Renzo Levi

Cyclic GMP regulates the Ca-channel current in guinea pig ventricular myocytes

The effect of intracellular perfusion with cyclic GMP (cGMP) on Ca current (ICa) was investigated in Cs-loaded isolated cells from guinea pig ventricle using the whole-cell patch-clamp technique and a perfused patch pipette. cGMP (5 μM) strongly reduced ICa which had been elevated by intracellular perfusion with 50 μM of either cyclic AMP (cAMP) or its hydrolysis-resistant analog 8-Bromo-cAMP. In addition, cGMP prevented the stimulation of ICa by IBMX, a phosphodiesterase inhibitor. The membrane permeant cGMP analog 8-Bromo-cGMP (100 μM), when applied outside the cell, also antagonized the stimulatory effect of IBMX on ICa. It is concluded that cGMP inhibits ICa in guinea pig ventricular cells by a mechanism different from the activation of a cGMP-stimulated phosphodiesterase recently found in frog ventricular cells.

Integrating Cardiac PIP3 and cAMP Signaling through a PKA Anchoring Function of p110γ

Adrenergic stimulation of the heart engages cAMP and phosphoinositide second messenger signaling cascades. Cardiac phosphoinositide 3-kinase p110γ participates in these processes by sustaining β-adrenergic receptor internalization through its catalytic function and by controlling phosphodiesterase 3B (PDE3B) activity via an unknown kinase-independent mechanism. We have discovered that p110γ anchors protein kinase A (PKA) through a site in its N-terminal region. Anchored PKA activates PDE3B to enhance cAMP degradation and phosphorylates p110γ to inhibit PIP(3) production. This provides local feedback control of PIP(3) and cAMP signaling events. In congestive heart failure, p110γ is upregulated and escapes PKA-mediated inhibition, contributing to a reduction in β-adrenergic receptor density. Pharmacological inhibition of p110γ normalizes β-adrenergic receptor density and improves contractility in failing hearts.

Guanylate-cyclase-mediated inhibition of cardiac ICa by carbachol and sodium nitroprusside.

We studied the role of cyclic guanosine monophosphate (cGMP) as a mediator of the reduction of L-type calcium current (ICa) induced by muscarinic receptor stimulation and by nitric oxide in isolated guinea-pig ventricular cells using the whole-cell patchclamp technique. Our results show that when the level of cyclic adenosine monophosphate was increased by the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX), stimulation of a pertussis-toxin (PTX)-sensitive muscarinic receptor by carbachol (1 μM) reduced the calcium current increase from 80.6±23.5% to 19.8±9.6% over the control and this effect was prevented by methylene blue (10 μM), an inhibitor of the soluble guanylate cyclase. Pipette solution containing 10 μM cGMP reduced the enhancement of ICa by IBMX from 121.9±11.6% to 14.2±5.4% above the control. Sodium nitroprusside (10 μM), a spontaneous donor of nitric oxide, and consequently a stimulator of soluble guanylate cyclase, also reduced IBMX-stimulated ICa from 115.2±13.2% to 32.2±6.9% above control and the sodium nitroprusside effect was also suppressed by methylene blue. The latter two reagents were ineffective on basal ICa.

M1 muscarinic receptors increase calcium current and phosphoinositide turnover in guinea-pig ventricular cardiocytes.

1. Physiological and molecular evidence for the presence and functional role of M1 muscarinic cholinergic receptors (mAChRs) in adult guinea‐pig ventricular cells is presented. 2. Whole‐cell clamp measurements of the L‐type calcium current (ICa) in isolated myocytes were performed. Caesium was used to suppress potassium currents. ICa was increased by the muscarinic agonist carbachol in cells pretreated with pertussis toxin which blocked the M2 mAChR‐triggered cascade of intracellular signalling, while it was not changed in untreated cells. 3. If the M2‐mediated regulation of ICa was blocked by directly saturating the cell with cyclic adenosine monophosphate (cAMP) through the patch pipette, application of carbachol induced a further small increase of the current above the level reached after cAMP perfusion. This increase was more pronounced in cells pretreated with pertussis toxin. 4. The carbachol‐induced increase of ICa was blocked by the selective M1 mAChR antagonist pirenzepine. 5. The application of high concentrations of carbachol increased the accumulation of [3H]inositol monophosphate up to 240% above control levels. This increase was reduced by application of pirenzepine. 6. The expression of M1 receptor mRNA in ventricular cardiocytes was shown by reverse transcriptase‐polymerase chain reaction. 7. These results suggest that M1 mAChR regulation of ICa can be a component of the paradoxical positive inotropism induced by high concentrations of muscarinic agonists.

Growth hormone-releasing hormone promotes survival of cardiac myocytes in vitro and protects against ischaemia–reperfusion injury in rat heart

AIMS The hypothalamic neuropeptide growth hormone-releasing hormone (GHRH) stimulates GH synthesis and release in the pituitary. GHRH also exerts proliferative effects in extrapituitary cells, whereas GHRH antagonists have been shown to suppress cancer cell proliferation. We investigated GHRH effects on cardiac myocyte cell survival and the underlying signalling mechanisms. METHODS AND RESULTS Reverse transcriptase-polymerase chain reaction analysis showed GHRH receptor (GHRH-R) mRNA in adult rat ventricular myocytes (ARVMs) and in rat heart H9c2 cells. In ARVMs, GHRH prevented cell death and caspase-3 activation induced by serum starvation and by the beta-adrenergic receptor agonist isoproterenol. The GHRH-R antagonist JV-1-36 abolished GHRH survival action under both experimental conditions. GHRH-induced cardiac cell protection required extracellular signal-regulated kinase (ERK)1/2 and phosphoinositide-3 kinase (PI3K)/Akt activation and adenylyl cyclase/cAMP/protein kinase A signalling. Isoproterenol strongly upregulated the mRNA and protein of the pro-apoptotic inducible cAMP early repressor, whereas GHRH completely blocked this effect. Similar to ARVMs, in H9c2 cardiac cells, GHRH inhibited serum starvation- and isoproterenol-induced cell death and apoptosis through the same signalling pathways. Finally, GHRH improved left ventricular recovery during reperfusion and reduced infarct size in Langendorff-perfused rat hearts, subjected to ischaemia-reperfusion (I/R) injury. These effects involved PI3K/Akt signalling and were inhibited by JV-1-36. CONCLUSION Our findings suggest that GHRH promotes cardiac myocyte survival through multiple signalling mechanisms and protects against I/R injury in isolated rat heart, indicating a novel cardioprotective role of this hormone.

Modulation of guinea‐pig cardiac L‐type calcium current by nitric oxide synthase inhibitors

1 Electrophysiological (whole‐cell clamp) techniques were used to study the effect of NO synthase (NOS) inhibitors on guinea‐pig ventricular calcium current (ICa), and biochemical measurements (Western blot and citrulline synthesis) were made to investigate the possible mechanisms of action. 2 The two NOS inhibitors, NG‐monomethyl‐L‐arginine (L‐NMMA, 1 mM) and NG‐nitro‐L‐arginine (L‐NNA, 1 mM), induced a rapid increase in ICa when applied to the external solution. D‐NMMA (1 mM), the stereoisomer of L‐NMMA, which has no effect on NOS, did not enhance ICa. 3 Western blot experiments gave no indication of the presence of inducible NOS protein (iNOS) in our cell preparation, neither immediately after dissociation nor after more than 24 h. Statistically, there was no significant difference between electrophysiological experiments performed on freshly dissociated cells and experiments performed the next day. Moreover cells prepared and kept in the presence of dexamethasone (3 μM), to inhibit the expression of iNOS, gave the same response to L‐NMMA as control cells. 4 The stimulatory effect of L‐NMMA (1 mM) on basal ICa was reversed by competition with higher doses (5 mM) of externally applied L‐arginine, the natural substrate of NOS. The effect of L‐NMMA was also eliminated by L‐arginine in the patch pipette solution. 5 Intracellular perfusion with GDPβS (0.5 mM), which stabilizes the G‐proteins in the inactive state, did not affect the L‐NMMA‐induced stimulation of ICa. 6 Carbachol (1 μM) reduced the ICa previously stimulated by L‐NMMA, and intracellular cGMP (10 μM) prevented L‐NMMA enhancement. 7 Simultaneous treatment with L‐NMMA and isoprenaline (1 μM) induced a non‐cumulative enhancement of ICa that could not be reversed by carbachol (1 μM). 8 NO synthesis, measured by the formation of [3H]citrulline from L‐[3H]arginine during a 15 min incubation, showed a relatively high basal NO production, which was inhibited by L‐NMMA but not affected by carbachol. 9 These results suggest that inhibitors of NOS are able to modulate the basal ventricular ICa in the absence of a receptor‐mediated pathway, and that NO might be required for the muscarinic reduction of ICa under isoprenaline stimulation, even if NO production is not directly controlled by the muscarinic pathway.

Limited plasticity of mesenchymal stem cells cocultured with adult cardiomyocytes.

In order to assess, in a controlled in vitro model, the differentiation potential of adult bone marrow derived stem cells we have developed a coculture procedure using adult rat cardiomyocytes and mesenchymal stem cells (MSCs) from transgenic GFP positive rats. We investigated in the cocultured MSCs the time course of cellular processes that are difficult to monitor in in vivo experiments. Adult rat cardiomyocytes and adult rat MSCs were cocultured for up to 7 days and analyzed by confocal microscopy. Several markers were studied by immunofluorescence technique. The fluorescent ST‐BODIPY‐Dihydropyridine was used to label calcium channels in living cells. Intracellular calcium was monitored with the fluorescent probe X‐Rhod‐1. Immunofluorescence experiments showed the presence of connexin‐43 between cardiomyocytes and MSCs and between MSCs, while no sarcomeric structures were observed at any time of the coculture. We looked at the expression of calcium channels and development of voltage‐dependent calcium signaling in cocultured MSCs. MSCs showed a time‐dependent increase of labeling of ST‐BODIPY‐Dihydropyridine, reaching a relatively strong level after 72 h of coculture. The treatment with a non‐fluorescent DHP, Nifedipine, completely abolished ST‐BODIPY labeling. We investigated whether depolarization could modulate intracellular calcium. Depolarization‐induced calcium transients increased in MSCs in relation to the coculture time. We conclude that MSCs cocultured with adult cardiomyocytes present preliminary evidence of voltage‐dependent calcium modulation uncoupled with the development of nascent or adult myofibrils, thus showing a limited lineage specification and a low plasticity to differentiate in a full cardiomyocyte‐like phenotype. J. Cell. Biochem. 100: 86–99, 2007.

The homologous rat chromogranin A1–64 (rCGA1–64) modulates myocardial and coronary function in rat heart to counteract adrenergic stimulation indirectly via endothelium-derived nitric oxide

Chromogranin A (CGA), produced by human and rat myocardium, generates several biologically active peptides processed at specific proteolytic cleavage sites. A highly conserved cleavage N‐terminal site is the bond 64–65 that reproduces the native rat CGA sequence (rCGA1–64), corresponding to human N‐terminal CGA‐derived vasostatin‐1. rCGA1–64 cardiotropic activity has been explored in rat cardiac preparations. In Langendorff perfused rat heart, rCGA1–64 (from 33 nM) induced negative inotropism and lusitropism as well as coronary dilation, counteracting isoproterenol (Iso)‐ and endothelin‐1 (ET‐1) ‐induced positive inotropic effects and ET‐1‐dependent coronary constriction. rCGA1–64 also depressed basal and Iso‐induced contractility on rat papillary muscles, without affecting calcium transients on isolated ventricular cells. Structure‐function analysis using three modified peptides on both rat heart and papillary muscles revealed the disulfide bridge requirement for the cardiotropic action. A decline in Iso intrinsic activity in the presence of the peptides indicates a noncompetitive antagonistic action. Experiments on rat isolated cardiomyocytes and bovine aortic endothelial cells indicate that the negative inotropism observed in rat papillary muscle is probably due to an endothelial phosphatidylinositol 3‐kinase‐dependent nitric oxide release, rather than to a direct action on cardiomyocytes. Taken together, our data strongly suggest that in the rat heart the homologous rCGA1–64 fragment exerts an autocrine/paracrine modulation of myocardial and coronary performance acting as stabilizer against intense excitatory stimuli.— Cerra, M. C., Gallo, M. P., Angelone, T., Quintieri, A. M., Pulera, E., Filice, E., Guerold, B., Shooshtarizadeh, P., Levi, R., Ramella, R., Brero, A., Boero, O., Metz‐Boutigue, M. H., Tota, B., Alloatti, G. The homologous rat chromogranin A1‐64 (rCGA1‐64) modulates myocardial and coronary function in rat heart to counteract adrenergic stimulation indirectly via endothelium‐derived nitric oxide. FASEB J. 22, 3992–4004 (2008)

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 (ICa) 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 (O2−). High concentrations of NO donors, SpermineNONOate (sp/NO, 300 μM) or SNAP (300 μM) increased basal ICa (50.3 ± 4.6%, n = 7 and 46.2 ± 5.0%, n = 13). The superoxide anion donor Pyrogallol (100 μM) also stimulated basal ICa (44.6 ± 2.8%, n = 11). At lower concentration sp/NO (10 nM) and Pyrogallol (1 μM), although separately ineffective on ICa, enhanced the current if applied together (33.5 ± 0.7%, n = 7). The simultaneous donor of O2− and NO, SIN‐1 (500 μM), also stimulated basal ICa (22.8 ± 2.1%, n = 13). In the presence of saturating cyclic GMP (cGMP, 50 μM) in the patch pipette or of extracellular dibutyryl cGMP (dbcGMP, 100 μM), ICa 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 μM) a ONOO− scavenger, and superoxide dismutase (SOD) (150 U/ml) reversed the stimulatory effect of SIN‐1 on ICa (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. J. Cell. Physiol. 197: 284–296, 2003© 2003 Wiley‐Liss, Inc.

Vasostatin 1 activates eNOS in endothelial cells through a proteoglycan-dependent mechanism

Accumulating evidences point to a significant role for the chromogranin A (CgA)‐derived peptide vasostatin 1 (VS‐1) in the protective modulation of the cardiovascular activity, because of its ability to counteract the adrenergic signal. We have recently shown that VS‐1 induces a PI3K‐dependent‐nitric oxide (NO) release by endothelial cells, contributing to explain the mechanism of its cardio‐suppressive and vasodilator properties. However, the cellular processes upstream the eNOS activation exerted by this peptide are still unknown, as typical high‐affinity receptors have not been identified. Here we hypothesize that in endothelial cells VS‐1 acts, on the basis of its cationic and amphipathic properties, as a cell penetrating peptide, binding to heparan sulfate proteoglycans (HSPGs) and activating eNOS phosphorylation (Ser1179) through a PI3K‐dependent, endocytosis‐coupled mechanism. In bovine aortic endothelial cells (BAE‐1 cells) endocytotic vesicles trafficking was quantified by confocal microscopy with a water‐soluble membrane dye; caveolin 1 (Cav1) shift from plasma membrane was studied by immunofluorescence staining; VS‐1‐dependent eNOS phosphorylation was assessed by immunofluorescence and immunoblot analysis. Our experiments demonstrate that VS‐1 induces a marked increase in the caveolae‐dependent endocytosis, (115 ± 23% endocytotic spots/cell/field in VS‐1‐treated cells with respect to control cells), that is significantly reduced by both heparinase III (HEP, 17 ± 15% above control) and Wortmannin (Wm, 7 ± 22% above control). Heparinase, Wortmannin, and methyl‐β‐cyclodextrin (MβCD) abolish the VS‐1‐dependent eNOS phosphorylation (PSer1179eNOS). These results suggest a novel signal transduction pathway for endogenous cationic and amphipathic peptides in endothelial cells: HSPGs interaction and caveolae endocytosis, coupled with a PI3K‐dependent eNOS phosphorylation. J. Cell. Biochem. 110: 70–79, 2010.