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* Division of Cardiology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA;
Department of Thoracic Surgery, Peking University Third Hospital, Beijing, China; and
Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
1Correspondence: 720 Rutland Ave., Ross 333, Baltimore, MD 21205, USA. E-mail: czheqin1{at}jhmi.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: ubiquitin-proteasome system • reperfusion injury • ubiquitin • phosphatase and tensin homologue deleted on chromosome 10 • PTEN • Akt
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. IPC activates several signaling pathways, including protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K) (2
3
4
5
6
7)
. Moreover, IPC inactivates the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) (8)
. There is also significant cross-talk between the phosphatase PTEN and the survival protein kinases (8
9
10)
. PTEN antagonizes PI3K by dephosphorylating its product phosphatidylinositol-3,4,5-triphosphate (PIP3), which activates Akt directly or through PIP3-dependent protein kinase 1 (PDK1) (10)
. PDK1 is a protein that functions upstream of PKC (11)
. Therefore, PTEN may play a pivotal role in controlling cell fate (4)
. IPC also activates the ubiquitin-proteasome system (UPS) in the brain (12)
, and proteasome inhibition leads to blockade of IPC protection in neurons (13
, 14)
. However, whether proteasome activity is necessary for IPC induction in the heart has not been investigated previously.
Cellular proteins are predominantly degraded through the UPS. The process of protein degradation can be divided into two distinct steps, polyubiquitination and proteasome digestion (15
16
17
18
19)
. Ubiquitins are first targeted to substrates through specific ligases. Polyubiquitinated substrates are rapidly degraded by the 26S proteasome, consisting of two regulatory 19S complexes and one catalytic 20S proteasome core (19)
. The 20S proteasome contains four stacked rings. The inner two rings are composed of two sets of seven β-subunits and two sets of seven 
-subunits are aligned over their corresponding β-subunits as outer rings. The β-subunits β1, β2, and β5 are linked to caspase-like (Cas-L), tryptic-like (Tryp-L), and chymotryptic-like (Chym-L) activities, respectively. These constitutive subunits can be replaced by the inducible immunoproteasome subunits low molecular mass polypeptide (LMP) -2/β1i, multicatalytic endopeptidase complex-like (MECL) -1/β2i, and LMP-7/β5i (20
, 21)
. After these subunits are incorporated and processed, the proteolytic activity and specificity of the proteasome are changed (22
23
24)
.
LMP-2 is induced by inflammatory stimuli, especially interferon-
; however, it is also constitutively expressed in several tissues (25
26
27)
, and its function is not limited to antigen presentation. LMP-2 has also been implicated in controlling the accumulation of damaged proteins. LMP-2 deficiency results in a decrease in 20S proteasome activity and accumulation of oxidized proteins in the brain and liver of mice (28)
. Moreover, LMP-2 regulates estrogen receptor-mediated gene transcription through the UPS (29)
. Finally, in addition to interferon-, other regulators of LMP-2 expression such as nitric oxide and heart shock have been reported (30
, 31)
. Therefore, LMP-2 may act as a modulator of proteasome activity in the cell. In this study, we have tested the hypothesis that LMP-2 plays a critical role in cardioprotection in response to IPC.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals
All experiments were performed with age-matched male mice that were either wild-type (WT) (C57BL6) or LMP-2 knockout (KO) (LMP-2–/–) (backcrossed 10 times to C57BL6). LMP-2–/– mice were generated by deletion of the corresponding gene using embryonic stem cell technology (27)
. At the time of the experiment, mice were 2–3 months old and weighed 22–32 g. All procedures were approved by The Johns Hopkins University Institutional Animal Care and Use Committee and conformed to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996).
In situ ischemia and reperfusion mouse model
Mice were anesthetized by intraperitoneal injection of pentobarbital (70 mg/kg) and were mechanically ventilated (32)
. A left thoracotomy was performed, and the mouse was then turned slightly to the right. The left coronary artery was identified visually, and a 7-0 suture was placed around the artery 
1 mm below the left auricle. The electrocardiogram (ECG) was recorded continuously. The mice were warmed to 37°C with heating lamps and kept continuously at this temperature, monitored with a rectal probe. A loop was made in the suture around the coronary artery and tightened over a small piece of polyethylene tubing. Occlusion of the left coronary artery resulted in changes in heart color (pallor) and ECG (ST-segment elevation and widening of the QRS complex). Reperfusion was accomplished by loosening the loop and confirmed by return of red color to the region and improved cardiac contraction. All mice were subjected to 30 min of ischemia (I-30) and 120 min of reperfusion (R-120) with or without preceding IPC, which consisted of a single episode of 10 min of ischemia (I-10) and 5 min of reperfusion (R-5). The PTEN inhibitor HOpic was administrated by intraperitoneal injection at a dose of 50 µg/kg (body weight) 15 min before ischemia-reperfusion. Saline was used as a control. At the end of the experiment, the animals were euthanized by transecting the aorta and removing the heart for infarct size determination.
Measurement of left ventricular pressure
After exposure of the heart, a 26-gauge needle was used to make a stab wound near the left ventricular apex. A Mikro-tip catheter (Millar Instruments, Houston, TX, USA) containing a tiny pressure sensor at the tip was inserted through the stab wound into the left ventricle. Left ventricular pressure was directly measured with the Powerlab Data Acquisition System and displayed on a computer. Left ventricular developed pressure (LVDP=systolic pressure–end-diastolic pressure), heart rate, positive maximal left ventricular pressure (LVP) derivative (+dp/dtm), and negative maximal LVP derivative (–dp/dtm) were automatically calculated using Chart 5 software.
Assessment of area at risk and infarct size
The loop around the coronary artery was retightened just before euthanasia. Then 1% Evans Blue solution was injected into the aorta, and the dye was passed into the coronary arteries and distributed throughout the nonrisk ventricular wall proximal to the coronary artery ligature. The heart was frozen and then cut transversely into five sections, each of which was weighed and incubated in 1.5% triphenyltetrazolium chloride (TTC) for 15 min at 37°C. The area not at risk appeared blue. Viable myocardium in the area at risk stained brick red and infarcted tissue was pale white. Two sides (A and B) of each section were photographed. The areas of blue, red, or white color were measured by computerized planimetry (Image J; National Institutes of Health, Bethesda, MD, USA). The following indices were calculated: infarct size as a percentage of left ventricle (IS/LV) = total weight of white tissue/weight of left ventricle (LV) x 100%; area at risk (AAR) as a percentage of LV (AAR/LV): total weight of white tissue + red tissue/weight of LV x 100%; and infarct size as a percentage of the area at risk (IS/AAR) = total weight of white tissue/total weight of white tissue + red tissue x 100%.
Mouse Langendorff preparation
Mice were anesthetized by intraperitoneal injection of pentobarbital (70 mg/kg). After the chest was opened, the heart was excised, and the ascending aorta was cannulated with a blunt needle. The heart was perfused at a constant pressure of 100 cm H2O with Krebs-Henseleit buffer (17 mM glucose, 120 mM NaCl, 25 mM NaHCO3, 2.5 mM CaCl2, 5.9 mM KCl, 1.2 mM MgSO4, and 0.5 mM EDTA), which was maintained at 37°C and bubbled continuously with a mixture of 95% O2 and 5% CO2. Global ischemia was induced by cessation of perfusion, followed by reperfusion. IPC was induced by a single episode of 10 min of ischemia and 5 min of reperfusion. HOpic (final concentration 100 nM) was injected into the perfusion line by a syringe pump according to the coronary flow rate.
Measurement of proteolytic activities
Hearts were homogenized on ice in the assay buffer (25 mM HEPES, pH 7.5; 0.5 mM EDTA; and 0.03% SDS) as described previously (33)
. Chymotryptic-like activity and caspase-like activity were measured using the fluorogenic substrates Suc-LLVY-aminomethycoumarin (AMC) and Z-LLE-AMC. Equal amounts (0.65 g) of protein were reacted with 50 µM concentrations of the substrates in 100 µl of assay buffer for 24 h at room temperature. The cleavage product AMC was analyzed in a fluorometer (excitation/emission: 355/460 nm). Background activity (caused by nonproteasomal degradation) was determined by addition of the proteasome inhibitor lactacystin at a final concentration of 50 µM. Proteolytic activities are presented as relative activity (RA) to wild-type control. A similar protocol was used to measure tryptic-like activity. In this assay, Boc-LLR-AMC was reacted with 6.5 g of protein in a separate buffer (25 mM HEPES, pH 7.5; 0.5 mM EDTA; 0.05% Nonidet P-40; and 0.001% SDS).
Immunoprecipitation and Western blot analysis
Hearts were homogenized in lysate buffer (20 mM Tris, pH 7.5; 150 mM NaCl; 1 mM EDTA; 1mM EGTA; 1 mM phenylmethylsulfonyl fluoride; 1 mM Na3VO4; and 1% Triton). PTEN was immunoprecipitated from 300 µg of heart lysates with 20 µl of anti-PTEN antibody overnight, followed by incubation with protein G-agarose beads (Santa Cruz Biotechnology, Inc.) for 2 h at 4°C. The precipitates were washed 3 times with lysate buffer and then dissolved in sample buffer. Proteins were separated on a precast NuPAGE Bis-Tris gel (Invitrogen, Carlsbad, CA, USA) and transferred to a nitrocellulose membrane. Proteins were detected using primary antibodies, followed by horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence.
Statistical analysis
Data are presented as mean ± SE values. The difference among groups was analyzed by ANOVA or Student’s t test. Differences were considered significant if P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), but their hearts were significantly smaller (147±7.1 vs. 178±10.5 mg, P<0.05) (Fig. 1A
). However, within each genotype (LMP-2+/+ or LMP-2–/–), control (CON) and IPC groups did not differ with respect to body weight or heart weight (Table 1)
. LMP-2 protein was undetectable in LMP-2–/– hearts (Fig. 1B
). LMP-2 deficiency did not significantly alter LVDP or cardiac contractility (+dp/dtm and –dp/dtm) but did reduce heart rate (346±16 vs. 412±24 beats/min, P<0.05) (Table 1)
. These data indicate that LMP-2 deficiency may result in anatomic and functional changes in the heart.
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Ischemic preconditioning is impaired in LMP-2–/– mice
To determine whether LMP-2 is involved in IPC, we subjected the LMP-2+/+ and LMP-2–/– mice to IPC, one cycle of I-10/R-5, followed by I-30/R-120. In LMP-2+/+ mice, IPC resulted in significantly higher LVDP at the end of reperfusion compared with mice without IPC (CON) (70±6 vs. 50±4 mmHg, P<0.05) (Fig. 2A
). However, in LMP-2–/– mice, IPC did not significantly improve LVDP compared with that in littermate controls (63±3 vs. 54±6 mmHg, P>0.05) (Fig. 2B
). Similarly, IPC increased cardiac contractility +dp/dtm in LMP-2+/+ mice (4060±529 vs. 2477±194 mmHg/s, P<0.05) (Fig. 2C
) but not in LMP-2–/– mice (3328±208 vs. 2997±360 mmHg/s, P>0.05) (Fig. 2D
). Consistent with the changes in cardiac function, IPC decreased myocardial infarct size in LMP-2+/+ mice compared with control mice (IS/AAR: 33±4 vs. 58±6%, P<0.01) (Fig. 3A, B
) but not in LMP-2–/– mice (IS/AAR: 50±4 vs. 47±6%, P>0.05) (Fig. 3A, C
). Similar results were obtained using IS/LV as an index of infarct size (LMP-2+/+ mice: 16±3 vs. 31±3%, P<0.01; LMP-2–/– mice: 27±2 vs. 22±3%, P>0.05) (Fig. 3B, C
). There was no significant difference in AAR/LV between CON and IPC in LMP-2+/+ or LMP-2–/– mice (Fig. 3A-C
). These data show that LMP-2 deficiency leads to impairment of IPC cardioprotection.
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PTEN degradation and Akt phosphorylation are decreased in preconditioned LMP-2–/– hearts
PTEN is down-regulated during ischemia and reperfusion by a proteasome-dependent mechanism (8)
. We therefore determined whether LMP-2 deficiency affects IPC-mediated PTEN inactivation. Isolated perfused hearts from LMP-2+/+ and LMP-2–/– mice were exposed to one episode of IPC (I-10/R-5) or CON (nonischemia), and then they were assayed for PTEN, p-Akt, and total Akt by Western blot. Under basal conditions, PTEN levels were significantly increased in LMP-2–/– hearts compared with those in LMP-2+/+ hearts [145±10 vs. 103±4 arbitrary units (AU), P<0.05] (Fig. 4A, B
). In LMP-2+/+ mice, IPC significantly decreased PTEN levels (70±3 vs. 102±4 AU, P<0.01) (Fig. 4B
) and increased Akt phosphorylation (117±4 vs. 40±2 AU, P<0.01) (Fig. 4C
). However, in LMP-2–/– mice, IPC did not significantly change PTEN or p-Akt levels (PTEN: 138±5 vs. 145±10 AU, P>0.05; p-Akt: 53±10 vs. 71±2 AU, P>0.05) (Fig. 4A-C
).
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Degradation of ubiquitinated proteins is impaired in LMP-2–/– hearts
To determine whether the impairment of PTEN down-regulation by IPC is due to deficient UPS function in LMP-2–/– hearts, we first detected ubiquitinated protein levels in isolated LMP-2+/+ and LMP-2–/– hearts with or without IPC. IPC decreased the levels of ubiquitinated proteins in LMP-2+/+ hearts, suggesting an increase in UPS function. In contrast, in LMP-2–/– hearts, the basal level of ubiquitinated proteins was increased compared with that in LMP-2+/+ hearts, but there was no change after IPC (Fig. 5A
). Next, to determine whether UPS-mediated PTEN degradation is impaired in LMP-2–/– hearts after IPC, we first precipitated PTEN with anti-rabbit PTEN antibody and then transferred it to a membrane, which was blotted with anti-mouse ubiquitin antibody. IPC decreased PTEN ubiquitination in LMP-2+/+ hearts, but in LMP-2–/– hearts, IPC increased PTEN ubiquitination (Fig. 5B
). Last, to determine whether the same amounts of PTEN were pulled down from the hearts, we stripped this Western blot. After no signal was detected from the membrane, we reblotted it with anti-mouse PTEN antibody. The PTEN bands (with molecular mass of 55 kDa) had a similar density in the lysates from CON and IPC hearts (Fig. 5C
). However, there was a second band with slightly higher molecular mass in the samples from LMP-2–/– hearts. This might be due to PTEN monoubiquitination. These results suggest that proteasome function is impaired in LMP-2–/– hearts.
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Proteolytic activities are decreased in LMP-2–/– hearts after IPC
To determine whether proteolytic activities are decreased in LMP-2–/– hearts, we exposed isolated LMP-2+/+ and LMP-2–/– hearts to IPC or CON and then measured Cas-L, Chym-L, and Tryp-L activities. IPC increased Cas-L activity in LMP-2+/+ hearts (1.15±0.02 vs. 1.00±0.02 RA, P<0.01) (Fig. 6
) compared with CON. However, in LMP-2–/– hearts, IPC significantly decreased this activity (0.90±0.03 vs. 1.17±0.02 RA, P<0.001) (Fig. 6)
. Under basal conditions, Chym-L activity and Tryp-L activity were decreased in LMP-2–/– hearts compared with LMP-2+/+ hearts (Chym-L activity: 0.73±0.02 vs. 1.00±0.03 RA, P<0.01; Tryp-L activity: 0.67±0.02 vs. 1.00±0.01 RA, P<0.01) (Fig. 6)
. IPC caused a decrease in Chym-L and Tryp-L activities in LMP-2+/+ hearts (Chym-L: 0.80±0.05 vs. 1.00±0.03 RA, P<0.01; Tryp-L: 0.83±0.02 vs. 1.00±0.01 RA, P<0.01) (Fig. 6)
and LMP-2–/– hearts (Chym-L: 0.52±0.04 vs. 0.73±0.02 RA, P<0.01; Tryp-L: 0.50±0.02 vs. 0.67±0.02 RA, P<0.01) (Fig. 6)
. Therefore, in LMP-2–/– hearts, proteolytic activities are dysregulated under basal conditions and worsened by IPC.
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The immunoproteasome is defective in LMP-2–/– hearts
Because proteolytic activities are executed in the 20S proteasome, we wondered whether the immunoproteasome is defective in LMP-2–/– hearts and whether IPC regulates the immunoproteasome in LMP-2+/+ hearts. We determined protein levels of LMP-2, LMP-7, MECL-1, and their precursors in LMP-2+/+ and LMP-2–/– hearts with or without preceding IPC. IPC significantly increased LMP-2 levels in LMP-2+/+ hearts (105±8 vs. 31±9 AU, P<0.01) (Fig. 7A, B
), but it decreased MECL-1 (68±5 vs. 134±10 AU, P<0.001) (Fig. 7C, D
) and LMP-7 (43±10 vs. 129±4 AU, P<0.001) (Fig. 7E, F
). Under basal conditions, LMP-2 was undetectable in LMP-2–/– hearts. Interestingly, MECL-1 and LMP-7 were also markedly decreased in these hearts compared with LMP-2+/+ hearts [MECL-1: 11±6 vs.134±10 AU, P<0.001 (Fig. 7C, D
); LMP-7: 8±6 vs. 129±4 AU, P<0.001 (Fig. 7E, F
)]. IPC did not significantly affect these protein levels in LMP-2–/– hearts. Together, the results show that the immunoproteasome is defective in LMP-2–/– hearts during CON and IPC and that IPC regulates immunoproteasome subunits in LMP-2+/+ hearts.
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Preconditioning with the PTEN inhibitor HOpic induces cardioprotection in LMP-2–/– mice
To determine whether PTEN inhibition is cardioprotective, we used the PTEN inhibitor HOpic to precondition LMP-2–/– hearts for 15 min, followed by I-30/R-120. HOpic significantly increased LVDP (71±3 vs. 55±2 mmHg, P<0.01) (Fig. 8A
) and maximal dp/dt (4499±371 vs. 3112±237 mmHg/s, P<0.01) (Fig. 8B
). Moreover, HOpic significantly reduced myocardial infarct size after I-30/R-120 compared with the saline control (IS/AAR: 28±2 vs. 48±3%, P<0.001) (Fig. 8C, D
), with no significant difference in the area at risk (AAR/LV: 53±4 vs. 48±3%, P>0.05) (Fig. 8D
). To confirm that PTEN is inactivated by HOpic in the heart, we measured p-Akt levels after isolated hearts were treated with 100 nM HOpic for 15 min. HOpic significantly increased Akt phosphorylation compared with saline (155±6 vs. 32±9 AU, P<0.01) (Fig. 9A, B
). These results indicate that PTEN inactivation induces cardioprotection.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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LMP-2 deficiency prevents immunoproteasome formation
The UPS plays a predominant role in the control of protein quality and quantity (15)
. Its dysfunction has been implicated in various cardiovascular diseases including ischemia and reperfusion injury, cardiac hypertrophy, and heart failure (16
17
18)
. However, little is known about the regulation of the UPS. The 20S proteasome executes protein digestion. Its proteolytic activities are generated from the constitutive β1, β2, and β5 subunits. In response to stimulation of interferon-, the 20S proteasome is modulated by incorporation of LMP-2, MECL-1, and LMP-7. The process includes assembly of their precursors and subsequent cleavage of prosequences (34)
. These inducible subunits assemble the immunoproteasome in a cooperative manner. LMP-2 plays an important role in this process. It is essential for incorporation and processing of MECL-1 (21
, 35)
. Consistent with prior observations, the present study showed that MECL-1 and LMP-7 are markedly reduced in LMP-2–/– hearts despite the presence of their precursors, suggesting that the immunoproteasome is defective in these mice. The decrease in MECL-1 and LMP-7 protein levels may explain the decreased Chym-L and Tryp-L activities in LMP-2–/– hearts. Decreased proteolytic activities are also found in the brain and liver of LMP-2–/– mice (27
, 28)
. LMP-2 may suppress Cas-L activity in the heart under basal conditions. Overexpression of LMP-2 causes decreased Cas-L activity in cells (23
, 24)
, and LMP-2 deficiency leads to increased Cas-L activity in certain tissues (27)
. We showed that Cas-L activity is increased in LMP-2–/– hearts. Therefore, LMP-2 is essential for immunoproteasome formation and normal proteolytic activities in the heart.
The immunoproteasome is necessary for normal UPS function in the heart
The immunoproteasome has been implicated in protein degradation by the UPS. It has been reported that decreased Chym-L and Tryp-L activities are associated with accumulation of oxidized proteins in LMP-2–/– mice (28)
. Moreover, LMP-2 inhibition blocks inhibitory NF-
B degradation and p50 generation during NF-B activation in a human invasive extravillous trophoblast cell line (36)
. In the present study, ubiquitinated proteins were increased in LMP-2–/– mice after IPC. Notably, the degradation of the proapoptotic protein PTEN was impaired after ubiquitination. IPC promotes degradation of ubiquinated proteins in LMP-2+/+ hearts. However, with the defective immunoproteasome induced by LMP-2 deficiency, the heart is unable to remove ubiquitinated proteins, consistent with an important role for the immunoproteasome in UPS function in the heart. IPC modulates proteolytic activities, probably by regulating immunoproteasome subunits. Decreased Chym-L and Tryp-L activities were associated with decreased LMP-7 and MECL-1 in LMP-2+/+ hearts after IPC (I-10/R-5) in our study. The result is consistent with results of a previous study, which showed that a longer period of ischemia and reperfusion (I-30/R-60) decreased Chym-L activity in the heart (44)
. The explanation for why we found decreased Chym-L activity after such a brief period of ischemia, unlike the early study, is uncertain but could be related to the more sensitive assay used in our study.
Interestingly, IPC increases endogenous LMP-2 levels and Cas-L activity in LMP-2+/+ hearts. The increased Cas-L activity may be necessary to degrade ubiquitinated proteins as part of IPC-induced cardioprotection. An increase in endogenous LMP-2 appears to have a different effect on Cas-L activity than exogenous overexpression of LMP-2 (23
, 24)
. The underlying mechanism for this is not known. LMP-2 deficiency also affects the regulation of the other inducible subunits mediated by IPC. There is no significant difference in LMP-7 and MECL-1 between CON and IPC in LMP-2–/– hearts. However, all three proteolytic activities are decreased in these samples after IPC. Because IPC produces reactive oxygen species (ROS), the decreased proteolytic activities in LMP-2–/– hearts may result from further damage of the 20S proteasome by ROS (37)
. Notably, the degradation of ubiquitinated proteins is increased in LMP-2+/+ hearts but impaired in LMP-2–/– hearts after IPC.
LMP-2 deficiency contributes to cardiac underdevelopment
The UPS is involved in cellular growth, differentiation, and survival. Proteasome inhibition attenuates cardiac hypertrophy induced by pressure overload and isoproterenol injection (38
, 39)
. In this study, we showed that PTEN levels are elevated in LMP-2–/– hearts. It has been reported that overexpression of wild-type PTEN promotes apoptosis in cardiomyocytes (40)
. Therefore, increased PTEN protein levels could be an explanation for impaired cardiac development in LMP-2–/– mice. Interestingly, despite a smaller heart size, these mice have a larger body size than LMP-2+/+ mice, as described previously (41)
. Although the underlying mechanism is not clear, the abnormality is associated with increased motor function (41)
. Heart rate is also lower in LMP-2–/– mice than in LMP-2+/+ mice. This finding is consistent with a recent report, in which proteasome inhibition decreased ventricular tachyarrhythmias caused by ischemia and reperfusion injury (42)
.
Impairment of PTEN degradation contributes to loss of IPC-induced cardioprotection
IPC generates profound cardioprotection in various models (2
3
4
5
6
7)
. In the IPC model used here, a single cycle of 10 min of ischemia followed by 5 min of reperfusion significantly decreased infarct size (in IS/AAR) by 42% and increased cardiac functional recovery (in LVDP) by 39%. However, IPC cardioprotection was lost in LMP-2–/– mice. Although several survival protein kinases have been implicated in IPC, including PI3K, PKC, and MAPK, new signaling molecules continue to emerge. Recently, we reported that PTEN inactivation by IPC is associated with its cardioprotection (8)
. PTEN protein is subjected to proteasomal degradation (8
, 43)
. In the present study, we demonstrated that PTEN degradation and Akt activation are impaired in LMP-2–/– hearts after IPC and that PTEN inhibition induces IPC-like cardioprotection in these hearts. These results provide additional evidence that PTEN plays an important role in IPC. Moreover, our study suggests that LMP-2 expression is necessary for proteasome-mediated PTEN degradation and IPC protection. Indeed, the down-regulation of proapoptotic proteins in IPC may be crucial for tissues to tolerate later lethal injury. In the brain, the proteasome mediates IPC protection through degradation of the proapoptotic protein Bim (14)
. Although the UPS can potentially regulate G protein-coupled receptors and receptor tyrosine kinases, it remains unclear whether the UPS is actually involved in the activation of the survival protein kinases through these pathways during IPC.
In conclusion, inducible LMP-2 is essential for immunoproteasome formation and function in the heart. IPC induces cardioprotection through UPS-mediated signaling, probably PTEN protein degradation and Akt activation. Our study also suggests that LMP-2 plays an important role in cardiac development. Therefore, by regulating LMP-2 activity, it might be possible to generate novel therapeutic treatments for various cardiovascular diseases, including ischemia-reperfusion injury, cardiac hypertrophy, and heart failure.
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ACKNOWLEDGMENTS |
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Received for publication January 22, 2008. Accepted for publication July 24, 2008.
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and 
in the heart of conscious rabbits without subcellular redistribution of total protein kinase C activity. Circ. Res. 81,404-414-inducible 11 S regulator (PA28) and the LMP2/LMP7 subunits govern the peptide production by the 20 S proteasome in vitro. J. Biol. Chem. 270,23808-23815-Interferon and expression of MHC genes regulate peptide hydrolysis by proteasomes. Nature 365,264-267[CrossRef][Medline] (IFN-)-inducible subunits. J. Exp. Med. 187,97-104This article has been cited by other articles:
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O. Tsukamoto, T. Minamino, and M. Kitakaze Functional alterations of cardiac proteasomes under physiological and pathological conditions Cardiovasc Res, January 15, 2010; 85(2): 339 - 346. [Abstract] [Full Text] [PDF]
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H. Su and X. Wang The ubiquitin-proteasome system in cardiac proteinopathy: a quality control perspective Cardiovasc Res, January 15, 2010; 85(2): 253 - 262. [Abstract] [Full Text] [PDF]
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S. R. Powell and A. Divald The ubiquitin-proteasome system in myocardial ischaemia and preconditioning Cardiovasc Res, January 15, 2010; 85(2): 303 - 311. [Abstract] [Full Text] [PDF]
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