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Cardiovascular Research 2004 63(4):673-681; doi:10.1016/j.cardiores.2004.05.009
© 2004 by European Society of Cardiology
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Copyright © 2004, European Society of Cardiology

Investigation of signaling pathways that mediate the inotropic effect of urotensin-II in human heart

Fraser D Russell* and Peter Molenaar

Department of Medicine, University of Queensland, The Prince Charles Hospital, Rode Road Chermside, 4032 Queensland, Australia

* Corresponding author. Tel.: +61-7-3350-8792; fax: +61-7-3359-2173. Email address: russell{at}medicine.uq.edu.au

Received 25 January 2004; revised 29 April 2004; accepted 16 May 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: This study investigated signaling pathways that may contribute to the potent positive inotropic effect of human urotensin-II (hU-II) in human isolated right atrial trabeculae obtained from patients with coronary artery disease. Methods: Trabeculae were set up in tissue baths and stimulated to contract at 1 Hz. Tissues were incubated with 20 nM hU-II with or without phorbol 12-myristate 13-acetate (PMA, 10 µM) to desensitize PKC, the PKC inhibitor chelerythrine (10 µM), 10 µM 4{alpha}-phorbol that does not desensitize PKC, the myosin light chain kinase inhibitor wortmannin (50 nM, 10 µM), or the Rho kinase inhibitor Y-27632 (0.1–10 µM). Activated RhoA was determined by affinity immunoprecipitation, and phosphorylation of signaling proteins was determined by SDS-PAGE. Results: hU-II caused a potent positive inotropic response in atrial trabeculae, and this was concomitant with increased phosphorylation of regulatory myosin light chain (MLC-2, 1.8±0.4-fold, P<0.05, n=6) and PKC{alpha}/βII (1.4±0.2-fold compared to non-stimulated controls, P<0.05, n=7). Pretreatment of tissues with PMA caused a marked reduction in the inotropic effect of hU-II, but did not affect hU-II-mediated phosphorylation of MLC-2. The inotropic response was inhibited by chelerythrine, but not 4{alpha}-phorbol or wortmannin. Although Y-27632 also reduced the positive inotropic response to hU-II, this was associated with a marked reduction in basal force of contraction. RhoA.GTP was immunoprecipitated in tissues pretreated with or without hU-II, with findings showing no detectable activation of RhoA in the agonist stimulated tissues. Conclusions: The findings indicated that hU-II increased force of contraction in human heart via a PKC-dependent mechanism and increased phosphorylation of MLC-2, although this was independent of PKC. The positive inotropic effect was independent of myosin light chain kinase and RhoA-Rho kinase signaling pathways.

KEYWORDS Urotensin-II; Contractile function; Protein kinase C; Signal transduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Human urotensin-II (hU-II) is an endogenous peptide ligand that interacts with the recently cloned human G-protein-coupled receptor, UT-II. The UT-II receptor system is widely distributed throughout the body, including the cardiovascular system where hU-II stimulates contraction of vascular smooth muscle, actin-cytoskeleton organization and proliferation of smooth muscle cells via a RhoA/Rho kinase-dependent mechanism [1]. hU-II also stimulates positive inotropy in isolated human myocardium [2]; however, the signaling mechanism involved in this response is unknown.

Stimulation of UT receptors with hU-II results in increased coupling to the guanine nucleotide binding protein, G{alpha}q. The amplitude of the Ca2+ transient that follows G{alpha}q-coupled receptor stimulation is dissociated from the amplitude of the contractile response. For example, the amplitude of Ca2+ transient was much lower in rabbit ventricular myocytes stimulated with endothelin-1 than with isoprenaline which activates G{alpha}s-protein-coupled β-adrenoceptors despite the use of concentrations that produced comparable increases in myocyte cell shortening [3]. These results suggest that the inotropic response to peptides such as endothelin-1 and hU-II may in part be mediated by sensitization of myofilaments to Ca2+.

Regulatory myosin light chain (MLC-2) reduces tension development in striated (skeletal and cardiac) muscle cells, and this inhibitory effect is diminished following phosphorylation by PKC [4,5] or myosin light chain kinase [4]. At sub-maximal concentrations of Ca2+, phosphorylation of MLC-2 orientates an increased number of cross-bridges in close proximity to actin [6] and an increased rate of formation of cross-bridges in a force-generating state [7]. A role of PKC [8,9] or myosin light chain kinase-dependent phosphorylation of MLC-2 [10,11] has been implicated in the positive inotropic effects of endothelin-1 and phenylephrine, respectively. In addition, phosphorylation of myosin light chain phosphatase (MLCP) by RhoA-GTP-activated Rho kinase may also increase force of contraction in cardiac muscle by preventing dephosphorylation of MLC-2 [12]. Consistent with this hypothesis, Rho kinase inhibitor-sensitive positive inotropic responses were observed to phenylephrine [10] and 17-phenyl trinor prostaglandin E2 [13] in isolated rat cardiac tissue. Together, the findings indicate a role of MLC-2 in the modulation of cardiac contraction to G{alpha}q-coupled receptor agonists. In this study, we investigated whether hU-II increases phosphorylation of MLC-2, and whether signaling pathways that are known to modulate MLC-2 also have a critical role in mediating the cardiostimulatory effect of hU-II.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Collection and preparation of tissues
Human right atrial appendages were obtained from 50 patients undergoing coronary artery bypass graft surgery for coronary artery disease (see Table 1 for patient details). Atrial appendages were placed immediately in cold, pre-oxygenated (5% CO2 in oxygen) modified Krebs' solution (125 mM Na+, 5 mM K+, 2.25 mM Ca2+, 0.5 mM Mg2+, 98.5 mM Cl–, 0.5 mM SO42–, 32 mM HCO3–, 1 mM HPO42–, 0.04 mM EDTA) and transported to the laboratory within ~5 min. Atrial trabeculae were dissected free from the appendage in continuously oxygenated modified Krebs' solution, with preference for long muscle preparations (typically ~5–10 mm). Trabeculae were set up in 50 ml tissue baths in oxygenated modified Krebs' solution supplemented with 15 mM Na+, 5 mM fumarate, 5 mM pyruvate, 5 mM L-glutamate, and 10 mM glucose at 37°C, stretched to 50% of Lmax, and driven with square wave pulses (5 ms duration, just over threshold voltage, 1 Hz). Tissues were not used if there was visual evidence of ischaemia, determined by pale tissue color. Trabeculae were pre-incubated with 200 nM (–)-propranolol (1 h, 37 °C), to block the effects of endogenously released noradrenaline at β1- and β2-adrenoceptors. Samples were collected with approval from the human ethics committees of The Prince Charles Hospital (EC2134) and The University of Queensland (2001002160). The investigation conforms to principles outlined in the Declaration of Helsinki.


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  		Table 1 Details relating to 50 patients from whom tissues were obtained for tissue bath and SDS-PAGE experiments

 
2.2. Determination of phospho-MLC-2 and phospho-PKC
Tissues were incubated with 20 nM hU-II until equilibration of contractile response (9 min), and then snap frozen in liquid nitrogen. This concentration of hU-II was used because it was shown to produce a maximal inotropic effect in human right atrial trabeculae [2]. Untreated tissues were used for a non-stimulated control group. Epicardial connective tissue was removed, cardiac muscle was matched for mass, minced with a scalpel blade and homogenized in 0.3 ml RIPA buffer (NaCl 150 mM, Tris–HCl 20 mM, Nonidet P40 1%, sodium dodecyl sulfate 0.1%, sodium deoxycholate 25 mM, activated orthovanadate 1.0 mM, sodium fluoride 1.0 mM and 1 tablet of Complete Mini protease inhibitor, pH 7.4) for MLC-2 experiments or 0.3 ml lysis buffer (Tris–HCl 20 mM, EDTA 2 mM, EGTA 2 mM, 2-mercaptoethanol 6 mM, activated orthovanadate 1.0 mM, sodium fluoride 1.0 mM and 1 tablet of Complete Mini protease inhibitor, pH 7.4) for PKC experiments, using a Polytron PT2100 with 5 mm aggregate (5 s at setting 30, Kinematica). For measurement of phospho-MLC-2, lysates were spun (500 x g, 2 x 2 min, 4 °C) and supernatants were collected. For measurement of phospho-PKC, lysates were spun (50,000 x g, 30 min, 4 °C), supernatants were collected (cytosolic fraction) while pellets were resuspended in lysis buffer containing 1% Triton X-100 and were incubated on ice for 10 min. The samples were spun (10,000 x g, 10 min, 4 °C) and supernatants collected (particulate fraction). All samples were matched for protein concentration using the Lowry method [14] and loaded onto 12.5% or 7.5% polyacrylamide gels for separation of MLC-2 and PKC, respectively. Gels were run at 150 V for 1 h and proteins were transferred to immobilon-P membranes. Proteins were detected using antibodies to human phospho-MLC-2 (kindly provided by Dr. Neal Epstein, National Heart, Lung and Blood Institute, NIH), phospho-PKC{alpha}/βII (Thr638/641) and phospho-PKC{delta} (Ser643) (Cell Signaling Technology, MA, USA), and phospho-PKC{varepsilon} (Ser729) (Upstate Biotechnology, NY, USA).

2.3. Tissue bath studies
2.3.1. Protein kinase C (PKC)
Inotropic response to hU-II was measured in the absence (0.01% dimethyl sulfoxide) or presence of 10 µM PMA or 10 µM 4{alpha}-phorbol (6.5 h at 25 °C). Tissues were equilibrated to 37 °C over 30 min prior to the addition of 20 nM hU-II. Additional tissues were pre-incubated in the absence or presence of 10 µM chelerythrine (2.5 h at 25 °C and then equilibrated to 37 °C over 30 min) before addition of 20 nM hU-II. Experiments were completed by measuring responses to endothelin-1 (10 nM) and (–)-isoprenaline (200 µM), and by increasing Ca2+ concentration to 9.25 mM.

2.3.2. Myosin light chain kinase (MLCK)
The role of MLCK was examined by incubating tissues with 50 nM wortmannin, to selectively inhibit phosphatidyl inositol 3-kinase, or 10 µM wortmannin for additional inhibition of MLCK (20 min at 37 °C). Data were corrected for fading baseline in tissues treated with 10 µM wortmannin.

2.3.3. Rho kinase
Concentration effect curves to hU-II (60 pM–20 nM) were constructed in the absence or presence of 10 µM Y-27632 (Calbiochem, CA, USA), which was added to the bath 20 min prior to the addition of hU-II. In a separate experiment, 20 nM hU-II was added to the bath and on equilibration of response, the Rho kinase inhibitor Y-27632 was added in a cumulative concentration-dependent manner (0.1–10.0 µM). To determine whether Y-27632 had a depressant effect on basal force of contraction, Y-27632 (0.1–10.0 µM) was added to the bath in the absence of cardiostimulation by hU-II.

2.4. RhoA activation assay
Right atrial trabeculae were incubated in tissue baths in the absence or presence of 20 nM hU-II or 50 nM endothelin-1, snap frozen and homogenized as described above, using lysis buffer (Tris–HCl, 25 mM, pH 7.5; 150 mM NaCl, 5 mM MgCl2, 1% NP-40, 1 mM DTT and 5% glycerol). Activated RhoA was affinity immunoprecipitated using a pull-down assay according to manufacturers protocols (Pierce Biotechnology, IL, USA). Briefly, GST-Rhotekin-RBD (400 µg) and lysate (supernatant from 16,000 x g spin, matched for protein) was added to immobilized glutathione discs in spin columns and incubated for 1 h at 4 °C. Additional untreated samples were incubated with EDTA (10 mM, pH 8.0), and 0.1 mM GTP{gamma}S for 30 min at 30 °C, then 60 mM MgCl2 for 5 min at 4 °C, prior to incubation with GST-Rhotekin-RBD. Samples were spun, RhoA-GTP was eluted using 2 x loading buffer which was loaded onto 12% polyacrylamide gel for Western blot analysis.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Urotensin-II stimulates phosphorylation of MLC-2 and increased levels of particulate phospho-PKC{alpha}/βII
Western blot analysis was used to investigate whether activation of UT-II receptors by hU-II increases phosphorylation of myosin regulatory light chain (MLC-2) in human right atrial trabeculae. A single band was detected for phosphorylated MLC-2 and hU-II increased phosphorylation by 1.76±0.40-fold (n=6, P<0.05) compared to non-stimulated controls (Fig. 1). The translocation of cardiac PKC isoenzymes to a particulate fraction was investigated using phosphorylation specific antibodies to differentiate activated—from inactive, non-primed-PKC. Stimulation of tissues with 20 nM hU-II caused a 1.42±0.18-fold increase in PKC within the particulate fraction using a phospho-PKC{alpha}/βII (Thr638/641)-specific antibody (Fig. 1, P<0.05, n=7), which was concomitant with increased force of contraction (basal force=9.7±2.1 mN, hU-II force=13.2±3.1 mN, n=7, P<0.05). A non-significant trend for decreased phospho-PKC{alpha}/βII was observed in the cytosolic fraction (0.90±0.09-fold compared to non-stimulated control, P>0.05, n=7). Particulate phospho-PKC{varepsilon} (Ser729) levels were unchanged following acute agonist stimulation (Fig. 1, P>0.05, n=8), while phospho-PKC{delta} (Ser643) was at the limit of detection for this assay and quantitation was therefore not attempted.


Figure 1
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  		Fig. 1 Western blot showing agonist stimulated phosphorylation of regulatory myosin light chain (MLC-2) and levels of particulate phospho-protein kinase C (PKC)-{alpha}/βII and PKC{varepsilon}, compared to non-stimulated controls. hU-II increased phosphorylation of MLC-2 (P<0.05, n=6), and levels of phospho-PKC{alpha}/βII in a particulate fraction of cardiac tissue (P<0.05, n=7), compared to non-stimulated control tissues. No change in levels of particulate phospho-PKC{varepsilon} was detected (n=8, P>0.05). Values are mean±S.E.M.; *P<0.05, Student's t-test.

 
3.2. Urotensin-II increases force of contraction in human right atrium via a protein kinase C-dependent pathway
hU-II increased force of contraction to 22.1±1.1% of the response produced by Ca2+ (9.25 mM total Ca2+ concentration, n=6). The role of protein kinase C in the transduction of hU-II response was investigated by preincubating tissues with 10 µM phorbol 12-myristate 13-acetate (PMA) for 7h to desensitize PKC. PMA pretreatment of tissues caused a marked reduction in hU-II-mediated inotropic response, consistent with a role of PKC (Fig. 2A,B,C, P<0.05). Similar findings were observed for atrial tissues stimulated with another G{alpha}q-coupled receptor agonist, endothelin-1 (DMSO, 55.0±5.4%; PMA, 30.8±5.0% of 9.25 mM Ca2+; n=6, P=0.02). Consistent with the direct effect of PMA on PKC, the inotropic response to hU-II was conserved in tissues treated with 4{alpha}-phorbol for 7 h (time matched control for the PMA experiment), which does not desensitize PKC (Fig. 2E, n=5, P>0.05). The role of PKC was supported by findings showing reduced inotropic response to hU-II in tissues that were pretreated with 10 µM chelerythrine, an inhibitor of the catalytic domain of PKC (Fig. 2F, n=7, P<0.05). Responses to the β-adrenoceptor agonist (–)-isoprenaline (200 µM) were not affected by pretreatment of tissues with PMA (Fig. 2D), 4{alpha}-phorbol or chelerythrine (P>0.05, not shown). No differences in basal force were observed between control and treated tissues (Table 2). The effect of hU-II on MLC-2 phosphorylation state was investigated by preincubating tissues with 10 µM PMA. Despite a marked reduction in force of contraction in PMA pretreated tissues, levels of phospho-MLC-2 were not reduced (not shown).


Figure 2
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  		Fig. 2 Effect of desensitization of protein kinase C, and inhibition of protein kinase C, phosphatidyl inositol 3-kinase or myosin light chain kinase on human urotensin-II (hU-II) and (–)-isoprenaline mediated contraction of human right atrial trabeculae. Original recordings showing increased force of contraction to 20 nM hU-II (arrow) in a right atrial trabeculum pretreated with vehicle (dimethyl sulfoxide) (A), and reduced functional responsiveness to 20 nM hU-II (arrow) in a separate tissue from the same patient pretreated with 10 µM phorbol 12-myristate 13-acetate (PMA) for 7h (B). PMA treatment caused a marked inhibition in contractile response to 20 nM hU-II (n=6) (C) but not 200 µM (–)-isoprenaline (n=6) (D). Responses to hU-II were unaffected by treatment of tissues with 4{alpha}-phorbol (10 µM, 7 h) (n=7) (E). Chelerythrine (10 µM, 3 h) reduced hU-II-mediated contraction of human right atrial trabeculae (n=7) (F), while responses were unaffected by 50 nM and 10 µM wortmannin (n=5) (G). Values are mean±S.E.M. *P<0.05, Student's t-test. Scale bars: vertical=8.0 mN (A), 3.0 mN (B); horizontal=2 min (A, B).

 

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  		Table 2 Absolute baseline force (mN) for tissue bath studies

 
Tissues were also incubated with wortmannin at concentrations used to inhibit phosphatidyl inositol 3-kinase (PI3K, 50 nM) or PI3K and myosin light chain kinase (10 µM). The cardiostimulatory effect of 20 nM hU-II was preserved in the presence of 50 nM and 10 µM wortmannin (Fig. 2G, n=5, P>0.05). Wortmannin (10 µM) reduced basal force of contraction by 3.04±0.53 mN (n=5, P<0.05). The findings indicated that the cardiostimulatory effect of hU-II was mediated by a PKC-dependent, myosin light chain kinase-independent pathway.

3.3. Effect of the Rho kinase inhibitor Y-27632 on the cardiostimulant response to hU-II in human right atrium
To investigate whether the cardiostimulatory effect of hU-II may also involve Rho kinase-dependent phosphorylation of myosin light chain phosphatase, concentration effect curves were constructed to hU-II in the absence or presence of the Rho kinase inhibitor, Y-27632 (10 µM). In control tissues, hU-II increased force of contraction to 22.0±2.2% of the response to 9.25 mM Ca2+ (–log EC50=9.1±0.1, maximal response at 20 nM hU-II, n=3). The response to hU-II was markedly attenuated in the presence of Y-27632 (force=5.7±0.1% of the response to 9.25 mM Ca2+, n=3, P<0.05), with no effect on potency (–log EC50=9.0±0.1) (Fig. 3A).


Figure 3
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  		Fig. 3 Effect of the Rho kinase inhibitor, Y-27632 on hU-II-mediated force of contraction in human right atrium. Concentration–effect curve to hU-II showing reduced amplitude of response (P<0.05), but no change in potency in tissues pretreated with 10 µM Y-27632 (A). Y-27632 had no effect on the inotropic response to 20 nM hU-II at 0.1 µM, but reduced force of contraction at 1.0 and 10.0 µM concentrations, compared to untreated tissues. Data is expressed as a proportion of the hU-II-stimulated value (B). In tissues not exposed to agonist, Y-27632 had no effect on basal force of contraction at 0.1 µM, but reduced basal force of contraction at 1.0 and 10.0 µM concentrations, compared to untreated tissues. Data is expressed as a proportion of basal force, determined prior to addition of Y-27632 (C). An original trace shows the reduction in basal force of contraction following addition of 10 µM Y-27632 to the bath (arrow) (D). Scale bars: vertical=1.0 mN; horizontal=2 min). Values are mean±S.E.M. *P<0.05, Student's t-test.

 
The effect of Y-27632 (0.1–10 µM) on force generated by 20 nM hU-II was determined in the tissue bath preparations. Force of contraction was not affected by 0.1 µM Y-27632, but was reduced with 1.0 µM and 10.0 µM Y-27632 compared to time-dependent fade control tissues (hU-II alone) (Fig. 3B). The force of atrial contraction determined in the presence of 20 nM hU-II and after the addition of 10 µM Y-27632 (1.6±0.6 mN, n=6) was lower than basal force determined prior to the addition of hU-II to the tissue bath (3.1±0.9 mN, n=6, P<0.05). This inhibitory effect of Y-27632 on basal force of contraction was confirmed by repeating the concentration effect curve to Y-27632 in the absence of hU-II. In these tissues, force of contraction was unchanged in the presence of 0.1 µM Y-27632, but was less than basal force at 1.0 µM (Fig. 3C) and 10.0 µM Y-27632 (Fig. 3C,D), suggesting an effect of the higher concentrations of Y-27632 on mechanisms intrinsic to excitation-contraction coupling. No difference was observed for absolute reduction in force (mN) at each concentration of Y-27632 for tissues preincubated with hU-II and tissues not preincubated with hU-II (Table 3).


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  		Table 3 Comparison of the absolute reduction in force, determined after equilibration of each concentration of Y-27632, in tissues pretreated or not pretreated with hU-II

 
3.4. Effect of hU-II on activation of RhoA
Activation of RhoA requires targeting of GTP-bound RhoA to the plasma membrane, whereupon it interacts with downstream effectors such as Rhotekin. Right atrial tissues were therefore incubated in the absence (untreated) or presence of hU-II, and GTP-bound RhoA was affinity immunoprecipitated using the GST-fusion protein mouse Rhotekin-Rho binding domain. Other tissues were also incubated with ET-1 as this agonist increases force of contraction in human right atrium with greater efficacy than hU-II. An intense 24-kDa band was observed in samples obtained from untreated tissues that were incubated with GTP{gamma}S, consistent with the formation of activated RhoA-GTP. This protein was not detected for samples obtained from untreated tissues or tissues incubated with hU-II or ET-1 where GTP{gamma}S was not added (Fig. 4). These findings indicate that although the RhoA-Rho kinase pathway may have an intrinsic role in contraction of electrically stimulated cardiac muscle, it does not have an obligatory role in the increase in force of contraction that is observed to hU-II. Consistent with this hypothesis, stimulation of right atrial tissues with hU-II did not cause phosphorylation of the myosin binding subunit (myosin phosphatase targeting subunits 1/2) of myosin light chain phosphatase (not shown). The findings indicated that the PKC-dependent effect of hU-II was independent of RhoA-Rho kinase signaling.


Figure 4
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  		Fig. 4 Investigation of activated RhoA (RhoA-GTP) formation in agonist stimulated (20 nM hU-II or 50 nM ET-1, to equilibration of contractile response), and non-stimulated tissues. GTP-bound RhoA was affinity immunoprecipitated using the GST-fusion protein mouse Rhotekin-Rho binding domain (see Section 2). RhoA-GTP was not detected in untreated tissues or in tissues exposed to 20 nM hU-II or 50 nM ET-1. A 24-kDa band, corresponding to the predicted molecular weight for RhoA, was observed in samples obtained from untreated tissues that were incubated with GTP{gamma}S.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
hU-II potently increases force of contraction in human right atrium through activation of G{alpha}q-coupled UT-II receptors [2]. In this study, we found that hU-II stimulated increased phosphorylation of MLC-2, suggesting a potential mechanism for positive inotropic response to hU-II. The phosphorylation state of MLC-2 may be modulated by activation of PKC, myosin light chain kinase, and by Rho kinase-dependent inhibition of myosin light chain phosphatase. The contribution of these pathways to the cardiostimulatory effect of hU-II was examined.

4.1. Urotensin-II increases force of contraction in human right atrium via a protein kinase C-dependent pathway
In the present study, pretreatment of tissues with 10 µM phorbol 12-myristate 13-acetate (PMA) attenuated the inotropic response to hU-II, in addition to the predicted reduction in endothelin-1-mediated response. These inhibitory effects are attributed to desensitization of PKC by PMA [15]. 4{alpha}-Phorbol, which does not desensitize PKC, was without effect and (–)-isoprenaline, which activates G{alpha}s-coupled β1/β2-adrenoceptors was also unaffected by PMA, consistent with specificity of PMA for PKC under the conditions used in this study. The inhibition of hU-II-mediated inotropic response by chelerythrine, an inhibitor of the catalytic domain of PKC, provided supporting evidence for the involvement of PKC in the cardiostimulatory effect. hU-II stimulated increased levels of phospho-PKC{alpha}/βII in the particulate fraction of samples obtained from non-failing human right atria. The increase in particulate PKC probably reflects translocation of PKC{alpha}, because expression of PKCβ was not detected in right atrium from patients with non-failing hearts [16]. In this respect, the signal transduction pathway activated by hU-II resembles that of endothelin-1, which caused translocation of PKC{alpha} to a particulate fraction in rat cardiac myocytes [17], and may be different to phenylephrine which caused translocation of PKC{delta} and-{varepsilon}, but not PKC{alpha} to a particulate fraction in neonatal rat myocardium [18].

The present findings did not support a role of PKC in the phosphorylation of MLC-2, despite an important role of PKC in the inotropic response to hU-II. Alternate PKC-dependent pathways may be involved in the inotropic effect of hU-II, including activation of the Na+–H+ exchanger, NHE1 to cause intracellular alkalinisation and sensitization of cardiac myofilaments to Ca2+ [3,19,20]. While cardiac troponin I is a down-stream target of PKC, its’ phosphorylation by PKC decreases Ca2+ sensitivity [4,5,21], indicating that tropinin I does not mediate the positive inotropic response to hU-II.

The role of other MLC-2-dependent signaling pathways in the cardiac effects of hU-II could not be excluded and were therefore investigated. The hU-II-mediated response was preserved in tissues incubated with a concentration of wortmannin that selectively inhibits phosphatidyl inositol 3-kinase (PI3K; 50 nM). PI3K has a role in formation of phosphatidylinositol 3,4,5-trisphosphate, which facilitates recruitment of phosphatidylinositol 3-phosphate-dependent kinase-1 (PDK-1) to the membrane [22]. Although PDK-1 phosphorylates the activation loop of PKC, an event that initiates priming of PKC for activity [23], cell culture experiments have shown that a high degree of basal phosphorylation of PKC exists and that reversal of this phosphorylation state is very slow, even after inhibition of the upstream pathway [24]. This reserve of primed yet inactive PKC may therefore explain the apparent insensitivity of hU-II-mediated response to 50 nM wortmannin. The inotropic response to hU-II was also insensitive to the higher concentration of wortmannin (10 µM), used to inhibit myosin light chain kinase. The signaling pathway involved in the cardiostimulatory effect of hU-II may therefore differ to that of phenylephrine, which has been shown to increase force of contraction in isolated rat and human cardiac tissue via a myosin light chain kinase inhibitor-sensitive pathway [10,11]. The difference in signaling pathways that culminate in positive inotropy for hU-II and phenylephrine is intriguing because both agonists stimulate G{alpha}q-coupled receptors. Possible explanations include differential activation of PKC isoenzymes, and differential coupling for {alpha}1-adrenoceptors and UT-II receptors to additional G-proteins. Interestingly, {alpha}1-adrenoceptors have been shown to stimulate RhoA through coupling to G{alpha}12/13, but not G{alpha}q [25]. Further studies are required to determine whether G{alpha}12/13 is also involved in the stimulation of myosin light chain kinase by {alpha}1-adrenoceptor agonists, and to identify the spectrum and efficiency of G-protein coupling for the urotensin-II receptor system. Nonetheless, the findings suggest that hU-II increased force of contraction through a PKC-dependent and myosin light chain kinase-independent pathway, and in this respect is similar to that described for ET-1 [8,9].

4.2. Effect of the Rho kinase inhibitor Y-27632 on the cardiostimulant response to hU-II in human right atrium
Force of contraction in striated muscle may also be modulated indirectly by attenuation of MLCP-mediated dephosphorylation of MLC-2. Phosphorylation of myosin phosphatase targeting subunits 1/2 of MLCP by Rho kinase is inhibitory on phosphatase activity, allowing unopposed phosphorylation of MLC-2. We therefore examined whether the inotropic response to hU-II was sensitive to the Rho kinase inhibitor, Y-27632. Y-27632 caused a marked reduction in amplitude of inotropic response to hU-II, with no effect on agonist potency, suggesting that hU-II may indeed increase force through a Rho kinase-dependent pathway. Previous findings had concluded that a Rho kinase-dependent pathway was involved in the inotropic response to phenylephrine [10] and 17-phenyl trinor prostaglandin E2 [13] in rat cardiac tissues. However, we noted that when Y-27632 was added in a concentration-dependent manner to hU-II-stimulated human right atrial trabeculae, force of contraction was reduced to below pre-hU-II-stimulated levels. In addition, it was noted that the lowest concentration of Y-27632 (0.1 µM), did not reduce force despite a predicted reduction in force based on the reported affinity of Y-27632 for Rho kinase (Ki=0.3 µM) [26]. The effect of Y-27632 on basal force was confirmed in non-agonist, electrically stimulated tissues where 1.0 and 10.0 µM Y-27632 reduced basal force of contraction. Interestingly, 1.0 and 10.0 µM Y-27632 also relaxed U46619 [GenBank] -preconstricted human internal mammary artery to levels below basal force [27], whereas 50 µM Y-27632 did not reduce basal contractility in rat papillary muscle [10], suggesting possible species differences.

In view of these observations, it is possible that Y-27632 modulated a mechanism that was intrinsic to myocardial contractility, in combination with or possibly independent of its' effect on Rho kinase. Experiments independent of the Rho kinase inhibitor were therefore carried out to determine whether the Rho kinase pathway was indeed involved in the increase in force of contraction to hU-II. The activated form of the small GTPase RhoA (RhoA-GTP), which stimulates Rho kinase, was immunoprecipitated in tissues treated in the absence and presence of hU-II. RhoA-GTP was not detected in the hU-II-treated tissues. This was unlikely to be a result of low efficacy of hU-II because RhoA-GTP was also undetected in tissues exposed to endothelin-1, a more efficacious agonist with respect to positive inotropy. The immunoprecipitation procedure was verified by treatment of tissue samples with the non-hydrolysable GTP analog GTP{gamma}S, where activated RhoA-GTP was readily detected.

The findings indicate that the RhoA-Rho kinase/myosin light chain phosphatase pathway does not have a major role in the inotropic effect of hU-II in human right atrium. While the mechanism involved in the reduction in basal force of contraction by Y-27632 has not been explored further here, it is of interest to note that the inhibitor was reported to have only 10–50-fold selectivity for the Rho kinase isoenzyme p160ROCK over PKC{varepsilon} [28]. Although protein kinase C-potentiated inhibitor (CPI-17) has also been shown to inhibit myosin phosphatase activity in smooth muscle preparations [29], its role in heart and the possibility that the inotropic effect of hU-II may be induced by phosphorylation of CPI-17, are not currently known.

In conclusion, this study has investigated signaling pathways involved in the positive inotropic response to hU-II in human right atrium. The findings revealed that hU-II potently increased force of contraction in human heart, concomitant with increased phosphorylation of MLC-2 and PKC{alpha}/βII. The positive inotropic effect, but not the phosphorylation of MLC-2, was PKC-dependent. The increase in force of contraction by hU-II was independent of myosin light chain kinase and RhoA-Rho kinase signaling pathways.


   Acknowledgements
 
This study was supported by a National Health and Medical Research Council of Australia New Investigator grant (FDR). The authors thank the surgeons and theatre staff of The Prince Charles Hospital, Ms. Anne Carle for assistance in patient recruitment and Dr. Neal Epstein, National Heart, Lung and Blood Institute, NIH, Bethesda, USA for providing the antibody to phosphorylated MLC-2.


   Notes
 
Time for primary review 34 days


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

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