Skip Navigation

Cardiovascular Research 2004 63(3):561-572; doi:10.1016/j.cardiores.2004.01.026
© 2004 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Petrashevskaya, N. N
Right arrow Articles by Schwartz, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Petrashevskaya, N. N
Right arrow Articles by Schwartz, A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2004, European Society of Cardiology

Effects of {alpha}1-adrenergic stimulation on normal and hypertrophied mouse hearts. Relation to caveolin-3 expression

Natalia N Petrashevskaya, Ilona Bodi, Sheryl E Koch, Shahab A Akhter and Arnold Schwartz*

Department of Surgery, Cardiovascular Research Center, Institute of Molecular Pharmacology and Biophysics, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati OH 45267-0828, USA

* Corresponding author. Tel.: +1-513-558-2400; fax: +1-513-558-1778. Email address: schwara{at}email.uc.edu

Received 13 August 2003; revised 7 January 2004; accepted 20 January 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Background: Modulation of the transduction efficiency through G-protein coupled receptors, caused by external stimulation, is essential in designing antihypertrophic treatment strategies in the dysfunctional heart. We compared protein-kinase C (PKC)-dependent regulation of positive inotropic effect via {alpha}1-adrenoreceptor (ADR)/Gq protein in hyperdynamic versus hypertrophied myocardium. Methods: Inotropic (work performing isolated heart) and cellular effects of {alpha}1-adrenoreceptor stimulation were studied in nontransgenic (Ntg) and transgenic (Tg) mice with cardiac specific overexpression of L-type voltage-dependent calcium channels (L-type VDCC). Results: Transgenic hyperdynamic and hypertrophic myocardium (due to overexpression of the L-type VDCC {alpha}1 subunit) were characterized by a lack of positive inotropic effect (PIE) to {alpha}1-ADR stimulation with phenylephrine (PE), as compared to a positive response in Ntg hearts. This was partially restored by PKC inhibition with chelerythrine and staurosporine only at the hyperdynamic stage. The inability of PKC inhibition to increase positive inotropy was associated with markedly decreased cardiac-specific caveolin-3 expression, and no changes in G{alpha}q, PLC-β1, caveolin-1 and {alpha}1-adrenoreceptor expression. Conclusion: In the hyperdynamic myocardium, PKC activation may be one of the switches responsible for an impaired {alpha}1-adrenergic positive inotropic response. In the hypertrophied myocardium, the interruption of the transduction from G{alpha}q-protein coupled receptors to downstream effectors may be due to the down-regulation of caveolin-3 expression.

KEYWORDS Myocardium; Receptors; Adrenergic; Alpha; Calcium; Hypertrophy; Signal transduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The calcium-dependent hypertrophic pathways (calcineurin/NFAT/GATA-4) [1–3], and calmodulin kinases II and IV, which activate the myocyte enhancer factor 2 (MEF2) [4], provide an essential part of {alpha}1-ADR/G{alpha}q-protein signaling by initiating and maintaining transcriptional responses leading to cardiomyocyte growth. Independent of G{alpha}q-protein coupled receptors, activation of Ca2+-dependent growth signal cascades, associated with a hyperdynamic state (which is an adaptive response in early heart disease), may converge with Gq-mediated cascades at the level of PLC [5] and PKC activation [6] thereby saturating the transduction mechanisms from G{alpha}q-coupled receptors to downstream effectors. It is unclear to what extent PKC activation, as a key signaling element in both pathways, may regulate the resultant functional output from G{alpha}q-coupled receptors in the hypercontractile myocardium. The extent and mechanisms of cross-talk between enhanced calcium handling and the {alpha}1-ADR/Gq-protein transduction pathway may be relevant in preventing maladaptive hypertrophy leading to heart failure.

The efficiency of signal transduction in the {alpha}1-ADR/Gq pathway can be modulated not only by calcium-dependent cascades, but also by caveolins. Related aromatic amino acid-rich protein sequences that function as caveolin-binding motifs have been identified in most G protein, in several peptide growth factors receptors, and in kinase domains of many tyrosine and serine/threonine kinases, including PKC isoforms (for review, see Ref. [7]). {alpha}1-ADR, and the signal transducers, Gq proteins and PLC-β1, are confined in the same microdomains as muscle specific caveolin-3, which serves to promote efficient and rapid coupling of agonist-occupied receptors to effector mechanisms and interaction between signal transducers and effectors [7–9]. Caveolae form a signaling module for PKC isoform, serving as a meeting place for activated PKC isoforms and their targets including the phosphorylation cascade of ERK activation [7]. Importance of PKC targeting to caveolae in the contractile and growth responses in the heart remains to be investigated.

In the present study, we examined {alpha}1-adrenergic/G{alpha}q-dependent cardiac function over a long period of sustained increased calcium loading and evaluated the role of PKC as a molecular switch regulating transduction efficiency from Gq-protein coupled receptors to inotropic effectors in the adapted and the hypertrophied Tg mouse hearts. We studied the potential association between the alteration in functional responses and protein abundance in signal transducers and modulators in the G{alpha}q-dependent pathway. A chronic hyperdynamic state, leading to hypertrophic growth, was achieved by genetically induced calcium ingress through the L-type VDCC [10].


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Experimental animals
Adult wild type (Ntg) and transgenic (Tg) mice were studied at 2 and 8 months of age. The generation of Tg mice overexpressing the {alpha}1-subunit of L-type VDCC was previously described [10]; these have remained consistent over numerous generations. 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). The animals in the present study were handled according to approved protocols and animal care regulations at the University of Cincinnati.

2.2. Experimental conditions for the work-performing mouse heart preparations
Mice of either sex were anesthetized with 100 mg/kg body weight pentobarbital sodium intraperitoneally. After thoracotomy, the hearts were first retrograde perfused with oxygenated Krebs–Henseleit solutions containing (in mmol): NaCl 118, CaCl2 2.5, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, Na2EDTA 0.5, NaHCO3 25 and glucose 11 at 37.4 °C. For pressure measurements, a custom-made polyethylene catheter PE 50 was inserted into the left ventricle and connected to a Cobe pressure transducer. Antegrade work-performing perfusion was carried out at a workload of 250 mm Hg ml/min, which was achieved with a venous return of 5 ml/min and the aortic pressure of 50 mm Hg [11].

We characterized two age groups of transgenic mice: 2 months without hypertrophic remodeling and 8 months with developed hypertrophy and functional impairment in cardiac performance.

2.3. Drug infusion
After a 15–20-min equilibration period, the hearts from 2- to 8-month-old Tg and Ntg animals were perfused with 1 µM of the β-adrenoreceptor blocker timolol to prevent a β-adrenergic indirect positive inotropic action of phenylephrine. After a 10-min perfusion with timolol, PE was added to the perfusion solution in concentrations from 10–10 to 10–4 M. Contractile responses were continuously monitored and inotropic effects were measured at maximal level after 10 min of perfusion with PE.

In a separate set of experiments, the {alpha}1-ADR antagonist prazosin was infused at a concentration of 1 µM in the presence of timolol for 10 min. Dose-dependent inotropic and chronotropic effects to PE were then examined.

To evaluate the role of PKC activation, a separate series of experiments were performed where the hearts from 2- to 8-month-old Tg animals were perfused with the PKC inhibitor chelerythrine (CE) 10 µM and staurosporine (ST) 0.01 µM. Hearts were then perfused with PE (10 µM) and CE or ST plus timolol and maximal contractile responses were assessed.

Effect of PKC activation on PIE to PE was studied using phorbol l,2-myristate 13-acetate (PMA). Heart were perfused with PMA at concentration from 10–10 to 10–6 M. Inotropic responses to PMA were evaluated after 10 min of PMA infusion. In a separate series of experiments, PE at concentration of (10–5 M) was infused after 10 min of perfusion with 10–7 M of PMA and sustained positive inotropic effect was measured.

2.4. Immunoblot analysis
Mouse left ventricular cardiac tissue was homogenized in ice-cold buffer containing 10 mM Tris–HCl (pH 7.4), 1 mM EDTA and 1% SDS with protease inhibitor cocktail tablets (Roche Diagnostics). The homogenates were boiled for 5 min, centrifuged at 16,000 x g for 20 min at 4 °C. The supernatant was removed and used for Western analysis. Proteins were boiled for 5 min and separated on 4–15% or 10–20% (caveolin-1 and -3) SDS-polyacrylamide gels (BioRad) and transferred to a nitrocellulose membrane (Hybond ECL, Amersham Pharmacia Biotech.). After nonspecific blocking with 5% nonfat dry milk in TBS-T, membranes were incubated overnight at 4 °C with either mouse monoclonal caveolin-1, -3 antibodies (Transduction Laboratories), PLC-β1 (Transduction Laboratories), at a final dilution of 1:200, or G{alpha}q and {alpha}1 ADR (Santa Cruz) at a final dilution of 1:100. Primary antibodies were detected with anti-mouse IgG horseradish peroxidase antibody diluted to 1:5000 (caveolin-1, caveolin-3, and PLC-β1; Amersham Life Science) or anti-rabbit IgG diluted to 1:2500 (G{alpha}q and {alpha}1ADR; Amersham Life Science).

2.5. Electrophysiological measurements
Cardiac ventricular myocyte isolation and electrophysiological measurements from 2- to 8-month-old Tg and Ntg mice were identical to those previously described by us [12]. Briefly, for action potential recordings, cardiomyocytes were perfused with normal Tyrode solution composed of (in mM): NaCl 138, KCl 4, CaCl2 2, MgCl2 1, glucose 10, HEPES 10, NaH2PO4 0.33, pH 7.4 with NaOH. The "physiological" pipette solution consisted of (in mM): KCl 140, MgCl2 1, Mg-ATP 4, NaCl 5, HEPES 10, EGTA 2, pH=7.4 with KOH. Action potentials were recorded in the current-clamp mode by injecting suprathreshold current pulses through the patch-clamp electrode. At least 15 steady-state action potentials were recorded at 1.0 Hz. The action potential duration (APD) at 50% and 90% repolarization were measured from the traces. All the recordings were carried out at room temperature (22–24 °C). Action potentials in the presence of PE were measured at 5–10 min after the addition of the drug when it had reached the steady state.

2.6. Statistical analysis
Data are presented as mean±S.E.M. Statistical analysis was carried out using Student's t-test for paired and unpaired observations. Concentration–responses to PE were compared using a one-way repeated measure ANOVA. Values of p<0.05 were regarded as statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Contractile phenotype of transgenic hearts overexpressing the L-VDCC {alpha}1-subunit
Comparison of age-dependent contractile performance in the L-VDCC overexpressors ({alpha}1-subunit) (Tg), revealed that at 2 months of age, the Tg animals demonstrated a selective increase in basal contractility (+dP/dt) (Table 1), which was not associated with a concomitant increase in maximal rate of pressure decline (–dP/dt). At 8–9 months, Tg animals developed a significant (p<0.05) increase in heart weight/body weight ratio, from 6.6±0.2 (10–3) to 11.5±0.5 (10–3), depressed relaxation (–dP/dt) and diastolic pressure (DP). There was also a significant decline in contractility (+dP/dt) in comparison with 2-month-old Tg mice (Table 1).


View this table:
[in this window]
[in a new window]

  		Table 1 Selected parameters of cardiac performance at two different age groups

 
3.2 Effect of {alpha}1-adrenoreceptor stimulation on cardiac function and action potential duration
Stimulation of {alpha}1-adrenoreceptors with PE, in the presence of the β-blocker timolol, in isolated work-performing non-transgenic (Ntg) hearts, resulted in a triphasic inotropic response (Fig. 1A), which was associated with a sustained decrease in the action potential duration (APD) (Table 2). The magnitude of the resultant positive inotropic response was age-dependent. As seen in Fig. 2A (upper and lower panels), the maximal positive inotropic effect (+dP/dt) after 10 µM PE, was significantly greater in hearts isolated from 2-month-old Ntg mice (5526±302 mm Hg/s) as compared with the 8-month-old Ntg mice (4144±201 mm Hg/s) and was associated with a concomitant positive lusitropy (increase in –dP/dt, Fig. 2B upper panel) without changes in the +dP/dt/–dP/dt ratio (Fig. 3A). In contrast, the positive inotropic response to PE was not associated with a concomitant increase in the relaxation index (Fig. 2B, lower panel) in the 8-month-old Ntg animals. This led to a different relaxation profile; –dP/dt was not increased proportionally to +dP/dt, producing a significant increase in the +dP/dt/–dP/dt ratio in 8-month-old Ntg animals (Fig. 3B). There was also a slight prolongation in the duration of half relaxation time (TR1/2) normalized to 1/2 relaxation pressure (Fig. 2C, lower panel). The positive inotropic effect and increase in RT1/2 were abolished by the {alpha}1-ADR antagonist prazosin (10–6 M, 10 min).


Figure 1
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Representative recording of left ventricular pressure from a 2-month-old Ntg and Tg heart in the presence of 10–5 M of the {alpha}1-adrenergic agonists PE and 10–5 M of β-blocker timolol. Hearts were perfused with timolol for 10 min following the initial equilibration and during the PE infusion to prevent the β-adrenergic action of PE in all experiments. An independent effect of timolol was not observed. (A) PE elicited a triphasic contractile response: a transient positive and negative inotropic response followed by a sustained positive inotropic response. (B) Hyperdynamic Tg myocardium was characterized by the lack of a PE-mediated positive inotropic effect. (C) The protein kinase inhibitor chelerythrine (10–5 M) partially restored sensitivity to {alpha}1-adrenergic stimulation in the hyperdynamic 2-month-old Tg myocardium.

 

View this table:
[in this window]
[in a new window]

  		Table 2 Effect of phenylephrine on action potential duration

 

Figure 2
View larger version (31K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Dose response effect of PE on isolated work-performing Ntg (closed circles) and Tg (open circles) in 2-month- (upper panel) and 8-month-old (bottom panel). The effect on contractility (A, +dP/dt), relaxation (B, –dP/dt) and time to half relaxation (C, TR1/2), normalized to 1/2 relaxation pressure. Positive inotropic effect of PE in 2-month-old Ntg hearts was associated with an increase in the lusitropic parameters (–dP/dT) and the shortening of relaxation time TR1/2. Tg hearts were characterized by the lack of a functional effect to {alpha}1-adreno-stimulation. *, **P<0.05 and 0.01, respectively, compared with base line.

 

Figure 3
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 The effect of PE (10 µM) on the +dP/dt/–dP/dt ratio in the isolated work-performing heart in 2- and 8-month-old mice. PE produced an increase in the +dP/dt/–dP/dt ratio only in the Ntg 8-month-old myocardium (n=5). Contraction/relaxation ratio was not affected by PE in Ntg 2-month-old myocardium (n=5) The +dP/dt/–dP/dt ratio was significantly greater in Tg myocardium at both ages (n=5) and was not affected by {alpha}1-adrenergic stimulation. **P<0.01 base line versus PE (10 µM).

 
In the Tg hearts from both age groups, PE in the presence of a β-blocker, surprisingly did not elicit significant hemodynamic effects (Figs. 1B and 2A, B, and C)Go. Logically, one expects that when the β-adrenergic system is blunted, as it is in this animal [10,11], the {alpha}1-adrenergic arm of the autonomic nervous system would "take over", but clearly this was not the case.

Electrophysiological responses to PE (30 µM) were examined in both Ntg and Tg cardiomyocytes. PE significantly shortened the ventricular APDs in both the Ntg and Tg 2-month-old age groups to a similar magnitude and produced a slight, but significant hyperpolarizing shift of the resting membrane potential (Fig. 4, Table 2). In contrast, PE did not produce any significant change in APD in the hypertrophic myocardium (8-month-old) compared to age-matched Ntg mice.


Figure 4
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Representative examples of superimposed ventricular action potential recordings at cycle length 1000 ms from Ntg and Tg hearts at two different age in the absence and the presence of {alpha}1-adrenoceptor agonist phenylephrine (30 µM). The action potentials were recorded in current-clamp. In each case, an action potential was recorded at the time of maximal drug effect. All experiments were performed in the presence of the β-blocker timolol (1 µM).

 
3.3 Effect of PKC blockade on {alpha}1-adrenoreceptor mediated contractile response
The mechanism(s) by which increased calcium influx affects {alpha}1-adrenergic transduction pathways may involve activation of several putative molecular switches, including activation of the calcium-dependent PKC-{alpha} isoform, which may serve as an initiator of the hypertrophic gene program [6]. The relative importance of PKC activation as a mechanism for the maladjustment in the signal transduction might help in defining strategies to modulate transduction efficiency in myocardial hypertrophy and failure.

In Ntg and Tg myocardium at both ages studied, PKC inhibition with chelerythrine did not produce significant changes in base line contractility (Table 3). At 2 months, PKC inhibition did not affect positive inotropic effect to PE in Ntg animals. In the presence of chelerythrine, the maximal inotropic effect to PE was from 4009±195 to 5477±314 mm Hg/s, which was similar to maximal PIE from 3896±5526 to 5526±302 mm Hg/s elicited by 10–5 M PE in the absence of PKC blockade. Initially blunted at 8-month-old animals, the PIE to phenylephrine was increased in hearts treated with chelerythrine. +dP/dt in the presence of CE increased from 3489±196 to 5143±294 mm Hg/s, whereas the change in +dP/dt, in the absence of CE at this age, was from 3418±196 to 4144±201 mm Hg/s (P<0.05 PE alone versus PE+CE).


View this table:
[in this window]
[in a new window]

  		Table 3 Effect of protein kinase C inhibitor chelerythrine on positive inotropic effect (PIE) to phenylephrine in NTG and TG isolated work-performing mouse heart

 
PKC inhibition resulted in different changes in response to {alpha}1-ADR stimulation in the adaptive (hypercontractile) phase of the growing Tg mouse heart (2 month) with increased calcium influx compared with the hypertrophied heart (8 month).

Chelerythrine (10–5 M, 10 min in the presence of the β-blocker timolol) partially restored PE-mediated inotropy in the 2-month-old animals (Figs. 1C and 5AGo, upper panel) but had no effect on {alpha}1-adrenergic responsiveness in the hypertrophied myocardium (Fig. 5A, lower panel). An increase in contractility in the 2-month Tg myocardium after PKC inhibition was not associated with concomitant changes in the lusitropic index (–dP/dt) and TR1/2 (Fig. 5A). Another PKC inhibitor, staurosporine (10–8 M, 10 min in the presence of the β-blocker timolol), produced similar effect on cardiac function in TG animals (Fig. 5B).


Figure 5
View larger version (49K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Effect of the PKC inhibitor chelerythrine (10–5 M, 10 min) and staurosporine (10–7 M, 10 min) on the positive inotropic effect of {alpha}1-ADR stimulation in Tg myocardium. (A) Chelerythrine, (B) Staurosporine. PKC inhibitors did not affect base line contractile-lusitropic properties in hyperdynamic Tg myocardium, but partially restored the positive inotropic response to PE in 2-month-old Tg hearts. PKC inhibition did not change the inotropic state or responsiveness to PE in hypertrophied Tg myocardium at 8 months. *P<0.05 by ANOVA.

 
This finding suggests that the blunted or uncoupled {alpha}1-adrenergic activity in the hyperdynamic myocardium was PKC-dependent and was dynamically regulated, while the hypertrophied myocardium developed a PKC-independent mechanism(s) leading to a blunting of an {alpha}1-ADR mediated functional response.

3.4 Effect of PMA on {alpha}1-ADR-mediated PIE
To assess effect of PKC activation on PIE following {alpha}1-ADR stimulation we used phorbol 12-myristate 13-acetate (PMA) [13]. In isolated work performing Ntg mouse heart, the PKC-activator produced a dose-dependent suppression of cardiac contractility and relaxation (Fig. 6A). At a concentration of 10–7 M, PMA decreased +dP/dt from 3151±152 to 1925±118 mm Hg/s (P<0.01) and –dP/dt from 2829±136 to 1390±216 mm Hg/s(P<0.01). The sustained maximal positive inotropic effect of phenylephrine was reduced after PMA treatment (Fig. 6B).


Figure 6
View larger version (29K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Effect of PMA on the inotropic response to {alpha}1-ADR stimulation. (A) PKC activation with PMA produced negative inotropic effect in the isolated work-performing mouse heart (n=5). (B) Maximal inotropic response to PE (10–5 M) in the presence of PMA (10–8 M) was significantly reduced *P<0.05 base line versus PMA, 10–8 M, #P<0.05 PE versus PE+PMA.

 
3.5. Changes in protein expression of Gq-pathway signal transducers and caveolins
We did not find significant changes in the myocardial protein expression of the G{alpha}q, PLC-β1, caveolin-1 and {alpha}1-adrenoreceptors in Tg versus Ntg animals at both ages studied. These results suggest that the sustained elevation in Ca2+ influx does not affect G{alpha}q, PLC-β1 and {alpha}1ADR protein expression (Fig. 7). No difference in abundance of these proteins was detected in Ntg and NTG between 2- and 8-month-old animals.


Figure 7
View larger version (42K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 Profiles of protein expression of G{alpha}q, PLC-β1, caveolin-1, -3 and {alpha}1-ADR in the hearts of Tg and Ntg mice at 2 and 8 months. (A) Representative Western blots from hearts of Tg (+) and Ntg (–) littermates at 2 and 8 months. Tg hearts were characterized by a dramatic decrease in the muscle-specific caveolin-3 protein expression during hypertrophic stages without significant changes in caveolin-1 expression. (B) Average changes in caveolin-1, -3, PLC-β, G{alpha}q and {alpha}1-ADR protein level at 8-month-old animals. Data are mean±S.E.M. n=4 for Ntg 8-month-old and n=4 for Tg 8-month-old hearts. **P<0.01 Tg 8-month-old versus Ntg 8 month old.

 
We found a significant decrease in the protein expression of the myocyte-specific caveolin-3 isoform in the Tg hypertrophied myocardium at 8-month-old animals in comparison with 2-month-old Tg and 2 and 8-month-old Ntg. Endothelial caveolin-1 expression, which is also abundant in fibroblasts, was unchanged in Tg versus Ntg myocardium at both ages.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
There are several findings from the present study. First, the issue as to whether activation of {alpha}1-adrenoreceptors causes a positive inotropic effect ("PIE") in murine work-loaded myocardium, which has been the subject of intense controversy [14,15], we feel is solved. The PIE evoked by phenylephrine in mouse heart is the same as that reported in other species [16–18] and is associated with a slight shortening of the late phase of the APD in ventricular myocytes. Interestingly, in our experimental setting, the shortening of the APD in 2- and 8-month-old Ntg mice was not accompanied with a sustained negative inotropic effect. Stimulation of G{alpha}q protein-coupled {alpha}1-ADRs is known to result in a complex effect on the distal and the proximal elements of excitation–contraction coupling through a dual mechanism: calcium mobilization during action potential, which results in an increase in the amplitude of Ca2+ transients and an amplification or regression of the myofilament Ca2+ sensitivity [17,18].

Several downstream kinases mediating contractile effects to {alpha}1-adrenoreceptor stimulation have been identified in cardiac muscle. Similarly, in the smooth and heart muscle, PIE to {alpha}1 ADR stimulation is mediated in part by the Ca2+–calmodulin-dependent myosin light chain kinase (MLKC-2), which phosphorylates myosin light chain-2 (MLC-2) and increases myofilament calcium sensitivity. MLC-2 is dephosphorylated by myosin phosphatase, which is inactivated by Rho-associated kinase. Rho kinase inhibitor decreased phenylephrine-induced calcium sensitization and contraction in smooth muscle [19], in the rat papillary muscle and human ventricular trabeculae [20]. Thus, MLCK and Rho-kinase act in concern to increase phosphorylation of MLC-2 and subsequently myofilament calcium sensitivity. Activated PKC phosphorylates numerous regulators of inotropic state such as ionic channel, Ca2+ storage proteins and components of the troponin complex, critically involved in regulation of heart muscle contractility. PKC has been shown to phosphorylate both the inhibitory subunit of troponin I (TnI) and the tropomyosin-binding subunit, troponin T (TnT) [21–24]. The net inotropic effect of PKC activation is determined by the balance between inhibitory and stimulating influences on cardiac contractility. It is possible that in mouse cardiomyocytes the phosphorylation of TnI by PKC outweighs the PE-induced alkalinizing effect, resulting in a net decrease in the sensitivity of the myofilaments, thus contributing to the overall negative inotropic effect of PKC activation. In the mouse myocardium, the resultant effect of PKC activation after {alpha}1-ADR stimulation varies from neutral to negative reflecting probably the state of age-dependent PKC{alpha} activation/translocation [6].

IP3 signaling and calcium mobilization, from a caffeine-insensitive intracellular pool may also account for at least part of the PIA evoked by {alpha}1-adrenostimulation and for the {alpha}1-adrenoreceptor initiated signaling process of hypertrophic growth, in addition to PKC-dependent mechanisms [17,18].

Second, does phenylephrine cause a sustained negative inotropic effect? A sustained negative action, described, recently, in the isolated mouse ventricular myocardium [14,15], which is revealed at low stimulation frequencies (1 Hz) is overcome by mechanisms of positive inotropy at intrinsic heart rates in the work-performing heart experimental setting. We feel that the frequency-dependent sarcoplasmic reticulum calcium loading and changes in the myofilament calcium sensitivity, (in the whole organ) but not calcium ingress during the depolarization cycle, are the leading factors in developing a positive cardiotonic effect following {alpha}1-ADR stimulation.

This observation is in accordance with the finding that cardiac-targeted {alpha}1A-ADR overexpression markedly enhanced cardiac contractility without changing –dP/dt, doubling the +dP/dt/–dP/dt ratio and prolonging the time of isovolumic contraction [25]. The {alpha}1B-ADR overexpression did not produce significant inotropic effects [26], so from the two populations of {alpha}1-adrenoreceptors, it is more likely that the {alpha}1A is involved in mediating the positive inotropy in the mouse heart.

Sustained signaling through the {alpha}1-ADR/G{alpha}q complex is believed to serve as a self-amplifying positive feedback mechanism, maintaining hypertrophic signaling over a long period of time and representing one of the features of a specific hypertrophic transcriptional program similar to the expression of hypertrophy genes; β-myosin heavy chain, and skeletal {alpha}-actin [27].

Third, signaling from {alpha}1-ADRs to contractile effectors is prevented in the hyperdynamic (2-month-old Tg) myocardium (i.e., there is no observable effect of phenylephrine administration) but a PIE can be restored by PKC inhibition. At this age, there was increase in most abundant Ca2+-dependent PKC{alpha} activation/translocation to membrane fraction [6] without significant changes in membrane/cytoplasmic ratio in novel PKC{varepsilon} [6], PKC{delta} and atypical PKC{eta} (data are not shown). Therefore, PKC{alpha} activation may be considered one of possible mechanisms selectively inhibiting the signal transduction from {alpha}1-ADR/G{alpha}q-proteins to effectors of positive inotropy in the hypercontractile, adapted Tg. Transduction from {alpha}1-ADR to electrophysiological effectors, including the Na+/Ca2+ exchanger (NCX), which mediate action potential shortening in the mouse ventricular myocytes [15], was not impaired in the hyperdynamic myocardium.

Fourth, in the hypertrophied myocardium, the lack of PIE following {alpha}1-adrenostimulation did not depend on PKC activation. This could conceivably reflect differing impairments in the intracellular transduction pathways caused by hypertrophic growth. For cellular and inotropic responses that involve G{alpha}q-mediated signaling, the preservation of the intracellular milieu, especially the calcium handling processes and intracellular alkalinization, seems to be required to observe physiological effects. Action potential prolongation and hemodynamic alteration, observed at the hypertrophy stage, are indicative of significant changes in calcium handling and sarcoplasmic reticulum function. However, this phenomenon of impaired signal transduction in cardiac hypertrophy may result from defective coupling between the G{alpha}q protein coupled receptors and their downstream effectors. As was shown in Refs. [28,29], changes in the expression of regulators of G-protein signaling (RGS) proteins negatively affected Gq-protein function The upregulation of RGS4 in failing human myocardium diminished Gq/11-mediated signaling and possibly was involved in the desensitization of Gq/11-mediated positive inotropic effects to endothelin. Apart from Gq-protein function, the lack of a selective action upon {alpha}1A-adrenergic contractile stimulation was ascribed to failure of PKC to couple to one of downstream effectors of positive inotropy, such as the Na+/H+ exchanger, in hypertrophied adult ventricular myocytes from ascending aortic-banded rats [30]. The early growth responses (protooncogene induction and protein synthesis) are blunted in the hypertrophied myocardium in response to stimulation with growth factors, angiotensin II, and {alpha}1-adrenergic agonists, as well as pressure overload [31,32]. Cessation of protein synthesis in spite of PKC activation can reflect more general impairments in signal transduction in cardiac hypertrophy, rather different from the selectively blunted {alpha}1ADR/G{alpha}q mediated inotropic effect in the hypercontractile (2-month-old Tgs) myocardium.

The lack of a PIE, following {alpha}1-adrenostimulation in the Tg myocardium at both the 2- and 8-month ages, cannot be related to changes in protein expression of the signal transducers {alpha}1ADR, G{alpha}q and PLC-β1, but may differentially depend on PKC inhibition. Most likely, the hypertrophied myocardium lacks a specific and efficient signal transmission from Gq-protein coupled receptors due to decreased caveolin-3 expression. Caveolin is required for the localization of certain signaling molecules to caveolae and for modulating the interaction between the signaling molecules [9]. Conventional calcium-sensitive PKC isoforms have been identified as a constituent component of caveolae [7]. Decreased caveolin may impair signaling via other G{alpha}q-coupled receptors, thereby diminishing the relative importance of Gq-coupled receptor activation in regulating functional and possibly transcriptional responses in hypertrophic myocardium.

The caveolin scaffolding domain has been shown to interact directly with many growth factor receptors and acts as a negative regulator of mitogenic growth by inactivating signaling molecules. Therefore, decreased caveolin protein expression might enhance basal signaling through other growth promoting pathways, including those for EGF, PDGF and VEGF and activation of the prohypertrophic p42/44 MAPK cascade [33,34]. Caveolin downregulation seems to be a necessary event for maintaining hypertrophic cytoplasmic signaling. Targeting the network of cytoplasmic signaling by modulating caveolin-3 abundance might therefore regulate hypertrophic transformation, rather than by inhibiting upstream signaling from {alpha}1-ADR/G{alpha}q proteins.

In conclusion, signaling from {alpha}1-ADR/G{alpha}q proteins to effectors of positive inotropy is prevented in both the hypercontractile, adapted 2-month Tg and the hypertrophied 8-month Tg mice myocardium by different mechanisms. In the hyperdynamic myocardium, PKC regulates the efficiency of signal transduction from the G{alpha}q-protein coupled receptors to effectors of positive inotropy. In the hypertrophied myocardium, however, the lack of a PIE to {alpha}1-ADR/G{alpha}q proteins is independent of PKC inhibition, reflecting a general impairment in compartmental organization, possibly related to decreased muscle-specific caveolin-3 expression.

4.1. Limitations of this study
One of the major limitations of this study is that we have not assessed functional consequences of caveolin-3 abundance to {alpha}1ADR/G{alpha}q transduction due to the lack of pharmacological tool to manipulate caveolin-3 activity. The significance of caveolin-3 abundance for impairment in cardiac signal transduction in hypertrophy and transition from hypertrophic stage to heart failure needs future study. However, the fact that caveolin-3 and eNOS [35] are downregulated in cardiac hypertrophy reflects the effect of chronic contractile stimulation on downstream transcriptional factors suppressing activity of negative regulators of myocyte growth while significance of external growth promoting signaling through {alpha}1/G{alpha}q-coupled receptors is diminished.


   Acknowledgements
 
This work was supported by Program Project Grant HL22619 (to A.S.) and Training Grant HL07382 (to N.N.P., and S.E.K., and A.S., Program Director) from National Institutes of Health.


   Notes
 
Time for primary review 27 days


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

  1. Sussman M.A, Lim H.W, Cude N, et al. Prevention of cardiac hypertrophy in mice by calcineurin inhibition. Science (1998) 281:1690–1693.[Abstract/Free Full Text]
  2. Mende U, Kagen A, Cohen A, et al. Transient cardiac expression of constitutively active G{alpha}q leads to hypertrophy and dilated cardiomyopathy by calcineurin-dependent and independent pathways. Proc. Natl. Acad. Sci. U. S. A. (1998) 95:13893–13898.[Abstract/Free Full Text]
  3. Fiedler B, Lohmann S.M, Smolenski A, et al. Inhibition of calcineurin-NFAT hypertrophy signaling by cGMP-dependent protein kinase type I in cardiac myocytes. Proc. Natl. Acad. Sci. U. S. A. (2002) 99:11363–11368.[Abstract/Free Full Text]
  4. Kato T, Sano M, Miyoshi S, et al. Calmodulin kinases II and IV and calcineurin are involved in leukemia inhibitory factor-induced cardiac hypertrophy in rats. Circ. Res. (2000) 87:937–945.[Abstract/Free Full Text]
  5. Matkovich S, Woodcock E. Ca2+-activated but not G-protein mediated inositol phosphate responses in rat neonatal cardiomyocytes involve inositol 1,4,5-trisphosphate generation. J. Biol. Chem. (2000) 275:10845–10850.[Abstract/Free Full Text]
  6. Muth J, Bodi I, Lewis W, et al. A Ca2+-dependent transgenic model of cardiac hypertrophy. A role for protein kinase C{alpha}. Circulation (2001) 103:140–147.[Abstract/Free Full Text]
  7. Rubin V, Xu X, Steinberg S, et al. Activated protein kinase C isoforms target to cardiomyocyte caveolae. Circ. Res. (1999) 84:980–988.[Abstract/Free Full Text]
  8. Hare J, Lofthouse R, Juang G, et al. Contribution of caveolin protein abundance to augment nitric oxide signaling in conscious dogs with pacing-induced heart failure. Circ. Res. (2000) 86:1085–1092.[Abstract/Free Full Text]
  9. Fujita T, Toya Y, Iwatsubo K, et al. Accumulation of molecules involved in {alpha}1-adrenergic signal within calveolae: caveolin expression and the development of cardiac hypertrophy. Cardiovasc. Res. (2001) 51:709–716.[Abstract/Free Full Text]
  10. Muth J, Yamaguchi H, Grupp I, et al. Cardiac-specific overexpression of the {alpha}1-subunit of the L-type voltage-dependent calcium channel in transgenic mice. J. Biol. Chem. (1999) 274:21503–21506.[Abstract/Free Full Text]
  11. Serikov V, Petrashevskaya N, Canning A, Schwartz A. Reduction of [Ca2+]i restores uncoupled β-adrenergic signaling in isolated perfused transgenic mouse heart. Circ. Res. (2001) 88:9–11.[Abstract/Free Full Text]
  12. Petrashevskaya N, Bodi I, Rubio M, Molkentin J, Schwartz A. Cardiac function and electrical remodeling of the calcineurin-overexpressed transgenic mouse. Cardiovasc. Res. (2002) 54:117–132.[Abstract/Free Full Text]
  13. Karmazyn M, Watson J, Moffat M. Mechanism for cardiac depression induced by phorbol myristate acetate in working rat heart. Br. J. Pharmacol. (1990) 1009:826–830.
  14. Tanaka H, Manita S, Matsuda T, et al. Sustained negative inotropism mediated by {alpha}-adrenoceptors in adult mouse myocardia: developmental conversion from positive response in the neonate. Br. J. Pharmacol. (1995) 114:673–677.[ISI][Medline]
  15. Nishimaru K, Kobayashi M, Matsuda T, et al. {alpha}-Adrenoreceptor stimulation-mediated negative inotropism and enhanced Na+/Ca2+ exchange in mouse ventricle. Am. J. Physiol. (2001) 280:H132–H141.[ISI]
  16. Korzick D.H, Holiman D.A, Boluyt M.O, et al. Diminished {alpha}1-adrenergic-mediated contraction and translocation of PKC in senescent rat heart. Am. J. Physiol. (2001) 281:H581–H589.[ISI]
  17. Endoh M, Hiramoto T, Ishihata A, et al. Myocardial {alpha}1-adrenoceptors mediate positive inotropic effect and changes in phosphatidylinositol metabolisms: species differences in receptor distribution and the intracellular coupling process in mammalian ventricular myocardium. Circ. Res. (1991) 68:1179–1190.[Abstract/Free Full Text]
  18. Endoh M, Blinks J.R. Actions of sympathomimetic amines on the Ca2+ transients and contractions of rabbit myocardium; reciprocal changes in myofibrillar responsiveness to Ca2+ mediated through {alpha}- and β-adrenoceptors. Circ. Res. (1988) 62:247–265.[Abstract/Free Full Text]
  19. Uehata M, Ishizaki T, Satoh H, et al. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature (1997) 389:990–994.[CrossRef][Medline]
  20. Andersen G.Q, Qvigstand E, Schiander I, et al. {alpha}1-AR-induced positive inotropic response in heart is dependent on myosin light chain phosphorylation. Am. J. Physiol. (2002) 283:H1471–H1480I.[ISI]
  21. Venema R.C, Kuo J.F. Protein kinase C-mediated phosphorylation of troponin I and C-protein in isolated myocardial cells is associated with inhibition of myofibrillar actomyosin MgATPase. J. Biol. Chem. (1993) 268:2705–27711.[Abstract/Free Full Text]
  22. Ding X.L, Akella A.B, Gulati J. Contribution of troponin I and troponin C to the acidic pH-induced depression of contractile Ca2+ sensitivity in cardiotrabeculae. Biochemistry (1995) 34:2309–2316.[CrossRef][ISI][Medline]
  23. Montgomery D.E, Chandra M, Huang Q, et al. Transgenic incorporation of skeletal TnT into cardiac myofilaments blunts PKC-mediated depression of force. Am. J. Physiol. (2001) 280:H1011–H1018.[ISI]
  24. Noland T.A, Guo X, Raynor R.L, et al. Cardiac troponin I mutants. Phosphorylation by protein kinases C and A and regulation of Ca2+-stimulated MgATPase of reconstituted actomyosin S-1. J. Biol. Chem. (1995) 270:25445–25454.[Abstract/Free Full Text]
  25. Lin F, Owens W.A, Chen S, et al. Targeted {alpha}1A-adrenergic receptor overexpression induces enhanced cardiac contractility but not hypertrophy. Circ. Res. (2001) 89:343–350.[Abstract/Free Full Text]
  26. Wang B.H, Du X.J, Autelitano D.J, et al. Adverse effects of constitutively active {alpha}1B-adrenergic receptors after pressure overload in mouse hearts. Am. J. Physiol. (2000) 279:H1079–H1086.[ISI]
  27. Rokosh D.G, Stewart A.F, Chang K.C, et al. {alpha}1-Adrenergic receptor subtype mRNAs are differentially regulated by {alpha}1-adrenergic and other hypertrophic stimuli in cardiac myocytes in culture and in vivo: repression of {alpha}1B and {alpha}1D but induction of {alpha}1C. J. Biol. Chem. (1996) 271:5839–5843.[Abstract/Free Full Text]
  28. Mittmann C, Chung C.H, Hoppner G, et al. Expression of ten RGS proteins in human myocardium: functional characterization an upregulation of RGS4 in heart failure. Cardiovasc. Res. (2002) 55:778–786.[Abstract/Free Full Text]
  29. Wieland T, Mittman C. Regulators of G-protein signaling: multifunctional proteins with impact on signaling in the cardiovascular system. Pharmacol. Ther. (2003) 97:95–115.[CrossRef][ISI][Medline]
  30. Ito N, Kagaya Y, Weinberg E.O, et al. Endothelin and angiotensin II stimulation of Na+–H+ exchange is impaired in cardiac hypertrophy. J. Clin. Invest. (1997) 99:125–135.[ISI][Medline]
  31. Schunkert H, Weinberg E.O, Bruckschlegel G, et al. Alteration in growth responses in established cardiac pressure overload hypertrophy in rats with aortic banding. J. Clin. Invest. (1995) 96:2768–2774.[ISI][Medline]
  32. Jalili T, Takeishi Y, Song G, et al. PKC translocation without changes in G{alpha}q and PLC-β protein abundance in cardiac hypertrophy and failure. Am. J. Physiol. (1999) 277:H2298–H2304.[ISI][Medline]
  33. Couet J, Sargiacomo M, Lisanti M.P. Interaction of a receptor tyrosine kinase, EGF-R, with caveolins. Caveolins binding negatively regulates tyrosine and serine/threonine kinase activities. J. Biol. Chem. (1997) 272:30429–30438.[Abstract/Free Full Text]
  34. Woodman S.E, Park D.S, Cohen A.W, et al. Caveolin-3 knock-out mice develop a progressive cardiomyopathy and show hyperactivation of the p42/44 MAPK cascade. J. Biol. Chem. (2002) 277:38988–38997.[Abstract/Free Full Text]
  35. Petrashevskaya N, Bodi I, Varadi G, Molkentin J, Schwartz A. Decreased eNOS expression in murine transgenic models of cardiac hypertrophy. Biophys. J. (2002) 82:602a. [Abstract].

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Petrashevskaya, N. N
Right arrow Articles by Schwartz, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Petrashevskaya, N. N
Right arrow Articles by Schwartz, A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?