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,1
* Department of Molecular Cardiovascular Biology Yamaguchi University School of Medicine;
Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi University Graduate School of Medicine, Ube, Japan; and
Medical Genetics Division, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
1Correspondence: Department of Molecular Cardiovascular Biology, Yamaguchi University School of Medicine, 1–1-1 Minami-Kogushi, Ube 755-8505 Japan. E-mail: ysikeda{at}yamaguchi-u.ac.jp
SPECIFIC AIMS
Protein phosphatase 1 (pp1), the major isotype of Ser/Thr protein phosphatase in cardiomyocytes, has been shown to be overactivated in the cytosol and in the preparations of sarcoplasmic reticulum (SR) in diseased hearts, although the relationship between PP1 and protein kinase A (PKA) signaling during heart failure (HF) progression has not been clearly demonstrated. We hypothesized that hyperactive PP1 is a maladaptive mechanism that occurs in conjunction with altered PKA signaling during HF progression, and inhibition of PP1 by inhibitor-2(INH-2) may be favorable for preserving cardiac function in HF. In the present study, we first characterized disease-stage-related changes in PP1 activities together with the PKA activities and phosphorylation status of several key proteins, and we further investigated whether in vivo PP1 inhibition by gene transfer of INH-2 prevents HF progression in the cardiomyopathic (CM) hamster, a well-established genetic HF model, which harbors the same genetic deficiencies as human dilated cardiomyopathy.
PRINCIPAL FINDINGS
1. Adenoviral mediated INH-2 gene delivery improves contractility in cardiomyocytes via increase in phosphorylation of Ser-16-PLN
AdV-INH-2 gene transfer significantly inhibited PP1 activity in adult rat cardiomyocytes without any effect on PP2A activity. Following adenovirus transfection, INH-2 significantly increased the phosphorylation of phospholamtan (PLN) at Ser-16, and enhanced Ca2+ transients and % cell shortening.
2. In vivo adenovirus-mediated INH-2 gene delivery improves LV systolic function
We tested the effect of in vivo AdV-mediated high-efficiency cardiac gene transfer of INH-2 on cardiac dysfunction at 14 wk in the midst of the transition phase of HF from moderate to severe dysfunction. At 7 days after gene transfer, the INH-2-transfected hamsters showed a significant reduction in LV chamber size (LV end-diastolic dimension and LV end-systolic dimension; LVDd and LVDs) and an improvement of percent fractional shortening of the LV (%FS) (Fig. 1
) compared with that before the gene transfer. Successful INH-2 gene transduction was confirmed by the immunoblotting for INH-2, as shown mainly in the cytosol. Interestingly, quantitative analysis revealed that the expression of INH-2 caused the changes in the subcellular distribution of PP1 catalytic subunit (PP1C) in the myocardium after the in vivo gene transfer. High level of INH-2 expression was accompanied with a higher amount of cytosolic PP1C than that in the LacZ-treated group, without inducing the corresponding increase in cytosolic PP1 activity. In contrast, higher INH-2 expression was accompanied with lower amount of microsomal PP1C and lower microsomal PP1 activity compared with that in the LacZ-treated group. Because INH-2 was found exclusively in the cytosol, these findings are interpreted that INH-2 induced an increase in inactive PP1C pools in the cytosol, thereby decreasing the active PP1C in the microsomes. This may account for the increase in phospho-Ser-16 PLN after in vivo INH-2 gene transfer (Fig. 2
).
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On the other hand, phosphorylation of ryanodine receptor (RyR) at Ser-2808 did not show significant change, and another intracellular PKA phosphorylation target, CREB, did not show any change in phosphorylation at Ser-133 by INH-2 gene transfer compared with the LacZ-treated group. The level of PKA activity in CM hamster heart at 14 wk was significantly higher than either the level of baseline PKA activity or that of maximally stimulated state by intravenous (i.v.) isoproterenol infusion in normal hamsters. Interestingly, INH-2 gene transfer significantly prevented an abnormal increase in PKA activity, although it did not affect the increase in PLN phosphorylation at Ser-16. Decrease in PKA activity in INH-2 treated hamsters paralleled with a decreasing trend in plasma catecholamine levels, suggesting that the reduction in the PKA activity was due to a decrease in sympathetic drive. Gene delivery of INH-2 into the CM hamster heart reduced brain natriuretic peptide (BNP) levels, a predictor of exacerbation of HF, compared with the LacZ-treated group. These results indicate that INH-2 gene transfer has a favorable effect on the failing heart, over the short term.
3. In vivo adeno-associated virus-2-(AAV-) mediated INH-2 gene delivery improves LV systolic function and extends survival time
Since in vivo AdV-INH-2 gene delivery resulted in improvement of cardiac function and ameliorated BNP expression over the short term, we further tested the long-term effect of INH-2 gene delivery by AAV-mediated gene transfer. We performed AAV-mediated gene transfer of INH-2 at 14 wk of age, followed by serial assessment of cardiac function for 12 wk after the gene transfer by echocardiography and hemodynamic analysis. AAV-INH-2 gene transfer preserved %FS with persistent suppression of microsomal PP1 activity and increased PLN phosphorylation at Ser-16 compared with the AAV-LacZ-treated group over the observation period (Fig. 3
). In addition, INH-2-treated hamster heart showed less interstitial fibrosis compared with that in LacZ-treated hearts. In the Kaplan-Meier plot, the AAV-INH-2-treated group showed a 54% survival rate at 12 wk after the gene transfer, whereas the AAV-LacZ-treated CM hamsters exhibited a deleterious time course, ending with a 29% survival rate. The difference between these survival curves was statistically significant (P = 0.03). These results clearly indicate that INH-2 gene transfer had a favorable effect on progressive HF in the long term.
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CONCLUSIONS AND SIGNIFICANCE
In the present study, we demonstrated that in vivo gene transfer of INH-2 effectively prevented HF progression throughout the one-week observation period in the experiment using adenoviral gene transfer and throughout three-month observation period in the experiment using AAV-mediated gene transfer. The INH-2 gene transfer not only preserved LV function but also reduced mRNA expression of BNP, a prognostic marker of HF, in the short-term experiment. Furthermore, long-term expression of exogenous INH-2 also slowed the progression of LV dysfunction and extended survival in CM hamsters. To our knowledge, this is the first report demonstrating the long-term therapeutic effect of PP1 inhibition in progressive HF using a high-efficiency cardiac gene transfer approach.
Our data suggested that the beneficial effect of INH-2 gene delivery is, at least in part, attributable to the increase in phosphorylation of PLN at Ser-16 (Fig. 2)
, because inhibition of endogenous PLN has been reported to be beneficial in treating certain types of HF, including HF in the other hamster model of cardiomyopathy (i.e., the BIO14.6 strain model).
In conclusion, we have demonstrated that in vivo myocardial PP1 inhibition by inhibitor-2 up-regulated SR calcium handling through PLN phosphorylation and protected cardiomyocyte damage, thereby ameliorating the long-term progression of HF in the cardiomyopathic hamster. Modulation of PP1 activity by INH-2 may constitute a new therapeutic strategy for the treatment of end-stage HF.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5299fje
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