Functional activity and morphology of isolated rat cardiac mitochondria under calcium overload. Effect of naringin.

The function of mitochondria as a regulator of myocyte calcium homeostasis has been extensively discussed. The aim of the present work was further clarification of the details of modulation of the functional activity of rat cardiac mitochondria by exogenous Ca2+ ions either in the absence or in the presence of the plant flavonoid naringin. Low free Ca2+ concentrations (40-250 nM) effectively inhibited the respiratory activity of heart mitochondria, remaining unaffected the efficacy of oxygen consumption. In the presence of high exogenous Ca2+ ion concentrations (Ca2+ free was 550 µM), we observed a dramatic increase in mitochondrial heterogeneity in size and electron density, which was related to calcium-induced opening of the mitochondrial permeability transition pores (MPTP) and membrane depolarization (Ca2+free ions were from 150 to 750 µM). Naringin partially prevented Ca2+-induced cardiac mitochondrial morphological transformations (200 µM) and dose-dependently inhibited the respiratory activity of mitochondria (10-75 µM) in the absence or in the presence of calcium ions. Our data suggest that naringin (75 µM) promoted membrane potential dissipation, diminishing the potential-dependent accumulation of calcium ions by mitochondria and inhibiting calcium-induced MPTP formation. The modulating effect of the flavonoid on Ca2+-induced mitochondria alterations may be attributed to the weak-acidic nature of the flavonoid and its protonophoric/ionophoric properties. Our results show that the sensitivity of rat heart mitochondria to Ca2+ ions was much lower in the case of MPTP opening and much higher in the case of respiration inhibition as compared to liver mitochondria.

Hypertonic pressure affects the pluripotency and self-renewal of mouse embryonic stem cells.

As an important mechanical cue in the extracellular microenvironment, osmotic stress directly affects the proliferation, migration, and differentiation of cells. In this paper, we focused on the influence of hypertonic pressure on the colony morphology, stemness, and self-renew of mouse embryonic stem cells (mESCs). Our results showed that culture media with hypertonic pressure are more conducive to the maintenance of 3D colony morphology and pluripotency of mESCs after withdrawing the glycogen synthase kinase 3ß (GSK3ß) inhibitor CHIR99021 and the mitogen-activated protein kinase (MEK) inhibitor PD0325901 (hereinafter referred to as 2i) for 48 h. Furthermore, we revealed the microscopic mechanisms of the this finding: hypertonic pressure resulted in the depolymerization of F-actin cytoskeleton and limits Yes-associated protein (hereinafter referred to as YAP) transmission into the nucleus which play a vital role in the regulation of cell proliferation, and resulting in cell-cycle arrest at last.

AFM-based indentation method for measuring the relaxation property of living cells.

Probing the mechanical properties of cells is critical for understanding their deformation behaviors and biological functions. Although some methods have been proposed to characterize the elastic properties of cells, it is still difficult to measure their time-dependent properties. This paper investigates the use of atomic force microscope (AFM) to determine the reduced relaxation modulus of cells. In principle, AFM is hard to perform an indentation relaxation test that requires a constant indenter displacement during load relaxation, whereas the real AFM indenter displacement usually varies with time during relaxation due to the relatively small bending stiffness of its cantilever. We investigate this issue through a combined theoretical, computational, and experimental effort. A protocol relying on the choice of appropriate cantilever bending stiffness is proposed to perform an AFM-based indentation relaxation test of cells, which enables the measurement of reduced relaxation modulus with high accuracy. This protocol is first validated by performing nanoindentation relaxation tests on a soft material and by comparing the results with those from independent measurements. Then indentation tests of cartilage cells are conducted to demonstrate this method in determining time-dependent properties of living cells. Finally, the change in the viscoelasticity of MCF-7 cells under hyperthermia is investigated.

Flavonoids modulate liposomal membrane structure, regulate mitochondrial membrane permeability and prevent erythrocyte oxidative damage.

In the present work, we investigated the interaction of flavonoids (quercetin, naringenin and catechin) with cellular and artificial membranes. The flavonoids considerably inhibited membrane lipid peroxidation in rat erythrocytes treated with tert-butyl hydroperoxide (700 µM), and the IC50 values for prevention of this process were equal to 9.7 ± 0.8 µM, 8.8 ± 0.7 µM, and 37.8 ± 4.4 µM in the case of quercetin, catechin and naringenin, respectively, and slightly decreased glutathione oxidation. In isolated rat liver mitochondria, quercetin, catechin and naringenin (10-50 µM) dose-dependently increased the sensitivity to Ca2+ ions - induced mitochondrial permeability transition. Using the probes TMA-DPH and DPH we showed that quercetin rather than catechin and naringenin strongly decreased the microfluidity of the 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomal membrane bilayer at different depths. On the contrary, using the probe Laurdan we observed that naringenin transfer the bilayer to a more ordered state, whereas quercetin dose-dependently decreased the order of lipid molecule packing and increased hydration in the region of polar head groups. The incorporation of the flavonoids, quercetin and naringenin and not catechin, into the liposomes induced an increase in the zeta potential of the membrane and enlarged the area of the bilayer as well as lowered the temperature and the enthalpy of the membrane phase transition. The effects of the flavonoids were connected with modification of membrane fluidity, packing, stability, electrokinetic properties, size and permeability, prevention of oxidative stress, which depended on the nature of the flavonoid molecule and the nature of the membrane.

A function of fascin1 in the colony formation of mouse embryonic stem cells.

Fascin1 is known to participate in the migration of cancer cells by binding to actin filaments. Recent studies evidenced that fascin1 also modulates processes such as the tumorigenesis and maintenance of pluripotency genes in cancer stem cells. However, the function of fascin1 in embryonic stem cells remains unclear. In this article, we report that fascin1 is highly expressed and widely distributed in mouse embryonic stem cells (mESCs), which are regulated by JAK-STAT3 and ß-catenin. We found that the overexpression of fascin1 impairs the formation of mESC colonies via the downregulation of intercellular adhesion molecules, and that mimicking the dephosphorylated mutation of fascin1 or inhibiting phosphorylation with Gö6983 significantly enhances colony formation. Hyperphosphorylated fascin1 can promote the maintenance of pluripotency in mESCs via nuclear localization and suppressing DNA methyltransferase expression. Our findings demonstrate a novel function of fascin1, as a vital regulator, in the colony formation and pluripotency of mESCs and provide insights into the molecular mechanisms underlying embryonic stem cell self-organization and development in vitro.

Mechanical Roles of F-Actin in the Differentiation of Stem Cells: A Review.

In the development and differentiation of stem cells, mechanical forces associated with filamentous actin (F-actin) play a crucial role. The present review aims to reveal the relationship among the chemical components, microscopic structures, mechanical properties, and biological functions of F-actin. Particular attention is given to the functions of the cytoplasmic and nuclear microfilament cytoskeleton and their regulation mechanisms in the differentiation of stem cells. The distributions of different types of actin monomers in mammal cells and the functions of actin-binding proteins are summarized. We discuss how the fate of stem cells is regulated by intra/extracellular mechanical and chemical cues associated with microfilament-related proteins, intercellular adhesion molecules, etc. In addition, we also address the differentiation-induced variation in the stiffness of stem cells and the correlation between the fate and geometric shape change of stem cells. This review not only deepens our understanding of the biophysical mechanisms underlying the fates of stem cells under different culture conditions but also provides inspirations for the tissue engineering of stem cells.

Substrate stiffness affects neural network activity in an extracellular matrix proteins dependent manner.

Neuronal growth, differentiation, extension, branching and neural network activity are strongly influenced by the mechanical property of extracellular matrix (ECM). However, the mechanism by which substrate stiffness regulates a neural network activity, and the importance of ECM composition in conferring substrate stiffness sensing have not been explored. To address this question, the hippocampal neurons were seeded on the polydimethylsiloxane (PDMS) substrate with different stiffness, which were coated with fibronectin and laminin respectively. Our results show that voltage-gated Ca2+ channel currents are greater in neurons on the stiff substrate than on the soft substrate. In addition, the neurons exhibit a greater increase of Ca2+ currents on laminin-coated stiff substrate than on those coated with fibronectin, indicating that the composition of ECM can modulate responses to substrate stiffness of neurons. Paired patch-clamp recordings have shown that upregulation of neural effective synaptic connectivity is greater on the laminin-coated stiff substrate than on the fibronectin-coated ones. Consistently, laminin-coated stiff substrate enhances Ca2+ oscillations of neurons is greater that compared with the fibronectin-coated ones. Our study demonstrates that a direct role for substrate stiffness in regulating neuronal network activity and indicate that this modulation is dependent on a specific type of ECM protein, which should be taken into account for the design of biomaterials for neuronal tissue engineering.

Involvement of BK channel in differentiation of vascular smooth muscle cells induced by mechanical stretch.

The differentiation of vascular smooth muscle cells (VSMCs), which are exposed to mechanical stretch in vivo, plays an important role in vascular remodeling during hypertension. Here, we demonstrated the mechanobiological roles of large conductance calcium and voltage-activated potassium (BK) channels in this process. In comparison with 5% stretch (physiological), 15% stretch (pathological) induced the de-differentiation of VSMCs, resulting in significantly decreased expressions of VSMC markers, i.e., α-actin, calponin and SM22. The activity of BK channels, assessed by patch clamp recording, was significantly increased by 15% stretch and was accompanied by an increased alternative splicing of BK channel α-subunit at the stress axis-regulated exons (STREX). Furthermore, transfection of whole BK or STREX-deleted BK plasmids revealed that STREX was important for BK channels to sense mechanical stretch. Using thapsigargin (TG) which induces endoplasmic reticulum (ER) stress, and xbp1-targeted siRNA transfection which blocks ER stress, the results revealed that ER stress was contribute to stretch-induced alternative splicing of STREX. Our results suggested that during hypertension, pathological stretch may induce the ER stress in VSMCs, which affects the alternative splicing and activity of BK channels, and subsequently modulates VSMC differentiation.

Uniaxial cyclic stretch promotes osteogenic differentiation and synthesis of BMP2 in the C3H10T1/2 cells with BMP2 gene variant of rs2273073 (T/G).

Ossification of the posterior longitudinal ligament of the cervical spine (OPLL) is characterized by the replacement of ligament tissues with ectopic bone formation, and this result is strongly affected by genetic and local factors. Two single nucleotide polymorphisms (SNPs) of rs2273073 (T/G) and rs235768 (A/T) of bone morphogenetic protein 2 (BMP2) gene which are associated with OPLL have been reported in our previous report. In this study, we confirmed the connection in 18 case samples analysis of BMP2 gene in OPLL patients; additionally, it was also shown from the OPLL patients with ligament tissues that enchondral ossification and expression of BMP2 were significantly higher compared with the non-OPLL patients by histological examination, immunohistochemistry and Western blotting analysis. To investigate the underlying mechanism, we studied the effect of SNPs in cell model. The C3H10T1/2 cells with different BMP2 gene variants were constructed and then subjected to uniaxial cyclic stretch (0.5 Hz, 10% stretch). In the presence of mechanical stress, the expression of BMP2 protein in C3H10T1/2 cells transfected by BMP2 (rs2273073 (T/G)) and BMP2 (rs2273073 (T/G), rs235768 (A/T)) were significantly higher than the corresponding static groups (P<0.05). In conclusion, these results suggested that BMP2 gene variant of rs2273073 (T/G) could not only increase cell susceptibility to bone transformation similar to pre-OPLL change, but also increase the sensibility to mechanical stress which might play an important role during the progression of OPLL.

Stiff substrates enhance cultured neuronal network activity.

The mechanical property of extracellular matrix and cell-supporting substrates is known to modulate neuronal growth, differentiation, extension and branching. Here we show that substrate stiffness is an important microenvironmental cue, to which mouse hippocampal neurons respond and integrate into synapse formation and transmission in cultured neuronal network. Hippocampal neurons were cultured on polydimethylsiloxane substrates fabricated to have similar surface properties but a 10-fold difference in Young's modulus. Voltage-gated Ca(2+) channel currents determined by patch-clamp recording were greater in neurons on stiff substrates than on soft substrates. Ca(2+) oscillations in cultured neuronal network monitored using time-lapse single cell imaging increased in both amplitude and frequency among neurons on stiff substrates. Consistently, synaptic connectivity recorded by paired recording was enhanced between neurons on stiff substrates. Furthermore, spontaneous excitatory postsynaptic activity became greater and more frequent in neurons on stiff substrates. Evoked excitatory transmitter release and excitatory postsynaptic currents also were heightened at synapses between neurons on stiff substrates. Taken together, our results provide compelling evidence to show that substrate stiffness is an important biophysical factor modulating synapse connectivity and transmission in cultured hippocampal neuronal network. Such information is useful in designing instructive scaffolds or supporting substrates for neural tissue engineering.

Dorsal root ganglion electrical stimulation promoted intertransverse process spinal fusion without decortications and bone grafting: a proof-of-concept study.

BACKGROUND CONTEXT: Periosteum, endosteum, and bone are innervated by sensory nerves expressing calcitonin gene-related peptide (CGRP), which is a known osteoanabolic peptide and plays an important role in fracture healing and spinal fusion. Synthesis and release of CGRP are found in sensory neurons located in the dorsal root ganglions (DRGs) and can be upregulated by electrical stimulation (ES) at DRG. PURPOSE: To prove our study hypothesis on the potential of precise ES at DRG through implantable microelectrical stimulation system (IMESS) for its effect on promoting spinal fusion in a rat model without decortications and bone grafting. STUDY DESIGN: An experimental animal study. METHODS: A novel IMESS was developed for stimulating L4-L6 DRG in rats. Sixteen rats were used and divided equally into the control group without ES and the ES group, with a daily 20 minutes ES to DRG for 6 weeks. At the end of 6 weeks, radiography and microcomputed tomography were conducted to evaluate new bone formation and spinal fusion. Bilateral L4-L6 DRGs were harvested for immunohistochemistry and quantification of neurons with upregulated CGRP expression. RESULTS: In the ES group, rate of radiographic fusion with complete and uninterrupted bony bridging was 100% (8/8) at the right L4/L5 transverse processes and 75% (6/8) at the right L5/L6 transverse processes. Bony callus formation was absent at the left L4-L6 transverse processes in the ES group and in bilateral L4-L6 transverse processes in the control group. CONCLUSIONS: We proved for the first time that precise ES at DRG through IMESS effectively promoted intertransverse process fusion in rat model without decortications and bone grafting. Electrical stimulation at DRG might be an attractive minimal invasive bioengineering approach and an alternative therapy for intertransverse process fusion that is increasingly being used for the treatment of degenerative spine disorders.

Membrane stretch and cytoplasmic Ca2+ independently modulate stretch-activated BK channel activity.

Large conductance Ca(2+)-activated K(+) (BK) channels are responsible for changes in chemical and physical signals such as Ca(2+), Mg(2+) and membrane potentials. Previously, we reported that a BK channel cloned from chick heart (SAKCaC) is activated by membrane stretch. Molecular cloning and subsequent functional characterization of SAKCaC have shown that both the membrane stretch and intracellular Ca(2+) signal allosterically regulate the channel activity via the linker of the gating ring complex. Here we investigate how these two gating principles interact with each other. We found that stretch force activated SAKCaC in the absence of cytoplasmic Ca(2+). Lack of Ca(2+) bowl (a calcium binding motif) in SAKCaC diminished the Ca(2+)-dependent activation, but the mechanosensitivity of channel was intact. We also found that the abrogation of STREX (a proposed mechanosensing apparatus) in SAKCaC abolished the mechanosensitivity without altering the Ca(2+) sensitivity of channels. These observations indicate that membrane stretch and intracellular Ca(2+) could independently modulate SAKCaC activity.

Exercise training changes the gating properties of large-conductance Ca2+-activated K+ channels in rat thoracic aorta smooth muscle cells.

Large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels play a critical role in regulating the cellular excitability in response to change in blood flow. It has been demonstrated that vascular BK(Ca) channel currents in both humans and rats are increased after exercise training. This up-regulation of the BK(Ca) channel activity in arterial myocytes may represent a cellular compensatory mechanism of limiting vascular reactivity to exercise training. However, the underlying mechanisms are not fully understood. In the present study, we examined the single channel activities and kinetics of the BK(Ca) channels in rat thoracic aorta smooth muscle cells. We showed that exercise training significantly increased the open probability (Po), decreased the mean closed time and increased the mean open time, and the sensitivity to Ca(2+) and voltage without altering the unitary conductance and the K(+) selectivity. Our results suggest a novel mechanism by which exercise training increases the K(+) currents by changing the BK(Ca) channel activities and kinetics.

Stress stimulus induced resistance to Cladosporium cucumerinum in cucumber seeding.

Up to date, the studies of plant induced resistance have become the focus in plant pathology and physiology. During the course of pathogens penetrating the plant cell, besides of chemical secretion, the pathogens may generate mechanical signal caused by the physical pressure on the plant cell. In the non-host resistance, both the chemical signal and the mechanical stress signal are considered to have contribution to the entire defense reaction acted by the plant. The penetration of pathogen Cladosporium cucumerinum to cucumber is thought to be one of the model in research of plant induced resistance. In the current study, as a mechanical signal elicitor, the appropriate stress stimulus was proved to effectually induce the resistance of cucumber seedling to C. cucumerinum. After the treatment of the stress stimulus on leaves, the activities of resistance-related enzymes were significantly increased, such as phenylanine ammonia lyase (PAL), peroxidase (POD). Also, we found that stress stimulation may cause synthesis of lignin, which acts as the physical barrier to defense the pathogens. The results suggest that stress stimulation may not only enhance ability of the plant cell resistance to pathogen penetration but also elicit the accumulation of pathogens suppression or antimicrobial chemical substance in the plant cell.

The accumulation of phytoalexin in cucumber plant after stress.

During the course of pathogens penetrating the plant cell, besides of chemical secretion, the pathogens may cause mechanical signal by the physical pressure on the plant cell. In the current study, we use the pressure as the stress signal to study the induction in plant resistance and the effect of accumulation of phytoalexin. We found that stress can induce the resistance in cucumber seeding significantly. Peptides contained RGD motif can specific block the adhesion between plant cell wall and plasma membrane. When breaking the plant cell wall and plasma membrane by using RGD peptides, the stress induction effect is almost absolutely eliminated. The results of assay with TLC and HPLC showed that stress stimulation could increase the accumulation of cucumber seeding phytoalexin. So, we can conclude that the accumulation of phytoalexin is one possible reason of improve the stress induced resistance. When block the adhesion between plant cell wall and plasma membrane by RGD, there are only part of accumulation of phytoalexin. The results suggest that stress induced resistance and accumulation of phytoalexin of plant is required for the adhesion of plant cell wall-plasma membrane.

Effect of local stress induction on resistance-related enzymes in cucumber seeding.

In the current study, we found that the stress stimulus can act as a kind of elicitor, which can efficiently induce the resistance of cucumber against fungal pathogen. After the treatment of the stress stimulus on leaves, the activities of resistance-related enzymes were increased significantly. Such as phenylamine ammonia lyase (PAL), peroxidase (POD) and polyphenoloxidase (PPO), which are strongly associated with the plant disease resistance. Also the expression of pathogenesis-related protein (PR protein) were activated by stress stimulus, with the results that the activities of chitinase and beta-l,3-glucanase were increased obviously. The data showed that one of the mechanism of stress stimulus induction plant resistance may act via eliciting the metabolism related disease resistance within plant, which can produce many suppressing and antimicrobial compounds to against pathogens infection efficiently.