Inositol Trisphosphate Receptors and Nuclear Calcium in Atrial Fibrillation

Rationale: The mechanisms underlying atrial fibrillation (AF), the most common clinical arrhythmia, are poorly understood. Nucleoplasmic Ca 2+ regulates gene-expression, but the nature and significance of nuclear Ca 2+ -changes in AF are largely unknown. Objective: To elucidate mechanisms by which AF alters atrial cardiomyocyte (CM) nuclear Ca 2+ ([Ca 2+ ] Nuc ) and Ca 2+ /calmodulin-dependent protein kinase-II (CaMKII)-related signaling. Methods and Results: Atrial CMs were isolated from control and AF-dogs (kept in AF by atrial tachypacing [600 bpm x 1 week]). [Ca 2+ ] Nuc and cytosolic [Ca 2+ ] (Ca 2+ ] Cyto ) were recorded via confocal microscopy. Diastolic [Ca 2+ ] Nuc was greater than [Ca 2+ ] Cyto under control conditions, while resting [Ca 2+ ] Nuc was similar to [Ca 2+ ] Cyto ; both diastolic and resting [Ca 2+ ] Nuc increased with AF. Inositol-trisphosphate-receptor (IP 3 R) stimulation produced larger [Ca 2+ ] Nuc increases in AF versus control CMs, and IP 3 R-blockade suppressed the AF-related [Ca 2+ ] Nuc -differences. AF upregulated nuclear protein-expression of IP 3 R-type 1 (IP 3 R1) and of phosphorylated CaMKII (immunohistochemistry and immunoblot), while decreasing the nuclear/cytosolic expression-ratio for histone deacetylase type-4 (HDAC4). Isolated atrial CMs tachypaced at 3 Hz for 24 hours mimicked AF-type [Ca 2+ ] Nuc changes and L-type calcium current (ICaL) decreases versus 1-Hz-paced CMs; these changes were prevented by IP3R knockdown with short-interfering RNA directed against IP 3 R1. Nuclear/cytosolic HDAC4 expression-ratio was decreased by 3-Hz pacing, while nuclear CaMKII and HDAC4 phosphorylation were increased. Either CaMKII-inhibition (by autocamtide-2-related peptide) or IP 3 R-knockdown prevented the CaMKII-hyperphosphorylation and nuclear-to-cytosolic HDAC4 shift caused by 3-Hz pacing. In human atrial CMs from AF patients, nuclear IP 3 R1-expression was significantly increased, with decreased nuclear/non-nuclear HDAC4 ratio. MicroRNA-26a was predicted to target ITPR1 (confirmed by Luciferase assay) and was downregulated in AF atrial CMs; microRNA-26a silencing reproduced AF-induced IP3R1 upregulation and nuclear diastolic Ca 2+ -loading. Conclusions: AF increases atrial CM nucleoplasmic Ca 2+ -handling by IP 3 R1-upregulation involving miR-26a, leading to enhanced IP 3 R1-CaMKII-HDAC4 signaling and I CaL -downregulation.

Steady-state Regulation of Secretory Cargo Export by Inositol Trisphosphate Receptors and Penta EF Hand Proteins

ABSTRACTER Ca2+ regulates ER-to-Golgi transport machinery. Sustained Ca2+ signaling by inositol trisphosphate receptors (IP3Rs) leads to depression of cargo export through activation of penta EF hand protein (PEF) ALG-2 which reduces outer COPII coat at ER exit sites (ERES). However, we do not know whether tonic Ca2+ signals during steady-state conditions affect ER export rates and if so by what mechanisms. Here we report that partial depletion of IP3 receptors from NRK epithelial cells causes a marked increase of basal ER export of the transmembrane glycoprotein cargo VSV-G. The increased ER-to-Golgi transport required ALG-2 and was actuated by decreased peflin and increased ALG-2 at ER exit sites (ERES) – a condition previously demonstrated to stimulate COPII-dependent ER export. Upon IP3R depletion the amount of outer coat at ERES increased, the opposite to what occurs during ALG-2-dependent inhibition of secretion during agonist-driven Ca2+ signaling. The increased ER export correlated with reduced spontaneous cytosolic Ca2+ oscillations caused by the reduced number of Ca2+ release channels. IP3R depletion also unexpectedly resulted in partial depletion of ER luminal Ca2+ stores. The low Ca2+ conditions appeared to decrease both ALG-2 and peflin expression levels somewhat, but these were the only detectable expression changes in COPII trafficking machinery and the Ca2+ decrease had no detectable impact on ER stress. We conclude that at steady state, IP3Rs produce tonic Ca2+ signals that suppress the basal rate of ER export by maintaining lower outer coat targeting to ERES.

IP3 mediated global Ca2+ signals arise through two temporally and spatially distinct modes of Ca2+ release

The ‘building-block’ model of inositol trisphosphate (IP3)-mediated Ca2+ liberation posits that cell-wide cytosolic Ca2+ signals arise through coordinated activation of localized Ca2+ puffs generated by stationary clusters of IP3 receptors (IP3Rs). Here, we revise this hypothesis, applying fluctuation analysis to resolve Ca2+ signals otherwise obscured during large Ca2+ elevations. We find the rising phase of global Ca2+ signals is punctuated by a flurry of puffs, which terminate before the peak by a mechanism involving partial ER Ca2+ depletion. The continuing rise in Ca2+, and persistence of global signals even when puffs are absent, reveal a second mode of spatiotemporally diffuse Ca2+ signaling. Puffs make only small, transient contributions to global Ca2+ signals, which are sustained by diffuse release of Ca2+ through a functionally distinct process. These two modes of IP3-mediated Ca2+ liberation have important implications for downstream signaling, imparting spatial and kinetic specificity to Ca2+-dependent effector functions and Ca2+ transport.

Allelic Variants of CRISPR/Cas9 Induced Mutation in an Inositol Trisphosphate 5/6 Kinase Gene Manifest Different Phenotypes in Barley

Inositol trisphosphate 5/6 kinases (ITPK) constitute a small group of enzymes participating in the sequential phosphorylation of inositol phosphate to inositol hexakisphosphate (IP6), which is a major storage form of phosphate in cereal grains. The development of lines with reduced IP6 content could enhance phosphate and mineral bioavailability. Moreover, plant ITPKs participate in abiotic stress signaling. To elucidate the role of HvITPK1 in IP6 synthesis and stress signaling, a barley itpk1 mutant was created using programmable nuclease Cas9. Homozygous single bp insertion and deletion mutant lines were obtained. The mutants contained altered levels of phosphate in the mature grains, ranging from 65% to 174% of the wild type (WT) content. Homozygous mutant lines were tested for their response to salinity during germination. Interestingly, insertion mutant lines revealed a higher tolerance to salinity stress than deletion mutants. Mature embryos of an insertion mutant itpk1-2 and deletion mutant itpk1-33 were cultivated in vitro on MS medium supplemented with NaCl at 50, 100, and 200 mM. While both mutants grew less well than WT on no or low salt concentrations, the itpk1-2 mutant was affected less than the WT and itpk33 when grown on the highest NaCl concentration. The expression of all ITPKs was induced in roots in response to salt stress. In shoots, the differential effect of high salt on IPTK expression in the two iptk1 mutants was consistent with their different sensitivities to salt stress. The results extend the evidence for the involvement of ITPK genes in phosphate storage and abiotic stress signaling.