Guangbin Shi

Obstructive Sleep Apnea and Circulating Potassium Channel Levels

Background Cardiac arrhythmias and sudden cardiac death are more frequent in patients with obstructive sleep apnea (OSA). OSA is associated with QT prolongation, and QT prolongation is an independent risk factor for sudden cardiac death. Because QT prolongation can be mediated by potassium channel loss of function, we tested whether OSA or continuous positive airway pressure therapy altered mRNA expression of circulating white blood cell potassium channels. Methods and Results In total, 28 patients with OSA newly diagnosed by polysomnogram and 6 participants without OSA were enrolled. Potassium channel levels in white blood cells at baseline and at a 4‐week follow‐up visit were compared. There was a significant inverse correlation between the severity of the OSA stratified by apnea–hypopnea index and mRNA expression of the main potassium channels assessed: KCNQ1 (r=−0.486, P=0.007), KCNH2 (r=−0.437, P=0.016), KCNE1 (r=−0.567, P=0.001), KCNJ2 (r=−0.442, P=0.015), and KCNA5 (r=−0.468, P=0.009). In addition, KCNQ1, KCNH2, and KCNE1 inversely correlated with the oxygen desaturation index 4. After 4 weeks of continuous positive airway pressure therapy, circulating KCNQ1 and KCNJ2 were increased 1.4±0.4‐fold (P=0.040) and 2.1±1.4‐fold (P=0.046) in the moderate OSA group. Compared with patients with mild or moderate OSA, patients with severe OSA had a persistently higher apnea–hypopnea index (mild 2.0±1.8, moderate 1.0±0.9, severe 5.8±5.6; P=0.015), perhaps explaining why the potassium channel changes were not seen in the severe OSA group. Conclusions The mRNA expression of most potassium channels inversely correlates with the severity of OSA and hypoxemia. Continuous positive airway pressure therapy improves circulating KCNQ1 and KCNJ2 in patients with moderate OSA.

Role of protein kinase C in metabolic regulation of the cardiac Na+ channel

BACKGROUND The reduced form of nicotinamide adenine dinucleotide (NADH) increases in cardiomyopathy, activates protein kinase C (PKC), up-regulates mitochondrial reactive oxygen species (mitoROS), and down-regulates the cardiac Na+ channel (NaV1.5). OBJECTIVE The purpose of this study was to determine how NADH signals down-regulation of NaV1.5. METHODS Isolated mouse cardiomyocytes were used for patch-clamp recording and for monitoring mitoROS with MitoSOX Red. HEK293 cells were used for transient transfections. HEK293 cells stably expressing human NaV1.5 were used for single channel recording, whole-cell patch-clamp recording, activity measurements of phospholipase C and phospholipase D (PLD), channel protein purification, and co-immunoprecipitation with PKC isoforms. HL-1 cells were used for mitochondria isolation. RESULTS NADH enhanced PLD activity (1.6- ± 0.1-fold, P <.01) and activated PKCδ. Activated PKCδ translocated to mitochondria and up-regulated mitoROS (2.8- ± 0.3-fold, P <.01) by enhancing the activities of mitochondrial complexes I, II, and IV (1.1- to 1.5-fold, P <.01). PKCδ also interacted with NaV1.5 to down-regulate Na+ current (INa). Reduction in INa by activated PKCδ was prevented by antioxidants and by mutating the known PKC phosphorylation site S1503. At the single channel level, the mechanism of current reduction by PKC and recovery by protein kinase A was a change in single channel conductance. CONCLUSION NADH activated PKCδ by enhancing PLD activity. PKCδ modulated both mitoROS and NaV1.5. PKCδ elevated mitoROS by enhancing mitochondrial oxidative phosphorylation complex activities. PKCδ-mediated channel phosphorylation and mitoROS were both required to down-regulate NaV1.5 and alter single channel conductance.

Activation of the unfolded protein response downregulates cardiac ion channels in human induced pluripotent stem cell-derived cardiomyocytes.

RATIONALE Heart failure is characterized by electrical remodeling that contributes to arrhythmic risk. The unfolded protein response (UPR) is active in heart failure and can decrease protein levels by increasing mRNA decay, accelerating protein degradation, and inhibiting protein translation. OBJECTIVE Therefore, we investigated whether the UPR downregulated cardiac ion channels that may contribute to arrhythmogenic electrical remodeling. METHODS Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were used to study cardiac ion channels. Action potentials (APs) and ion channel currents were measured by patch clamp recording. The mRNA and protein levels of channels and the UPR effectors were determined by quantitative RT-PCR and Western blotting. Tunicamycin (TM, 50 ng/mL and 5 μg/mL), GSK2606414 (GSK, 300 nmol/L), and 4μ8C (5 μmol/L) were utilized to activate the UPR, inhibit protein kinase-like ER kinase (PERK) and inositol-requiring protein-1 (IRE1), respectively. RESULTS TM-induced activation of the UPR caused significant prolongation of the AP duration (APD) and a reduction of the maximum upstroke velocity (dV/dtmax) of the AP phase 0 in both acute (20-24 h) and chronic treatment (6 days). These changes were explained by reductions in the sodium, L-type calcium, the transient outward and rapidly/slowly activating delayed rectifier potassium currents. Nav1.5, Cav1.2, Kv4.3, and KvLQT1 channels showed concomitant reductions in mRNA and protein levels under activated UPR. Inhibition of PERK or IRE1 shortened the APD and reinstated dV/dtmax. The PERK branch regulated Nav1.5, Kv4.3, hERG, and KvLQT1. The IRE1 branch regulated Nav1.5, hERG, KvLQT1, and Cav1.2. CONCLUSIONS Activated UPR downregulates all major cardiac ion currents and results in electrical remodeling in hiPSC-CMs. Both PERK and IRE1 branches downregulate Nav1.5, hERG, and KvLQT1. The PERK branch specifically downregulates Kv4.3, while the IRE1 branch downregulates Cav1.2. Therefore, the UPR contributed to electrical remodeling, and targeting the UPR might be anti-arrhythmic.

HuR-mediated SCN5A messenger RNA stability reduces arrhythmic risk in heart failure

BACKGROUND Downregulated sodium currents in heart failure (HF) have been linked to increased arrhythmic risk. Reduced expression of the messenger RNA (mRNA)-stabilizing protein HuR (also known as ELAVL1) may be responsible for the downregulation of sodium channel gene SCN5A mRNA. OBJECTIVE The purpose of this article was to investigate whether HuR regulates SCN5A mRNA expression and whether manipulation of HuR benefits arrhythmia control in HF. METHODS Quantitative real-time reverse-transcriptase polymerase chain reaction was used to investigate the expression of SCN5A. Optical mapping of the intact heart was adopted to study the effects of HuR on the conduction velocity and action potential upstroke in mice with myocardial infarct and HF after injection of AAV9 viral particles carrying HuR. RESULTS HuR was associated with SCN5A mRNA in cardiomyocytes, and expression of HuR was downregulated in failing hearts. The association of HuR and SCN5A mRNA protected SCN5A mRNA from decay. Injection of AAV9 viral particles carrying HuR increased SCN5A expression in mouse heart tissues after MI. Optical mapping of the intact heart demonstrated that overexpression of HuR improved action potential upstroke and conduction velocity in the infarct border zone, which reduced the risk of reentrant arrhythmia after MI. CONCLUSION Our data indicate that HuR is an important RNA-binding protein in maintaining SCN5A mRNA abundance in cardiomyocytes. Reduced expression of HuR may be at least partially responsible for the downregulation of SCN5A mRNA expression in ischemic HF. Overexpression of HuR may rescue decreased SCN5A expression and reduce arrhythmic risk in HF. Increasing mRNA stability to increase ion channel currents may correct a fundamental defect in HF and represent a new paradigm in antiarrhythmic therapy.

Mitochondrial Ca2+ flux modulates spontaneous electrical activity in ventricular cardiomyocytes

Introduction Ca2+ release from sarcoplasmic reticulum (SR) is known to contribute to automaticity via the cytoplasmic Na+-Ca2+ exchanger (NCX). Mitochondria participate in Ca2+ cycling. We studied the role of mitochondrial Ca2+ flux in ventricular spontaneous electrical activity. Methods Spontaneously contracting mouse embryonic stem cells (ESC)-derived ventricular cardiomyocytes (CMs) were differentiated from wild type and ryanodine receptor type 2 (RYR2) knockout mouse ESCs and differentiated for 19–21 days. Automaticity was also observed in human induced pluripotent stem cell (hiPSC)-derived ventricular CMs differentiated for 30 days, and acute isolated adult mouse ventricular cells in ischemic simulated buffer. Action potentials (APs) were recorded by perforated whole cell current-clamp. Cytoplasmic and mitochondrial Ca2+ transients were determined by fluorescent imaging. Results In mouse ESC-derived ventricular CMs, spontaneous beating was dependent on the L-type Ca2+ channel, cytoplasmic NCX and mitochondrial NCX. Spontaneous beating was modulated by SR Ca2+ release from RYR2 or inositol trisphosphate receptors (IP3R), the pacemaker current (If) and mitochondrial Ca2+ uptake by the mitochondrial Ca2+ uniporter (MCU). In RYR2 knockout mouse ESC-derived ventricular CMs, mitochondrial Ca2+ flux influenced spontaneous beating independently of the SR Ca2+ release from RYR2, and the mitochondrial effect was dependent on IP3R SR Ca2+ release. Depolarization of mitochondria and preservation of ATP could terminate spontaneous beating. A contribution of mitochondrial Ca2+ flux to automaticity was confirmed in hiPSC-derived ventricular CMs and ischemic adult mouse ventricular CMs, confirming the findings across species and cell maturity levels. Conclusions Mitochondrial and sarcolemma NCX fluxes are required for ventricular automaticity. Mitochondrial Ca2+ uptake plays a modulatory role. Mitochondrial Ca2+ uptake through MCU is influenced by IP3R-dependent SR Ca2+ release.

RNA Binding Protein, HuR, Regulates SCN5A Expression Through Stabilizing MEF2C transcription factor mRNA

Background Although transcription is the initial process of gene expression, posttranscriptional gene expression regulation has also played a critical role for fine‐tuning gene expression in a fast, precise, and cost‐effective manner. Although the regulation of sodium channel α‐subunit (SCN5A) mRNA expression has been studied at both transcriptional and pre‐mRNA splicing levels, the molecular mechanisms governing SCN5A mRNA expression are far from clear. Methods and Results Herein, we show that, as evidenced by ribonucleoprotein immunoprecipitation assay, RNA binding protein Hu antigen R/ELAV like RNA binding protein 1 (HuR/ELAVL1) and myocyte enhancer factor‐2C (MEF2C) transcription factor mRNA are associated. HuR positively regulated transcription factor MEF2C mRNA expression by protecting its mRNA from degradation. As demonstrated by both chromatin immunoprecipitation–quantitative polymerase chain reaction assay and an electrophoretic mobility shift assay, MEF2C enhanced SCN5A transcription by binding to a putative MEF2C binding site within SCN5A promoter region. Overexpression of HuR increased the expression of SCN5A mRNA, and this effect was attenuated by the presence of MEF2C small interfering RNA in cardiomyocytes. Conclusions In conclusion, our results suggested that HuR participates in a combined network at the DNA and RNA levels that regulates SCN5A mRNA expression. HuR upregulates MEF2C mRNA expression by protecting MEF2C mRNA from degradation, and consequently, the elevated MEF2C enhances SCN5A mRNA transcription.