Flecainide induces a sustained countercurrent dependent effect on RyR2 in permeabilized WT ventricular myocytes but not in intact cells.

Background and purpose: While flecainide is now an accepted treatment for arrhythmias associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), its mechanism of action remains controversial. In studies on myocytes from CPVT mice, inhibition of proarrhythmic Ca2+ waves was initially attributed to a novel action on the type-2 ryanodine receptor (RyR2). However, subsequent work on wild type (WT) myocytes questioned the conclusion that flecainide has a direct action on RyR2. In the present study, the effects of flecainide were compared in intact and permeabilized WT myocytes. Experimental approach: Intracellular Ca2+ was measured using confocal microscopy in intact or saponin permeabilized adult rat ventricular myocytes (ARVM). In some experiments on permeabilized cells, flecainide was studied following partial inhibition of the sarcoplasmic reticulum (SR) counter-current. Key results: Flecainide induced sustained changes Ca2+ sparks and waves in permeabilized ARVM, which were comparable to those reported in intact or permeabilized myocytes from CPVT mice. However, a relatively high level of flecainide (25 µM) was required to induce these effects. Inhibition of the SR counter-current potentiated the effects of flecainide on SR Ca2+ waves. In intact field stimulated ARVM, prolonged exposure to 15 µM flecainide decreased wave frequency but RyR2 dependent effects on Ca2+ sparks were absent; higher drug concentrations blocked field stimulation, consistent with inhibition of Nav1.5. Conclusions and implications: In intact ARVM, the absence of effects on Ca2+ sparks suggests that the intracellular flecainide concentration was insufficient to influence RyR2. Wave inhibition in intact ARVM may reflect secondary effects of Nav1.5 inhibition. Potentiation of flecainide's action by counter-current inhibition can be explained if transient polarization of the SR membrane during SR Ca2+ release facilitates its action on RyR2.

Super-Resolution Analysis of the Origins of the Elementary Events of ER Calcium Release in Dorsal Root Ganglion Neurons.

Coordinated events of calcium (Ca2+) released from the endoplasmic reticulum (ER) are key second messengers in excitable cells. In pain-sensing dorsal root ganglion (DRG) neurons, these events can be observed as Ca2+ sparks, produced by a combination of ryanodine receptors (RyR) and inositol 1,4,5-triphosphate receptors (IP3R1). These microscopic signals offer the neuronal cells with a possible means of modulating the subplasmalemmal Ca2+ handling, initiating vesicular exocytosis. With super-resolution dSTORM and expansion microscopies, we visualised the nanoscale distributions of both RyR and IP3R1 that featured loosely organised clusters in the subplasmalemmal regions of cultured rat DRG somata. We adapted a novel correlative microscopy protocol to examine the nanoscale patterns of RyR and IP3R1 in the locality of each Ca2+ spark. We found that most subplasmalemmal sparks correlated with relatively small groups of RyR whilst larger sparks were often associated with larger groups of IP3R1. These data also showed spontaneous Ca2+ sparks in <30% of the subplasmalemmal cell area but consisted of both these channel species at a 3.8-5 times higher density than in nonactive regions of the cell. Taken together, these observations reveal distinct patterns and length scales of RyR and IP3R1 co-clustering at contact sites between the ER and the surface plasmalemma that encode the positions and the quantity of Ca2+ released at each Ca2+ spark.

Action potential responses to changes in stimulation frequency and isoproterenol in rat ventricular myocytes.

PURPOSE: Current understanding of ventricular action potential adaptation to physiological stress is generally based on protocols using non-physiological rates and conditions isolating rate effects from escalating adrenergic stimulation. To permit refined understanding, ventricular action potentials were assessed across physiological pacing frequencies in the presence and absence of adrenergic stimuli. Isolated and combined effects were analyzed to assess their ability to replicate in-vivo responses. METHODS: Steady-state action potentials from ventricular myocytes isolated from male Wistar rats (3 months; N = 8 animals) were recorded at 37°C with steady-state pacing at 1, 2, 4, 6, 8 and 10 Hz using whole-cell patch-clamp. Action potential repolarization to 25, 50, 75, 90 and 100% of full repolarization (APD25-100 ) was compared before and after 5 nM, 100 nM and 1 µM isoproterenol doses. RESULTS: A Repeated measures ANOVA found APD50-90  shortened with 5 nM isoproterenol infusion by 6-25% (but comparable across doses) (p ≤ 0.03). Pacing frequencies emulating a normal rat heart rate (6 Hz) prolonged APD50 23% compared with 1 Hz pacing. Frequencies emulating exercise or stress (10 Hz) shortened APD90 (29%). CONCLUSION: These results demonstrate modest action potential shortening in response to adrenergic stimulation and elevations in pacing beyond physiological resting rates. Our findings indicate changes in action potential plateau and late repolarization predominantly underlie simulated exercise responses in the rat heart. This work provides novel action potential reference data and will help model cardiac responses to physiological stimuli in the rat heart via computational techniques.

A correlative super-resolution protocol to visualise structural underpinnings of fast second-messenger signalling in primary cell types.

Nanometre-scale cellular information obtained through super-resolution microscopies are often unaccompanied by functional information, particularly transient and diffusible signals through which life is orchestrated in the nano-micrometre spatial scale. We describe a correlative imaging protocol which allows the ubiquitous intracellular second messenger, calcium (Ca2+), to be directly visualised against nanoscale patterns of the ryanodine receptor (RyR) Ca2+ channels which give rise to these Ca2+ signals in wildtype primary cells. This was achieved by combining total internal reflection fluorescence (TIRF) imaging of the elementary Ca2+ signals, with the subsequent DNA-PAINT imaging of the RyRs. We report a straightforward image analysis protocol of feature extraction and image alignment between correlative datasets and demonstrate how such data can be used to visually identify the ensembles of Ca2+ channels that are locally activated during the genesis of cytoplasmic Ca2+ signals.

Three-Dimensional and Chemical Mapping of Intracellular Signaling Nanodomains in Health and Disease with Enhanced Expansion Microscopy.

Nanodomains are intracellular foci which transduce signals between major cellular compartments. One of the most ubiquitous signal transducers, the ryanodine receptor (RyR) calcium channel, is tightly clustered within these nanodomains. Super-resolution microscopy has previously been used to visualize RyR clusters near the cell surface. A majority of nanodomains located deeper within cells have remained unresolved due to limited imaging depths and axial resolution of these modalities. A series of enhancements made to expansion microscopy allowed individual RyRs to be resolved within planar nanodomains at the cell periphery and the curved nanodomains located deeper within the interiors of cardiomyocytes. With a resolution of ∼ 15 nm, we localized both the position of RyRs and their individual phosphorylation for the residue Ser2808. With a three-dimensional imaging protocol, we observed disturbances to the RyR arrays in the nanometer scale which accompanied right-heart failure caused by pulmonary hypertension. The disease coincided with a distinct gradient of RyR hyperphosphorylation from the edge of the nanodomain toward the center, not seen in healthy cells. This spatial profile appeared to contrast distinctly from that sustained by the cells during acute, physiological hyperphosphorylation when they were stimulated with a ß-adrenergic agonist. Simulations of RyR arrays based on the experimentally determined channel positions and phosphorylation signatures showed how the nanoscale dispersal of the RyRs during pathology diminishes its intrinsic likelihood to ignite a calcium signal. It also revealed that the natural topography of RyR phosphorylation could offset potential heterogeneity in nanodomain excitability which may arise from such RyR reorganization.

A key role for peroxynitrite-mediated inhibition of cardiac ERG (Kv11.1) K+ channels in carbon monoxide-induced proarrhythmic early afterdepolarizations.

Exposure to CO causes early afterdepolarization arrhythmias. Previous studies in rats have indicated that arrhythmias arose as a result of augmentation of the late Na+ current. The purpose of the present study was to examine the basis for CO-induced arrhythmias in guinea pig myocytes in which action potentials (APs) more closely resemble those of human myocytes. Whole-cell current- and voltage-clamp recordings were made from isolated guinea pig myocytes as well as from human embryonic kidney 293 (HEK293) cells that express wild-type or a C723S mutant form of ether-a-go-go-related gene (ERG; Kv11.1). We also monitored the formation of peroxynitrite (ONOO-) in HEK293 cells fluorimetrically. CO-applied as the CO-releasing molecule, CORM-2-prolonged the APs and induced early afterdepolarizations in guinea pig myocytes. In HEK293 cells, CO inhibited wild-type, but not C723S mutant, Kv11.1 K+ currents. Inhibition was prevented by an antioxidant, mitochondrial inhibitors, or inhibition of NO formation. CO also raised ONOO- levels, an effect that was reversed by the ONOO- scavenger, FeTPPS [5,10,15,20-tetrakis-(4-sulfonatophenyl)-porphyrinato-iron(III)], which also prevented the CO inhibition of Kv11.1 currents and abolished the effects of CO on Kv11.1 tail currents and APs in guinea pig myocytes. Our data suggest that CO induces arrhythmias in guinea pig cardiac myocytes via the ONOO--mediated inhibition of Kv11.1 K+ channels.-Al-Owais, M. M., Hettiarachchi, N. T., Kirton, H. M., Hardy, M. E., Boyle, J. P., Scragg, J. L., Steele, D. S., Peers, C. A key role for peroxynitrite-mediated inhibition of cardiac ERG (Kv11.1) K+ channels in carbon monoxide-induced proarrhythmic early afterdepolarizations.

Epac2-Rap1 Signaling Regulates Reactive Oxygen Species Production and Susceptibility to Cardiac Arrhythmias.

AIMS: In the heart, ß1-adrenergic signaling involves cyclic adenosine monophosphate (cAMP) acting via both protein kinase-A (PKA) and exchange protein directly activated by cAMP (Epac): a guanine nucleotide exchange factor for the small GTPase Rap1. Inhibition of Epac-Rap1 signaling has been proposed as a therapeutic strategy for both cancer and cardiovascular disease. However, previous work suggests that impaired Rap1 signaling may have detrimental effects on cardiac function. The aim of the present study was to investigate the influence of Epac2-Rap1 signaling on the heart using both in vivo and in vitro approaches. RESULTS: Inhibition of Epac2 signaling induced early afterdepolarization arrhythmias in ventricular myocytes. The underlying mechanism involved an increase in mitochondrial reactive oxygen species (ROS) and activation of the late sodium current (INalate). Arrhythmias were blocked by inhibition of INalate or the mitochondria-targeted antioxidant, mitoTEMPO. In vivo, inhibition of Epac2 caused ventricular tachycardia, torsades de pointes, and sudden death. The in vitro and in vivo effects of Epac2 inhibition were mimicked by inhibition of geranylgeranyltransferase-1, which blocks interaction of Rap1 with downstream targets. INNOVATION: Our findings show for the first time that Rap1 acts as a negative regulator of mitochondrial ROS production in the heart and that impaired Epac2-Rap1 signaling causes arrhythmias due to ROS-dependent activation of INalate. This has implications for the use of chemotherapeutics that target Epac2-Rap1 signaling. However, selective inhibition of INalate provides a promising strategy to prevent arrhythmias caused by impaired Epac2-Rap1 signaling. CONCLUSION: Epac2-Rap1 signaling attenuates mitochondrial ROS production and reduces myocardial arrhythmia susceptibility. Antioxid. Redox Signal. 27, 117-132.

The Golgi apparatus is a functionally distinct Ca2+ store regulated by the PKA and Epac branches of the ß1-adrenergic signaling pathway.

Ca(2+) release from the Golgi apparatus regulates key functions of the organelle, including vesicle trafficking. We found that the Golgi apparatus was the source of prolonged Ca(2+) release events that originated near the nuclei of primary cardiomyocytes. Golgi Ca(2+) release was unaffected by depletion of sarcoplasmic reticulum Ca(2+), and disruption of the Golgi apparatus abolished Golgi Ca(2+) release without affecting sarcoplasmic reticulum function, suggesting functional and spatial independence of Golgi and sarcoplasmic reticulum Ca(2+) stores. ß1-Adrenoceptor stimulation triggers the production of the second messenger cAMP, which activates the Epac family of Rap guanine nucleotide exchange factors and the kinase PKA (protein kinase A). Phosphodiesterases (PDEs), including those in the PDE3 and PDE4 families, degrade cAMP. Activation of ß1-adrenoceptors stimulated Golgi Ca(2+) release, an effect that required activation of Epac, PKA, and the kinase CaMKII. Inhibition of PDE3s or PDE4s potentiated ß1-adrenergic-induced Golgi Ca(2+) release, which is consistent with compartmentalization of cAMP signaling near the Golgi apparatus. Interventions that stimulated Golgi Ca(2+) release appeared to increase the trafficking of vascular endothelial growth factor receptor-1 (VEGFR-1) from the Golgi apparatus to the surface membrane of cardiomyocytes. In cardiomyocytes from rats with heart failure, decreases in the abundance of PDE3s and PDE4s were associated with increased Golgi Ca(2+) release events. These data suggest that the Golgi apparatus is a focal point for ß1-adrenergic-stimulated Ca(2+) signaling and that the Golgi Ca(2+) store functions independently from the sarcoplasmic reticulum and the global Ca(2+) transients that trigger contraction in cardiomyocytes.

Transient overexpression of genes in neurons using nucleofection.

Nucleofection is a transfection method used to introduce substrates such as cDNA plasmids into primary cells or other cell lines. The method can be successfully applied to cells that are considered difficult to transfect or suffer from low transfection efficiency as seen with traditional transfection techniques. Neurons in primary cultures retain many properties of their in vivo state and therefore, in many instances, are considered better experimental systems than immortalized cell lines, thus becoming increasingly desirable cell types for biomedical research. However, being post-mitotic, primary neuronal cultures are particularly difficult to transfect using routine transfection reagents. There is therefore a growing need for the efficient delivery of expression vectors into such neuronal cultures. In this chapter we will discuss the application of nucleofection for the heterologous expression of genes in primary neuronal cultures. We also discuss the advantage of this technique relative to other conventional methods, and describe a reliable method for transfection of cultured rat dorsal root ganglion (DRG) and trigeminal (TG) neurons.