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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
ABSTRACT
The type 1 protein phosphatase (PP1) has been reported to be overactivated in the failing heart, leading to a depression in cardiac function. We investigated whether in vivo PP1 inhibition by myocardial gene transfer of inhibitor-2 (INH-2), an endogenous PP1 inhibitor, alleviates heart failure (HF) progression in the cardiomyopathic (CM) hamster, a well-established HF model. Adenoviral INH-2 gene delivery improved % fractional shortening of the left ventricle (LV) accompanied by reduced chamber size at 1 wk. In vivo myocardial INH-2 gene delivery induced an increase in cytosolic PP1 catalytic subunit 
(PP1C) without inducing the corresponding increase in cytosolic PP1 activity. On the other hand, INH-2 delivery induced a decrease in microsomal PP1C, resulting in a preferential decrease in microsomal PP1 activity, thereby increasing in phospholamban phosphorylation at Ser16. INH-2 gene transfer alleviated brain natriuretic peptide expression, presumably reflecting improved cardiac function. Moreover, adeno-associated virus-mediated INH-2 gene delivery significantly extended the survival time for 3 mo. These results indicate that increased PP1 activity is an exacerbating factor during progression of genetic cardiomyopathy and modulation of PP1 activity by INH-2 provides a potential new treatment for HF without activating protein kinase A signaling in cardiomyocytes.— Yamada, M., Ikeda, Y., Yano, M., Yoshimura, K., Nishino, S., Aoyama, H., Wang, L., Aoki, H., and Matsuzaki, M. Inhibition of protein phosphatase 1 by inhibitor-2 gene delivery ameliorates heart failure progression in genetic cardiomyopathy.
Key Words: gene transfer • protein kinase A • phosphorylation • phospholamban
HEART FAILURE (HF) is a leading cause of death in developed countries. Morbidity and mortality rates remain high despite the recent significant progress in medical and surgical therapy (1)
. Development of a new therapeutic strategy, which will complement the underlying biological process within the failing heart is awaited to alleviate the high mortality in patients with HF.
In this context, recent studies have revealed that defective regulation of Ca2+ cycling in the failing heart is a major determinant of progressive cardiac dysfunction and fatal arrhythmia (2
, 3)
. Importantly, the amplitude and the velocity of Ca2+ cycling in cardiomyocytes are finely regulated by dynamic balance of phosphorylation and dephosphorylation through kinases and phosphatases in response to a variety of extrinsic stimuli. For example, sympathetic stimulation leads to activation of the ß-adrenergic receptor, which, in turn, stimulates the production of cyclic AMP (cAMP) by adenylate cyclase, resulting in the activation of protein kinase A (PKA). Activated PKA phosphorylates phospholamban (PLN) and ryanodine receptor (RyR) in the sarcoplasmic reticulum (SR), thereby augmenting the amplitude and the velocity of SR Ca2+ cycling and cellular contractility (4)
in a beat-by-beat manner, whereas protein phosphatase (PP)1 and 2A counterbalance phosphorylation of these proteins (5)
.
In the failing heart, chronic overactivation of the ß-adrenergic system leads to maladaptation of these cellular signaling via PKA hyperactivation with hyperphosphorylation of RyR (6)
and/or hyperactivation of protein phosphatases with hypophosphorylation of PLN (7
, 8)
in the corresponding subcellular domain. These abnormalities cause impaired sarcoplasmic reticulum (SR) Ca2+ loading and cytosolic Ca2+ overload, resulting in myocyte damage.
Protein phosphatase 1 (PP1), the major isotype of Ser/Thr protein phosphatase in cardiomyocytes (5)
, has been shown to be overactivated in the cytosol and in the preparations of SR in diseased hearts, (7
, 9
10
11)
, although the relationship between PP1 and PKA signaling during HF progression has not been clearly demonstrated. The role of PP1 hyperactivity is also supported by the finding of Carr et al. (12)
that overexpression of the PP1 catalytic subunit (PP1C) in the mouse heart caused dilated cardiomyopathy. In this regard, overexpression of inhibitor-1 (INH-1), an endogenous PP1 inhibitor, or constitutively active INH-1 has been shown to restore Ca2+ cycling and cell contraction/relaxation in in vitro cardiomyocytes (12
, 13)
and in vivo aorta-constricted rat hearts (14)
. In addition, inhibitor-2 (INH-2), another endogenous PP1 inhibitor, has also been shown to increase cardiac contractility by augmenting Ca2+ cycling in a transgenic model (15)
.
Accordingly, we hypothesized that hyperactive PP1 is a maladaptation mechanism in conjunction with altered PKA signaling during HF progression, and inhibition of PP1 by 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 PKA activities and phosphorylation status of several key proteins. 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 deficiency as human dilated cardiomyopathy (16
, 17)
.
MATERIALS AND METHODS
Animals
Animal experiments were carried out on normal male hamsters (6 to 28 wk old) obtained from Japan SLC and UMX7.1 CM hamsters bred at Yamaguchi University. Male adult rats (8 wk old) for isolation of adult rat cardiomyocyte were obtained from Japan SLC. All animal protocols were approved by the Yamaguchi University School of Medicine Animal Subject Committee.
Hemodynamic measurement
Hamsters of three different ages (6, 10, and 28 wk old) underwent transthoracic echocardiography and left ventricular (LV) pressure measurement as described previously (18
, 19)
. Briefly, hamsters were anesthetized by an intraperitoneal (i.p.) injection of sodium pentobarbital (50 mg/kg), followed by measurement of LV diameter by a HDI-5000 ultrasound machine (Philips, Netherlands) equipped with a 15-MHz probe. Pressure in the LV was measured using a fidelity pressure manometer (Millar, Houston, TX) introduced through the LV apex by a small midline thoracotomy. Maximum and minimum LV dP/dt and the time constant of relaxation, 
(using an exponential function), were calculated from LV pressure as described (20)
.
Cell fractionation and immunoblottings
The LV specimens were homogenized with the buffer containing (in mM): 25 Tris-HCl (pH 7.4), 50 NaCl, 300 sucrose, 1 EDTA, 1 EGTA, 50 NaF, 1 Na3VO4, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS, 0.02% 2-mercaptoethanol, as well as 1% protease inhibitor cocktail (PIC) (Sigma, St. Louis, MO). Protein concentrations were calibrated by Bradford assay, and an equal amount of samples was loaded onto each lane for quantitative immunoblot analysis by using LAS-1000 (Fuji-Film, Tokyo, Japan). Part of the LV specimens underwent subcellular fractionation to obtain cytosolic and microsomal fractions, as described previously (19)
in the buffer containing (in mM): 25 Tris-HCl (pH 7.4), 50 NaCl, 300 sucrose, 1 EDTA, 1 EGTA, 50 NaF, 1 Na3VO4, as well as 0.02% 2-mercaptoethanol and 1% PIC.
Protein phosphatase activities
Specimens for PP assay were prepared from both the cytosol and the microsome fraction in the buffer containing (in mM):25 Tris-HCl (pH7.4), 50 NaCl, 300 sucrose, 0.1 EDTA, 0.1 EGTA, 1% Triton-X 100 0.02% 2-mercaptoethanol, and 1% PIC. The PP1 activity was measured in the absence or presence of 1 nM okadaic acid (OA) to block PP2A activity or 1 µM OA to block both PP1 and PP2A activities with [32P]phosphorylase a as a substrate (21)
. The specificity of measurement for PP1 activity with use of 1 nM OA was confirmed by addition of 500 nM recombinant inhibitor-2 peptide (Sigma) to block entire PP1 activity into the reaction solution.
Protein kinase A activity, cyclic AMP content, and plasma catecholamine measurements
The PKA activity was measured from the LV homogenates using a kit (V7480; Promega, Madison, WI) according to the manufacturer’s instructions. The plasma norepinephrine, epinephrine, and tissue cyclic AMP levels were measured as described previously (22)
. The tissue homogenates from isoproterenol-stimulated hearts were prepared as described previously (23)
.
Construction of adenoviruses and adeno-associated viruses
Recombinant human adenovirus-5 (AdV) encoding nuclear localizing ß-galactosidase (LacZ) was amplified and purified as described previously (20)
. The human INH-2 cDNA (NM_006241) was amplified by RT-PCR with the primer set of 5'-GCAGAATTCACCATGGCGGCCTCGACGGCCTCG-3' and 5'-GCATCTAGATGAACTTCGTAATTTGTTTTGCTG-3'. The adenovirus encoding INH-2 was created with or without a FLAG tag at the carboxyl-terminal, amplified, and purified as described previously (20)
. Recombinant adeno-associated virus serotype-2 vectors (AAV) encoding LacZ or INH-2 were produced in the vector core facility at the University of Pennsylvania.
Analysis of isolated adult rat cardiomyocytes
Adult rat cardiomyocytes were prepared from 8-wk-old Wistar rats, as described previously (24)
. Isolated rat cardiomyocytes were transfected with AdV at an MOI of 100 and incubated for 36 h at 37°C in 5%CO2/95%O2 atmosphere. Cardiomyocyte shortening and intracellular Ca2+ transients were simultaneously recorded, as described previously (25)
. A total of 40 cells were analyzed from 8 wk-old male Wistar rats.
Cardiac in vivo AdV/AAV transfection protocol
Fourteen-week-old male normal and CM hamsters underwent in vivo high-efficiency cardiac gene delivery, according to the previously described protocol (20)
. Briefly, preoperative echocardiography was performed in all animals, hamsters were anesthetized with sodium pentobarbital (75 mg/kg), and ventilated, and the right carotid artery was cannulated with a catheter placed at the aortic root. The animals were subjected to general hypothermia by external cooling until the core temperature reached below 26°C. The pulmonary artery and the ascending aorta were occluded, histamine pretreatment was administered into the aorta (20 mM, vol 2.5 µl/gram body wt for 3 min), followed by the injection of the viral vector solution and subsequent resuscitation. A total of 1–6 x 1011 viral particles of AdV or AAV particles per 100 g of body wt were injected through the transcoronary route. The animals were terminated at 7 days after gene transfer in the AdV protocol, and at 12 wk after gene transfer in the AAV protocol.
The number of operated animals was as follows: normal hamsters, n = 6 for AdV-LacZ and n = 6 for AdV-INH-2; CM hamsters, n = 20 for AdV-LacZ, n = 20 for AdV-INH-2, n = 20 for AAV-LacZ, and n = 17 for AAV-INH-2 Death of the animals within 5 days after gene transfer procedure was regarded as improper recovery and not incorporated into the survival study (AAV-LacZ-treated group; n=3, AAV-INH-2-treated group, n=4). Overall operational mortality in 14-week-old CM hamsters subjected to the hypothermic gene delivery by AdV or AAV vectors was 18%.
Postgene transfer analysis
Before termination, the left ventricular dimension and systolic function were assessed in all animals with echocardiography and hemodynamic measurement by an operator who was unaware of the transduced gene. The heart tissue was then acquired for biochemical analyses. The gene transfer efficiency was estimated in AdV-LacZ- or AAV-LacZ-treated hearts, as described previously (20)
. Total RNA was prepared by using an RNeasy kit (Qiagen, Hilden, Germany). Northern blot analysis and real-time RT-PCR (Roche Diagnostics, Basel, Switzerland) were performed as described previously (26)
. The RT-PCR primer sets for PP1C and GAPDH were (5'-TCTGACCCTGACAAGGATG-3', 5'-CTTCTACAACCTGATGTGCTC-3') and (5'-TGCGGAAGAAAACTGCCTGG-3', 5'-CGGCTTGGTAAGAAGTCAGACG-3'), respectively.
Antibodies
Antibodies for PP1C and Rho-GDI (BD Biosciences, San Jose, CA), PP1 catalytic subunit ß (PP1Cß) and INH-2 (Calbiochem), phosphorylated-PLN at Ser16, PLN and phosphorylated-cyclic AMP response element binding protein (CREB) at Ser133 (Upstate Biotechnology, Lake Placid, NY), phosphorylated-PLN at Thr17 (Badrilla, UK), RyR(Sigma), CREB(Cell Signaling Technology, Beverly, MA), were obtained from commercially available sources. An antibody (Ab) for phosphorylated-RyR at Ser2808 was generously provided by Dr. Marks, Columbia University. Ab for INH-1 was generated by immunizing New Zealand White rabbits with polypeptides (DNSPRKIQFTVPLLEC and CGEEPEGATESTGNQE) of rat INH-1, and affinity-purified with the peptides.
Statistical analysis
Comparisons between two groups were performed by Student’s t test. Comparisons between repeated measurements were done by ANOVA, followed by post hoc testing (the Student-Newman-Keuls method was used to compare the two groups when appropriate). The cumulative survival curve was plotted by the Kaplan-Meier method, and statistical difference was evaluated by a log-rank test. A value of P < 0.05 was considered statistically significant. Data are expressed as the mean ± SEM
RESULTS
PP1 activities in CM hamster hearts
The time course of LV function was assessed by serial echocardiography, as shown in Fig. 1
. CM hamsters showed almost normal cardiac function at 6 wk with normal LV pressure (Fig. 1A-C
). At 10 wk, % fractional shortening (%FS) started to show a significant decrease, indicating progression of LV dysfunction. At 28 wk, LV diastolic dimension (LVDd) was markedly dilated with severe depression of %FS, decrease in LV systolic pressure, and elevation of LV end-diastolic pressure (Fig. 1A-C
), indicating overt signs of HF.
|
Biochemical analyses were performed at 6, 10, and 28 wk for the PP activities (Fig. 1D
–E) and PP1-associated protein expressions in the cytosol (Fig. 1F
) and the microsome (Fig. 1G
), respectively, and to determine the phosphorylation levels of key phosphoproteins in normal and CM hamsters (Fig. 2
). CM hamsters showed a gradual increase in PP1 activity both in the cytosol (Fig. 1D
) and in the microsome (Fig. 1E
) over the course of disease, while there was no change in normal hamsters. There was no change in PP2A activity in CM hamster throughout the observation period (Supplemental Fig. 1A–B). Immunoblotting of PP1 isoforms revealed a significant increase in PP1Cß (Fig. 1F
and Supplemental Fig. 1D) in the cytosol throughout the observation period. Increased expression levels of PP1C and ß in the microsome showed a trend to increase at 6 and 10 wk and significantly increased at 28 wk (Fig. 1G
and Supplemental Fig. 1E--F). INH-1 expression in CM hamsters was decreased compared with that in age-matched normal hamsters throughout the observation period, and there was no change in INH-2 expression compared with normal hamsters or among the different age groups (Supplemental Figs. 1G–H and 2). These results suggest that increased expression levels of PP1Cs may account for the increase in the PP1 activity in each fraction, and other regulatory molecules, including INH-1 and INH-2 may play an additional role.
|
The phosphorylation levels of PLN at Ser16 and RyR at Ser2808 (27)
(corresponding to the Ser2809 RyR reported by Reiken et al. (28)
) declined over the time course in normal hamsters (Fig. 2)
. Despite the increase in PP1 activity, the phosphorylation levels of PLN at Ser16 and RyR at Ser2808 were increased at 10 wk, a period of compensatory LV dysfunction, in CM hamsters compared to those in the age-matched normal hamsters. Phosphorylation levels of PLN at Ser16 further decreased at 28 wk compared to those assessed at 6 and 10 wk in the same strain (Fig. 2A
and 2B
), and became insignificant compared to that in age-matched normal hamsters. Phosphorylation levels of RyR at Ser2808 also showed a declining pattern at 28 wk (Fig. 2A
and 2D
) but still significantly higher than that in age-matched normal hamsters. Phosphorylation of PLN at Thr17 did not change significantly throughout the observation period between CM and normal hamsters or among the different age groups (Fig. 2A
and 2C
), although the phosphorylation showed a tendency to increase at 6 wk and a tendency to decrease at 28 wk in CM hamsters compared with normal hamsters. Phosphorylation of CREB at Ser133 in CM hamsters was decreased at 6 wk and became insignificant at 10 and 28 wk compared with that in normal hamsters (Fig. 2A
and 2E
). As phosphorylation of Ser16 PLN and of Ser2808 RyR was increased despite the increase in PP1 activity in CM hamsters, we hypothesized that PKA is hyperactive in CM hamster hearts. Indeed, the increases in phosphorylation of PLN at Ser16 and RyR at 2808 were roughly correlated with increased PKA activity (Fig. 2F
) and cAMP content (Fig. 2G
) during the transition to HF, whereas other phosphorylation targets such as Thr17 of PLN or Ser133 of CREB did not correlate with PKA activity. These data suggest that phosphorylation levels in Ser16-PLN and Ser2808-RyR are largely affected by the balance between PKA and PP1 activity during a period of compensatory LV dysfunction.
In vitro evaluation of overexpressing INH-2 in isolated adult cardiomyocytes
We used adenovirus (AdV)-mediated overexpression of INH-2 in cardiomyocytes to inhibit PP1 activity. Transduced INH-2 was detected in the cytosol in adult rat cardiomyocytes (Fig. 3
A). AdV-INH-2 gene transfer significantly inhibited PP1 activity without any effect on PP2A activity (Fig. 3B
). There was no change in PP1C expression with INH-2 overexpression in cardiomyocytes (Fig. 3C
). INH-2 gene transfer significantly increased the phosphorylation of PLN at Ser16 and of RyR at Ser2808 (Fig. 3C
), and enhanced Ca2+ transients and % cell shortening, as shown in the representative traces (Fig. 3D
) and summarized in Figure 3E
. INH-2 induced a 2.2- fold increase in % cell shortening, along with augmentation of the Ca2+ amplitude and decay time constant of the descending limb of the Ca2+ transient. These results indicate that INH-2 gene transfer can efficiently modulate the PP1 activity in cardiomyocytes.
|
In vivo effect of AdV-INH-2 gene transfer on cardiac function
We first tested the effect of in vivo AdV-mediated high efficiency cardiac gene transfer of INH-2 on cardiac function in normal hamsters. Hamsters into which the INH-2 gene was transferred showed hypercontractile LV function compared with LacZ-treated hamsters (Fig. 4
A). Successful INH-2 gene transduction was demonstrated by immunoblotting for INH-2, as shown in the cytosol (Fig. 4B
). Interestingly, expression of exogenous INH-2 caused a 120% increase in cytosolic PP1C and a 63% decrease in microsomal PP1C (Fig. 4B
). Although the expression of exogenous INH-2 caused an increase in cytosolic PP1C protein levels, there was no increase in cytosolic PP1 activity compared with the LacZ-treated group, suggesting that INH-2 inhibits PP1C in the cytosol. Indeed, the expression levels of exogenous INH-2 showed an inverse correlation with the cytosolic PP1 activity within the INH-2 treated group (Supplemental Fig. 3A). In contrast, the 63% decrease in microsomal PP1C expression coincided with a 47% decrease in microsomal PP1 activity and an increase in PLN phosphorylation at Ser16 after INH-2 gene delivery (Fig. 4B
--D and Supplemental Fig. 3C--F). These data suggest that overexpressed INH-2 induced an increase in inactive PP1Ca pools in the cytosol and promoted a translocation of active PP1C from the microsomes to the cytosol. These changes account for the decrease in microsomal PP1 activity, and concurrent increase in PLN phosphorylation at Ser16.
|
INH-2- gene transfer in CM hamsters was performed at 14 wk in the midst of the transition phase of HF from moderate to severe dysfunction. We chose this time point for the in vivo gene transfer study in order to test whether in vivo inhibition of PP1 is effective in preserving cardiac function after cardiac dysfunction becomes overt. At 14 wk, the CM hamsters showed a marked decrease in %FS and dilation in LVDd, as assessed by echocardiography (Fig. 5
A). We evaluated a total of 40 CM hamsters at 14 wk by echocardiography and randomly assigned them into two groups (20 CM hamsters received AdV-LacZ treatment and 20 received AdV-INH-2 treatment). The overall transfection efficiency at 7 days after gene transfer was 
40%, as assessed by LacZ staining of the LV (Fig. 5B
). At 7 days after gene transfer, the INH-2 transfected hamsters showed a significant reduction in LV chamber size (LVDd and LVDs; Fig. 5C
and 5D
) and an improvement of %FS (Fig. 5F
) compared with those before the gene transfer, whereas LacZ-treated hamsters showed slight deterioration of %FS and enlargement of LVDd, although these changes in the LacZ group did not reach the level of statistical significance. The index of LV diastolic wall stress, LVDd/PWT, was also significantly attenuated in the AdV-INH-2-treated group compared with the LacZ group (Fig. 5E
).
|
INH-2 gene transduction in the CM hamster hearts was confirmed by the immunoblotting for INH-2 (Supplemental Fig. 4A). Changes in PP1C expression levels and PP1 activities in the cytosol and microsomes were almost identical with those in normal hamsters transfected with the INH-2 gene (an 80% increase in cytosolic PP1C and a 30% decrease in microsomal PP1C), except for the slight increase in PP1 activity in the cytosol (Supplemental Fig. 4C
). Phosphorylation of PLN at Ser16 was increased and that at Thr17 also showed an increasing tendency (Fig. 6
A). On the other hand, phosphorylation of RyR at Ser2808 did not show significant change (Fig. 6A
), and another intracellular PKA phosphorylation target, CREB, did not show any change in phosphorylation at Ser133 by INH-2 gene transfer compared with the LacZ-treated group (Fig. 6A
). The PKA activity in CM hamster heart at 14 wk was significantly higher than that in normal hamsters. Increased PKA activity in the CM hamster was abnormally higher than that of PKA activity stimulated by intravenous (i.v.) isoproterenol infusion in normal hamsters (Fig. 16B). Interestingly, INH-2 gene transfer significantly prevented an abnormal increase in PKA activity, despite the significant increase in PLN phosphorylation at Ser16. Decrease in PKA activity in INH-2-treated hamsters paralleled a decreasing tendency of plasma catecholamine levels (LacZ vs. INH-2 group: epinephrine, 490±291 vs. 56±21 pg/ml, P = 0.08; norepinephrine, 9862±6001 vs. 4754±1609 pg/ml, P = 0.22; n=5 in each group), suggesting that the reduction in the PKA activity was due to the decrease in sympathetic drive. Gene delivery of INH-2 into the CM hamster heart reduced brain natriuretic peptide (BNP) levels (Fig. 6C
), a predictor of exacerbation of HF, compared with the LacZ-treated group (P<0.05, n=6 in each group). These results indicate that INH-2 gene transfer has a favorable effect on the failing heart, at least in the short term.
|
In vivo effect of AAV-INH-2 gene transfer on cardiac function and survival
Because in vivo AdV-INH-2 gene delivery resulted in improvement of cardiac function and ameliorated BNP expression in the short term, we further tested the long-term effect of INH-2 gene delivery by AAV-mediated gene transfer, as described previously (19
, 25)
. 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. Comparison of the survival time course between the AAV-INH-2- and AAV-LacZ-treated groups was made during this 12 wk follow-up period. As shown in Figure 7
A, the transfection efficiency of AAV-LacZ was 40% of the LV cross-sectional area, which was similar to that by adenoviral gene delivery. The expression of exogenous INH-2 was confirmed by immunoblotting in the cardiac homogenate as shown in Figure 7B
. AAV-INH-2 gene transfer preserved %FS compared with the AAV-LacZ-treated group over the observation period (Fig. 7C
). The left ventricular end-diastolic diameter showed decreasing tendency at 1 and 3 mo following gene transfer, but did not reach the level of statistical significance (data not shown). The maximum dP/dt at 26 wk of age in the AAV-INH-2 group also showed a tendency toward preservation (P=0.051) compared with the AAV-LacZ group, whereas LV peak systolic pressure and heart rate did not show any significant difference (Table 1)
. Other hemodynamic parameters also did not show statistical significance as summarized in Table 1
. AAV-INH-2 gene transfer significantly decreased microsomal PP1 activity (Fig. 7D
) and increased PLN phosphorylation at Ser16 (Fig. 7E
), as observed in the short-term adenoviral gene transfer, although PKA activity showed no differences between in the AAV-INH-2-treated and the AAV-LacZ-treated groups (data not shown). In addition, there was significantly less interstitial fibrosis in INH-2-treated CM hamster hearts (Fig. 7F
). It should be noted that the difference between the AAV-INH-2- and AAV-LacZ-treated groups in the hemodynamic data at 26 wk of age may have been biased toward dissipation. This is because 71% of the AAV-LacZ-treated animals had died before 26 wk of age, presumably because of the worsening cardiac function, and could not be assessed (see below).
|
|
In the Kaplan-Meier plot (Fig. 7G
), 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.
DISCUSSION
In the present study, we demonstrated that in vivo gene transfer of INH-2 effectively prevented HF progression in the observation period of one week in adenoviral gene transfer and three months in 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 caused less progression of LV dysfunction and interstitial fibrosis, and the 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 the high efficiency cardiac gene transfer approach (19
, 20
, 25)
.
Our biochemical characterization suggested that hyperactivation of PKA and increase in phosphorylation of PLN at younger age in CM hamsters may represent the compensatory mechanism against deteriorating cardiac function. However, the compensatory mechanism eventually fails in the late phase, as demonstrated by rapid deterioration of cardiac function, which coincided with the increase in PP1 activity and decrease in phosphorylation of Ser16-PLN, even though PKA activity and cAMP content were still higher than those in normal hamsters. Because changes in PKA activities did not directly correlate with changes in PLN phosphorylation at the terminal stage of HF (i.e., 28 wk of age), it is suggested that defects in local regulation of PKA through a variety of A-kinase-anchoring proteins (AKAP) may also be critically involved in the phosphorylation balance in CM hamsters (29)
. In addition, local regulation of PP1C and its regulatory proteins may also participate in altered phosphorylation balance of phosphoproteins in the corresponding microdomains. The increase in phosphorylation of Ser16-PLN together with PKA activation during the transition to HF in the CM heart is somewhat different from previous reports (7
, 8
, 30)
. However, other studies have reported that the phosphorylation status of Ser16-PLN did not change in progressive HF (31
, 32)
. As HF is a disease with multiple etiologies, which progresses through complicated biochemical alterations, it is expected that the phosphoprotein status of Ser16-PLN and PKA activity may differ depending on the etiology and/or the time point examined. Nonetheless, our data support the notion that hyperactive PP1 is an exacerbating factor against the compensatory mechanism of HF, because suppression of PP1 activity by INH-2 gene transfer showed beneficial effects in CM hamster where phosphorylation of PLN is still preserved compared with that in normal hamsters. Therefore, INH-2 gene transfer seems to support the compensatory mechanism in the failing heart, resulting in better cardiac function and survival.
Our data suggested that the beneficial effect of INH-2 gene delivery is, at least in part, attributable to the increase in phosphorylation of Ser16-PLN at Ser16, because inhibition of endogenous PLN has been reported to be beneficial in treating certain types of HF (19
, 25
, 33
34
35)
, including HF in the other hamster model of cardiomyopathy (i.e., the BIO14.6 strain model) (19)
. Interestingly, INH-2 gene transfer in vivo caused a decrease in microsomal PP1 activity, which may be explained by a translocation of the PP1C protein from the microsome to the cytosol. Increase in cytosolic PP1C in normal and CM hamster, increasing trend of cytosolic PP1 activity in CM hamster may also be explained by the function of INH-2 to stabilize PP1C in the cytosol, as suggested previously (15)
. Although the molecular mechanisms by which INH-2 induces a translocation of PP1C remain to be determined, the subsequent decrease in microsomal PP1 activity appeared to be well coupled with the increase in Ser16 PLN phosphorylation by INH-2 overexpression.
Intriguingly, INH-2 gene transfer normalized the hyperactive PKA in CM hamsters in the short-term experiment. This was interpreted that INH-2 augmented PLN phosphorylation, thereby enhancing cardiac contractility in the failing heart without PKA activation. Enhanced cardiac performance by INH-2 gene transfer relieved the hyperactive sympathetic tone and concomitant ß-adrenergic signaling in these CM hamsters, thereby decreasing PKA activity in the heart. This interpretation is supported by the trend of a decrease in serum catecholamine levels in the I-2-treated CM hamsters. Although hyperactivation of the ß-adrenergic system enhances cardiac output in the short term, it may be cardiotoxic over the long run, according to the failed clinical trials of phosphodiesterase III inhibitors (36)
and in vivo transgenic animal studies (37
, 38)
. In addition, sympathetic hyperactivation results in the increase in the peripheral vascular resistance, thereby increasing the afterload of the failing heart (1)
. Therefore, the enhanced cardiac function via PLN phosphorylation without activating PKA may explain the extended survival time in CM hamsters by INH-2 gene transfer.
The phosphorylation status of RyR appeared to be less affected in in vivo INH-2-treated hamsters, whereas in vitro adenoviral INH-2 gene transfer was associated with an increase in RyR phosphorylation, as well as an increase in PLN phosphorylation in the cardiomyocytes. This difference of the effect of INH-2 on RyR phosphorylation between in vivo and in vitro experiments may be explained by the extent of PP1 inhibition and the difference of PP1C expression levels between in vitro and in vivo, and local regulation of PKA and PP1 activities as discussed above (29)
. These findings suggest that PLN phosphorylation is more sensitive to the modulation of PP1 activity. This differential effect of INH-2 on RyR and PLN may be beneficial because hyperphosphorylation of RyR may cause Ca2+ leak leading to arrhythmogenicity (39)
and cardiac dysfunction (40)
, although the effect of INH-2 gene transfer on arrhythmogenicity remains to be determined. It also remains to be determined whether INH-2 gene delivery causes some adverse effects, such as carcinogenesis or metabolic disorder, since systemic PP inhibition by chemicals has been known to be hazardous (41)
.
Recently, Pathak et al. (14)
have demonstrated that transgenic PP1 inhibition by constitutively active INH-1 suppressed the LV remodeling in an aortic constriction model and prevented the progression of cardiac failure in aorta-banded rats in the short-term using adenoviral mediated in vivo gene transfer approach. Although the long-term benefit of PP1 inhibition by gene transfer was not evaluated in their study, their results are in agreement with the present study regarding the benefit of selective PP1 inhibition and the enhancement of PLN phosphorylation in the failing heart.
In conclusion, we have demonstrated that in vivo myocardial PP1 inhibition by inhibitor-2 up-regulated SR calcium handling through PLN phosphorylation and corrected the abnormally increased PKA activity, thereby ameliorating the long-term progression of HF in the cardiomyopathic hamster. PP1 inhibition in the sarcoplasmic reticulum and sarcolemma may constitute a new therapeutic target for the treatment of end-stage HF.
ACKNOWLEDGMENTS
We thank Hiromi Nakagawa, Tomoko Hozawa, Megumi Ohishi, and Rie Ishihara for technical assistance, and Tomoko Ohkusa for her maintenance of the CM hamster colony. This study was supported by grants from the Ministry of Education, Science and Technology of Japan [Nos. 14370228 (M.M.), 15590754 (Y.I.), 16209026 (M.M.)] and by Grants-in-Aid from the Japan Heart Foundation Molecular Cardiovascular Research Group (Y.I.), the Mitsubishi Pharma Research Foundation (M.M.), the Japan Cardiovascular Research Foundation (Y.I.), the Takeda Science Foundation (Y.I.), and the Kanae Foundation for Life & Socio-Medical Science (YI), and by a Grant from the Sankyo Pharmaceutical Inc. for the Department of Molecular Cardiovascular Biology, Yamaguchi University, School of Medicine.
Received for publication October 23, 2005. Accepted for publication January 24, 2006.
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