AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 293: H3001-H3007, 2007. First published August 31, 2007; doi:10.1152/ajpheart.01276.2006
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Atrial myocardium is the predominant inotropic target of adrenomedullin in the human heart

Egbert Bisping,1,* Gero Tenderich,2,* Paul Barckhausen,1 Burkhard Stumme,1 Sebastian Bruns,1 Dirk von Lewinski,1 and Burkert Pieske3

1Department of Cardiology and Pneumology, University of Göttingen, Göttingen, 2Department of Cardio-Thoracic Surgery, Heart Center Nordrhein-Westfalen, Bad Oeynhausen, Germany; and 3Department of Cardiology, University of Graz, Graz, Austria

Submitted 21 November 2006 ; accepted in final form 27 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Adrenomedullin (ADM) is an endogenous peptide with favorable hemodynamic effects in vivo. In this study, we characterized the direct functional effects of ADM in isolated preparations from human atria and ventricles. In electrically stimulated human nonfailing right atrial trabeculae, ADM (0.0001–1 µmol/l) increased force of contraction in a concentration-dependent manner, with a maximal increase by 35 ± 8% (at 1 µmol/l; P < 0.05). The positive inotropic effect was accompanied by a disproportionate increase in calcium transients assessed by aequorin light emission [by 76 ± 20%; force/light ratio ({Delta}F/{Delta}L) 0.58 ± 0.15]. In contrast, elevation of extracellular calcium (from 2.5 to 3.2 mmol/l) proportionally increased force and aequorin light emission ({Delta}F/{Delta}L 1.0 ± 0.1; P < 0.05 vs. ADM). Consistent with a cAMP-dependent mechanism, ADM (1 µmol/l) increased atrial cAMP levels by 90 ± 12%, and its inotropic effects could be blocked by the protein kinase A (PKA) inhibitor H-89. ADM also exerted positive inotropic effects in failing atrial myocardium and in nonfailing and failing ventricular myocardium. The inotropic response was significantly weaker in ventricular vs. atrial myocardium and in failing vs. nonfailing myocardium. In conclusion, ADM exerts Ca2+-dependent positive inotropic effects in human atrial and less-pronounced effects in ventricular myocardium. The inotropic effects are related to increased cAMP levels and stimulation of PKA. In heart failure, the responsiveness to ADM is reduced in atria and ventricles.

contractility; Ca2+ handling; human myocardium

ADRENOMEDULLIN (ADM) is an endogenous vasoactive peptide consisting of 52 amino acids that exerts long-lasting vasodilating effects (11), stimulates diuresis and natriuresis (13), and exerts substantial hemodynamic effects in vivo. These hemodynamic effects are characterized by increased cardiac output and decreased afterload (19, 29). ADM as well as specific ADM-binding sites are expressed in atrial and ventricular cardiac myocytes (2, 14, 15), suggesting a potential autocrine and paracrine functional role of ADM in the heart.

Although vasodilation may unequivocally contribute to increased cardiac output, a potential direct functional effect of ADM in the heart is controversial. In a variety of different species, direct positive inotropic effects have been reported (9, 17, 29, 33), but no (2, 18) or even negative inotropic effects (10, 16) have also been observed. In humans, direct evidence for a positive inotropic effect of ADM is lacking. ADM plasma levels (14) and myocardial ADM expression (25) are elevated in patients with heart failure, and it exerts beneficial hemodynamic effects in heart-failure patients (23) and in patients after myocardial infarction (22). Three different mechanisms might contribute to these hemodynamic effects: 1) decreased afterload due to vasodilation, 2) normalization of volume overload by increased natriuresis and diuresis, and 3) direct positive inotropy.

Therefore, we directly assessed functional effects of ADM in human nonfailing and failing atria and ventricles. We found that ADM exerts clear positive inotropic effects in human nonfailing atrial myocardium. The effects were associated with increased calcium transients and elevated levels of cAMP and were dependent on activation of protein kinase A (PKA). ADM's effects were smaller in nonfailing ventricle and were also significantly reduced in failing tissue.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Human myocardium and patient characteristics. Isolated trabeculae were obtained from four different types of human myocardium: 1) right atria from nonfailing hearts, 2) right atria from failing hearts, 3) left ventricles from nonfailing donor hearts, and 4) left ventricles from failing hearts.

Normal atrial myocardium were derived from 24 patients (mean age 61 ± 3 yr, 76% male, 24% female) with normal ventricular function and sinus rhythm who underwent coronary artery bypass surgery. Failing atrial myocardium was obtained from three patients (mean age 50 ± 4 yr, 100% male) with end-stage failing hearts (2 ischemic, 1 dilated cardiomyopathy). These hearts had been explanted at the time of cardiac transplantation and had an average ejection fraction of 24 ± 1%. Two patients were in sinus rhythm, one in atrial fibrillation; all showed significant atrial dilatation.

Isolated nonfailing ventricular trabeculae were obtained from three patients (mean age 44 ± 3 yr, 67% male, 33% female) with nonfailing hearts that could not be transplanted for technical reasons. Failing ventricular muscle strips came from 19 transplanted patients (mean age 57 ± 2 yr, 88% male, 22% female) with end-stage failing hearts (9 ischemic, 10 dilated cardiomyopathy). On transplantation, these failing hearts were characterized by a mean ejection fraction of 25 ± 2%, pulmonary wedge pressure of 23 ± 2 mmHg, and a mean cardiac index of 2.2 ± 0.1 l·min–1·m–2. The use of human myocardium was approved by the Ethical Committee of the University of Göttingen.

Muscle-strip preparation and experimental protocol. Muscle strips were prepared as previously described (28). After an initial equilibration period of 15 min in the organ chamber, muscles were paced at 1.0 Hz and the extracellular Ca2+ concentration ([Ca2+]o) was gradually increased to 2.5 mmol/l. Then muscles were stretched along their length-tension curve until maximal isometric twitch force was reached. After complete mechanical stabilization, the bioluminescent intracellular Ca2+ indicator aequorin was macroinjected as described into a subset of the muscle strips (see Assessment of intracellular Ca2+ transients). Isometric twitch force and aequorin light emission were recorded at 5-min intervals. After a stabilization period of 60 min, mechanical rundown of the muscle strips was constant and was quantified over a subsequent period of 30 min. This allowed us to determine the individual rundown of force and aequorin light signal in each muscle-strip preparation and to use it for rundown correction of the obtained data. For rundown correction, resulting data were multiplied with a correction factor (100% + %rundown within the given time interval). The correction factor was determined individually for each muscle preparation. With and without rundown correction, solvent alone (NaCl) had no effect on contractile force (rundown-corrected force decreased by 1 ± 1%; not significant).

Drug application. ADM was added to the organ bath either in the form of cumulative concentration-response curves or as a bolus of a single concentration of either 0.1 µmol/l or 1.0 µmol/l. The resulting effects on force of contraction and aequorin light signal were recorded over a time period of 30 min or until the resulting functional effects had reached a stable maximum. Pharmacological inhibition of PKA was achieved by preincubation with H-89 (5 µmol/l) for 30 min before application of ADM. In additional experiments, the inotropic response to an increase in [Ca2+]o from 2.5 to 3.2 mmol/l was tested for comparison. Active force of contraction (i.e., the amplitude of the isometric twitch), diastolic tension, the maximum rates of tension rise and fall (+dF/dtmax and –dF/dtmax), time to peak tension, and time to 50% and 95% relaxation were quantified from the recordings.

Assessment of intracellular Ca2+ transients. For simultaneous assessment of force development and intracellular Ca2+ transients, muscle strips were loaded with the Ca2+-regulated bioluminescent photoprotein aequorin as previously described (26). Aequorin was kindly provided in lyophilized form from J. R. Blinks (Friday Harbor Laboratories, University of Washington, Friday Harbor, WA). The photoprotein was dissolved in Ca2+-free distilled water, and 3 µl of this aqueous solution was carefully macroinjected into the quiescent muscle within the organ chamber. Aequorin light emission, reflecting intracellular Ca2+ transients, was monitored with the help of a photomultiplier placed just above the muscle strips. Data were digitally recorded and analyzed as previously reported (26).

Measurement of cAMP. Atrial trabecular muscle strips were electrically stimulated at 1 Hz and were incubated in carbogen-bubbled Tyrode solution for 20 min without (control) or with addition of ADM (1 µmol/l). Thereafter, muscle strips were shock frozen and stored at –80°C before further processing. Samples were weighted and homogenized in a buffer solution. The cAMP levels were measured by using a commercially available [3H]cAMP radioimmunoassay kit (Amersham Pharmacia Biotech, Piscataway, NJ) following the protocol provided by the manufacturer. The cAMP levels are reported as picomoles per milligram of protein.

Drugs. Human ADM was kindly provided by Dr. Klaus Dembowsky (Bayer, Leverkusen, Germany; current address: Ingenium Pharmaceuticals, Martinsried, Germany). ADM was dissolved in 0.9% NaCl to obtain a 100 µmol/l stock solution. H-89 (Calbiochem, catalog no. 371962) was dissolved in DMSO at 10–2 mol/l stock solution. All other compounds were obtained from Sigma Aldrich. Experiments with elevated [Ca2+]o were performed in phosphate-free Tyrode solution to avoid Ca2+ complexation.

Statistical analysis. Average values are given as means ± SE. Differences depending on one independent factor were tested by a one-way ANOVA followed by Bonferroni-Holmes correction. Differences depending on two independent factors were analyzed by two-way ANOVA followed by post hoc testing by the Fisher least significant difference method. A P value <0.05 was considered as statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
ADM exerts positive inotropic effects in nonfailing atrial myocardium. Figure 1A shows the typical inotropic response to 1 µmol/l ADM in an atrial muscle strip. Figure 1B depicts the results from cumulative concentration-response curves (n = 4 from 3 hearts). Because negative inotropic effects of ADM were reported at low concentrations (21), we started these experiments at 0.0001 µmol/l. ADM exerted neither negative nor positive inotropic effects at low concentrations but exerted significant positive inotropic effects at concentrations >0.1 µmol/l. Developed force increased from 4.0 ± 0.6 mN/mm2 (basal value) to 5.7 ± 0.8 mN/mm2 at 1 µmol/l (highest concentration tested; P < 0.05 vs. basal value).


Figure 1
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  		Fig. 1. Inotropic effects of adrenomedullin (ADM) in human atrial myocardium. A: original recording of force of contraction in an isolated human atrial trabecula. Arrow indicates application of ADM (1 µmol/l). B: concentration-response curve for ADM in atrial myocardium (n = 4 from 3 hearts). *P < 0.05 vs. basal value.

 
We also tested the effect of single concentrations of ADM. At 0.1 µmol/l (n = 6 from 6 hearts), ADM increased developed force by 11 ± 5% (P < 0.05) and at 1 µmol/l (n = 5 from 5 hearts) by 35 ± 8% (P < 0.05). Diastolic tension did not significantly change after ADM. Maximum rate of force increase (+dF/dt) and maximum rate of force decline (–dF/dt) significantly increased with ADM (1 µmol/l): +dF/dt increased by 39 ± 10% (P < 0.05), and –dF/dt increased by 32 ± 8% (P < 0.05). Twitch time parameters of contraction and relaxation tended to decrease: time to peak tension was 89 ± 10 at baseline and 84 ± 9 ms after 1 µmol/l ADM, and time to 95% relaxation was 187 ± 20 at baseline and 186 ± 20 after 1 µmol/l ADM (not significant).

ADM disproportionately increases calcium transients in nonfailing atrial myocardium. To determine if the inotropic effects of ADM are calcium dependent, we simultaneously assessed aequorin light emission and twitch force (n = 5 from 5 hearts; Fig. 2, A and B). We also compared the relative increases in aequorin light signals and twitch-force amplitude with ADM (1 µmol/l) to the effect of an approximately equally effective [Ca2+]o, i.e., elevation from 2.5 to 3.2 mmol/l (Fig. 2C). The average 35 ± 8% increase in twitch force with ADM was associated with a disproportionate increase (by 76 ± 20%) in aequorin light amplitude (P < 0.05; Fig. 2C). In contrast, elevation of [Ca2+]o to 3.2 mmol/l induced commensurate increases in force (by 52 ± 8%; P < 0.05) and aequorin light amplitude (by 50 ± 6%; P < 0.05; Fig. 2C). These differences in intracellular Ca2+ handling after ADM or elevated [Ca2+]o also became apparent by the ratio of changes in force ({Delta}F) and changes in aequorin light amplitude ({Delta}L). The average ratio of {Delta}F/{Delta}L with ADM (0.58 ± 0.13) was significantly different from the ratio with [Ca2+]o ({Delta}F/{Delta}L 1.05 ± 0.08; P < 0.05 between groups).


Figure 2
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  		Fig. 2. Effects of ADM on intracellular calcium handling in atrial myocardium. A: superimposed original tracings of effect of ADM (1 µmol/l) on isometric twitch tension in an atrial trabecula. B: corresponding calcium transients (aequorin light signals). C: effects of ADM (1 µmol/l; n = 5 from 5 hearts; left) and of extracellular calcium concentration ([Ca2+]o; 3.2 mmol/l; n = 6 from 6 hearts; right) on twitch force (closed bar) and aequorin light emission (open bar) in atrial myocardium. *P < 0.05 vs. basal value.

 
cAMP- and PKA-dependent effects of ADM. To further study the mechanism of action of ADM, we directly measured cAMP levels in human nonfailing atrial myocardium by radioimmunoassay. cAMP content in shock-frozen atrial trabeculae under basal functional conditions (control; n = 7 from 7 hearts) was 0.617 ± 0.187 pmol/mg. In trabeculae from the same hearts, application of 1 µmol/l ADM for 20 min to the organ bath increased cAMP content to 1.172 ± 0.139 pmol/mg; i.e., 190% of untreated control (P < 0.05 vs. control; Fig. 3A).


Figure 3
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  		Fig. 3. cAMP and protein kinase A (PKA) dependence in atrial myocardium. A: cAMP content in contracting atrial trabeculae. Values are normalized to protein content. Control, without ADM (n = 7 from 7 hearts). ADM, after 20 min incubation with 1 µmol/l ADM (n = 7 from 7 hearts). B: inotropic effect of ADM in atrial myocardium without (n = 4 from 4 hearts) and with preincubation of H-89 (n = 4 from identical hearts). NS, not significant. *P < 0.05 between groups.

 
Next we tested whether the functional effects of ADM were mediated by PKA activation. For this purpose, the inotropic response to ADM (1 µmol/l) was assessed with and without 30 min preincubation of the PKA inhibitor H-89 (paired experiments in trabeculae from the same hearts; each n = 4 from 4 hearts). ADM alone increased force by 43 ± 7% (P < 0.05 vs. base). However, in the presence of H-89, this increase was significantly blunted (5 ± 7%; not significant vs. base and P < 0.05 vs. ADM alone; Fig. 3B).

ADM exerts small functional effects in ventricular myocardium. We also examined the functional effects of ADM in ventricular preparations from nonfailing and failing human hearts. At 0.1 µmol/l (single concentration), ADM tended to increase twitch force marginally by 2 ± 2% in nonfailing (n = 5 from 3 hearts; not significant) and by 1 ± 1% in failing myocardium (n = 7 from 6 hearts; not significant; Fig. 4A).


Figure 4
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  		Fig. 4. Functional effects of ADM in ventricular myocardium. A: average increase in isometric twitch force with ADM in ventricular nonfailing (n = 5 from 3 hearts for 0.1 and 1 µmol/l) and failing trabeculae (n = 7 from 6 hearts for 0.1 µmol/l; n = 11 from 10 hearts for 1 µmol/l ADM). B: effects of ADM (1 µmol/l; n = 9 from 9 hearts) and of [Ca2+]o (3.2 mmol/l; n = 8 from 8 hearts) on twitch force (closed bars) and aequorin light emission (open bars) in failing ventricular myocardium. *P < 0.05 vs. basal value. #P < 0.05 vs. nonfailing.

 
At 1 µmol/l, ADM significantly increased force of contraction by 11 ± 3% in nonfailing (n = 5 from 3 hearts; P < 0.05) and by 6 ± 1% in failing ventricular myocardium (n = 11 from 10 hearts; P < 0.05). Interestingly, the response at 1 µmol/l was significantly stronger in nonfailing vs. failing myocardium (P < 0.05). Lower concentrations (0.0001–0.01 µmol/l) resulted in neither positive nor negative inotropic effects (n = 3 from 3 failing hearts; data not shown).

Simultaneous aequorin light emission was measured in 9 (from 9 hearts) failing ventricular muscle-strip preparations and (due to limited availability) in 1 single nonfailing muscle strip with 1 µmol/l ADM. In nonfailing hearts, force increased by 17% and aequorin light by 55%. In failing hearts, force increased by 5 ± 1% and aequorin light by 16 ± 5% (both P < 0.05). In contrast, an elevation of [Ca2+]o from 2.5 to 3.2 mmol/l (n = 8) resulted in proportional increases of force (by 29 ± 3%) and aequorin light amplitude (by 26 ± 3%). Accordingly, as in atrial preparations, {Delta}F/{Delta}L after ADM (0.63 ± 0.20) was significantly different from elevated [Ca2+]o (1.12 ± 0.07; P < 0.05; Fig. 4B) in failing human ventricular myocardium.

ADM effects depend on heart-chamber type and functional state. Comparison of the inotropic effects of ADM at 1 µmol/l showed significantly weaker responses in nonfailing and failing ventricular myocardium than in nonfailing atrial myocardium (Fig. 5A). To properly differentiate the effects of heart chamber (atrial vs. ventricular) and the effect of functional state (nonfailing vs. failing), we performed experiments in muscle strips (n = 5 from 3 hearts) from the atria of failing human hearts to compare these with the nonfailing atrial and the ventricular failing and nonfailing group (Fig. 5A). A two-way ANOVA testing revealed that the inotropic response to ADM depends on both the heart-chamber type and the functional state (P < 0.01 for both factors). Functional parameters as well as the corresponding patient characteristics of this comparison are summarized in Table 1.


Figure 5
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  		Fig. 5. Dependence of inotropic effects on heart-chamber type and functional state. A: average increase in isometric twitch force with 1 µmol/l ADM in nonfailing atrial (n = 5 from 5 hearts), failing atrial (n = 5 from 3 hearts), nonfailing ventricular (n = 5 from 3 hearts), and failing ventricular (n = 11 from 10 hearts) trabeculae. *P < 0.05 for failing vs. nonfailing. #P < 0.05 for atrial vs. ventricular. B: linear regression for changes in force and light signals with ADM (1.0 µmol/l) in atrial (n = 5) and ventricular failing (n = 9) trabeculae. For comparison, data are shown for [Ca2+]o (3.2 mmol/l) in atrial (n = 6) and ventricular failing (n = 8) trabeculae; r, correlation coefficients.

 

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  		Table 1. ADM dependence on functional state and chamber type

 
According to Fig. 5A, nonfailing atrial myocardium and failing ventricular myocardium are at the ends of the scale of inotropic responses to ADM. Interestingly, the disproportionate effects of ADM on force and calcium transients were consistent in these two types of preparations. When plotting {Delta}F vs. {Delta}L of all individual experiments, a close linear correlation between {Delta}F and {Delta}L became apparent within each set of experiments (correlation coefficients ranging from 0.63 to 0.91; P < 0.05 for all; Fig. 5B). The linear regression lines showed markedly less-steep slopes for ADM than for [Ca2+]o in both atrial and ventricular myocardium. In contrast, within all ADM experiments regression lines between atrial and ventricular myocardium were very similar. Accordingly, a two-way ANOVA of the {Delta}F/{Delta}L ratio revealed a significant difference for ADM vs. [Ca2+]o (P = 0.007) but no difference between atrial and ventricular myocardium (P = 0.71).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study demonstrates that 1) ADM exerts direct positive inotropic effects in isolated human myocardium, 2) these effects are substantial in atrial but small in ventricular myocardium and are diminished in heart failure, and 3) the inotropic effects are related to increased Ca2+ transients and to an increase in intracellular cAMP level and depend on stimulation of PKA.

Functional effects of ADM in atrial myocardium. Our study reports for the first time a direct positive inotropic effect of ADM in isolated human atrial myocardium. In other species, ADM has shown variable inotropic effects (9, 10, 16–18, 29, 33). Also, the results within one species differed depending on experimental conditions, e.g., different preparations of rat heart (2, 9, 16, 17, 33) or different effects of ADM observed at different time points due to dual stimulation of two distinct pathways (20).

In human atria, the inotropic effect of ADM was associated with a significant increase in intracellular Ca2+ transients. This is in agreement with previous data from isolated rat myocardium (33). The relationship between increases in force and increases in aequorin light emission ({Delta}F/{Delta}L) is established as an indirect parameter for myofilament calcium responsiveness (1). {Delta}F/{Delta}L is near 1 if calcium responsiveness is unchanged, below this value if calcium responsiveness is decreased, and higher if the responsiveness is increased (27). Our data demonstrate that the inotropic effect of ADM was associated with an disproportionate increase in aequorin light emission ({Delta}F/{Delta}L = 0.58), whereas elevation of [Ca2+]o resulted in a proportional increase in force and aequorin light emission ({Delta}F/{Delta}L = 1.05). This observation with ADM is reminiscent of the behavior of calcium transients under beta-adrenergic agonists such as isoproterenol (5). beta-Adrenergic agonists increase intracellular cAMP levels with a resulting activation of PKA and subsequent increases in Ca2+ turnover and troponin I phosphorylation, which promotes a decrease in myofilament calcium responsiveness (6). Thus the similar qualitative observations in our experiments suggest that ADM both increases Ca2+ turnover and decreases calcium myofilament responsiveness, potentially by a cAMP-dependent mechanism. To confirm this hypothesis, we measured cAMP content by radioimmunoassay in atrial preparations. The experiments revealed a clear increase in cAMP with ADM. In addition, our experiments with preincubation of the PKA inhibitor H-89 revealed that stimulation of PKA is necessary for the inotropic effect of ADM. These data are consistent with previous reports demonstrating cAMP-dependent effects of ADM in several animal species (9, 18, 23). Also, elevated cAMP levels have been reported in human patients after infusion of ADM (23) but could not be separated from potential reactive effects with an increase in sympathetic tone due to vasodilation. Our data can rule out such potential confounders and therefore give insight into the direct mechanism of ADM in human atrial myocardium.

Functional ADM effects in ventricular myocardium. We demonstrate small, albeit significant, Ca2+-dependent positive inotropic effects of ADM in both nonfailing and failing ventricular myocardium. In a previous study, Saetrum Opgard et al. (30) detected neither positive nor negative inotropic effects of ADM in human myocardium. In common with their study, we used isolated multicellular preparations, but in contrast to their study, we used higher concentrations of ADM. This allowed us to identify a positive inotropic effect that might have been undetectable at lower concentrations. At low concentrations, we also did not detect any positive or negative inotropic effect of ADM. However, a negative inotropic effect was described in isolated human ventricular myocytes (21).

Of interest, Ca2+ transients increased disproportionately with ADM in both nonfailing atrial and ventricular failing myocardium. This was apparent from the almost identical {Delta}F/{Delta}L ratios in these groups. Because these two groups represent the opposite ends of the scale of inotropic response to ADM, it is tempting to speculate that signal-transduction pathways of ADM for functional effects are similar in atrial nonfailing vs. ventricular failing myocardium but are less effective in the latter.

ADM concentrations in the intact organism. The concentrations of ADM examined in this study exceed the physiological range of plasma concentrations in humans (14). For a direct comparison, however, the tissue and not the plasma concentrations of ADM have to be considered. There is reason to assume that tissue concentrations of ADM are markedly higher than its plasma concentrations: ADM is an autocrine/paracrine factor (15) that is produced and secreted under the control of angiotensin II from cardiac myocytes and fibroblasts (12). In addition, ADM plasma levels (24) as well as myocardial ADM expression (25) were reported to be elevated in human and experimental heart failure. Therefore, local ADM concentrations at the level of the myocytes may be similar to the range examined in our study and sufficient to mediate the reported functional effects under in vivo conditions.

Influence of heart-chamber type and functional state on the effects of ADM. We found that both heart chamber and the functional state of the heart determine the strength of the inotropic response to ADM. The finding that ADM effects are larger in atrial vs. ventricular human myocardium resembles the observations made for several other vasoactive peptides. Endothelin (32), calcitonin gene-related peptide (30), vasoactive intestinal peptide (31), and angiotensin II (8) all show more pronounced inotropic effects in atrial and smaller or even no (angiotensin II) effects in ventricular myocardium. Besides a higher receptor density in atrial myocardium, an increased strength of the receptor coupling to signal transduction has been discussed (31, 32), which might be true for ADM as well. In fact, receptor-binding studies suggest that ADM receptor density is higher in atrial myocardium (3). Moreover, ADM expression is markedly higher in atria than in ventricles (14). Unfortunately, the groups of our comparisons could not be matched in age, because the heart donors from whom nonfailing ventricular samples were derived were younger than the patients in the other groups (Table 1). Thus our conclusion that the differences between atrial and ventricular nonfailing samples reflect different chamber-specific signal-transduction efficiencies is confounded by this age mismatch.

In heart failure, the response to ADM was diminished in both atrial and ventricular myocardium. This observation is likely explained by the diminished responsiveness of the failing heart to cAMP stimulation associated with upregulation of Gi protein levels (4) and is shared by other cAMP-elevating compounds (7).

Conclusion. ADM increases cardiac output in humans, but the underlying mechanisms were poorly characterized. We could demonstrate that ADM exerts concentration- and Ca2+-dependent positive inotropic effects in atrial, and to a minor extent in ventricular, myocardium. Heart failure diminishes the inotropic responsiveness to ADM in both atrial and ventricular myocardium. Mechanistically, the inotropic effect involves elevation of intracellular cAMP levels and activation of PKA. Improved ventricular filling by increased atrial contraction and slightly enhanced ventricular contractility might contribute to increased cardiac output with ADM, but hemodynamic effects of ADM may primarily be related to afterload reduction.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG PI 414/1-4) to B. Pieske.


   ACKNOWLEDGMENTS
 
We thank Dr. Klaus Dembowsky, Ingenium Pharmaceuticals, Martinsried, Germany for providing us with ADM and Andre Wilken for excellent technical support.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. Pieske, Dept. of Cardiology, Medical Univ. Graz, Auenbrugger Platz 15, 8036 Graz, Austria (e-mail: burkert.pieske{at}meduni-graz.at)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* E. Bisping and G. Tenderich contributed equally to this work. Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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