Prognostic value of N-terminal natriuretic peptides in systemic sclerosis: a single centre study.

OBJECTIVES: Cardiac involvement is an important determinant of prognosis in systemic sclerosis (SSc). The identification of patients with high risk is of great importance. Our aim was to investigate the diagnostic and prognostic value of circulating concentrations of N-terminal fragments of A- and B-type natriuretic peptides (NT-proANP and NT-proBNP) in patients with SSc. METHODS: We prospectively studied 144 patients with SSc and followed them up for five years. Blood was collected for natriuretic peptide measurement at the time of the yearly scheduled cardiological check-up. The occurrence of clinically significant cardiac disease was measured as the composite of pulmonary arterial hypertension, cardiac revascularisation, development of left ventricular dysfunction or death. RESULTS: Patients diagnosed with heart involvement during the study had significantly higher levels of NT-proANP and NT-proBNP (791.4 ± 379.9 pmol/l vs. 608.0 ± 375.8 pmol/l, p<0.05 and 183.1 ± 162.6 vs. 125.7 ± 117.5 pmol/l, p<0.05, respectively). Receiver-operator-characteristic analysis identified <822.5 pmol/l as the best NT-proANP and <154.5 pmol/l as the best NT-proBNP threshold (sensitivity 56.3%, specificity 79.5%, negative predictive value: 86.4% and sensitivity 50.0%, specificity 76.8%, negative predictive value: 83.7%, respectively). During the follow-up, lower NT-proANP levels were significantly associated with a longer event-free survival (p<0.05), similar but a non-significant trend regarding NT-proBNP levels was also shown (p=0.052). CONCLUSIONS: In our cohort, NT-proANP had a supplementary prognostic value for cardiac involvement in systemic sclerosis. In addition, the high negative predictive value of natriuretic peptides supports the more extensive use in identifying SSc patients with high risk of future cardiac involvement.

Regulation of cardiac melusin gene expression by hypertrophic stimuli in the rat.

AIM: Melusin is an integrin ß1-interacting protein proposed to act as a biomechanical sensor in the heart. We characterized mechanisms and signalling pathways regulating cardiac melusin expression. METHODS: Infusion of arginine(8) -vasopressin (AVP) in Sprague-Dawley (SD) rats, spontaneously hypertensive rats (SHR) and double transgenic rats (dTGR) harbouring both human angiotensinogen and renin genes as well as infusion of angiotensin II (Ang II) in SD rats were used. The effect of direct left ventricular (LV) wall stretch was analysed by using isolated perfused rat heart preparation. For the cell culture studies, mouse atrial HL-1 cell line and neonatal rat ventricular myocytes (NRVMs) were used. RESULTS: Left atrial melusin mRNA levels increased already after 30 min of AVP infusion. Ang II caused significant upregulation of left atrial melusin mRNA (2.1-fold at 6 h, P < 0.05) and protein (1.9-fold at 72 h, P < 0.05) levels. In contrast, LV melusin mRNA levels remained unchanged in response to both infusions, as well as to aortic banding-induced pressure overload. Direct LV wall stress or late-stage hypertensive heart disease did not modify LV melusin gene expression either. Interestingly, in atrial HL-1 cells, cyclic stretching increased melusin mRNA levels. Stretching and treatments with hypertrophic agonists increased melusin mRNA and protein levels in NRVMs, endothelin-1 being the most potent. PD98059, an extracellular signal-regulated protein kinase 1/2 inhibitor, markedly attenuated the endothelin-1-induced upregulation of melusin gene expression in NRVMs. CONCLUSION: Multiple hypertrophic stimuli regulate melusin expression predominately in the atria, which may represent a necessary initial step in early adaptive remodelling processes.

Physiological regulation of cardiac contractility by endogenous reactive oxygen species.

Increased production of reactive oxygen species (ROS) has been linked to the pathogenesis of congestive heart failure. However, emerging evidence suggests the involvement of ROS in the regulation of various physiological cellular processes in the myocardium. In this review, we summarize the latest findings regarding the role of ROS in the acute regulation of cardiac contractility. We discuss ROS-dependent modulation of the inotropic responses to G protein-coupled receptor agonists (e.g. ß-adrenergic receptor agonists and endothelin-1), the potential cellular sources of ROS (e.g. NAD(P)H oxidases and mitochondria) and the proposed end-targets and signalling pathways by which ROS affect contractility. Accumulating new data supports the fundamental role of endogenously generated ROS to regulate cardiac function under physiological conditions.

Factors derived from adrenals are required for activation of cardiac gene expression in angiotensin II-induced hypertension.

The mechanisms mediating the activation of cardiac gene expression during pressure overload are not fully understood. We examined whether angiotensin II-induced activation of ventricular gene expression is related to blood pressure and ventricular mass or requires other factors by infusing angiotensin II in sham-operated and adrenalectomized rats. In sham-operated rats, angiotensin II (33 microg/kg x h, sc) produced a significant increase in mean arterial pressure (measured by telemetry) within 3 h. Mean arterial pressure (up to 45 h) and the increase in left ventricular hypertrophy in adrenalectomized rats during angiotensin II infusion were similar to those in sham-operated rats. Angiotensin II produced 3.6-fold (P < 0.01) and 20.4-fold (P < 0.001) increases in ventricular atrial natriuretic peptide mRNA levels at 12 and 72 h, respectively. Angiotensin II infusion for 12 h also significantly increased the ventricular mRNA levels of B-type natriuretic peptide (5.2-fold) and adrenomedullin (1.4-fold). Adrenalectomy either abolished (atrial natriuretic peptide and adrenomedullin) or blunted (B-type natriuretic peptide) the early activation of ventricular gene expression by angiotensin II. The baseline synthesis of atrial natriuretic peptide, B-type natriuretic peptide, and adrenomedullin in the ventricle remained unchanged in adrenalectomized rats. In conclusion, our results indicate that factors derived from the adrenals are required for angiotensin II-induced early activation of cardiac gene expression.

AM reverses pressor response to ET-1 independently of NO in rat coronary circulation.

Endothelin-1 (ET-1) elicits a vasoconstrictor response via ET(A) receptors, whereas simultaneous activation of ET(B) receptors triggers the release of nitric oxide (NO), which may limit the constrictor effect of ET-1. Recently, stimulation of ET(B) receptors has been shown to increase the secretion of adrenomedullin (AM), a newly identified vasorelaxing peptide. The present study was designed to see whether AM can oppose the vasoconstrictor response to ET-1. In the isolated perfused paced rat heart preparation, infusion of ET-1 at concentrations of 1 nmol/l for 30 min induced a significant coronary vasoconstriction, whereas it had no effect on perfusion pressure at a dose of 0.08 nmol/l. N(omega)-nitro-L-arginine methyl ester (L-NAME; 300 micromol/l), a potent inhibitor of NO synthase (NOS), did not change the perfusion pressure when added alone to the perfusion fluid but it unmasked the constrictor effect of ET-1 at both concentrations. In the presence of L-NAME, AM (0.03 to 1 nmol/l) markedly reversed the pressor response to ET-1 at both concentrations. Administration of AM (0.03 and 1 nmol/l) alone resulted in a dose-dependent decrease in perfusion pressure, which was not modified in the presence of L-NAME. In conclusion, the coronary vasoconstrictor response to ET-1 is markedly augmented in the presence of a NOS inhibitor. This constrictor response is substantially reversed by AM. Our results indicate that AM may serve as a paracrine modulator of ET-1-induced vasoconstriction independently of the NO pathway.

Impact of NO on ET-1- and AM-induced inotropic responses: potentiation by combined administration.

We characterize herein the impact of myocardial nitric oxide (NO) synthesis on the inotropic response to two cardioactive peptides, endothelin-1 (ET-1) and adrenomedullin (AM). In the isolated perfused rat heart preparation, intracoronary infusion of AM (0.03 and 1 nmol/l) and ET-1 (0.08 and 1 nmol/l) for 30 min induced a dose-dependent, gradual increase in developed tension, the maximal responses being equal. Inhibition of myocardial NO synthase (NOS) by N(omega)-nitro-L-arginine methyl ester (L-NAME; 300 micromol/l) enhanced the inotropic response to ET-1 at a concentration of 1 nmol/l; meanwhile, the effect of AM was not augmented significantly. The inotropic response to simultaneous administration of low, equipotent doses of AM (0.03 nmol/l) and ET-1 (0.08 nmol/l) was significantly smaller than that of either peptide alone. This depressed response was more than overcome by concomitant administration of L-NAME. In conclusion, this study reveals that the maximal inotropic response to ET-1 can be augmented by inhibition of myocardial NOS, whereas it has only a minor impact on the effect of AM. The inotropic response to combined administration of low doses of AM and ET-1 is substantially suppressed by endogenous NO, whereas the individual effects of the peptides at these doses are not the subject of secondary modulation by NO.

Enhanced accumulation of pericardial fluid ferritin in patients with coronary artery disease.

BACKGROUND: Ferritin is a storage protein for iron that can either represent a source of iron or perform a cytoprotective action as an iron sequestrant. OBJECTIVE: To compare the concentrations of ferritin in pericardial fluid of patients with valvular heart disease, serving as controls, and in patients with coronary artery disease. DESIGN: We studied a total of 59 consecutive male patients undergoing elective heart valve replacement (group 1: n = 22, mean +/- SD age 55 +/- 11 years) or elective coronary artery bypass grafting (group 2: n = 37, mean +/- SD age 59 +/- 9 years). METHODS: Iron status indicators, total protein and albumin concentrations, and lactate dehydrogenase activities were determined in pericardial fluid and serum samples obtained from patients during surgery. RESULTS: Pericardial fluid concentrations of ferritin in both patient populations were significantly (P < 0.001) greater than the concentrations in sera: group 1, 375 (107-2030) micrograms/l compared with 146.5 (21-407) micrograms/l; group 2, 1115 (226-2500) micrograms/l compared with 152.0 (16-398) micrograms/l (median (range)), respectively. Moreover, pericardial fluid ferritin concentration was significantly (P < 0.01) greater in patients undergoing coronary artery bypass grafting than in those undergoing heart valve replacement, whereas serum ferritin concentrations did not differ between the two patient populations. CONCLUSIONS: As pericardial fluid reflects the composition of the myocardial interstitium, we suggest that ferritin released can serve as a potential source of iron in the cardiac interstitium that may promote the generation of oxygen free radicals. Conversely, we presume that induction of ferritin synthesis, representing an important mechanism by which tissue adapts to hypoxic damage, can afford myocardial cytoprotection.

Intrapericardial infusion of endothelin-1 induces ventricular arrhythmias in dogs.

OBJECTIVES: Recently, extremely high levels of endothelin-1 (ET-1) were detected in the pericardial fluid of patients with heart disease; however, the pathophysiological importance of this finding is not known. The present study was designed to characterize ET-1 levels in canine pericardial fluid and to investigate the effects of local high concentrations of exogenous ET-1 in vivo. METHODS: In anesthetized, open-chest dogs ET-1 (Groups 1 and 2: 11 and 33 pmol.kg-1.min-1; n = 6 and 6, respectively) or physiological saline (Group 3, n = 5) were infused into the closed pericardial sac for 40 min. In serial pericardial fluid and aortic blood plasma samples, ET-1 levels were measured by radioimmunoassay, and analysed by high-performance liquid chromatography (HPLC). Systemic arterial blood pressure, heart rate, cardiac output (CO), standard ECG and right ventricular endocardial monophasic action potentials (MAPs) were recorded. RESULTS: Basal pericardial fluid ET-1 levels were significantly higher than respective plasma levels (342 +/- 210 vs. 8.0 +/- 5.2 pmol.l-1, n = 14, P < 0.001. In HPLC analysis pericardial fluid ET-1 was indistinguishable from ET-1(1-21). Infusion of exogenous ET-1 into the pericardial space induced ventricular arrhythmias in all instances, which were associated with 9.7-fold increase in pericardial fluid ET-1 levels. Ventricular tachycardias developed in 9 of 12 animals. The arrhythmogenic effect of ET-1 was more apparent in dogs with the larger dose. Before the onset of arrhythmias, intrapericardial infusion of ET-1 increased QT time (Group 1: 207 +/- 18 to 230 +/- 23 ms, P < 0.01; Group 2: 220 +/- 12 to 277 +/- 17 ms, P < 0.01) and MAP duration at 90% repolarization (at 300 ms cycle length) (Group 1: 192 +/- 9 to 216 +/- 9 ms, P < 0.01; Group 2: 205 +/- 9 to 255 +/- 9 ms, P < 0.001). Hemodynamic variables did not change significantly prior to the onset of ventricular tachyarrhythmias. In Group 3, arrhythmias were not observed and all electrophysiological and hemodynamic parameters remained unchanged. CONCLUSIONS: Administration of exogenous ET-1 into the pericardial space induces ventricular arrhythmias associated with prolongation of QT time and MAP duration. Whether pericardial fluid ET-1 under pathophysiological conditions can ever reach sufficiently high levels to induce ventricular arrhythmias remains to be elucidated.

Endothelin does not interact with angiotensin II in the coronary vascular bed of anesthetized dogs.

We postulated that individually subthreshold circulating levels of angiotension II (Ang II) and endothelin-1 (ET-1) might induce fulminant coronary vasoconstriction when both are present. In 16 pentobarbital-anesthetized, open-chest mongrel dogs, blood pressure, heart rate, and standard ECG were registered continuously. Coronary blood flow (CBF) was measured in the left anterior descending coronary artery (LAD) by an electromagnetic flow probe. Drugs were administered into the LAD via an indwelling catheter. Bolus injections of Ang II (7.8 x 10(-13) to 3.9 x 10(-11) M) and ET-1 (10(-12) to 10(-9) M) induced a dose-dependent decrease in CBF (delta CBFmax -82 +/- 10% for Ang II and -91 +/- 8% for ET-1). Simultaneous Ang II and ET-1 boluses had slightly smaller effects on CBF than the calculated additive figure. Five-minute infusions of Ang II (10(-12) to 10(-10) M/min) and ET-1 (5 x 10(-12) to 2 x 10(-10) M/min) induced a slight decrease in CBF (delta CBFmax -12 +/- 9% for Ang II and -19 +/- 9% for ET). Background ET-1 or Ang II infusions did not alter the dose-response curve of the other drug. Simultaneous Ang II and ET-1 infusions at different rates (10(-12) to 10(-10) M/min for Ang II and 5 x 10(-12) to 2 x 10(-10) M/min for ET-1) over 5 min had similar effects on CBF as the calculated additive figure (delta CBFmax -35 +/- 17% for the joint administration of the highest doses). We conclude that after simultaneous administration into the dog coronary artery, Ang II and ET-1 do not interact sufficiently to induce fulminant vasoconstriction.

High pericardial fluid levels of endothelin are not caused by altered neutral endopeptidase activity in cardiac patients.

We have previously detected in cardiac patients severalfold higher levels of endothelin (ET) in the pericardial fluid (PF) than in the plasma (PL). We postulated that this is due to different activities of neutral endopeptidase (NEP) in the two compartments. With approval of the ethical committee and informed consent by 32 patients (18 men, 14 women, aged 62 +/- 2 years; NYHA II-IV), PF was taken during cardiac surgery. PL samples were obtained on the day of surgery before premedication. ET was measured by radioimmunoassay after extraction (SepPakC18). NEP activity was measured by a microplate-based kinetic enzyme assay over 120 min. PF ET (78 +/- 11 pg/ml) was significantly (p < 0.05) higher than PL ET (3.38 +/- 0.48 pg/ml). The PF/PL ratio was 38 +/- 14, range 7-200. PF ET was inversely related to the NYHA state of the patients, whereas a similar relation was not found with PL ET. PL and PF ET levels did not correlate. In HPLC, the total immunoreactive ET activity co-eluted with the human ET standard. PF NEP activity (2.26 +/- 0.12 U/l) was lower (p < 0.05) than PL NEP (3.62 +/- 0.22 U/I). PL NEP was not different from that of healthy controls (3.28 +/- 0.22 U/L; n = 50). No correlation was found between NEP in either compartment and the NYHA state of the patients. ET concentration and NEP activity did not correlate in PF or PL. We conclude that ET is extremely high in the PF of cardiac patients and that this is not caused by altered NEP activity.

Verapamil reduces the arrhythmogenic effect of endothelin.

In a previous study we established that endothelin-1 (ET-1) can induce characteristic ventricular tachycardias (VT) with significant prolongation of QT and QTc time. In this investigation we studied the role of CA2+ channels in the pro-arrhythmic effects of ET-1. In 24 anesthetized, open-chest mongrel dogs, ET-1 was administered into the left anterior descending coronary artery at a comparatively low dose (60 pmol/min) for 30 min. Twelve dogs received the Ca(2+)-channel blocker verapamil (0.4 mg/kg) before ET-1 application. The following parameters were recorded continuously over the infusion period: systemic arterial blood pressure, coronary blood flow, surface ECG leads, epicardial atrial and ventricular electrograms, and right and left ventricular endocardial monophasic action potentials (MAP). Electrophysiologic studies were performed by programmed electrical stimulation of the heart. Blockade of myocardial Ca2+ channels attenuated the arrhythmogenic action of ET-1. After verapamil administration to ET-1-treated dogs, sustained VT did not appear and ventricular fibrillation (VF) developed only in two dogs. In the control group serious and sustained VT and VF developed in nine animals. It is noteworthy that verapamil did not prevent ET-1-induced prolongation of QT time. The results appear to prove that myocardial Ca2+ channels are involved in the proarrhythmic effect of ET-1.

Characterization of canine pericardial fluid endothelin-1 levels.

Recently, extremely high concentrations of endothelin-1 (ET-1) were detected in the pericardial fluid of patients undergoing cardiac surgery. This study was designed to characterize ET-1 levels in plasma and pericardial fluid of dogs. Plasma and pericardial fluid samples were extracted using SepPak C18 cartridges and ET-1 levels were measured by a specific radioimmunoassay. Pericardial fluid ET-1 levels (739 +/- 81 pg/ml; n = 10) were significantly higher than respective plasma levels (22.4 +/- 4.0 pg/ml; p < 0.05). The mean pericardial fluid: plasma ratio of ir-ET-1 concentration was 54.7 +/- 16.8. In HPLC analysis, the total ET-1-like immunoreactivity of pericardial fluid co-eluted with ET-1 standard. In anesthetized dogs (n = 6), disappearance of [125I]ET-1 was sixfold slower in the pericardial space than in plasma. Our results show that high concentrations of ir-ET-1 can be found in canine pericardial fluid. Slow elimination of ET-1 from the pericardial fluid compartment may contribute to high peptide levels.

Potential pathophysiologic role of endothelin-1 in canine pericardial fluid.

Recently, extremely high levels of endothelin-1 (ET-1) were detected in the pericardial fluid of patients undergoing cardiac surgery. This study was designed to assess the pathophysiologic importance of this finding by infusing ET-1 into the closed pericardial sac of anesthetized dogs. Systemic arterial blood pressure, heart rate, and standard ECG were recorded. Intrapericardial infusion of ET-1 (11 and 33 pmol/kg/min; n = 4/4) for 40 min induced ventricular arrhythmias in all instances. The lower dose of ET-1 induced a substantial number of ventricular extrasystoles, couplets, and triplets. In one instance, ventricular extrasystoles accelerated into nonsustained ventricular tachycardia (VT). In animals receiving the higher dose, nonsustained VTs occurred regularly, whereas sustained VTs were detected in two of four animals. Before the onset of arrhythmias, QT time was significantly prolonged [ET-1 (11 pmol/kg/min) 180 +/- 12 to 198 +/- 10 ms, p < 0.05; ET-1 (33 pmol/kg/min) 192 +/- 15 to 233 +/- 13 ms, p < 0.01]. Hemodynamic variables did not change significantly before the onset of ventricular arrhythmias. Our results show that administration of exogenous ET-1 into the pericardial space induces ventricular arrhythmias associated with prolongation of QT time.

Bradycardia increases the arrhythmogenic effect of endothelin.

The effect of permanent bradycardia on the proarrhythmic action of endothelin-1 (ET) was investigated in 24 open-chest anesthetized mongrel dogs. In 12 dogs, permanent bradycardia was induced by radiofrequency ablation of the AV node and the hearts were paced at 70 beats/min. ET (60 pmol/min) was infused into the left anterior descending coronary artery. Blood pressure, coronary blood flow (CBF), and atrial and ventricular epicardial surface ECG were recorded continuously. Polymorphous ventricular tachycardia developed in every dog with permanent bradycardia, and ventricular fibrillation terminated the experiments in 11 cases. Bradycardia prolonged the basal QT but there was no difference in the frequency corrected QTc time between the two groups. ET prolonged the QT time in a similar fashion in both groups. In the control group, six dogs developed sustained ventricular tachycardias and ventricular fibrillation occurred in nine cases. EADP was found in six cases of eight registered. Signs of myocardial ischemia did not accompany the development of arrhythmias. We conclude that permanent bradycardia augments the direct proarrhythmic effect of ET in dogs.

Evidence for cAMP-independent mechanisms mediating the effects of adrenomedullin, a new inotropic peptide.

BACKGROUND: Adrenomedullin (ADM), a new vasorelaxing and natriuretic peptide, may function as an endogenous regulator of cardiac function, because ADM and its binding sites have been found in the heart. We characterize herein the cardiac effects of ADM as well as the underlying signaling pathways in vitro. METHODS AND RESULTS: In isolated perfused, paced rat heart preparation, infusion of ADM at concentrations of 0.1 to 1 nmol/L for 30 minutes induced a dose-dependent, gradual increase in developed tension, whereas proadrenomedullin N-20 (PAMP; 10 to 100 nmol/L), a peptide derived from the same gene as ADM, had no effect. The ADM-induced positive inotropic effect was not altered by a calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP8-37, or H-89, a cAMP-dependent protein kinase inhibitor. ADM also failed to stimulate ventricular cAMP content of the perfused hearts. Ryanodine (3 nmol/L), a sarcoplasmic reticulum Ca2+ release channel opener, suppressed the overall ADM-induced positive inotropic effect. Pretreatment with thapsigargin (30 nmol/L), which inhibits sarcoplasmic reticulum Ca2+ ATPase and depletes intracellular Ca2+ stores, attenuated the early increase in developed tension produced by ADM. In addition, inhibition of protein kinase C by staurosporine (10 nmol/L) and blockade of L-type Ca2+ channels by diltiazem (1 micromol/L) significantly decreased the sustained phase of ADM-induced increase in developed tension. Superfusion of atrial myocytes with ADM (1 nmol/L) in isolated left atrial preparations resulted in a marked prolongation of action potential duration between 10 and -50 mV transmembrane voltage, consistent with an increase in L-type Ca2+ channel current during the plateau. CONCLUSIONS: Our results show that ADM enhances cardiac contractility via cAMP-independent mechanisms including Ca2+ release from intracellular ryanodine- and thapsigargin-sensitive Ca2+ stores, activation of protein kinase C, and Ca2+ influx through L-type Ca2+ channels.

Presence of immunoreactive endothelin-1 and atrial natriuretic peptide in human pericardial fluid.

This study was undertaken to characterize endothelin-1 (ET-1) and atrial natriuretic peptide (ANP) concentrations in human pericardial fluid, blood plasma, right atrial appendage and papillary muscle by use of specific radioimmunoassays. In patients undergoing cardiac surgery (n=16) pericardial fluid mean immunoreactive (ir-) ET-1 and ir-ANP levels were 36-fold and 4-fold higher than corresponding plasma levels, respectively. In high performance liquid chromatography (HPLC) pericardial fluid ir-ET-1 was indistinguishable from human ET-1[1-21] and the majority of pericardial fluid ir-ANP coeluted with human ANP[99-126]. Atrial tissue ir-ET-1 and ir-ANP concentrations were 17-fold and 870-fold higher than in ventricular tissue. Our present study demonstrated for the first time the presence of ir-ET-1 in the pericardial fluid in humans. Human pericardial fluid contained far the highest concentrations of ET-1 among all biological fluids tested thus far. The functions of pericardial fluid ET-1 and ANP on cardiac performance and coronary vascular tone require further investigations.

Electrophysiological effects of intrapericardial infusion of endothelin-1.

Recently, extremely high levels of endothelin-1 (ET-1) were detected in the pericardial fluid of patients undergoing open-heart surgery. ET-1 has been suggested to have direct arrhythmogenic effect on myocardium. The aim of the present study was to examine the putative arrhythmogenic effect of intrapericardial infusion of ET-1 in anesthetized dogs (n = 15). In preliminary experiments, ET-1 (0.125-1.0 nmol/min, n = 7) was infused into the closed pericardial sack for 40 min. ET-1 induced non-sustained and/or sustained ventricular tachyarrhythmias in all but the lowest dose. For detailed arrhythmia analysis in addition to standard ECG ventricular endocardial and epicardial monophasic action potentials (MAP) were recorded. ET-1 (0.250 nmol/min, n = 7) induced mono- and polymorphic ventricular tachycardias, which degenerated into ventricular fibrillation in two instances. Moderate if any ischemic signs could be detected before the onset of arrhythmias. The arrhythmias spontaneously disappeared in all instances with the exception when ventricular fibrillation terminated the experiment. QT interval (260 +/- 23 ms vs. 317 +/- 31 ms, P < 0.05), and endo- and epicardial MAPD90 (at 300 ms cycle length) prolonged significantly (in average 182 +/- 12 ms vs. 224 +/- 25 ms, P < 0.05). Using MAP recording afterdepolarizations were detected in three instances. In control animals (n = 3) arrhythmias were not observed and all electrophysiological parameters remained unchanged. The present results show that intrapericardial administration of ET-1 can induce ventricular arrhythmias in dogs. The arrhythmogenic effect of ET-1 may be based on prolongation of MAP duration and development of afterdepolarizations. However, the elucidation of the precise mechanism needs further investigation.

Adrenomedullin gene expression in the rat heart is stimulated by acute pressure overload: blunted effect in experimental hypertension.

The levels of adrenomedullin (ADM), a newly discovered vasodilating and natriuretic peptide, are elevated in plasma and ventricular myocardium in human congestive heart failure suggesting that cardiac synthesis may contribute to the plasma concentrations of ADM. To examine the time course of induction and mechanisms regulating cardiac ADM gene expression, we determined the effect of acute and short-term cardiac overload on ventricular ADM mRNA and immunoreactive ADM (ir-ADM) levels in conscious rats. Acute pressure overload was produced by infusion of arginine8-vasopressin (AVP, 0.05 microg/kg/min, i.v.) for 2 h into 12-week-old hypertensive TGR(mREN-2)27 rats and normotensive Sprague-Dawley (SD) rats. Hypertension and marked left ventricular hypertrophy were associated with 2.2-times higher ir-ADM levels in the left ventricular epicardial layer (178 +/- 36 vs. 81 +/- 23 fmol/g, P<0.05) and 2.6-times higher ir-ADM levels in the left ventricular endocardial layer (213 +/- 23 vs. 83 +/- 22 fmol/g, P<0.01). The infusion of AVP for 2 h in normotensive rats produced rapid increases in the levels of left ventricular ADM mRNA (epicardial layer: 1.6-fold, P<0.05) and ir-ADM (endocardial layer: from 83 +/- 22 to 140 +/- 12 fmol/g, P<0.05), whereas ventricular ADM mRNA and ir-ADM levels did not change significantly in hypertensive rats. Short-term cardiac overload, induced by administration of angiotensin II (33.3 microg/kg/h, s.c., osmotic minipumps) for two weeks in normotensive SD rats resulted in left ventricular hypertrophy (3.05 +/- 0.17 vs. 2.75 +/- 0.3 mg/g, P<0.05) and a 1.5-fold increase (P<0.05) in ventricular ADM mRNA levels. In conclusion, the present results show that pressure overload acutely stimulated ventricular ADM gene expression in conscious normotensive rats suggesting a potential beneficial role for endogenous ADM production in the heart against cardiac overload. Since pressure overload-induced increase in ADM synthesis was attenuated in hypertensive rats, alterations in the ADM system may contribute to the pathogenesis of hypertension in the TGR(mREN-2)27 rat.

Characterization and stimuli for production of pericardial fluid atrial natriuretic peptide in dogs.

Recently high immunoreactive atrial natriuretic peptide (ir-ANP) levels have been found in the pericardial fluid of patients undergoing cardiac surgery. The present study was designed to characterize pericardial fluid ANP in anesthetized dogs. Pericardial fluid ir-ANP levels were 3.4-fold higher than plasma levels and the molecular form, revealed by high performance liquid chromatography, was indistinguishable from ANP[99-126]. Elimination of [125I]ANP was 5-fold slower in the pericardial space than in plasma. Activity of the major ANP degrading enzyme, neutral endopeptidase (NEP, EC 3.4.24.11), was 15-times higher in the pericardial fluid than in plasma. Right atrial balloon distension and rapid right ventricular pacing induced maximally 2.3-fold and 1.5-fold increases of pericardial fluid ir-ANP, respectively. Pericardial fluid ir-ANP concentrations and right atrial pressure values showed significant correlation during the stimuli. Our present results show that high concentrations of ir-ANP can be found in the dog pericardial fluid even under unstimulated conditions. Slow elimination of ANP from the pericardial fluid compartment may contribute to the high peptide levels. However this slow elimination cannot be attributed to a lower NEP activity. High basal levels of ANP in the pericardial fluid could be further increased by atrial balloon stretch and rapid ventricular pacing. The increase of pericardial fluid ir-ANP appeared to be a stretch-dependent response. ANP released into the pericardial fluid may be involved in the regulation of cardiac function and coronary vascular tone.