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 regulates gene expression, but the nature and significance of nuclear Ca-changes in AF are largely unknown.

Objective: To elucidate mechanisms by which AF alters atrial-cardiomyocyte nuclear Ca ([Ca]) and CaMKII (Ca/calmodulin-dependent protein kinase-II)-related signaling.

Caged ligands to study the role of intracellular GPCRs.

In addition to cell surface membranes, numerous G protein-coupled receptors (GPCRs) are located on intracellular membranes including the nuclear envelope. Although the role of numerous GPCRs at the cell surface has been well characterized, the physiological function of these same receptors located on intracellular membranes remains to be determined. Here, we employ a novel caged Ang-II analog, cAng-II, to compare the effects of the activation of cell surface versus intracellular angiotensin receptors in intact cardiomyocytes.

Photoreleasable ligands to study intracrine angiotensin II signalling.

Key Points: The renin-angiotensin system plays a key role in cardiovascular physiology and its overactivation has been implicated in the pathogenesis of several major cardiovascular diseases. There is growing evidence that angiotensin II (Ang-II) may function as an intracellular peptide to activate intracellular/nuclear receptors and their downstream signalling effectors independently of cell surface receptors. Current methods used to study intracrine Ang-II signalling are limited to indirect approaches because of a lack of selective intracellularly-acting probes.

Using caged ligands to study intracrine endothelin signaling in intact cardiac myocytes.

Intracrine signaling refers to the activation of receptors located within the cell and many intracrine receptors have been localized to the nuclear membrane. The presence and function of nuclear receptors have been studied in isolated nuclei. Much less information is available concerning the function of these receptors within the context of intact cells due, in part, to difficulties in accessing the intracellular receptor without activating those at the cell surface.

Intracrine endothelin signaling evokes IP3-dependent increases in nucleoplasmic Ca²⁺ in adult cardiac myocytes.

Endothelin receptors are present on the nuclear membranes in adult cardiac ventricular myocytes. The objectives of the present study were to determine 1) which endothelin receptor subtype is in cardiac nuclear membranes, 2) if the receptor and ligand traffic from the cell surface to the nucleus, and 3) the effect of increased intracellular ET-1 on nuclear Ca(2+) signaling. Confocal microscopy using fluorescently-labeled endothelin analogs confirmed the presence of ETB at the nuclear membrane of rat cardiomyocytes in skinned-cells and isolated nuclei.

Regulation of cardiac nitric oxide signaling by nuclear β-adrenergic and endothelin receptors.

At the cell surface, βARs and endothelin receptors can regulate nitric oxide (NO) production. β-adrenergic receptors (βARs) and type B endothelin receptors (ETB) are present in cardiac nuclear membranes and regulate transcription. The present study investigated the role of the NO pathway in the regulation of gene transcription by these nuclear G protein-coupled receptors.

Post-translational modifications, a key process in CD36 function: lessons from the spontaneously hypertensive rat heart.

CD36, a multifunctional protein, is involved in cardiac long chain fatty acid (LCFA) metabolism and in the etiology of heart diseases, yet the functional impact of Cd36 gene variants remains unclear. In 7-week-old spontaneously hypertensive rats (SHR), which, like humans, carry numerous mutations in Cd36, we tested the hypothesis that their restricted cardiac LCFA utilization occurs prior to hypertrophy due to defective CD36 post-translational modifications (PTM), as assessed by ex vivo perfusion of (13)C-labeled substrates and biochemical techniques. Compared to their controls, SHR hearts displayed a lower (i) contribution of LCFA to β-oxidation (-40%) and triglycerides (+2.

Hypoxia in early life is associated with lasting changes in left ventricular structure and function at maturity in the rat.

Background: There is a growing population of adults with repaired cyanotic congenital heart disease. These patients have increased risk of impaired cardiac health and premature death. We hypothesized that hypoxia in early life before surgical intervention causes lasting changes in left ventricular structure and function with physiological implications in later life.

Effect of pressure overload-induced hypertrophy on the expression and localization of p38 MAP kinase isoforms in the mouse heart.

p38 mitogen-activated protein kinases (MAPKs) are serine/threonine specific protein kinases that respond to cellular stress and regulate a broad range of cellular activities. There are four major isoforms of p38 MAPK: alpha, beta, gamma, and delta. To date, the prominent isoform in heart has been thought to be p38alpha.

Characterization of a novel MK3 splice variant from murine ventricular myocardium.

p38 MAP kinase (MAPK) isoforms alpha, beta, and gamma, are expressed in the heart. p38alpha appears pro-apoptotic whereas p38beta is pro-hypertrophic. The mechanisms mediating these divergent effects are unknown; hence elucidating the downstream signaling of p38 should further our understanding.

Nuclear-delimited angiotensin receptor-mediated signaling regulates cardiomyocyte gene expression.

Angiotensin-II (Ang-II) from extracardiac sources and intracardiac synthesis regulates cardiac homeostasis, with mitogenic and growth-promoting effects largely due to altered gene expression. Here, we assessed the possibility that angiotensin-1 (AT1R) or angiotensin-2 (AT2R) receptors on the nuclear envelope mediate effects on cardiomyocyte gene expression. Immunoblots of nucleus-enriched fractions from isolated cardiomyocytes indicated the presence of AT1R and AT2R proteins that copurified with the nuclear membrane marker nucleoporin-62 and histone-3, but not markers of plasma (calpactin-I), Golgi (GRP-78), or endoplasmic reticulum (GM130) membranes.

Gbetagamma is a negative regulator of AP-1 mediated transcription.

Following stimulation of G protein-coupled receptors (GPCRs) at the cell surface, heterotrimeric G proteins are activated. Both Galpha and Gbetagamma subunits regulate specific effectors to transmit signals received by the receptor. Recent data suggest potential nuclear localization or translocation of the Gbetagamma subunit.

Characterization of the expression and regulation of MK5 in the murine ventricular myocardium.

MK5, a member of the MAPK-activated protein kinase family, is highly expressed in the heart. Whereas MK2 and MK3 are activated by p38 MAPK, MK5 has also been shown to be activated by ERK3 and ERK4. We studied the regulation of MK5 in mouse heart.

Association of neonatal hypoxia with lasting changes in left ventricular gene expression: an animal model.

Objective: Innovations in pediatric cardiovascular surgery have resulted in significant improvements in survival for children with congenital heart disease. In adults with such disease, however, surgical morbidity and mortality remain significant. We hypothesized that hypoxemia in early life causes lasting changes in gene expression in the developing heart and that such changes may persist into later life, affecting the physiology of the adult myocardium.

GABA-B(1) receptors are coupled to the ERK1/2 MAP kinase pathway in the absence of GABA-B(2) subunits.

In the current model of gamma-aminobutyric acid (GABA) B receptor function, there is a requirement for GABA-B(1/2) heterodimerisation for targetting to the cell surface. However, different lines of evidence suggest that the GABA-B(1) subunit can form a functional receptor in the absence of GABA-B(2). We observed coupling of endogenous GABA-B(1) receptors in the DI-TNC1 glial cell line to the ERK pathway in response to baclofen even though these cells do not express GABA-B(2).

Mechanisms underlying rate-dependent remodeling of transient outward potassium current in canine ventricular myocytes.

Transient outward K+ current (I to) downregulation following sustained tachycardia in vivo is usually attributed to tachycardiomyopathy. This study assessed potential direct rate regulation of cardiac I(to) and underlying mechanisms. Cultured adult canine left ventricular cardiomyocytes (37 degrees C) were paced continuously at 1 or 3 Hz for 24 hours.

Cellular signaling underlying atrial tachycardia remodeling of L-type calcium current.

Atrial tachycardia (AT) downregulates L-type Ca(2+) current (I(CaL)) and causes atrial fibrillation-promoting electric remodeling. This study assessed potential underlying signal transduction. Cultured adult canine atrial cardiomyocytes were paced at 0, 1, or 3 Hz (P0, P1, P3) for up to 24 hours.

Heterodimers of adenylyl cyclases 2 and 5 show enhanced functional responses in the presence of Galpha s.

Recent studies have demonstrated that adenylyl cyclase isoforms can form both homo- and heterodimers and that this may be the basic functional unit of these enzymes (see Cooper, D.M.F.

Angiopoietin-mediated endothelial P-selectin translocation: cell signaling mechanisms.

Recently identified, angiopoietin-1 (Ang1) and -2 (Ang2) bind to the tyrosine kinase receptor Tie2 and contribute to orchestrate blood vessel formation during angiogenesis. Ang1 mediates vessel maturation and integrity by favoring the recruitment of pericytes and smooth muscle cells. Ang2, initially identified as a Tie2 antagonist, may under certain circumstances, induce Tie2 phosphorylation and biological activities.

Seven transmembrane receptor core signaling complexes are assembled prior to plasma membrane trafficking.

Much is known about beta2-adrenergic receptor trafficking and internalization following prolonged agonist stimulation. However, less is known about outward trafficking of the beta2-adrenergic receptor to the plasma membrane or the role that trafficking might play in the assembly of receptor signaling complexes, important for targeting, specificity, and rapidity of subsequent signaling events. Here, by using a combination of bioluminescence resonance energy transfer, bimolecular fluorescence complementation, and confocal microscopy, we evaluated the steps in the formation of the core receptor-G protein heterotrimer complex.

Interactions between GABA-B1 receptors and Kir 3 inwardly rectifying potassium channels.

gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the mammalian brain. It acts via both ionotropic GABA-A and metabotropic GABA-B receptors. We evaluated the interaction of receptors with members of the inwardly rectifying potassium (Kir 3) channel family, which also play an important role in neuronal transmission and membrane excitability.

Restoring depressed HERG K+ channel function as a mechanism for insulin treatment of abnormal QT prolongation and associated arrhythmias in diabetic rabbits.

Abnormal QT prolongation (QT-P) in diabetic patients has become a nonnegligible clinical problem and has attracted increasing attention from basic scientists, because it increases the risk of lethal ventricular arrhythmias. Correction of QT-P may be an important measure in minimizing sudden cardiac death in diabetic patients. Here we report the efficacy of insulin in preventing QT-P and the associated arrhythmias and the mechanisms underlying the effects in a rabbit model of type 1 insulin-dependent diabetes mellitus (IDDM).

Sub-cellular distribution of endothelin signaling pathway components in ventricular myocytes and heart: lack of preformed caveolar signalosomes.

Stimulation of endothelin receptors (ETRs) leads to activation of the extracellular signal-regulated protein kinase (ERK) cascade. It is unclear whether compartmentalization to lipid rafts is necessary for proper endothelin signaling, as methodologies employed to isolate and study caveolae involve detergent extraction, which may induce aggregation of membrane-associated proteins. The present study was to determine if components of the endothelin-1 (ET-1) pathway leading to ERK activation localize to caveolae and constitute preformed signalosomes.

VEGF-mediated endothelial P-selectin translocation: role of VEGF receptors and endogenous PAF synthesis.

The acute increase in vascular permeability produced by vascular endothelial growth factor (VEGF-A(165)) requires activation of endothelial Flk-1 receptors (VEGFR-2) and stimulation of platelet-activating factor (PAF) synthesis. Like PAF, VEGF-A(165) promotes translocation of P-selectin to the endothelial cell (EC) surface. However, the mechanisms involved remain unknown.

Modulation of Ca2+-dependent Cl- channels by calcineurin in rabbit coronary arterial myocytes.

The role of the Ca2+-dependent phosphatase calcineurin (CaN) in the modulation of Ca2+-dependent Cl- channels (ClCa) was studied in freshly isolated rabbit coronary arterial myocytes. Immunocytochemical experiments showed that calmodulin-dependent protein kinase II (CaMKII) and CaN were distributed evenly throughout the cytoplasm of coronary myocytes at rest and translocated to the plasmalemma when intracellular Ca2+ was increased. ClCa currents (ICl(Ca)) elicited by cell dialysis with fixed intracellular Ca2+ levels up to 500 nM were inhibited by 10 microM cyclosporin A (CsA), a specific inhibitor of CaN, in a voltage-dependent manner, whereas currents evoked by 1 microM Ca2+ were not affected.