Nicotinic acid adenine dinucleotide phosphate (NAADP) potently releases Ca 2+ from acidic intracellular endolysosomal Ca 2+ stores. It is widely accepted that two types of two-pore channels, termed TPC1 and TPC2, are responsible for the NAADP-mediated Ca 2+ release but the Samantha Pitt gained her PhD degree from the University of Cambridge. Following two postdoctoral positions studying mechanisms of ion-channel regulation, one at University College London (Department of Pharmacology) and one at the University of Bristol (Department of Pharmacology) she moved to her current position as a Royal Society of Edinburgh Biomedical Fellow in the School of Medicine, University of St Andrews. Her research group investigates intracellular calcium dynamics and the molecular function of intracellular ion channels. At present, she has a particular interest in understanding the molecular mechanisms of NAADP-regulated signalling via two-pore channels. Benedict Reilly-O'Donnell is a second year PhD student at the University of St Andrews. He is interested in the single channel properties of two-pore channels and other intracellular calcium-release channels. Using a combination of electrophysiological, molecular and biochemical methods, he is currently investigating the role of zinc in intracellular calcium release. Benedict completed his undergraduate studies in pharmacology at University College London in 2013. Rebecca Sitsapesan obtained her PhD at the University of Strathclyde and following appointment as British Heart Foundation Basic Science Lecturer at Imperial College London moved to Bristol University and then to the University of Oxford where she is currently Professor of Pharmacology. Her group investigates the biophysical properties of RyR and other ion channels that are present on intracellular organelles and are involved in the process of intracellular Ca 2+ release, particularly in regard to cardiac physiology and pathophysiology.This review was presented at the symposium "Non-selective cationic channels in chemical and physical stress", which took place at Physiology 2015 in Cardiff, UK, 6-8 July 2015.C 2016 The Authors. Abstract figure legend TPC1 and TPC2: NAADP-activated Ca 2+ -release pathways. TPC1 and TPC2 both play a role in the release of Ca 2+ from lysosomes and endolysosomes. Evidence suggests that TPC1 and TPC2 exhibit subtle but significant differences in ion conduction and selectivity, and although both channels appear to be activated by NAADP, regulation of gating by various additional modulators (such as cytosolic and luminal Ca 2+ , PI(3,5)P 2 , luminal pH, voltage) is also different. Thus, TPC1 and TPC2 may play complementary physiological roles. AbbreviationsNAADP, nicotinic acid adenine dinucleotide phosphate; TPC, two-pore channel; PI(3,5)P 2 , phosphatidylinositol 3,5-bisphosphate.

Fibrosis is a significant global health problem associated with many inflammatory and degenerative diseases affecting multiple organs, individually or simultaneously. Fibrosis develops when extracellular matrix (ECM) remodeling becomes excessive or uncontrolled and is associated with nearly all forms of heart disease. Cardiac fibroblasts and myofibroblasts are the main effectors of ECM deposition and scar formation. The heart is a complex multicellular organ, where the various resident cell types communicate between themselves and with cells of the blood and immune systems. Exosomes, which are small extracellular vesicles, (EVs), contribute to cell-to-cell communication and their pathophysiological relevance and therapeutic potential is emerging. Here, we will critically review the role of endogenous exosomes as possible fibrosis mediators and discuss the possibility of using stem cell-derived and/or engineered exosomes as anti-fibrotic agents.

Aberrant Zn2+ homeostasis is associated with dysregulated intracellular Ca2+ release, resulting in chronic heart failure. In the failing heart a small population of cardiac ryanodine receptors (RyR2) displays sub-conductance-state gating leading to Ca2+ leakage from sarcoplasmic reticulum (SR) stores, which impairs cardiac contractility. Previous evidence suggests contribution of RyR2-independent Ca2+ leakage through an uncharacterized mechanism. We sought to examine the role of Zn2+ in shaping intracellular Ca2+ release in cardiac muscle. Cardiac SR vesicles prepared from sheep or mouse ventricular tissue were incorporated into phospholipid bilayers under voltage-clamp conditions, and the direct action of Zn2+ on RyR2 channel function was examined. Under diastolic conditions, the addition of pathophysiological concentrations of Zn2+ (≥2 nm) caused dysregulated RyR2-channel openings. Our data also revealed that RyR2 channels are not the only SR Ca2+-permeable channels regulated by Zn2+. Elevating the cytosolic Zn2+ concentration to 1 nm increased the activity of the transmembrane protein mitsugumin 23 (MG23). The current amplitude of the MG23 full-open state was consistent with that previously reported for RyR2 sub-conductance gating, suggesting that in heart failure in which Zn2+ levels are elevated, RyR2 channels do not gate in a sub-conductance state, but rather MG23-gating becomes more apparent. We also show that in H9C2 cells exposed to ischemic conditions, intracellular Zn2+ levels are elevated, coinciding with increased MG23 expression. In conclusion, these data suggest that dysregulated Zn2+ homeostasis alters the function of both RyR2 and MG23 and that both ion channels play a key role in diastolic SR Ca2+ leakage.

Changes in cardiac ryanodine receptor (RyR2) phosphorylation are thought to be important regulatory and disease related post-translational protein modifications. The extent of RyR2 phosphorylation is mainly determined by the balance of the activities of protein kinases and phosphatases, respectively. Increased protein phosphatase-1 (PP1) activity has been observed in heart failure (HF), but the regulatory role of this enzyme on intracellular Ca 2þ handling remains poorly understood. To determine the physiological and pathophysiological significance of increased PP1 activity, we investigated the effect of the PP1 catalytic subunit on Ca 2þ sparks in permeabilized cardiomyocytes. We used wild-type (WT) and transgenic mice in which the highly phosphorylated site RyR2-S2808 has been ablated to investigate its involvement in RyR2 modulation. In WT myocytes, where cytosolic Ca 2þ was clamped at 60nM, 2U/ml of PP1 initially increased Ca 2þ spark frequency (CaSpF) by 2.2-fold, followed by a second phase during which CaSpF returned to control. Spark mass was decreased, but due to the high CaSpF, spark-mediated leak was increased by PP1. This was accompanied by depletion of the sarcoplasmic reticulum (SR) Ca 2þ stores, as determined by application of caffeine. Changes in Ca 2þ release and SR Ca 2þ load were prevented by 5uM of okadaic acid, an inhibitor of PP1. S2808A mutant myocytes showed lower resting CaSpF compared to WT (1.1850.23 vs 2.350.35 sparks/100um/s) and 2U/ml of PP1 failed to generate changes in CaSpF as well as in SR Ca 2þ load. A higher concentration of PP1 (10U/ml) increased CaSpF 4-fold compared to control in WT, and 2.8-fold in S2808A cells, indicating a concentration-dependence. Our results suggest that increased intracellular PP1 activity stimulates RyR2-mediated SR Ca 2þ release and that de-phosphorylation of RyR2-S2808 and at least one not yet identified phosphorylation site may be important in RyR2 modulation.

Cardiac fibrosis occurs in a wide range of cardiac diseases and is characterised by the transdifferentiation of cardiac fibroblasts (FB) into myofibroblasts (MFB). Myofibroblasts produce large quantities of extracellular matrix proteins, resulting in myocardial scar. The antifibrotic effect of the bile acid ursodeoxycholic acid (UDCA) is established in cases of liver fibrosis but not the adult myocardium.Our hypothesis is: UDCA is antifibrotic in the adult heart, mediated by the membrane bile acid receptor Takeda G protein-coupled receptor 5 (TGR5).We constructed a predictive network of fibrosis using RNA-seq datasets. We found that UDCA and it’s analogue INT-777, both reduced MFB markers in rat and human FBs and living myocardial slices (LMS). Utilising a knock-out mouse model, we show that the antifibrotic effect of UDCA is mediated by TGR5. Finally, we performed RNA-seq upon UDCA-treated human FB and integrated with our network of fibrosis, establishing the mechanism of TGR5 agonists.