The total xanthones extracted from Gentianella acuta alleviates HFpEF by activating the IRE1α/Xbp1s pathway.

Heart failure with preserved ejection fraction (HFpEF) is a clinical syndrome characterized by pulmonary and systemic congestion resulting from left ventricular diastolic dysfunction and increased filling pressure. Currently, however, there is no evidence on effective pharmacotherapy for HFpEF. In this study, we aimed to investigate the therapeutic effect of total xanthones extracted from Gentianella acuta (TXG) on HFpEF by establishing an high-fat diet (HFD) + L-NAME-induced mouse model. Echocardiography was employed to assess the impact of TXG on the cardiac function in HFpEF mice. Haematoxylin and eosin staining, wheat germ agglutinin staining, and Masson's trichrome staining were utilized to observe the histopathological changes following TXG treatment. The results demonstrated that TXG alleviated HFpEF by reducing the expressions of genes associated with myocardial hypertrophy, fibrosis and apoptosis. Furthermore, TXG improved cardiomyocyte apoptosis by inhibiting the expression of apoptosis-related proteins. Mechanistic investigations revealed that TXG could activate the inositol-requiring enzyme 1α (IRE1α)/X-box-binding protein 1 (Xbp1s) signalling pathway, but the knockdown of IRE1α using the IRE1α inhibitor STF083010 or siRNA-IRE1α impaired the ability of TXG to ameliorate cardiac remodelling in HFpEF models. In conclusion, TXG alleviates myocardial hypertrophy, fibrosis and apoptosis through the activation of the IRE1α/Xbp1s signalling pathway, suggesting its potential beneficial effects on HFpEF patients.

Electrophysiological evaluation of an anticancer drug gemcitabine on cardiotoxicity revealing down-regulation and modification of the activation gating properties in the human rapid delayed rectifier potassium channel.

Gemcitabine is an antineoplastic drug commonly used in the treatment of several types of cancers including pancreatic cancer and non-small cell lung cancer. Although gemcitabine-induced cardiotoxicity is widely recognized, the exact mechanism of cardiac dysfunction causing arrhythmias remains unclear. The objective of this study was to electrophysiologically evaluate the proarrhythmic cardiotoxicity of gemcitabine focusing on the human rapid delayed rectifier potassium channel, hERG channel. In heterologous hERG expressing HEK293 cells (hERG-HEK cells), hERG channel current (IhERG) was reduced by gemcitabine when applied for 24 h but not immediately after the application. Gemcitabine modified the activation gating properties of the hERG channel toward the hyperpolarization direction, while inactivation, deactivation or reactivation gating properties were unaffected by gemcitabine. When gemcitabine was applied to hERG-HEK cells in combined with tunicamycin, an inhibitor of N-acetylglucosamine phosphotransferase, gemcitabine was unable to reduce IhERG or shift the activation properties toward the hyperpolarization direction. While a mannosidase I inhibitor kifunensine alone reduced IhERG and the reduction was even larger in combined with gemcitabine, kifunensine was without effect on IhERG when hERG-HEK cells were pretreated with gemcitabine for 24 h. In addition, gemcitabine down-regulated fluorescence intensity for hERG potassium channel protein in rat neonatal cardiomyocyte, although hERG mRNA was unchanged. Our results suggest the possible mechanism of arrhythmias caused by gemcitabine revealing a down-regulation of IhERG through the post-translational glycosylation disruption possibly at the early phase of hERG channel glycosylation in the endoplasmic reticulum that alters the electrical excitability of cells.

miR-223-3p inhibits the progression of atherosclerosis via down-regulating the activation of MEK1/ERK1/2 in macrophages.

BACKGROUND: microRNAs (miRNAs) have drawn more attention to the progression of atherosclerosis (AS), due to their noticeable inflammation function in cardiovascular disease. Macrophages play a crucial role in disrupting atherosclerotic plaque, thereby we explored the involvement of miR-223-3p in the inflammatory response in macrophages. METHODS: RT-qPCR was used to analyze the miR-223-3p levels in carotid arteries and serum of AS patients. ROC curve was used to assess the diagnostic value of miR-223-3p. Movat staining was applied to evaluate the morphological differences. FISH was used to identify the expression of miR-223-3p in macrophages of atherosclerotic lesions. Bioinformatic analysis was performed. Double-immunofluorescence and western blot were performed to assess the inflammatory cytokine secretion and p-ERK1/2. C16-PAF was injected into the culture medium of the miR-223-3p mimic/NC-transfected macrophages with ox-LDL. RESULTS: MiR-223-3p was up-regulated in AS patients and was associated with a higher overall survival rate. MiR-223-3p was co-localized with CD68+ macrophages in vulnerable atherosclerotic lesions. MiR-223-3p mimics decreased atherosclerotic lesions, macrophages numbers whereas increased SMCs numbers in the lesions. The TNF-a immune-positive areas were reduced by miR-223-3p mimics. MAP2K1 was negatively associated with miR-223-3p. MiR-223-3p mimics reduced the inflammation and the MEK1/ERK1/2 signaling pathway in vivo and in vitro. C16-PAF reversed the effects of miR-223-3p mimics on inflammation and ERK1/2 signaling pathway. CONCLUSIONS: MiR-223-3p negatively regulates inflammatory responses by the MEK1/ERK1/2 signaling pathway. Our study provides new insight into how miR-223-3p protects against atherosclerosis, representing a broader therapeutic prospect for treating atherosclerosis by miR-223-3p.

The Role of METTL3-Mediated N6-Methyladenosine (m6A) of JPH2 mRNA in Cyclophosphamide-Induced Cardiotoxicity.

Cyclophosphamide (CYP)-induced cardiotoxicity is a common side effect of cancer treatment. Although it has received significant attention, the related mechanisms of CYP-induced cardiotoxicity remain largely unknown. In this study, we used cell and animal models to investigate the effect of CYP on cardiomyocytes. Our data demonstrated that CYP-induced a prolonged cardiac QT interval and electromechanical coupling time courses accompanied by JPH2 downregulation. Moreover, N6-methyladenosine (m6A) methylation sequencing and RNA sequencing suggested that CYP induced cardiotoxicity by dysregulating calcium signaling. Importantly, our results demonstrated that CYP induced an increase in the m6A level of JPH2 mRNA by upregulating methyltransferases METTL3, leading to the reduction of JPH2 expression levels, as well as increased field potential duration and action potential duration in cardiomyocytes. Our results revealed a novel mechanism for m6A methylation-dependent regulation of JPH2, which provides new strategies for the treatment and prevention of CYP-induced cardiotoxicity.

Nitric oxide down-regulates voltage-gated Na+ channel in cardiomyocytes possibly through S-nitrosylation-mediated signaling.

Nitric oxide (NO) is produced from endothelial cells and cardiomyocytes composing the myocardium and benefits cardiac function through both vascular-dependent and-independent effects. This study was purposed to investigate the possible adverse effect of NO focusing on the voltage-gated Na+ channel in cardiomyocytes. We carried out patch-clamp experiments on rat neonatal cardiomyocytes demonstrating that NOC-18, an NO donor, significantly reduced Na+ channel current in a dose-dependent manner by a long-term application for 24 h, accompanied by a reduction of Nav1.5-mRNA and the protein, and an increase of a transcription factor forkhead box protein O1 (FOXO1) in the nucleus. The effect of NOC-18 on the Na+ channel was blocked by an inhibitor of thiol oxidation N-ethylmaleimide, a disulfide reducing agent disulfide 1,4-Dithioerythritol, or a FOXO1 activator paclitaxel, suggesting that NO is a negative regulator of the voltage-gated Na+ channel through thiols in regulatory protein(s) for the channel transcription.

Disruption of asparagine-linked glycosylation to rescue and alter gating of the NaV1.5-Na+ channel.

SCN5A gene encodes the voltage-gated sodium channel NaV1.5 which is composed of a pore-forming α subunit of the channel. Asparagine (N)-linked glycosylation is one of the common post-translational modifications in proteins. The aim of this study was to investigate impact of N-linked glycosylation disruption on the Na+ channel, and the mechanism by which glycosylation regulates the current density and gating properties of the Na+ channel. The NaV1.5-Na+ channel isoform (α submit) derived from human was stably expressed in human embryonic kidney (HEK)-293 cells (Nav1.5-HEK cell). We applied the whole-cell patch-clamp technique to study the impact of N-linked glycosylation disruption in Nav1.5-HEK cell. Inhibition of the N-glycosylation with tunicamycin caused a significant increase of NaV1.5 channel current (INa) when applied for 24 h. Tunicamycin shifted the steady-state inactivation curve to the hyperpolarization direction, whereas the activation curve was unaffected. Recovery from inactivation was prolonged, while the fast phase (τfast) and the slow phase (τslow) of the current decay was unaffected by tunicamycin. INa was unaffected by tunicamycin in the present of a proteasome inhibitor MG132 [N-[(phenylmethoxy)carbonyl]-L-leucy-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide], while it was significantly increased by tunicamycin in the presence of a lysosome inhibitor butyl methacrylate (BMA). These findings suggest that N-glycosylation disruption rescues the NaV1.5 channel possibly through the alteration of ubiquitin-proteasome activity, and changes gating properties of the NaV1.5 channel by modulating glycan milieu of the channel protein.

Asparagine-linked glycosylation modifies voltage-dependent gating properties of CaV3.1-T-type Ca2+ channel.

T-type channels are low-voltage-activated channels that play a role in the cardiovascular system particularly for pacemaker activity. Glycosylation is one of the most prevalent post-translational modifications in protein. Among various glycosylation types, the most common one is asparagine-linked (N-linked) glycosylation. The aim of this study was to elucidate the roles of N-linked glycosylation for the gating properties of the CaV3.1-T-type Ca2+ channel. N-linked glycosylation synthesis inhibitor tunicamycin causes a reduction of CaV3.1-T-type Ca2+ channel current (CaV3.1-ICa.T) when applied for 12 h or longer. Tunicamycin (24 h) significantly shifted the activation curve to the depolarization potentials, whereas the steady-state inactivation curve was unaffected. Use-dependent inactivation of CaV3.1-ICa.T was accelerated, and recovery from inactivation was prolonged by tunicamycin (24 h). CaV3.1-ICa.T was insensitive to a glycosidase PNGase F when the channels were expressed on the plasma membrane. These findings suggest that N-glycosylation contributes not only to the cell surface expression of the CaV3.1-T-type Ca2+ channel but to the regulation of the gating properties of the channel when the channel proteins were processed during the folding and trafficking steps in the cell.

Short- and long-term roles of phosphatidylinositol 4,5-bisphosphate PIP2 on Cav3.1- and Cav3.2-T-type calcium channel current.

T-type calcium (Ca2+) channels play important physiological functions in excitable cells including cardiomyocyte. Phosphatidylinositol-4,5-bisphosphate (PIP2) has recently been reported to modulate various ion channels' function. However the actions of PIP2 on the T-type Ca2+ channel remain unclear. To elucidate possible effects of PIP2 on the T-type Ca2+ channel, we applied patch clamp method to investigate recombinant CaV3.1- and CaV3.2-T-type Ca2+ channels expressed in mammalian cell lines with PIP2 in acute- and long-term potentiation. Short- and long-term potentiation of PIP2 shifted the activation and the steady-state inactivation curve toward the hyperpolarization direction of CaV3.1-ICa.T without affecting the maximum inward current density. Short- and long-term potentiation of PIP2 also shifted the activation curve toward the hyperpolarization direction of CaV3.2-ICa.T without affecting the maximum inward current density. Conversely, long-term but not short-term potentiation of PIP2 shifted the steady-state inactivation curve toward the hyperpolarization direction of CaV3.2-ICa.T. Long-term but not short-term potentiation of PIP2 blunted the voltage-dependency of current decay CaV3.1-ICa.T. PIP2 modulates CaV3.1- and CaV3.2-ICa.T not by their current density but by their channel gating properties possibly through its membrane-delimited actions.