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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 2, 2003 as doi:10.1096/fj.02-0057fje. |
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Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Universitätsklinikum Hamburg-Eppendorf, Germany;
* Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany;
Dana-Farber Cancer Institute, Boston, USA;
Imperial College School of Medicine, Cardiac Medicine, NHLI, London, UK; and
$ Institut für Pharmakologie und Toxikologie, Westfälische Wilhelms-Universität Münster, Münster, Germany
2Correspondence: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany. E-mail: t.eschenhagen{at}uke.uni-hamburg.de
SPECIFIC AIMS
In vitro, the protein phosphatase inhibitor-1 (PPI-1) is a highly specific and potent inhibitor of type 1 phosphatases (PP1) that is active only in its PKA-phosphorylated form, and so could play a positive feedback role in cAMP-dependent signaling. The present study used an adenovirus approach and two cardiac cell models to test the hypothesis that overexpression of PPI-1 sensitizes cardiac myocytes to ß-adrenergic stimulation. A potential role in human pathophysiology was studied by determining PPI-1 expression in ventricular samples from failing and nonfailing human hearts.
PRINICIPAL FINDINGS
1. The sequence of rat heart PPI-1 differs from published sequences in one codon
PPI-1 was cloned from rat heart by RT-PCR and sequenced by two standard methods from both sides. Compared with published sequences of rat skeletal, rabbit, or human PPI-1, a single nucleotide exchange results in an amino acid exchange at position 27 (lysine instead of glutamine) near the regulatory threonine-35 phosphorylation site. The recombinant protein generated in Escherichia coli exhibited potent inhibition of cardiac phosphatase activity when phosphorylated by PKA (IC50 75 nM) and much less in its unphosphorylated form (IC50 1402 nM). Thus, rat heart PPI-1 shares classical features of PPI-1.
2. Adenovirus-mediated overexpression of wild-type, full-length rat PPI-1 in neonatal rat cardiac myocytes suppressed phosphatase-1 activity
Monolayer cultures of neonatal rat cardiac myocytes (NRCM) were infected with adenovirus encoding both PPI-1 and GFP under the control of two separate CMV promotors (AdPPI-1/GFP) or GFP-only virus (AdGFP) for control. Adenovirus gene transfer was virus/dose dependent and reached 100% at a multiplicity of infection of 1. Protein phosphatase activity was measured after 5 min stimulation with 100 nM isoprenaline. Compared with AdGFP, PP1 activity was reduced by 73% in AdPPI-1/GFP-infected NRCM, indicating the expected biological activity of adenoviral PPI-1.
3. Adenovirus-mediated overexpression of PPI-1 sensitizes engineered heart tissue to ß-adrenergic stimulation
Engineered heart tissue (EHT) from neonatal rat cardiac myocytes was infected with AdPPI-1/GFP or AdGFP and assayed for basal force of contraction as well as inotropic responses to calcium and isoprenaline. Compared with AdGFP, AdPPI-1/GFP-infected EHTs exhibited slightly higher force of contraction under basal and stimulated conditions. The concentration/response curve for isoprenaline, but not for calcium, was shifted to the left by a factor of 5. Relaxation time was shorter in AdPPI-1/GFP- than in AdGFP-infected EHTs.
4. Adenovirus-mediated overexpression of PPI-1 sensitizes adult rat cardiac myocytes to ß-adrenergic stimulation
To investigate the effect of PPI-1 overexpression in a second cardiac model, adult rat cardiac myocytes (ARCM) were infected with adenovirus and evaluated for single-cell shortening as well as for phosphorylation of phospholamban (PLB), one of the main target proteins of PKA involved in regulation of cardiac contractility. Basal cell shortening was unaffected, but on stimulation with 1 nM isoprenaline, cell shortening rate was 37% higher in AdPPI-1/GFP-infected than in AdGFP-infected ARCM (Fig. 1
A). Under identical conditions, phosphorylation of PLB was markedly higher in AdPPI-1/GFP than in AdGFP-infected ARCM (Fig. 1B
), suggesting that the more pronounced increase in (isoprenaline-stimulated) PLB phosphorylation in PPI-1 overexpressing ARCM participates in the increased effect on shortening rate.
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5. PPI-1 expression is reduced by > 50% in failing human hearts
Given the well-known desensitization of the ß-adrenergic signaling pathway in human heart failure, we hypothesized that changes in the expression of PPI-1 may participate in this process and determined PPI-1 mRNA levels in samples from terminally failing and nonfailing human hearts by Northern blot (Fig. 2
). PPI-1 transcript levels were reduced by > 50% in failing human hearts in both dilated and ischemic cardiomyopathy.
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CONCLUSIONS AND SIGNIFICANCE
The data summarized above demonstrate that overexpression of PPI-1 can sensitize the ß-adrenergic signaling pathway in cardiac myocytes. The result is in accordance with data from biochemical assays, in which PPI-1 acted as a potent and highly specific inhibitor of PP1 that is active only after its phosphorylation by the cAMP-dependent PKA. Thus, our data support a model in which PPI-1 acts as an amplifier in the ß-adrenergic signaling cascade that, once activated by PKA-mediated phosphorylation, enhances steady-state phosphorylation of other PKA substrates by inhibiting their dephosphorylation through PP1 (Fig. 3
). Such a role in cardiac signaling has been postulated earlier because isoprenaline had been found to increase PPI-1 phosphorylation and concomitant inhibition of PP1 activity in the myocardium. However, direct evidence for this hypothesis was lacking. Moreover, evaluation of mice with targeted inactivation of the PPI-1 gene yielded surprising results in that neither smooth muscle cell function nor learning behavior was altered by the lack of PPI-1. Thus, the role of PPI-1 in general and in cardiac myocytes in particular remained largely unknown.
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The present study investigated the effect of enhanced PPI-1 expression on contractile properties in two in vitro model systems, EHT and ARCM, through adenoviral gene transfer. The principal feasibility of the adenovirus approach was tested in neonatal rat cardiac myocytes in which infection with AdPPI-1/GFP resulted in a 73% reduction of phosphatase activity. The EHT model was chosen because EHTs allow determination of twitch tension, resting tension, and relaxation time under isometric conditions; they provide cells with a relatively physiological 3-dimensional environment; and they are easily transfected with adenovirus. On the other hand, EHTs are derived from neonatal cells and do not represent a pure cardiac myocyte population, which may obscure some of the cardiac myocyte specific effects. Consequently, isolated ARCM were used as an additional model.
Overexpression of PPI-1 in EHTs resulted in a fivefold higher potency of isoprenaline to increase twitch tension. Similarly, cell shortening of ARCM at an EC50 concentration of isoprenaline was significantly higher in PPI-1-overexpressing ARCM. Under this condition, phosphorylation of PLB was enhanced compared with control-infected ARCM. In our view, this is an important new finding that provides a plausible link between overexpressed PPI-1, enhanced inhibition of phosphatases, less dephosphorylation of PLB, disinhibition of SERCA, and improved diastolic and systolic contractile function. Accordingly, phosphorylation of PLB was undetectable in PPI-1-overexpressing and control-infected ARCM in the absence of isoprenaline; the cell shortening rate was similar, strengthening the idea that regulation of the phosphorylation level of PLB by PPI-1 is central to the effects of PPI-1 overexpression observed. The data in the EHT model were different in that force of contraction and time of relaxation were enhanced already under unstimulated conditions. This was unexpected, because PPI-1 needs to be phosphorylated for its activity. However, protein kinases and phosphatases are active in cardiac myocytes under basal, unstimulated conditions; experiments with a antibody that specifically recognizes phosphorylated PPI-1 demonstrated that adenovirus-overexpressed PPI-1 is phosphorylated in EHTs under basal conditions (data not shown).
Given that PPI-1 appears to sensitize cardiac myocytes to ß-adrenergic stimulation and desensitization of the ß-adrenergic signaling pathway is a hallmark of human heart failure, it was tempting to analyze expression levels of PPI-1 in failing human hearts. PPI-1 transcript levels were decreased in both dilated and ischemic cardiomyopathy. The degree of this reduction is marked (-57%) compared with other known alterations in gene expression and is comparable to the level of down-regulation of ß1-adrenergic receptors. The decrease in PPI-1 could contribute to the 2.5-fold increase in phosphatase activity and thereby to the reduced phosphorylation level of PLB consistently observed in human heart failure. Reduced phosphorylation of PLB in response to ß-adrenergic stimulation is likely due to desensitization of the upstream adenylyl cyclase system, but increased phosphatase activity is assumed to contribute. On the other hand, recent data provided evidence for hyperphosphorylation of ryanodine receptors in human heart failure and for a decreased amount of phosphatase 1 physically associated with the ryanodine receptor. The group of A. Marks explains the apparent contrast of this finding to ß-adrenergic desensitization and increased global PP1 activity by the existence of two compartments: a junctional SR (ryanodine receptor) and a free SR (PLB). PPI-1 as a cytoplasmic protein is likely to regulate predominantly the free SR and may contribute to functional compartmentalization. Taken together, the present findings suggest that a decrease in PPI-1 contributes to desensitization of the ß-adrenergic signaling pathway in human heart failure and thereby identifies a new regulatory element in this pathway. PPI-1 is dephosphorylated and inactivated by protein phosphatase type 2B or calcineurin. PPI-1 could therefore represent a link between cAMP and calcium-dependent signaling pathways in the heart.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0057fje; to cite this article, use FASEB J. (January 2, 2003) 10.1096/fj.02-0057fje 
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