Conventional root canal treatment replaces the infected pulp with defined materials. Alternative cell-based tissue engineering strategies aim to regenerate a fully functional pulp within the root ...canal. Despite recent advances in this area, however, the regeneration of an innervated pulp remains a major challenge in the field. Both graphene (2DG) and pulsed electromagnetic fields (PEMFs) independently have been shown to promote diverse cellular developmental programs. The present study showed that 2DG promoted the neurogenic induction of human dental pulp stem cells (hDPSCs) by upregulating and accelerating the expression of mature neuronal markers. Notably, 2DG induced the highest expression of transient receptor potential canonical cation channel type 1 (TRPC1) during early neurogenesis. As brief PEMF exposure promotes in vitro differentiation by activating a TRPC1-mitochondrial axis, an opportunity to combine 2DG with developmentally targeted PEMF exposure for synergistic effects was realizable. Neurogenic gene expression, neurotransmitter release, and reactive oxygen species (ROS) production were greatly enhanced by a brief (10 min) and low amplitude (2 mT) PEMF exposure timed to coincide with the highest TRPC1 expression from hDPSCs on 2DG. In contrast, hDPSCs on glass were less responsive to PEMF exposure. The capacity of PEMFs to promote neurogenesis was precluded by the administration of penicillin/streptomycin, mirroring previous studies demonstrating that aminoglycoside antibiotics block TRPC1-mediated calcium entry and verifying the contribution of TRPC1 in this form of magnetoreception. Hence, graphene created a more conducive environment for subsequent PEMF-stimulated neurogenic induction of hDPSCs through their mutual capacity to activate TRPC1with subsequent ROS production.
Myoblast therapy relies on the integration of skeletal muscle stem cells into distinct muscular compartments for the prevention of clinical conditions such as heart failure, or bladder dysfunction. ...Understanding the fundamentals of myogenesis is hence crucial for the success of these potential medical therapies. In this report, we followed the rearrangement of the surface membrane structure and the actin cytoskeletal organization in C2C12 myoblasts at different stages of myogenesis using atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM). AFM imaging of living myoblasts undergoing fusion unveiled that within minutes of making cell-cell contact, membrane tubules appear that unite the myoblasts and increase in girth as fusion proceeds. CLSM identified these membrane tubules as built on scaffolds of actin filaments that nucleate at points of contact between fusing myoblasts. In contrast, similarly behaving membrane tubules are absent during cytokinesis. The results from our study in combination with recent findings in literature further expand the understanding of the biochemical and membrane structural rearrangements involved in the two fundamental cellular processes of division and fusion.
Purpose: The goal of this work is to investigate the anticancer potency of magnetically induced muscle secretome on breast cancer in vitro, in vivo and ex vitro. The work also examines the function ...of magnetically mediated preconditioning paradigm to enhance muscle secretome release and production.
Briefly (10 min) exposing C2C12 myotubes to low amplitude (1.5 milliTesla) pulsed electromagnetic fields (PEMFs) generated a conditioned media (pCM) that mitigated breast cancer cell growth, migration and invasiveness in vitro, whereas the conditioned media harvested from unexposed myotubes, representing constitutively released secretome (cCM), was less effective. Ex vivo, administering pCM to breast cancer microtumors engrafted onto the chorioallantoic membrane of the chicken egg reduced tumor volume and vascularity. In vivo, blood serum collected from PEMF-exposed or exercised mice allayed breast cancer cell growth, migration and invasiveness. Based on PEMF-induction of the muscle secretome, a preconditioning methodology was developed to enhance secretome production and release. HTRA1 (High-Temperature Requirement A Serine Peptidase 1) was upregulated in the pCM and was shown necessary for myogenic progression and anticancer potency. HTRA1 expression hence embodied key adaptive features of the pCM preconditioning paradigm and emulating an exercise-like effect. Brief and non-invasive PEMF stimulation may represent a method to commandeer the secretome response of muscle for clinical exploitation in cancer.
Brief and non-invasive pulsed electromagnetic field (PEMF) stimulation was shown to induce the secretome response of muscles akin to exercise to impact cancer viability and epithelial-mesenchymal transition (EMT). Moreover, the preconditioning of cells to PEMF may represent a method to induce the secretome response of muscles for clinical exploitation in cancer.
1. Calcium currents were recorded from smooth muscle cells dispersed from conduit and resistance rabbit pulmonary arteries.
We tested the hypothesis that Ca2+ channel activity was regulated by ...environmental O2 tension. 2. Conduit (proximal) and resistance
(distal) myocytes differ in their Ca2+ channel density and responses to low PO2. Ca2+ current density in distal myocytes (20.7
+/- 7.4 pA pF-1, n = 10) is almost twice the value in proximal myocytes (12.6 +/- 5.5 pA pF-1, n = 39). In proximal myocytes,
the predominant response to reductions in PO2 is inhibition of the calcium current (n = 12) at membrane potentials below 0
mV, whereas potentiation of current amplitude is observed in distal myocytes (n = 24). 3. Hypoxia also produces opposite shifts
in the conductance-voltage relationships along the voltage axis. The average displacements induced by low PO2 are +5.05 +/-
2.98 mV (n = 5) in proximal myocytes and -6.06 +/- 2.45 (n = 10) in distal myocytes. 4. These findings demonstrate longitudinal
differences in Ca2+ channel density and O2 sensitivity in myocytes along the pulmonary arterial tree. These results may help
to understand the differential reactivity to hypoxia of the pulmonary vasculature: vasodilatation in conduit arteries and
vasoconstriction in resistance vessels.
1. We examined the activity of single mechanosensitive ion channels in recordings from cell-attached patches on myoblasts,
differentiated myotubes and acutely isolated skeletal muscle fibres from ...wild-type and mdx and dy mutant mice. The experiments
were concerned with the role of these channels in the pathophysiology of muscular dystrophy. 2. The predominant form of channel
activity recorded with physiological saline in the patch electrode arose from an approximately 25 pS mechanosensitive ion
channel. Channel activity was similar in undifferentiated myoblasts isolated from all three strains of mice. By contrast,
channel activity in mdx myotubes was approximately 3-4 times greater than in either wild-type or dy myotubes and arose from
a novel mode of mechanosensitive gating. 3. Single mechanosensitive channels in acutely isolated flexor digitorum brevis fibres
had properties indistinguishable from those of muscle cells grown in tissue culture. The channel open probability in mdx fibres
was approximately 2 times greater than the activity recorded from wild-type fibres. The overall level of activity in fibres,
however, was roughly an order of magnitude smaller than in myoblasts or myotubes. 4. Histological examination of the flexor
digitorum brevis fibres from mdx mice showed no evidence of myonecrosis or regenerating fibres, suggesting that the elevated
channel activity in dystrophin-deficient muscle precedes the onset of fibre degeneration. 5. An early step in the dystrophic
process of the mdx mouse, which leads to pathophysiological Ca2+ entry, may be an alteration in the mechanisms that regulate
mechanosensitive ion channel activity.
We have investigated the modifications of cytosolic Ca2+ and the activity of Ca2+channels in freshly dispersed arterial myocytes to test whether lowering O2tension (PO2) directly influences ...Ca2+homeostasis in these cells. Unclamped cells loaded with fura-2 AM exhibit oscillations of cytosolic Ca2+whose frequency depends on extracellular Ca2+influx. Switching from a PO2of 150 to 20 mmHg leads to a reversible attenuation of the Ca2+oscillations. In voltage-clamped cells, hypoxia reversibly reduces the influx of Ca2+through voltage-dependent channels, which can account for the inhibition of the Ca2+oscillations. Low PO2selectively inhibits L-type Ca2+channel activity, whereas the current mediated by T-type channels is unaltered by hypoxia. The effect of low PO2on the L-type channels is markedly voltage dependent, being more apparent with moderate depolarizations. These findings demonstrate the existence of O2-sensitive, voltage-dependent, Ca2+channels in vascular smooth muscle that may critically contribute to the local regulation of circulation.
Oxygen (O(2)) tension is a major regulator of blood flow in the coronary circulation. Hypoxia can produce vasodilation through activation of ATP regulated K(+) (K(ATP)) channels in the myocyte ...membrane, which leads to hyperpolarization and closure of voltage-gated Ca(2+) channels. However, there are other O(2)-sensitive mechanisms intrinsic to the vascular smooth muscle since hypoxia can relax vessels precontracted with high extracellular K(+), a condition that prevents hyperpolarization following opening of K(+) channels. The objective of the present study was to determine whether inhibition of Ca(2+) influx through voltage-dependent channels participates in the response of coronary myocytes to hypoxia.
Experiments were performed on porcine anterior descendent coronary arterial rings and on enzymatically dispersed human and porcine myocytes of the same artery. Cytosolic Ca(2+) was measured by microfluorimetry and whole-cell currents were recorded with the patch clamp technique.
Hypoxia (O(2) tension approximately 20 mmHg) dilated endothelium-denuded porcine coronary arterial rings precontracted with high K(+) in the presence of glibenclamide (5 microM), a blocker of K(ATP) channels. In dispersed human and porcine myocytes, low O(2) tension decreased basal cytosolic Ca(2+) and transmembrane Ca(2+) influx independently of K(+) channel activation. In patch clamped cells, hypoxia reversibly inhibited L-type Ca(2+) channels. RT-PCR indicated that rHT is the predominant mRNA variant of the alpha(1C) Ca(2+) channel subunit in human coronary myocytes.
Our study demonstrates, for the first time in a human preparation, that voltage-gated Ca(2+)channels in coronary myocytes are under control of O(2) tension.
Exercise modulates metabolism and the gut microbiome. Brief exposure to low mT‐range pulsing electromagnetic fields (PEMFs) was previously shown to accentuate in vitro myogenesis and ...mitochondriogenesis by activating a calcium‐mitochondrial axis upstream of PGC‐1α transcriptional upregulation, recapitulating a genetic response implicated in exercise‐induced metabolic adaptations. We compared the effects of analogous PEMF exposure (1.5 mT, 10 min/week), with and without exercise, on systemic metabolism and gut microbiome in four groups of mice: (a) no intervention; (b) PEMF treatment; (c) exercise; (d) exercise and PEMF treatment. The combination of PEMFs and exercise for 6 weeks enhanced running performance and upregulated muscular and adipose Pgc‐1α transcript levels, whereas exercise alone was incapable of elevating Pgc‐1α levels. The gut microbiome Firmicutes/Bacteroidetes ratio decreased with exercise and PEMF exposure, alone or in combination, which has been associated in published studies with an increase in lean body mass. After 2 months, brief PEMF treatment alone increased Pgc‐1α and mitohormetic gene expression and after >4 months PEMF treatment alone enhanced oxidative muscle expression, fatty acid oxidation, and reduced insulin levels. Hence, short‐term PEMF treatment was sufficient to instigate PGC‐1α‐associated transcriptional cascades governing systemic mitohormetic adaptations, whereas longer‐term PEMF treatment was capable of inducing related metabolic adaptations independently of exercise.
1. The effects of hypoxia on cytosolic Ca2+ ¿Ca2+i) and spontaneous cytosolic Ca2+ spikes were examined in fura 2-loaded
myocytes isolated from conduit and resistance branches of the rabbit ...pulmonary artery. In all myocyte classes, generation
of the Ca2+ spikes was modulated by basal Ca2+i which, in turn, was influenced by the influx of Ca2+ through L-type Ca2+
channels of the plasmalemma. 2. Conduit and resistance myocytes responded distinctly to hypoxia. In most conduit myocytes
(approximately 82% of total; n = 23) exposure to hypoxia reduced basal Ca2+i. This effect was often associated with the
abolition of the Ca2+ spikes. Hypoxia gave rise to two main responses in resistance myocytes. In a subset of resistance myocytes
(41 % of total; n = 34) hypoxia incremented basal Ca2+i but reduced Ca2+ spike amplitude. This response mimicked the effect
of membrane depolarization with K+ and was reverted by nifedipine or the removal of extracellular Ca2+. In a second subset
of resistance myocytes (59% of total; n = 34) hypoxia decreased basal Ca2+i and, in most cases, increased spike amplitude;
a response counteracted by depolarization with K+. 3. These results indicate that hypoxia can differentially modulate Ca2+i
in smooth muscle cells from large and small diameter pulmonary vessels through a dual effect on transmembrane Ca2+ influx.
Our observations further demonstrate the longitudinal heterogeneity of myocytes along the pulmonary arterial tree and help
to explain the hypoxic vasomotor responses in the pulmonary circulation.
Single-channel activity was recorded from cell-attached patches on skeletal muscle cells isolated from wild-type mice and from mice carrying the dy or mdx mutations. Spontaneous openings of the ...nicotinic acetylcholine receptor channel (nAChR) were detected in virtually all recordings from either dy/dy or dy/+ myotubes, but only infrequently from wild-type or mdx myotubes. Spontaneous openings were also present in most recordings from undifferentiated myoblasts from all of the mouse strains studied. The biophysical properties of the spontaneous activity were similar to those of the embryonic form of the nAChR in the presence of acetylcholine (ACh). Examination of the single-channel currents evoked by low concentrations of ACh showed a reduced sensitivity to the agonist in the dystrophic dy and mdx myotubes, but not in wild-type myotubes. The results suggest that alterations in nAChR function are associated with the pathogenesis of muscular dystrophy in the dy mouse.
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