosin light chain kinase (MLCK) may play a key role in cellular contraction, paracellular permeability and lung water homeostasis. In vitro, thyroid hormones (THs) potently inhibit MLCK activation and, hence, MLC phosphorylation. Whether similar effect is exerted by THs in in vivo systems is not known. Therefore, we investigated the effects of hypothyroid (HO) and hyperthyroid (HR) states on the level of phospho-MLC, aquaporin 5 (AQP5) protein expression, and water holding capacity in the rat lung. Alterations in thyroid state were induced by adding methimazole or levothyroxine (L-T4) to animals' drinking water. Serum TH concentration and thyroid gland histomorphology were assessed to verify the onset of the thyroid state. Lung phospho-MLC and AQP5 proteins were assessed by Western blotting and immunohistochemistry. Lung extravascular water content was estimated by the tissue wet weight-to-dry weight (W/D) ratio. The HO state induced significant increases in the expression of lung phospho-MLC and AQP5 proteins. In contrast, the HR state caused moderate decreases in lung phospho-MLC and AQP5 proteins. While lung water holding capacity was significantly increased in HO animals, it was significantly reduced in HR animals. The data of this study show that THs are able to modulate MLC phosphorylation in in vivo systems. Besides, they suggest that the circulating level of THs may alter lung fluid balance not only through expression of water channels but also through regulation of cellular contraction and paracellular permeability.

Effects of hydrotalcites and tris (1-chloro-2-propyl) phosphate on thermal stability, cellular structure and fire resistance of isocyanate-based polyimide foams

Isocyanate-based polyimide foams (PIFs) with different dosages of liquid tri (1-chloro-2-propyl) phosphate (TCPP) and micro-sized hydrotalcites (LDHs) particles alone, as well as different mixing ratios of TCPP and LDHs, were prepared via a one-step process in this work. Limiting oxygen index (LOI) and cone calorimeter test (CCT) results indicated that TCPP exhibited more pronounced flame retardant efficiency than LDHs for isocyanate-based PIFs. However, scanning electron microscopy images and digital macrostructural images results showed that, in contrast to LDHs, when the dosage of TCPP exceeded 10% it caused a clear cracking effects on the macro-cellular structure and opening cell effects on the micro-cellular structure for isocyanate-based PIFs. Because the dramatically volatilization of TCPP during the postcuring process caused obvious cellular contraction in the isocyanate-based PIFs. Meanwhile, the use of TCPP also obviously decreased the thermal stability of isocyanate-based PIFs unlike LDHs. However, when these two flame retardants were used in combination, they could effectively enhance the fire resistance and ensure macro- and micro-cellular structures of the isocyanate-based PIFs, unlike standalone use of TCPP and LDHs. When 10% TCPP was simultaneously used with 10% LDHs, the macro- and micro-cellular structures of the resultant foams were clearly improved compared with foams prepared using TCPP only. These results were believed to be attributable to LDHs dispersion in the foams, which enhanced the strength of cellular windows and skeletons, then restrained the cell contraction. Compared with foams without flame retardants, the fire resistance of the isocyanate-based PIFs prepared with 10% TCPP and 10% LDHs was obviously enhanced; specially, the LOI was enhanced by 29.4%, and the peak of heat release rate (PHRR) decreased by 36.1%. Thus, the use of liquid and solid flame retardants in combination may effectively yield isocyanate-based PIFs with high quality cellular structures and excellent fire resistance.

The effect of Rac1 inhibition to retinal pigment epithelial cellular behavior change induced by transforming growth factor β

Objective To study the role of Rac1 in the epithelial-mesenchymal transition (EMT) process of retinal pigment epithelial cells (RPE) induced by transforming growth factor β (TGF-β). Methods Human ARPE-19 cells were divided into 4 groups including control group, TGF-β group, TGF-β + NSC23766 group, NSC23766 group. NSC23766 was added to medium 2 hours before TGF-β treatment to block the Rac1 receptors. α-smooth muscle actin (α-SMA) expression was measured by immunofluorescence and Western blot. Cell scratch assay, invasion assay and gel contraction experiments were used to measure cell migration, invasion, cell contraction. Results The expression of α-SMA was higher in TGF-β group, compared with the control group, TGF-β + NSC23766 group (F=825.314, P<0.05). Cell scratch assay showed that the cellular gap was less in GF-β group, compared with the control group, TGF-β + NSC23766 group, NSC23766 group (F=177.351, P<0.05). Cell invasion assay showed that, the number of cells pass through the fiber membrane was the same in TGF-β group and other 3 groups (F=0.371, P=0.055). Gel contraction assay showed that TGF-β can promote the cellular contraction, compare to the control group, TGF-β + NSC23766 group, NSC23766 group, the difference was statistically significant (F=40.473, P<0.05). Conclusion Rac1 play a role in TGF-β-induced behavioral changes of RPE cells; NSC23766 inhibit RPE cellular behavior change by regulating Rac1 activation. Key words: Retinal pigment epithelium; Cell transdifferentiation; Transforming growth factor beta; rac1 GTP-binding protein

Thermal energy enhances cell-mediated contraction of lax rat tail tendon fascicles following exercise

the application of thermal energy (TE) has shown promise in the treatment of tendinopathy. However, the precise mechanism(s) of action of this therapy is unclear. The loss of tendon cell homeostatic tension, due to loading-induced laxity, produces catabolic changes associated with tendinopathy. This catabolic activity can be inhibited through the re-establishment of a normal tensile environment via a cellular contraction mechanism. We hypothesized that application of TE will enhance the contraction rate of lax rat tail tendon fascicles (RTTfs) in an in vitro model. following loading, 10 lax RTTfs from each mature rat (n=5) were treated once daily for 7 days with TE by replacing the culture media at 37°C (control) with 42°C media. Using calibrated photographs, RTTf lengths were measured daily. Additional RTTfs were utilized to investigate any changes in material (n=12) and/or histological (n=12) properties with TE. TE significantly increased the contraction rate of RTTfs (p>0.001) without altering the material or histological properties. these results demonstrate that TE significantly enhances the contraction rate of previously exercised tendons. The ability to more quickly re-establish a normal mechanobiological environment, thus minimizing any catabolic changes, may explain the beneficial effects reported with applied TE in tendinopathy treatment.

Coupling of Apical Contractions and Adherens Junction Maturation by Synaptopodin-Depedent Recruitment of A-Actinin-4

Cell-cell adhesive contact is a central player in mechanotransduction, providing both mechanical support and a hub for signaling. Yet, the molecular mechanism is poorly defined, partly due to a lack of cell culture model to mechanically manipulate tension at cell-cell contacts. Here, using live cell imaging of α-actinin, we showed that MDCK cells exhibit many cellular behaviors of epithelial cells in vivo, including (1) oscillating contractility of the adherens junction, (2) pulsatile centripetal cellular contractions, and (3) ratchet constrictions of apical junctional domain. We hypothesized that pulsatile contractions participate in the maturation of junctional complexes. To test our hypothesis, we applied cyclic pulsatile tension to cell-cell contacts in confluent cell monolayers. We found that pulsatile intercellular tension induces recruitment of α-actinin-4 to the adherens junction in a time and tension-dependent manner without changing the localization of canonical adherens junction proteins, suggesting that a missing factor is involved. We have identified this missing factor as synaptopodin. Synaptopodin recruitment to the adherens junction is also tension-sensitive, which is necessary for generation of contractility at cell-cell adhesions. Recruitment of a-actinin-4 strengthens cell-cell adhesion and promote epithelial permeability barrier. Thus, by controlling the mechanical force generated through apical contractility, epithelial cells can adjust the strength of cell-cell adhesion and the maturation process of adherens junction maturation. Conversely, synaptopodin dictates the efficiency of mechanical-biochemical coupling between contractility and junction assembly through the recruitment of a-actinin-4. Our studies reveal a tunable molecular system with both mechanical and biochemical inputs and underscore the complexity of junction assembly in epithelial monolayers in vivo and in cultured cell systems.

MiR-155 augments CD8+ T-cell antitumor activity in lymphoreplete hosts by enhancing responsiveness to homeostatic γc cytokines

Lymphodepleting regimens are used before adoptive immunotherapy to augment the antitumor efficacy of transferred T cells by removing endogenous homeostatic "cytokine sinks." These conditioning modalities, however, are often associated with severe toxicities. We found that microRNA-155 (miR-155) enabled tumor-specific CD8(+) T cells to mediate profound antitumor responses in lymphoreplete hosts that were not potentiated by immune-ablation. miR-155 enhanced T-cell responsiveness to limited amounts of homeostatic γc cytokines, resulting in delayed cellular contraction and sustained cytokine production. miR-155 restrained the expression of the inositol 5-phosphatase Ship1, an inhibitor of the serine-threonine protein kinase Akt, and multiple negative regulators of signal transducer and activator of transcription 5 (Stat5), including suppressor of cytokine signaling 1 (Socs1) and the protein tyrosine phosphatase Ptpn2. Expression of constitutively active Stat5a recapitulated the survival advantages conferred by miR-155, whereas constitutive Akt activation promoted sustained effector functions. Our results indicate that overexpression of miR-155 in tumor-specific T cells can be used to increase the effectiveness of adoptive immunotherapies in a cell-intrinsic manner without the need for life-threatening, lymphodepleting maneuvers.

Gene expression analysis of uterine smooth muscle cells exposed to bisphenol A

To evaluate the effect of bisphenol A (BPA) on gene expression changes in uterine myometrium, uterine smooth muscle cells were incubated with 2.28 ng/mL BPA for 48 h. Total RNA was extracted and quantified spectrophotometrically. Gene expression changes were analyzed using oligonucleotide microarray hybridization comparing the treated and control groups (1.5-fold change, p<0.05). Data indicated that 560 genes were upregulated and 231 were downregulated in response to BPA exposure. The biological processes positively associated with gene upregulation included the regulation of cell differentiation, cellular metabolic processes, cell proliferation and smooth muscle contraction. In contrast, genes involved in mitosis, lipid metabolism, regulation of muscle cell differentiation and cell adhesion were significantly downregulated. In the KEGG pathway analysis, MAPK signaling pathway, insulin signaling pathway and extracellular matrix (ECM)-mediated interactions were positively associated with gene upregulation.

Superresolution Modeling of Calcium Release in the Heart

Stable calcium-induced calcium release (CICR) is critical for maintaining normal cellular contraction during cardiac excitation-contraction coupling. The fundamental element of CICR in the heart is the calcium (Ca2+) spark, which arises from a cluster of ryanodine receptors (RyR). Opening of these RyR clusters is triggered to produce a local, regenerative release of Ca2+ from the sarcoplasmic reticulum (SR). The Ca2+ leak out of the SR is an important process for cellular Ca2+ management, and it is critically influenced by spark fidelity, i.e., the probability that a spontaneous RyR opening triggers a Ca2+ spark. Here, we present a detailed, three-dimensional model of a cardiac Ca2+ release unit that incorporates diffusion, intracellular buffering systems, and stochastically gated ion channels. The model exhibits realistic Ca2+ sparks and robust Ca2+ spark termination across a wide range of geometries and conditions. Furthermore, the model captures the details of Ca2+ spark and nonspark-based SR Ca2+ leak, and it produces normal excitation-contraction coupling gain. We show that SR luminal Ca2+-dependent regulation of the RyR is not critical for spark termination, but it can explain the exponential rise in the SR Ca2+ leak-load relationship demonstrated in previous experimental work. Perturbations to subspace dimensions, which have been observed in experimental models of disease, strongly alter Ca2+ spark dynamics. In addition, we find that the structure of RyR clusters also influences Ca2+ release properties due to variations in inter-RyR coupling via local subspace Ca2+ concentration ([Ca2+]ss). These results are illustrated for RyR clusters based on super-resolution stimulated emission depletion microscopy. Finally, we present a believed-novel approach by which the spark fidelity of a RyR cluster can be predicted from structural information of the cluster using the maximum eigenvalue of its adjacency matrix. These results provide critical insights into CICR dynamics in heart, under normal and pathological conditions.

Long-Range Force Transmission in Fibrous Matrices Enabled by Tension-Driven Alignment of Fibers

Cells can sense and respond to mechanical signals over relatively long distances across fibrous extracellular matrices. Recently proposed models suggest that long-range force transmission can be attributed to the nonlinear elasticity or fibrous nature of collagen matrices, yet the mechanism whereby fibers align remains unknown. Moreover, cell shape and anisotropy of cellular contraction are not considered in existing models, although recent experiments have shown that they play crucial roles. Here, we explore all of the key factors that influence long-range force transmission in cell-populated collagen matrices: alignment of collagen fibers, responses to applied force, strain stiffening properties of the aligned fibers, aspect ratios of the cells, and the polarization of cellular contraction. A constitutive law accounting for mechanically driven collagen fiber reorientation is proposed. We systematically investigate the range of collagen-fiber alignment using both finite-element simulations and analytical calculations. Our results show that tension-driven collagen-fiber alignment plays a crucial role in force transmission. Small critical stretch for fiber alignment, large fiber stiffness and fiber strain-hardening behavior enable long-range interaction. Furthermore, the range of collagen-fiber alignment for elliptical cells with polarized contraction is much larger than that for spherical cells with diagonal contraction. A phase diagram showing the range of force transmission as a function of cell shape and polarization and matrix properties is presented. Our results are in good agreement with recent experiments, and highlight the factors that influence long-range force transmission, in particular tension-driven alignment of fibers. Our work has important relevance to biological processes including development, cancer metastasis, and wound healing, suggesting conditions whereby cells communicate over long distances.

Abstract 3160: ROCK inhibition promotes microtentacle formation and reattachment of breast cancer cells

Abstract Circulating Tumor Cells (CTCs) predict poor patient outcome and increased CTC detection is correlated with higher risk of metastasis. The efficiency of cancers to metastasize depends on the survival and reattachment of CTCs in distant tissues. Our lab has demonstrated the significance of microtubule based cellular extensions, termed microtentacles, in enhancing CTC aggregation and reattachment to the endothelial wall. Microtentacles (McTNs) are highly dynamic protrusions formed in free-floating suspended cells and occur at higher frequencies in more metastatic cell lines. Recently, there has been interest generated in using ROCK as a therapeutic target in cancers based on emerging data that over-activation of Rho/ROCK pathway plays a role in tumor development and metastasis. ROCK inhibitors are being considered for clinical trials because of their pre-clinical success in reducing tumor cell migration, invasion and metastasis. However, the effect of Rho/ROCK inhibition on CTC reattachment and metastasis has not been well established. ROCK promotes cellular contraction by phosphorylating non-muscle myosin II (NMII), allowing it to crosslink actin, as well as by phosphorylating and inhibiting the activity of Myosin Light Chain Phosphatase (MLCP). In a normal, noncancerous cell, the outward expansion of microtubules is balanced by the inward contraction of the actin cortex. ROCK inhibition decreases cell contractility and shifts the balance towards a looser, more relaxed actin cortex that is unable to contain the outward growth of microtubules. Western blot analysis of attached and suspended cells treated with a ROCK inhibitor showed a decrease in phosphorylated MLCP and NMII in the breast cancer cell lines, BT549 and Hs578T. Treatment with ROCK inhibitor significantly increased the number of cells expressing McTNs. The increase in McTNs corresponded with increased in vitro cell reattachment, measured by the xCelligence, Real-Time Cell Analyser. Knocking down ROCK1 and ROCK2 with silencing RNAs also resulted in increased McTN counts. Conversely, increasing cell contractility by treating cells with a Rho pathway activator led to a reduction in McTNs and cell attachment. These results show that ROCK inhibitors could actually promote metastasis by increasing attachment of CTCs to the vasculature. A better understanding of the effect of cancer therapies on all stages of metastasis will prevent the use of drugs that could reduce tumor growth and motility in the short-term but result in increasing circulating tumor cells and elevating metastatic potential in the long-term. Citation Format: Lekhana Bhandary, Michele I. Vitolo, Rebecca A. Bettes, Monica S. Charpentier, Amanda E. Boggs, Jana Slovic, Keyata Thompson, Stuart S. Martin. ROCK inhibition promotes microtentacle formation and reattachment of breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3160. doi:10.1158/1538-7445.AM2014-3160

Detecting calcium in cardiac muscle: fluorescence to dye for.

THE IMPORTANCE OF Ca 2 as a second messenger in general and as a mediator between electrical excitation and cellular contraction in the heart is well accepted. Accordingly, numerous Ca 2 -sensitive fluorescent dyes are available nowadays, rendering the choice of the appropriate dye for a particular application a challenge. It appears to be important to provide calibrated Ca 2 signals rather than fluorescence transients, especially when comparing data between studies. In case calibrated signals cannot be provided, then the time course of fluorescence transients should be interpreted with caution and such signals should be referred to as fluorescence transients rather than Ca 2 transients. Additionally, we discuss how major properties of the fluorescence dyes should be considered with a special emphasis on their Ca 2 dissociation properties. Furthermore, we highlight putative risks of tripping, especially in the context of multicellular recordings, and how to circumvent them. As an ubiquitous intracellular messenger, Ca 2 ions play an important role in almost all living cells (4). A particularly dynamic role has been assigned to Ca 2 in muscle cells because here they not only control gene expression and other slower signaling processes but serve as a direct transducer of the electrical excitation of the plasma membrane into the activation of the physiological response of the cell, the contraction, by activation of the contractile machinery (5). Almost 50 years ago, Jobsis and O’Connor (10) reported the measurements of intracellular Ca 2 transients in skeletal muscle using the Ca 2 -sensitive dye murexide, which changes its absorbance upon binding of Ca 2 ions. Later, these absorbance dyes were improved and an important member of this family, arsenazo III, was widely used to study intracellular Ca 2 in

The actomyosin machinery is required for Drosophila retinal lumen formation.

Multicellular tubes consist of polarized cells wrapped around a central lumen and are essential structures underlying many developmental and physiological functions. In Drosophila compound eyes, each ommatidium forms a luminal matrix, the inter-rhabdomeral space, to shape and separate the key phototransduction organelles, the rhabdomeres, for proper visual perception. In an enhancer screen to define mechanisms of retina lumen formation, we identified Actin5C as a key molecule. Our results demonstrate that the disruption of lumen formation upon the reduction of Actin5C is not linked to any discernible defect in microvillus formation, the rhabdomere terminal web (RTW), or the overall morphogenesis and basal extension of the rhabdomere. Second, the failure of proper lumen formation is not the result of previously identified processes of retinal lumen formation: Prominin localization, expansion of the apical membrane, or secretion of the luminal matrix. Rather, the phenotype observed with Actin5C is phenocopied upon the decrease of the individual components of non-muscle myosin II (MyoII) and its upstream activators. In photoreceptor cells MyoII localizes to the base of the rhabdomeres, overlapping with the actin filaments of the RTW. Consistent with the well-established roll of actomyosin-mediated cellular contraction, reduction of MyoII results in reduced distance between apical membranes as measured by a decrease in lumen diameter. Together, our results indicate the actomyosin machinery coordinates with the localization of apical membrane components and the secretion of an extracellular matrix to overcome apical membrane adhesion to initiate and expand the retinal lumen.

Superpulsed (Ga-As, 904 nm) low-level laser therapy (LLLT) attenuates inflammatory response and enhances healing of burn wounds.

Low-level laser therapy (LLLT) using superpulsed near-infrared light can penetrate deeper in the injured tissue and could allow non-pharmacological treatment for chronic wound healing. This study investigated the effects of superpulsed laser (Ga-As 904 nm, 200 ns pulse width; 100 Hz; 0.7 mW mean output power; 0.4 mW/cm(2) average irradiance; 0.2 J/cm(2) total fluence) on the healing of burn wounds in rats, and further explored the probable associated mechanisms of action. Irradiated group exhibited enhanced DNA, total protein, hydroxyproline and hexosamine contents compared to the control and silver sulfadiazine (reference care) treated groups. LLLT exhibited decreased TNF-α level and NF-kB, and up-regulated protein levels of VEGF, FGFR-1, HSP-60, HSP-90, HIF-1α and matrix metalloproteinases-2 and 9 compared to the controls. In conclusion, LLLT using superpulsed 904 nm laser reduced the inflammatory response and was able to enhance cellular proliferation, collagen deposition and wound contraction in the repair process of burn wounds. Photomicrographs showing no, absence inflammation and faster wound contraction in LLLT superpulsed (904 nm) laser treated burn wounds as compared to the non-irradiated control and silver sulfadiazine (SSD) ointment (reference care) treated wounds.

Malaria and hypertension. Another co-evolutionary adaptation?

Arterial hypertension is a complex multifactorial disease and a global public health concern. It is responsible for at least 45% of deaths due to heart disease and 51% of deaths due to stroke, adding up the tremendous number of 9.4 million deaths every year (Lim et al., 2012; World Health Organization, 2013). The renin-angiotensin-aldosterone system (RAAS) is one of the most important regulatory systems of blood volume, arterial pressure and cardiovascular homeostasis. Angiotensin II (Ang II) is the principal effector hormone of the RAAS in vascular biology, mediating effects via two main receptors: Angiotensin receptor type 1 (AT1) and type 2 (AT2) (Callera et al., 2007). When Ang II binds to AT1 on vascular smooth muscle cells, it mobilizes intracellular Ca2+, leading to cellular contraction. Sustained cellular contraction increases peripheral vascular resistance, resulting in high blood pressure (Touyz and Schiffrin, 2000). Among others, genetic factors are closely related with the development of hypertension. People with African American and South Asian genetic background have higher prevalence of hypertension compared to Caucasians, independently of their socioeconomic status (Cappuccio, 1997; Sampson et al., 2014). These differences in the ethnic background can increase the prevalence of hypertension by 2 fold until the age of 55, when the differences decline, presumably because of the interfering effect of age-related factors (Wolz et al., 2000). Several polymorphisms have been associated with higher prevalence of arterial hypertension; there are 2 polymorphisms in the Angiotensin Converting Enzyme (ACE) and Angiotensin Converting Enzyme 2 (ACE2) that lead to elevate circulating Ang II levels (Giner et al., 2000; Di Pasquale et al., 2004; Fan et al., 2007). Interestingly, these same genetic variations (the “D” allele of ACE I/D polymorphism and the ACE2 C→T substitution) have been associated with a lower incidence of cerebral malaria (CM) in Indian adults, although the later one only in women (Dhangadamajhi et al., 2010). Malaria is still a major public health problem world wide, causing approximately 600,000 deaths, mostly among African children (World Health Organization, 2012a). A high proportion of these deaths are caused by CM, a syndrome characterized by impaired consciousness, generalized convulsions, coma and neurological sequelae (Idro et al., 2005). CM is caused by the interaction between Plasmodium falciparum infected erythrocytes and host brain endothelial cells. Parasite proteins that are expressed on the surface of infected erythrocytes (PfEMP1), interact with host endothelial cell receptors (Protein C receptor, ICAM1; Newbold et al., 1997; Turner et al., 2013) leading to their sequestration within the brain microcirculation. Disruption of the blood-brain barrier is observed producing the characteristic petechiae and ring hemorrhages found on the brain of dead patients with CM (Rasti et al., 2004). Although it is still not well-established that Ang II has beneficial effects onmalaria and particularly on CM, different lines of evidence suggest a possible ‘protective’ effect that could be mediated by different, non-exclusive mechanisms that could affect parasite development and/or host susceptibility to Plasmodium-induced pathology. Recent reports have shown that angiotensin peptides can induce impairment of the erythrocytic cycle of Plasmodium, reducing the parasite growth in vitro (Maciel et al., 2008; Saraiva et al., 2011). Since the development of CM depends on the initial levels of parasitemia in mice (Amani et al., 1998) and possibly in humans (Bejon et al., 2005), this could be a potential explanation for Ang II protection from CM. Our unpublished results using mice infected with Plasmodium berghei seem to confirm that the elevation of Ang II levels results in modestly decreased parasitemias in this animal model. It is possible that Ang II could modulate malaria severity through additional mechanisms, specially, since the inhibitory effect observed in parasite growth is modest (Maciel et al., 2008; Saraiva et al., 2011). Sodium conservation could provide an alternative explanation to the protective effect of the polymorphisms associated with higher levels of Ang II. In this case, Ang II could be acting to counterbalance hyponatremia by stimulating the secretion of aldosterone from the adrenal cortex, the major regulator of Na+ reabsorption. Hyponatremia (serum sodium < 135mmol/L) is frequent in malaria patients and correlates with disease severity in imported malaria (van Wolfswinkel et al., 2010). However, other studies have shown that circulating Na+

Introduction to cell-hydrogel mechanosensing.

The development of hydrogel-based biomaterials represents a promising approach to generating new strategies for tissue engineering and regenerative medicine. In order to develop more sophisticated cell-seeded hydrogel constructs, it is important to understand how cells mechanically interact with hydrogels. In this paper, we review the mechanisms by which cells remodel hydrogels, the influence that the hydrogel mechanical and structural properties have on cell behaviour and the role of mechanical stimulation in cell-seeded hydrogels. Cell-mediated remodelling of hydrogels is directed by several cellular processes, including adhesion, migration, contraction, degradation and extracellular matrix deposition. Variations in hydrogel stiffness, density, composition, orientation and viscoelastic characteristics all affect cell activity and phenotype. The application of mechanical force on cells encapsulated in hydrogels can also instigate changes in cell behaviour. By improving our understanding of cell-material mechano-interactions in hydrogels, this should enable a new generation of regenerative medical therapies to be developed.

Acute slowing of cardiac conduction in response to myofibroblast coupling to cardiomyocytes through N-cadherin

The electrophysiological consequences of cardiomyocyte and myofibroblast interactions remain unclear, and the contribution of mechanical coupling between these two cell types is still poorly understood. In this study, we examined the time course and mechanisms by which addition of myofibroblasts activated by transforming growth factor-beta (TGF-β) influence the conduction velocity (CV) of neonatal rat ventricular cell monolayers. We observed that myofibroblasts affected CV within 30min of contact and that these effects were temporally correlated with membrane deformation of cardiomyocytes by the myofibroblasts. Expression of dominant negative RhoA in the myofibroblasts impaired both myofibroblast contraction and myofibroblast-induced slowing of cardiac conduction, whereas overexpression of constitutive RhoA had little effect. To determine the importance of mechanical coupling between these cell types, we examined the expression of the two primary cadherins in the heart (N- and OB-cadherin) at cell–cell contacts formed between myofibroblasts and cardiomyocytes. Although OB-cadherin was frequently found at myofibroblast–myofibroblast contacts, very little expression was observed at myofibroblast–cardiomyocyte contacts. The myofibroblast-induced slowing of cardiac conduction was not prevented by silencing of OB-cadherin in the myofibroblasts, and could be reversed by inhibitors of mechanosensitive channels (gadolinium or streptomycin) and cellular contraction (blebbistatin). In contrast, N-cadherin expression was commonly observed at myofibroblast–cardiomyocyte contacts, and silencing of N-cadherin in myofibroblasts prevented the myofibroblast-dependent slowing of cardiac conduction. We propose that myofibroblasts can impair the electrophysiological function of cardiac tissue through the application of contractile force to the cardiomyocyte membrane via N-cadherin junctions.

Ucp2 Modulates Cellular Excitation Contraction Coupling via Mitochondrial Calcium Uptake

Introduction: Mitochondrial Ca2+ handling modulates the spatial and temporal profile of intracellular Ca2+ signaling. It is mainly mediated by the mitochondrial calcium uniporter (MCU). Uncoupling protein-2 (UCP2) a mitochondrial membrane protein has been proposed to be fundamental for MCU activity. Whether cellular excitation contraction (EC) coupling is modulated by UCP2 remains to be determined. Methods: To investigate this scenario, we isolated intact cardiomyocytes from adult wild-type (WT) and B6.129S4-Ucp2tm1Lowl/J (UCP2-/-) mice to analyze dual Ca2+ transients from the mitochondrial ((Ca2+)m) and intracellular compartment ((Ca2+)c) in the whole cell configuration using Rhod2-AM and Fluo4 pentapotassium salt. We also tested whether the previously described inhibitory effect of ATP on mitochondrial Ca2+ uptake could be mediated by UCP2. To detect possible regulatory effects on sarcolemmal Ca2+ uptake in UCP2-/- mice, trigger Ca2+ influx was measured using Ryanodine. Results: In cardiomyocytes of WT mice (Ca2+)m was selectively decreased by Ruthenium360 (Ru360) while (Ca2+)c was upregulated. The (Ca2+)m was decreased in UCP2-/- myocytes compared to WT while (Ca2+)c was unchanged suggesting a compensational modulation of EC coupling. Trigger Ca2+ influx was reduced in UCP2-/- vs. WT myocytes, presumably to prevent cytosolic Ca2+ overload. Additionally, we observed an inhibitory effect of ATP on mitochondrial Ca2+ uptake in myocytes of WT mice while no effect was observed in UCP2-/- mice. Conclusion: Our experiments suggest a possible role of MCU and UCP2 interaction in controlling mitochondrial Ca2+ uptake as well as EC coupling.

Cell-Based Multi-Parametric Model of Cleft Progression during Submandibular Salivary Gland Branching Morphogenesis

Cleft formation during submandibular salivary gland branching morphogenesis is the critical step initiating the growth and development of the complex adult organ. Previous experimental studies indicated requirements for several epithelial cellular processes, such as proliferation, migration, cell-cell adhesion, cell-extracellular matrix (matrix) adhesion, and cellular contraction in cleft formation; however, the relative contribution of each of these processes is not fully understood since it is not possible to experimentally manipulate each factor independently. We present here a comprehensive analysis of several cellular parameters regulating cleft progression during branching morphogenesis in the epithelial tissue of an early embryonic salivary gland at a local scale using an on lattice Monte-Carlo simulation model, the Glazier-Graner-Hogeweg model. We utilized measurements from time-lapse images of mouse submandibular gland organ explants to construct a temporally and spatially relevant cell-based 2D model. Our model simulates the effect of cellular proliferation, actomyosin contractility, cell-cell and cell-matrix adhesions on cleft progression, and it was used to test specific hypotheses regarding the function of these parameters in branching morphogenesis. We use innovative features capturing several aspects of cleft morphology and quantitatively analyze clefts formed during functional modification of the cellular parameters. Our simulations predict that a low epithelial mitosis rate and moderate level of actomyosin contractility in the cleft cells promote cleft progression. Raising or lowering levels of contractility and mitosis rate resulted in non-progressive clefts. We also show that lowered cell-cell adhesion in the cleft region and increased cleft cell-matrix adhesions are required for cleft progression. Using a classifier-based analysis, the relative importance of these four contributing cellular factors for effective cleft progression was determined as follows: cleft cell contractility, cleft region cell-cell adhesion strength, epithelial cell mitosis rate, and cell-matrix adhesion strength.

β-Arrestin Regulation of Myosin Light Chain Phosphorylation Promotes AT1aR-mediated Cell Contraction and Migration

Over the last decade, it has been established that G-protein-coupled receptors (GPCRs) signal not only through canonical G-protein-mediated mechanisms, but also through the ubiquitous cellular scaffolds β-arrestin-1 and β-arrestin-2. Previous studies have implicated β-arrestins as regulators of actin reorganization in response to GPCR stimulation while also being required for membrane protrusion events that accompany cellular motility. One of the most critical events in the active movement of cells is the cyclic phosphorylation and activation of myosin light chain (MLC), which is required for cellular contraction and movement. We have identified the myosin light chain phosphatase Targeting Subunit (MYPT-1) as a binding partner of the β-arrestins and found that β-arrestins play a role in regulating the turnover of phosphorylated myosin light chain. In response to stimulation of the angiotensin Type 1a Receptor (AT1aR), MLC phosphorylation is induced quickly and potently. We have found that β-arrestin-2 facilitates dephosphorylation of MLC, while, in a reciprocal fashion, β-arrestin 1 limits dephosphorylation of MLC. Intriguingly, loss of either β-arrestin-1 or 2 blocks phospho-MLC turnover and causes a decrease in the contraction of cells as monitored by atomic force microscopy (AFM). Furthermore, by employing the β-arrestin biased ligand [Sar1,Ile4,Ile8]-Ang, we demonstrate that AT1aR-mediated cellular motility involves a β-arrestin dependent component. This suggests that the reciprocal regulation of MLC phosphorylation status by β-arrestins-1 and 2 causes turnover in the phosphorylation status of MLC that is required for cell contractility and subsequent chemotaxic motility.

A model and simulation of uterine contractions

We present a model of uterine contractions that consists of tissue mechanics, electrical activity, and intrauterine pressure. Uterine contractions were simulated to measure the impact that ± 3% changes in mechanical and electrical properties have on peak intrauterine pressure and the duration above 90% peak pressure. Peak pressure was overwhelmingly affected by changes in one mechanical property (maximum stress generated from cellular contraction), whereas duration above 90% peak pressure was overwhelmingly affected by changes in two electrical properties (the recovery rate and activation threshold of the action potential). In contrast, changes in other properties (e.g. pacemaker location and diffusion coefficient) had an impact on intrauterine pressure one order of magnitude smaller. Additionally, intrauterine pressure was strengthened by decreasing the fraction of fasciculi aligned in the longitudinal rather than the circumferential direction.