Long Noncoding RNA Kcna2 Antisense RNA Contributes to Ventricular Arrhythmias via Silencing Kcna2 in Rats With Congestive Heart Failure.
ABSTRACT: Congestive heart failure (CHF) is a common cardiovascular disease that is often accompanied by ventricular arrhythmias. The decrease of the slow component of the delayed rectifier potassium current (IKs) in CHF leads to action potential (AP) prolongation, and the IKs is an important contributor to the development of ventricular arrhythmias. However, the molecular mechanisms underlying ventricular arrhythmias are still unknown.Kcna2 and Kcna2 antisense RNA (Kcna2 AS) transcript expression was measured in rat cardiac tissues using quantitative real-time reverse transcription-polymerase chain reaction and Western blotting. There was a 43% reduction in Kcna2 mRNA in the left ventricular myocardium of rats with CHF. Kcna2 knockdown in the heart decreased the IKs and prolonged APs in cardiomyocytes, consistent with the changes observed in heart failure. Conversely, Kcna2 overexpression in the heart significantly attenuated the CHF-induced decreases in the IKs, AP prolongation, and ventricular arrhythmias. Kcna2 AS was upregulated ≈1.7-fold in rats with CHF and with phenylephrine-induced cardiomyocyte hypertrophy. Kcna2 AS inhibition increased the CHF-induced downregulation of Kcna2. Consequently, Kcna2 AS mitigated the decrease in the IKs and the prolongation of APs in vivo and in vitro and reduced ventricular arrhythmias, as detected using electrocardiography.Ventricular Kcna2 AS expression increases in rats with CHF and contributes to reduced IKs, prolonged APs, and the occurrence of ventricular arrhythmias by silencing Kcna2. Thus, Kcna2 AS may be a new target for the prevention and treatment of ventricular arrhythmias in patients with CHF.
Project description:BACKGROUND:The slow and rapid delayed rectifier K+ currents (IKs and IKr, respectively) are responsible for repolarizing the ventricular action potential (AP) and preventing abnormally long APs that may lead to arrhythmias. Although differences in biophysical properties of the 2 currents have been carefully documented, the respective physiological roles of IKr and IKs are less established. In this study, we sought to understand the individual roles of these currents and quantify how effectively each stabilizes the AP and protects cells against arrhythmias across multiple species. METHODS:We compared 10 mathematical models describing ventricular myocytes from human, rabbit, dog, and guinea pig. We examined variability within heterogeneous cell populations, tested the susceptibility of cells to proarrhythmic behavior, and studied how IKs and IKr responded to changes in the AP. RESULTS:We found that (1) models with higher baseline IKs exhibited less cell-to-cell variability in AP duration; (2) models with higher baseline IKs were less susceptible to early afterdepolarizations induced by depolarizing perturbations; (3) as AP duration is lengthened, IKs increases more profoundly than IKr, thereby providing negative feedback that resists excessive AP prolongation; and (4) the increase in IKs that occurs during ?-adrenergic stimulation is critical for protecting cardiac myocytes from early afterdepolarizations under these conditions. CONCLUSIONS:Slow delayed rectifier current is uniformly protective across a variety of cell types. These results suggest that IKs enhancement could potentially be an effective antiarrhythmic strategy.
Project description:<h4>Aims</h4>Cardiac sympathetic overactivation is an important trigger of ventricular arrhythmias in patients with chronic heart failure (CHF). Our previous study demonstrated that N-type calcium (Cav2.2) currents in cardiac sympathetic post-ganglionic (CSP) neurons were increased in CHF. This study investigated the contribution of Cav2.2 channels in cardiac sympathetic overactivation and ventricular arrhythmogenesis in CHF.<h4>Methods and results</h4>Rat CHF was induced by surgical ligation of the left coronary artery. Lentiviral Cav2.2-? shRNA or scrambled shRNA was transfected in vivo into stellate ganglia (SG) in CHF rats. Final experiments were performed at 14?weeks after coronary artery ligation. Real-time polymerase chain reaction and western blot data showed that in vivo transfection of Cav2.2-? shRNA reduced the expression of Cav2.2-? mRNA and protein in the SG in CHF rats. Cav2.2-? shRNA also reduced Cav2.2 currents and cell excitability of CSP neurons and attenuated cardiac sympathetic nerve activities (CSNA) in CHF rats. The power spectral analysis of heart rate variability (HRV) further revealed that transfection of Cav2.2-? shRNA in the SG normalized CHF-caused cardiac sympathetic overactivation in conscious rats. Twenty-four-hour continuous telemetry electrocardiogram recording revealed that this Cav2.2-? shRNA not only decreased incidence and duration of ventricular tachycardia/ventricular fibrillation but also improved CHF-induced heterogeneity of ventricular electrical activity in conscious CHF rats. Cav2.2-? shRNA also decreased susceptibility to ventricular arrhythmias in anaesthetized CHF rats. However, Cav2.2-? shRNA failed to improve CHF-induced cardiac contractile dysfunction. Scrambled shRNA did not affect Cav2.2 currents and cell excitability of CSP neurons, CSNA, HRV, and ventricular arrhythmogenesis in CHF rats.<h4>Conclusions</h4>Overactivation of Cav2.2 channels in CSP neurons contributes to cardiac sympathetic hyperactivation and ventricular arrhythmogenesis in CHF. This suggests that discovering purely selective and potent small-molecule Cav2.2 channel blockers could be a potential therapeutic strategy to decrease fatal ventricular arrhythmias in CHF.
Project description:BACKGROUND:In the heart, slow delayed rectifier channels provide outward currents (IKs) for action potential (AP) repolarization in a region- and context-dependent manner. In diseased hearts, chronic elevation of angiotensin II (Ang II) may remodel IKs in a region-dependent manner, contributing to atrial and ventricular arrhythmias of different mechanisms. OBJECTIVE:The purpose of this study was to study whether/how chronic in vivo Ang II administration remodels IKs in atrial and ventricular myocytes. METHODS:We used the guinea pig (GP) model whose myocytes express robust IKs. GPs were implanted with minipumps containing Ang II or vehicle. Treatment continued for 4-6 weeks. We used patch clamp, immunofluorescence/confocal microscopy, and immunoblots to evaluate changes in IKs function and to explore the underlying mechanisms. RESULTS:We confirmed the pathologic state of the heart after chronic Ang II treatment. IKs density was increased in atrial myocytes but decreased in ventricular myocytes in Ang II- vs vehicle-treated animals. The former was correlated with an increase in KCNQ1/KCNE1 colocalization in myocyte periphery, whereas the latter was correlated with a decrease in KCNQ1 protein level. Interestingly, these changes in IKs were not translated into expected alterations in AP duration or plateau voltage, indicating that other currents were involved. In atrial myocytes from Ang II-treated animals, the L-type Ca channel current was increased, contributing to AP plateau elevation and AP duration prolongation. CONCLUSION:IKs is differentially modulated by chronic in vivo Ang II administration between atrial and ventricular myocytes. Other currents remodeled by Ang II treatment also contribute to changes in action potentials.
Project description:Cardiac sympathetic overactivation is involved in arrhythmogenesis in patients with chronic heart failure (CHF). Inflammatory infiltration in the stellate ganglion (SG) is a critical factor for cardiac sympathoexcitation in patients with ventricular arrhythmias. This study aims to investigate if macrophage depletion in SGs decreases cardiac sympathetic overactivation and ventricular arrhythmogenesis in CHF. Surgical ligation of the coronary artery was used for induction of CHF. Clodronate liposomes were microinjected into bilateral SGs of CHF rats for macrophage depletion. Using cytokine array, immunofluorescence staining, and Western blot analysis, we found that macrophage expansion and expression of TNFα and IL-1β in SGs were markedly increased in CHF rats. Flow cytometry data confirmed that the percentage of macrophages in SGs was higher in CHF rats than that in sham rats. Clodronate liposomes significantly reduced CHF-elevated proinflammatory cytokine levels and macrophage expansion in SGs. Clodronate liposomes also reduced CHF-increased N-type Ca<sup>2+</sup> currents and excitability of cardiac sympathetic postganglionic neurons and inhibited CHF-enhanced cardiac sympathetic nerve activity. ECG data from 24-h, continuous telemetry recording in conscious rats demonstrated that clodronate liposomes not only restored CHF-induced heterogeneity of ventricular electrical activities, but also decreased the incidence and duration of ventricular tachycardia/fibrillation in CHF. Macrophage depletion with clodronate liposomes attenuated CHF-induced cardiac sympathetic overactivation and ventricular arrhythmias through reduction of macrophage expansion and neuroinflammation in SGs.
Project description:BACKGROUND:Electrophysiological remodeling and increased susceptibility for cardiac arrhythmias are hallmarks of heart failure (HF). Ventricular action potential duration (APD) is typically prolonged in HF, with reduced repolarization reserve. However, underlying K+ current changes are often measured in nonphysiological conditions (voltage clamp, low pacing rates, cytosolic Ca2+ buffers). METHODS AND RESULTS:We measured the major K+ currents (IKr, IKs, and IK1) and their Ca2+- and ?-adrenergic dependence in rabbit ventricular myocytes in chronic pressure/volume overload-induced HF (versus age-matched controls). APD was significantly prolonged only at lower pacing rates (0.2-1 Hz) in HF under physiological ionic conditions and temperature. However, when cytosolic Ca2+ was buffered, APD prolongation in HF was also significant at higher pacing rates. Beat-to-beat variability of APD was also significantly increased in HF. Both IKr and IKs were significantly upregulated in HF under action potential clamp, but only when cytosolic Ca2+ was not buffered. CaMKII (Ca2+/calmodulin-dependent protein kinase II) inhibition abolished IKs upregulation in HF, but it did not affect IKr. IKs response to ?-adrenergic stimulation was also significantly diminished in HF. IK1 was also decreased in HF regardless of Ca2+ buffering, CaMKII inhibition, or ?-adrenergic stimulation. CONCLUSIONS:At baseline Ca2+-dependent upregulation of IKr and IKs in HF counterbalances the reduced IK1, maintaining repolarization reserve (especially at higher heart rates) in physiological conditions, unlike conditions of strong cytosolic Ca2+ buffering. However, under ?-adrenergic stimulation, reduced IKs responsiveness severely limits integrated repolarizing K+ current and repolarization reserve in HF. This would increase arrhythmia propensity in HF, especially during adrenergic stress.
Project description:Gain-of-function mutations in the pore-forming subunit of IKs channels, KCNQ1, lead to short QT syndrome (SQTS) and lethal arrhythmias. However, how mutant IKs channels cause SQTS and the possibility of IKs-specific pharmacological treatment remain unclear. V141M KCNQ1 is a SQTS associated mutation. We studied its effect on IKs gating properties and changes in the action potentials (AP) of human ventricular myocytes. Xenopus oocytes were used to study the gating mechanisms of expressed V141M KCNQ1/KCNE1 channels. Computational models were used to simulate human APs in endocardial, mid-myocardial, and epicardial ventricular myocytes with and without β-adrenergic stimulation. V141M KCNQ1 caused a gain-of-function in IKs characterized by increased current density, faster activation, and slower deactivation leading to IKs accumulation. V141M KCNQ1 also caused a leftward shift of the conductance-voltage curve compared to wild type (WT) IKs (V1/2 = 33.6 ± 4.0 mV for WT, and 24.0 ± 1.3 mV for heterozygous V141M). A Markov model of heterozygous V141M mutant IKs was developed and incorporated into the O'Hara-Rudy model. Compared to the WT, AP simulations demonstrated marked rate-dependent shortening of AP duration (APD) for V141M, predicting a SQTS phenotype. Transmural electrical heterogeneity was enhanced in heterozygous V141M AP simulations, especially under β-adrenergic stimulation. Computational simulations identified specific IK1 blockade as a beneficial pharmacologic target for reducing the transmural APD heterogeneity associated with V141M KCNQ1 mutation. V141M KCNQ1 mutation shortens ventricular APs and enhances transmural APD heterogeneity under β-adrenergic stimulation. Computational simulations identified IK1 blockers as a potential antiarrhythmic drug of choice for SQTS.
Project description:OBJECTIVES:Postherpetic neuralgia (PHN) is the most common complication of herpes zoster, but the mechanism of PHN is still unclear. Activation of spinal astrocytes is involved in PHN. Our study aims to explore whether lncRNA KCNA2 antisense RNA (KCNA2-AS) regulates spinal astrocytes in PHN through signal transducers and activators of transcription 3 (STAT3). METHODS:Varicella zoster virus (VZV)-infected CV-1 cells were injected into rats to construct a PHN model. Primary spinal cord astrocytes were activated using S-Nitrosoglutathione (GSNO). Glial fibrillary acidic protein (GFAP; marker of astrocyte activation), phosphorylated STAT3 (pSTAT3), and KCNA2-AS were analyzed by immunofluorescence and RNA fluorescence in situ hybridization. RNA pull-down and RNA immunoprecipitation were used to detect binding of KCNA2-AS to pSTAT3. RESULTS:KCNA2-AS was highly expressed in the spinal cord tissue of PHN model rats, and was positively correlated with GFAP expression. GFAP was significantly increased in GSNO-induced cells, but the knockdown of KCNA2-AS reversed this result. Meanwhile, pSTAT3 was significantly increased in GSNO-induced cells, but knockdown of KCNA2-AS reduced pSTAT3 within the nucleus while the total pSTAT3 did not change significantly. pSTAT3 bound to KCNA2-AS and this binding increased with GSNO treatment. Furthermore, knockdown of KCNA2-AS in PHN model rats relieved mechanical allodynia. CONCLUSION:Down-regulation of KCNA2-AS alleviates PHN partly by reducing the translocation of pSTAT3 cytoplasm to the nucleus and then inhibiting the activation of spinal astrocytes.
Project description:Some cardiovascular and non-cardiovascular drugs frequently cause excessive prolongation of the cardiac action potential (AP) and lead to the development of early afterdepolarisations (EADs), which trigger lethal ventricular arrhythmias. Combining computer simulations in APs with numerical calculations based on dynamical system theory, we investigated stability changes of APs observed in a paced human ventricular myocyte model by decreasing and/or increasing the rapid (I <sub>Kr</sub>) and slow (I <sub>Ks</sub>) components of delayed rectifying K<sup>+</sup> current. Upon reducing I <sub>Kr</sub>, the APs without EADs (no-EAD response) showed gradual prolongation of AP duration (APD), and were annihilated without AP configuration changes due to the occurrence of saddle-node bifurcations. This annihilation caused a transition to an AP with EADs as a new stable steady state. Furthermore, reducing repolarisation currents (repolarisation reserve attenuation) evoked multi-stable states consisting of APs with different APDs, and caused multiple hysteretic dynamics. Depending on initial ion circumstances within ventricular myocytes, these multi-stable AP states might increase the local/global heterogeneity of AP repolarisations in the ventricle. Thus, the EAD-induced arrhythmias with repolarisation reserve attenuation might be attributed to the APD variability caused by multi-stability in cardiac AP dynamics.
Project description:Introduction:Long QT syndrome type 1 (LQT1) is caused by mutations in KCNQ1 coding slowly-activating delayed-rectifier K+ channels. We identified the novel missense mutation M437V of KCNQ1 in a LQT1 patient. Here, we employed iPS cell (iPSC)-derived cardiomyocytes to investigate electrophysiological properties of the mutant channel and LQT1 cardiomyocytes. Methods:To generate iPSCs from the patient and a healthy subject, peripheral blood T cells were reprogrammed by Sendai virus vector encoding human OCT3/4, SOX2, KLF4, and c-MYC. Cardiomyocytes were prepared from iPSCs and human embryonic stem cells using a cytokine-based two-step differentiation method and were subjected to patch clamp experiments. Results:LQT1 iPSC-derived cardiomyocytes exhibited prolongation of action potential duration (APD), which was due to a reduction of the KCNQ1-mediated current IKs; Na+, Ca2+ and other K+ channel currents were comparable. When expressed in HEK293 and COS7 cells, the mutant KCNQ1 was normally expressed in the plasma membrane but generated smaller currents than the wild type. Isoproterenol significantly prolonged APDs of LQT1 cardiomyocytes, while shortening those of healthy ones. A mathematical model for IKs-reduced human ventricular myocytes reproduced APD prolongation and generation of early afterdepolarizations (EADs) under β-adrenergic stimulation. Conclusions:QT prolongation of the LQT1 patient with the mutation M437V of KCNQ1 was caused by IKs reduction, which may render the patient vulnerable to generation of EADs and arrhythmias.
Project description:Early afterdepolarization (EAD) is known to cause lethal ventricular arrhythmias in long QT syndrome (LQTS). In this study, dynamical mechanisms of EAD formation in human ventricular myocytes (HVMs) were investigated using the mathematical model developed by ten Tusscher and Panfilov (Am J Physiol Heart Circ Physiol 291, 2006). We explored how the rapid (IKr) and slow (IKs) components of delayed-rectifier K+ channel currents, L-type Ca2+ channel current (ICa L), Na+/Ca2+ exchanger current (INCX), and intracellular Ca2+ handling via the sarcoplasmic reticulum (SR) contribute to initiation, termination and modulation of phase-2 EADs during pacing in relation to bifurcation phenomena in non-paced model cells. Parameter-dependent dynamical behaviors of the non-paced model cell were determined by calculating stabilities of equilibrium points (EPs) and limit cycles, and bifurcation points to construct bifurcation diagrams. Action potentials (APs) and EADs during pacing were reproduced by numerical simulations for constructing phase diagrams of the paced model cell dynamics. Results are summarized as follows: (1) A modified version of the ten Tusscher-Panfilov model with accelerated ICaL inactivation could reproduce bradycardia-related EADs in LQTS type 2 and ?-adrenergic stimulation-induced EADs in LQTS type 1. (2) Two types of EADs with different initiation mechanisms, ICaL reactivation-dependent and spontaneous SR Ca2+ release-mediated EADs, were detected. (3) Termination of EADs (AP repolarization) during pacing depended on the slow activation of IKs. (4) Spontaneous SR Ca2+ releases occurred at higher Ca2+ uptake rates, attributable to the instability of steady-state intracellular Ca2+ concentrations. Dynamical mechanisms of EAD formation and termination in the paced model cell are closely related to stability changes (bifurcations) in dynamical behaviors of the non-paced model cell, but they are model-dependent. Nevertheless, the modified ten Tusscher-Panfilov model would be useful for systematically investigating possible dynamical mechanisms of EAD-related arrhythmias in LQTS.