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European Journal of Heart Failure 2007 9(11):1077-1080; doi:10.1016/j.ejheart.2007.09.006
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© 2007 European Society of Cardiology

Long-term β-adrenergic stimulation leads to downregulation of protein phosphatase inhibitor-1 in the heart

Ali El-Armouchea,*,1, Fabian Gochta,1, Elmar Jaeckelb, Katrin Wittköppera, Micha Peecka and Thomas Eschenhagena,*

a Institute of Experimental and Clinical Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf Hamburg, Germany
b Department of Gastroenterology, Hepatology and Endocrinology, Medical School of Hannover Germany

* Corresponding authors. E-mail address: a.el-armouche{at}uke.uni-hamburg.de t.eschenhagen{at}uke.uni-hamburg.de


   Abstract
Top
 Notes
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Desensitization of the β-adrenoceptor/cAMP/PKA pathway is a hallmark of heart failure. Inhibitor-1 (I-1) acts as a conditional amplifier of β-adrenergic signalling downstream of PKA by inhibiting type-1 phosphatases in the PKA-phosphorylated form. I-1 is downregulated in failing hearts and thus presumably contributes to β-adrenergic desensitization. To test whether I-1 downregulation is a consequence of excessive adrenergic drive in heart failure, rats were treated via minipumps with isoprenaline 2.4mg/kg/day (ISO) or 0.9% NaCl for 4days. As expected, chronic ISO increased heart-to-body weight ratio by ~40% and abolished the inotropic response to acute ISO in papillary muscles by ~50%. In the ISO-treated hearts I-1 mRNA and protein levels were decreased by 30% and 54%, respectively. This was accompanied by decreased phospholamban phosphorylation (–40%), a downstream target of I-1, and a reduction in 45Ca2+ uptake (–54%) in membrane vesicles. Notably, phospholamban phosphorylation correlated significantly with I-1 protein levels indicating a causal relationship. We conclude that I-1 downregulation in heart failure is likely a consequence of the increased sympathetic adrenergic drive and participates in desensitization of the β-adrenergic signalling cascade.

Key Words: Phosphatase inhibitor-1 • Phospholamban • Beta-adrenergic signalling

Received June 14, 2007; Revised August 22, 2007; Accepted September 13, 2007


   1. Introduction
Top
 Notes
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
While β-adrenergic receptor stimulation is ideally suited for short-term increases in cardiac performance, long-term activation in chronic heart failure is detrimental and leads to desensitization of these receptors [1,2]. A downstream element of the β-adrenergic signalling pathway in the heart is the specific protein phosphatase-1 inhibitor-1 (I-1). I-1 becomes active upon phosphorylation at Thr-35 by cAMP-dependent PKA. Activation of I-1 results in inhibition of protein phosphatase type-1 (PP1), the major isoform of Ser-Thr-protein phosphatases in the heart, and inhibition of PP1 enhances PKA-mediated protein phosphorylation [3,4]. In adult rat and in failing human cardiac myocytes, overexpression of either wild-type I-1 or a constitutively active I-1 mutant protein, has been shown to enhance β-adrenergic/cAMP/PKA-dependent contractile responses, emphasizing its amplifier role in this system [5,6]. I-1 is sufficient to modulate SR Ca2+ uptake by enhancing PKA-mediated PLB phosphorylation through inhibition of phospholamban (PLB) dephosphorylating PP1 [5-8]. The I-1 protein has been shown to be markedly downregulated in failing human hearts (–60%) [9], which is consistent with increased PP1 activity and decreased PLB phosphorylation in human heart failure [9,10]. By using the isoprenaline infusion model in rats, this study tested the hypothesis that I-1 downregulation is, like β1-AR downregulation, a consequence of prolonged β-adrenergic stimulation and thus may represent an protective mechanism against excessive adrenergic drive in heart failure.


   2. Methods
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 Notes
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Animal treatment, contraction experiments and 45Ca2+ uptake
Male Wistar rats (240-300 g) were treated for 4 days with either isoprenaline (2.4 mg/kg/day, ISO) or vehicle (0.9% NaCl, Ctr) administered via osmotic minipumps (Alzet, USA). All procedures regarding care and use of animals were in accordance with institutional guidelines. Contraction experiments were performed on electrically driven left papillary muscles (1 Hz, 37 °C) as described previously [11]. Oxalate-stimulated Ca2+ uptake was determined in crude frozen homogenates prepared from frozen tissue essentially as described before [11] in the presence 30 mM of the phosphatase inhibitor NaF.

2.2. Enrichment of I-1, Western blotting and RNAse protection assay
I-1 was enriched from myocardial tissue by an optimized trichloroacetic acid (TCA) extraction procedure as described before [9]. Western blotting was performed with primary antibodies against inhibitor-1 (custom-made, Eurogentec, Brussels), calsequestrin (CSQ, Dianova, Germany) and Ser16-phosphorylated PLB (PhosphoProtein Research, UK). RNAse protection assay was performed by using the RPA II– Ribonuclease Protection Assay Kit (Ambion Inc., Austin, Texas, USA) as described before [11] with a 520 bp I-1 and a 110 bp G{alpha}s-probe.

2.3. Statistical analysis
Values presented are arithmetic means±SEM. Statistical significance was determined by Student's T-test and correlation analysis with Spearman-Rank Correlation; A p value <0.05 was considered significant.


   3. Results
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Chronic ISO-infusion causes cardiac hypertrophy, diminished β-adrenergic inotropic response and decreased Ca2+-uptake
Subcutaneous infusion of ISO for 4 days induced an increase in heart-/body weight ratio by +39±3% (n≥12, p<0.05, Fig. 1A) and total RNA yield by +33±6% (n≥8, p<0.05, data not shown). Contractility measurements of papillary muscles from ISO-treated rats revealed a markedly reduced positive inotropic response to increasing ISO concentrations (maximal change in force of contraction:+2.0±0.6 vs.+4.6±0.8 mN in Ctr, n≥15, p<0.05, Fig. 1B) with similar baseline force of contraction (twitch amplitude: 3.8±0.7 vs. 3.0±0.4 mN in Ctr). In addition, chronic ISO reduced the rate of oxalate-stimulated 45Ca-uptake in isolated membrane vesicles by 54±5% (n=8, p<0.05, Fig. 1C).


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  		Fig. 1 Long-term β-adrenergic stimulation reduces inhibitor-1 mRNA and protein levels. Influence of a 4-day infusion of isoprenaline (ISO, 2.4 mg/kg/day) in rats on heart-/body weight ratio (A), contractility of papillary muscles in response to increased ISO concentrations (B), oxalate-stimulated 45Ca-uptake in isolated membrane vesicles (C), cardiac inhibitor-1 (I-1) mRNA (D) and protein levels (E, top), phospholamban (PLB) phosphorylation (E, bottom) and correlation between I-1 protein and PLB phosphorylation (F) *p<0.05 vs. NaCl (Ctr).

 
3.2. Decreased I-1 mRNA, I-1 protein and PLB-phosphorylation levels after chronic ISO
I-1 mRNA was analyzed in RNAse protection assays and normalized to G{alpha}s, which was not influenced by ISO (in pg/µg total RNA; NaCl: 40±4; ISO: 42±6, n≥8; data not shown). ISO reduced I-1 transcript levels by 29±6% (p<0.05, n≥8, Fig. 1D). I-1 protein levels were determined in TCA-extracts. 721±12 mg LV-tissue were used for each sample and yielded 21±2 µg/µl protein in the initial homogenate and 1.23±0.12 µg/µl protein in the TCA-extracts, with no differences between the groups. Immunoblots of these extracts (20 µg/lane) demonstrated that the signal intensity was >50% smaller in ISO-treated hearts (NaCl: 1±0.1; ISO 0.5±0.1, n≥15, p<0.05, Fig. 1E, top). PLB phosphorylation at Ser16 (PKA-site) was analyzed in standard homogenates (30 µg/lane) and normalized to CSQ. Phospho-PLB/CSQ ratio was reduced by ~40% after ISO (NaCl 1.0±0.1, ISO 0.6±0.1, n≥15, p<0.05, Fig. 1E, bottom). I-1 protein levels and phospholamban phosphorylation obtained from the same hearts correlated significantly (Fig. 1F).


   4. Discussion
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In human heart failure there is a pronounced activation of the sympathetic system that is inversely correlated with survival [12]. Human heart failure is associated with diminished β-adrenergic responsiveness, increased protein phosphatase activity and altered contractile protein phosphorylation [2]. Since I-1 is a PKA-dependent amplifier of β-adrenergic signalling by specific inhibition of the dephosphorylating type-1 phosphatases (PP1), its marked downregulation in failing human hearts (–60%) may contribute to these alterations [6,9]. The present study shows that excessive adrenergic drive itself is sufficient to downregulate I-1 to a similar level as observed in human failing hearts. This phenomenon was accompanied by the well-known β-adrenergic subsensitivity and decreased PLB-phosphorylation. The reduction in I-1 mRNA levels by 30% indicates that suppression of I-1 gene expression is at least partially involved.

Downregulation of I-1 is expected to be associated with a decreased potency of isoprenaline (because its amplifier function is reduced) and no change in efficacy (because the signalling cascade β-AR-Gs-AC-cAMP-PKA-target proteins per se is not affected). This is exactly what we have recently observed in I-1-KO mice (unpublished data). In the present isoprenaline-infusion model, however, the main effect was a reduction in efficacy with no significant change in potency. Two reasons may explain the differences. First, isoprenaline infusion, in addition to the now established downregulation of I-1, leads to downregulation of β-AR, upregulation of G{alpha}i and β-ARK as well as the induction of a fetal gene expression program [13]. These changes are all affecting the contractile response to acutely administered isoprenaline and likely lead to a reduction in isoprenaline efficacy. The second, related and partially technical reason is that changes in potency are difficult to see when the maximal response is largely reduced.

Reduction in I-1 protein will cause less inhibition of PP1 and will thereby contribute to the increase in PP1 activity as reported in this model by Boknik et al. and in failing human hearts [10,14]. PP1 is the major protein phosphatase dephosphorylating PLB [15]. Thus the reduced phosphorylation of PLB phosphorylation observed here is, besides a reduction in the cAMP-signalling pathway, likely a consequence of increased PP1 activity. This view is supported by the good correlation between PLB phosphorylation and I-1 protein levels. The phosphorylation state of PLB is the predominant mechanism regulating the rate of Ca2+-uptake by the SR-Ca2+-ATPase (SERCA) [16]. Unphosphorylated PLB reduces the Ca2+ affinity of SERCA and thus inhibits it. This is well compatible with our finding of reduced Ca2+-uptake and is in accordance with observations in human failing hearts [17,18].

Our main finding of marked I-1-downregulation establishes I-1 as a new member of the group of proteins involved in β-adrenergic desensitization in heart failure [2]. The question arises, whether this is a beneficial mechanism that protects the heart from the detrimental effects of chronic β-adrenergic stimulation (e.g. arrhythmia and hypertrophy), or whether it compromises the contractile performance of the heart or both. Based on these considerations, therapeutic strategies might attempt either to further inhibit I-1 or to over activate it. Pathak et al. [8] demonstrated that overexpression of a truncated constitutively active form of I-1 suppressed remodelling in an aortic constriction model (TAC) and prevented the progression of cardiac failure after TAC in rats by a short-term gene transfer approach. Interestingly, the truncated form of I-1 selectively increased phosphorylation of PLB, but not of other PKA substrates such as the ryanodine receptor. These data indicate that gene transfer of truncated I-1 may offer the possibility to intervene in the β-adrenergic signalling pathway downstream of cAMP to increase force without increasing the risk of arrhythmia. Further investigations are now needed to clarify this issue.


   Acknowledgements
 
We thank Jutta Starbatty and Thomas Schulze for providing expert technical assistance. These studies were supported by the Deutsche Forschungsgemeinschaft (FOR 604 to A.E.A. and T.E.), and by the European Commission (EUGeneHeart to T. E.).


   Notes
Top
 Notes
Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
1 These authors contributed equally to this work. Back


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

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