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Cardiovascular Research 2006 69(2):402-411; doi:10.1016/j.cardiores.2005.10.015
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Copyright © 2005, European Society of Cardiology

Urocortin II enhances contractility in rabbit ventricular myocytes via CRF2 receptor-mediated stimulation of protein kinase A

Li-Zhen Yanga,1, Jens Kockskämperb,1, Frank R. Heinzelb, Michael Hauberb, Stefanie Waltherb, Joachim Spiessa,* and Burkert Pieskeb

aDepartment of Molecular Neuroendocrinology, Max Planck Institute for Experimental Medicine, Hermann-Rein-Str. 3, D-37075 Göttingen, Germany
bDepartment of Cardiology and Pneumology, Georg-August-Universität Göttingen, Robert-Koch-Str. 40, D-37075 Göttingen, Germany

* Corresponding author. Tel.: +49 551 3899 308; fax: +49 551 3899 359. Email address: spiess{at}em.mpg.de

Received 30 May 2005; revised 21 October 2005; accepted 31 October 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Objective: Urocortin II (UcnII), a peptide of the corticotropin-releasing factor (CRF) family, exerts profound actions on the cardiovascular system. Direct effects of UcnII on adult cardiomyocytes have not been evaluated before. Our aim was to characterize functional effects of UcnII on cardiomyocytes and to elucidate the underlying signaling pathway(s) and cellular mechanisms.

Methods: Rabbit ventricular cardiomyocytes were stimulated at 0.5 Hz (22–25 °C). Unloaded cell shortening (FS, edge detection), [Ca2+]i transients (Fluo-4), and L-type Ca2+ currents (ICa, whole-cell patch clamping) were measured. Sarcoplasmic reticulum (SR) Ca2+ load was assessed by rapid application of caffeine (20 mmol/L).

Results: UcnII increased cell shortening and accelerated relaxation in a time- and concentration-dependent manner (EC50: 10.7 nmol/L). The inotropic effect of UcnII was maximal at 100 nmol/L (35% ± 11% increase in FS, n=8, P<0.05). The inotropic and lusitropic actions of UcnII were largely eliminated by inhibition of CRF2 receptors (10 nmol/L antisauvagine-30, n=5) or protein kinase A (PKA, 500 nmol/L H-89, n=5). UcnII increased [Ca2+]i transient amplitude (by 63% ± 35%, n=7, P<0.05) and decreased the time constant for decay (from 800 ± 63 to 218 ± 27 ms, n=7, P<0.001). UcnII also increased SR Ca2+ load (by 19% ± 7%, n=7, P<0.05) and fractional Ca2+ release (from 57% ± 7% to 98% ± 2%, n=7, P<0.01). ICa was augmented by 32.7% ± 10.0% (n=9, P<0.05) and the ICa–V relationship was shifted by –15 mV during UcnII treatment.

Conclusion: UcnII exerts positive inotropic and lusitropic effects in cardiomyocytes via CRF2 receptor-mediated stimulation of PKA which augments ICa and SR Ca2+ load to increase SR Ca2+ release and [Ca2+]i transients.

KEYWORDS Peptide hormones; Myocytes; E–c coupling; Protein kinase A; SR (function)


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Corticotropin-releasing factor (CRF) is a 41 amino acid peptide that plays important roles in the regulation of the hypothalamic–pituitary–adrenal axis and in endocrine, behavioral, and autonomic responses to stress [1,2]. Two G protein-coupled CRF receptors encoded by distinct genes have been cloned. CRF receptor type 1 is mainly expressed in the brain, whereas CRF receptor type 2 (CRF2) is found also in the gastrointestinal tract, skeletal muscle and heart [3].

The CRF family comprises a number of distinct peptides including urocortin I, II, and III. While urocortin I can activate both CRF receptors, urocortin II and III are selective agonists for CRF2 [4,5]. Urocortins and CRF2 are expressed throughout the cardiovascular system [6,7]. Expression levels may be altered in human cardiovascular disease [8–10], suggesting important physiological and pathophysiological roles of urocortins and their cognate CRF2. Evidence from animal models indicates that urocortins exert profound hemodynamic effects in vivo [11–13], including increases in heart rate and cardiac contractility and vasodilation. These beneficial cardiovascular actions of urocortins were also present in experimental models of heart failure [13,14], suggesting a potential use for treatment of the disease. In this regard, the use of CRF2-selective agonists, such as urocortin II (UcnII), would be preferable over unselective CRF agonists, such as urocortin I, as the former lack the concomitant CRF1-mediated activation of the stress hormone ACTH [9].

Despite recent advances in our understanding of the cardiovascular actions of the peripheral urocortin–CRF2 system in health and disease, the cellular mechanisms underlying the UcnII-mediated changes in cardiac function are largely unknown. Direct effects of UcnII on cardiomyocytes have not been characterized in detail and it remains to be shown which of the complex cardiovascular effects observed in vivo are caused by direct effects on the myocardium rather than indirect effects mediated through decreases in peripheral resistance or increases in coronary blood flow. Therefore, the aim of the present study was to characterize in detail the effects of UcnII on the contractile activity of ventricular myocytes isolated from hearts of adult rabbits. Employing contractility measurements, confocal Ca2+ imaging, and patch clamp recordings in conjunction with pharmacological interventions, we identified receptor subtype, signal transduction pathway, and the cellular mechanisms mediating the UcnII-dependent changes in contractility. The results revealed that UcnII exerted profound positive inotropic and lusitropic actions via activation of CRF2. CRF2 activation stimulated protein kinase A (PKA) activity to increase Ca2+ influx via L-type Ca2+ channels (ICa) and Ca2+ sequestration in the SR mediated by the SR Ca2+ pump (SERCA).


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusions
 References
 
2.1 Myocyte isolation and contractility measurements
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Ventricular myocytes were isolated from adult rabbit hearts as described previously [15].

A dish containing freshly isolated ventricular cardiomyocytes was positioned on the stage of an inverted microscope. Cells were bathed in Tyrode solution containing (in mmol/L): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, pH 7.4 (NaOH). Cardiomyocytes were field-stimulated at 0.5 Hz. Cell length was measured by video edge detection (Crescent Electronics, Sandy, UT, USA). Data acquisition and analysis was performed using FeliX software (Photon Technology International, USA). All experiments were conducted at room temperature (22–24 °C). Contractile parameters analyzed included: diastolic and systolic cell length, unloaded fractional shortening (FS), maximal rates of cell shortening (+dL/dt) and relengthening (–dL/dt), and time-to-peak shortening (TTP).

2.2 Electrophysiology
An EPC9 patch clamp amplifier (HEKA, Germany) was used to deliver voltage clamp pulses in whole-cell mode. Pipette solution contained (in mmol/L): 135 CsCl, 2 MgCl2, 10 HEPES, 10 EGTA, 4 MgATP, pH 7.2 (adjusted with tetraethylammonium hydroxide). The pipette resistance was 2–4 M{Omega}. The bath solution contained (in mmol/L): 135 NaCl, 5 CsCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, pH 7.4 (adjusted with NaOH). L-type Ca2+ currents (ICa) were identified as nifedipine-sensitive inward currents (n=4) and measured every 2 s as the difference between the peak and the late current during a 300 ms step from –45 to +10 mV.

2.3 Confocal Ca2+ measurements
[Ca2+]i transients were recorded on a Nipkow disc-based confocal imaging setup (VisiTech International Ltd., UK) [16]. The setup used an XR/MEGA-10 ICCD camera (Stanford Photonics Inc., USA) with a temporal resolution of 120 Hz. Cells were loaded with Fluo-4/AM (Molecular Probes; 8 µmol/L; 25 min loading; >15 min de-esterification). Myocytes were field-stimulated at 0.5 Hz. Fluo-4 was excited by the 488 nm line of an argon-ion laser and emitted fluorescence collected at >505 nm. Changes of [Ca2+]i are expressed as changes of normalized fluorescence, F/F0, where F0 denotes the resting fluorescence at the beginning of an experiment.

2.4 Drugs
UcnII and antisauvagine-30, a selective CRF2 antagonist [17], were synthesized and analyzed at the Department of Molecular Neuroendocrinology (Max Planck Institute for Experimental Medicine, Göttingen) [18]. Caffeine, H-89, and U-73122 were from Sigma-Aldrich, Germany.

2.5 Application of drugs
Because of limited availability of UcnII and antisauvagine-30, cells were not superfused with Tyrode solution and all drugs were applied directly to the bath solution (except for caffeine experiments, see below) using a needle attached to a syringe positioned close (~5 mm) to the cell under study. Control experiments revealed that the magnitude and time course of the positive inotropic effect of UcnII were not significantly affected by this means of application when compared with cells directly superfused with Tyrode solution (not shown).

In experiments using rapid application of caffeine to assess SR Ca2+ content (Fig. 6), cardiomyocytes were superfused directly with Tyrode solution by means of a gravity-driven superfusion pipette. The pipette was positioned in close proximity (~100–300 µm) to the cells under investigation. Laminar solution flow was ~3 mL/min and solution exchange was 90% complete within ~1 s as determined by the fluorescence change evoked by fluorescein-containing Tyrode solution.


Figure 6
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  		Fig. 6 Urocortin II elevates SR Ca2+ load and fractional Ca2+ release. A, Original recordings of [Ca2+]i transients evoked by electrical stimulation or rapid application of 20 mmol/L caffeine to assess SR Ca2+ load. Recordings were obtained before (left; control, recovery) and after 10 min exposure (right) of the myocyte to 100 nmol/L UcnII. B, Comparison of peak [Ca2+]i transient (left), peak caffeine transient (middle), and fractional SR Ca2+ release (right) before (ctrl, open bars) and after (UcnII, filled bars) 10 min application of UcnII. n=7 myocytes; * = P<0.05, ** = P<0.01 versus control.

 
2.6 Data analysis and statistics
Average data are expressed as the mean ± SEM. Differences between groups were evaluated by Student's t-tests. P ≥ 0.05 was considered to indicate significance.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusions
 References
 
3.1 UcnII enhances cardiomyocyte contractility
Fig. 1A shows recordings of cell shortening of a ventricular cardiomyocyte stimulated at 0.5 Hz and challenged with 100 nmol/L UcnII. Following application of the peptide, myocyte contractility was enhanced progressively. Comparison of twitches obtained at the time of UcnII application (a) and after 25 min in the presence of the peptide (b) revealed that UcnII increased the shortening amplitude by 5.5 µm or to 134% of the initial control (a) and, in addition, altered the twitch kinetics (right panel). TTP was reduced and the +dL/dt and –dL/dt were increased with UcnII. Fig. 1A also shows that UcnII decreased diastolic cell length by ~8 µm.


Figure 1
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  		Fig. 1 Urocortin II enhances contractility in rabbit ventricular myocytes. A, Recording of cell shortening of a myocyte treated with 100 nmol/L UcnII. Resting cell length at the beginning of the recording was 186 µm. Right, Superimposed shortening traces (aligned to resting cell length) obtained at the beginning (a) and end (b) of the experiment. B, Mean time course of fractional shortening during 0.5 Hz stimulation in myocytes treated with UcnII (100 nmol/L, circles) or isoprenaline (10 nmol/L, squares). C, Comparison of time-to-peak (TTP) and maximal shortening (+dL/dt) and relengthening (–dL/dt) velocities following 15 min UcnII treatment versus initial control (ctrl). B, C Mean ± SEM of n=5–8 myocytes; *, ** = P<0.05, P<0.01 versus ctrl; # = P<0.05 versus UcnII.

 
Fig. 1B and C summarizes the effects of 100 nmol/L UcnII on FS (Fig. 1B) and twitch kinetics (Fig. 1C). UcnII increased FS to 135% ± 11% of the initial control (from 12.8% ± 0.9% to 17.9% ± 1.4% after 15 min; Fig. 1B; n=8; P<0.05). The peptide also accelerated shortening and relaxation. TTP of the twitch was decreased, whereas +dL/dt and –dL/dt were increased after 15 min UcnII (Fig. 1C, n=8, P<0.05 for all values). Moreover, UcnII caused a reduction of diastolic cell length by 2.9% ± 0.7% (n=8, P<0.05). Comparison with time-matched control cells (n=12) revealed the same results (Table 1; control versus UcnII). Taken together, these findings indicate that UcnII exerted clear positive inotropic and positive lusitropic effects in isolated rabbit ventricular myocytes.


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  		Table 1 Kinetic parameters of unloaded cell shortening

 
The time course of the UcnII-induced positive inotropic effect was rather slow with a maximal response after 15 min. This was a consistent finding and also observed in cells directly superfused with UcnII-containing solution (not shown, n=5). For comparison, the time course of the positive inotropic effect of an equi-effective concentration of bath-applied isoprenaline (10 nmol/L) was studied. Isoprenaline is a β-adrenergic agonist that acts via the cAMP–PKA cascade, the same signaling pathway that is thought to underlie the positive inotropic effect of UcnII (see below). Fig. 1B shows that 10 nmol/L isoprenaline increased cell shortening to a similar extent but with a much faster time course than UcnII.

3.2 Concentration dependence of the positive inotropic effect of UcnII
To determine the concentration dependence of the positive inotropic effect of UcnII, myocytes were exposed to various concentrations of UcnII (1–500 nmol/L) and FS 15 min after application of UcnII was taken as an index of contractility. FS was normalized to the initial control obtained at the beginning of the experiment. Untreated cells showed a decrease of FS to 77% ± 4% (n=12) of the initial control (base of the curve in Fig. 2B). By contrast, UcnII prevented this rundown and increased FS in a concentration-dependent manner. Original recordings from three different myocytes are presented in Fig. 2Aa–c. Increasing [UcnII] from 1 to 10 nmol/L, and to 100 nmol/L increased FS from 84% to 112%, and to 140%, respectively, of the initial control (ctrl). Average values obtained from 5–8 ventricular myocytes at each concentration (1, 10, 30, 100, 500 nmol/L) and the resulting concentration–response curve are shown in Fig. 2B. The maximal positive inotropic effect was observed at 100 nmol/L UcnII and amounted to 135% ± 11% of the initial control (n=8, P<0.05). The EC50 value and the Hill coefficient were 10.7 nmol/L and 1.4, respectively.


Figure 2
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  		Fig. 2 Concentration dependence of the positive inotropic effect of urocortin II. A, Original recordings of cell shortening obtained before (ctrl) and 15 min after application of 1 nmol/L (a), 10 nmol/L (b), and 100 nmol/L (c, d) UcnII. In Ad, UcnII was applied in the presence of 10 nmol/L antisauvagine-30. Shortening traces were aligned to diastolic cell length. Resting cell lengths under control conditions were 159 µm (Aa), 153 µm (Ab), 145 µm (Ac), and 209 µm (Ad). B, Fractional shortening (% of control; mean ± SEM of n=5–8 cells) as a function of UcnII concentration in the absence (circles) and presence (squares) of 10 nmol/L antisauvagine-30. The curve is a fit of the Hill equation to the data yielding an EC50 value of 10.7 nmol/L UcnII and a Hill coefficient of 1.4. # = P<0.05 versus baseline; * = P<0.05 versus UcnII only.

 
3.3 The positive inotropic effect of UcnII is mediated via CRF2
UcnII binds with high affinity to the CRF2 [5]. To test whether the positive inotropic effect of UcnII in rabbit ventricular myocytes was mediated via CRF2, UcnII (100 nmol/L) was applied in the presence of 10 nmol/L antisauvagine-30, a selective antagonist of CRF2 [17]. Fig. 2Ad shows that, in the presence of antisauvagine-30, UcnII did not enhance myocyte contractility. Cell shortening even decreased during UcnII treatment, which is in clear contrast to the increase observed in the absence of the CRF2 antagonist (Fig. 2Ac). Antisauvagine-30 (10 nmol/L) reduced the positive inotropic effect of UcnII at both 10 nmol/L (n=5) and at 100 nmol/L (n=5; P<0.02; Fig. 2B, squares). These results indicate that UcnII enhances contractility in rabbit ventricular myocytes via activation of CRF2.

3.4 Signaling pathways involved in the positive inotropic effect of UcnII
CRF2 couples to Gs–cAMP–PKA signaling [8]. Therefore, we reasoned that the positive inotropic and lusitropic actions of UcnII were mediated by stimulation of PKA and conducted experiments in the presence of H-89, an inhibitor of PKA. Furthermore, to probe for the possible involvement of phospholipase C (PLC) signaling (with subsequent generation of inositol-1,4,5-trisphosphate and activation of the diacylglycerol–protein kinase C cascade), additional experiments were carried out in the presence of the PLC inhibitor U-73122. In the presence of 500 nmol/L H-89 (n=5) or 1 µmol/L U-73122 (n=5), FS declined by ~25–30% within 20 min, which was not significantly different from the rundown of FS observed in untreated control myocytes (n=12, not shown). Fig. 3 illustrates two recordings of the inotropic effects of 100 nmol/L UcnII applied 15 min after inhibition of PKA or PLC by 500 nmol/L H-89 (Fig. 3A) and 1 µmol/L U-73122 (Fig. 3B), respectively. In the presence of H-89, UcnII did not enhance cell shortening (Fig. 3A). Instead, a time-dependent rundown of shortening amplitude was observed. Superposition of shortening traces obtained at the beginning (a) and at the end (b) of the 20 min recording period revealed that shortening amplitude decreased under UcnII. Twitch kinetics, however, were hardly altered. By contrast, U-73122 did not affect the ability of UcnII to enhance cell shortening (Fig. 3B). In the presence of U-73122, cell shortening was increased to 174% of the initial control, and shortening as well as relengthening were accelerated by UcnII. Average values presented in Table 1 confirm these findings. H-89, but not U-73122, could largely reduce the increase in FS and the acceleration of the twitch kinetics elicited by UcnII.


Figure 3
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  		Fig. 3 Inhibition of PKA by H-89 reduces the positive inotropic effect of urocortin II. Original recordings of cell shortening following application of 100 nmol/L UcnII either in the presence of 500 nmol/L H-89 to inhibit PKA (A) or 1 µmol/L U-73122 to inhibit PLC (B). H-89 and U-73122 were applied for 15 min before UcnII exposure was begun. Resting cell lengths under control conditions were 135 µm (A) and 152 µm (B). Right, Superimposed shortening traces (aligned to resting cell length) obtained at the beginning (a) and at the end (b) of the 20 min recordings.

 
3.5 UcnII augments [Ca2+]i transients
To analyze whether altered [Ca2+]i regulation underlies the UcnII-induced changes in contractility, we measured electrically evoked [Ca2+]i transients. Fig. 4A shows [Ca2+]i transients of a ventricular myocyte recorded before and during exposure to 100 nmol/L UcnII. The peptide induced a time-dependent increase in the [Ca2+]i transient amplitude that was maximal 10–15 min following UcnII application. In addition to systolic [Ca2+]i, there was also an increase in diastolic [Ca2+]i which was most pronounced at 15–20 min. Finally, UcnII caused a dramatic acceleration of the decaying phase of the [Ca2+]i transient. Average data from seven cells obtained 15 min after UcnII application are presented in Fig. 4B. Both diastolic and systolic [Ca2+]i increased significantly under UcnII from 1.0 ± 0.0 to 1.18 ± 0.05 F/F0 (n=7; P<0.02) and from 2.04 ± 0.10 to 2.69 ± 0.17 F/F0 (n=7; P<0.02), respectively. TTP [Ca2+]i transient was unchanged (~300 ms), but the time constant {tau} for the [Ca2+]i transient decay was greatly reduced from 800 ± 63 to 218 ± 27 ms (n=7; P<0.001). The effects of UcnII on the [Ca2+]i transient were blocked completely by pre-application (15 min) of the PKA inhibitor H-89 (500 nmol/L, n=6, not shown) suggesting that they were mediated by cAMP–PKA signaling.


Figure 4
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  		Fig. 4 Urocortin II increases the [Ca2+]i transient. A, [Ca2+]i transients recorded immediately before and following application of 100 nmol/L UcnII. B, Average values for diastolic [Ca2+]i, systolic [Ca2+]i, time-to-peak, and the time constant {tau} for the decay of the [Ca2+]i transient (from left to right) recorded before (ctrl, open bars) and 15 min after UcnII application (black bars). n=7 myocytes, N.S. = not significant, ** = P<0.01, *** = P<0.001 versus ctrl.

 
3.6 UcnII increases L-type Ca2+ current and SR Ca2+ content
In order to identify the possible sources of the [Ca2+]i increase during UcnII treatment, we measured the L-type Ca2+ current (ICa) by means of the patch clamp technique and SR Ca2+ content using rapid caffeine applications.

ICa was elicited by voltage steps from –45 to +10 mV. Untreated control cells exhibited a steady rundown in ICa that averaged 45% in 20 min (Fig. 5F, open circles, n=8). By contrast, in the presence of UcnII there was an increase in ICa that started after ~10 min and reached a maximum between 15 and 20 min (Fig. 5F, filled circles, n=9). Original current traces of a ventricular myocyte exposed to 100 nmol/L UcnII are illustrated in Fig. 5A and B. Fig. 5A shows ICa before the onset (control) and Fig. 5B during the maximum of the UcnII effect, respectively. UcnII augmented ICa from –0.69 nA (Fig. 5A, control) to –1.03 nA (Fig. 5B, 100 nmol/L UcnII) or by 49%. In a total of 9 myocytes, UcnII increased ICa by 32.7% ± 10.0% (P<0.05) from –3.30 ± 0.39 pA/pF (at 10 min) to –4.39 ± 0.68 pA/pF (at 20 min; P<0.05). Compared to time-matched controls (Fig. 5F), the increase in ICa at 20 min averaged 60% (–4.39 ± 0.68 pA/pF in the presence of UcnII versus –2.74 ± 0.54 pA/pF in control (–UcnII), P<0.05). To study the voltage dependence of this UcnII action, ICa–V relationships were recorded. Original current traces acquired during voltage steps from –45 mV to test potentials of –30, –20, –10, 0, +10, and +20 mV are illustrated in Fig. 5C (control) and Fig. 5D (UcnII). Average normalized ICa–V relationships from a total of 9 untreated control cells and 5 cells treated with UcnII are shown in Fig. 5E. UcnII shifted the maximum of the ICa–V relationship by –15 mV from +10 to –5 mV.


Figure 5
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  		Fig. 5 Urocortin II increases ICa in a voltage-dependent manner. A, B ICa after voltage steps from –45 to +10 mV in a myocyte (CM: 141 pF) exposed to 100 nmol/L UcnII. A, control; B, UcnII. C, D, Current traces in a cardiomyocyte recorded before (C) and after (D) the onset of the UcnII effect. E, Normalized mean ICa–V relationships of cells challenged with UcnII (filled circles, n=5) and time-matched control cells (open circles, n=9). F, Time course of ICa current density (normalized to ICa at t=0 min, ICa_0) in myocytes treated with UcnII (filled circles, +UcnII, n=9) and untreated controls (open circles, –UcnII, n=8). ICa at t=0 min amounted to –4.97 ± 0.54 pA/pF (–UcnII) and to –4.93 ± 0.32 pA/pF (+UcnII), respectively. * = P<0.05, ** = P<0.01 versus control.

 
Increased Ca2+ influx during the action potential is expected to increase the [Ca2+]i transient by at least two mechanisms. First, it provides more trigger Ca2+ to induce larger Ca2+ release from the SR. Second, it contributes some extra Ca2+i that can be stored in the SR to elevate SR Ca2+ load, which in turn increases the amount and the fraction of Ca2+ released from the SR [19]. Therefore, we assessed whether UcnII increased SR Ca2+ content in rabbit ventricular myocytes. SR Ca2+ content was estimated as the transient [Ca2+]i increase evoked by rapid application of 20 mmol/L caffeine. SR Ca2+ content was measured before (control) and during exposure of the cells to 100 nmol/L UcnII. Fig. 6A shows original [Ca2+]i transients of a myocyte challenged with UcnII. Before the application of the peptide, electrically evoked [Ca2+]i transients exhibited systolic [Ca2+]i values of ~2.0 F/F0. Caffeine increased [Ca2+]i to ~3.9 F/F0. Thus, under control conditions, fractional SR Ca2+ release, defined as the amplitude of the [Ca2+]i transient normalized to the amplitude of the caffeine transient, amounted to 34% (1.0/2.9). After washout of caffeine by superfusion (5 min) with Tyrode solution, the [Ca2+]i transient recovered completely (recovery). UcnII caused a large increase of both the [Ca2+]i and the caffeine transient to ~5.5 and ~5.6 F/F0, respectively, indicating that also fractional release was enhanced to 98% (4.5/4.6). Average values from 7 cells presented in Fig. 6B confirm that UcnII increased the peaks of the [Ca2+]i transients evoked by electrical stimulation and caffeine by 75% ± 23% and 19% ± 7%, respectively (P<0.05). Moreover, fractional SR Ca2+ release was enhanced from 57% ± 7% to 98% ± 2% (P<0.01). It should be noted that under the current experimental conditions the Na+/Ca2+ exchanger was not blocked during caffeine application. Therefore, it is likely that SR Ca2+ content was slightly underestimated and, in turn, that fractional SR Ca2+ release was overestimated. Nevertheless, the data clearly indicate that UcnII caused an increase in fractional SR Ca2+ release.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusions
 References
 
The cellular mechanisms underlying the in vivo hemodynamic effects of UcnII observed in previous studies [11–13] have remained elusive. Direct effects of UcnII on cardiomyocyte contractility have not been demonstrated before. Therefore, we characterized the signaling pathways and cellular mechanisms involved in the inotropic and lusitropic actions of UcnII in rabbit ventricular myocytes. Our results provide evidence that UcnII acts via CRF2 to stimulate PKA activity. PKA, in turn, increases L-type Ca2+ current and elevates SR Ca2+ content via increased SERCA activity to augment [Ca2+]i transients and enhance cardiomyocyte contractility.

4.1 UcnII enhances contractility of ventricular myocytes
The present study is the first to provide direct evidence for positive inotropic and lusitropic effects of UcnII in adult ventricular myocytes. UcnII significantly increased FS and accelerated myocyte shortening and relaxation. These effects were concentration-dependent. Maximal and half-maximal positive inotropic effects were observed at 100 and 10.7 nmol/L UcnII, respectively. The positive inotropic and lusitropic effects in isolated ventricular myocytes compare well with the UcnII-induced increases in left ventricular function in mice [13] and suggest that the latter were caused mainly by direct effects of UcnII on the ventricular myocardium.

4.2 Receptor subtype and signaling pathways
Until recently, few data have been available regarding the cellular signaling mechanisms underlying the inotropic and lusitropic actions of UcnII in the heart. Cardiomyocytes express CRF2 [7], which couples to the Gs–cAMP–PKA cascade. Studies in cultured rat neonatal myocytes have shown that activation of CRF2 increases cAMP [8]. Furthermore, UcnII is a selective ligand of CRF2 [5]. Therefore, we hypothesized that the inotropic and lusitropic actions of UcnII reported in the present study were mediated via CRF2 activation leading to increased PKA activity. Indeed, pretreatment of myocytes with either antisauvagine-30, a selective CRF2 antagonist, or H-89, an inhibitor of PKA, largely reduced the inotropic and lusitropic effects of UcnII. By contrast, inhibition of the Gq-PLC pathway (using the PLC inhibitor U-73122), another signaling cascade frequently activated by G protein-coupled receptors, did not affect the response of the myocytes to UcnII. Taken together, these results indicate that UcnII exhibited its positive inotropic and lusitropic effects via CRF2-mediated stimulation of PKA.

4.3 Cellular mechanisms underlying functional effects of UcnII
The cellular mechanisms underlying the inotropic and lusitropic actions of UcnII in adult cardiomyocytes have not been studied before. Stimulation of PKA activity causes phosphorylation of several key proteins involved in cardiac excitation–contraction coupling including the L-type Ca2+ channel, the ryanodine receptor Ca2+ release channel (RyR), phospholamban and troponin I [20]. Troponin I phosphorylation, however, does not play a significant role for the functional effects of PKA under unloaded conditions [21]. Accordingly, we focused on possible alterations in Ca2+ handling as a likely cause for the functional effects of UcnII. UcnII increased electrically evoked [Ca2+]i transients with a time course similar to the positive inotropic effect. The increase in [Ca2+]i transients was paralleled by elevations of SR Ca2+ content and fractional SR Ca2+ release. The latter were caused, in part, by increased Ca2+ influx via L-type Ca2+ channels. PKA phosphorylation of cardiac L-type Ca2+ channels augments ICa and induces a leftward shift of the ICa–V relationship [22]. The same changes in ICa were observed in the present study after treatment of myocytes with UcnII, suggesting that they were caused by PKA-dependent phosphorylation of the Ca2+ channels. The voltage-dependent increase in ICa elicited by UcnII is at variance with a recent study in rat ventricular myocytes where urocortin induced a concentration-dependent decrease in ICa (by 35% at 100 nmol/L) which was voltage-independent [23]. Whether this difference reflects true differences between the actions of urocortin and UcnII or species-dependent differences in the effects of urocortins remains to be determined.

The functional effects of PKA phosphorylation of RyR on SR Ca2+ release in intact myocytes are still under debate [24,25]. Recent evidence indicates that the primary effect of PKA phosphorylation of RyR (when trigger Ca2+ and SR Ca2+ load are controlled and matched) is to increase the rate rather than the amount or fraction of SR Ca2+ release [25]. Our experiments do not permit any conclusions as to this issue, since we studied UcnII effects on Ca2+ homeostasis in intact myocytes where conditions of controlled trigger Ca2+ and SR Ca2+ load could not be met. It should be pointed out though, that any alterations in RyR activity can only translate into steady-state changes of the [Ca2+]i transient when concomitant changes in SR Ca2+ load occur [26] as observed in the present study.

Several lines of evidence indicate that increased L-type Ca2+ current in combination with increased SERCA activity mediated by phospholamban phosphorylation contributed to the elevations of SR Ca2+ load and fractional SR Ca2+ release and that these effects were mediated by stimulation of PKA. First, inhibition of PKA activity abolished the positive inotropic and lusitropic effects of UcnII. Second, ICa was increased by UcnII in a way characteristic for PKA-dependent phosphorylation of the channel [22]. Third, acceleration of the [Ca2+]i transient decay after stimulation of PKA is mediated in large part by stimulation of SERCA [27]. The latter occurs predominantly through decreased inhibition of SERCA by PKA-phosphorylated phospholamban. Increased ICa and SERCA activity combine to elevate SR Ca2+ load. Elevated SR Ca2+ load per se [19] as well as increased ICa then lead to augmented fractional Ca2+ release from the SR and increased [Ca2+]i transients.

4.4 Unresolved questions
The observed time course of the functional effects of UcnII was slow reaching a maximum only after ~15 min. Direct comparison with β-adrenergic signaling revealed that UcnII effects lagged behind the isoprenaline effects by at least 5 min. Thus, although both CRF2 and β-adrenergic receptors couple to cAMP–PKA signaling, their time courses are strikingly different. Furthermore, despite clear evidence for an involvement of the cAMP–PKA cascade in the functional effects of UcnII, there was an increase in diastolic [Ca2+]i and a decrease in diastolic cell length. This can not be readily accounted for by cAMP–PKA signaling. It is known though that CRF receptors in cardiomyocytes couple to signaling cascades other than the cAMP–PKA pathway. For example, urocortin-dependent stimulation of p44/42-MAPK [28] or PKCepsilon [29] has been reported. Therefore, it is possible that in our experiments, in addition to the cAMP–PKA cascade, other signaling pathways were activated by UcnII in parallel to contribute to the changes in diastolic [Ca2+]i and cell length as well as the delayed inotropic response. Future studies will have to test this hypothesis and evaluate the possible involvement of other signaling pathways in the inotropic effects of UcnII in cardiomyocytes.


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
References
 
UcnII produced acute positive inotropic and lusitropic effects in adult rabbit ventricular myocytes. These effects were mediated via activation of CRF2 and subsequent stimulation of PKA activity. PKA augmented ICa and SERCA-mediated Ca2+ uptake into the SR, thus elevating SR Ca2+ content and the [Ca2+]i transient to mediate the positive inotropic effect. The positive lusitropic effect, on the other hand, resulted in large part from the accelerated decay of the [Ca2+]i transient.


   Acknowledgements
 
The authors are grateful for support from the Deutsche Forschungsgemeinschaft (DFG PI 414, BP and JK), the Ernst und Berta Grimmke-Stiftung, Düsseldorf (JK and BP), and the Kompetenznetz Herzinsuffizienz, Teilprojekt 8 (BP and FRH).


   Notes
 
1 These authors contributed equally to this work. Back

Time for primary review 17 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 

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