How Does a Cell Change Flow Direction Due to a Micro Groove?

The change in direction of a cell flowing over an oblique micro groove has been analyzed in vitro. The micro flow-channel (0.05 mm height x 1 mm width x 25 mm length) with oblique micro grooves (4.5 μm depth) was manufactured on a polydimethylsiloxane (PDMS) disk by the micromachining technique. The angle between the main flow direction and the longitudinal axis of the groove is 45 degrees. The effect of variation of the groove width (0.03 mm, 0.04 mm, and 0.05 mm) was studied. Myoblasts (C2C12: mouse myoblast cell line) were used in the test. The main flow velocity (0.02 mm/s < vx < 0.23 mm/s) of the medium was controlled by the pressure difference between the inlet and the outlet. The shape of each flowing cell was tracked on a movie recorded by the camera attached to the eyepiece of the microscope. The experimental results show that the change of the direction of each cell by each groove depends on the shape of the cell, which depends on both the shape of the cell and the width of the groove.

20-Hydroxyecdysone activates the protective arm of the RAAS via Mas receptor

20-Hydroxyecdysone (20E) is a steroid hormone that plays a key role in insect development through nuclear ecdysteroid receptors (EcR/RXR complex) and at least one membrane GPCR receptor (DopEcR). It also displays numerous pharmacological effects in mammals, where its mechanism of action is still debated, involving either an unidentified GPCR or the estrogen ERβ receptor. The goal of this study was to better understand 20E mechanism of action in mammals. A mouse myoblast cell line (C2C12) and the gene expression of myostatin (a negative regulator of muscle growth) was used as a reporter system of anabolic activity. Experiments using protein-bound 20E established the involvement of a membrane receptor. 20E-like effects were also observed with angiotensin-(1-7), the endogenous ligand of Mas. Additionally, the effect on myostatin gene expression was abolished by Mas receptor knock-down using small interfering RNA (siRNA) or pharmacological inhibitors. 17β-Estradiol (E2) also inhibited myostatin gene expression, but protein-bound E2 was inactive, and E2 activity was not abolished by angiotensin-(1-7) antagonists. A mechanism involving cooperation between the Mas receptor and a membrane-bound palmitoylated estrogen receptor is proposed. The possibility to activate the Mas receptor with a safe steroid molecule is consistent with the pleiotropic pharmacological effects of ecdysteroids in mammals and, indeed, the proposed mechanism may explain the close similarity between angiotensin-(1-7)’s and 20E’s effects. Our findings open up many possible therapeutic developments involving stimulation of the protective arm of the renin-angiotensin-aldosterone system (RAAS) with 20E.

Hysteresis Effect of Tangential Force Field With Centrifuge on Myoblast: Cultured on Striped Pattern of Micro Ridge for Direction Control

Hysteresis effects of the direction of mechanical stimulation on the cell behavior have been examined in vitro. A micro ridge pattern was made on the surface of the scaffold to align the directions of the cells being stimulated. The stripe pattern (0.7 μm heigh, 3 μm wide, and 3 μm interval) was created by the photolithography technique. Three regions, which have the uniform value of the angle between the longitudinal direction of the ridge and the direction of the tangential force, were set: 0, 45, and 90 degrees in each region. Myoblasts (C2C12: mouse myoblast cell line) were used in the experiment. The scaffold plate with cells was set in the tube of a conventional centrifuge placed in an incubator to apply the tangential force field to each cell. After the cell culture for 5 hours with centrifugation, the behavior of each cell was analyzed on time-lapse microscopic images for 10 hours. Experimental results show that cell activities (migration and deformation) are enhanced after stimulation of tangential forces perpendicular to the long axis of myoblasts.

Movement of Myoblast Flowing Through Electric Field Perpendicular to Flow Channel

The sorting technology with little invasion to cells would be applied to regenerative medicine and diagnosis. In this study, dielectrophoresis is focused on. The dielectrophoretic effect on the flowing myoblasts was maximized by adjusting several parameters: the shape of the electrodes, the amplitude and frequency of the alternating current. The suspension of C2C12 (mouse myoblast cell line) was injected into the channel, and the movement of each flowing cell was analyzed at the microscopic movie image. A pair of titanium-coated (200 nm thick) asymmetric surface electrodes (a triangular electrode with a tip angle of 0.35 rad and a rectangular reference electrode with a flat edge) was manufactured by photolithography technique. With the alternating square cyclic wave at the frequency of 3 MHz and the amplitude of current of ± 7.5 mA, 70 μm movement along the electric field (perpendicular to the main flow direction) of the cell was obtained. The movement along the electric field is governed by several parameters of the cell: the diameter, the deformation ratio, and the direction of the major axis. The method can be applied to cell sorting.

Behavior of Cell Passing Through Micro Slit Between Micro Machined Plates

Deformation of each cell, as it passes through the micro-slit in the flow channel, has been investigated in vitro. A slit with a rectangular cross section (height 10 μm, width 0.4 mm, length 0.1 mm) was made in the center of the flow path by photolithography technique. Myoblasts (C2C12: mouse myoblast cell line) were used for the test. The flow rate of the medium, in which the cells were suspended, was controlled by a pressure head between the inlet and the outlet. Deformation of each cell passing through the micro-slit was observed with an inverted phase contrast microscope. Using the contour of the image of each cell passing through the slit intermittently, several parameters were analyzed: the two-dimensional projected area, the degree of deformation by ellipse approximation, and the deformation direction. The experimental results show that elongation of the cell in the slit tends to decrease the area of the cell.

Hormonally regulated myogenic miR-486 influences sex-specific differences in cancer-induced skeletal muscle defects

Cancer-induced skeletal muscle defects show sex-specific differences in severity with men performing poorly compared to women. Hormones and sex chromosomal differences are suggested to mediate these differences, but the functional skeletal muscle markers to document these differences are unknown. We show that the myogenic microRNA miR-486 is a marker of sex-specific differences in cancer-induced skeletal muscle defects. Cancer-induced loss of circulating miR-486 was more severe in men with bladder, lung and pancreatic cancers compared to women with the same cancer types. In syngeneic model of pancreatic cancer, circulating and skeletal muscle loss of miR-486 was more severe in male mice compared to female mice. Estradiol (E2) and the clinically used selective estrogen receptor modulator toremifene increased miR-486 in undifferentiated and differentiated myoblast cell line C2C12 and E2-inducible expression correlated with direct binding of estrogen receptor alpha (ERα) to the regulatory region of miR-486 gene. E2 and toremifene reduced the actions of cytokines such as myostatin, TGFβ and TNFα, which mediate cancer-induced skeletal muscle wasting. E2 and toremifene treated C2C12 myoblast/myotube cells contained elevated levels of active AKT with corresponding decrease in the levels of its negative regulator PTEN, which is a target of miR-486. We propose an ERα:E2-miR-486-AKT signaling axis, which reduces the deleterious effects of cancer-induced cytokines/chemokines on skeletal muscle mass and/or function.

CRISPR mediated targeting of DUX4 distal regulatory element represses DUX4 target genes dysregulated in Facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is a debilitating muscle disease that currently does not have an effective cure or therapy. The abnormal reactivation of DUX4, an embryonic gene that is epigenetically silenced in somatic tissues, is causal to FSHD. Disease-specific reactivation of DUX4 has two common characteristics, the presence of a non-canonical polyadenylation sequence within exon 3 of DUX4 that stabilizes pathogenic transcripts, and the loss of repressive chromatin modifications at D4Z4, the macrosatellite repeat which encodes DUX4. We used CRISPR/Cas9 to silence DUX4 using two independent approaches. We deleted the DUX4 pathogenic polyadenylation signal, which resulted in downregulation of pathogenic DUX4-fl transcripts. In another approach, we transcriptionally repressed DUX4 by seeding heterochromatin using the dCas9-KRAB platform within exon 3. These feasibility of targeting DUX4 experiments were initially tested in a non-myogenic carcinoma cell line that we have previously characterized. Subsequently, in an immortalized patient myoblast cell line, we demonstrated that targeting DUX4 by either approach led to substantial downregulation of not only pathogenic DUX4 transcripts, but also a subset of its target genes that are known biomarkers of FSHD. These findings offer proof-of-concept of the effect of silencing the polyadenylation sequence on pathogenic DUX4 expression.

Zebrafish Melanophores Suggest Novel Functions of Cell Chirality in Tissue Formation

Several types of cells show left–right asymmetric behavior, unidirectional rotation, or spiral movements. For example, neutrophil-like differentiated HL60 (dHL60) cells show leftward bias in response to chemoattractant. Neurons extend neurites, creating a clockwise spiral. Platelet cells shows unidirectional spiral arrangements of actin fibers. In the microfabricated culture environment, groups of C2C12 cells (mouse myoblast cell line) were autonomously aligned in a counter-clockwise spiral pattern, and isolated C2C12 cells showed unidirectional spiral pattern of the actin skeleton. This biased directionality suggested that these cells have inherent cell chirality. In addition to these cells, we recently found that melanophores of zebrafish also have an intrinsic cellular chirality that was shown by their counter-clockwise self-rotation. Although this cell chirality is obvious, the function of the cell chirality is still unclear. In this review, we compare the cell chirality of melanophores of zebrafish with other cell chirality and consider the function of cell chirality in morphogenesis.

A Theacrine-Based Supplement Increases Cellular NAD+ Levels and Affects Biomarkers Related to Sirtuin Activity in C2C12 Muscle Cells In Vitro

There is evidence in rodents to suggest that theacrine-based supplements modulate tissue sirtuin activity as well as other biological processes associated with aging. Herein, we examined if a theacrine-based supplement (termed NAD3) altered sirtuin activity in vitro while also affecting markers of mitochondrial biogenesis. The murine C2C12 myoblast cell line was used for experimentation. Following 7 days of differentiation, myotubes were treated with 0.45 mg/mL of NAD3 (containing ~2 mM theacrine) for 3 and 24 h (n = 6 treatment wells per time point). Relative to control (CTL)-treated cells, NAD3 treatments increased (p < 0.05) Sirt1 mRNA levels at 3 h, as well as global sirtuin activity at 3 and 24 h. Follow-up experiments comparing 24 h NAD3 or CTL treatments indicated that NAD3 increased nicotinamide phosphoribosyltransferase (NAMPT) and SIRT1 protein levels (p < 0.05). Cellular nicotinamide adenine dinucleotide (NAD+) levels were also elevated nearly two-fold after 24 h of NAD3 versus CTL treatments (p < 0.001). Markers of mitochondrial biogenesis were minimally affected. Although these data are limited to select biomarkers in vitro, these preliminary findings suggest that a theacrine-based supplement can modulate select biomarkers related to NAD+ biogenesis and sirtuin activity. However, these changes did not drive increases in mitochondrial biogenesis. While promising, these data are limited to a rodent cell line and human muscle biopsy studies are needed to validate and elucidate the significance of these findings.