Julie Bossuyt

Local InsP3-dependent perinuclear Ca2+ signaling in cardiac myocyte excitation-transcription coupling

Previous work showed that calmodulin (CaM) and Ca2+-CaM-dependent protein kinase II (CaMKII) are somehow involved in cardiac hypertrophic signaling, that inositol 1,4,5-trisphosphate receptors (InsP3Rs) in ventricular myocytes are mainly in the nuclear envelope, where they associate with CaMKII, and that class II histone deacetylases (e.g., HDAC5) suppress hypertrophic gene transcription. Furthermore, HDAC phosphorylation in response to neurohumoral stimuli that induce hypertrophy, such as endothelin-1 (ET-1), activates HDAC nuclear export, thereby regulating cardiac myocyte transcription. Here we demonstrate a detailed mechanistic convergence of these 3 issues in adult ventricular myocytes. We show that ET-1, which activates plasmalemmal G protein-coupled receptors and InsP3 production, elicits local nuclear envelope Ca2+ release via InsP3R. This local Ca2+ release activates nuclear CaMKII, which triggers HDAC5 phosphorylation and nuclear export (derepressing transcription). Remarkably, this Ca2+-dependent pathway cannot be activated by the global Ca2+ transients that cause contraction at each heartbeat. This novel local Ca2+ signaling in excitation-transcription coupling is analogous to but separate (and insulated) from that involved in excitation-contraction coupling. Thus, myocytes can distinguish simultaneous local and global Ca2+ signals involved in contractile activation from those targeting gene expression.

Phospholemman-Phosphorylation Mediates the β-Adrenergic Effects on Na/K Pump Function in Cardiac Myocytes

Cardiac sympathetic stimulation activates β-adrenergic (β-AR) receptors and protein kinase A (PKA) phosphorylation of proteins involved in myocyte Ca regulation. The Na/K-ATPase (NKA) is essential in regulating intracellular [Na] ([Na]i), which in turn affects [Ca]i via Na/Ca exchange. However, how PKA modifies NKA function is unknown. Phospholemman (PLM), a member of the FXYD family of proteins that interact with NKA in various tissues, is a major PKA substrate in heart. Here we tested the hypothesis that PLM phosphorylation is responsible for the PKA effects on cardiac NKA function using wild-type (WT) and PLM knockout (PLM-KO) mice. We measured NKA-mediated [Na]i decline and current (IPump) to assess β-AR effects on NKA function in isolated myocytes. In WT myocytes, 1 &mgr;mol/L isoproterenol (ISO) increased PLM phosphorylation and stimulated NKA activity mainly by increasing its affinity for internal Na (Km decreased from 18.8±1.4 to 13.6±1.5 mmol/L), with no significant effect on the maximum pump rate. This led to a significant decrease in resting [Na]i (from 12.5±1.8 to 10.5±1.4 mmol/L). In PLM-KO mice under control conditions Km (14.2±1.5 mmol/L) was lower than in WT, but comparable to that for WT in the presence of ISO. Furthermore, ISO had no significant effect on NKA function in PLM-KO mice. ATPase activity in sarcolemmal vesicles also showed a lower Km(Na) in PLM-KO versus WT (12.9±0.9 versus 16.2±1.5). Thus, PLM inhibits NKA activity by decreasing its [Na]i affinity, and this inhibitory effect is relieved by PKA activation. We conclude that PLM modulates the NKA function in a manner similar to the way phospholamban affects the related SR Ca-ATPase (inhibition of transport substrate affinity, that is relieved by phosphorylation).

Ca2+/Calmodulin-Dependent Protein Kinase IIδ and Protein Kinase D Overexpression Reinforce the Histone Deacetylase 5 Redistribution in Heart Failure

Cardiac hypertrophy and heart failure (HF) are associated with reactivation of fetal cardiac genes, and class II histone deacetylases (HDACs) (eg, HDAC5) have been strongly implicated in this process. We have shown previously that inositol trisphosphate, Ca2+/calmodulin-dependent protein kinase II (CaMKII), and protein kinase (PK)D are involved in HDAC5 phosphorylation and nuclear export in normal adult ventricular myocytes and also that CaMKII&dgr; and inositol trisphosphate receptors are upregulated in HF. Here we tested whether, in our rabbit HF model, nucleocytoplasmic shuttling of HDAC5 was altered either at baseline or in response to endothelin-1, which would indicate HDAC5 phosphorylation and transcription effects. The fusion protein HDAC5–green fluorescent protein (HDAC5-GFP) was more cytosolic in HF myocytes (Fnuc/Fcyto 3.3±0.3 vs 7.2±0.4 in control), and HDAC5 was more phosphorylated. Despite this baseline cytosolic HDAC5 shift, endothelin-1 produced more rapid HDAC5-GFP nuclear export in HF versus control myocytes. We also find that PKD and CaMKII&dgr;C expression and activation state are increased in both rabbit and human HF. Inhibition of either CaMKII or PKD in HF myocytes partially restored the HDAC5-GFP Fnuc/Fcyto toward control, and simultaneous inhibition restored Fnuc/Fcyto to that in control myocytes. Moreover, adenovirus-mediated overexpression of PKD, CaMKII&dgr;B, or CaMKII&dgr;C reduced baseline HDAC5 Fnuc/Fcyto in control myocytes (3.4±0.5, 3.8±0.5, and 5.2±0.5, respectively), approaching that seen in HF. We conclude that chronic upregulation and activation of inositol trisphosphate receptors, CaMKII, and PKD in HF shifts HDAC5 out of the nucleus, derepressing transcription of hypertrophic genes. This may directly contribute to the development and/or maintenance of HF.

Regulation of Ca2+ and Na+ in Normal and Failing Cardiac Myocytes

Abstract:  Ca2+ in cardiac myocytes regulates contractility and relaxation, and Ca2+ and Na +regulation are linked via Na+/Ca2+ exchange (NCX). Heart failure (HF) is accompanied by contractile dysfunction and arrhythmias, both of which may be due to altered cellular Ca2+ handling. Smaller Ca2+ transient and sarcoplasmic reticulum (SR) Ca2+ content cause systolic dysfunction in HF. The reduced SR Ca2+ content is due to: (a) reduced SR Ca2+‐ATPase function (which also contributes to diastolic dysfunction), (b) increased expression and function of NCX (which competes with SR Ca2+‐ATPase during relaxation, but preserves diastolic function), and (c) enhanced diastolic SR Ca2+ leak. Relative contributions of these may vary with HF etiology and stage. Triggered arrhythmias (e.g., delayed afterdepolarizations [DADs]) are prominent in HF. DADs are due to spontaneous SR Ca2+ release and consequent activation of transient inward NCX current, which in HF allows DADs to more readily trigger arrhythmogenic action potentials. Thus NCX and Na+ are critical in systolic and diastolic function and arrhythmias. [Na+]i is elevated in HF, which may limit SR unloading and provide some Ca2+ influx during the HF action potential, thus limiting the depression of systolic function. High [Na+]i in HF is due to enhanced Na+ influx. Cellular Na+/K+‐ATPase (NKA) function appears unaltered, despite reduced NKA expression. This dichotomy led us to test NKA regulation by phospholemman (PLM). We find that PLM regulates NKA in a manner analogous to phospholamban regulation of SR Ca2+‐ATPase (i.e., inhibition that is relieved by PLM phosphorylation). We measured intermolecular FRET between PLM and NKA, which is reduced upon PLM phosphorylation. The lower expression level of more phosphorylated PLM in HF may explain the above dichotomy. Thus, altered Ca2+ and Na+ handling contributes to altered contractile function and arrhythmogenesis in HF.

Biosensors to Measure Inositol 1,4,5-Trisphosphate Concentration in Living Cells with Spatiotemporal Resolution

Phosphoinositides participate in many signaling cascades via phospholipase C stimulation, which hydrolyzes phosphatidylinositol 4,5-bisphosphate, producing second messengers diacylglycerol and inositol 1,4,5-trisphosphate (InsP3). Destructive chemical approaches required to measure [InsP3] limit spatiotemporal understanding of subcellular InsP3 signaling. We constructed novel fluorescence resonance energy transfer-based InsP3 biosensors called FIRE (fluorescent InsP3-responsive element) by fusing plasmids encoding the InsP3-binding domain of InsP3 receptors (types 1–3) between cyan fluorescent protein and yellow fluorescent protein sequences. FIRE was expressed and characterized in COS-1 cells, cultured neonatal cardiac myocytes, and incorporated into an adenoviral vector for expression in adult cardiac ventricular myocytes. FIRE-1 exhibits an ∼11% increase in the fluorescence ratio (F530/F480) at saturating [InsP3] (apparent Kd = 31.3 ± 6.7 nm InsP3). In COS-1 cells, neonatal rat cardiac myocytes and adult cat ventricular myocytes FIRE-1 exhibited comparable dynamic range and a 10% increase in donor (cyan fluorescent protein) fluorescence upon bleach of yellow fluorescent protein, indicative of fluorescence resonance energy transfer. In FIRE-1 expressing ventricular myocytes endothelin-1, phenylephrine, and angiotensin II all produced rapid and spatially resolved increases in [InsP3] using confocal microscopy (with free [InsP3] rising to ∼30 nm). Local entry of intracellular InsP3 via membrane rupture by a patch pipette (containing InsP3)in myocytes expressing FIRE-1 allowed detailed spatiotemporal monitoring of intracellular InsP3 diffusion. Both endothelin-1-induced and direct InsP3 application (via pipette rupture) revealed that InsP3 diffusion into the nucleus occurs with a delay and blunted rise of [InsP3] versus cytosolic [InsP3]. These new biosensors allow studying InsP3 dynamics at high temporal and spatial resolution that will be powerful in under-standing InsP3 signaling in intact cells.

Expression and Phosphorylation of the Na-Pump Regulatory Subunit Phospholemman in Heart Failure

Intracellular [Na] is ≈3 mmol/L higher in heart failure (HF; in our arrhythmogenic rabbit model;3), and this can profoundly affect cardiac Ca and contractile function via Na/Ca exchange and Na/H exchange. Na/K-ATPase is the primary mechanism of Na extrusion. We examine here in HF rabbits (and human hearts) expression of Na/K-ATPase isoforms and phospholemman (PLM), a putative Na/K-ATPase regulatory subunit that inhibits pump function and is a major cardiac phosphorylation target. Na/K-ATPase &agr;1- and &agr;2-isoforms were reduced in HF in rabbit ventricular homogenates (by 24%) and isolated myocytes (by 30% and 17%), whereas &agr;3 was increased (50%) in homogenates and decreased (52%) in myocytes (P<0.05). Homogenate Na/K-ATPase activity in left ventricle was also decreased in HF. However, we showed previously that Na/K-ATPase characteristics in intact ventricular myocytes were unaltered in HF. To reconcile these findings, we assessed PLM expression, phosphorylation, and association with Na/K-ATPase. PLM coimmunoprecipitated with Na/K-ATPase &agr;1 and &agr;2 in control and HF rabbit myocytes. PLM expression was reduced in HF by 42% in isolated rabbit left ventricular (LV) myocytes, by 48% in rabbit LV homogenates, and by 24% in human LV homogenate. The fraction of PLM phosphorylated at Ser-68 was increased dramatically in HF. Our results are consistent with a role for PLM analogous to that of phospholamban for SR Ca-ATPase (SERCA): inhibition of Na/K-ATPase function that is relieved on PLM phosphorylation. So reduced Na/K-ATPase expression in HF may be functionally offset by lower inhibition by PLM (because of reduced PLM expression and higher PLM phosphorylation).

RhoA protects the mouse heart against ischemia/reperfusion injury.

The small GTPase RhoA serves as a nodal point for signaling through hormones and mechanical stretch. However, the role of RhoA signaling in cardiac pathophysiology is poorly understood. To address this issue, we generated mice with cardiomyocyte-specific conditional expression of low levels of activated RhoA (CA-RhoA mice) and demonstrated that they exhibited no overt cardiomyopathy. When challenged by in vivo or ex vivo ischemia/reperfusion (I/R), however, the CA-RhoA mice exhibited strikingly increased tolerance to injury, which was manifest as reduced myocardial lactate dehydrogenase (LDH) release and infarct size and improved contractile function. PKD was robustly activated in CA-RhoA hearts. The cardioprotection afforded by RhoA was reversed by PKD inhibition. The hypothesis that activated RhoA and PKD serve protective physiological functions during I/R was supported by several lines of evidence. In WT mice, both RhoA and PKD were rapidly activated during I/R, and blocking PKD augmented I/R injury. In addition, cardiac-specific RhoA-knockout mice showed reduced PKD activation after I/R and strikingly decreased tolerance to I/R injury, as shown by increased infarct size and LDH release. Collectively, our findings provide strong support for the concept that RhoA signaling in adult cardiomyocytes promotes survival. They also reveal unexpected roles for PKD as a downstream mediator of RhoA and in cardioprotection against I/R.

Phospholemman Phosphorylation Mediates the Protein Kinase C–Dependent Effects on Na+/K+ Pump Function in Cardiac Myocytes

Because phospholemman (PLM) regulates the Na+/K+ pump (NKA) and is a major cardiac phosphorylation target for both protein kinase A (at Ser68) and protein kinase C (PKC) (at both Ser63 and Ser68), we evaluated whether PLM mediates the PKC-dependent regulation of NKA function and protein kinase A/PKC crosstalk in ventricular myocytes. PKC was activated by PDBu (300 nmol/L), and we measured NKA-mediated [Na+]i decline (fluorescence measurements) and current (Ipump) (voltage clamp). In wild-type mouse myocytes, PDBu increased PLM phosphorylation at Ser63 and Ser68, Ipump (both at 10 and 100 mmol/L Na+ in the pipette solution) and maximal NKA-mediated Na+ extrusion rate (Vmax) from 7.9±1.1 to 12.7±1.9 mmol·L−1 per minute without altering NKA affinity for internal Na+ (K0.5). In PLM knockout mice, PDBu had no effect on either Vmax or K0.5. After pretreatment with isoproterenol (ISO) (1 &mgr;mol/L), PDBu still increased the NKA Vmax and PLM phosphorylation at Ser63 and Ser68. Conversely, after pretreatment with PDBu, ISO further increased the Na+ affinity of NKA and phosphorylation at Ser68, as it did alone without PDBu. The final NKA activity was independent of the application sequence. The NKA activity in PLM knockout myocytes, after normalizing the protein level, was similar to that after PDBu and ISO treatment. We conclude that (1) PLM mediates the PKC-dependent activation of NKA function in cardiac myocytes, (2) PDBu and ISO effects are additive in the mouse (affecting mainly Vmax and K0.5, respectively), and (3) PDBu and ISO combine to activate NKA in wild-type to the level found in the PLM knockout mouse.

Isoform Specificity of the Na/K-ATPase Association and Regulation by Phospholemman

Phospholemman (PLM) phosphorylation mediates enhanced Na/K-ATPase (NKA) function during adrenergic stimulation of the heart. Multiple NKA isoforms exist, and their function/regulation may differ. We combined fluorescence resonance energy transfer (FRET) and functional measurements to investigate isoform specificity of the NKA-PLM interaction. FRET was measured as the increase in the donor fluorescence (CFP-NKA-α1 or CFP-NKA-α2) during progressive acceptor (PLM-YFP) photobleach in HEK-293 cells. Both pairs exhibited robust FRET (maximum of 23.6 ± 3.4% for NKA-α1 and 27.5 ± 2.5% for NKA-α2). Donor fluorescence depended linearly on acceptor fluorescence, indicating a 1:1 PLM:NKA stoichiometry for both isoforms. PLM phosphorylation induced by cAMP-dependent protein kinase and protein kinase C activation drastically reduced the FRET with both NKA isoforms. However, submaximal cAMP-dependent protein kinase activation had less effect on PLM-NKA-α2 versus PLM-NKA-α1. Surprisingly, ouabain virtually abolished NKA-PLM FRET but only partially reduced co-immunoprecipitation. PLM-CFP also showed FRET to PLM-YFP, but the relationship during progressive photobleach was highly nonlinear, indicating oligomers involving ≥3 monomers. Using cardiac myocytes from wild-type mice and mice where NKA-α1 is ouabain-sensitive and NKA-α2 is ouabain-resistant, we assessed the effects of PLM phosphorylation on NKA-α1 and NKA-α2 function. Isoproterenol enhanced internal Na+ affinity of both isoforms (K½ decreased from 18.1 ± 2.0 to 11.5 ± 1.9 mm for NKA-α1 and from 16.4 ± 2.5 to 10.4 ± 1.5 mm for NKA-α2) without altering maximum transport rate (Vmax). Protein kinase C activation also decreased K½ for both NKA-α1 and NKA-α2 (to 9.4 ± 1.0 and 9.1 ± 1.1 mm, respectively) but increased Vmax only for NKA-α2 (1.9 ± 0.4 versus 1.2 ± 0.5 mm/min). In conclusion, PLM associates with and modulates both NKA-α1 and NKA-α2 in a comparable but not identical manner.

Fluorescence Resonance Energy Transfer–Based Sensor Camui Provides New Insight Into Mechanisms of Calcium/Calmodulin-Dependent Protein Kinase II Activation in Intact Cardiomyocytes

Rationale: Calcium/calmodulin-dependent protein kinase II (CaMKII) is a key mediator of intracellular signaling in the heart. However, the tools currently available for assessing dynamic changes in CaMKII localization and activation in living myocytes are limited. Objective: We use Camui, a novel FRET-based biosensor in which full-length CaMKII is flanked by CFP and YFP, to measure CaMKII activation state in living rabbit myocytes. Methods and Results: We show that Camui and mutant variants that lack the sites of CaMKII autophosphorylation (T286A) and oxidative regulation (CM280/1VV) serve as useful biosensors for CaMKII&dgr; activation state. Camui (wild-type or mutant) was expressed in isolated adult cardiac myocytes, and localization and CaMKII activation state were determined using confocal microscopy. Camui, like CaMKII&dgr;, is concentrated at the z-lines, with low baseline activation state. Camui activation increased directly with pacing frequency, but the maximal effect was blunted with the T286A, consistent with frequency-dependent phosphorylation of CaMKII at T286 mainly at high-frequency and high-amplitude Ca transients. Camui was also activated by 4 neurohormonal agonists. Angiotensin II and endothelin-1 activated Camui, largely through an oxidation-dependent mechanism, whereas isoproterenol- and phenylephrine-mediated mechanisms had a significant autophosphorylation-dependent component. Conclusions: Camui is a novel, nondestructive tool that allows spatiotemporally resolved measurement of CaMKII activation state in physiologically functioning myocytes. This represents a first step in using Camui to elucidate key mechanistic details of CaMKII signaling in live hearts and myocytes.Rationale Calcium/calmodulin dependent protein kinase II (CaMKII) is a key mediator of intracellular signaling in the heart. However, the tools currently available for assessing dynamic changes in CaMKII localization and activation in living myocytes are limited.