Targeted ‘knockdown’ of channel expression in vivo with an antisense morpholino oligonucleotide

Circadian rhythms are generated by cell autonomous circadian clocks that perform a ubiquitous cellular time-keeping function and cell type-specific functions important for normal physiology. Studies show inducing the deletion of the core circadian clock transcription factor Bmal1 in adult mouse cardiomyocytes disrupts cardiac circadian clock function, cardiac ion channel expression, slows heart rate, and prolongs the QT-interval at slow heart rates. This study determined how inducing the deletion of Bmal1 in adult cardiomyocytes impacted the in vivo electrophysiological phenotype of a knock-in mouse model for the arrhythmogenic long QT syndrome (Scn5a+/ΔKPQ). Electrocardiographic telemetry showed inducing the deletion of Bmal1 in the cardiomyocytes of mice with or without the ΔKPQ-Scn5a mutation increased the QT-interval at RR-intervals that were ≥130 ms. Inducing the deletion of Bmal1 in the cardiomyocytes of mice with or without the ΔKPQ-Scn5a mutation also increased the day/night rhythm-adjusted mean in the RR-interval, but it did not change the period, phase or amplitude. Compared to mice without the ΔKPQ-Scn5a mutation, mice with the ΔKPQ-Scn5a mutation had reduced heart rate variability (HRV) during the peak of the day/night rhythm in the RR-interval. Inducing the deletion of Bmal1 in cardiomyocytes did not affect HRV in mice without the ΔKPQ-Scn5a mutation, but it did increase HRV in mice with the ΔKPQ-Scn5a mutation. The data demonstrate that deleting Bmal1 in cardiomyocytes exacerbates QT- and RR-interval prolongation in mice with the ΔKPQ-Scn5a mutation.

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Visualization in zebrafish larvae of Na+ uptake in mitochondria-rich cells whose differentiation is dependent on foxi3a

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00200.2006 ◽

2007 ◽

Vol 292 (1) ◽

pp. R470-R480 ◽

Cited By ~ 73

Author(s):

Masahiro Esaki ◽

Kazuyuki Hoshijima ◽

Sayako Kobayashi ◽

Hidekazu Fukuda ◽

Koichi Kawakami ◽

...

Keyword(s):

Transcription Factor ◽

Cell Differentiation ◽

Direct Evidence ◽

Zebrafish Embryos ◽

Morpholino Oligonucleotide ◽

Gastrula Stage ◽

Zebrafish Larvae ◽

Transport Inhibitors ◽

Antisense Morpholino ◽

Na Uptake

Uptake of Na+ from the environment is an indispensable strategy for the survival of freshwater fish, as they easily lose Na+ from the plasma to a diluted environment. Nevertheless, the location of and molecules involved in Na+ uptake remain poorly understood. In this study, we utilized Sodium Green, a Na+-dependent fluorescent reagent, to provide direct evidence that Na+ absorption takes place in a subset of the mitochondria-rich (MR) cells on the yolk sac surface of zebrafish larvae. Combined with immunohistochemistry, we revealed that the Na+-absorbing MR cells were exceptionally rich in vacuolar-type H+-ATPase (H+-ATPase) but moderately rich in Na+-K+-ATPase. We also addressed the function of foxi3a, a transcription factor that is specifically expressed in the H+-ATPase-rich MR cells. When foxi3a was depleted from zebrafish embryos by antisense morpholino oligonucleotide injection, differentiation of the MR cells was completely blocked and Na+ influx was severely reduced, indicating that MR cells are the primary sites for Na+ absorption. Additionally, foxi3a expression is initiated at the gastrula stage in the presumptive ectoderm; thus, we propose that foxi3a is a key gene in the control of MR cell differentiation. We also utilized a set of ion transport inhibitors to assess the molecules involved in the process and discuss the observations.

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Downregulation of miR-503 in Activated Kidney Fibroblasts Disinhibits KCNN4 in an in Vitro Model of Kidney Fibrosis

Kidney & Blood Pressure Research ◽

10.1159/000498875 ◽

2019 ◽

Vol 44 (1) ◽

pp. 113-122 ◽

Cited By ~ 2

Author(s):

Christoph Mann ◽

Brajesh Pratap Kaistha ◽

Michael Kacik ◽

Thorsten Stiewe ◽

Joachim Hoyer

Keyword(s):

Kidney Fibrosis ◽

Calcium Dependent ◽

Channel Current ◽

Channel Expression ◽

Pharmacological Blockade ◽

Vitro Model ◽

End Stage ◽

Activated Fibroblasts

Background/Aims: Activated fibroblasts are key controllers of extracellular matrix turnover in kidney fibrosis, the pathophysiological end stage of chronic kidney disease. The proliferation of activated fibroblasts depends on the expression of the calcium-dependent potassium channel KCNN4. Expression of this ion channel is upregulated in fibrotic kidneys. Genetic and pharmacological blockade of KCNN4 inhibits fibrosis in vitro and in vivo. Methods: We studied the regulation of KCNN4 and possible involvement of miRNAs in an in-vitro fibrosis model using murine kidney fibroblasts. We tested fibroblast proliferation, channel function, channel expression and expression regulation after FGF-2 stimulation. Results: Proliferation was significantly increased by FGF-2, channel current and expression were almost doubled (+ 91% and +125%, respectively). MiRNA microarray identified upregulation of miRNA-503, which targets RAF1 and thereby controls KCNN4-expression via disinhibition of the Ras/Raf/MEK/ ERK-cascade. Conclusion: This data show a) a profound upregulation of KCNN4 in stimulated fibroblast and b) identifies miR-503 as a regulator of KCNN4 expression.

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Regulation of voltage-gated sodium channel expression in cancer: hormones, growth factors and auto-regulation

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2013.0105 ◽

2014 ◽

Vol 369 (1638) ◽

pp. 20130105 ◽

Cited By ~ 77

Author(s):

Scott P. Fraser ◽

Iley Ozerlat-Gunduz ◽

William J. Brackenbury ◽

Elizabeth M. Fitzgerald ◽

Thomas M. Campbell ◽

...

Keyword(s):

Growth Factors ◽

Voltage Gated ◽

Metastatic Cells ◽

Channel Expression ◽

Expression Activity ◽

Hormone Sensitive ◽

Breast And Prostate Cancer ◽

Activity Dependent

Although ion channels are increasingly being discovered in cancer cells in vitro and in vivo , and shown to contribute to different aspects and stages of the cancer process, much less is known about the mechanisms controlling their expression. Here, we focus on voltage-gated Na + channels (VGSCs) which are upregulated in many types of carcinomas where their activity potentiates cell behaviours integral to the metastatic cascade. Regulation of VGSCs occurs at a hierarchy of levels from transcription to post-translation. Importantly, mainstream cancer mechanisms, especially hormones and growth factors, play a significant role in the regulation. On the whole, in major hormone-sensitive cancers, such as breast and prostate cancer, there is a negative association between genomic steroid hormone sensitivity and functional VGSC expression. Activity-dependent regulation by positive feedback has been demonstrated in strongly metastatic cells whereby the VGSC is self-sustaining, with its activity promoting further functional channel expression. Such auto-regulation is unlike normal cells in which activity-dependent regulation occurs mostly via negative feedback. Throughout, we highlight the possible clinical implications of functional VGSC expression and regulation in cancer.

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Altered expression and modulation of the two-pore-domain (K2P) mechanogated potassium channel TREK-1 in overactive human detrusor

AJP Renal Physiology ◽

10.1152/ajprenal.00638.2016 ◽

2017 ◽

Vol 313 (2) ◽

pp. F535-F546 ◽

Cited By ~ 4

Author(s):

Ricardo H. Pineda ◽

Balachandar Nedumaran ◽

Joseph Hypolite ◽

Xiao-Qing Pan ◽

Shandra Wilson ◽

...

Keyword(s):

Close Association ◽

Cytoskeletal Proteins ◽

Bladder Smooth Muscle ◽

Altered Expression ◽

Channel Expression ◽

Filling Phase ◽

And Function ◽

Pore Domain

Detrusor overactivity (DO) is the abnormal response of the urinary bladder to physiological stretch during the filling phase of the micturition cycle. The mechanisms of bladder smooth muscle compliance upon the wall stretch are poorly understood. We previously reported that the function of normal detrusor is regulated by TREK-1, a member of the mechanogated subfamily of two-pore-domain potassium (K2P) channels. In the present study, we aimed to identify the changes in expression and function of TREK-1 channels under pathological conditions associated with DO, evaluate the potential relationship between TREK-1 channels and cytoskeletal proteins in the human bladder, and test the possibility of modulation of TREK-1 channel expression by small RNAs. Expression of TREK-1 channels in DO specimens was 2.7-fold decreased compared with control bladders and was associated with a significant reduction of the recorded TREK-1 currents. Isolated DO muscle strips failed to relax when exposed to a TREK-1 channel opener. Immunocytochemical labeling revealed close association of TREK-1 channels with cell cytoskeletal proteins and caveolins, with caveolae microdomains being severely disrupted in DO specimens. Small activating RNA (saRNA) tested in vitro provided evidence that expression of TREK-1 protein could be partially upregulated. Our data confirmed a significant downregulation of TREK-1 expression in human DO specimens and provided evidence of close association between the channel, cell cytoskeleton, and caveolins. Upregulation of TREK-1 expression by saRNA could be a future step for the development of in vivo pharmacological and genetic approaches to treat DO in humans.

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