Ca²⁺ Overload Decreased Cellular Viability in Magnetic Hyperthermia without a Macroscopic Temperature Rise

Magnetic hyperthermia is a crucial medical engineering technique for treating diseases, which usually uses alternating magnetic fields (AMF) to interplay with magnetic substances to generate heat. Recently, it has been found that in some cases, there is no detectable temperature increment after applying an AMF, which caused corresponding effects surprisingly. The mechanisms involved in this phenomenon are not yet fully understood. In this study, we aimed to explore the role of Ca2+ overload in the magnetic hyperthermia effect without a perceptible temperature rise. A cellular system expressing the fusion proteins TRPV1 and ferritin was prepared. The application of an AMF (518 kHz, 16 kA/m) could induce the fusion protein to release a large amount of iron ions, which then participates in the production of massive reactive oxygen radicals (ROS). Both ROS and its induced lipid oxidation enticed the opening of ion channels, causing intracellular Ca2+ overload, which further led to decreased cellular viability. Taken together, Ca2+ overload triggered by elevated ROS and the induced oxidation of lipids contributes to the magnetic hyperthermia effect without a perceptible temperature rise. These findings would be beneficial for expanding the application of temperature-free magnetic hyperthermia, such as in cellular and neural regulation, design of new cancer treatment methods

Reactive Oxygen Species Activate a Ferritin-Linked TRPV4 Channel under a Static Magnetic Field

Magnetogenetics has shown great potential for cell function and neuromodulation using heat or force effects under different magnetic fields; however, there is still a contradiction between experimental effects and underlying mechanisms by theoretical computation. In this study, we aimed to investigate the role of reactive oxygen species (ROS) in mechanical force-dependent regulation from a physicochemical perspective. The transient receptor potential vanilloid 4 (TRPV4) cation channels fused to ferritin (T4F) were overexpressed in HEK293T cells and exposed to static magnetic fields (sMF, 1.4–5.0 mT; gradient: 1.62 mT/cm). An elevation of ROS levels was found under sMF in T4F-overexpressing cells, which could lead to lipid oxidation. Compared with the overexpression of TRPV4, ferritin in T4F promoted the generation of ROS under the stimulation of sMF, probably related to the release of iron ions from ferritin. Then, the resulting ROS regulated the opening of the TRPV4 channel, which was attenuated by the direct addition of ROS inhibitors or an iron ion chelator, highlighting a close relationship among iron release, ROS production, and TRPV4 channel activation. Taken together, these findings indicate that the produced ROS under sMF act on the TRPV4 channel, regulating the influx of calcium ions. The study would provide a scientific basis for the application of magnetic regulation in cellular or neural regulation and disease treatment and contribute to the development of the more sensitive regulatory technology

Model for Acquisition of Dorsoventral Patterning in the Trunk and the Role of <i>Tbx15</i>

<div><p>(A) A tricolor pigmentation pattern is generated by the combination of distinct mechanisms that affect distribution of <i>Agouti</i> mRNA and histochemical staining for melanocytes; effects of the latter mechanism by itself are evident in <i>a^e</i>/<i>a^e</i> mice (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020003#pbio-0020003-g001" target="_blank">Figure 1</a>). In <i>a^t/a^t</i> mice, reduced hair melanocyte activity and high levels of <i>Agouti</i> mRNA in the ventrum lead to a cream color; as melanocyte activity gradually increases towards the dorsum, a lateral stripe is apparent on the flank. The distributions of <i>Agouti</i> mRNA and histochemical staining for melanocytes are both affected by <i>Tbx15</i> and are externally evident by a widening of the lateral stripe and an increased proportion of total skin occupied by the cream-colored area.</p> <p>(B) The lateral yellow stripe in <i>a^t/a^t</i> mice lies at the same level as the limb dorsoventral boundary. As described in the text, we propose that distinct dorsoventral compartments in ectoderm of the trunk provide an instructional cue to the mesoderm, leading to expression of <i>Tbx15</i> in dorsal trunk mesenchyme and acquisition of dorsal dermis character. In the absence of <i>Tbx15</i>, dorsal mesenchyme assumes ventral characteristics instead.</p></div

Comparison of the Dorsoventral <i>a^t/a^t</i> Pigmentation Boundary to the Lateral Somitic Frontier

<div><p>(A) Dorsoventral slices of skin from at the midtrunk region prepared such that the dorsal midline lies in the center of the slice. Sections were taken at P1.5 (a) or P4.5 (b–e) from <i>a^t/a^t</i> or <i>R26R</i>/+; <i>Tg.Hoxb6-Cre</i>/+ mice (the latter were stained with X-Gal), as described in Materials and Methods. For purposes of comparison, images were proportionally scaled. The boundary of X-Gal staining marks dermis derived from lateral plate versus dermis derived from mesoderm (the lateral somitic frontier) and lies more dorsal than the <i>a^t/a^t</i> pigmentation boundary.</p> <p>(B) Quantitation of mean (± SEM) dorsal pigmentation area (<i>n</i> = 5) and somite-derived dermis area (<i>n</i> = 3) shows a significant difference (<i>p</i> < 0.005, <i>t</i>-test).</p> <p>(C) RNA in situ hybridization showing that <i>Tbx15</i> expression at E11.5 is complementary to <i>En1</i> expression on the flank (scale bars = 200 μm). The arrow indicates the boundary between the expression domains of the two genes.</p></div

Embryonic Expression of <i>Tbx15</i> Compared to <i>Agouti</i> in <i>a^t</i>/<i>a^t</i> Mice

<p>(A and C) <i>Tbx15</i>. (B and D) <i>Agouti</i>. At E12.5, expression of <i>Tbx15</i> in dorsal skin is approximately complementary to that of <i>Agouti</i> in ventral skin. At E14.5, the levels of expression for both genes are lower, but <i>Tbx15</i> expression has expanded ventrally and overlaps extensively with that of <i>Agouti</i>. In all four panels, arrows mark the approximate ventral limit of <i>Tbx15</i> and the approximate dorsal limit of <i>Agouti</i> (scale bars = 500 μm).</p

The <i>de^H</i> Pigmentation Phenotype

<div><p>(A) 10-wk-old <i>de^H/de^H</i> and nonmutant animals on a <i>a^t</i> background. A thin stripe of yellow hair normally separates the dorsal black hairs from the ventral cream hairs. In <i>de^H</i>, the yellow stripe is extended dorsally, and the boundary between the yellow and the black hairs is fuzzier.</p> <p>(B) Skin slices taken from 1.5-mo-old <i>de^H/de^H</i> and nonmutant littermates (scale bar = 0.5 cm).</p> <p>(C) Proportion of total skin area as determined by observation of pelts taken from the interlimb region. The proportion occupied by the yellow lateral compartment (± SEM) differs between mutant and nonmutant littermate flanks (<i>p</i> < 0.0005, paired <i>t</i>-test, <i>n</i> = 6 pairs). There is also (data not shown) a small increase in the proportion of total skin area occupied by the ventral cream-colored compartment, 47.9 % in mutant compared to 37.8% in nonmutant (<i>p</i> < 0.005, paired <i>t</i>-test, <i>n</i> = 6 pairs).</p> <p>(D) On an <i>a^e/a^e</i> background, the extent of dorsal skin pigmentation is reduced in <i>de^H/de^H</i> neonates (P3.5).</p> <p>(E) Hair length in a representative pair of 1.5-mo-old <i>de^H/de^H</i> and nonmutant littermates, averaged over three skin slices at different rostrocaudal levels, and plotted as a function of the absolute distance from middorsum or the percentage of total slice length.</p></div

Dorsoventral Skin Characteristics

<div><p>(A) Skin slices from animals of different age and genotype demonstrate similar patterns of hair-length variation along the dorsoventral axis (scale bar = 1 cm).</p> <p>(B) Enlarged area from (A), demonstrating the transition in hair length and color in <i>a^t</i>/<i>a^t</i> mice (scale bar = 0.375 cm).</p> <p>(C) Proportional hair length for (A) plotted as a function of relative position along the dorsoventral axis.</p> <p>(D) Hair length plotted as a function of absolute position along the dorsoventral axis for 8-wk-old BA strain mice.</p> <p>(E) Proportion of zigzag hairs (± SEM) differs slightly between dorsum and ventrum of inbred mice (<i>p</i> < 0.0001, χ² test, <i>n</i> = 1,958, 1,477, 1,579, 1,502).</p> <p>(F) Differences in dorsal and ventral skin development at P4.5 (scale bar = 1 mm, upper; 200 μm, lower).</p> <p>(G) Differences in hair melanin content and DOPA staining for dorsum (d), flank (f), and ventrum (v) in <i>a^e</i>/<i>a^e</i> and <i>a^t</i>/<i>a^t</i> mice. The upper panel also demonstrates a cream-colored appearance of the <i>a^t</i>/<i>a^t</i> ventrum. The middle panel shows representative awls (scale bar = 100 μm). The lower panel shows DOPA-stained dermis (scale bar = 200 μm).</p></div

Molecular Genetics of <i>de^H</i> and <i>Tbx15</i>

<div><p>(A) Genetic and physical map, as described in the text. Markers M1 to M3 are SSCP markers generated from a BAC contig of the region; marker M4 is STS 16.MMHAP32FLF1 and was also used as an SSCP marker. M2 and M3, which flank the <i>Tbx15</i> and <i>M6pr-ps</i> on the UCSC genome browser map and lie 634 kb apart, were nonrecombinant with <i>de^H</i> in 2340 meioses.</p> <p>(B) The <i>de^H</i> mutation is a deletion that starts in <i>Tbx15</i> intron 1 and ends in the <i>M6pr-ps</i>.</p> <p>(C) Sequence of deletion breakpoints.</p> <p>(D) Diagram of <i>Tbx15^LacZ</i> allele constructed by gene targeting. As described in the text, this allele is predicted to give rise to a protein truncated after approximately 154 codons and is lacking critical residues of the T box. Heterozygotes for the targeted allele exhibit normal size, morphology, and hair-color patterns, but homozygotes and <i>Tbx15^LacZ</i>/<i>de^H</i> compound heterozygotes are identical to <i>de^H</i> homozygotes.</p></div

Effect of <i>de^H</i> on <i>Agouti</i> Expression

<div><p>Comparable sections from <i>a^t/a^t</i>; <i>de^H</i>/<i>de^H</i> and <i>a^t/a^t</i>; +/+ littermates.</p> <p>(A) At E14.5, <i>de^H</i>/<i>de^H</i> embryos have a smaller body cavity and loose skin within which <i>Agouti</i> expression appears to be shifted dorsally, as marked by arrows (scale bars = 500 μm).</p> <p>(B) At P4.5, <i>Agouti</i> expression in both dorsal and ventral skin is similar in <i>de^H</i>/<i>de^H</i> compared to nonmutant, but in the midflank region, there is increased <i>Agouti</i> expression in <i>de^H</i>/<i>de^H</i>, especially in the upper dermis (scale bars = 200 μm). Sections shown are representative of two mutant and two nonmutant samples examined at each time.</p></div

Developmental Expression of <i>Tbx15</i>

<div><p>(A) At E12.5, transverse sections at different levels show expression in head mesenchyme (a and b); myotome, occipital, and periocular mesenchyme (b); palatal shelf, cervical sclerotome, and nasal cartilage (c); maxillary and mandibular processes (d); limbs (e); and myotome and lateral mesenchyme (e and f) (scale bars = 500 μm).</p> <p>(B) Transverse sections through the flank at different times show expression in lateral mesenchyme (E11.5), expanding dorsally at E12.5, and both ventrally and dorsally at E13.5, detectable in loose mesenchyme underlying the dermis and the abdominal and subcutaneous muscles (scale bar = 500 μm). At P3.5, <i>Tbx15</i> is expressed in the entire dermis and is most strongly expressed in dermal sheaths (scale bar = 200 μm).</p></div