Abstract The inward rectifier Kir2.1 current (IKir2.1) was reported to be facilitated by fluid flow. However, the mechanism underlying this facilitation remains uncertain. We hypothesized that during K + influx or efflux, [K + ] adjacent to the outer mouth of the Kir2.1 channel might decrease or increase, respectively, compared with the average [K + ] of the bulk extracellular solution, and that fluid flow could restore the original [K + ] and result in the apparent facilitation of IKir2.1. We recorded the IKir2.1 in RBL-2H3 cells and HEK293T cells that were ectopically over-expressed with Kir2.1 channels by using the whole-cell patch-clamp technique. Fluid-flow application immediately increased the IKir2.1, which was not prevented by either the pretreatment with inhibitors of various protein kinases or the modulation of the cytoskeleton and caveolae. The magnitudes of the increases of IKir2.1 by fluid flow were driving force-dependent. Simulations performed using the Nernst-Planck mass equation indicated that [K + ] near the membrane surface fell markedly below the average [K + ] of the bulk extracellular solution during K + influx, and, notably, that fluid flow restored the decreased [K + ] at the cell surface in a flow rate-dependent manner. These results support the “convection-regulation hypothesis” and define a novel interpretation of fluid flow-induced modulation of ion channels.
Abstract Angiotensin II (AngII) has potent cardiac hypertrophic effects mediated through activation of hypertrophic signaling like Wnt/b-Catenin signaling. In the current study, we examined the role of protein arginine methyltransferase 7 (PRMT7) in cardiac function. PRMT7 was greatly decreased in hypertrophic hearts chronically infused with AngII and cardiomyocytes treated with AngII. PRMT7 depletion in rat cardiomyocytes resulted in hypertrophic responses. Consistently, mice lacking PRMT7 exhibited displayed the cardiac hypertrophy and fibrosis. PRMT7 overexpression abrogated the cellular hypertrophy elicited by AngII, while PRMT7 depletion exacerbated the hypertrophic response caused by AngII. Similar with AngII treatment, the cardiac transcriptome analysis of PRMT7-deficient hearts revealed the alteration in gene expression profile related to Wnt signaling pathway. Inhibition of PRMT7 by gene deletion or an inhibitor treatment enhanced the activity of b-Catenin. PRMT7 deficiency decreases symmetric dimethylation of b-Catenin. Mechanistic studies reveal that methylation of arginine residue 93 in b-Catenin decreases the activity of b-Catenin. Taken together, our data suggest that PRMT7 is important for normal cardiac function through suppression of b-Catenin activity.
The peroxisome proliferator-activated receptor gamma (PPARγ) gene plays an important role in the biosynthesis process controlled by a number of fatty acid transcription factors. This study investigates the relationships between 130 single-nucleotide polymorphisms (SNPs) in the PPARγ gene and the fatty acid composition of muscle fat in the commercial population of Korean native cattle. We identified 38 SNPs and verified relationships between 3 SNPs (g.1159-71208 A>G, g.42555-29812 G>A, and g.72362 G>T) and the fatty acid composition of commercial Korean native cattle (n = 513). Cattle with the AA genotype of g.1159-71208 A>G and the GG genotype of g.42555-29812 G>A and g.72362 G>T had higher levels of monounsaturated fatty acids and carcass traits (p<0.05). The results revealed that the 3 identified SNPs in the PPARγ gene affected fatty acid composition and carcass traits, suggesting that these 3 SNPs may improve the flavor and quality of beef in commercial Korean native cattle. Keywords: Korean Native Cattle; PPARγ; SNP; Fatty Acid Composition; Marbling Score
Sonic hedgehog (Shh) signaling plays an important role for early heart development, such as heart looping and cardiomyogenesis of pluripotent stem cells. A multifunctional receptor Cdo functions as a Shh coreceptor together with Boc and Gas1 to activate Shh signaling and these coreceptors seem to play compensatory roles in early heart development. Thus in this study, we examined the role of Cdo in cardiomyogenesis by utilizing an in vitro differentiation of pluripotent stem cells. Here we show that Cdo is required for efficient cardiomyogenesis of pluripotent stem cells by activation of Shh signaling. Cdo is induced concurrently with Shh signaling activation upon induction of cardiomyogenesis of P19 embryonal carcinoma (EC) cells. Cdo -depleted P19 EC and Cdo –/– mouse embryonic stem (ES) cells display decreased expression of key cardiac regulators, including Gata4, Nkx2.5 and Mef2c and this decrease coincides with reduced Shh signaling activities. Furthermore Cdo deficiency causes a stark reduction in formation of mature contractile cardiomyocytes. This defect in cardiomyogenesis is overcome by reactivation of Shh signaling at the early specification stage of cardiomyogenesis. The Shh agonist treatment restores differentiation capacities of Cdo -deficient ES cells into contractile cardiomyocytes by recovering both the expression of early cardiac regulators and structural genes such as cardiac troponin T and Connexin 43. Therefore Cdo is required for efficient cardiomyogenesis of pluripotent stem cells and an excellent target to improve the differentiation potential of stem cells for generation of transplantable cells to treat cardiomyopathies.
Continuous identification and validation of novel drug targets require the development of rapid, reliable, and sensitive cell-based high-throughput screening (HTS) methods for proposed targets. Recently, the 5-HT6 receptor (5- HT6R), a member of the class of recently discovered 5-HT receptors, has received considerable attention for its possible implications in depression, cognition, and anxiety. However, the cellular signaling mechanisms of 5-HT6R are poorly understood due to the lack of selective 5-HT6R ligands. In the present study, we examined functional coupling of the human 5-HT6R, 5-HT7AR, or 5-HT7BR with various Gα-proteins (Gα15, Gαqs5, or GαqG66Ds5) to develop a reliable cell-based HTS method for 5-HT receptors. Among variable couplings between 5-HT receptors and G-proteins, we found that functional coupling of human 5-HT6R with GαqG66Ds5 produced the highest levels of Ca2+ signaling in HEK293 cells as measured by the fluorescence-based HTS plate reader, FDSS6000. After validation of this new 5-HT6R HTS system (Z´-factor = 0.56) in 96-well plates and characterization of the pharmacological profile of the 5-HT6R, we screened ∼500 synthetic chemical compounds including butanamide and benzenesulfonamide derivatives. Based on this preliminary screening, we found that the butanamide derivative LSG11104 produced an IC50 value of 6.3 μM. This compound will serve as a lead structure for further chemical modification to develop novel 5-HT6R ligands. Furthermore, we demonstrated that this HTS method can be utilized to identify proteins that modulate 5-HT6R function and present Fyn tyrosine kinase as an example, which is already known as a 5-HT6R interacting protein. Taken together, these results suggest that the 5-HT6R/GαqG66Ds5 FDSS6000 system can be utilized to screen for selective 5-HT6R ligands and to examine any functional relationships between 5-HT6R and its binding proteins. Keywords: 5-hyrdoxytryptamine (5-HT), 5-HT6 receptor, HTS, FDSS6000, chimeric G-protein, fluoresce Ca2+ imaging, Z´-factor, Fyn
Abstract HCN channels regulate excitability and rhythmicity in the hippocampal CA1 pyramidal cells. Perturbation in the HCN channel current ( I h ) is associated with neuropsychiatric disorders, such as autism spectrum disorders. Recently, protein arginine methyltransferase 7 (PRMT7) was shown to be highly expressed in the hippocampus, including the CA1 region. However, the physiological function of PRMT7 in the CA1 neurons and the relationship to psychiatric disorders are unclear. Here we showed that PRMT7 knockout (KO) mice exhibit hyperactivity and deficits in social interaction. The firing frequency of the CA1 neurons in the PRMT7 KO mice was significantly higher than that in the wild-type (WT) mice. Compared with the WT CA1 neurons, the PRMT7 KO CA1 neurons showed a more hyperpolarized resting potential and a higher input resistance, which were occluded by the I h -current inhibitor ZD7288; these findings were consistent with the decreased I h and suggested the contribution of I h -channel dysfunction to the PRMT7 KO phenotypes. The HCN1 protein level was decreased in the CA1 region of the PRMT7 KO mice in conjunction with a decrease in the expression of Shank3 , which encodes a core scaffolding protein for HCN channel proteins. A brief application of the PRMT7 inhibitor DS437 did not reproduce the phenotype of the PRMT7 KO neurons, further indicating that PRMT7 regulates I h by controlling the channel number rather than the open probability. Moreover, shRNA-mediated PRMT7 suppression reduced both the mRNA and protein levels of SHANK3, implying that PRMT7 deficiency might be responsible for the decrease in the HCN protein levels by altering Shank3 expression. These findings reveal a key role for PRMT7 in the regulation of HCN channel density in the CA1 pyramidal cells that may be amenable to pharmacological intervention for neuropsychiatric disorders.
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