Ligand-independent function of ß2-adrenergic receptor affects IgE-mediated Ca2+ influx in mast cells.

BACKGROUND: Mast cells are key effector cells that elicit immunoglobulin E (IgE)-mediated allergic inflammations. Allergen cross-linking of IgE bound to the high-affinity IgE receptor, FcεRI, on mast cells triggers signaling cascades that activate signal proteins and evoke extracellular Ca2+ influx, which are crucial for cytokine production. The ß2-adrenergic receptor (Adrb2) on mast cells negatively regulates FcεRI signaling, as demonstrated by the inhibition of IgE/antigen (Ag)-induced activation by Adrb2 agonists. OBJECTIVE: Although ß2-adrenergic-related reagents are known to influence mast cell functions, the specific intrinsic role of Adrb2 in these cells is not fully understood, potentially because of off-target effects. In this study, the additional roles of Adrb2 in mast cells were investigated, specifically the involvement of Adrb2 in FcεRI signaling, using Adrb2-/- mice. METHODS: Adrb2-/- mice were used to investigate the roles of Adrb2 in mast cells by examining bone marrow-derived mast cells (BMMCs) for surface expression of mast cell markers, granule numbers, and gene expression of mast cell proteases. Cytokine production, Ca2+ influx, and nuclear factor of activated T cells (NFAT) nuclear translocation were measured in Adrb2-/- and Adrb2+/+ BMMCs upon IgE/Ag stimulation. RESULTS: Adrb2-/- did not affect the generation of BMMCs, their surface expression of mast cell markers, granule numbers, or gene expression of mast cell proteases, indicating that the absence of Adrb2 had no adverse effect on mast cell development. However, Adrb2-/- BMMCs exhibited reduced tumor necrosis factor α (TNFα) production and diminished Ca2⁺ influx upon IgE/Ag stimulation, which correlated with decreased NFAT translocation. Restoration of Adrb2 in Adrb2-/- BMMCs rescued cytokine production. Notably, FcεRI-mediated phosphorylation of the phospholipase PLCγ1 and mitogen-activated protein kinases (MAPKs) remained unchanged in the absence of Adrb2. CONCLUSION: These results suggest that Adrb2 has a novel ligand-independent function, increasing Ca2+ entry in mast cells when stimulated with IgE/Ag.

Stretch-induced reactive oxygen species contribute to the Frank-Starling mechanism.

Myocardial stretch physiologically activates NADPH oxidase 2 (NOX2) to increase reactive oxygen species (ROS) production. Although physiological low-level ROS are known to be important as signalling molecules, the role of stretch-induced ROS in the intact myocardium remains unclear. To address this, we investigated the effects of stretch-induced ROS on myocardial cellular contractility and calcium transients in C57BL/6J and NOX2-/- mice. Axial stretch was applied to the isolated cardiomyocytes using a pair of carbon fibres attached to both cell ends to evaluate stretch-induced modulation in the time course of the contraction curve and calcium transient, as well as to evaluate maximum cellular elastance, an index of cellular contractility, which is obtained from the end-systolic force-length relationship. In NOX2-/- mice, the peak calcium transient was not altered by stretch, as that in wild-type mice, but the lack of stretch-induced ROS delayed the rise of calcium transients and reduced contractility. Our mathematical modelling studies suggest that the augmented activation of ryanodine receptors by stretch-induced ROS causes a rapid and large increase in the calcium release flux, resulting in a faster rise in the calcium transient. The slight increase in the magnitude of calcium transients is offset by a decrease in sarcoplasmic reticulum calcium content as a result of ROS-induced calcium leakage, but the faster rise in calcium transients still maintains higher contractility. In conclusion, a physiological role of stretch-induced ROS is to increase contractility to counteract a given preload, that is, it contributes to the Frank-Starling law of the heart. KEY POINTS: Myocardial stretch increases the production of reactive oxygen species by NADPH oxidase 2. We used NADPH oxidase 2 knockout mice to elucidate the physiological role of stretch-induced reactive oxygen species in the heart. We showed that stretch-induced reactive oxygen species modulate the rising phase of calcium transients and increase myocardial contractility. A mathematical model simulation study demonstrated that rapid activation of ryanodine receptors by reactive oxygen species is important for increased contractility. This response is advantageous for the myocardium, which must contract against a given preload.

High hydrostatic pressure induces slow contraction in mouse cardiomyocytes.

Cardiomyocytes are contractile cells that regulate heart contraction. Ca2+ flux via Ca2+ channels activates actomyosin interactions, leading to cardiomyocyte contraction, which is modulated by physical factors (e.g., stretch, shear stress, and hydrostatic pressure). We evaluated the mechanism triggering slow contractions using a high-pressure microscope to characterize changes in cell morphology and intracellular Ca2+ concentration ([Ca2+]i) in mouse cardiomyocytes exposed to high hydrostatic pressures. We found that cardiomyocytes contracted slowly without an acute transient increase in [Ca2+]i, while a myosin ATPase inhibitor interrupted pressure-induced slow contractions. Furthermore, transmission electron microscopy showed that, although the sarcomere length was shortened upon the application of 20 MPa, this pressure did not collapse cellular structures such as the sarcolemma and sarcomeres. Our results suggest that pressure-induced slow contractions in cardiomyocytes are driven by the activation of actomyosin interactions without an acute transient increase in [Ca2+]i.

Role of the TRPM4 channel in mitochondrial function, calcium release, and ROS generation in oxidative stress.

Ischemic heart disease is one of the most common causes of death worldwide. Mitochondrial dysfunction, excessive reactive oxygen species (ROS) generation, and calcium (Ca2+) overload are three key factors leading to myocardial death during ischemia-reperfusion (I/R) injury. Inhibition of TRPM4, a Ca2+-activated nonselective cation channel, protects the rat heart from I/R injury, but the specific mechanism underlying this effect is unclear. In this study, we investigated the mechanism of cardioprotection against I/R injury via TRPM4 using hydrogen peroxide (H2O2), a major contributor to oxidative stress, as an I/R injury model. We knocked out the TRPM4 gene in the rat cardiomyocyte cell line H9c2 using CRISPR/Cas9. Upon H2O2 treatment, intracellular Ca2+ level and ROS production increased in wild type (WT) cells but not in TRPM4 knockout (TRPM4KO) cells. With this treatment, two indicators of mitochondrial function, mitochondrial membrane potential (ΔΨm) and intracellular ATP levels, decreased in WT but not in TRPM4KO cells. Taken together, these findings suggest that blockade of the TRPM4 channel might protect the myocardium from oxidative stress by maintaining the mitochondrial membrane potential and intracellular ATP levels, possibly through preventing aberrant increases in intracellular Ca2+ and ROS.

The Mechanisms of the Development of Atherosclerosis in Prediabetes.

Lifestyle changes, such as overeating and underexercising, can increase the risk of prediabetes. Diabetes is one of the leading causes of atherosclerosis, and recently it became clear that the pathophysiology of atherosclerosis progresses even before the onset of diabetic symptoms. In addition to changes in platelets and leukocytes in the hyperglycemic state and damage to vascular endothelial cells, extracellular vesicles and microRNAs were found to be involved in the progression of prediabetes atherosclerosis. This review discusses the cellular and molecular mechanisms of these processes, with an intention to enable a comprehensive understanding of the pathophysiology of prediabetes and atherosclerosis.

Treatment of Oxidative Stress with Exosomes in Myocardial Ischemia.

A thrombus in a coronary artery causes ischemia, which eventually leads to myocardial infarction (MI) if not removed. However, removal generates reactive oxygen species (ROS), which causes ischemia-reperfusion (I/R) injury that damages the tissue and exacerbates the resulting MI. The mechanism of I/R injury is currently extensively understood. However, supplementation of exogenous antioxidants is ineffective against oxidative stress (OS). Enhancing the ability of endogenous antioxidants may be a more effective way to treat OS, and exosomes may play a role as targeted carriers. Exosomes are nanosized vesicles wrapped in biofilms which contain various complex RNAs and proteins. They are important intermediate carriers of intercellular communication and material exchange. In recent years, diagnosis and treatment with exosomes in cardiovascular diseases have gained considerable attention. Herein, we review the new findings of exosomes in the regulation of OS in coronary heart disease, discuss the possibility of exosomes as carriers for the targeted regulation of endogenous ROS generation, and compare the advantages of exosome therapy with those of stem-cell therapy. Finally, we explore several miRNAs found in exosomes against OS.

Increased hydrostatic pressure induces nuclear translocation of DAF-16/FOXO in C. elegans.

Mechanical stimulation is well known to be important for maintaining tissue and organ homeostasis. Here, we found that hydrostatic pressure induced nuclear translocation of a forkhead box O (FOXO) transcription factor DAF-16, in C. elegans within minutes, whereas the removal of this pressure resulted in immediate export of DAF-16 to the cytoplasm. We also monitored DAF-16-dependent transcriptional changes by exposure to 1 MPa pressure for 5 min, and found significant changes in collagen and other genes in a DAF-16 dependent manner. Lifespan was markedly prolonged with exposure to cyclic pressure treatment (1 MPa once a day for 5 min from L1 larvae until death). Furthermore, age-dependent decline in locomotor activity was suppressed by the treatment. In contrast, the nuclear translocation of the yes-associated protein YAP-1 was not induced under the same pressure conditions. Thus, moderate hydrostatic pressure improves ageing progression through activation of DAF-16/FOXO in C. elegans.

Mechanical strain attenuates cytokine-induced ADAMTS9 expression via transient receptor potential vanilloid type 1.

The synovial fluids of patients with osteoarthritis (OA) contain elevated levels of inflammatory cytokines, which induce the expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and of the matrix metalloproteinase (MMP) in chondrocytes. Mechanical strain has varying effects on organisms depending on the strength, cycle, and duration of the stressor; however, it is unclear under inflammatory stimulation how mechanical strain act on. Here, we show that mechanical strain attenuates inflammatory cytokine-induced expression of matrix-degrading enzymes. Cyclic tensile strain (CTS), as a mechanical stressor, attenuated interleukin (IL)-1ß and tumor necrosis factor (TNF)-α-induced mRNA expression of ADAMTS4, ADAMTS9, and MMP-13 in normal chondrocytes (NHAC-kn) and in a chondrocytic cell line (OUMS-27). This effect was abolished by treating cells with mechano-gated channel inhibitors, such as gadolinium, transient receptor potential (TRP) family inhibitor, ruthenium red, and with pharmacological and small interfering RNA-mediated TRPV1 inhibition. Furthermore, nuclear factor κB (NF-κB) translocation from the cytoplasm to the nucleus resulting from cytokine stimulation was also abolished by CTS. These findings suggest that mechanosensors such as the TRPV protein are potential therapeutic targets in treating OA.

The Inhibitory Role of Rab11b in Osteoclastogenesis through Triggering Lysosome-Induced Degradation of c-Fms and RANK Surface Receptors.

Rab11b, abundantly enriched in endocytic recycling compartments, is required for the establishment of the machinery of vesicle trafficking. Yet, no report has so far characterized the biological function of Rab11b in osteoclastogenesis. Using in vitro model of osteoclasts differentiated from murine macrophages like RAW-D cells or bone marrow-derived macrophages, we elucidated that Rab11b served as an inhibitory regulator of osteoclast differentiation sequentially via (i) abolishing surface abundance of RANK and c-Fms receptors; and (ii) attenuating nuclear factor of activated T-cells c1 (NFATc-1) upstream signaling cascades, following RANKL stimulation. Rab11b was localized in early and late endosomes, Golgi complex, and endoplasmic reticulum; moreover, its overexpression enlarged early and late endosomes. Upon inhibition of lysosomal function by a specific blocker, chloroquine (CLQ), we comprehensively clarified a novel function of lysosomes on mediating proteolytic degradation of c-Fms and RANK surface receptors, drastically ameliorated by Rab11b overexpression in RAW-D cell-derived osteoclasts. These findings highlight the key role of Rab11b as an inhibitor of osteoclastogenesis by directing the transport of c-Fms and RANK surface receptors to lysosomes for degradation via the axis of early endosomes-late endosomes-lysosomes, thereby contributing towards the systemic equilibrium of the bone resorption phase.

Development of a model of ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells.

Ischemic heart disease remains the largest cause of death worldwide. Accordingly, many researchers have sought curative options, often using laboratory animal models such as rodents. However, the physiology of the human heart differs significantly from that of the rodent heart. In this study, we developed a model of ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs). After optimizing the conditions of ischemia, including the concentration of oxygen and duration of application, we evaluated the consequent damage to hiPS-CMs. Notably, exposure to 2% oxygen, 0 mg/ml glucose, and 0% fetal bovine serum increased the percentage of nuclei stained with propidium iodide, an indicator of membrane damage, and decreased cellular viability. These conditions also decreased the contractility of hiPS-CMs. Furthermore, ischemic conditioning increased the mRNA expression of IL-8, consistent with observed conditions in the in vivo heart. Taken together, these findings suggest that our hiPS-CM-based model can provide a useful platform for human ischemic heart disease research.

Spiral Trajectory Modulation of Rheotaxic Motile Human Sperm in Cylindrical Microfluidic Channels of Different Inner Diameters.

We investigated the relationship between human sperm rheotaxis and motile sperm trajectories by using poly-(dimethylsiloxane) (PDMS)-based cylindrical microfluidic channels with inner diameters of 100 µm, 50 µm, and 70 µm, which corresponded to the inner diameter of the human isthmus, the length of a sperm and a diameter intermediate between the two, respectively. We counted the number of rheotaxic sperm and sperm with spiral motion. We also analyzed motile sperm trajectories. As the cylindrical channel diameter was decreased, the percentage of sperm cells exhibiting rheotaxis, the percentage of sperm cells exhibiting spiral motion, the frequency-to-diameter ratio of the sperm cells' spiral trajectories, and the surface area of the microfluidic channel increased, while the flagellar motion at the channel wall decreased. The percentage of sperm exhibiting a spiral trajectory and the frequency-to-diameter ratio of the sperm cells' spiral trajectories were thus affected by the channel diameter. Our findings suggest that the oviduct structure affects the swimming properties of sperm cells, guiding them from the uterus to the ampulla for egg fertilization. These results could contribute to the development of motile sperm-sorting microfluidic devices for assisted reproductive technologies.

The effects of load on transmural differences in contraction of isolated mouse ventricular cardiomyocytes.

Mechanical properties of cardiomyocytes from different transmural regions are heterogeneous in the left ventricular wall. The cardiomyocyte mechanical environment affects this heterogeneity because of mechano-electric feedback mechanisms. In the present study, we investigated the effects of the mechanical load (preload and afterload) on transmural differences in contraction of subendocardial (ENDO) and subepicardial (EPI) single cells isolated from the murine left ventricle. Various preloads imposed via axial stretch and afterloads (unloaded and heavy loaded conditions) were applied to the cells using carbon fiber techniques for single myocytes. To simulate experimentally obtained results and to predict mechanisms underlying the cellular response to change in load, our mathematical models of the ENDO and EPI cells were used. Our major findings are the following. Our results show that ENDO and EPI cardiomyocytes have different mechanical responses to changes in preload to the cells. Under auxotonic contractions at low preload (unstretched cells), time to peak contraction (Tmax) and the time constant of [Ca2+]i transient decay were significantly longer in ENDO cells than in EPI cells. An increase in preload (stretched cells) prolonged Tmax in both cell types; however, the prolongation was greater in EPI cells, resulting in a decrease in the transmural gradient in Tmax at high preload. Comparing unloaded and heavy loaded (isometric) contractions of the cells we found that transmural gradient in the time course of contraction is independent of the loading conditions. Our mathematical cell models were able to reproduce the experimental results on the distinct cellular responses to changes in the mechanical load when we accounted for an ENDO/EPI difference in the parameters of cooperativity of calcium activation of myofilaments.

Human gingival fibroblast feeder cells promote maturation of induced pluripotent stem cells into cardiomyocytes.

The use of human induced pluripotent stem (iPS) cells has been investigated in multiple regenerative medicine studies. However, although methods for efficient differentiation of iPS cells into heart tissues have been devised, it remains difficult to obtain cardiac tissue with high contractility. Herein, we established a method for differentiating iPS cells into highly contractile cardiomyocytes (CMs), and demonstrate that the use of human gingival fibroblasts (HGFs) as a feeder cells promotes maturation of iPS-derived CMs (iPS-CMs) in vitro. After CM differentiation of iPS cells, iPS-CMs showed increased mRNA expression of the CM specific maker cardiac troponin T (cTnT) in the absence and presence (on-feeder condition) of cocultured HGFs, and decreased expression of pluripotent markers was observed under both conditions. Protein expression of cTnT was also observed in immunocytochemical analyses, although on-feeder CMs showed comparatively robust sarcomere structure and significantly stronger contractility than feederless cardiomyocytes, suggesting that HGF feeder cells facilitate CM differentiation of iPS cells.

Stretching of single DNA molecules caused by accelerating flow on a microchip.

DNA elongation induced by fluidic stress was investigated on a microfluidic chip composed of a large inlet pool and a narrow channel. Through single-DNA observation with fluorescence microscopy, the manner of stretching of individual T4 DNA molecules (166 kbp) was monitored near the area of accelerating flow with narrowing streamlines. The results showed that the DNA long-axis length increased in a sigmoidal manner depending on the magnitude of flow acceleration, or shear, along the DNA chain. To elucidate the physical mechanism of DNA elongation, we performed a theoretical study by adopting a model of a coarse-grained nonlinear elastic polymer chain elongated by shear stress due to acceleration flow along the chain direction.

Effect of Oxidative Stress on Cardiovascular System in Response to Gravity.

Long-term habitation in space leads to physiological alterations such as bone loss, muscle atrophy, and cardiovascular deconditioning. Two predominant factors-namely space radiation and microgravity-have a crucial impact on oxidative stress in living organisms. Oxidative stress is also involved in the aging process, and plays important roles in the development of cardiovascular diseases including hypertension, left ventricular hypertrophy, and myocardial infarction. Here, we discuss the effects of space radiation, microgravity, and a combination of these two factors on oxidative stress. Future research may facilitate safer living in space by reducing the adverse effects of oxidative stress.

[Response to mechanical stimulus and cardiovascular homeostasis.]

Response to mechanical stimulus, including blood pressure regulation as a typical example, is essential for cardiovascular homeostasis. Traditionally, mechanism of blood pressure regulation can be divided into two categories:short term neural regulation via arterial baroreceptor reflex and long term humoral regulation via renin-angiotensin system. Recent studies have revealed that sensitivity of baroreceptor reflex is impaired in metabolic syndrome and aging. Moreover, it is suggested that the baroreceptor reflex is involved in long term blood pressure regulation. This review introduces the mechanism of mechanotransduction in the baroreceptor reflex.

Impaired viability of muscle precursor cells in muscular dystrophy with glycosylation defects and amelioration of its severe phenotype by limited gene expression.

A group of muscular dystrophies, dystroglycanopathy is caused by abnormalities in post-translational modifications of dystroglycan (DG). To understand better the pathophysiological roles of DG modification and to establish effective clinical treatment for dystroglycanopathy, we here generated two distinct conditional knock-out (cKO) mice for fukutin, the first dystroglycanopathy gene identified for Fukuyama congenital muscular dystrophy. The first dystroglycanopathy model-myofiber-selective fukutin-cKO [muscle creatine kinase (MCK)-fukutin-cKO] mice-showed mild muscular dystrophy. Forced exercise experiments in presymptomatic MCK-fukutin-cKO mice revealed that myofiber membrane fragility triggered disease manifestation. The second dystroglycanopathy model-muscle precursor cell (MPC)-selective cKO (Myf5-fukutin-cKO) mice-exhibited more severe phenotypes of muscular dystrophy. Using an isolated MPC culture system, we demonstrated, for the first time, that defects in the fukutin-dependent modification of DG lead to impairment of MPC proliferation, differentiation and muscle regeneration. These results suggest that impaired MPC viability contributes to the pathology of dystroglycanopathy. Since our data suggested that frequent cycles of myofiber degeneration/regeneration accelerate substantial and/or functional loss of MPC, we expected that protection from disease-triggering myofiber degeneration provides therapeutic effects even in mouse models with MPC defects; therefore, we restored fukutin expression in myofibers. Adeno-associated virus (AAV)-mediated rescue of fukutin expression that was limited in myofibers successfully ameliorated the severe pathology even after disease progression. In addition, compared with other gene therapy studies, considerably low AAV titers were associated with therapeutic effects. Together, our findings indicated that fukutin-deficient dystroglycanopathy is a regeneration-defective disorder, and gene therapy is a feasible treatment for the wide range of dystroglycanopathy even after disease progression.

A simple method for decreasing the liquid junction potential in a flow-through-type differential pH sensor probe consisting of pH-FETs by exerting spatiotemporal control of the liquid junction.

The liquid junction potential (LJP), the phenomenon that occurs when two electrolyte solutions of different composition come into contact, prevents accurate measurements in potentiometry. The effect of the LJP is usually remarkable in measurements of diluted solutions with low buffering capacities or low ion concentrations. Our group has constructed a simple method to eliminate the LJP by exerting spatiotemporal control of a liquid junction (LJ) formed between two solutions, a sample solution and a baseline solution (BLS), in a flow-through-type differential pH sensor probe. The method was contrived based on microfluidics. The sensor probe is a differential measurement system composed of two ion-sensitive field-effect transistors (ISFETs) and one Ag/AgCl electrode. With our new method, the border region of the sample solution and BLS is vibrated in order to mix solutions and suppress the overshoot after the sample solution is suctioned into the sensor probe. Compared to the conventional method without vibration, our method shortened the settling time from over two min to 15 s and reduced the measurement error by 86% to within 0.060 pH. This new method will be useful for improving the response characteristics and decreasing the measurement error of many apparatuses that use LJs.