Optogenetic stimulation of Gs-signaling in the heart with high spatio-temporal precision.

The standard technique for investigating adrenergic effects on heart function is perfusion with pharmaceutical agonists, which does not provide high temporal or spatial precision. Herein we demonstrate that the light sensitive Gs-protein coupled receptor JellyOp enables optogenetic stimulation of Gs-signaling in cardiomyocytes and the whole heart. Illumination of transgenic embryonic stem cell-derived cardiomyocytes or of the right atrium of mice expressing JellyOp elevates cAMP levels and instantaneously accelerates spontaneous beating rates similar to pharmacological ß-adrenergic stimulation. Light application to the dorsal left atrium instead leads to supraventricular extrabeats, indicating adverse effects of localized Gs-signaling. In isolated ventricular cardiomyocytes from JellyOp mice, we find increased Ca2+ currents, fractional cell shortening and relaxation rates after illumination enabling the analysis of differential Gs-signaling with high temporal precision. Thus, JellyOp expression allows localized and time-restricted Gs stimulation and will provide mechanistic insights into different effects of site-specific, long-lasting and pulsatile Gs activation.

Frequency-dependent drug screening using optogenetic stimulation of human iPSC-derived cardiomyocytes.

Side effects on cardiac ion channels are one major reason for new drugs to fail during preclinical evaluation. Herein we propose a simple optogenetic screening tool measuring extracellular field potentials (FP) from paced cardiomyocytes to identify drug effects over the whole physiological heart range, which is essential given the rate-dependency of ion channel function and drug action. Human induced pluripotent stem cell-derived cardiomyocytes were transduced with an adeno-associated virus to express Channelrhodopsin2 and plated on micro-electrode arrays. Global pulsed illumination (470 nm, 1 ms, 0.9 mW/mm2) was applied at frequencies from 1 to 2.5 Hz, which evoked FP simultaneously in all cardiomyocytes. This synchronized activation allowed averaging of FP from all electrodes resulting in one robust FP signal for analysis. Field potential duration (FPD) was ~25% shorter at 2.5 Hz compared to 1 Hz. Inhibition of hERG channels prolonged FPD only at low heart rates whereas Ca2+ channel block shortened FPD at all heart rates. Optogenetic pacing also allowed analysis of the maximum downstroke velocity of the FP to detect drug effects on Na+ channel availability. In principle, the presented method is well scalable for high content cardiac toxicity screening or personalized medicine for inherited cardiac channelopathies.

Macrophage-dependent IL-1ß production induces cardiac arrhythmias in diabetic mice.

Diabetes mellitus (DM) encompasses a multitude of secondary disorders, including heart disease. One of the most frequent and potentially life threatening disorders of DM-induced heart disease is ventricular tachycardia (VT). Here we show that toll-like receptor 2 (TLR2) and NLRP3 inflammasome activation in cardiac macrophages mediate the production of IL-1ß in DM mice. IL-1ß causes prolongation of the action potential duration, induces a decrease in potassium current and an increase in calcium sparks in cardiomyocytes, which are changes that underlie arrhythmia propensity. IL-1ß-induced spontaneous contractile events are associated with CaMKII oxidation and phosphorylation. We further show that DM-induced arrhythmias can be successfully treated by inhibiting the IL-1ß axis with either IL-1 receptor antagonist or by inhibiting the NLRP3 inflammasome. Our results establish IL-1ß as an inflammatory connection between metabolic dysfunction and arrhythmias in DM.

Integrin Based Isolation Enables Purification of Murine Lineage Committed Cardiomyocytes.

In contrast to mature cardiomyocytes which have limited regenerative capacity, pluripotent stem cells represent a promising source for the generation of new cardiomyocytes. The tendency of pluripotent stem cells to form teratomas and the heterogeneity from various differentiation stages and cardiomyocyte cell sub-types, however, are major obstacles to overcome before this type of therapy could be applied in a clinical setting. Thus, the identification of extracellular markers for specific cardiomyocyte progenitors and mature subpopulations is of particular importance. The delineation of cardiomyocyte surface marker patterns not only serves as a means to derive homogeneous cell populations by FACS, but is also an essential tool to understand cardiac development. By using single-cell expression profiling in early mouse embryonic hearts, we found that a combination of integrin alpha-1, alpha-5, alpha-6 and N-cadherin enables isolation of lineage committed murine cardiomyocytes. Additionally, we were able to separate trabecular cardiomyocytes from solid ventricular myocardium and atrial murine cells. These cells exhibit expected subtype specific phenotype confirmed by electrophysiological analysis. We show that integrin expression can be used for the isolation of living, functional and lineage-specific murine cardiomyocytes.

Systemic gene transfer enables optogenetic pacing of mouse hearts.

AIMS: Optogenetic pacing of the heart has been demonstrated in transgenic animals expressing channelrhodopsin-2 (ChR2). However, for the clinical use of optogenetics to treat cardiac arrhythmias, gene transfer to non-transgenic hearts is required. The aim of this study was to describe a reliable method for gene transfer of ChR2 into a sufficient percentage of cardiomyocytes to overcome the electrical sink of all the coupled non-expressing cardiomyocytes during optical pacing of the whole heart in vivo. METHODS AND RESULTS: Adeno-associated virus (AAV) with cardiac tropism for expression of ChR2 in fusion with mCherry was systemically injected into wild-type mouse hearts. Bright mCherry fluorescence was detected in the whole heart 4-10 weeks later. Single-cell dissociation revealed that on average 58% cardiomyocytes were mCherry-positive. These showed light-induced inward currents, action potentials, and contractions. Pulsed illumination of the left ventricle induced ventricular pacing in vivo in 74% of mice, and higher light intensities were required for reduced pulse duration or size of illumination. Non-responding hearts showed low AAV expression, and the threshold for optical pacing was estimated to be 35-40% ChR2-expressing cardiomyocytes. Optical pacing in vivo was stable over extended periods without negative effects on normal sinus rhythm and ECG parameters after termination of stimulation indicating sufficient cardiac output during pacing. CONCLUSIONS: Gene transfer generates sufficient ChR2 photocurrent for reliable optogenetic pacing in vivo and lays out the basis for future optogenetic pacemaker and pain-free defibrillation therapies.

Ultrasound-mediated stimulation of microbubbles after acute myocardial infarction and reperfusion ameliorates left-ventricular remodelling in mice via improvement of borderzone vascularization.

AIMS: Post-infarction remodelling (PIR) determines left-ventricular (LV) function and prognosis after myocardial infarction. The aim of this study was to evaluate transthoracic ultrasound-mediated microbubble stimulation (UMS) as a novel gene- and cell-free therapeutic option after acute myocardial infarction and reperfusion (AMI/R) in mice. METHODS AND RESULTS: For myocardial delivery of UMS, a novel therapeutic ultrasound-system (TIPS, Philips Medical) and commercially available microbubbles (BR1, Bracco Suisse SA) were utilized in a closed-chest mouse model. UMS was performed as myocardial post-conditioning (PC) on day four after 30 minutes of coronary occlusion and reperfusion. LV-morphology, as well as global and regional function were measured repeatedly with reconstructive 3-dimensional echocardiography applying an additional low-dose dobutamine protocol after two weeks. Scar size was quantified by means of histomorphometry. A total of 41 mice were investigated; 17 received PC with UMS. Mean ejection fraction (EF) prior UMS was similar in both groups 53%±10 (w/o UMS) and 53%±14 (UMS, p = 0.5), reflecting comparable myocardial mass at risk 17%±8 (w/o UMS), 16%±13 (UMS, p = 0.5). Two weeks after AMI/R, mice undergoing UMS demonstrated significantly better global LV-function (EF = 53%±7) as compared to the group without PC (EF = 39%±11, p<0.01). The fraction of akinetic myocardial mass was significantly lower among mice undergoing UMS after AMI/R [27%±10 (w/o UMS), 13%±8 (UMS), p<0.001)]. Our experiments showed a fast onset of transient, UMS-induced upregulation of vascular-endothelial and insulin-like growth factor (VEGF-a, IGF-1), as well as caveolin-3 (Cav-3). The mice undergoing PC with UMS after AMI/R showed a significantly lower scar size. In addition, the microvascular density was significantly higher in the borderzone of UMS-treated animals. CONCLUSION: UMS following AMI/R ameliorates PIR in mice via up-regulation of VEGF-a, IGF-1 and Cav-3, and consecutive improvement of myocardial borderzone vascularization.

FACS-based isolation, propagation and characterization of mouse embryonic cardiomyocytes based on VCAM-1 surface marker expression.

Purification of cardiomyocytes from the embryonic mouse heart, embryonic stem (ES) or induced pluripotent stem cells (iPS) is a challenging task and will require specific isolation procedures. Lately the significance of surface markers for the isolation of cardiac cell populations with fluorescence activated cell sorting (FACS) has been acknowledged, and the hunt for cardiac specific markers has intensified. As cardiomyocytes have traditionally been characterized by their expression of specific transcription factors and structural proteins, and not by specific surface markers, this constitutes a significant bottleneck. Lately, Flk-1, c-kit and the cellular prion protein have been reported to specify cardiac progenitors, however, no surface markers have so far been reported to specify a committed cardiomyocyte. Herein show for the first time, that embryonic cardiomyocytes can be isolated with 98% purity, based on their expression of vascular cell adhesion molecule-1 (VCAM-1). The FACS-isolated cells express phenotypic markers for embryonic committed cardiomyocytes but not cardiac progenitors. An important aspect of FACS is to provide viable cells with retention of functionality. We show that VCAM-1 positive cardiomyocytes can be isolated with 95% viability suitable for in vitro culture, functional assays or expression analysis. In patch-clamp experiments we provide evidence of functionally intact cardiomyocytes of both atrial and ventricular subtypes. This work establishes that cardiomyocytes can be isolated with a high degree of purity and viability through FACS, based on specific surface marker expression as has been done in the hematopoietic field for decades. Our FACS protocol represents a significant advance in which purified populations of cardiomyocytes may be isolated and utilized for downstream applications, such as purification of ES-cell derived cardiomyocytes.

Modulation of calcium-activated potassium channels induces cardiogenesis of pluripotent stem cells and enrichment of pacemaker-like cells.

BACKGROUND: Ion channels are key determinants for the function of excitable cells, but little is known about their role and involvement during cardiac development. Earlier work identified Ca(2+)-activated potassium channels of small and intermediate conductance (SKCas) as important regulators of neural stem cell fate. Here we have investigated their impact on the differentiation of pluripotent cells toward the cardiac lineage. METHODS AND RESULTS: We have applied the SKCa activator 1-ethyl-2-benzimidazolinone on embryonic stem cells and identified this particular ion channel family as a new critical target involved in the generation of cardiac pacemaker-like cells: SKCa activation led to rapid remodeling of the actin cytoskeleton, inhibition of proliferation, induction of differentiation, and diminished teratoma formation. Time-restricted SKCa activation induced cardiac mesoderm and commitment to the cardiac lineage as shown by gene regulation, protein, and functional electrophysiological studies. In addition, the differentiation into cardiomyocytes was modulated in a qualitative fashion, resulting in a strong enrichment of pacemaker-like cells. This was accompanied by induction of the sino-atrial gene program and in parallel by a loss of the chamber-specific myocardium. In addition, SKCa activity induced activation of the Ras-Mek-Erk signaling cascade, a signaling pathway involved in the 1-ethyl-2-benzimidazolinone-induced effects. CONCLUSIONS: SKCa activation drives the fate of pluripotent cells toward mesoderm commitment and cardiomyocyte specification, preferentially into nodal-like cardiomyocytes. This provides a novel strategy for the enrichment of cardiomyocytes and in particular, the generation of a specific subtype of cardiomyocytes, pacemaker-like cells, without genetic modification.

Perlecan is critical for heart stability.

AIMS: Perlecan is a heparansulfate proteoglycan found in basement membranes, cartilage, and several mesenchymal tissues that form during development, tumour growth, and tissue repair. Loss-of-function mutations in the perlecan gene in mice are associated with embryonic lethality caused primarily by cardiac abnormalities probably due to hemopericards. The aim of the present study was to investigate the mechanism underlying the early embryonic lethality and the pathophysiological relevance of perlecan for heart function. METHODS AND RESULTS: Perlecan-deficient murine embryonic stem cells were used to investigate the myofibrillar network and the electrophysiological properties of single cardiomyocytes. The mechanical stability of the developing perlecan-deficient mouse hearts was analysed by microinjecting fluorescent-labelled dextran. Maturation and formation of basement membranes and cell-cell contacts were investigated by electron microscopy, immunohistochemistry, and western blotting. Sarcomere formation and cellular functional properties were unaffected in perlecan-deficient cardiomyocytes. However, the intraventricular dye injection experiments revealed mechanical instability of the early embryonic mouse heart muscle wall before embryonic day 10.5 (E10.5). Accordingly, perlecan-null embryonic hearts contained lower amounts of the critical basement membrane components, collagen IV and laminins. Furthermore, basement membranes were absent in perlecan-null cardiomoycytes whereas adherens junctions formed and matured around E9.5. Infarcted hearts from perlecan heterozygous mice displayed reduced heart function when compared with wild-type hearts. CONCLUSION: We propose that perlecan plays an important role in maintaining the integrity during cardiac development and is important for heart function in the adult heart after injury.

Mechanisms underlying nebivolol-induced endothelial nitric oxide synthase activation in human umbilical vein endothelial cells.

1. Nebivolol (NEB) has been shown to be a selective blocker of beta1-adrenoceptors with additional vasodilating properties that are mediated, at least in part, by an endothelial-dependent liberation of nitric oxide (NO). In the present study, we investigated the underlying mechanisms of NEB-induced vasodilation. 2. Immunohistochemical staining of endothelial nitric oxide synthase (eNOS) was performed in the absence and presence of NEB in human umbilical vein endothelial cells (HUVEC). In addition, we measured the release of nitric oxide (NO) using diaminofluorescein. Metoprolol (MET) was used for comparison. 3. Nebivolol, but not MET (each at 10 micromol/L), caused a time-dependent increase in NO release from HUVEC, as demonstrated by an increase in DAF fluorescence at 0 versus 10 min (+234 +/- 7 and 55 +/- 22% basal, respectively). Blockade of beta3-adrenoceptors by SR 59230A (1 micromol/L) partially reduced the NEB-induced increase in DAF fluorescence. Complete inhibition of NEB-induced NO liberation was achieved by the simultaneous blockade of beta3-adrenoceptors and oestrogen receptors (with 1 micromol/L ICI 182,780). 4. Application of NEB significantly increased eNOS translocation and serine 1177 phosphorylation of eNOS. However, NEB did not alter eNOS-phosphorylation at threonine 495 and at serine 114. 5. In conclusion, the endothelium-dependent NO liberation induced by NEB is due to stimulation of beta3-adrenoceptors and oestrogen receptors and coincides with eNOS translocation and a phosphorylation at eNOS-serine 1177. These characteristics of NEB may be beneficial not only when treating patients suffering from cardiovascular disease, but may also prevent further deterioration of endothelial dysfunction.

Phosphoinositide 3-kinase gamma controls autonomic regulation of the mouse heart through Gi-independent downregulation of cAMP level.

Cardiac beta-adrenergic and the muscarinic receptors control contractility and heart rate by triggering multiple signaling events involving downstream targets like the phosphoinositide 3-kinase gamma (PI3Kgamma). We thus investigated whether the lack of PI3Kgamma could play a role in the autonomic regulation of the mouse heart. Contractility and ICaL of mutant cardiac preparations appeared increased in basal conditions and after beta-adrenergic stimulation. However, basal and beta-adrenergic stimulated heart rate were normal. Conversely, muscarinic inhibition of heart rate was reduced without alteration of the Gbetagamma-dependent stimulation of IK,ACh current. In addition, muscarinic-mediated anti-adrenergic effect on papillary muscle contractility and ICaL was significantly depressed. Consistently, cAMP level of PI3Kgamma-null ventricles was always higher than wild-type controls. Thus, PI3Kgamma controls the cardiac function by reducing cAMP concentration independently of Gi-mediated signaling.

Nitric oxide, a key signaling molecule in the murine early embryonic heart.

Nitric oxide (NO) is thought to play an important role as a signaling molecule in embryonic and adult cardiomyocytes; however, its involvement in muscarinic signaling is still unclear. The aim of the present work was to analyze the muscarinic modulation of the L-type Ca2+ current (ICa) in early- and late-stage embryonic ventricular cardiomyocytes. Muscarinic stimulation depressed basal ICa by 30.1 +/- 3.2% (n=27) in early-stage cardiomyocytes. Pharmacological evidence suggested that the muscarinic modulation was mediated through generation of NO, activation of cGMP-dependent phosphodiesterase (PDE) 2, and ensuing lowering of cyclic AMP/protein kinase A (cAMP/PKA) levels. Conversely, in late-stage cardiomyocytes, muscarinic regulation of ICa occurred in a NO-independent manner via inhibition of prestimulated adenylyl cyclase (AC). To unequivocally prove the involvement of NO and to identify the nitric oxide synthase (NOS) isoform(s), we analyzed muscarinic signaling in embryonic ventricular cardiomyocytes of NOS2 (-/-) and NOS3 (-/-) mice. The early-stage NOS3 (-/-) cardiomyocytes lacked muscarinic modulation, whereas it was preserved in NOS2 (-/-) cells. Moreover, at the late embryonic stage, muscarinic modulation of ICa was intact in both strains. Thus, NO is the key regulator of muscarinic signaling in the early embryonic ventricle, whereas at later stages, signaling occurs through a NO-independent pathway.

Phosphoinositide 3-kinase gamma-deficient hearts are protected from the PAF-dependent depression of cardiac contractility.

OBJECTIVES: Following an ischemic insult, cardiac contractile recovery might be perturbed by the release of autacoids, like platelet-activating factor (PAF), that depress heart function by acting through G protein-coupled receptors (GPCRs). The signaling events downstream the PAF receptor that lead to the negative inotropic effect are still obscure. We thus investigated whether the GPCR-activated phosphoisositide 3-kinase gamma (PI3Kgamma) could play a role in the cardiac response to PAF. METHODS: The negative inotropic effect of PAF was studied ex vivo, in isolated electrically driven atria and in Langendorff-perfused whole hearts derived from wild-type and PI3Kgamma-null mice. Postischemic recovery of contractility was analyzed in normal and mutant whole hearts subjected to 30 min of ischemia and 40 min of reperfusion in the presence or absence of a PAF receptor antagonist. RESULTS: While wild-type hearts stimulated with PAF showed increased nitric oxide (NO) production and a consequent decreased cardiac contractility, PI3Kgamma-null hearts displayed reduced phosphorylation of nitric oxide synthase 3 (NOS3), blunted nitric oxide production and a complete protection from the PAF-induced negative inotropism. In addition, Langendorff-perfused PI3Kgamma-null hearts showed a better contractile recovery after ischemia/reperfusion, a condition where PAF is known to be an important player in depressing contractility. In agreement with a role of PI3Kgamma in this PAF-mediated signaling, postischemic contractile recovery in PI3Kgamma-null mice appeared overlapping with that of normal hearts treated with the PAF receptor antagonist WEB 2170. CONCLUSION: These data indicate a novel PAF-dependent signaling pathway that, involving PI3Kgamma and NOS3, contributes to postischemic contractile depression.

Cardiac effects of ghrelin and its endogenous derivatives des-octanoyl ghrelin and des-Gln14-ghrelin.

The mechanisms underlying the cardiac activities of synthetic growth hormone secretagogues (GHS) are still unclear. The natural ligand of the GHS receptors, i.e. ghrelin, classically binds the GHS receptor and exerts endocrine actions in acylated forms only; its cardiovascular actions still need to be investigated further. In order to clarify these aspects, we studied the effects of either the synthetic peptidyl GHS hexarelin (1 microM), or the natural ghrelin (50 nM) and the endogenous ghrelin derivatives des-Gln14-ghrelin (1-100 nM) and des-octanoyl ghrelin (50 nM), on the tension developed by guinea pig papillary muscle and on L-type Ca2+ current (ICa) of isolated ventricular cells. The binding of these molecules to ventricular cell membrane homogenates was also studied. We observed that all peptides reduced the tension developed at low frequencies (60-120 beats/min) in a dose-dependent manner. No alteration in cardiac contractility was induced by des-Gln14-ghrelin or des-octanoylated ghrelin when the endocardial endothelium had been removed or after cyclooxygenase blockade. Pretreatment with tyramine (2 microM) had no effect on the inotropic response induced by des-Gln(14)-ghrelin. No significant effect on I(Ca) of isolated ventricular cells was observed in the presence of des-Gln14-ghrelin (100 nM). The order of potency on the tension of papillary muscle was: des-octanoyl ghrelin > ghrelin = des-Gln14-ghrelin > hexarelin. This gradient of potency was consistent with the binding experiments performed on ventricular membranes where either acylated or unacylated ghrelin forms, and hexarelin, recognized a common high-affinity binding site. In conclusion, ghrelin, des-Gln14-ghrelin and des-octanoyl ghrelin, show similar negative inotropic effect on papillary muscle; as des-octanoyl ghrelin is peculiarly devoid of any GH-releasing activity, the cardiotropic action of these molecules is independent of GH release. The binding studies and the experiments performed both on the isolated cells and on papillary muscle after endothelium removal or cyclooxygenase blockade indicate that the cardiotropic action of natural and synthetic ghrelin analogues reflects the interaction with a novel GHS receptor (peculiarly common for ghrelin and des-octanoyl ghrelin), leading to release of cyclooxygenase metabolites from endothelial cells, as indicated by direct measurement of prostacyclin metabolite 6-keto-PGF(1alpha).

Cyclic AMP and cyclic GMP independent stimulation of ventricular calcium current by peroxynitrite donors in guinea pig myocytes.

We investigated the potential involvement of peroxynitrite (ONOO(-)) in the modulation of calcium current (I(Ca)) in guinea pig ventricular myocytes with the whole-cell patch clamp technique and with cyclic AMP (cAMP) measurements. Because of the short half-life of ONOO(-) at physiological pH, we induced an increase in its intracellular levels by using donors of the precursors, nitric oxide (NO) and superoxide anion (O(2) (-)). High concentrations of NO donors, SpermineNONOate (sp/NO, 300 microM) or SNAP (300 microM) increased basal I(Ca) (50.3 +/- 4.6%, n = 7 and 46.2 +/- 5.0%, n = 13). The superoxide anion donor Pyrogallol (100 microM) also stimulated basal I(Ca) (44.6 +/- 2.8%, n = 11). At lower concentration sp/NO (10 nM) and Pyrogallol (1 microM), although separately ineffective on I(Ca), enhanced the current if applied together (33.5 +/- 0.7%, n = 7). The simultaneous donor of O(2) (-) and NO, SIN-1 (500 microM), also stimulated basal I(Ca) (22.8 +/- 2.1%, n = 13). In the presence of saturating cyclic GMP (cGMP, 50 microM) in the patch pipette or of extracellular dibutyryl cGMP (dbcGMP, 100 microM), I(Ca) was still increased by SIN-1 (32.0 +/- 6.1%, n = 4 and 30.0 +/- 5.4%, n = 8). Both Manganese(III)tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP, 100 microM) a ONOO(-) scavenger, and superoxide dismutase (SOD) (150 U/ml) reversed the stimulatory effect of SIN-1 on I(Ca) (respectively -0.6 +/- 4.1%, n = 4 and 3.6 +/- 4.3%, n = 4). Intracellular cAMP level was unaltered by SIN-1, while it was enhanced by blocking the NO-cGMP pathway with the NO synthase inhibitor L-NMMA. These results suggest that peroxynitrite donors increase cardiac calcium current without the involvement of cAMP and cGMP.

Microtubules mobility affects the modulation of L-type I(Ca) by muscarinic and beta-adrenergic agonists in guinea-pig cardiac myocytes.

To investigate the interaction of cytoskeleton with the receptor modulation of ionic currents, we studied the effect of muscarinic and beta-adrenergic stimulation in adult guinea-pig ventricular cardiac myocytes treated with paclitaxel and colchicine, two drugs that respectively stabilize or destabilize microtubules. We observed that the stabilization of microtubules with paclitaxel (1 microM for 1-4 h) did not markedly affect either the kinetics of I(Ca), or the stimulatory effect of isoproterenol (Iso, 1 microM); however paclitaxel significantly blunted the response to carbachol (CCh, 1 microM). In agreement with the electrophysiological measurements, Iso induced a similar enhancement of intracellular cAMP levels in both control and paclitaxel-treated cells, while the response to CCh 1 microM was significantly reduced in paclitaxel-treated cells. The reduction of muscarinic response induced by paclitaxel was also evident in atrial cells, in which the stimulation of I(KACh) by CCh 1 microM was reduced to about 10%. Compared to the muscarinic response, paclitaxel did not have significant effect on the purinergic (adenosine 1-10 microM) modulation of I(Ca). In contrast to paclitaxel, in colchicine-treated cells, I(Ca) was not enhanced by beta-adrenergic stimulation, but instead reduced by CCh, even in the absence of previous stimulation. In conclusion, our data suggest that microtubule stabilization significantly affects the muscarinic modulation of I(Ca), by interacting with the receptor or the G-protein rather than on the intracellular signaling cascade.

Ghrelin and des-acyl ghrelin inhibit cell death in cardiomyocytes and endothelial cells through ERK1/2 and PI 3-kinase/AKT.

Ghrelin is an acyl-peptide gastric hormone acting on the pituitary and hypothalamus to stimulate growth hormone (GH) release, adiposity, and appetite. Ghrelin endocrine activities are entirely dependent on its acylation and are mediated by GH secretagogue (GHS) receptor (GHSR)-1a, a G protein-coupled receptor mostly expressed in the pituitary and hypothalamus, previously identified as the receptor for a group of synthetic molecules featuring GH secretagogue (GHS) activity. Des-acyl ghrelin, which is far more abundant than ghrelin, does not bind GHSR-1a, is devoid of any endocrine activity, and its function is currently unknown. Ghrelin, which is expressed in heart, albeit at a much lower level than in the stomach, also exerts a cardio protective effect through an unknown mechanism, independent of GH release. Here we show that both ghrelin and des-acyl ghrelin inhibit apoptosis of primary adult and H9c2 cardiomyocytes and endothelial cells in vitro through activation of extracellular signal-regulated kinase-1/2 and Akt serine kinases. In addition, ghrelin and des-acyl ghrelin recognize common high affinity binding sites on H9c2 cardiomyocytes, which do not express GHSR-1a. Finally, both MK-0677 and hexarelin, a nonpeptidyl and a peptidyl synthetic GHS, respectively, recognize the common ghrelin and des-acyl ghrelin binding sites, inhibit cell death, and activate MAPK and Akt.These findings provide the first evidence that, independent of its acylation, ghrelin gene product may act as a survival factor directly on the cardiovascular system through binding to a novel, yet to be identified receptor, which is distinct from GHSR-1a.