Application Of Isoproterenol Research Articles

Control of βAR- and N-methyl-D-aspartate (NMDA) Receptor-Dependent cAMP Dynamics in Hippocampal Neurons.

Norepinephrine, a neuromodulator that activates β-adrenergic receptors (βARs), facilitates learning and memory as well as the induction of synaptic plasticity in the hippocampus. Several forms of long-term potentiation (LTP) at the Schaffer collateral CA1 synapse require stimulation of both βARs and N-methyl-D-aspartate receptors (NMDARs). To understand the mechanisms mediating the interactions between βAR and NMDAR signaling pathways, we combined FRET imaging of cAMP in hippocampal neuron cultures with spatial mechanistic modeling of signaling pathways in the CA1 pyramidal neuron. Previous work implied that cAMP is synergistically produced in the presence of the βAR agonist isoproterenol and intracellular calcium. In contrast, we show that when application of isoproterenol precedes application of NMDA by several minutes, as is typical of βAR-facilitated LTP experiments, the average amplitude of the cAMP response to NMDA is attenuated compared with the response to NMDA alone. Models simulations suggest that, although the negative feedback loop formed by cAMP, cAMP-dependent protein kinase (PKA), and type 4 phosphodiesterase may be involved in attenuating the cAMP response to NMDA, it is insufficient to explain the range of experimental observations. Instead, attenuation of the cAMP response requires mechanisms upstream of adenylyl cyclase. Our model demonstrates that Gs-to-Gi switching due to PKA phosphorylation of βARs as well as Gi inhibition of type 1 adenylyl cyclase may underlie the experimental observations. This suggests that signaling by β-adrenergic receptors depends on temporal pattern of stimulation, and that switching may represent a novel mechanism for recruiting kinases involved in synaptic plasticity and memory.

Relative Contribution of Local Ca2+ Releases (LCRS) and AP-Induced Ca2+ Transient Decay to Diastolic Depolarization in Rabbit Sa Node Cells

Background: Intracellular Ca2+ dynamics contribute to cardiac pacemaker cell function via activation of electrogenic Na+/Ca2+ exchanger during diastolic depolarization. Two fundamental mechanisms have been implicated for the diastolic Ca2+ signaling: LCRs and the decaying AP-induced Ca2+ transients. Their relative contributions, however, are unknown.Methods: We evaluated the relative contributions based on simultaneous high-speed camera recordings of Ca2+ signal, alongside membrane potential recordings by perforated patch clamp in single isolated rabbit SA node cells prior to and during beta-adrenergic receptor stimulation with isoproterenol (100 nM). We obtained further insights by employing simulations of our recent numerical model featuring local Ca2+ dynamics in 3D in these cells.Results: All cells tested (n=5) exhibited both LCRs and AP-induced-transient residuals during diastolic depolarization. Individual LCRs have been characterized in the 2D movies using our novel computer algorithm (see our poster on automated analysis of LCRs). The values of LCR signal mass and Ca2+ transient residuals were integrated within the entire cell perimeter in the focal plane of video-recording and compared. Both our analysis of the experimental data and model simulations demonstrated that LCRs provide a major part of the net Ca2+ diastolic signal, both prior to and during isoproterenol exposure. The relative contribution of LCRs, however, substantially increased after isoproterenol application. Specifically, while the contribution of the transient residuals into the net diastolic Ca2+ signal prior to isoproterenol is about 30%, the net signal following isoproterenol becomes almost completely driven by the LCRs, Ca2+ transient decays are accelerated and almost fully complete prior the maximum diastolic depolarization.Conclusions: Both LCRs and local Ca2+ transient residuals contribute to diastolic depolarization. As pacemaker rate increases LCR signal waxes, but the contribution of Ca2+ transient residuals wanes.

Combination of isoproterenol and length oscillations in relaxing porcine airway smooth muscles

Treatments for asthma are largely pharmaceutical, with some therapies also utilising alternative breathing techniques. The objective of both medical and alternative methods is to relax contracted airway smooth muscle (ASM). In normal subjects, tidal breathing- and deep inspiration-oscillations are believed to have a bronchodilatory effect. Similarly, application of length oscillations to isolated, contracted ASM also elicits muscle relaxation. As a means of investigating more-effective alternative treatment methods for contracted airways, we analyse the combined effects of bronchodilators and length oscillations on isolated, contracted ASM. The contractile state of the muscle tissue prior to treatment is of primary interest. Thereafter, the effect of applying a combination of small superimposed length oscillations with tidal breathing-like oscillations to ASM is studied alone and in combination with a common bronchodilator, isoproterenol (ISO). This work suggests that relaxation of isolated, contracted ASM following application of combined oscillations and ISO is larger than treatments of either combined oscillations or ISO alone. Further, the observed oscillation-associated relaxation is found to be amplitude- rather than frequency-dependent. This study gives additional insight into the role of oscillations and bronchodilators on contracted airways.

Abstract 17193: Inhibiting Late Sodium Current Attenuates Isoproterenol-Induced Transient Inward Current and Delayed Afterdepolarizations in Ventricular Myocytes

Introduction: The β-adrenergic agonist isoproterenol (ISO) is known to induce the arrhythmogenic transient inward current (I Ti ) and delayed afterdepolarization (DAD) via a stimulation of L-type Ca 2+ current. Recent studies found that ISO-induced DADs in cardiac tissues are inhibited by GS967, a selective blocker of the late Na + current (I NaL ). Thus, we hypothesize that I NaL contributes to the actions of ISO, and selective inhibition of this current will reduce ISO-induced I Ti and DADs. Methods: Transmembrane currents and action potentials of rabbit and guinea pig (GP) ventricular myocytes were recorded using the whole-cell patch-clamp technique. ISO (0.1 μM), GS967 (1 μM) and the Na + channel blocker tetrodotoxin (TTX, 3 μM) were used in the experiments. Results: In rabbit myocytes, application of ISO caused an increase in the amplitude of I NaL from -0.10±0.03 to -0.32±0.04 pA/pF (n = 17, p < 0.05). The ISO-stimulated I NaL was inhibited by GS967 and TTX. In one series of experiments, ISO increased the I NaL from -0.14±0.04 to -0.35±0.06 pA/pF, and GS967 applied in the presence of ISO reduced the current to -0.14±0.03 pA/pF (n = 9, p < 0.05). In another series of experiments, the amplitude of I NaL was increased by ISO from -0.17±0.08 to -0.41±0.09 pA/pF, and was decreased to -0.09±0.08 pA/pF when TTX was applied with ISO (n = 5, p < 0.05). Application of ISO also induced I Ti and DADs. GS967 applied in the presence of ISO inhibited the amplitude of I Ti by 52±6%, from -1.79±0.30 to -0.87±0.16 pA/pF (n = 8, p < 0.05). Consistent with the inhibition of I Ti , GS967 suppressed the amplitude of ISO-induced DADs by 56±12%, from 6.54±1.59 to 3.22±1.27 mV (n = 5, p < 0.05). Similarly, in GP myocytes ISO-induced I Ti and DADs were decreased by GS967 from -1.14±0.21 to -0.73±0.16 pA/pF (n = 7, p < 0.05) and from 7.16±0.59 to 4.67±0.24 mV (n = 5, p < 0.05), respectively. Conclusions: An increased I NaL is likely to contribute to the proarrhythmic effects of ISO in cardiac myocytes. GS967 significantly attenuated ISO-induced I NaL , I Ti and DADs, suggesting that inhibiting this current could be an effective strategy to antagonize the arrhythmogenic actions of β-adrenergic stimulation.

Noradrenergic modulation of glutamate-induced excitatory responses in single neurons of the red nucleus: An electrophysiological study

The effect induced by noradrenaline (NA) on the spiking activity evoked by glutamate (Glu) on single neurons of the mesencephalic red nucleus (RN) of the rat was studied extracellularly. Long-lasting microiontophoretic applications of the amine induced a significant and reversible depression of the responsiveness of RN neurons to Glu. This effect was mediated by noradrenergic alpha2 receptors since it was mimicked by application of clonidine, an alpha2 adrenoceptor agonist, and blocked or at least reduced by application of yohimbine, an antagonist of NA for the same receptors. The effect appears homogeneously throughout the nucleus and is independent of the effect of NA on baseline firing rate. Application of isoproterenol, a beta adrenoceptor agonist, either enhanced or depressed neuronal responses to Glu in a high percentage (86%) of the tested neurons. Moreover, application of timolol, a beta adrenoceptor antagonist, was able to strengthen the depressive effects induced by NA application on neuronal responsiveness to Glu.Although these data suggest some involvement of beta adrenergic receptors in the modulation of neuronal responsiveness to Glu, the overall results indicate a short-term depressive action of NA, mediated by alpha2 receptors, on the responsiveness of RN neurons and suggest that stress initially leads to an attenuation of the relay function of the RN.

Ectopic automaticity induced in ventricular myocytes by transgenic overexpression of HCN2

Hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) are expressed in the ventricles of fetal hearts but are normally down-regulated as development progresses. In the hypertrophied heart, however, these channels are re-expressed and generate a hyperpolarization-activated, nonselective cation current (Ih), which evidence suggests may increase susceptibility to arrhythmia. To test this hypothesis, we generated and analyzed transgenic mice overexpressing HCN2 specifically in their hearts (HCN2-Tg). Under physiological conditions, HCN2-Tg mice exhibited no discernible abnormalities. After the application of isoproterenol (ISO), however, ECG recordings from HCN2-Tg mice showed intermittent atrioventricular dissociation followed by idioventricular rhythm. Consistent with this observation, 0.3μmol/L ISO-induced spontaneous action potentials (SAPs) in 76% of HCN2-Tg ventricular myocytes. In the remaining 24%, ISO significantly depolarized the resting membrane potential (RMP), and the late repolarization phase of evoked action potentials (APs) was significantly longer than in WT myocytes. Analysis of membrane currents revealed that these differences are attributable to the Ih tail current. These findings suggest HCN2 channel activity reduces the repolarization reserve of the ventricular action potential and increases ectopic automaticity under pathological conditions such as excessive β-adrenergic stimulation.

Abstract 18592: Anti-arrhythmic Effect of a Novel Rycal, S44121 / Arm036, in a Post-myocardial Infarction Mouse Model of Heart Failure

Introduction: Targeting leaky cardiac ryanodine receptors (RyR2) to prevent diastolic Ca2+ release from the sarcoplasmic reticulum (SR) is a promising pharmacological approach, to rescue the impaired cardiac contraction and prevent Ca2+-dependent arrhythmias in heart failure (HF) and disease. Hypothesis: Based on prior work from the Marks group, the Rycal S44121 (also known as ARM036) is an experimental small molecule stabilizer of RyR. We investigated the effects of S44121 in a post-myocardial infarction (PMI) mouse model of HF. Methods and results: Mice were randomly assigned to 3 groups: Sham, PMI (subjected to left coronary artery ligation), and PMI-S (treated for 3 weeks with S44121 by subcutaneous osmotic pumps on day 7 post-MI, 10 mg/kg/day). Intracellular Ca2+ was measured on single left ventricular myocytes. PMI mice exhibited a 4-fold increase in the frequency of spontaneous Ca2+ release events, Ca2+ sparks, as measured in quiescent cells using the fluorescent Ca2+ indicator Fluo-4. PMI mice also exhibited higher global diastolic Ca2+, measured with the ratiometric fluorescent probe, Indo-1 AM, and increased the occurrence of ectopic diastolic Ca2+ waves. Acute application of S44121 (10 μM for 15 min) reduced Ca2+ sparks frequency. Chronic treatment of mice with S44121 also normalized the frequency of Ca2+ sparks and of ectopic Ca2+ waves, and corrected diastolic cellular Ca2+ overload. Effects were maximal at 20 mg/kg/day. Furthermore, treatment with S44121 abolished Ca2+ waves promoted by β-adrenergic challenge (acute application of isoproterenol, 10 nM). The potential anti-arrhythmic benefit of S44121 was assessed in vivo using telemetric surface electrocardiograms. S44121 had no effect on ECG intervals and did not alter the heart rate. However, anti-arrhythmic effects were confirmed by observation of a dose-dependent reduction of spontaneous ventricular extrasystoles and ventricular tachycardia. Near maximum benefits were observed at 10 mg/kg/day, both in basal conditions or following a challenge with acute treatment of isoproterenol (0.5 mg/kg, dosed ip). Conclusion: In mice with post-ischemic HF, treatment with S44121 prevented the abnormal diastolic SR Ca2+ leak and ectopic Ca2+ waves, and reduced ventricular arrhythmias.

Molecular dependence of hippocampal bidirectional plasticity

Hippocampal plasticity inducing protocols vary in their activation of signal transduction pathways and resulting dependence on signaling molecules. Many long-term potentiation (LTP) inducing paradigms require both memory kinases: calcium-calmodulin dependent protein kinase II (CaMKII) and protein kinase A (PKA) signaling pathways. Both CaMKII and PKA are activated by calcium bound calmodulin, CaMKII directly, and PKA indirectly via adenylyl cyclase production of cAMP. The adenylyl cyclase is also activated by stimulation of beta-adrenergic receptor (βAR). Curiously, not all forms of cAMP dependent LTP require PKA. Specifically, LTP induced with isoproterenol (ISO) bath application followed by 100 Hz stimulation is PKA independent [1,2], whereas both ISO followed by 3 min of 5 Hz [2] and four trains of 100 Hz stimulation do require PKA [3]. Furthermore, recent experiments suggest a novel βAR signaling pathway, in which PKA phosphorylation prevents activation of G protein subtype s (Gs), and switches the βAR to the Gi subtype of G [4], leading to recruitment of another memory kinase: extracellular signal-regulated kinase (ERK). To investigate how temporal pattern of synaptic activation determines which signaling pathways are activated, we employed a multi-compartmental stochastic reaction-diffusion model of calcium and cAMP activated signaling pathways. We investigated two model variants, one which took into account only βARcoupling to Gs, and the other which included the novel switching pathway. We performed simulations of four trains of 100 Hz, and isoproterenol application followed by either 1 train of 100 Hz or 3 min of 5 Hz, all of which produce long lasting LTP. We also simulated 3 stimulation paradigms which do not produce LTP, including 1 Hz stimulation which produces LTD. Our results using the first model could not explain and predict the direction of synaptic plasticity based only on CaMKII, PKAc and phosphatases activity, especially for paradigms that do not induce plasticity, such as bath application of isoproteronol. Experimentally, isoproteronol does not cause LTP despite βAR activation resulting in cAMP synthesis [2], suggesting that βAR undergo PKA dependent desensitization, that changes their coupling from Gs (stimulating adenylyl cyclases and cAMP synthesis) to Gi. Gi coupling both inhibits adenylyl cyclase, limiting the elevation in cAMP in response to bath application, and provides free Giβγ, which recruits molecules leading to ERK [5]. Including PKA dependent switching from Gs to Gi resulted in a model which could explain which molecules are activated in 5 LTP induction paradigms, 1 LTD induction paradigms, and 2 induction paradigms that do not produce plasticity. The model also allowed for proposing a measure of plasticity direction. The measure took into account activation of CaMKII, PKA, Giβγ (representing ERK), Epac and influence of phosphatase activity.

How does β-adrenergic signalling affect the transitions from ventricular tachycardia to ventricular fibrillation?

Ventricular tachycardia (VT) and fibrillation (VF) are the most lethal cardiac arrhythmias. The degeneration of VT into VF is associated with the breakup of a spiral wave of the action potential in cardiac tissue. β-Adrenergic (βAR) signalling potentiates the L-type Ca current (ICaL) faster than the slow delayed rectifier potassium current (IKs), which transiently prolongs the action potential duration (APD) and promotes early after depolarizations. In this study, we aimed at investigating how βAR signalling affects the transition from VT to VF. We used a physiologically detailed computer model of the rabbit ventricular myocyte in a two-dimensional tissue to determine how spiral waves respond to βAR activation following administration of isoproterenol. A simplified mathematical model was also used to investigate the underlying dynamics. We found that the spatiotemporal behaviour of spiral waves strongly depends on the kinetics of βAR activation. When βAR activation is rapid, a stable spiral wave turns into small fragments and its electrocardiogram reveals the transition from VT to VF. This is due to the transiently steepened APD restitution induced by the faster activation of ICaL vs. IKs upon sudden βAR activation. The spiral wave may also disappear if its transient wavelength is too large to be supported by the tissue size upon sudden strong βAR activation that prolongs APD transiently. When βAR activation is gradual, a stable spiral wave remains such, because of more limited increase in both APD and slope of APD restitution due to more contemporaneous ICaL and IKs activation. Changes in APD restitution during βAR activation revealed a novel transient spiral wave dynamics; this spatiotemporal characteristic strongly depends on the protocol of isoproterenol application.

Targeted ablation of the histidine-rich Ca2+-binding protein (HRC) gene is associated with abnormal SR Ca2+-cycling and severe pathology under pressure-overload stress

The histidine-rich Ca(2+)-binding protein (HRC) is located in the lumen of the sarcoplasmic reticulum (SR) and exhibits high-capacity Ca(2+)-binding properties. Overexpression of HRC in the heart resulted in impaired SR Ca(2+) uptake and depressed relaxation through its interaction with SERCA2a. However, the functional significance of HRC in overall regulation of calcium cycling and contractility is not currently well defined. To further elucidate the role of HRC in vivo under physiological and pathophysiological conditions, we generated and characterized HRC-knockout (KO) mice. The KO mice were morphologically and histologically normal compared to wild-type (WT) mice. At the cellular level, ablation of HRC resulted in significantly enhanced contractility, Ca(2+) transients, and maximal SR Ca(2+) uptake rates in the heart. However, after-contractions were developed in 50 % of HRC-KO cardiomyocytes, compared to 11 % in WT mice under stress conditions of high-frequency stimulation (5 Hz) and isoproterenol application. A parallel examination of the electrical activity revealed significant increases in the occurrence of Ca(2+) spontaneous SR Ca(2+) release and delayed afterdepolarizations with ISO in HRC-KO, compared to WT cells. The frequency of Ca(2+) sparks was also significantly higher in HRC-KO cells with ISO, consistent with the elevated SR Ca(2+) load in the KO cells. Furthermore, HRC-KO cardiomyocytes showed significantly deteriorated cell contractility and Ca(2+)-cycling caused possibly by depressed SERCA2a expression after transverse-aortic constriction (TAC). Also HRC-null mice exhibited severe cardiac hypertrophy, fibrosis, pulmonary edema and decreased survival after TAC. Our results indicate that ablation of HRC is associated with poorly regulated SR Ca(2+)-cycling, and severe pathology under pressure-overload stress, suggesting an essential role of HRC in maintaining the integrity of cardiac function.

Exercise enhances inotropic responsiveness of cardiac myocytes

Exercise‐induced cardiac structural and functional remodeling has recently been shown to vary between the sexes. In this study we sought to examine the sex‐based differences in the cardiac remodeling after swim training.MethodsMale and female Swiss Webster mice swam for 70 minutes two times daily for 4 weeks. Myocytes were isolated from control male (CM) and control female (CF) and swim‐trained male (STM) and swim‐trained female (STF) mice, field stimulated, and Fluo‐3 transients and sarcomere shortening were measured.ResultsSwim training resulted in significant cardiac hypertrophy in both males and females (16% and 12%, respectively). STF myocytes exhibited lower basal contractile properties (2Hz, 1.5mM Ca) compared to CF (% resting sarcomere length 4.5±.29% STF vs 7.1±.31% CF; fluo‐3 fluorescence 1.66±.04 STF vs 2.18±.09CF) while there were no differences between CM and STM contractile properties. Contractile reserve responses of myocytes were studied by application of isoproterenol (ISO, 10−7M). ISO caused a significantly greater increase in the contractile properties of STF (333±37%) and STM (325±39%) myocytes than in CF (209±13%) or CM (230±29%) myocytes.ConclusionSwim training induces physiological cardiac hypertrophy and causes increased contractile responses to catecholamines in both males and females which should enhance cardiac pump function during exercise.

Sulfur Dioxide Derivative Prevents Left Ventricular Hypertrophy and Electrophysiological Alterations

SO2and derivatives generally accepted as toxic gasseous molecules and inhalation of itdrives biological systems severe conditions. On the other hand, they are used as safepreservative in foods. Recently, SO2was discovered to be produced in thecardiovascular system and to influence important biological processes.It was reported by Liang Y.et al that endogenous SO2progressively inhibit hypertrophycaused by isoproterenol application to rats via increasing antioxidation capacity. In this study we aimed to investigate underlying molecular and electro physiological mechanisms of this inhibition of ventricular hypertrophy. Wistar rats were administrated 20 mg/kg isoproterenolfor seven days subcutaneuosly to drive hypertropic cardiomyopathy and some animals was taken SO2 donor sodium metabisulfite together with isoproterenol through i.p. at a dose of 85 mg/kg. At the end of the experiment, animals were sacrified by heart exicition and followed by enzimatic dissociation of single left ventricular myocytes. Iso admistrated animal heart (6.69±0.41 mg/kg bodyweight) were hypertrofied compared to control animals hearts(3.94±0.12 mg/kg). On the other hand, metabisulfite inhibits hypertrothy of the rats hearts (4.27±0.32 mg/kg).Single cardiomyocytes were examined electrophysiologicaly by voltage clamp in order to examine effect of SO2on potassium currents (IK1, Ito and Iss), action potential (AP) and L‐type calcium current (ICaL). While metabisulfite had no effect on potassium currents, prolonged AP and reduced ICaL ameliorate to the control level. In conclusion, SO2derivative sodium metabisulfite prevent cardiac hypertrophy generated by adrenergicstimulation both electrophysiologicaly and histologicaly. This work supported by Akdeniz University Scientific Research Unit.

β-adrenergic stimulation activates early afterdepolarizations transiently via kinetic mismatch of PKA targets

Sympathetic stimulation regulates cardiac excitation–contraction coupling in hearts but can also trigger ventricular arrhythmias caused by early afterdepolarizations (EADs) in pathological conditions. Isoproterenol (ISO) stimulation can transiently cause EADs which could result from differential kinetics of L-type Ca current (ICaL) vs. delayed rectifier potassium current (IKs) effects, but multiple PKA targets complicate mechanistic analysis. Utilizing a biophysically detailed model integrating Ca and β-adrenergic signaling, we investigate how different phosphorylation kinetics and targets influence β-adrenergic-induced transient EADs. We found that: 1) The faster time course of ICaL vs. IKs increases recapitulates experimentally observed ISO-induced transient EADs (which are due to ICaL reactivation). These EADs disappear at steady state ISO and do not occur during more gradual ISO application. 2) This ICaL vs. IKs kinetic mismatch with ISO can also induce transient EADs due to spontaneous sarcoplasmic reticulum (SR) Ca release and Na/Ca exchange current. The increased ICaL, SR Ca uptake and action potential duration (APD) raise SR Ca to cause spontaneous SR Ca release, but eventual IKs activation and APD shortening abolish these EADs. 3) Phospholemman (PLM) phosphorylation decreases both types of EADs by increasing outward Na/K-ATPase current (INaK) for ICaL-mediated EADs, and reducing intracellular Na and Ca loading for SR Ca-release-mediated EADs. Slowing PLM phosphorylation kinetics abolishes this protective effect. 4) Blocking phospholamban (PLB) phosphorylation has little effect on ICaL-mediated transient EADs, but abolishes SR Ca-release-mediated transient EADs by limiting SR Ca loading. 5) RyR phosphorylation has little effect on either transient EAD type. Our study emphasizes the importance of understanding non-steady state kinetics of several systems in mediating β-adrenergic-induced EADs and arrhythmias. This article is part of a Special Issue entitled "Calcium Signaling in Heart".

β-Adrenergic receptor stimulation causes cardiac hypertrophy via a Gβγ/Erk-dependent pathway

Activation of the β(1)-adrenergic receptor and its G protein, G(s), induces cardiac hypertrophy. However, activation of classic Gα(s) effectors, adenylyl cyclases (AC) and protein kinase A, is not sufficient for induction of hypertrophy, which suggests the involvement of additional pathway(s) activated by G(s). Recently, we discovered that βγ subunits of G(q) induce phosphorylation of the extracellular regulated kinases 1 and 2 (Erk1/2) at threonine188 and thereby induce hypertrophy. Here we investigated whether β-adrenergic receptors might also induce cardiac hypertrophy via Erk(Thr188) phosphorylation. β-Adrenergic receptor activation induced Erk(Thr188) phosphorylation in mouse hearts and in neonatal cardiomyocytes. Inhibition of Erk1/2 or overexpression of Erk(Thr188) phosphorylation-deficient mutants (Erk2(T188A) and Erk2(T188S)) significantly attenuated β-adrenergic cardiomyocyte hypertrophy in vitro. Erk activity was stimulated by both isoproterenol and the direct AC activator forskolin, but only isoproterenol induced Erk(Thr188) phosphorylation. Erk(Thr188) phosphorylation required Gβγ released from G(s) and was prevented by Gβγ inhibition. Similarly, isoproterenol, but not forskolin, induced nuclear accumulation of Erk and cardiomyocyte hypertrophy. Long-term application of isoproterenol in mice caused left ventricular hypertrophy and cardiac remodelling, and this was reduced in Erk2(T188S) transgenic mice, supporting the physiological relevance of Erk(Thr188) phosphorylation. Activation of G(s) by β-adrenergic receptors leads to (i) canonical Erk1/2 activation via AC, and (ii) release of Gβγ, which then associates with activated Erk1/2 and induces Erk(Thr188) phosphorylation, causing nuclear accumulation of Erk and ultimately cardiomyocyte hypertrophy. These findings reveal a new pathway critically involved in β-adrenergically mediated cardiac hypertrophy and may yield new therapeutic strategies against hypertrophic remodelling.

Reactive oxygen species contribute to the development of arrhythmogenic Ca2+ waves during β‐adrenergic receptor stimulation in rabbit cardiomyocytes

While β-adrenergic receptor (β-AR) stimulation leads to positive inotropic effects, it can also induce arrhythmogenic Ca2+ waves. β-AR stimulation increases mitochondrial oxygen consumption and, thereby, the production of reactive oxygen species (ROS). We therefore investigated the role of ROS in the generation of Ca2+ waves during β-AR stimulation in rabbit ventricular myocytes. Isoproterenol (ISO) increased Ca2+ transient amplitude during systole, sarcoplasmic reticulum (SR) Ca2+ load and the occurrence of Ca2+ waves during diastole. These effects, however, developed at different time points during ISO application.While SR Ca2+ release and load reached a maximum level after 3 min, Ca2+ waves occurred at the highest frequency only after 6 min of ISO application.Measurement of intra-SR-free Ca2+ concentration ([Ca2+]SR) showed an initial increase of SR Ca2+ load followed by a gradual decline over time during ISO application. This decline of [Ca2+]SR was not due to decreased SR Ca2+ uptake, but instead was the result of increased SRCa2+ leak mainly in the form of Ca2+ waves. ISO application led to significant RyR phosphorylation at the protein kinase A (PKA)-specific site, which remained relatively stable throughout β-AR activation.Moreover, β-AR stimulation significantly increased ROS production after 4–6 min of ISO application. The ROS scavenger Tiron and the superoxide dismutase mimetic MnTBPA abolished the ISO-mediated ROS production. The mitochondria-specific antioxidant Mito-Tempo and an inhibitor of the electron transport chain, rotenone, also effectively prevented the ISO-mediated ROS production. Scavenging ROS during ISO application decreased the occurrence of Ca2+ waves and partially prevented augmentation of SRCa2+ leak, but did not affect the increase of Ca2+ transient amplitude. Treatment of myocytes with ISO for 15 min significantly reduced the free thiol content in RyRs. These data suggest that increased mitochondrial ROS production during β-AR stimulation causes RyR oxidation. Together with RyR phosphorylation, oxidation of RyRs increases diastolic SR Ca2+ leak to a critical level leading to the generation of arrhythmogenic Ca2+ waves.

β-Adrenergic modulation of spontaneous spatiotemporal activity patterns and synchrony in hyperexcitable hippocampal circuits

A description of healthy and pathological brain dynamics requires an understanding of spatiotemporal patterns of neural activity and characteristics of its propagation between interconnected circuits. However, the structure and modulation of the neural activation maps underlying these patterns and their propagation remain elusive. We investigated effects of β-adrenergic receptor (β-AR) stimulation on the spatiotemporal characteristics of emergent activity in rat hippocampal circuits. Synchronized epileptiform-like activity, such as interictal bursts (IBs) and ictal-like events (ILEs), were evoked by 4-aminopyridine (4-AP), and their dynamics were studied using a combination of electrophysiology and fast voltage-sensitive dye imaging. Dynamic characterization of the spontaneous IBs showed that they originated in dentate gyrus/CA3 border and propagated toward CA1. To determine how β-AR modulates spatiotemporal characteristics of the emergent IBs, we used the β-AR agonist isoproterenol (ISO). ISO significantly reduced the spatiotemporal extent and propagation velocity of the IBs and significantly altered network activity in the 1- to 20-Hz range. Dual whole cell recordings of the IBs in CA3/CA1 pyramidal cells and optical analysis of those regions showed that ISO application reduced interpyramidal and interregional synchrony during the IBs. In addition, ISO significantly reduced duration not only of the shorter duration IBs but also the prolonged ILEs in 4-AP. To test whether the decrease in ILE duration was model dependent, we used a different hyperexcitability model, zero magnesium (0 Mg(2+)). Prolonged ILEs were readily formed in 0 Mg(2+), and addition of ISO significantly reduced their durations. Taken together, these novel results provide evidence that β-AR activation dynamically reshapes the spatiotemporal activity patterns in hyperexcitable circuits by altering network rhythmogenesis, propagation velocity, and intercellular/regional synchronization.

Genetic suppression of Gαs protein provides rate control in atrial fibrillation

Gene therapy-based modulation of atrioventricular (AV) conduction by overexpression of a constitutively active inhibitory Gα(i) protein effectively reduced heart rates in atrial fibrillation (AF). However, catecholamine stimulation caused an excessive increase in ventricular rate. We hypothesized that modest genetic suppression of a stimulatory G protein in the AV node would allow persistent rate control in acute AF and would prevent undesired heart rate acceleration during β-adrenergic activation. Atrial fibrillation was induced in 12 pigs by atrial burst pacing via an implanted cardiac pacemaker. Study animals were then assigned to receive either Ad-siRNA-Gα(s) gene therapy to inactivate Gα(s) protein or Ad-β-gal as control. Gα(s) protein inactivation resulted in a 20 % heart rate reduction (P < 0.01). AH and HV intervals were prolonged by 37 ms (P < 0.001) and 28 ms (P < 0.001), respectively, demonstrating atrioventricular conduction delay. Impairment of left ventricular ejection fraction (LVEF) during AF was attenuated by Gα(s) suppression (LVEF 49 %) compared with controls (LVEF 34 %; P = 0.03). Isoproterenol application accelerated ventricular heart rate from 233 to 281 bpm (P < 0.001) in control animals but did not significantly affect pigs treated with Ad-siRNA-Gα(s) (192 vs. 216 bpm; P = 0.19). In conclusion, genetic inhibition of Gα(s) protein in the AV node reduced heart rate and prevented AF-associated reduction of cardiac function in a porcine model. Rate control by gene therapy may provide an alternative to current pharmacological treatment of AF.

Modulation of SRCa2+release by the triadin-to-calsequestrin ratio in ventricular myocytes

Calsequestrin (CSQ) is a Ca(2+) storage protein that interacts with triadin (TRN), the ryanodine receptor (RyR), and junctin (JUN) to form a macromolecular tetrameric Ca(2+) signaling complex in the cardiac junctional sarcoplasmic reticulum (SR). Heart-specific overexpression of CSQ in transgenic mice (TG(CSQ)) was associated with heart failure, attenuation of SR Ca(2+) release, and downregulation of associated junctional SR proteins, e.g., TRN. Hence, we tested whether co-overexpression of CSQ and TRN in mouse hearts (TG(CxT)) could be beneficial for impaired intracellular Ca(2+) signaling and contractile function. Indeed, the depressed intracellular Ca(2+) concentration ([Ca](i)) peak amplitude in TG(CSQ) was normalized by co-overexpression in TG(CxT) myocytes. This effect was associated with changes in the expression of cardiac Ca(2+) regulatory proteins. For example, the protein level of the L-type Ca(2+) channel Ca(v)1.2 was higher in TG(CxT) compared with TG(CSQ). Sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) expression was reduced in TG(CxT) compared with TG(CSQ), whereas JUN expression and [(3)H]ryanodine binding were lower in both TG(CxT) and TG(CSQ) compared with wild-type hearts. As a result of these expressional changes, the SR Ca(2+) load was higher in both TG(CxT) and TG(CSQ) myocytes. In contrast to the improved cellular Ca(2+), transient co-overexpression of CSQ and TRN resulted in a reduced survival rate, an increased cardiac fibrosis, and a decreased basal contractility in catheterized mice, working heart preparations, and isolated myocytes. Echocardiographic and hemodynamic measurements revealed a depressed cardiac performance after isoproterenol application in TG(CxT) compared with TG(CSQ). Our results suggest that co-overexpression of CSQ and TRN led to a normalization of the SR Ca(2+) release compared with TG(CSQ) mice but a depressed contractile function and survival rate probably due to cardiac fibrosis, a lower SERCA2a expression, and a blunted response to β-adrenergic stimulation. Thus the TRN-to-CSQ ratio is a critical modulator of the SR Ca(2+) signaling.

Pull-Push Neuromodulation of LTP and LTD Enables Bidirectional Experience-Induced Synaptic Scaling in Visual Cortex

Neuromodulatory input, acting on G protein-coupled receptors, is essential for the induction of experience-dependent cortical plasticity. Here we report that G-coupled receptors in layer II/III of visual cortex control the polarity of synaptic plasticity through a pull-push regulation of LTP and LTD. In slices, receptors coupled to Gs promote LTP while suppressing LTD; conversely, receptors coupled to Gq11 promote LTD and suppress LTP. In vivo, the selective stimulation of Gs- or Gq11-coupled receptors brings the cortex into LTP-only or LTD-only states, which allows the potentiation or depression of targeted synapses with visual stimulation. The pull-push regulation of LTP/LTD occurs via direct control of the synaptic plasticity machinery and it is independent of changes in NMDAR activation or neuronal excitability. We propose these simple rules governing the pull-push control of LTP/LTD form a general metaplasticity mechanism that may contribute to neuromodulation of plasticity in other cortical circuits.

Activation of Ca2+ Sparks during β-Adrenergic Stimulation in Resting Cardiomyocytes May Involve CAMKII and No, But Not ROS

During β-adrenergic stimulation of cardiac myocytes, phosphorylation of the sarcoplasmic reticulum (SR) Ca2+ release channels (ryanodine receptors, RyRs) by PKA and CaMKII has been linked to the observed diastolic Ca2+ leak (as Ca2+ sparks) from the SR. Using confocal Ca2+ imaging, we previously showed that β-adrenergic stimulation with 1 μM isoproterenol (ISO) increases the spark frequency in quiescent voltage-clamped guinea-pig cardiomyocytes, without altering the SR Ca2+ content. Protein kinase inhibitors (KN-93 and H89) indicated an involvement of CaMKII in the change of spark frequency, but not PKA. Additional experiments showed that the change in sensitivity of the RyRs upon β-adrenergic stimulation may involve reactive nitrogen intermediates (RNI), as incubation with the NOSs inhibitor L-NAME (500 μM) and specific nNOS inhibitor AAAN (100 μM) prevented the increase in spark frequency without changes in SR Ca2+ content. This observation was confirmed by using the NO scavenger PTIO (130 μM). Furthermore, recent evidence suggests that redox activation of CaMKII or redox modifications of RyRs may contribute to the SR Ca2+ leakage. Therefore, we examined the effect of reactive oxygen species (ROS) on the spark frequency upon ISO application. Incubation with the SOD mimetic (and peroxynitrite decomposition catalyst) Mn-TBAP (100 μM) did not prevent the increase in spark frequency in cells with constant SR Ca2+ content. Similarly, acute application of the ROS scavenger TIRON (200 μM) could not suppress the progressive increase in spark frequency upon ISO application. Our observations suggest that CaMKII and RNI, but not ROS, are involved in the upregulation of spark frequency during β-adrenergic stimulation.