The role of inositol 1,4,5-trisphosphate receptors in Ca2+ signalling and the generation of arrhythmias in rat atrial myocytes

Various cardio-active stimuli, including endothelin-1 (ET-1), exhibit potent arrhythmogenicity, but the underlying cellular mechanisms of their actions are largely unclear. We used isolated rat atrial myocytes and related changes in their subcellular Ca2+ signalling to the ability of various stimuli to induce diastolic, premature extra Ca2+ transients (ECTs). For this, we recorded global and spatially resolved Ca2+ signals in indo-1- and fluo-4-loaded atrial myocytes during electrical pacing. ET-1 exhibited a higher arrhythmogenicity (arrhythmogenic index; ratio of number of ECTs over fold-increase in Ca2+ response, 8.60; n = 8 cells) when compared with concentrations of cardiac glycosides (arrhythmogenic index, 4.10; n = 8 cells) or the β-adrenergic agonist isoproterenol (arrhythmogenic index, 0.11; n = 6 cells) that gave similar increases in the global Ca2+ responses. Seventy-five percent of the ET-1-induced arrhythmogenic Ca2+ transients were accompanied by premature action potentials, while for digoxin this proportion was 25 %. The β-adrenergic agonist failed to elicit a significant number of ECTs. Direct activation of inositol 1,4,5-trisphosphate (InsP3) receptors with a membrane-permeable InsP3 ester (InsP3 BM) mimicked the effect of ET-1 (arrhythmogenic index, 14.70; n = 6 cells). Inhibition of InsP3 receptors using 2 μM 2-aminoethoxydiphenyl borate, which did not display any effects on Ca2+ signalling under control conditions, specifically suppressed the arrhythmogenic action of ET-1 and InsP3 BM. Immunocytochemistry indicated a co-localisation of peripheral, junctional ryanodine receptors with InsP3Rs. Thus, the pronounced arrhythmogenic potency of ET-1 is due to the spatially specific recruitment of Ca2+ sparks by subsarcolemmal InsP3Rs. Summation of such sparks efficiently generates delayed afterdepolarisations that trigger premature action potentials. We conclude that the particular spatial profile of cellular Ca2+ signals is a major, previously unrecognised, determinant for arrhythmogenic potency and that the InsP3 signalling cassette might therefore be a promising new target for understanding and managing atrial arrhythmia

MLL-rearranged acute lymphoblastic leukemias activate BCL-2 through H3K79 methylation and are sensitive to the BCL-2-specific antagonist ABT-199

Targeted therapies designed to exploit specific molecular pathways in aggressive cancers are an exciting area of current research. Mixed Lineage Leukemia (MLL) mutations such as the t(4;11) translocation cause aggressive leukemias that are refractory to conventional treatment. The t(4;11) translocation produces an MLL/AF4 fusion protein that activates key target genes through both epigenetic and transcriptional elongation mechanisms. In this study, we show that t(4;11) patient cells express high levels of BCL-2 and are highly sensitive to treatment with the BCL-2-specific BH3 mimetic ABT-199. We demonstrate that MLL/AF4 specifically upregulates the BCL-2 gene but not other BCL-2 family members via DOT1L-mediated H3K79me2/3. We use this information to show that a t(4;11) cell line is sensitive to a combination of ABT-199 and DOT1L inhibitors. In addition, ABT-199 synergizes with standard induction-type therapy in a xenotransplant model, advocating for the introduction of ABT-199 into therapeutic regimens for MLL-rearranged leukemias

Contractile responses to endothelin-1 are regulated by PKC phosphorylation of cardiac myosin binding protein-C in rat ventricular myocytes

The shortening of sarcomeres that co-ordinates the pump function of the heart is stimulated by electrically-mediated increases in [Ca2+]. This process of excitation-contraction coupling (ECC) is subject to modulation by neurohormonal mediators that tune the output of the heart to meet the needs of the organism. Endothelin-1 (ET-1) is a potent modulator of cardiac function with effects on contraction amplitude, chronotropy and automaticity. The actions of ET-1 are evident during normal adaptive physiological responses and increased under pathophysiological conditions, such as following myocardial infarction and during heart failure, where ET-1 levels are elevated. In myocytes, ET-1 acts through ETA- or ETB-G protein-coupled receptors (GPCRs). Although well studied in atrial myocytes, the influence and mechanisms of action of ET-1 upon ECC in ventricular myocytes are not fully resolved. We show in rat ventricular myocytes that ET-1 elicits a biphasic effect on fractional shortening (initial transient negative and sustained positive inotropy) and increases the peak amplitude of systolic Ca2+ transients in adult rat ventricular myocytes. The negative inotropic phase was ETB receptor-dependent, whereas the positive inotropic response and increase in peak amplitude of systolic Ca2+ transients required ETA receptor engagement. Both effects of ET-1 required phospholipase C (PLC)-activity, although distinct signalling pathways downstream of PLC elicited the effects of each ET receptor. The negative inotropic response involved inositol 1,4,5-trisphosphate (InsP3) signalling and protein kinase C epsilon (PKCε). The positive inotropic action and the enhancement in Ca2+ transient amplitude induced by ET-1 were independent of InsP3 signalling, but suppressed by PKCε. Serine 302 in cardiac myosin binding protein-C was identified as a PKCε substrate that when phosphorylated contributed to the suppression of contraction and Ca2+ transients by PKCε following ET-1 stimulation. Thus, our data provide a new role and mechanism of action for InsP3 and PKCε in mediating the negative inotropic response and in restraining the positive inotropy and enhancement in Ca2+ transients following ET-1 stimulation