Catestatin (chromogranin A_344-364) is a novel cardiosuppressive agent: inhibition of isoproterenol and endothelin signaling in the frog heart

Abstract

The catecholamine release-inhibitory catestatin [Cts; human chromogranin (Cg) A_352-372, bovine CgA_344-364] is a vasoreactive and anti-hypertensive peptide derived from CgA. Using the isolated avascular frog heart as a bioassay, in which the interactions between the endocardial endothelium and the subjacent myocardium can be studied without the confounding effects of the vascular endothelium, we tested the direct cardiotropic effects of bovine Cts and its interaction with β-adrenergic (isoproterenol, ISO) and endothelin-1 (ET-1) signaling. Cts dose-dependently decreased stroke volume and stroke work, with a threshold concentration of 11 nM, approaching the in vivo level of the peptide. Cts reduced contractility by inhibiting phosphorylation of phospholamban (PLN). Furthermore, the Cts effect was abolished by pretreatment with either nitric oxide synthase (N^G-monomethyl-l-arginine) or guanylate cyclase (ODQ) inhibitors, or an ET_B receptor (ET_BR) antagonist (BQ-788). Cts also noncompetitively inhibited the positive inotropic action of ISO. In addition, Cts inhibited the positive inotropic effect of ET-1, mediated by ET_A receptors, and did not alter the negative inotropic ET-1 influence mediated by ET_BR. Cts action through ET_BR was further suggested when, in the presence of BQ-788, Cts failed to inhibit the positive inotropism of both ISO and ET-1 stimulation and PLN phosphorylation. We concluded that the cardiotropic actions of Cts, including the β-adrenergic and ET-1 antagonistic effects, support a novel role of this peptide as an autocrine-paracrine modulator of cardiac function, particularly when the stressed heart becomes a preferential target of both adrenergic and ET-1 stimuli.

chromogranin a (CgA), an acidic secretory proprotein, is the chromogranin/secretogranin protein family's index member (24, 53, 57). CgA proteolytic processing gives rise to several peptides of biological importance, including such counterregulatory agents as the dysglycemic hormone pancreastatin (52), vasodilator vasostatin 1 (VS-1) (2), and catecholamine release-inhibitory peptide catestatin (Cts; human CgA_352-372, bovine CgA_344-364) (35–40). Plasma CgA levels and epinephrine levels are increased in patients with essential hypertension or heart failure (15), whereas Cts plasma levels are significantly lower (44), suggesting that Cts has an inhibitory effect on chromaffin cells in vivo. Consistent with human studies, genetic ablation of the CgA gene (Chga) results in high blood pressure in mice (34), and the high blood pressure in Chga−/− mice can be rescued either by pretreatment with Cts or introduction of the human CgA gene (CHGA+/+) in the mice Chga−/− background by bacterial artificial chromosome (BAC) transgenic technology. Furthermore, low Cts levels predict augmented adrenergic responses to stressors (44), suggesting that a reduction in Cts may increase the risk of hypertension.

It has recently been reported that human recombinant CgA (10–100 nmol/l) stimulates the release of endothelin (ET; by ∼4-fold) from human vein endothelial cells, suggesting interaction between the sympathochromaffin system and the endothelium (29).

We have shown previously that the NH₂-terminal CgA fragment VS-1 exerts profound myocardiosuppressive and antiadrenergic action in frog, eel, and rat hearts (54, 17, 25, 16). Unlike in eel (25) and rat (20), in frog, the VS-1-induced negative inotropism appears independent from the endocardial endothelium (EE) and the nitric oxide (NO)-cGMP pathway, indicating species- and tissue-specific targets and signaling mechanisms (17). Of note, in the eel and frog hearts, the VS-1-induced negative inotropism is abolished by Ca²⁺ and K⁺ channel antagonists and inhibitors of cytoskeletal reorganization (42). Similarly, involvements of either Ca²⁺ and K⁺ channels or cytoskeleton rearrangement appear also involved in VS-induced changes detected in vascular preparations (1, 8) or pulmonary and coronary arterial endothelial cells (5), respectively. These findings imply that the highly conserved NH₂-terminal VS-1 domain of CgA modulates cardiovascular functions at least in three classes of vertebrates. Interestingly, another remarkable vasoreactive (26) and antihypertensive (34) peptide, Cts, is present in the COOH-terminal domain of CgA, but nothing is known regarding the direct cardiac actions of Cts. We hypothesized that this fragment could also act as a cardioactive agent modulating heart performance both under basal and stimulated conditions. If our hypothesis proves to be correct, then CgA would further epitomize that the processing of more than one peptide from a single prohormone rather than from two precursors may be homeostatically advantageous provided both peptides regulate the same physiological response.

Therefore, in the present study, we investigated Cts' direct cardiotropic action and the possible EE-NO-cGMP pathway involvement, as well as Cts' counteracting actions against both β-adrenergic and ET-1-elicited stimulations. We used the isolated working frog heart, which allowed a hemodynamic evaluation independent from coronary reactivity and extrinsic neuronal and endocrine influences. In fact, in the hearts of homeotherms (e.g., mammals), the coronary vascular endothelium and EE act in concert to modulate humorally myocardial activity, making EE contribution in the paracrine regulation of myocardial function difficult to define (10). In contrast, in the avascular frog heart, EE is the only barrier between the superfusing blood and the subjacent myocardial microenvironment and is therefore a unique model to analyze its autocrine/paracrine role in the transduction of blood-borne endoluminal chemical stimuli that can target the myocardium (21).

On the whole, the data suggest a new role of Cts in cardiac physiology, particularly under conditions of heightened neuroendocrine activation and consequent myocardial damage.

MATERIALS AND METHODS

Isolated and Perfused Working Heart Preparation

Frog hearts were isolated from both male and female specimens of Rana esculenta (weighing 16 ± 0.8 g) and connected to a perfusion apparatus as previously described (51). Animal maintenance and experimental procedures approved by the Italian University Minister were in accordance with the Guide for the Use and Care of Laboratory Animals (European Communities Council Directive 1986). Efforts were made to minimize animal suffering and reduce the number of species used. Experiments were performed at room temperature (18–20°C). A Grass S44 stimulator was used to electrically stimulate and pace heart preparations with single pulses of 20 V for 0.1 s. The stimulation rate was identical to that of the control, unpaced rate. The hearts were perfused with saline that was equilibrated with air and was composed (in mM) of 115 NaCl, 2.5 KCl, 1.0 CaCl₂, 2.15 Na₂HPO₄, 0.85 NaH₂PO₄, and 5.6 glucose. The buffer pH was adjusted to 7.30–7.35 by the addition of Na₂HPO₄.

Measurements and Calculations

Pressures were measured through two MP-20D pressure transducers (Micron Instruments, Simi Valley, CA) that were connected to a PowerLab data acquisition system and analyzed using Chart software (ADInstruments, Ugo Basile, Comerio, Italy). Pressures were expressed in millimeters mercury and corrected for cannula resistance. The afterload (mean aortic pressure) was calculated as 2/3 diastolic pressure +1/3 maximum pressure. Cardiac output (CO) was collected over 1 min and weighed. The CO was corrected for fluid density and expressed as volume measurements (ml/min) that were normalized to the wet body weight in kilograms. Stroke volume [SV, CO/heart rate (HR)], at constant pre- and afterload in paced hearts, was used as a measure of ventricular performance (i.e., as an index of inotropism). Ventricular stroke work (SW), an index of systolic functionality, was calculated (in mJ/g) as (afterload-preload, mmHg) × SV (ml)/ventricle weight (g).

Drugs and Chemicals

Bovine Cts (bCgA_344-364) peptide was synthesized by the solid-phase method, using 9H-(f)louren-9-yl(m)eth(o)xy(c)arbonyl protection chemistry, as previously described (37). Peptides were purified to >95% homogeneity by preparative reverse-phase HPLC (RP-HPLC) on C-18 silica columns. Authenticity and purity of peptides were further verified by analytical chromatography (RP-HPLC), and electrospray-ionization or matrix-assited laser desorption/ionization mass spectrometry. Isoproterenol (ISO), ET-1 (human porcine), the ET_AR inhibitor BQ-123 sodium salt [cyclo(d-Asp–Pro-d-Val-Leu-d-Trp)], the ET_BR inhibitor BQ-788 sodium salt (N-cis-2,6-dimethylpiperidinocarbonyl-l-gamma-methylleucyl-d-I-methoxycorbonyltryptophanyl- d-norleucine, Na), the nonspecific nitric oxide synthase (NOS) inhibitor N^G-monomethyl-l-arginine (l-NMMA), the guanylate cyclase inhibitor ODQ [1H-(1,2,4) oxadiazolo-(4,3-a)quinoxalin-1-one], and Triton X-100 (t-octylphenoxypolyethoxyethanol) were purchased from Calbiochem. ISO, BQ-123, l-NMMA and Triton X-100 were prepared as stock solutions in double-distilled water; ET-1 was prepared in acetic acid; BQ-788 was prepared in methanol; ODQ was prepared in dimethyl sulfoxide and, being light sensible, used in a darkened perfusion apparatus to prevent degradation. Dilutions were made in Ringer solution immediately before use.

Experimental Protocols

Basal conditions.

In all experiments, the minimal output pressure (diastolic afterload) was set at 29.5 mmHg, and the input pressure (preload) was regulated to obtain a CO of ∼110 ml·min⁻¹·kg⁻¹ (wet body wt) and are within the physiological range (51). The heart generated its own rhythm. CO, HR, and pressures were measured simultaneously during the experiments. Hearts that did not stabilize within 10–15 min from the onset of perfusion were discarded. These parameters are stable for >1 h (21). All of the experiments were carried out within this period. To analyze the inotropic effects without confounding chronotropic action of substances, the preparations were electrically paced.

Drug application.

After the 15-min control period, the hearts were perfused for 10–15 min with Ringer solution enriched with increasing concentrations of either Cts (11–110 nM), ISO (0.1–1,000 nM), or ET-1 (0.01–10 nM) to generate concentration-response curves. Repetitive exposure of each heart to a single drug concentration revealed the absence of desensitization and the recovery from drug application after washing with Ringer (data not shown). Thus concentration-response curves were generated by perfusing cardiac preparations with increasing concentrations of the drug. The effects of ET-1 (0.01–10 nM) were also analyzed, exposing each heart preparation to a single concentration of the drug.

Cardiac signaling.

Hearts were perfused with and without 110 nM Cts as before. At the end of the experiment, hearts were snap-frozen under liquid nitrogen and homogenized with 1 ml of ice-cold 0.2 M sucrose, Tris maleate (pH 7.0) buffer supplemented with 2 mM EDTA, pH 8.0, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 0.1 mM 3-isobutyl-1-methylxanthine. Sarcoplasmic reticulum (SR) membrane fractions were isolated from cytosolic proteins using methods described previously (50, 7). Protein content was determined by Bradford assay (Bio-Rad), and 100 μg of cytosolic protein were subjected to SDS-PAGE immunoblot analysis for phosphorylated (P)-extracellular signal-regulated kinase (ERK; Santa Cruz) and total ERK (Santa Cruz). For assessment of total phospholamban and phosphorylated PLN (P-PLN-Ser¹⁶) levels, 20 μg of SR membranes were subjected to electrophoresis, and the immunoblots were probed with anti-mouse PLN antibody (Affinity Bioreagents) and anti-P-PLN-Ser¹⁶.

Cts' effects on ISO stimulation.

To analyze the effects of Cts on the ISO response, Cts-stimulated hearts were perfused with Ringer containing a single concentration of Cts (33, 65, or 110 nM) plus ISO (10 nM) in the presence or absence of BQ-788 (1 μM), an antagonist of ET_BR. To further investigate the antagonistic action of Cts against the positive inotropic action of ISO, the concentration-response curve of ISO (from 0.1 to 1,000 nM) was performed in the presence of single concentrations of Cts (33, 65, or 110 nM). The time exposure to each drug concentration was 10–15 min.

ET-1- or Cts-stimulated preparations.

To analyze the involvement of the EE and NOS-cGMP pathway in mediation of the effects of Cts and ET-1, hearts were perfused with ET-1 or Cts after pretreatment with Triton X-100 (0.05%), l-NMMA (NOS inhibitor, 10 μM), and ODQ (guanylate cyclase inhibitor, 10 μM).

For functional impairment of the EE, 0.1 ml of Triton X-100 (0.05%) was injected in the heart through a needle inserted in the output cannula in the ventricle, avoiding damage of the atrium (for protocol details see Ref. 21). Subsequently, variables of cardiac performance were measured after 20 min of perfusion with the saline.

To investigate the role of the ET_AR and ET_BR subtypes in mediation of the effects of ET-1 or Cts, cardiac preparations were pretreated with either 1 μM of BQ-123, an antagonist of the ET_AR, or 1 μM of BQ-788, an antagonist of ET_BR. Subsequently, the hearts were perfused with Ringer solution containing increasing concentrations of ET-1 (from 0.01 to 10 nM) or Cts (from 11 to 110 nM), with and without supplementation of BQ-123 or BQ-788. The time exposure to each drug concentration was 10–15 min.

The effects of Cts on ET-1-mediated cardiac effects.

To analyze the effects of Cts on the ET-1 response, Cts-stimulated hearts (110 nM) were perfused with increasing concentrations of ET-1 (from 0.01 to 1 nM) both in presence or absence of BQ-788. The time exposure to each drug concentration was 10–15 min.

Statistics.

All data are expressed as means ± SE. Curve fitting was accomplished in the program Kaleidagraph (Synergy Software, Reading, PA). The EC₅₀ values of a peptide were interpolated as the concentration that achieved 50% stimulation. For analysis of phosphorylated proteins, levels were quantified using Bio-Rad QuantifyOne, and the volumes of phosphorylated proteins were divided by the levels of nonphosphorylated proteins to calculate the degree of activation. Stimulation of protein activity was expressed as fold increase over the vehicle-treated control. Multiple comparisons between groups were made using either one-way ANOVA followed by Bonferroni's post hoc test or two-way ANOVA. Statistical significance was concluded at P < 0.05. Statistics were computed with the program InStat (GraphPad Software, San Diego, CA).

RESULTS

Basal Conditions

As previously described (21, 51), the in vitro isolated, perfused whole frog heart preparation generates cardiac hemodynamic responses that mimic those obtained in vivo. After 20 min of equilibration, the following basal recordings were obtained: SV = 1.9 ± 0.1 ml/kg, SW = 3.5 ± 0.2 mJ/g, HR = 59.6 ± 1.8 beat/min, preload = 1.6 ± 0.1 mmHg, afterload = 27.0 ± 0.3 mmHg, and CO = 112.5 ± 2.3 ml·min⁻¹·kg⁻¹.

Influence of Cts on Myocardial Performance

Cts concentrations ranging from 11 to 165 nM caused a concentration-dependent decrease in both SV and SW that reached a maximum reduction of ∼20% at 165 nM of Cts (Fig. 1A). A significant 7.3% decrease of SV was induced by 33 nM of Cts (Fig. 1A).

Fig. 1.

A: concentration-dependent response. Effects of Cts (11–165 nM) on stroke volume (SV) and stroke work (SW) in paced frog heart preparations. B and C: immunoblot analysis. Total and phosphorylated phospholamban (PLN; B) and extracellular signal-regulated kinase (ERK) 1/2 (C) in control and catestatin (Cts)-treated hearts both in presence and absence of BQ-123 and BQ-788. α, Comparison of control vs. treated groups; β, comparison of Cts alone vs. Cts plus inhibitors.

Signaling Mechanisms by Which Cts Modulates Cardiac Function

It is known that both PLN and ERK1/2 are involved in the coordination of cardiac contraction and relaxation and that they are expressed in frog heart (56, 19, 3, 4). Therefore, we checked whether Cts modulates PLN and ERK1/2. Cts inhibited phosphorylation of PLN at the protein kinase A specific site, Ser¹⁶ (by ∼50%; Fig. 1B), and augmented phosphorylation of ERK1/2 (by ∼53%; Fig. 1C). These effects were modified by BQ-788 (ET_BR antagonist) but not by BQ-123 (ET_AR antagonist), suggesting an involvement of ET_BR in Cts signaling to cardiac function (Fig. 1, B and C). The involvement of the NO-cGMP pathway in the Cts-mediated depression of SV has been demonstrated. In fact, we found that the negative inotropic effects of Cts (110 nM) were abolished by pretreatment with either NOS (l-NMMA; 10 μM) or guanylate cyclase (ODQ; 10 μM) inhibitors (Fig. 2A). In addition, the EE integrity is necessary for the cardioinhibitory action of Cts because functional damage of the EE with Triton X-100 (0.05%) abrogated this effect (Fig. 2A). We have previously shown that l-NMMA, ODQ, and Triton X-100 alone cause significant increments in SV and SW (21, 51).

Fig. 2.

A: nitric oxide (NO)-cGMP pathway. The effects of 110 nM Cts on SV before and after treatment with either the nitric oxide synthase (NOS) inhibitor [N^G-monomethyl-l-arginine (l-NMMA), 10 μM], guanylate cyclase inhibitor (ODQ, 10 μM), or Triton X-100 (0.05%). B: involvement of endothelin A (ET_AR) and B (ET_BR) receptor. Concentration-dependent effects of Cts (11–110 nM) after treatment with the ET_AR (BQ-123, 1 μM) or ET_BR antagonists (BQ-788; 1 μM) on SV in paced frog heart preparations.

The role of the ET_R subtypes in the Cts response (11, 33, 65, and 110 nM) was also analyzed. Although the pretreatment with ET_AR inhibitor (BQ-123; 1 μM) failed to modify the negative inotropism of Cts at all concentration tested, BQ-788 (1 μM), the ET_BR antagonist, completely blocked this response (Fig. 2B). At the concentration tested, ET receptor antagonists alone did not induce significant changes on SV (1.08 ± 0.66 for BQ-788 and −2.37 ± 1.15 for BQ-123).

Cts Modulation of ISO-Induced Cardiac Changes

Cardiac frog preparations exposed to ISO (10 nM) confirmed the classical positive inotropic response (21, 49) as reflected by significant increments in SV (by 10.3%; Fig. 3). Although the low dose of Cts (33 nM) failed to inhibit the ISO-induced increase in SV, Cts at higher concentrations of 65 and 110 nM completely abolished the ISO-mediated increase of SV (Fig. 3). Cts failed to inhibit the ISO-induced response in the presence of ET_BR antagonist (Fig. 3).

Fig. 3.

Effects of isoproterenol (ISO, 10 nM) on SV before and after treatment with Cts (33, 65, and 110 nM) or Cts plus BQ-788 (1 μM) in paced frog heart preparations.

The antagonistic effect of Cts (33, 65, and 110 nM) toward the ISO (from 0.1 to 1,000 nM)-mediated increase in cardiac function was investigated. ISO alone caused a concentration-dependent increment in SV (Fig. 4). The analysis of the percentage of variations of SV permitted deduction of EC₅₀ values with ISO alone or with ISO plus a single concentration of Cts (33, 65, or 110 nM). The EC₅₀ values (in log M) were as follows: ISO alone 7.71 ± 0.15 (r² = 0.98), ISO plus 33, 65, and 110 nM Cts −7.98 ± 0.04 (r² = 0.99), −6.97 ± 0.04 (r² = 0.99) and −7.3 ± 0.07 (r² = 0.99), respectively. Two-way ANOVA revealed significant interactions between ISO and Cts peptides [ISO vs. ISO + Cts (65 nM): P < 0.02; ISO vs. ISO + Cts (110 nM): P < 0.016]. Because increasing concentrations of ISO failed to overcome the antagonistic effects of Cts, these effects are considered as a noncompetitive antagonism (Fig. 4).

Fig. 4.

Concentration-response curves of ISO alone (from 10⁻¹⁰ to 10⁻⁶ M) and ISO + a single concentration of Cts (33, 65, or 110 nM) on SV in paced frog heart preparations. The EC₅₀ values were as follows (in log M): ISO alone, −7.71 ± 0.15 (r² = 0.98); ISO + Cts (33, 65, or 110 nM), −7.98 ± 0.04 (r² = 0.99), −6.97 ± 0.04 (r² = 0.99), and −7.3 ± 0.07 (r² = 0.99), respectively.

Concentration-Dependent Effects of ET-1 in the Frog Heart

Exposure of the isolated heart to ET-1 alone caused a dose-dependent biphasic effect on cardiac function. ET-1 exerted both negative and positive inotropic effects as documented by changes in SV (Fig. 5). Although the low dose of ET-1 (0.01 nM) always caused a negative inotropic effect (∼8%), the higher concentrations of ET-1 (0.1–10 nM) triggered either the negative (50% preparations; by −7.9 to −11%) or positive (50% preparations; by ∼7 to ∼10%) inotropic effects. Pretreatment with l-NMMA (10 μM) or ODQ (10 μM) completely abolished the ET-1 (0.01 nM)-induced negative inotropic effects (Fig. 6A). Negative inotropic effects of a low dose of ET-1 (0.01 nM) were also abolished by the damage of the EE with Triton X-100 (0.05%) (Fig. 6A). In contrast, the positive inotropic effects caused by higher doses of ET-1 (1 nM) remained unaffected by pretreatment with l-NMMA (Fig. 6B). Hence, we did not pursue the effects of ODQ and Triton X-100 on the positive inotropic effects of ET-1.

Fig. 5.

Negative and positive effects of ET-1 (0.01–10 nM) on SV in paced frog heart preparations.

Fig. 6.

A: effects of ET-1 (0.01 nM) on SV before and after treatment with either the NOS inhibitor (l-NMMA, 10 μM), guanylate cyclase inhibitor (ODQ, 10 μM), or Triton X-100 (0.05%). B: effects of ET-1 (1 nM) on SV before and after treatment with l-NMMA (10 μM).

Chemical blockade of the ET_AR by BQ-123 caused complete inhibition of the positive inotropic effects of ET-1 (0.1–10 nM) (Fig. 7A). After chemical inhibition of ET_AR, ET-1 exerted only the negative inotropic effects through the unopposed ET_BR (Fig. 7A). In contrast, the chemical inhibition of ET_BR by BQ-788 inhibited the negative inotropic effects of ET-1 at all doses tested. In the absence of ET_BR, ET-1 induced only the positive inotropic effects (0.1–10 nM) through the unopposed ET_AR (Fig. 7B).

Fig. 7.

A: concentration-dependent effects of ET-1 (0.01–10 nM) on SV in paced frog heart preparations after treatment with ET_AR antagonist (BQ-123, 1 μM). α, Comparison of ET-1 negative vs. ET-1 + BQ-123; β, comparison of ET-1 positive vs. ET-1 + BQ-123. B: ET_BR antagonist (BQ-788, 1 μM). α, Comparison of ET-1 negative vs. ET-1 + BQ-788. C: Cts (110 nM) + BQ-788. α, Comparison of ET-1 negative vs. ET-1 + Cts; β, comparison of ET-1 + Cts vs. ET-1 + Cts + BQ-788.

Cts-ET-1 Interaction

Cts (110 nM) completely abolished the positive inotropic effects of ET-1 on SV (0.1–1 nM) but did not modulate ET-1's negative inotropic effects on SV, indicating that ET-1 and Cts act through a convergent signaling pathway to exert the latter effect (Fig. 7C). Interestingly, Cts failed to abolish ET-1's positive inotropic effects in the presence of the ET_BR antagonist BQ-788 (Fig. 7C). These findings point out that Cts activates the ET_BR to antagonize the positive inotropic effects of ET-1 on SV (Fig. 7C).

DISCUSSION

Cts and Basal Mechanical Performance

It is well established that Cts acts as an endogenous noncompetitive antagonist of nicotine-evoked catecholamine secretion from rat PC-12 or bovine chromaffin cells (36–39). Cts was also shown to act as a potent in vivo vasodilator by stimulating histamine release through H₁ receptor activation (26). Furthermore, clinical and human studies indicated that Cts may function as an antihypertensive agent (44), whereas, on the contrary, nothing is known about its direct effects on myocardial physiology.

We used the avascular frog heart, which is exclusively supplied from the luminal (lacunae) blood, to investigate Cts' direct myocardiotropic action in the absence of coronary reactivity. At the same time, this natural heart model is ideally suited for analyzing the distinct paracrine role of the EE in the signal transduction of blood-borne chemical stimuli (exogenous Cts) targeting the heart. The isolated and perfused heart, working at physiological loads, showed a cardiac performance stable for >1 h, as indicated by typical time course experiments (21). With this preparation, we demonstrated that Cts dose-dependently modulates the basal mechanical performance of the isolated working frog heart, acting as a direct cardiodepressing peptide. Because preliminary experiments revealed the absence of desensitization, concentration-response curves were generated.

We recently demonstrated that peptides derived from the NH₂ terminus of CgA (VS-1-related fragments) exert notable cardiosuppressive action on the isolated rat, frog, and eel hearts (17, 25, 16). It is interesting to note that amphipathic VS-1 and amphiphilic Cts peptides, representing the NH₂- and COOH-terminal domains of CgA, respectively, exhibit analogous negative contractile responses at similar concentrations, reaching a maximum (by ∼20%) reduction of SV and SW. Despite the analogous cardiosuppressive effects of both VS-1 and Cts, only Cts was able to rescue Chga null mice from high blood pressure (34). Thus these results further document that CgA, as being the precursor of cardioactive peptides, modulates the mechanical performance of the heart under nonstimulated conditions. In the present study, Cts causes marked reduction in SV with an EC₅₀ ∼50 nM. In normotensive individuals, circulating Cts (∼1.5–2 nmol/l) is inversely proportional to intact CgA (∼5–10 nmol/l) (44). Because CgA and CgA-derived fragments are expressed in rat heart tissue extracts (23) and in human ventricular myocardium (46), the local concentration of Cts in the myocardium may reach 50 nM and regulate cardiac function in an autocrine/paracrine manner.

Signaling Mechanisms of Cts Modulation

Intracellular Ca²⁺ handling is the central coordinator of cardiac contraction and relaxation. PLN is a reversible inhibitor of sarco(endo)plasmic reticulum (SERCA) 2's Ca²⁺ affinity and cardiac contractility. Studies in genetically altered mouse models have demonstrated that the levels of phosphorylated PLN are critical in modulating basal Ca²⁺ handling and contractility. The net result is increased SR Ca²⁺ release via ryanodine receptor 2 and enhanced SR Ca²⁺ uptake by the SR Ca²⁺ pump (SERCA2a), resulting in larger intracellular Ca²⁺ transients (22). Furthermore, phosphorylation of ERKs is implicated in mediating the cardiac contractile response (12). In the isolated perfused frog heart, ERKs are reported to be activated in response to pressure overload (3) or in response to α₁- and β-adrenoceptor stimulation (4). Therefore, the interaction between Cts and PLN and/or ERK1/2 may be of potential interest in relation to further work on Cts-induced changes of cardiac contractility.

Here we demonstrate that Cts directly inhibits PLN-Ser¹⁶ and augments P-ERK1/2 levels. It may be postulated that the Cts-mediated increment in P-ERK1/2 levels led to the stimulation of endothelial NOS (eNOS) with consequent production of NO (Fig. 8). In fact, a recent work (45) suggests that ERK1/2 directly activates the NO signal transduction pathway. Another study (58) suggested an interaction between ET_BR and ERK at the level of the caveolae. At present, the receptor that mediates the effects of Cts on the heart is unknown. However, we postulate that Cts mediates its effects through a G protein-coupled ET_BR (see Fig. 8 for putative mechanisms of action). This hypothesis is supported by our results in which the inhibitory and stimulatory effects of Cts on PLN and ERK1/2, respectively, appear mediated by ET_BR, but not by ET_AR, receptors.

Fig. 8.

Schematic diagram showing the putative ET-1, ISO, or Cts signaling in endothelial and myocardial cells. AC, adenylate cyclase; β-AR, β-adrenergic receptor; β-ARK, β-adrenergic receptor kinase; DHPR, dihydropyridine receptor; eNOS, endothelial NOS; IP₃, inositol trisphosphate; NCX, Na⁺/Ca²⁺ exchangers; PKA, protein kinase A; PI 3-K, phosphoinositide 3-kinase; PLC, phospholipase C; PMCA, plasma membrane Ca²⁺-ATPases; RyR, ryanodine receptor; SERCA, sarco(endo)plasmic reticulum Ca²⁺-ATPases; SR, sarcoplasmic reticulum; +, stimulation; −, inhibition; ±, no effect.

In the frog heart, we have previously demonstrated the presence of a constitutive NOS (eNOS) located within EE, the only barrier between the cardiac lumen and the subjacent myocardium (51). The EE is able to modulate ventricular performance under both basal (i.e., nonstimulated) (51) and chemically (i.e., cholinergic)-stimulated conditions (21) through a NO-cGMP signaling pathway. In the present study, we found that Cts-mediated negative inotropism is abolished by pretreatment with NOS or guanylate cyclase inhibitors, consistent with Cts-induced stimulation of the NO-cGMP pathway. In addition, the results obtained with Triton X-100, which made EE dysfunctional (10, 21), implicated an EE-mediated mechanism in Cts signaling transduction. Notably, this contrasts with the VS-1-induced negative inotropism reported in the frog heart, which, unlike the pattern demonstrated in eel and rat hearts (25, 13), involved neither the EE nor the G protein nor the NO-cGMP-protein kinase G mechanism (17). Therefore, the signal transduction mechanisms underlying the cardiosuppressive action of the two CgA-derived peptides are not identical. As discussed later, the question remains open as to how and why an amphipathic peptide VS-1 and an amphiphilic peptide Cts, representing NH₂- and COOH-terminal domains of CgA, respectively, utilize divergent signaling pathways to converge on their effects on cardiosuppression. The analogous effects of VS-1 and Cts on cardiosuppression raise the intriguing question regarding the biological significance of such apparently redundant molecular strategy. Further studies will clarify whether, behind this similarity, the two peptides may subserve subtly different functions, e.g., summation and synergism, or distinct spatiotemporal compartmentation (cell- and tissue-specific proteolytic processing and release).

Cts and ISO Signaling

Loewi (32) initially described adrenergic regulation of cardiac contractility in the denervated frog heart using Accellerans-Stoff or Sympathin, which was later identified as epinephrine (31). It has long been known that epinephrine is the predominant neurotransmitter in the frog heart, and in frog plasma under resting conditions it is five times more concentrated than norepinephrine; the latter, however, may increase considerably under stress, following release from the adrenal medulla (6). In excessive amounts, these two endogenous catecholamines are well known to be cardiotoxic for both amphibian and mammalian (including human) hearts, since they induce dose-dependent myocardial lesions. For example, exogenous ISO has been frequently used to produce myocardial necrosis both in frog (14) and mammalian hearts (47).

In a previous study, it has been established that ISO induces positive inotropic responses in frog heart via β-adrenergic receptors (49). In this work, the complete inhibition of ISO (10 nM)-induced positive inotropic effects by Cts indicates that Cts is acting as an antiadrenergic peptide, and Cts signaling pathways dominate over the adrenergic one. The costimulation experiments with both Cts and ISO in the presence of BQ-788 reinforce the hypothesis that Cts mediates its dominant antiadrenergic action through the activation of the ET_BR. Because the concentration-dependent positive inotropism of ISO failed to overcome the inhibitory effects of Cts on positive response, we concluded that the Cts inhibition of ISO is noncompetitive. Interestingly, the Cts-mediated inhibition of nicotine-evoked catecholamine secretion is also noncompetitive (39, 36, 33). Similar to Cts, analogous antiadrenergic actions of several CgA NH₂-terminal fragments have been demonstrated in frog, eel, and rat hearts (17, 25, 16). Tota and coworkers (54) also reported that the homologous frog CgA peptides (CgA_4-16 and CgA_47-66) are able to counteract the positive inotropism exerted by ISO in the frog heart. Thus, in addition to the pharmacological implications of our data, it is possible to hypothesize that the autocrine-paracrine role of Cts, as well as of the other CgA-derived peptides, may play a physiological role acting as a natural protective mechanism against excessive adrenergic stimulation.

ET-1-Cts Interaction

ETs are essential physiological regulators of normal cardiac and vascular functions, and an excessive generation or activity of ETs has been linked to several cardiovascular pathologies, including hypertension (9). The recent finding that human CHGA gene influences endothelial ET-1 secretion in vivo suggests an interaction between the sympatochromaffin system and the endothelium (29).

In the present study, we have demonstrated that ET-1 induces negative inotropism at lower concentrations (0.01 nM). In contrast, higher concentrations of ET-1 (0.1–10 nM) induced both positive (50% of hearts) and negative inotropism (50% of hearts). In mammals, either positive or negative inotropic responses of ET-1 have been reported in cardiac preparations of different species and experimental models (cell, tissue, organ). For example, in whole cardiac preparations, the effects of ET are often complicated by concomitant influences of coronary perfusion (9). By using the avascular frog heart, we analyzed the direct myocardial effects of ET-1 independently from the coronary-induced changes occurring in coronary-supplied hearts. The findings that l-NMMA, ODQ, and Triton X-100 completely abolished the negative inotropic effects of ET-1 indicate that ET-1 utilizes the functionally intact EE and the NO-cGMP pathway to exert its negative inotropic effects.

The ET-1 actions occur via G protein-coupled ET_AR and ET_BR subtypes. Although vascular smooth muscle cells, cardiomyocytes, fibroblasts, hepatocytes, and neurons express ET_AR, ET_BR are mainly expressed in endothelial and vascular smooth muscle cells. ET-1 binding to ET_AR activates phospholipase C (PLC), leading to the generation of inositol trisphosphate, which in turn stimulates intracellular calcium release (43) (Fig. 8). Stimulation of ET_AR also activates the mitogen-activated protein kinase cascade (41). ET_BR are coupled to a G_q/11 protein. Activation of ET_BR recruits pathway-dependent PLC, Na⁺/H⁺ exchanger, adenylate and guanylate cyclases (18), in addition to intracellular Ca²⁺ and Ca²⁺ entry (48) (Fig. 8). It has recently been shown that ET-1 binding to the ET_BR causes eNOS phosphorylation and NO synthesis (30). In our preparations, the chemical blockade of ET_AR by BQ-123 completely inhibited ET-1-induced positive inotropic effects, suggesting that ET-1 utilizes the ET_AR to exert its positive inotropic effects. However, BQ-123 failed to inhibit the negative inotropic effect of ET-1 at a concentration of 0.01 nM, indicating that ET-1, at this concentration, acts exclusively through the ET_BR. Interestingly, since after chemical blockade of ET_AR, ET-1 exerted only negative inotropism, we suggest that ET-1 action via ET_BR could now act unopposed without the influence of the ET_AR. Furthermore, inhibition of the ET_BR by BQ-788 completely inhibited the negative (0.01–10 nM) inotropic effects of ET-1 that appear mediated by NO and an intact EE. Of note, our results agree with a recent study (28) that demonstrated, in rabbit papillary muscles, that higher concentrations (10⁻⁹ M) of ET-1 produced a sustained positive inotropic effect, whereas the negative inotropic effect was observed only when ET-1 was given in the presence of an ET_AR antagonist. In addition, the same study demonstrated that negative inotropism induced by ET_BR stimulation is EE and NO dependent.

In the frog heart, Cts blocked ET-1's positive, but not the negative, inotropic effects. Because BQ-788, but not BQ-123, completely abolished Cts' negative inotropic effects, we can conclude that Cts acts through ET_BR (Fig. 8). It is interesting to note that, while Cts acts through ET_BR to exert its negative inotropic effect, it completely abolished ET-1's positive inotropic effect mediated via ET_AR. This indicates that activation of ET_BR by Cts dominates over the activation of ET_AR by ET-1. Cts action through ET_BR was reinforced when Cts failed to inhibit ET-1-induced positive inotropism in the presence of BQ-788. The demonstration that Cts is able to counteract the stimulatory influence of ET-1 may represent another potentially beneficial attribute of this CgA-derived peptide.

Significance of Two Cardioactive Peptides in one Precursor

The analogous cardiosuppressive effects of VS-1 and Cts raise the question regarding the biological significance of two peptides processed from one precursor. Further studies will clarify whether, behind such apparently redundant molecular strategy, there is an advantage of having two peptides regulating the same physiological response. Because of coordinate actions, they may act on overlapping or different sites, subserving subtly different functions, e.g., summation and synergism, or distinct spatiotemporal compartmentation (cell- and tissue-specific proteolytic processing and release). With specific reference to cardiovascular prohormones, an example is provided by the two peptide hormones processed from the pro-atrial natriuretic peptide (ANP) precursor, i.e., vessel dilator and ANP (55), both showing vasodilatory, diuretic, and natriuretic properties.

Conclusions

The avascular frog heart, used as bioassay to investigate the role of EE-dependent transduction of the ET-1 and Cts endoluminal stimuli, has contributed to reveal the importance of this tissue in mediating the interaction between the sympatochromaffin system and the myocardium. Should this mechanism be experimentally extended to the mammalian heart, it could contribute to account for the antihypertensive effects of Cts. In conclusion, the present study suggests a novel paracrine/autocrine role of Cts in the frog heart, since Cts depresses cardiac function and inhibits both β-adrenergic and ET-1-elicited stimuli. These Cts' cardiotropic features could be homeostatically relevant, particularly under stress conditions, when the heart becomes a preferential target of both adrenergic and endothelin stimuli, thus emphasizing the concept of “zero steady-state error” homeostasis proposed for other CgA-derived peptides (27).

GRANTS

This work was supported by the Ministero dell'lstruzione e dell'Università of Italy (60%: B. Tota, A. Gattuso, R. Mazza) and the “Dottorato di Biologia Animale” University of Calabria-Italy (S. F. Barbieri). S. K. Mahata was supported by grants from the Dept. of Veterans Affairs and the National Institutes of Health (R01 DA-011311 and P01 HL-58120).