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,1
* Department of Cell Biology and
Department of Pharmaco-Biology, University of Calabria, Calabria, Italy;
Department of Animal and Human Biology, University of Turin, Turin, Italy; and
Unity INSERM 575, Physiopathology of Nervous System, University "Louis Pasteur," Strasbourg, France
1 Correspondence: B.T., Department of Cell Biology, University of Calabria, 87030 Arcavacata di Rende (CS), Calabria, Italy. E-mail: tota{at}unical.it; A.G., Department of Animal and Human Biology, University of Turin (TO), Italy. E-mail: giuseppe.alloatti{at}unito.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Key Words: endocrine heart • peptide hormones • endothelial cells • stress response
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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, is costored with catecholamines, nucleotides, calcium, and other peptide hormones in the secretory granules of several endocrine and neuronal cells and is released in the extracellular environment by exocytosis (2)
. Apart from an intracellular role in secretory vesicle biogenesis (3)
, CGA exerts an important extracellular function as a prohormone for a number of shorter biologically active peptides produced by tissue-specific proteolytic processing (for references, see ref. 4
). In the specific context of cardiocirculatory homeostasis, these include vasostatin (VS) -1, a potent vasodilator and cardioinhibitory agent (5)
and catestatin, an inhibitor of catecholamine release characterized by antihypertensive properties (6)
. In response to stress, CGA and its derived fragments are released in the blood, reaching nanomolar concentrations in the peripheral circulation in humans (7)
. It is notable that in patients with chronic heart failure, higher CGA concentrations correlate with the severity of the disease, representing a prognostic indicator of mortality (7
8
9)
. Elevated circulating levels have also been reported in patients with neuroendocrine tumors (10
, 11)
as well as in renal (12)
and liver (13)
failure. This information and the finding that CGA knockout mice show hypertension and cardiac enlargement (14)
point to the importance of CGA in cardiocirculatory physiology. Interestingly, CGA-derived VSs appear to function as endocrine/paracrine cardiac stabilizers, particularly in the presence of intense adrenergic stimuli, e.g., under stress responses (5)
, and elicit a protective effect against the extension of myocardial infarction (15)
. In the rat heart, CGA is stored in nonadrenergic myoendocrine atrial cells containing atrial natriuretic peptide (16)
, in Purkinje fibers of the atrium and ventricle containing the calcium channel 
1E subunit (17)
; in sheep, it is stored in the sympathetic nerve termini (18)
. Moreover, in the rat heart, in addition to intact CGA and larger fragments containing the C terminus, four VS-containing CGA peptides have been identified, i.e., CGA4–113, CGA1–124 (vasostatin-2), CGA1–135, and CGA1–199 (19)
. More recently, Pieroni et al. (20)
demonstrated that CGA is produced by the human myocardium, in which it is colocalized with brain natriuretic peptide and overexpressed in dilated and hypertrophic cardiomyopathy, suggesting a neuroendocrine role in the regulation of cardiac function and a potential therapeutic target in heart failure. The possibility exists that locally derived VSs may exert autocrine/paracrine regulation of cardiac function, because in the normal or stressed heart a specific stimulus-induced proteolytic activation could conceivably generate and locally increase lower molecular mass CGA-derived VSs; production of some of them could also result from extracellular processing, as reported for CGA in the adrenal gland (4)
.
Rat CGA is a 448-amino acid protein with an isoelectric point (pI) of 4.5–5.0, containing a disulfide bridge and displaying numerous monobasic and dibasic (nine) residues serving as a possible recognition signal for endoproteolytic processing (subtilisin-like and trypsin-like) enzymes (21)
. In rat heart, the detected PC1/3, PC2, and carboxypeptidase H/E (22)
might be involved in the CGA maturation process. Metz-Boutigue et al. (23)
have previously characterized intragranular and extracellular processing of CGA in chromaffin granules from bovine adrenal medulla, showing that the processing starts at both the N terminus and the C terminus of the protein. Among the fragments generated, CGA1–76 (VS-1) and prochromacin (CGA79–431) represent the major products of proteolytic processing in bovine adrenal medulla (23)
. Conversely, in rat CGA (rCGA), because of the lack of the first dibasic site (24
, 25)
, the first N-terminal cleavage product is β-granin (rCGA1–128), corresponding to vasostatin-2. The alignment of bovine, human, and rat VS-1 (88, 86, 89, and 82% homology between rat and bovine/human CGA1–76, CGA17–38, CGA1–64, and CGA65–76, respectively), depicted in Table 1
, shows that, in contrast to the human sequence, the N-terminal sequence of rat CGA does not contain the Lys77Lys78 dibasic site, whereas it does contain an insertion of 15 glutamine residues. Thus, proteolytic processing in humans and in rats is probably different.
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Because CGA fragments are likely to be secreted by cardiomyocytes, extracellular processing can be expected to occur in the secreted medium, as shown for CGA and proenkephalin-A secreted from chromaffin cells (23
, 26)
. The presence of extracellular proteases both on cardiomyocyte cell membranes and in the extracellular matrix suggests that extracellular processing does occur, as proposed for angiotensin II. In fact, in the heart, angiotensin-converting enzyme and/or renin, present on cell membranes, are involved in the conversion of angiotensinogen into angiotensin II (27)
.
Among the major proteolytic cleavage sites involved in CGA processing, the bond 64–65, present in the N-terminal moiety of the protein and included in the VS sequence, is highly conserved (23)
. For the first time we synthesized this N-terminal fragment, reproducing the native rat sequence (rCGA1–64 with S-S bridge: rCGA1–64S-S) to determine its putative cardiotropic activity on rat cardiac preparations. Similarly, we also synthesized and tested the (12-amino acid) endogenous CGA fragment (65–76), flanked by a dibasic pair on the C-terminal side, which was previously identified and detected by Metz-Boutigue et al. (23)
in the extracellular space of stimulated cultured chromaffin cells. Using both the Langendorff perfused rat heart and isolated papillary muscle, we demonstrate here that rCGA1–64S-S induces inotropic, lusitropic, and coronary actions and that the peptide-elicited negative inotropism is endothelium dependent.
Furthermore, to clarify the structure-function relationship, we compared the actions of two modified peptides, i.e., rCGA1–64 without S-S bridge (rCGA1–64SH) and rCGA1–64 oxidized (rCGA1–64OX), revealing the disulfide bridge requirement for their cardiotropic action. Finally, to define the signaling pathways activated by rCGA1–64S-S, using rat papillary muscle, isolated rat ventricular cardiomyocytes, and cultured bovine aortic endothelial (BAE-1) cells, we demonstrated that the rCGA1–64-induced negative inotropism is mainly due to an endothelial phosphatidylinositol 3-kinase (PI3K) -dependent NO release, rather than to a direct action on cardiomyocytes. In contrast, the CGA 65–76 fragment failed to show any cardiotropic activity. Taken together, our data point to rCGA1–64 as a novel cardiac autocrine/paracrine modulator of basal myocardial performance, able, at the same time, to exert a counterregulatory role against excessive excitatory stimuli, particularly heightened by β-adrenergic activity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Isolated Langendorff heart preparation
Hearts were rapidly excised and transferred to ice-cold buffered Krebs-Henseleit solution (KHS) after the rats had been anesthetized with an intraperitoneal injection of ethyl carbamate (2 g/kg of weight). As described previously (5)
, the aorta was immediately cannulated with a glass cannula and connected to the Langendorff apparatus to start perfusion at a constant flow rate (12 ml/min). In brief, the apex of the left ventricle (LV) was pierced to avoid fluid accumulation. A water-filled latex balloon, connected to a BLPR gauge (WPI, Inc. Sarasota, FL, USA), was inserted through the mitral valve into the LV to allow isovolumic contractions and to continuously record mechanical parameters. Another pressure transducer located just above the aorta recorded coronary pressure (CP). Hemodynamic parameters were assessed using a PowerLab data acquisition system and analyzed using Chart software (both purchased from ADInstruments, Basile, Italy).
Basal conditions
The cardiac performance of the Langendorff-perfused rat heart was evaluated for inotropic effects by analyzing the left ventricular pressure (LVP) (mmHg), which is an index of contractile activity, the rate-pressure product (RPP), which is an index of cardiac work (28)
, the maximal value of the first derivative of LVP [+(LVdP/dt)max] (mmHg/s), which indicates the maximal rate of LV contraction, and time to peak tension of isometric twitch and for lusitropic effects by analyzing the maximal rate of LVP decline of [–(LVdP/dt)max] (mmHg/s), the half-time relaxation (HTR) (s), which is the time required for tension to fall from the peak to 50% (29)
, and the T/–t ratio obtained by +(LVdP/dt)max/–(LVdP/dt)max (30)
. Mean CP (mmHg) was calculated as the average of values obtained during several cardiac cycles (31)
.
rCGA1–64-stimulated preparations
Preliminary experiments (data not shown) obtained by repetitive exposure of each heart to one concentration of rCGA1–64S-S (65 nM) revealed the absence of desensitization. In fact, each peptide dose produced a LVP percentage reduction of 17.7 ± 3.61. Thus, concentration-response curves were generated by perfusing the cardiac preparations with KHS with increasing concentrations of either rCGA1–64S-S or rCGA1–64SH or rCGA1–64OX (from 11 to 165 nM) for 10 min.
Isoproterenol (Iso) -stimulated preparations
To obtain preliminary information on the antagonistic action of either rCGA1–64S-S (11, 33, and 65 nM), rCGA1–64SH (65 and 165 nM), or rCGA1–64OX (65 and 165 nM) toward the Iso-dependent stimulation, dose-response curves were generated by perfusing heart preparations with KHS enriched with increasing concentrations of Iso (0.1 nM-1 µM) alone. These curves were then compared with those obtained by exposing other cardiac preparations to the same perfusion medium containing increasing concentrations of Iso (0.1 nM-1 µM) plus a single concentration of either rCGA1–64S-S (11, 33, and 65 nM), rCGA1–64SH (65 and 165 nM), or rCGA1–64OX (65 and 165 nM).
Gi/o protein involvement
To evaluate the involvement of Gi/o protein in the mechanism of action of rCGA1–64S-S, the hearts were preincubated for 60 min with KHS enriched with pertussis toxin (PTx) (0.01 nM) and then exposed for 10 min to 65 nM rCGA1–64S-S. As shown in the rat heart (see ref. 31
and references therein), PTx catalyzes the ADP-ribosylation of the -subunit of Gi/o proteins and uncouples the interaction between Gi and inhibitory receptors of adenylate cyclase, such as adrenergic receptors.
Involvement of PI3K activity on basal performance
To establish the involvement of PI3K activity in the action mechanism of rCGA1–64S-S, we used wortmannin, a potent inhibitor of PI3K. Cardiac preparations were perfused with 65 nM rCGA1–64 for 10 min and then washed out with KHS. After a return to control conditions, each heart was perfused with KHS containing wortmannin (10 nM) for another 10 min. Then the hearts were exposed to the specific drug plus 65 nM rCGA1–64S-S.
Nitric oxide (NO) -cGMP-protein kinase G (PKG) pathway
To obtain preliminary information about NO pathway involvement in the cardiotropic and vasomotory action of rCGA1–64S-S, cardiac preparations were stabilized for 20 min with KHS. Thus, the hearts were perfused with 65 nM rCGA1–64S-S for 10 min and then washed out with KHS. After a return to control conditions, each heart was perfused with KHS containing the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), the nonspecific nitric oxide synthase (NOS) inhibitor NG-monomethyl-L-arginine (L-NMMA), the soluble guanylate cyclase inhibitor 1H-(1,2,4)oxadiazole-(4,4-a)quinoxalin-1-one (ODQ), or the protein kinase G blocker KT5823 for another 10 min. Then the hearts were exposed to the specific drug plus 65 nM rCGA1–64S-S.
Endothelin-1 (ET-1) -stimulated preparations
To verify the ability of rCGA1–64S-S to counteract the ET-1 stimulation, the hearts, stabilized for 20 min with KHS, were perfused for 10 min with ET-1 (1 nM) and then washed out with KHS. After a return to control conditions, each heart was perfused with KHS containing a single concentration of rCGA1–64S-S (65 nM) plus 1 nM ET-1 for another 10 min.
cGMP and cAMP measurements
Acid extracts of frozen heart tissue used for cGMP and cAMP determinations (endocardial tissue and cardiomyocytes from atria and ventricles) (200–300 mg) were treated with 6% trichloroacetic acid at 0°C and centrifuged at 10,000 g for 10 min. The supernatant was extracted 3 times with 3 ml of diethyl ether saturated with water, and the aqueous phase was collected and stored at –80°C. cGMP and cAMP concentrations were measured using a commercial enzyme immunoassay (Biotrak enzyme immunoassay system; Amersham Biosciences, Piscataway, NJ, USA).
Isolated papillary muscle
Papillary muscles were dissected free from the left ventricle under a stereomicroscope and superfused with oxygenated Tyrode’s solution at 36°C. Papillary muscles were driven at constant frequency (120 beats/min) with a pair of electrodes connected to a 302-T Anapulse Stimulator via a 305-R Stimulus Isolator (W. P. Instruments, New Haven, CT, USA) operating in constant current mode. Isometric twitches were evaluated with a Harvard transducer (60-2997) and continuously acquired and recorded with a PowerMac computer, using LabVIEW software (National Instruments Corp., Austin, TX, USA). Before each experiment, papillary muscles were equilibrated in oxygenated (100% O2) Tyrode’s solution for at least 30 min (32)
. To verify whether rCGA1–64S-S, rCGA1–64SH, or rCGA1–64OX affects mammalian basal cardiac performance, rat papillary muscles were exposed to increasing concentrations of either rCGA1–64S-S (1–100 nM), rCGA1–64SH (50–100 nM), or rCGA1–64OX (50–100 nM) to generate concentration-response curves. Each treatment lasted for 20 min, and then the perfusion was switched to Tyrode’s solution alone, to study the reversibility of the effects. To test the antiadrenergic effect of CGA-derived peptides, they were applied in the presence of 1 µM Iso. All solutions containing drugs were prepared immediately before the experiments. NG-Nitro-L-arginine methyl ester (L-NAME) (1 mM) was used to block the synthesis of NO. Wortmannin (100 nM) was used to block the activity of PI3K.
Ventricular cardiomyocytes
The hearts were explanted, washed in modified calcium-free Tyrode’s solution (for this and other solutions, see the next section), and cannulated via the aorta. All of the following operations were carried on under a laminar flow hood. The heart was perfused at a constant flow rate of 10 ml/min with calcium-free Tyrode’s solution with a peristaltic pump for 
5 min (37°C) to wash away the blood and then with 10 ml of calcium-free Tyrode’s solution supplemented with collagenase (0.3 mg/ml) and protease (0.02 mg/ml). Hearts were then perfused and enzymatically dissociated with 30 ml of calcium-free Tyrode’s solution containing 50 µM CaCl2 and the same enzymatic concentration mentioned before. Atria and ventricles were then separated, and the ventricles were cut in small pieces and shaken for 10 min in 20 ml of calcium-free Tyrode’s solution in the presence of 50 µM CaCl2, collagenase, and protease (32)
.
Measurement of calcium transients
Cardiomyocytes were loaded with indo-1 AM (2 µM; Invitrogen, Carlsbad, CA, USA) with a 40-min incubation at 37°C. Cells were then washed in control Tyrode’s solution and placed on the stage of an inverted microscope. Cells were stimulated electrically at a frequency of 2 Hz by a two platinum electrode insert (RC-37W; Warner Instruments, Hamden, CT, USA) connected to a bipolar stimulator (SIU-102, Warner Instruments). Calcium transients were evaluated as the fluorescence ratio at 400 nm/490 nm emitted by the cells exited at 350 nm. During the experiments, cardiomyocytes were maintained in standard Tyrode’s solution, and solution changes were performed with a microperfusion system. The experiments were recorded by AxoGraph software (Molecular Devices, Sunnyvale, CA, USA) on an Apple PowerMac computer and analyzed with IGOR software (Wavemetrics, Inc., Lake Oswego, OR, USA).
BAE-1 cells
The effects induced by rCGA1–64S-S on intracellular calcium concentration and NO production were studied in BAE-1 cells by confocal microscopy. BAE-1 cells (European Collection of Cell Cultures, Salisbury, Wiltshire, UK) were maintained in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich Corp., St. Louis, MO, USA) supplemented with 10% heat-inactivated fetal calf serum (lot 1SB0019; Biowhittaker, Verviers, Belgium), 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, and 0.25 mg/ml Fungizone at 37°C in a humidified atmosphere of 5% CO2 in air. Cells were used at passages 2–6.
Confocal fluorimetric measurements were performed using an Olympus FluoView 200 laser scanning confocal system (Olympus America Inc., Melville, NY, USA) mounted on an inverted IX70 Olympus microscope, equipped with an x60 oil-immersion objective (NA 1.2). Cells were seeded on glass-bottom dishes (35/22 mm 
, WillCo Wells, Amsterdam, The Netherlands) at a density of 5000 cells/cm2. For simultaneous calcium and NO measurements, cells were loaded simultaneously with fluo-3 AM (2 µM; Invitrogen) and DAR-4M AM (2 µM; Invitrogen) for 30 min at 37°C and excited at 488 and 568 nm. Emission signals were filtered with 515-nm (for fluo-3 AM) and 610-nm (for DAR-4M AM) bandpass filters. During the experiments, BAE-1 cells were maintained in standard Tyrode’s solution. Solutions were applied with a microperfusion system (pipette inner diameter 200 µm) (32)
. X-Y plane images (resolution 512x512 pixels) were acquired every 3.3 s and subsequently analyzed with ImageJ, a public domain Java image-processing software tool (ImageJ, version 1.36; W. Rasband, U.S. National Institutes of Health, Bethesda, MD, USA). Changes in intracellular calcium concentration were represented as (F–F0)/F0 to normalize the traces. As the reaction of DAR-4M AM with NO forming the corresponding fluorescent triazole compound is not reversible, changes in intracellular NO synthesis were both expressed as (F–F0)/F0 and as the first derivative of the fluorescence signal, to highlight accumulating NO synthesis over slope changes.
Solutions and drugs
rCGA1–64 was synthesized in the INSERM U575, Physiopathology of Nervous System, Strasbourg, France. Peptides were synthesized as follows. Rat CGA1–64 was synthesized on an ABI 431A peptide synthesizer (Applied Biosystems, Inc., Foster City, CA, USA) using the standard procedures of 9- fluorenylmethoxycarbonyl chemistry (33)
. Formation of the disulfide bridge between C17 and C38 was obtained in the presence of N-ethyldiisopropylamine at pH 8.5, leaving the mixture to stand under open atmosphere for 24 h until the reaction was complete, according to the Ellman test (34)
. To oxidize CGA1–64, the oxidizing agent was prepared by addition of formic acid to 30% hydrogen peroxide (v/v, 9:1) and stirring at 20°C for 45 min. The mixture was chilled at 0°C for 30 min, and the reaction was stopped by dilution with water (500 µl). Samples were then concentrated by evaporation but not to dryness. This washing step was repeated 3 times to eliminate completely performic acid.
The perfusion solution consisted of a modified nonrecirculating KHS containing 113 mM NaCl, 4.7 mM KCl, 25 mM NaHCO3, 1.2 mM MgSO4, 1.8 mM CaCl2, 1.2 KH2PO4, 11 mM glucose, 1.1 mM mannitol, and 5 mM sodium pyruvate (pH 7.4; 37°C; 95%O2:5%CO2). Calcium-free Tyrode’s solution contained 135 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM HEPES, 10 mM glucose, 10 mM butanedione monoxime, and 5 mM taurine, pH 7.40, adjusted with NaOH. Iso, ET-1, L-NMMA, PTIO, ODQ, KT5823, and wortmannin were purchased from Sigma-Aldrich Corp. All drug-containing solutions were freshly prepared before the experiments.
Statistical analysis
All values are presented as the mean ± SE. All data were subjected to ANOVA followed by the Bonferroni correction for post hoc t tests. Significance was accepted at P < 0.05. The concentration-response curves of the stimulation of LVP induced by Iso alone and by Iso plus different peptides were fitted using GraphPad Prism 4.02 (GraphPad Software Inc., San Diego, CA, USA). This provided for each curve the concentration (in –logM) of Iso alone and of Iso plus different peptides that induced a 50% effect (EC50).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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. Performance variables measured every 10 min showed that the heart is stable for up to 180 min.
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Previous work revealed that human recombinant VS-1 (STA-CGA1–76; hereafter hrVS-1) exerts a negative inotropic effect in isolated rat heart (5)
as well in rat papillary muscle (32)
. To verify whether rCGA1–64S-S, rCGA1–64SH, or rCGA1–64OX affect mammalian basal cardiac performance, rat cardiac preparations were exposed to increasing concentrations of either rCGA1–64S-S, rCGA1–64SH, or rCGA1–64OX to generate concentration-response curves. The effects of all three peptides remained stable until 15–20 min. Accordingly, cardiac parameters were measured at 10 min.
RCGA1–64S-S caused a concentration-dependent negative inotropic effect, showed by a decrement of LVP, significant starting from 33 nM. The peptide also caused a reduction of RPP and +(LVdP/dt)max, significant from 65 nM, without changing HR (Fig. 1A
). Analysis of the lusitropic changes revealed a reduction of –(LVdP/dt)max and an increase in T/–t from 65 nM. rCGA1–64S-S did not affect HTR (Fig. 1A
). Notably, the peptide induced a significant vasodilation starting from 33 nM (Fig. 1A
). rCGA1–64SH significantly decrease LVP at 65, 110, and 165 nM. No significant changes were detected in the case of the other parameters tested (Fig. 1A
). rCGA1–64OX significantly increased HR at 11 and 33 nM and +(LVdP/dt)max at 65, 110 and 165 nM, without affecting the other parameters (Fig. 1A
).
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Isolated papillary muscle
We observed that rCGA1–64S-S reduced papillary muscle contractility under both basal conditions and with β-adrenergic stimulation (1 µM Iso). Under basal conditions, low concentrations (1–5 nM) of rCGA1–64S-S had no effect on myocardial contractility (Fig. 1B
), whereas higher concentrations of rCGA1–64S-S reduced developed tension in a dose-dependent manner. In contrast, neither rCGA1–64SH nor rCGA1–64OX affected papillary muscle contractility (Fig. 1B
).
Structure-function relationship of three CGA1–64 peptides on Iso- and ET-1-induced myocardial inotropy
Langendorff perfused heart
To verify the possible antiadrenergic action of the three peptides, heart preparations were perfused with KHS containing increasing concentrations of Iso (0.1 nM-1 µM), either alone or in combination with one of the three rCGA peptides. Iso stimulation induced a significant increase of LVP from 5 nM to 1 µM (Fig. 2A
). The subsequent analysis of the percentage of variations of LVP provided EC50 values in the presence of increasing concentrations of either Iso alone or of Iso plus rCGA1–64S-S (11, 33, and 65 nM), rCGA1–64SH (65 and 165 nM), or rCGA1–64OX (65 and 165 nM). Results showed that rCGA1–64S-S exerts a noncompetitive antagonism on adrenergic stimulation, inducing a dose-dependent reduction of Iso intrinsic activity, which is abolished by a 65 nM concentration of the peptide. rCGA1–64SH and rCGA1–64OX (both at 65 nM) did not modify the intrinsic Iso activity, but they only reduced it at the highest concentration tested (165 nM) (Fig. 2A
). EC50 values (in logM) and the intrinsic activity of Iso alone and of Iso in the presence of the peptides are shown in Table 3
.
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ET-1, a potent coronary constrictor (35)
, is able to induce a positive inotropic effect in isolated perfused mammalian heart (36)
. To establish whether rCGA1–64S-S is able to counteract the ET-1-mediated effects, hearts were perfused with KHS containing ET-1 alone or in combination with rCGA1–64S-S at 65 nM. ET-1 alone induces positive inotropism on LVP and RPP and vasoconstriction. The costimulation with rCGA1–64S-S abolished the positive inotropism and significantly reduced the coronary constriction (Fig. 3A
).
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Isolated papillary muscle
As shown in Fig. 2B
, the enhanced contractile response to Iso was blunted by rCGA1–64S-S at and above 50 nM (P<0.01).
Effects of rCGA65–76 on the Langendorff perfused heart
The treatment with rCGA65–76 did not modify cardiac performance at any concentrations tested except for a small chronotropic effect at and above 11 nM. At 165 nM rCGA65–76 did not modify the intrinsic effect of Iso (results not shown).
Effects of rCGA1–64S-S in rat ventricles
To verify the involvement of cGMP and cAMP pathways in the mechanism of action of rCGA1–64S-S, we performed intracellular cGMP and cAMP measurements on extracts of rat heart. In Fig. 3B
, 165 nM rCGA1–64S-S increased cGMP while reducing the Iso-induced increase in cAMP in the heart extracts, as is to be expected for negative inotropism and counteraction of adrenergic activation in the perfused hearts, respectively.
Effects of signal pathway inhibitors on basal response to rCGA1–64S-S
Involvement of Gi/o protein and PI3 kinases activity on basal performance
Langendorff perfused heart
To verify the involvement of the Gi/o protein system, cardiac preparations were perfused with KHS containing PTx in the presence of rCGA1–64S-S. PTx abolished rCGA1–64S-S-mediated negative inotropism [i.e., LVP and +(LVdP/dt)max], –(LVdP/dt)max reduction, and vasodilation] (Fig. 4A
). To verify the involvement of PI3K activity in the mechanism of action of the peptide, hearts were perfused with rCGA1–64S-S alone and in the presence of wortmannin (37)
. Wortmannin abolished both the rCGA1–64-mediated inotropic effect and vasodilation (Fig. 4B
).
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Isolated papillary muscle
On the basis of previous experiments suggesting that the negative inotropic effect of hrVS-1 is mainly due to the activation of the PI3K-Akt-NO pathway and the consequent release of NO from endothelial cells (32)
, we studied the effect of rCGA1–64S-S in papillary muscles pretreated with either L-NAME or with wortmannin. We observed that the negative inotropic effect of rCGA1–64S-S was abrogated (Fig. 5A
) after pharmacological blockade of NO synthesis. Similarly, pretreatment of papillary muscles with wortmannin before rCgA1–64S-S at and above 50 nM did not reveal a basal negative inotropy as exerted by the peptide alone (Fig. 5A
).
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NO-cGMP-PKG signal transduction pathway
Langendorff perfused heart
The involvement of the NO-cGMP-PKG signaling in rCGA1–64S-S-dependent cardiomodulation was examined by perfusing the cardiac preparations with either PTIO, L-NMMA, ODQ, or KT5823 in the presence of the peptide. The concentration of the antagonist was selected on the basis of preliminary dose-response curves as the first dose that does not significantly affect cardiac performance. rCGA1–64S-S-induced reduction of LVP, +(LVdP/dt)max, and –(LVdP/dt)max; and CP was abolished by both removal of NO by the NO scavenger, PTIO, and NOS inhibition by L-NMMA (Fig. 5
B). It was also abrogated by pretreatment with ODQ and KT5823 (Fig. 5
B).
Calcium transients in ventricular myocytes
To exclude the presence of a direct inotropic effect of rCGA1–64S-S on cardiomyocytes, we performed calcium transient measurements on electric field-stimulated cardiomyocytes, loaded with the fluorescent calcium probe Indo-1.
As shown in Fig. 6
, rCGA1–64S-S was without effect on amplitude of calcium transients at basal conditions (Fig. 6A
, arrow 2) and the Iso-stimulated response (Fig. 6B
, arrow 3) (94.2±2.1 and 96.5±2.9% of the calcium transient amplitude in the control condition; 50 and 100 nM, n=8 and 6, respectively, P>0.05; at 100 nM: 98.1±10.7% of the Iso-stimulated calcium transient amplitude, n=5. P>0.05). Basal and Iso-stimulated transients were not affected by either rCGA1–64SH (2.2±0.8%, n=5 and 2.2±1.1%, n=5, respectively) or rCGA1–64OX (–3.1±1.9%, n=5 and 1.9±2.8%, n=3, respectively).
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NO production by BAE-1 cells
Because previous experiments on isolated papillary muscle and on isolated ventricular myocytes suggested an indirect, endothelium-derived NO-dependent effect of rCGA1–64S-S, we performed simultaneous NO and intracellular calcium measurements with fluorescent probes on BAE-1 cells. Cells were observed by confocal microscopy after loading with both fluo-3 AM and DAR-4M AM. As a positive control, we used 100 µM ATP to induce calcium-dependent NO production.
We found that, in contrast with ATP, rCGA1–64S-S enhanced NO production with a mechanism that was independent from the intracellular calcium concentration. To distinguish the increase in DAR-4M AM fluorescence intensity from the accumulating basal NO synthesis, we measured the slope changes by calculating the derivative of the fluorescence intensity. Figure 6C-H
shows representative experiments with rCGA1–64S-S on intracellular calcium and NO production in single cells from selected fields. Figure 6C, D, F, G
shows normalized fluorescence intensities of fluo-3 and DAR-4M, respectively. The peptide did not affect intracellular calcium, whereas NO production was enhanced (Fig. 6D, E
). Figure 6F, G
shows the normalized fluorescence intensity of fluo-3 and DAR-4M in BAE-1 cells pretreated with 100 nM wortmannin before addition of rCGA1–64S-S. Wortmannin abolished the peptide effect on NO production (Fig. 6G, H
). Figure 6E, H
shows the slope of the fluorescence intensity in Fig.6D, G
.
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DISCUSSION |
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. The fact that the inotropic and lusitropic effects are similar to those exerted by the hrVS-1 on the rat heart (5)
highlights the negative inotropic property of the VS domain. At the same time, the demonstration that rCGA1–64 induces a vasodilatation already significant at 33 nM suggests its involvement also in the regulation of coronary activity. This coronary reactivity agrees with the VS-induced vasodilation reported in human thoracic artery and saphenous vein and in bovine coronary arteries independent of the endothelium (38
, 39)
. However, it is in contrast with the absence of coronary activity reported for the hrVS-1 in the rat heart (5)
. Therefore, distinct species-specific vascular sensitivities against homologous vs. heterologous VS peptides may account for the observed differences in coronary responses. We have recently shown that hrVS-1 has no effect on basal papillary muscle contractility (32)
; thus, the negative inotropism exerted by rCGA1–64 suggests a more specific action of the homologous peptide on the rat heart.
Antiadrenergic and antiendothelin influences
The analysis of the interactions between rCGA1–64 and the adrenergic signaling on the Langendorff rat heart has revealed that the peptide exerts a functional noncompetitive antagonism, confirming previous results obtained with hrVS-1 (5)
. Accordingly, the antiadrenergic action of rCGA1–64 tested on the isolated papillary muscle preparation also shows that the peptide with the intact S-S bridge reduces the effect of Iso stimulation. Similar data were recently documented for hrVS-1 (32)
.
Comparison between rCGA1–64S-S and hrVS-1 revealed similar negative inotropic abilities and antiadrenergic action [EC50 of rCGA1–64S-S (65 nM) –7.8±1.15 and of hrVS-1 (65 nM) –7.8 ± 0.78). The intracellular cardiac cGMP and cAMP measurements indicated that rCGA1–64S-S under basal heart performance increases intracardiac cGMP levels on the unstimulated heart while reducing the Iso-induced increased cAMP levels, according to its anti-β-adrenergic action.
An open question concerns the possible mechanisms responsible for the rCGA1–64-induced inhibition of the Iso-mediated positive inotropy. As discussed for other VS peptides (4)
, at present it is not known whether the cardiac effects of rCGA1–64 on its cellular targets (endocardial and coronary endothelium, smooth muscle, myocardiocytes, intracardiac nerve terminals, fibroblasts, and others) occur via a classic receptor-ligand interaction or, alternatively, through hydrophobic interactions between specific peptide domains, e.g., CGA1–40 and CGA47–66, and spatially localized regions of the lipid bilayer with consequent modulation of cellular effectors (40)
. In line with this hypothesis, at least several possible mechanisms underlying the inhibition of the Iso-mediated positive inotropy stimulation may be suggested, with one being via an allosteric modulation of the β-adrenergic receptor independent from the ligand binding site and the other being via modulation of the downstream intracellular signaling triggered by activation of the β-adrenoreceptor itself. Moreover, the observed cAMP reduction might be the result of a direct activation of the PTX-sensitive Gi/o in a receptor-independent manner under basal conditions, independent from a direct, although noncompetitive, action via the adrenergic and ET-1 receptors.
An important feature of the rCGA1–64S-S cardioactive profile is its ability to counteract ET-1-induced positive inotropic and coronary constrictor effects. ET-1 exerts diverse and important cardiovascular actions, including autocrine/paracrine myocardial contractile and powerful vasoconstrictor coronary effects, which are also of physiopathological concern in relation to the stressed and ischemic heart (36)
. In particular, the demonstration that rCGA1–64S-S acts as a potent vasodilator on ET-1-preconstricted coronary arteries, which illustrates for the first time on an intact coronary bed the peptide vasoreactivity responsible for the name vasostatin (4)
, may represent another potentially beneficial attribute of this CGA-derived peptide. Therefore, in addition to the antiadrenergic action, this anti-ET-1 influence of rCGA1–64 strongly supports its role as a cardiac counter-regulatory modulator in "zero steady-state error" homeostasis postulated for other CGA-derived peptides (41)
. This novel function of rCGA1–64 as a cardiostatin may be of notable relevance in the natural defense against exaggerated cardiac hyperactivity, such as the "neuroendocrine storm" responsible for neuroendocrine cardiomyopathy and myocardial necrosis (42)
.
Structure-function relationship
Analyses using loop-modified peptides have revealed an order of potency for the cardiotropic and antiadrenergic actions: rCGA1–64S-S > rCGA1–64SH > rCGA1–64OX, confirming the significance of the intact disulfide bridge loop. In fact, a previous study on the isolated frog heart using several VS-derived peptides, e.g., CGA1–40 and CGA7–57, has shown the crucial role of the disulfide bridge for the cardiotropic effect (43)
. Notably, the residues 64–65 correspond to a highly conserved, potential cleavage site in a flexible region of VS-1 (23)
. On the other hand, the synthetic rCGA65–76 failed to modify basal and adrenergic inotropism, further supporting our conclusion that rCGA1–64S-S is sufficient for the cardiotropic effects of VS-1. The eventual biological function of the rat CGA 65–76 fragment remains to be established.
Signaling pathways for rCGA1–64S-S
Preliminary analysis of the mechanism of action of rCGA1–64 showed an involvement of the Gi/o protein-PI3K-NO signal transduction pathway. In fact, in the isolated heart, rCGA1–64S-S-induced negative inotropism was mediated by the NO-cGMP-PKG pathway. The involvement of NO is further corroborated by the findings obtained on the papillary muscle, indicating that the action of rCGA1–64S-S depends on a PI3K-dependent NO release by endothelial cells. In agreement with our previous data obtained using hrVS-1 (32)
, the present study shows that the effect of rCGA1–64 is not due to a direct action on cardiomyocytes but is mediated by calcium-independent/PI3K-dependent NO release by endothelial cells. Indeed, rCGA1–64, although it had no effect on the intracellular calcium concentration in isolated ventricular cells, promoted the release of NO from BAE-1 cells by means of a calcium-independent mechanism. Further study is necessary for clarifying the eventual involvement of the endocardial endothelium in this mechanism.
NOS-NO-cGMP-PKG intracardiac signaling is known to be involved in the control of the contractile myocardial performance. For example, in rat ventricular myocytes, NOS-produced NO through a soluble GC-PKG mechanism induces a decrease of L-type Ca2+ current (44)
and troponin I phosphorylation, thus negatively affecting contractility (45)
. Indeed, in their cardiac preconditioning study on the Langendorff rat heart, Cappello et al. (15)
proposed that both L-type Ca2+ current reduction and PKG-mediated myofilament desensitization to Ca2+ may account for hrVS-1-induced negative inotropy and ischemic cardioprotection. Of note, the finding that rCGA1–64S-S-induced coronary dilation also was abolished by inhibitors of the NO-cGMP-PKG pathway is consistent with the possible endothelial release of vasodilator autacoids, e.g., NO (46)
.
Calcium-independent activation of endothelial NOS (eNOS) was already reported after stimulation of endothelium with insulin, insulin-like growth factor-1, and estrogens (47)
. As proposed by Hartell et al. (48)
and Shaul (49)
, such a mechanism might involve Akt-dependent NOS phosphorylation. In this perspective, our experiments performed on papillary muscles and on BAE-1 cells treated with wortmannin strongly suggest that the rCGA1–64-induced NO synthesis in both the endocardium-containing rat papillary muscle and the bovine aorta endothelial cells depends on PI3K activation. Interestingly, a recent report proposes a novel calcium-independent mechanism of eNOS activation involving caveolae-mediated endocytosis induced by the albumin-binding protein gp60 and activation of downstream Src, Akt, and PI3K pathways in endothelial cells (50)
. Because it has been proposed that VSs may interact with the caveolar domain (refs. 4
, 51
and references therein) and that endothelial cells internalize CGA1–78 (52)
, we suggest that a similar mechanism may explain the rCGA1–64S-S-dependent NOS activation in the BAE-1 cells.
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and our previous evidence that intact CGA in the rat heart is processed to smaller N-terminal peptides (19)
, this study makes it evident that rCGA1–64S-S contains the potent cardiovascular principle of hrVS-1 and CGA7–57 previously demonstrated in eel, frog, and rat hearts (4)
, involving PI3K-dependent NO release from endocardial cells. It is hoped that increased knowledge on the physiopathological significance of locally produced and/or circulating CGA-derived peptides, particularly in reference to the myocardium and the coronary protection against prolonged and excessive stress (adrenergic and ET stimuli), may pave the way for medically oriented cardiovascular research.
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ACKNOWLEDGMENTS |
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Received for publication March 26, 2008. Accepted for publication July 10, 2008.
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in heart failure. Adv. Exp. Med. Biol. 482,351-359[Medline]1E voltage-gated Ca2+ channel in chromogranin-positive muscle cells of rat heart and in distal tubules of human kidney. J. Histochem. Cytochem. 48,807-819-induced vascular leakage. FASEB J. 18,554-556This article has been cited by other articles:
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  K. B. Helle The chromogranin A-derived peptides vasostatin-I and catestatin as regulatory peptides for cardiovascular functions Cardiovasc Res, January 1, 2010; 85(1): 9 - 16. [Abstract] [Full Text] [PDF]
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R. Granata, L. Trovato, M. P. Gallo, S. Destefanis, F. Settanni, F. Scarlatti, A. Brero, R. Ramella, M. Volante, J. Isgaard, et al. Growth hormone-releasing hormone promotes survival of cardiac myocytes in vitro and protects against ischaemia-reperfusion injury in rat heart Cardiovasc Res, July 15, 2009; 83(2): 303 - 312. [Abstract] [Full Text] [PDF]
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, control, no peptide; 
, cGMP (+rCGA1–64S-S); 
