Long-Term Alcohol-Activated c-Jun N-terminal Kinase Isoform 2 Preserves Cardiac Function but Drives Ca2+-Triggered Arrhythmias.

Long-term alcohol consumption leads to cardiac arrhythmias including atrial fibrillation (AF), the most common alcohol-related arrhythmia. While AF significantly increases morbidity and mortality in patients, it takes years for an alcoholic individual undergoing an adaptive status with normal cardiac function to reach alcoholic cardiomyopathy. The underlying mechanism remains unclear to date. In this study, we assessed the functional role of JNK2 in long-term alcohol-evoked atrial arrhythmogenicity but preserved cardiac function. Wild-type (WT) mice and cardiac-specific JNK2dn mice (with an overexpression of inactive dominant negative (dn) JNK2) were treated with alcohol (2 g/kg daily for 2 months; 2 Mo). Confocal Ca2+ imaging in the intact mouse hearts showed that long-term alcohol prolonged intracellular Ca2+ transient decay, and increased pacing-induced Ca2+ waves, compared to that of sham controls, while cardiac-specific JNK2 inhibition in JNK2dn mice precluded alcohol-evoked Ca2+-triggered activities. Moreover, activated JNK2 enhances diastolic SR Ca2+ leak in 24 h and 48 h alcohol-exposed HL-1 atrial myocytes as well as HEK-RyR2 cells (inducible expression of human RyR2) with the overexpression of tGFP-tagged active JNK2-tGFP or inactive JNK2dn-tGFP. Meanwhile, the SR Ca2+ load and systolic Ca2+ transient amplitude were both increased in ventricular myocytes, along with the preserved cardiac function in 2 Mo alcohol-exposed mice. Moreover, the role of activated JNK2 in SR Ca2+ overload and enhanced transient amplitude was also confirmed in long-term alcohol-exposed HL-1 atrial myocytes. In conclusion, our findings suggest that long-term alcohol-activated JNK2 is a key driver in preserved cardiac function, but at the expense of enhanced cardiac arrhythmogenicity. Modulating JNK2 activity could be a novel anti-arrhythmia therapeutic strategy.

The role of P21-activated kinase (Pak1) in sinus node function.

Sinoatrial node (SAN) dysfunction (SND) and atrial arrhythmia frequently occur simultaneously with a hazard ratio of 4.2 for new onset atrial fibrillation (AF) in SND patients. In the atrial muscle attenuated activity of p21-activated kinase 1 (Pak1) increases the risk for AF by enhancing NADPH oxidase 2 dependent production of reactive oxygen species (ROS). However, the role of Pak1 dependent ROS regulation in SAN function has not yet been determined. We hypothesize that Pak1 activity maintains SAN activity by regulating the expression of the hyperpolarization activated cyclic nucleotide gated cation channel (HCN). To determine Pak1 dependent changes in heart rate (HR) regulation we quantified the intrinsic sinus rhythm in wild type (WT) and Pak1 deficient (Pak1-/-) mice of both sexes in vivo and in isolated Langendorff perfused hearts. Pak1-/- hearts displayed an attenuated HR in vivo after autonomic blockage and in isolated hearts. The contribution of the Ca2+ clock to pacemaker activity remained unchanged, but Ivabradine (3 µM), a blocker of HCN channels that are a membrane clock component, eliminated the differences in SAN activity between WT and Pak1-/- hearts. Reduced HCN4 expression was confirmed in Pak1-/- right atria. The reduced HCN activity in Pak1-/- could be rescued by class II HDAC inhibition (LMK235), ROS scavenging (TEMPOL) or attenuation of Extracellular Signal-Regulated Kinase (ERK) 1/2 activity (SCH772984). No sex specific differences in Pak1 dependent SAN regulation were determined. Our results establish Pak1 as a class II HDAC regulator and a potential therapeutic target to attenuate SAN bradycardia and AF susceptibility.

Subcellular Propagation of Cardiomyocyte ß-Adrenergic Activation of Calcium Uptake Involves Internal ß-Receptors and AKAP7.

ß-adrenergic receptor (ß-AR) signaling in cardiac myocytes is central to cardiac function, but spatiotemporal activation within myocytes is unresolved. In rabbit ventricular myocytes, ß-AR agonists or high extracellular [Ca] were applied locally at one end, to measure ß-AR signal propagation as Ca-transient (CaT) amplitude and sarcoplasmic reticulum (SR) Ca uptake. High local [Ca]o, increased CaT amplitude under the pipette faster than did ISO, but was also more spatially restricted. Local isoproterenol (ISO) or norepinephrine (NE) increased CaT amplitude and SR Ca uptake, that spread along the myocyte to the unexposed end. Thus, local [Ca]i decline kinetics reflect spatio-temporal progression of ß-AR end-effects in myocytes. To test whether intracellular ß-ARs contribute to this response, we used ß-AR-blockers that are membrane permeant (propranolol) or not (sotalol). Propranolol completely blocked NE-dependent CaT effects. However, blocking surface ß-ARs only (sotalol) suppressed only ∼50% of the NE-induced increase in CaT peak and rate of [Ca]i decline, but these changes spread more gradually than NE alone. We also tested whether A-kinase anchoring protein 7γ (AKAP7γ; that interacts with phospholamban) is mobile, such that it might contribute to intracellular spatial propagation of ß-AR signaling. We found AKAP7γ to be highly mobile using fluorescence recovery after photobleach of GFP tagged AKAP7γ, and that PKA activation accelerated AKAP7γ-GFP wash-out upon myocyte saponin-permeabilization, suggesting increased AKAP7γ mobility. We conclude that local ß-AR activation can activate SR Ca uptake at remote myocyte sites, and that intracellular ß-AR and AKAP7γ mobility may play a role in this spread of activation.

Stress-driven cardiac calcium mishandling via a kinase-to-kinase crosstalk.

Calcium homeostasis in the cardiomyocyte is critical to the regulation of normal cardiac function. Abnormal calcium dynamics such as altered uptake by the sarcoplasmic reticulum (SR) Ca2+-ATPase and increased diastolic SR calcium leak are involved in the development of maladaptive cardiac remodeling under pathological conditions. Ca2+/calmodulin-dependent protein kinase II-δ (CaMKIIδ) is a well-recognized key molecule in calcium dysregulation in cardiomyocytes. Elevated cellular stress is known as a common feature during pathological remodeling, and c-jun N-terminal kinase (JNK) is an important stress kinase that is activated in response to intrinsic and extrinsic stress stimuli. Our lab recently identified specific actions of JNK isoform 2 (JNK2) in CaMKIIδ expression, activation, and CaMKIIδ-dependent SR Ca2+ mishandling in the stressed heart. This review focuses on the current understanding of cardiac SR calcium handling under physiological and pathological conditions as well as the newly identified contribution of the stress kinase JNK2 in CaMKIIδ-dependent SR Ca2+ abnormal mishandling. The new findings identifying dual roles of JNK2 in CaMKIIδ expression and activation are also discussed in this review.

JNK2, a Newly-Identified SERCA2 Enhancer, Augments an Arrhythmic [Ca2+]SR Leak-Load Relationship.

RATIONALE: We recently discovered pivotal contributions of stress kinase JNK2 (c-Jun N-terminal kinase isoform 2) in increased risk of atrial fibrillation through enhanced diastolic sarcoplasmic reticulum (SR) calcium (Ca2+) leak via RyR2 (ryanodine receptor isoform 2). However, the role of JNK2 in the function of the SERCA2 (SR Ca2+-ATPase), essential in maintaining SR Ca2+ content cycling during each heartbeat, is completely unknown. OBJECTIVE: To test the hypothesis that JNK2 increases SERCA2 activity SR Ca2+ content and exacerbates an arrhythmic SR Ca2+ content leak-load relationship. METHODS AND RESULTS: We used confocal Ca2+ imaging in myocytes and HEK-RyR2 (ryanodine receptor isoform 2-expressing human embryonic kidney 293 cells) cells, biochemistry, dual Ca2+/voltage optical mapping in intact hearts from alcohol-exposed or aged mice (where JNK2 is activated). We found that JNK2, but not JNK1 (c-Jun N-terminal kinase isoform 1), increased SERCA2 uptake and consequently elevated SR Ca2+ content load. JNK2 also associates with and phosphorylates SERCA2 proteins. JNK2 causally enhances SERCA2-ATPase activity via increased maximal rate, without altering Ca2+ affinity. Unlike the CaMKII (Ca2+/calmodulin-dependent kinase II)-dependent JNK2 action in SR Ca2+ leak, JNK2-driven SERCA2 function was CaMKII independent (not prevented by CaMKII inhibition). With CaMKII blocked, the JNK2-driven SR Ca2+ loading alone did not significantly raise leak. However, with JNK2-CaMKII-driven SR Ca2+ leak present, the JNK2-enhanced SR Ca2+ uptake limited leak-induced reduction in SR Ca2+, normalizing Ca2+ transient amplitude, but at a higher arrhythmogenic SR Ca2+ leak. JNK2-specific inhibition completely normalized SR Ca2+ handling, attenuated arrhythmic Ca2+ activities, and alleviated atrial fibrillation susceptibility in aged and alcohol-exposed myocytes and intact hearts. CONCLUSIONS: We have identified a novel JNK2-induced activation of SERCA2. The dual action of JNK2 in CaMKII-dependent arrhythmic SR Ca2+ leak and a CaMKII-independent uptake exacerbates atrial arrhythmogenicity, while helping to maintain normal levels of Ca2+ transients and heart function. JNK2 modulation may be a novel therapeutic target for atrial fibrillation prevention and treatment.

Expression and function of voltage gated proton channels (Hv1) in MDA-MB-231 cells.

Expression of the voltage gated proton channel (Hv1) as identified by immunocytochemistry has been reported previously in breast cancer tissue. Increased expression of HV1 was correlated with poor prognosis and decreased overall and disease-free survival but the mechanism of its involvement in the disease is unknown. Here we present electrophysiological recordings of HV1 channel activity, confirming its presence and function in the plasma membrane of a breast cancer cell line, MDA-MB-231. With western blotting we identify significant levels of HV1 expression in 3 out of 8 "triple negative" breast cancer cell lines (estrogen, progesterone, and HER2 receptor expression negative). We examine the function of HV1 in breast cancer using MDA-MB-231 cells as a model by suppressing the expression of HV1 using shRNA (knock-down; KD) and by eliminating HV1 using CRISPR/Cas9 gene editing (knock-out; KO). Surprisingly, these two approaches produced incongruous effects. Knock-down of HV1 using shRNA resulted in slower cell migration in a scratch assay and a significant reduction in H2O2 release. In contrast, HV1 Knock-out cells did not show reduced migration or H2O2 release. HV1 KO but not KD cells showed an increased glycolytic rate accompanied by an increase in p-AKT (phospho-AKT, Ser473) activity. The expression of CD171/LCAM-1, an adhesion molecule and prognostic indicator for breast cancer, was reduced in HV1 KO cells. When we compared MDA-MB-231 xenograft growth rates in immunocompromised mice, tumors from HV1 KO cells grew less than WT in mass, with lower staining for the Ki-67 marker for cell proliferation rate. Therefore, deletion of HV1 expression in MDA-MB-231 cells limits tumor growth rate. The limited growth thus appears to be independent of oxidant production by NADPH oxidase molecules and to be mediated by cell adhesion molecules. Although HV1 KO and KD affect certain cellular mechanisms differently, both implicate HV1-mediated pathways for control of tumor growth in the MDA-MB-231 cell line.

Loss of p21-activated kinase 1 (Pak1) promotes atrial arrhythmic activity.

BACKGROUND: Atrial fibrillation (AF) is initiated through arrhythmic atrial excitation from outside the sinus node or remodeling of atrial tissue that allows reentry of excitation. Angiotensin II (AngII) has been implicated in the initiation and maintenance of AF through changes in Ca2+ handling and production of reactive oxygen species (ROS). OBJECTIVE: We aimed to determine the role of p21-activated kinase 1 (Pak1), a downstream target in the AngII signaling cascade, in atrial electrophysiology and arrhythmia. METHODS: Wild-type and Pak1-/- mice were used to determine atrial function in vivo on the organ and cellular level by quantification of electrophysiological and Ca2+ handling properties. RESULTS: We demonstrate that reduced Pak1 activity increases the inducibility of atrial arrhythmia in vivo and in vitro. On the cellular level, Pak1-/- atrial myocytes (AMs) exhibit increased basal and AngII (1 µM)-induced ROS production, sensitivity to the NADPH oxidase-2 (NOX2) inhibitors gp91ds-tat and apocynin (1 µM), and enhanced membrane translocation of Ras-related C3 substrate 1 (Rac1) that is part of the multimolecular NOX2 complex. Upon stimulation with AngII, Pak1-/- AMs exhibit an exaggerated increase in the intracellular Calcium concentration ([Ca2+]i) and arrhythmic events that were sensitive to sodium-calcium exchanger (NCX) inhibitors (KB-R7943 and SEA0400; 1 µM) and suppressed in AMs from NOX2-deficient (gp91phox-/-) mice. Pak1 stimulation (FTY720; 200 nM) in wild-type AMs and AMs from a canine model of ventricular tachypacing-induced AF prevented AngII-induced arrhythmic Ca2+ overload by attenuating NCX activity in a NOX2-dependent manner. CONCLUSION: The experimental results support that Pak1 stimulation can attenuate NCX-dependent Ca2+ overload and prevent triggered arrhythmic activity by suppressing NOX2-dependent ROS production.

ß-Adrenergic induced SR Ca2+ leak is mediated by an Epac-NOS pathway.

Cardiac ß-adrenergic receptors (ß-AR) and Ca2+-Calmodulin dependent protein kinase (CaMKII) regulate both physiological and pathophysiological Ca2+ signaling. Elevated diastolic Ca2+ leak from the sarcoplasmic reticulum (SR) contributes to contractile dysfunction in heart failure and to arrhythmogenesis. ß-AR activation is known to increase SR Ca2+ leak via CaMKII-dependent phosphorylation of the ryanodine receptor. Two independent and reportedly parallel pathways have been implicated in this ß-AR-CaMKII cascade, one involving exchange protein directly activated by cAMP (Epac2) and another involving nitric oxide synthase 1 (NOS1). Here we tested whether Epac and NOS function in a single series pathway to increase ß-AR induced and CaMKII-dependent SR Ca2+ leak. Leak was measured as both Ca2+ spark frequency and tetracaine-induced shifts in SR Ca2+, in mouse and rabbit ventricular myocytes. Direct Epac activation by 8-CPT (8-(4-chlorophenylthio)-2'-O-methyl-cAMP) mimicked ß-AR-induced SR Ca2+ leak, and both were blocked by NOS inhibition. The same was true for myocyte CaMKII activation (assessed via a FRET-based reporter) and ryanodine receptor phosphorylation. Inhibitor and phosphorylation studies also implicated phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt) downstream of Epac and above NOS activation in this pathway. We conclude that these two independently characterized parallel pathways function mainly via a single series arrangement (ß-AR-cAMP-Epac-PI3K-Akt-NOS1-CaMKII) to mediate increased SR Ca2+ leak. Thus, for ß-AR activation the cAMP-PKA branch effects inotropy and lusitropy (by effects on Ca2+ current and SR Ca2+-ATPase), this cAMP-Epac-NOS pathway increases pathological diastolic SR Ca2+leak. This pathway distinction may allow novel SR Ca2+ leak therapeutic targeting in treatment of arrhythmias in heart failure that spare the inotropic and lusitropic effects of the PKA branch.

p21-Activated kinase1 (Pak1) is a negative regulator of NADPH-oxidase 2 in ventricular myocytes.

Ischemic conditions reduce the activity of the p21-activated kinase (Pak1) resulting in increased arrhythmic activity. Triggered arrhythmic activity during ischemia is based on changes in cellular ionic balance and the cells Ca(2+) handling properties. In the current study we used isolated mouse ventricular myocytes (VMs) deficient for the expression of Pak1 (Pak1(-/-)) to determine the mechanism by which Pak1 influences the generation of arrhythmic activity during simulated ischemia. The Ca(2+) transient amplitude and kinetics did not significantly change in wild type (WT) and Pak1(-/-) VMs during 15 min of simulated ischemia. However, Pak1(-/-) VMs exhibited an exaggerated increase in [Ca(2+)]i, which resulted in spontaneous Ca(2+) release events and waves. The Ca(2+) overload in Pak1(-/-) VMs could be suppressed with a reverse mode blocker (KB-R7943) of the sodium calcium exchanger (NCX), a cytoplasmic scavenger of reactive oxygen species (ROS; TEMPOL) or a RAC1 inhibitor (NSC23766). Measurements of the cytoplasmic ROS levels revealed that decreased Pak1 activity in Pak1(-/-) VMs or VMs treated with the Pak1 inhibitor (IPA3) enhanced cellular ROS production. The Pak1 dependent increase in ROS was attenuated in VMs deficient for NADPH oxidase 2 (NOX2; p47(phox-/-)) or in VMs where NOX2 was inhibited (gp91ds-tat). Voltage clamp recordings showed increased NCX activity in Pak1(-/-) VMs that depended on enhanced NOX2 induced ROS production. The exaggerated Ca(2+) overload in Pak1(-/-) VMs could be mimicked by low concentrations of ouabain. Overall our data show that Pak1 is a critical negative regulator of NOX2 dependent ROS production and that a latent ROS dependent stimulation of NCX activity can predispose VMs to Ca(2+) overload under conditions where no significant changes in excitation-contraction coupling are yet evident.

Functional integrity of the T-tubular system in cardiomyocytes depends on p21-activated kinase 1.

p21-activated kinase (Pak1), a serine-threonine protein kinase, regulates cytoskeletal dynamics and cell motility. Recent experiments further demonstrate that loss of Pak1 results in exaggerated hypertrophic growth in response to pathophysiological stimuli. Calcium (Ca) signaling plays an important role in the regulation of transcription factors involved in hypertrophic remodeling. Here we aimed to determine the role of Pak1 in cardiac excitation-contraction coupling (ECC). Ca transients were recorded in isolated, ventricular myocytes (VMs) from WT and Pak1(-/-) mice. Pak1(-/-) Ca transients had a decreased amplitude, prolonged rise time and delayed recovery time. Di-8-ANNEPS staining revealed a decreased T-tubular density in Pak1(-/-) VMs that coincided with decreased cell capacitance and increased dis-synchrony of Ca induced Ca release (CICR) at individual release units. These changes were not observed in atrial myocytes of Pak1(-/-) mice where the T-tubular system is only sparsely developed. Experiments in cultured rabbit VMs supported a role of Pak1 in the maintenance of the T-tubular structure. T-tubular density in rabbit VMs significantly decreased within 24h of culture. This was accompanied by a decrease of the Ca transient amplitude and a prolongation of its rise time. However, overexpression of constitutively active Pak1 in VMs attenuated the structural remodeling as well as changes in ECC. The results provide significant support for a prominent role of Pak1 activity not only in the functional regulation of ECC but for the structural maintenance of the T-tubular system whose remodeling is an integral feature of hypertrophic remodeling.

Ischemia/Reperfusion injury protection by mesenchymal stem cell derived antioxidant capacity.

Mesenchymal stem cell (MSC) transplantation after ischemia/reperfusion (I/R) injury reduces infarct size and improves cardiac function. We used mouse ventricular myocytes (VMs) in an in vitro model of I/R to determine the mechanism by which MSCs prevent reperfusion injury by paracrine signaling. Exposure of mouse VMs to an ischemic challenge depolarized their mitochondrial membrane potential (Ψmito), increased their diastolic Ca(2+), and significantly attenuated cell shortening. Reperfusion of VMs with Ctrl tyrode or MSC-conditioned tyrode (ConT) resulted in a transient increase of the Ca(2+) transient amplitudes in all cells. ConT-reperfused cells exhibited a decreased number early after depolarization (EADs) (ConT: 6.3% vs. Ctrl: 28.4%) and prolonged survival (ConT: 58% vs. Ctrl: 33%). Ψmito rapidly recovered in Ctrl as well as ConT-treated VMs on reperfusion; however, in Ctrl solution, an exaggerated hyperpolarization of Ψmito was determined that preceded the collapse of Ψmito. The ability of ConT to attenuate the hyperpolarization of Ψmito was suppressed on inhibition of the PI3K/Akt signaling pathway or IK,ATP. However, protection of Ψmito was best mimicked by the reactive oxygen species (ROS) scavenger mitoTEMPO. Analysis of ConT revealed a significant antioxidant capacity that was linked to the presence of extracellular superoxide dismutase (SOD3) in ConT. In conclusion, MSC ConT protects VMs from simulated I/R injury by its SOD3-mediated antioxidant capacity and by delaying the recovery of Ψmito through Akt-mediated opening of IK,ATP. These changes attenuate reperfusion-induced ROS production and prevent the opening of the permeability transition pore and arrhythmic Ca(2+) release.

Mesenchymal stem cells improve cardiac conduction by upregulation of connexin 43 through paracrine signaling.

Mesenchymal stem cells (MSCs) were shown to improve cell survival and alleviate cardiac arrhythmias when transplanted into cardiac tissue; however, little is known about the mechanism by which MSCs modify the electrophysiological properties of cardiac tissue. We aimed to distinguish the influence of cell-cell coupling between myocytes and MSCs from that of MSC-derived paracrine factors on the spontaneous activity and conduction velocity (θ) of multicellular cardiomyocyte preparations. HL-1 cells were plated on microelectrode arrays and their spontaneous activity and θ was determined from field potential recordings. In heterocellular cultures of MSCs and HL-1 cells the beating frequency was attenuated (t(0h): 2.26 ± 0.18 Hz; t(4h): 1.98 ± 0.26 Hz; P < 0.01) concomitant to the intercellular coupling between MSCs and cardiomyocytes. In HL-1 monolayers supplemented with MSC conditioned media (ConM) or tyrode (ConT) θ significantly increased in a time-dependent manner (ConT: t(0h): 2.4 cm/s ± 0.2; t(4h): 3.1 ± 0.4 cm/s), whereas the beating frequency remained constant. Connexin (Cx)43 mRNA and protein expression levels also increased after ConM or ConT treatment over the same time period. Enhanced low-density lipoprotein receptor-related protein 6 (LRP6) phosphorylation after ConT treatment implicates the Wnt signaling pathway. Suppression of Wnt secretion from MSCs (IWP-2; 5 µmol/l) reduced the efficacy of ConT to induce phospho-LRP6 and to increase θ. Inhibition of ß-catenin (cardamonin; 10 µmol/l) or GSK3-α/ß (LiCl; 5 mmol/l) also suppressed changes in θ, further supporting the hypothesis that MSC-mediated Cx43 upregulation occurs in part through secreted Wnt ligands and activation of the canonical Wnt signaling pathway.

Differential localization of neuregulin-1 type III in the central and peripheral nervous system.

In the developing PNS, axonal neuregulin-1 (NRG1) type III is the key determinant for myelination. However, the specific role for NRG1 (III) in the CNS has not been established. To address this issue, isotype-specific antibodies were generated, characterized, and used for the immunofluorescent localization of NRG1 (III) in the developing and adult CNS of rat. In contrast to adult peripheral nerve, which showed robust axonal staining, no immunoreactivity was observed in CNS myelinated tracts during the period of active myelination or in the adult CNS. Surprisingly, NRG1 (III) was prominently expressed on dendrites and soma in both the developing and adult CNS. These findings were corroborated through the subcellular fractionation of adult rat brain combined with an immunoblotting analysis. The immunolocalization of NRG1 (III) suggests that it plays a novel role in the myelination fate of CNS axons possibly through undetermined roles in neuronal maturation, or dendritic development and activation.

IP3-dependent nuclear Ca2+ signalling in the mammalian heart.

In cardiac myocytes the type-2 inositol 1,4,5-trisphosphate receptor (IP(3)R2) is the predominant isoform expressed. The IP(3)R2 channel is localized to the SR and to the nuclear envelope. We studied IP(3)-dependent nuclear Ca(2+) signals ([Ca(2+)](Nuc)) in permeabilized atrial myocytes and in isolated cardiac nuclei. In permeabilized myocytes IP(3) (20 microm) and the more potent IP(3)R agonist adenophostin (5 microm) caused an elevation of [Ca(2+)](Nuc). An IP(3)-dependent increase of [Ca(2+)](Nuc) was still observed after pretreatment with tetracaine to block Ca(2+) release from ryanodine receptors (RyRs), and the effect of IP(3) was partially reversed or prevented by the IP(3)R blockers heparin and 2-APB. Isolated nuclei were superfused with an internal solution containing the Ca(2+) indicator fluo-4 dextran. Exposure to IP(3) (10 microm) and adenophostin (0.5 microm) increased [Ca(2+)](Nuc) by 25 and 27%, respectively. [Ca(2+)](Nuc) increased to higher levels than [Ca(2+)](Cyt) immediately adjacent to the outer membrane of the nuclear envelope, suggesting that a significant portion of nuclear IP(3) receptors are facing the nucleoplasm. When nuclei were pretreated with heparin or 2-APB, IP(3) failed to increase [Ca(2+)](Nuc). Isolated nuclei were also loaded with the membrane-permeant low-affinity Ca(2+) probe fluo-5N AM which compartmentalized into the nuclear envelope. Exposure to IP(3) and adenophostin resulted in a decrease of the fluo-5N signal that could be prevented by heparin. Stimulation of IP(3)R caused depletion of the nuclear Ca(2+) stores by approximately 60% relative to the maximum depletion produced by the ionophores ionomycin and A23187. The fluo-5N fluorescence decrease was particularly pronounced in the nuclear periphery, suggesting that the nuclear envelope may represent the predominant nuclear Ca(2+) store. The data indicate that IP(3) can elicit Ca(2+) release from cardiac nuclei resulting in localized nuclear Ca(2+) signals.

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 approximately 11% increase in the fluorescence ratio (F530/F480) at saturating [InsP3] (apparent K(d) = 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 approximately 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.

Cardiac type 2 inositol 1,4,5-trisphosphate receptor: interaction and modulation by calcium/calmodulin-dependent protein kinase II.

The type 2 inositol 1,4,5-trisphosphate receptor (InsP(3)R2) was identified previously as the predominant isoform in cardiac ventricular myocytes. Here we reported the subcellular localization of InsP(3)R2 to the cardiomyocyte nuclear envelope (NE). The other major known endo/sarcoplasmic reticulum calcium-release channel (ryanodine receptor) was not localized to the NE, indicating functional segregation of these channels and possibly a unique role for InsP(3)R2 in regulating nuclear calcium dynamics. Immunoprecipitation experiments revealed that the NE InsP(3)R2 associates with Ca(2+)/calmodulin-dependent protein kinase IIdelta (CaMKIIdelta), the major isoform expressed in cardiac myocytes. Recombinant InsP(3)R2 and CaMKIIdelta(B) also co-immunoprecipitated after co-expression in COS-1 cells. Additionally, the amino-terminal 1078 amino acids of the InsP(3)R2 were sufficient for interaction with CaMKIIdelta(B) and associated upon mixing following separate expression. CaMKII can also phosphorylate InsP(3)R2, as demonstrated by (32)P labeling. Incorporation of CaMKII-treated InsP(3)R2 into planar lipid bilayers revealed that InsP(3)-mediated channel open probability is significantly reduced ( approximately 11 times) by phosphorylation via CaMKII. We concluded that the InsP(3)R2 and CaMKIIdelta likely represent two central components of a multiprotein signaling complex, and this raises the possibility that calcium release via InsP(3)R2 in the myocyte NE may activate local CaMKII signaling, which may feedback on InsP(3)R2 function.

Structure of the type 1 inositol 1,4,5-trisphosphate receptor revealed by electron cryomicroscopy.

The three-dimensional structure of the type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1) has been determined by electron cryomicroscopy and single-particle reconstruction. The receptor was immunoaffinity-purified and formed functional InsP3- and heparin-sensitive channels with a unitary conductance similar to native InsP3Rs. The channel structure exhibits the expected 4-fold symmetry and comprises two morphologically distinct regions: a large pinwheel and a smaller square. The pinwheel region has four radial curved spokes interconnected by a central core. The InsP3-binding core domain has been localized within each spoke of the pinwheel region by fitting its x-ray structure into our reconstruction. A structural mapping of the amino acid sequences to several functional domains is deduced within the structure of the InsP3R1 tetramer.