Pulsed electromagnetic fields synergize with graphene to enhance dental pulp stem cell-derived neurogenesis by selectively targeting TRPC1 channels.

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.

Time-lapse imaging of in vitro myogenesis using atomic force microscopy.

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.

Reduction of Ca(2+) channel activity by hypoxia in human and porcine coronary myocytes.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Distinct ion channel classes are expressed on the outer nuclear envelope of T- and B-lymphocyte cell lines.

The outer nuclear membrane, endoplasmic reticulum, and mitochondrial membrane ion channels are poorly understood, although they are important in the control of compartmental calcium levels, cell division, and apoptosis. Few direct recordings of these ion channels have been made because of the difficulty of accessing these intracellular membranes. Using patch-clamp techniques on isolated nuclei, we measured distinct ion channel classes on the outer nuclear envelope of T-cell (human Jurkat) and BFL5 cell (murine promyelocyte) lines. We first imaged the nuclear envelopes of both Jurkat and FL5 cells with atomic force microscopy to determine the density of pore proteins. The nuclear pore complex was intact at roughly similar densities in both cell types. In patch-clamp recordings of Jurkat nuclear membranes, Cl channels (105 +/- 5 pS) predominated and inactivated with negative pipette potentials. Nucleotides transiently inhibited the anion channel. In contrast, FL5 nuclear channels were cation selective (52 +/- 2 pS), were inactivated with positive membrane potentials, and were insensitive to GTPgammaS applied to the bath. We hypothesize that T- and B-cell nuclear membrane channels are distinct, and that this is perhaps related to their unique roles in the immune system.

Low PO2 inhibits calcium channel activity in arterial smooth muscle cells.

We studied the effect of O2 tension (PO2) on the activity of voltage-gated Ca2+ channels recorded in whole cell patch-clamped smooth muscle cells enzymatically dispersed from rabbit cerebral, celiac, femoral, and main pulmonary arteries, as well as from the porcine coronary artery. In all myocyte classes examined, a reduction of PO2 (hypoxia) produced a rapid and reversible inhibition of the macroscopic L-type Ca2+ current of similar general characteristics. The hypoxic inhibition of Ca2+ channel activity closely followed the time course of bath exchange, first becoming apparent at below approximately 80 mmHg PO2. The interaction of O2 with the Ca2+ channels was strongly voltage dependent. At -30 mV the average extent of current inhibition was approximately 80%; however, no effect or even potentiation of current amplitude was observed at potentials more positive than +30 mV. Hypoxia selectively slowed activation kinetics (approximately 1.5 times at -20 mV); however, channel deactivation and inactivation were unaltered by low PO2. In addition, hypoxia produced a reversible shift (8.1 +/- 1.0 mV, n = 12) of the Ca2+ conductance-voltage curve toward positive membrane potentials. We propose that the O2 sensitivity of Ca2+ channels may contribute to the well-known hypoxic dilatation of systemic and the main pulmonary arteries.

Contrasting effects of hypoxia on cytosolic Ca2+ spikes in conduit and resistance myocytes of the rabbit pulmonary artery.

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.

Differential oxygen sensitivity of calcium channels in rabbit smooth muscle cells of conduit and resistance pulmonary arteries.

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.

Spontaneous opening of the acetylcholine receptor channel in developing muscle cells from normal and dystrophic mice.

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.

Oxygen-sensitive calcium channels in vascular smooth muscle and their possible role in hypoxic arterial relaxation.

We have investigated the modifications of cytosolic [Ca2+] and the activity of Ca2+ channels in freshly dispersed arterial myocytes to test whether lowering O2 tension (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 PO2 of 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 PO2 selectively inhibits L-type Ca2+ channel activity, whereas the current mediated by T-type channels is unaltered by hypoxia. The effect of low PO2 on 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.

Mechanosensitive ion channels in skeletal muscle from normal and dystrophic mice.

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.