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 β-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.

Substance P (SP) is a prominent neuromodulator, which is produced and released by peripheral damage-sensing (nociceptive) neurons; these neurons also express SP receptors. However, the mechanisms of peripheral SP signaling are poorly understood. We report a signaling pathway of SP in nociceptive neurons: Acting predominantly through NK1 receptors and G i/o proteins, SP stimulates increased release of reactive oxygen species from the mitochondrial electron transport chain. Reactive oxygen species, functioning as second messengers, induce oxidative modification and augment M-type potassium channels, thereby suppressing excitability. This signaling cascade requires activation of phospholipase C but is largely uncoupled from the inositol 1,4,5-trisphosphate sensitive Ca 2+ stores. In rats SP causes sensitization of TRPV1 and produces thermal hyperalgesia. However, the lack of coupling between SP signaling and inositol 1,4,5-trisphosphate sensitive Ca 2+ stores, together with the augmenting effect on M channels, renders the SP pathway ineffective to excite nociceptors acutely and produce spontaneous pain. Our study describes a mechanism for neurokinin signaling in sensory neurons and provides evidence that spontaneous pain and hyperalgesia can have distinct underlying mechanisms within a single nociceptive neuron.G protein coupled receptors | inflammatory pain | intracellular signaling | KCNQ | M current E xcitation of peripheral terminals of sensory neurons is a primary event in somatosensation, including pain. A large proportion of sensory neurons (mostly TRPV1 + damage-sensing or "nociceptive" neurons) produce neuropeptides such as substance P (SP), which are released in response to nociceptive stimulation both at spinal cord synapses and in the periphery (1). In the spinal cord SP acts as an excitatory neurotransmitter or cotransmitter (2, 3), whereas peripheral SP release is thought to underlie the inflammatory response known as "neurogenic inflammation" (4). Many peripheral nociceptors also express receptors for SP [neurokinin receptors (NKR) 1-3 (NK1-3)]; this expression may suggest paracrine or autocrine actions of this peptide. However, the nature of such actions is controversial, and the molecular events triggered by NKR in peripheral nociceptors are not well established. The NKR traditionally are classed as G q/11 -coupled G protein-coupled receptors (GPCR) that activate phospholipase C (PLC), with subsequent hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and release of inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DAG) (5, 6). Common downstream steps of G q/11 signaling include IP 3 -mediated release of Ca 2+ from intracellular stores and DAG-mediated activation of PKC. However, it was hitherto unknown whether NKR in peripheral sensory neurons couple fully to this signaling cascade, because a lack of coupling between NKR and Ca 2+ release in dorsal root ganglia (DRG) neurons has been noted (ref. 7 and 8, but cf. ref. 9). Nevertheless, it is accepted that peripherally act...

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 steadystate 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 (APD 25-100 ) was compared before and after 5 nM, 100 nM and 1 µM isoproterenol doses.Results: A Repeated measures ANOVA found APD 50-90 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 APD 50 23% compared with 1 Hz pacing. Frequencies emulating exercise or stress (10 Hz) shortened APD 90 (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.

Aims: In the heart, β1-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.

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 (Ca 2+ ), to be directly visualised against nanoscale patterns of the ryanodine receptor (RyR) Ca 2+ channels which give rise to these Ca 2+ signals in wildtype primary cells. This was achieved by combining total internal reflection fluorescence (TIRF) imaging of the elementary Ca 2+ 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 Ca 2+ channels that are locally activated during the genesis of cytoplasmic Ca 2+ signals.

Ca 2+ release from the Golgi apparatus regulates key functions of the organelle, including vesicle trafficking. However, the signaling pathways that control this form of Ca 2+ release are poorly understood and evidence of discrete Golgi Ca 2+ release events is lacking. Here, we identified the Golgi apparatus as the source of prolonged Ca 2+ release events that originate from the nuclear 'poles' of primary cardiac cells. Once initiated, Golgi Ca 2+ release was unaffected by global 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 anatomical independence of Golgi and sarcoplasmic reticulum Ca 2+ stores. Maximal activation of β 1 -adrenoceptors had only a small stimulating effect on Golgi Ca 2+ release. However, inhibition of phosphodiesterase (PDE) 3 or 4, or downregulation of PDE 3 and 4 in heart failure markedly potentiated β 1 -adrenergic stimulation of Golgi Ca 2+ release, consistent with compartmentalization of cAMP signaling within the Golgi apparatus microenvironment. β 1 -adrenergic stimulation of Golgi Ca 2+ release involved activation of both Epac and PKA signaling pathways and CaMKII. Interventions that stimulated Golgi Ca 2+ release induced trafficking of vascular growth factor receptor-1 (VEGFR-1) from the Golgi apparatus to the surface membrane. These data establish the Golgi apparatus as a juxtanuclear focal point for Ca 2+ and β 1 -adrenergic signaling, which functions independently from the sarcoplasmic reticulum and the global Ca 2+ transients that underlie the primary contractile function of the cell. *To whom correspondence should be addressed: d.steele@leeds.ac.uk. Author contributions: Yang: confocal Ca 2+ imaging and DAF-2 experiments; Kirton: Ca 2+ imaging and immunofluorescence experiments. MacDougall: Western blot analysis; Boyle: Immunofluorescence measurements; Deuchars: electron microscopy; Frater: electron microscopy; Ponnambalan immunofluorescence experiments and experimental design; Hardy: preparation of heart failure model; White: preparation of heart failure model; Calaghan Western blot analysis and experimental design; Peers: experimental design, immunofluorescence and editing of draft manuscript; Steele: Experimental design, manuscript preparation and overall project management.

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.

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.