A Novel Treatment for Arrhythmias via the Control of the Degradation of Ion Channel Proteins

Although there are many reports on the regulation of ion channel expression in transcription and translation, few drugs have been studied to influence post-translational modification of ion channel proteins. The Kv1.5 channel is a potassium ion channel expressed in atrial muscle, belongs to the voltage-gated K+ channel superfamily, and forms an ultrarapid delayed rectifier potassium ion current. It is important to understand the fate of these channel proteins, as cardiac Kv1.5 mutations can cause arrhythmias. Disruption of quantitative and qualitative control mechanisms of channels leads to stagnation and degradation of intracellular channel proteins. As a result, ion channel proteins are not transported to the cell membrane and are involved in the development of atrial fibrillation. This review takes the Kv1.5 channel as an example and focuses on the degradation mechanism of ion channel proteins, and discusses its application to the treatment of arrhythmia by drugs that control the mechanism of ion channel protein degradation

Establishment of a Novel In Situ Rat Model for Direct Measuring of Intestinal Drug Absorption: Confirmation of Inhibitory Effects of Daijokito on the Absorption of Ranitidine

【Background】 Daijokito (DJKT), a classical traditional Kampo and Chinese medicine, has been used to treat acute pancreatitis in China. In our previous study, DJKT was found to reduce the area under the plasma concentration-time curve (AUC) of ranitidine in humans. Therefore, we established a novel rat model to examine the direct absorption of ranitidine after daijokito administration. 【Methods】 An in situ intestinal injection with portal vein sampling (IIPS) model was created to determine the rate of intestinal drug absorption. Rats were divided into two groups: the ranitidine group (R, n = 6) or the ranitidine and daijokito group (RD, n = 6). Blood was collected after intestinal injection of drugs. After the experiment, the concentrations of ranitidine were measured by LC/MS/MS analysis. 【Results】 The concentrations of ranitidine increased linearly with time in both groups. Compared with the R group, the concentrations of ranitidine in RD group significantly decreased throughout the experiment. 【Conclusion】 Co-administration of ranitidine with DJKT resulted in significant decreases in intestinal absorption in rats. The reduction of the systemic ranitidine concentration by co-administration of DJKT may be due, at least in part, to the inhibition of intestinal absorption of ranitidine

Characterization of the novel mutant A78T-HERG from a long QT syndrome type 2 patient: Instability of the mutant protein and stabilization by heat shock factor 1

Background:The human ether-a-go-go-related gene (HERG) encodes the α-subunit of rapidly activating delayed-rectifier potassium channels. Mutations in this gene cause long QT syndrome type 2 (LQT2). In most cases, mutations reduce the stability of the channel protein, which can be restored by heat shock (HS). Methods: We identified the novel mutant A78T-HERG in a patient with LQT2. The purpose of the current study was to characterize this mutant protein and test whether HS and heat shock factors (HSFs) could stabilize the mutant protein. A78T-HERG and wild-type HERG (WT-HERG) were expressed in HEK293 cells and analyzed by immunoblotting, immunoprecipitation, immunofluorescence, and whole-cell patch clamping. Results: When expressed in HEK293 cells, WT-HERG gave rise to immature and mature forms of the protein at 135 and 155 kDa, respectively. A78T-HERG gave rise only to the immature form, which was heavily ubiquitinated. The proteasome inhibitor MG132 increased the expression of immature A78T-HERG and increased both the immature and mature forms of WT-HERG. WT-HERG, but not A78T-HERG, was expressed on the plasma membrane. In whole-cell patch clamping experiments, depolarizing pulses evoked E4031-sensitive HERG channel currents in cells transfected with WT-HERG, but not in cells transfected with A78T-HERG. The A78V mutant, but not A78G mutant, remained in the immature form similarly to A78T. Maturation of the A78T-HERG protein was facilitated by HS, expression of HSF-1, or exposure to geranyl geranyl acetone. Conclusions: A78T-HERG was characterized by protein instability and reduced expression on the plasma membrane. The stability of the mutant was partially restored by HSF-1, indicating that HSF-1 is a target for the treatment for LQT2 caused by the A78T mutation in HERG

MicroRNA-494-3p inhibits formation of fast oxidative muscle fibres by targeting E1A-binding protein p300 in human-induced pluripotent stem cells.

MYOD-induced microRNA-494-3p expression inhibits fast oxidative myotube formation by downregulating myosin heavy chain 2 (MYH2) in human induced pluripotent stem cells (hiPSCs) during skeletal myogenesis. However, the molecular mechanisms regulating MYH2 expression via miR-494-3p remain unknown. Here, using bioinformatic analyses, we show that miR-494-3p potentially targets the transcript of the E1A-binding protein p300 at its 3\u27-untranslated region (UTR). Myogenesis in hiPSCs with the Tet/ON-myogenic differentiation 1 (MYOD1) gene (MyoD-hiPSCs) was induced by culturing them in doxycycline-supplemented differentiation medium for 7 days. p300 protein expression decreased after transient induction of miR-494-3p during myogenesis. miR-494-3p mimics decreased the levels of p300 and its downstream targets MYOD and MYH2 and myotube formation efficiency. p300 knockdown decreased myotube formation efficiency, MYH2 expression, and basal oxygen consumption rate. The binding of miR-494-3p to the wild type p300 3\u27-UTR, but not the mutated site, was confirmed using luciferase assay. Overexpression of p300 rescued the miR-494-3p mimic-induced phenotype in MyoD-hiPSCs. Moreover, miR-494-3p mimic reduced the levels of p300, MYOD, and MYH2 in skeletal muscles in mice. Thus, miR-494-3p might modulate MYH2 expression and fast oxidative myotube formation by directly regulating p300 levels during skeletal myogenesis in MyoD-hiPSCs and murine skeletal muscle tissues

Functional role of inward rectifier current in heart probed by Kir2.1 overexpression and dominant-negative suppression

The inward rectifier current I(K1) is tightly regulated regionally within the heart, downregulated in heart failure, and genetically suppressed in Andersen syndrome. We used in vivo viral gene transfer to dissect the role of I(K1) in cardiac repolarization and maintenance of the resting membrane potential (RMP) in guinea pig ventricular myocytes. Kir2.1 overexpression boosted Ba(2+)-sensitive I(K1) by more than 100% (at –50mV), significantly shortened action potential durations (APDs), accelerated phase 3 repolarization, and hyperpolarized RMP compared with control cells (nongreen cells from the same hearts and green cells from GFP-transduced hearts). The dominant-negative Kir2.1AAA reduced I(K1) by 50–90%; those cells with less than 80% reduction of I(K1) exhibited prolonged APDs, decelerated phase 3 repolarization, and depolarization of the RMP. Further reduction of I(K1) resulted in a pacemaker phenotype, as previously described. ECGs revealed a 7.7% ± 0.9% shortening of the heart rate–corrected QT interval (QTc interval) in Kir2.1-transduced animals (n = 4) and a 16.7% ± 1.8% prolongation of the QTc interval (n = 3) in Kir2.1AAA-transduced animals 72 hours after gene delivery compared with immediate postoperative recordings. Thus, I(K1) is essential for establishing the distinctive electrical phenotype of the ventricular myocyte: rapid terminal repolarization to a stable and polarized resting potential. Additionally, the long-QT phenotype seen in Andersen syndrome is a direct consequence of dominant-negative suppression of Kir2 channel function

Uric Acid as a Risk Factor for Chronic Kidney Disease and Cardiovascular Disease: Japanese Guideline on the Management of Asymptomatic Hyperuricemia

Serum uric acid (UA) is taken up by endothelial cells and reduces the level of nitric oxide (NO) by inhibiting its production and accelerating its degradation. Cytosolic and plasma xanthine oxidase (XO) generates superoxide and also decreases the NO level. Thus, hyperuricemia is associated with impaired endothelial function. Hyperuricemia is often associated with vascular diseases such as chronic kidney disease (CKD) and cardiovascular disease (CVD). It has long been debated whether hyperuricemia is causally related to the development of these diseases. The 2020 American College of Rheumatology Guideline for the Management of Gout (ACR2020) does not recommend pharmacological treatment of hyperuricemia in patients with CKD/CVD. In contrast, the Japanese Guideline on Management of Hyperuricemia and Gout (JGMHG), 3rdedition, recommends pharmacological treatment of hyperuricemia in patients with CKD. In a FREED study on Japanese hyperuricemic patients with CVD, an XO inhibitor, febuxostat, improved the primary composite endpoint of cerebro-cardio-renovascular events, providing a rationale for the use of urate-lowering agents (ULAs). Since a CARES study on American gout patients with CVD treated with febuxostat revealed increased mortality, ACR2020 recommends switching to different ULAs. However, there was no difference in the mortality of Japanese patients between the febuxostat-treated group and the placebo or allopurinol-treated groups in either the FEATHER or FREED studies

Cited4 is related to cardiogenic induction and maintenance of proliferation capacity of embryonic stem cell-derived cardiomyocytes during in vitro cardiogenesis.

Cardiac progenitor cells have a limited proliferative capacity. The CREB-binding protein/p300-interacting transactivator, with the Glu/Asp-rich carboxy-terminal domain (Cited) gene family, regulates gene transcription. Increased expression of the Cited4 gene in an adult mouse is associated with exercise-induced cardiomyocyte hypertrophy and proliferation. However, the expression patterns and functional roles of the Cited4 gene during cardiogenesis are largely unknown. Therefore, in the present study, we investigated the expression patterns and functional roles of the Cited4 gene during in vitro cardiogenesis. Using embryoid bodies formed from mouse embryonic stem cells, we evaluated the expression patterns of the Cited4 gene by quantitative reverse transcriptase-polymerase chain reaction. Cited4 gene expression levels increased and decreased during the early and late phases of cardiogenesis, respectively. Moreover, Cited4 gene levels were significantly high in the cardiac progenitor cell population. A functional assay of the Cited4 gene in cardiac progenitor cells using flow cytometry indicated that overexpression of the Cited4 gene significantly increased the cardiac progenitor cell population compared with the control and knockdown groups. A cell proliferation assay, with 5-ethynyl-2'-deoxyuridine incorporation and Ki67 expression during the late phase of cardiogenesis, indicated that the number of troponin T-positive embryonic stem cell-direived cardiomyocytes with proliferative capacity was significantly greater in the overexpression group than in the control and knockdown groups. Our study results suggest that the Cited4 gene is related to cardiac differentiation and maintenance of proliferation capacity of embryonic stem cell-derived cardiomyocytes during in vitro cardiogenesis. Therefore, manipulation of Cited4 gene expression may be of great interest for cardiac regeneration