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Circulation. 2006;113:1787-1798
Published online before print April 3, 2006, doi: 10.1161/CIRCULATIONAHA.105.578278
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(Circulation. 2006;113:1787-1798.)
© 2006 American Heart Association, Inc.

Molecular Cardiology

Role of p90 Ribosomal S6 Kinase–Mediated Prorenin-Converting Enzyme in Ischemic and Diabetic Myocardium

Seigo Itoh, MD, PhD; Bo Ding, MD; Tetsuro Shishido, MD, PhD; Nicole Lerner-Marmarosh, PhD; Nadan Wang, MS; Naoya Maekawa, PhD; Bradford C. Berk, MD, PhD; Yasuchika Takeishi, MD, PhD; Chen Yan, PhD; Burns C. Blaxall, PhD; Jun-ichi Abe, MD, PhD

From the Cardiovascular Research Institute (S.I., B.D., T.S., N.L.-M., N.W., N.M., B.C. Berk, C.Y., B.C. Blaxall, J.-i.A.), University of Rochester, Rochester, NY; and Department of Internal Medicine, Yamagata University (Y.T.), Yamagata, Japan.

Correspondence to Jun-ichi Abe, MD, PhD, Cardiovascular Research Institute, 601 Elmwood Ave, Box 679, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642. E-mail jun-ichi_abe{at}urmc.rochester.edu

Received July 25, 2005; revision received February 7, 2006; accepted February 9, 2006.


*    Abstract
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Background— Epidemiological data strongly indicate that diabetes increases the incidence of heart failure. Although the benefit of angiotensin-converting enzyme inhibitor (ACE-I) treatment during and after myocardial infarction has been found to be greater in diabetics than nondiabetics and activation of the renin-angiotensin system (RAS) has been implicated, the molecular basis of these actions remains unclear.

Methods and Results— We generated transgenic mice with cardiac-specific overexpression of wild-type p90 ribosomal S6 kinase (WT-p90RSK-Tg) and a dominant-negative form of p90RSK (DN-p90RSK-Tg). Recovery of cardiac function after ischemia/reperfusion in WT-p90RSK-Tg isolated mouse hearts was significantly impaired. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry revealed specific induction of prorenin-converting enzyme (PRECE) in WT-p90RSK-Tg mice. mRNA induction of PRECE was confirmed with serial angiotensinogen protein reduction after perfusion in WT-p90RSK-Tg mice, suggesting an increase of angiotensinogen cleavage and subsequent RAS activation in WT-p90RSK-Tg mice. We investigated the role of the RAS in WT-p90RSK-Tg animals after ischemia/reperfusion with the use of an ACE-I (captopril) and an angiotensin II type 1 receptor blocker (olmesartan). We did not observe any effect of these inhibitors in non-Tg littermate controls, thus corroborating other reports in rodents. In contrast, both captopril and olmesartan significantly improved cardiac function and reduced infarct size in WT-p90RSK-Tg mice. At 8 months of age, WT-p90RSK-Tg mice developed cardiac dysfunction. p90RSK activity and PRECE mRNA were both increased by streptozotocin-induced hyperglycemia in non-Tg littermate controls, whereas DN-p90RSK-Tg animals exposed to streptozotocin did not have PRECE induction.

Conclusions— This study demonstrates the critical role of p90RSK in hyperglycemia-mediated myocardial PRECE induction, which may explain the augmentation of the RAS in diabetic hearts and provide an alternative therapeutic approach to treat diabetic cardiomyopathy.


Key Words: angiotensin • diabetes mellitus • ischemia • renin • signal transduction


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowDiscussion
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Diabetes is an independent risk factor for both mortality and morbidity after myocardial infarction (MI).1 A number of clinical studies have shown that post-MI left ventricular function is significantly worse in diabetic compared with nondiabetic patients.2,3 In addition, several clinical studies strongly indicate that activation of the renin-angiotensin system (RAS) in diabetic patients is a critical factor for developing heart failure after MI.2,3 Although these clinical studies indicate that there is a greater benefit with angiotensin-converting enzyme inhibitor (ACE-I) treatment after MI in diabetic than in nondiabetic patients, the molecular basis for this difference is unclear. During the past several decades, investigators in several laboratories have examined the levels and activity of elements of the RAS in plasma and various tissues during diabetes. Measurement of angiotensin (Ang) II and its upstream components of the RAS has been complicated by the rapid degradation of these peptides4,5 and the local regulation of this production within specific vascular tissues and lesions.6 Therefore, reports on the effects of diabetes on plasma and the tissue RAS, including Ang II levels, are controversial,7–9 and interpretation of these changes is limited by the potential downstream modulation of renin and Ang II production and stability.

Clinical Perspective p 1798

The importance of protein kinase C (PKC)-ß activation during diabetes has been demonstrated by studies reporting that the specific PKC-ß inhibitor LY333531 inhibits many abnormalities, such as renal mesangial expansion, cardiomyopathy, and monocyte activation in diabetic rats.10,11 Interestingly, it has been reported that cardiac-specific overexpression of PKC-ßII12 but not of PKC-{epsilon} in transgenic mice decreases cardiac function.13 Previously, we showed that H2O2-mediated p90 ribosomal S6 kinase (p90RSK) activation is partially dependent on PKC activation in Jurkat cells.14 Interestingly, p90RSK activation is specifically upregulated during overexpression of PKC-ßII in transgenic mice, which is thought to be relevant to diabetic cardiomyopathy.15 p90RSK is a serine/threonine kinase that is involved in the activation of nuclear factor-{kappa}B by phosphorylation of the inhibitor I{kappa}-B16 or the phosphorylation of transcription factors, including c-Fos,17 Nur77,18 and cAMP response element–binding protein.19 However, the role of p90RSK and its relation to the RAS in diabetic hearts remain largely unknown.

To determine the role of p90RSK activation in the heart, we generated transgenic (Tg) mice in our laboratory with cardiac-specific overexpression of wild-type p90RSK (WT-p90RSK-Tg) and overexpression of a dominant-negative form of p90RSK (DN-p90RSK-Tg). We found that expression of prorenin-converting enzyme (PRECE) was specifically upregulated in WT-p90RSK-Tg mice compared with non-Tg littermate control (NLC) mice by analyzing the 2-dimensional gel images integrated with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Interestingly, we found that both cardiac p90RSK activation and PRECE expression were significantly increased in mice whose diabetes was induced by streptozotocin (STZ), and this PRECE induction was completely abolished in DN-p90RSK-Tg mice. Furthermore, after reaching 8 months of age, WT-p90RSK-Tg mice developed cardiac dysfunction with increased interstitial fibrosis and hypertrophied cardiomyocytes, which resemble features of diabetic cardiomyopathy.20 p90RSK-induced PRECE and the subsequent RAS activation in the heart may present a new mechanism to regulate cardiac function, especially in the diabetic heart.


*    Methods
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An expanded Methods section is available in the online-only Data Supplement. Details on protein extracts from heart tissue, p90RSK in vitro kinase assays, measurement of cardiac damage, Western blot analysis, animals, 2-dimensional gel electrophoresis, MALDI-TOF MS analysis, measurement of left ventricular function in Langendorff preparations, echocardiographic analysis, hemodynamic analysis, relative quantitative reverse transcription–polymerase chain reaction (RT-PCR), and analysis of apoptosis are also provided in the online Data Supplement.

Statistical Analysis
Values are presented as mean±SD, unless indicated otherwise. Statistical analysis was performed with the StatView 4.0 package (Abacus Concepts, Berkeley, Calif). We performed a repeated-measures nonparametric 1-way ANOVA, a non–repeated-measures nonparametric 1-way ANOVA, and a non–repeated-measures nonparametric 2-way ANOVA, as indicated in the online Data Supplement. These analyses were followed by Scheffé correction. A value of P<0.05 was considered significant.

The authors had full access to the data and take full responsibility for their integrity. All authors have read and agree to the manuscript as written.


*    Results
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 *Results
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Functional Role for p90RSK in Ischemia/Reperfusion Injury in Cardiac-Specific WT-p90RSK-Tg Mice at 4 Months of Age
To examine the effect of p90RSK activation at the whole-organ level, we created Tg mice with cardiac-specific expression of WT-p90RSK. The level of Tg protein expression in 3 different lines of Tg mice was determined by Western blotting with an anti-p90RSK antibody. Because all 3 lines showed similar p90RSK expression levels and phenotypes, including the response to ischemia/reperfusion (I/R) in Langendorff preparations, we describe the data from line Tg-03 only as a representative result for all WT-p90RSK-Tg mouse lines. We found a 5- to 8-fold increase in total p90RSK expression relative to NLC mice (Figure 1A). The WT-p90RSK-Tg lines exhibited normal feeding, activity, and weight gain up to 4 months of age compared with the NLC group.


Figure 1
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  		Figure 1. Cardiac-selective expression of WT-p90RSK accelerates I/R-induced contractile dysfunction and postischemic cardiac injury. A, Lysates were prepared from 10-week-old NLC and WT-p90RSK-Tg mouse hearts and immunoblotted (IB) with a p90RSK (upper) and an actin (lower) antibody. B, Measurements of left ventricular developed pressure before, during, and after global (no-flow) ischemia, followed by reperfusion. C, Measurement of left ventricular dP/dtmax performed at the times indicated. All experimental values calculated for NLC (n=5) and WT-p90RSK-Tg mouse hearts (n=5) are represented as mean±SD. D, CK and LDH cardiac enzymes measured in the superfusate from mouse hearts after ischemia (n=4), reported as mean±SD.

We next examined the basal phenotype and cardiac function of NLC and WT-p90RSK-Tg hearts. Cardiac structure and function in 10-week-old mice were normal, as assessed by gross morphometric, histological, and noninvasive echocardiographic measurements. A cross section from both NLC and p90RSK-Tg hearts showed no change in ventricular wall thickness suggestive of cardiomyopathy (data not shown), and M-mode echocardiographic images (Table 1) confirmed normal basal ventricular dimensions and function in live hearts until 4 months of age.


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  		TABLE 1. Cardiac Parameters in NLC and WT-p90RSK-Tg Mice

We investigated the potential functional consequence of overexpression of WT-p90RSK in Langendorff preparations. Because we wished to exclude the heart from circulatory effects, we used an isolated-heart preparation to determine the "local" effect of p90RSK on cardiac function and on creatine kinase (CK) and lactate dehydrogenase (LDH) release, especially after I/R. No difference in basal contractile function was noted between NLC and WT-p90RSK-Tg hearts. Recovery of left ventricular developed pressure after 20 minutes of I/R was >90% of baseline for the NLC hearts. In contrast, developed pressure recovered to only <30% of baseline in the WT-p90RSK-Tg hearts at all time points after ischemia and during reperfusion (Figure 1B). A similar trend was seen in dP/dtmax values, with a significantly lower recovery of this parameter observed in WT-p90RSK-Tg hearts on reperfusion (Figure 1C). In addition, the end-diastolic pressures of NLC and WT-p90RSK-Tg hearts after 20 minutes of ischemia were 7.5±1.3 and 17.7±1.8 mm Hg, respectively (n=5, P<0.01). The results strongly suggest that although WT-p90RSK-Tg hearts are functionally normal, they display significantly weaker contractile recovery compared with NLC hearts after 20 minutes of ischemia.

To assess total cardiac damage incurred in the post-I/R heart, we measured the levels of CK and LDH released from the heart, which were collected at 5, 15, and 25 minutes after reperfusion, and estimated the total release of CK and LDH. Perfusates collected from NLC hearts documented no CK and modest LDH release (Figure 1D). WT-p90RSK-Tg mouse hearts subjected to the same insult demonstrated greater CK and LDH elevations, suggesting that p90RSK activation induced more severe I/R damage.

PRECE Is Upregulated in WT-p90RSK-Tg Hearts
To characterize proteins that are specifically regulated by p90RSK activation, homogenates were prepared from NLC and WT-p90RSK-Tg hearts and then analyzed by 2-dimensional electrophoresis and subsequent MALDI-TOF MS, as described previously.21 As shown in Figure 2A, increased expression of a specific protein in WT-p90RSK-Tg hearts was detected by silver staining on 2-dimensional gels. Among the spots on the 2-dimensional gel, one of the most highly regulated spots was at 28 kDa, with an isoelectric point of 6.4. This spot was identified as PRECE by MALDI-TOF MS analysis, with 100% fragment matching covering 40% of the total amino acid sequences of mouse PRECE (Figure 2B). To confirm the enhanced PRECE expression in WT-p90RSK-Tg hearts, RT-PCR was performed. As shown in Figure 2C, we found that the mRNA expression of PRECE was significantly increased in WT-p90RSK-Tg hearts compared with that in NLC hearts. Because kallikrein-like PRECE can cleave not only prorenin to renin but also angiotensinogen to generate Ang II directly,22 we examined the angiotensinogen protein level in NLC and WT-p90RSK-Tg mice. As shown in Figure 2D, in NLC mice, angiotensinogen levels declined slowly after Krebs-Henseleit buffer perfusion in the Langendorff model. In contrast, a significant rapid reduction of angiotensinogen content after perfusion was observed in WT-p90RSK-Tg mice. Taken together with the increase of PRECE expression in WT-p90RSK-Tg mice, these data suggest increased angiotensinogen cleavage in WT-p90RSK-Tg mice, which is associated with increased I/R damage.


Figure 2
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  		Figure 2. Protein expression profiles of NLC and WT-p90RSK-Tg mouse hearts. A, 2-dimensional gels of NLC (upper) and WT-p90RSK-Tg (lower) cardiac proteins, stained with silver; IPG NL 4 to 7; 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. After being stained with silver, the gel images were compared. We selected spots that were significantly increased in WT-p90RSK-Tg samples, digested them with trypsin, and then analyzed them with MALDI-TOF MS. B, Analysis of MALDI-TOF MS data, demonstrating the 40% match with the PRECE-2 (mKLK26) amino acid sequence. Boldface characters in the mouse PRECE-2 amino acid sequence indicate matched amino acids. C, PRECE mRNA expression was increased in WT-p90RSK-Tg mouse hearts. PRECE mRNA levels were determined by relative quantitative RT-PCR. 18S rRNA was used as an internal control (left). Densitometric analysis (right) of PRECE mRNA expression in NLC and WT-p90RSK-Tg mouse hearts. Results were normalized for all experiments by arbitrarily setting the mean densitometry of NLC heart samples to 1.0 (shown as mean±SD, n=6, **P<0.01). D, Serial Ang levels in NLC and WT-p90RSK-Tg mice after perfusion. Lysates were prepared from 10-week-old NLC and WT-p90RSK-Tg mouse hearts and immunoblotted (IB) with an angiotensinogen (upper) and a tubulin (middle) antibody. Densitometric analysis (bottom) of serial angiotensinogen protein levels in NLC and WT-p90RSK-Tg mouse hearts after perfusion. Results were normalized for all experiments by arbitrarily setting the mean densitometry of NLC heart samples to 1.0 at 3 minutes after Krebs-Henseleit buffer perfusion (shown as mean±SD, n=4, *P<0.01). M.W. indicates molecular weight.

Role of the RAS in p90RSK-Mediated Enhancement of Cardiac Injury by I/R
Because we found that PRECE protein and mRNA expression were significantly increased in WT-p90RSK-Tg hearts, we sought to determine whether upregulation of the RAS by p90RSK-mediated PRECE could significantly enhance cardiac injury after I/R in WT-p90RSK-Tg mice. However, owing to the rapid degradation of cardiac renin and Ang II, along with residual contamination from serum, it is well recognized that accurate measurement of these proteins is very difficult.5 Therefore, we investigated the contribution of the RAS to p90RSK-mediated cardiac dysfunction by evaluating the effect of an ACE-I and an Ang II type 1 (AT1) receptor blocker on the recovery of cardiac function after I/R. Under condition of 20 minutes of ischemia, developed pressures in NLC hearts could almost completely recover (Figure 3A), but the recovery of developed-pressure after reperfusion in WT-p90RSK-Tg hearts was {approx}30% of the basal level, as previously shown in Figure 2 (Figure 3C). As shown in Figure 3A and 3B, pretreatment with an ACE-I (captopril, 50 µmol/L) had no effect on recovery after I/R in NLC mice. Of note, because in NLC mice we observed almost full recovery of cardiac function after 20 minutes of ischemia, we also performed a prolonged 40 minutes of ischemia in NLC hearts (Figure 3B). Forty minutes of ischemia in NLC hearts reduced cardiac function to {approx}30% of basal levels and resulted in recovery similar to that of WT-p90RSK-Tg hearts subjected to a shorter, 20-minute ischemic episode. However, we could not detect any beneficial effect of the ACE-I even after 40 minutes of ischemia in NLC hearts (Figure 3B and 3D), which is consistent with previous reports in rodents from several different laboratories.23–25 In contrast, pretreatment with the ACE-I in WT-p90RSK-Tg mice resulted in a significant improvement in recovery of cardiac function after 20 minutes of ischemia (Figure 3C and 3D). We also found similar protective effects with an AT1 receptor blocker (olmesartan, 10 µmol/L) in WT-p90RSK-Tg mice (Figure I in the online Data Supplement). We also measured levels of the cardiac enzymes CK and LDH released from the ischemic heart (Figure 4). Perfusates collected from NLC mouse hearts after 40 minutes of global ischemia had elevated CK and LDH levels, but pretreatment with captopril showed no beneficial effect on the release of CK and LDH in NLC hearts. Because after 40 minutes of global ischemia WT-p90RSK-Tg mouse hearts could not regain any contractile function, we selected a 20-minute ischemic period in WT-p90RSK-Tg animals. In contrast to the NLC group, captopril significantly reduced release of these cardiac enzymes after 20 minutes of ischemia in WT-p90RSK-Tg hearts, consistent with the cardiac function data shown in Figure 3. Because {alpha}-myosin heavy-chain promoter–derived p90RSK expression is selectively induced in cardiomyocytes and our data are demonstrated in isolated-heart preparations, these data suggest enhancement of the local cardiac RAS in WT-p90RSK-Tg mice. Activation of the local cardiac RAS is consistent with the increase of PRECE expression in WT-p90RSK-Tg hearts.


Figure 3
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  		Figure 3. The ACE-I captopril (50 µmol/L) protected WT-p90RSK-Tg but not NLC hearts from I/R-induced contractile dysfunction. A and B, Measurements of left ventricular developed pressure (left) and dP/dtmax (right) before, during, and after global (no-flow) ischemia (Isc) followed by reperfusion with vehicle or captopril pretreatment in NLC hearts. Short 20-minute (A) or prolonged 40-minute (B) ischemia was induced. C, Measurements of left ventricular developed pressure (left) and dP/dtmax (right) before, during, and after global (no-flow) ischemia followed by reperfusion with vehicle or captopril pretreatment in WT-p90RSK-Tg mouse hearts after 20-minute ischemia. D, Measurements of left ventricular developed pressure (left) and dP/dtmax (right) after prolonged 40-minute ischemia in NLC hearts and short 20-minute ischemia in WT-p90RSK-Tg hearts, followed by 25-minute reperfusion with vehicle or captopril (50 µmol/L) pretreatment (shown as mean±SD, n=5, **P<0.01).


Figure 4
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  		Figure 4. The ACE-I captopril (50 µmol/L) protected WT-p90RSK-Tg but not NLC hearts from I/R-induced cardiac injury. CK and LDH cardiac enzymes measured in the superfusate from NLC hearts after prolonged 40-minute ischemia (Isc) (n=4) and from WT-p90RSK-Tg mouse hearts after short 20-minute ischemia (n=4) are reported as mean±SD (*P<0.05, **P<0.01). N.S. indicates not significant; Rep, reperfusion.

WT-p90RSK-Tg Mice Show Cardiac Dysfunction at 8 Months of Age, With Increasing Apoptosis and Interstitial Fibrosis
Although no significant pathological phenotype was observed in WT-p90RSK-Tg mice up to 4 months old, we found that WT-p90RSK-Tg mice displayed significant impairment in cardiac contractility, as assessed by decreased dP/dt and developed pressure values, at 10 months of age (Figure 5A). In addition, we determined that –dP/dt and {tau} (the time constant of exponential decay)26 were increased by 10 months in WT-p90RSK-Tg mice, which might suggest diastolic dysfunction (Table 2). Because we did not find a significant difference in heart rate (NLC, 480±23 bpm; WT-p90RSK-Tg, 455±21 bpm; mean±SD, P=NS), these differences are most likely not due to the depth of anesthesia. To confirm the functional invasive hemodynamic alterations, echocardiographic measurements were performed, and these showed that both fractional shortening and the velocity of circumferential fiber shortening were reduced in WT-p90RSK-Tg mice at 8 to 10 months of age (Table 1 and Figure 5B and 5C), again indicating impairment of contractile function. Because we observed impairment of contractile function in WT-p90RSK-Tg mice, we examined whether apoptosis was increased in them. As shown in Figure 5D, there was a significant increase in apoptotic cells in WT-p90RSK-Tg compared with NLC by mice, as assessed by terminal deoxynucleotidyl transferase nick end-labeling (TUNEL) assay. Bcl-2 is a well-known antiapoptotic molecule, and its expression can be repressed by Ang II.27 We observed decreased Bcl-2 expression levels in WT-p90RSK-Tg mice. These data also support the concept that p90RSK activation promotes apoptosis, probably via repression of Bcl-2 expression (Figure 5E).


Figure 5
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  		Figure 5. Cardiac dysfunction in WT-p90RSK-Tg mice at age 10 months. A, Hemodynamic measurements in NLC (n=6) and WT-p90RSK-Tg (n=6) mice. All data are expressed as mean±SD (**P<0.01, *P<0.05). DP indicates developed pressure; LV, left ventricle. B, Representative M-mode echocardiographic images of contracting hearts in 10-month-old NLC and WT-p90RSK-Tg mice. C, Percent fractional shorting (%FS) and velocity of circumferential fiber shortening (Vcfs) in 3- and 10-month-old NLC (n=6) and WT-p90RSK-Tg (n=5) mice. Values (mean±SEM) were determined by echocardiography. **P<0.01 between groups. D, Detection of apoptosis by TUNEL. Green fluorescence shows apoptotic cardiomyocytes stained by the TUNEL method; nuclei were counterstained with Hoechst3 3342 (blue), and cardiomyocytes were stained with anti–{alpha}-actinin (sarcomeric) (clone EA-53, red). Overlay images are shown. Right panel shows quantitative analysis of apoptotic cells. Units along the vertical axis indicate the ratio (in %) of TUNEL-positive cells to Hoechst 33342–positive nuclei, which are clearly overlaid by EA-53 stain (indicated by arrows). We did not count cells that were not clearly counterstained with EA-53 (indicated by asterisk). More than 1000 cells were screened per section. E, Bcl-2 expression in NLC and WT-p90RSK-Tg mice. Lysates were prepared from 10-month-old NLC and WT-p90RSK-Tg mouse hearts and immunoblotted (IB) with a Bcl-2 (upper) and an actin (lower) antibody.


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  		TABLE 2. Hemodynamic Parameters in NLC and WT-p90RSK-Tg Mice

Normalized cardiac mass (heart weight–body weight ratio) was slightly increased in WT-p90RSK-Tg mice at 8 to 10 months of age but not at 3 months of age (Figure 6A). Expression of molecular markers of cardiac hypertrophy, such as atrial natriuretic factor (ANF) and brain natriuretic protein (BNP), were also increased in WT-p90RSK-Tg compared with NLC mice at 8 to 10 months. We observed increased heart size in WT-p90RSK-Tg animals at 10 months of age (Figure 6A and 6C). Histologically, an increase in overall heart size was observed (data not shown), characterized by interstitial fibrosis and hypertrophied cardiomyocytes in WT-p90RSK-Tg compared with NLC mice (Figure 6D). These data demonstrate an increase in interstitial fibrosis with apoptosis in WT-p90RSK-Tg mice at 8 months, which resembles diabetic cardiomyopathy as previously described.20


Figure 6
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  		Figure 6. Cardiac hypertrophy, interstitial fibrosis, and apoptosis in WT-p90RSK-Tg mice at 10 months of age. A, Ratios of heart weight (HW) to body weight (BW) in 3- and 10-month-old NLC and WT-p90RSK-Tg mice. B, ANF and BNP mRNA expression in 10-month-old NLC and WT-p90RSK-Tg mice. ANF and BNP mRNA levels were determined by relative quantitative RT-PCR. 18S rRNA was used as an internal control. Bottom panel shows densitometric analysis of ANF and BNP mRNA expression. Results were normalized for all experiments by arbitrarily setting the densitometry of NLC 10-month-old heart samples to 1.0 (shown as mean±SD, n=4, **P<0.01). C, Top, Representative photographs of NLC and WT-p90RSK-Tg hearts at 10 months of age. Histological images (x200, Masson’s trichrome stain) of hearts from NLC and WT-p90RSK-Tg mice at 10 months of age.

p90RSK Activation in STZ-Induced Diabetic Mice
Previously, we reported that PKC-ß activation is critical in H2O2-mediated p90RSK activation. In addition, we found that p90RSK activity was significantly increased in cardiac-specific PKC-ß–overexpressing mice.15 Because a critical role for PKC-ß activation in diabetes has been extensively studied, we investigated whether p90RSK is activated in STZ-induced hyperglycemic mice. In the present study, we used STZ-induced diabetic mice, a known useful model for the study of diabetes.28 STZ treatment significantly increased fasting blood glucose levels after 2 weeks of injection (vehicle, 107±8 mg/dL versus STZ, 224±5 mg/dL; P<0.01). As shown in Figure 7A, we found that PKC-{alpha}/-ßII, but not extracellular signal–regulated kinase (ERK) 1/2, phosphorylation was significantly increased in STZ-induced hyperglycemic mice. p90RSK activation was also increased in hyperglycemic mice (Figure 7B), supporting the possible contribution of p90RSK in diabetic cardiomyopathy.


Figure 7
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  		Figure 7. PKC-{alpha}/-ßII and p90RSK but not ERK1/2 activations were increased in STZ-mediated hyperglycemic mice. A, ERK1/2 and PKC-{alpha}/-ßII activities were measured by Western blot analysis (IB) with a phosphospecific (P-) ERK1/2 antibody (second from bottom) and PKC-{alpha}/-ßII (top). No difference in the amount of ERK1/2 and PKC-ß was observed in lysates from any heart sample by Western blot analysis with anti-ERK1/2 (bottom) and PKC-ßII (second from top). B, p90RSK activation in STZ-mediated hyperglycemic mice. p90RSK activity was detected by an in vitro kinase assay with the S6 kinase substrate peptide, as described in Methods. Data (n=3) are expressed as mean±SD, **P<0.01.

p90RSK Activation and PRECE Expression in the Diabetic Heart and Involvement of p90RSK Activation in Hyperglycemia-Mediated PRECE Expression
Because we found that p90RSK activation was increased in STZ-induced hyperglycemic mice (Figure 7), we determined whether PRECE expression was also increased in this diabetic model. PRECE mRNA expression was significantly increased in STZ-induced diabetic mice (Figure 8). To determine the role of p90RSK activation in diabetes-mediated PRECE expression in the heart, we utilized cardiac-specific DN-p90RSK-Tg mice, which showed no change in basal cardiac phenotype but demonstrated a cardioprotective effect against I/R injury, as previously described.29 p90RSK activation was increased by STZ injection in NLC mice, but it was significantly inhibited in DN-p90RSK-Tg mice (Figure 7B) and NLC+STZ mice: 12991±1810 cpm; DN-p90RSK-Tg+STZ, 8009±797 cpm; mean±SD, P<0.05. As shown in Figure 8, PRECE mRNA expression was increased by STZ injection in NLC but not in DN-p90RSK-Tg mice, suggesting a critical role for p90RSK activation in STZ-induced PRECE expression in the heart.


Figure 8
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  		Figure 8. Diabetes-mediated PRECE mRNA expression is inhibited in DN-p90RSK-Tg mouse hearts. Upper shows that STZ injection–mediated diabetes increased PRECE mRNA expression after 2 weeks of STZ injection, which was inhibited in DN-p90RSK-Tg mouse hearts. 18S rRNA was used as an internal control. Bottom panel shows densitometric analysis of PRECE mRNA expression in STZ-injected diabetic NLC and DN-p90RSK-Tg mice. Results were normalized for all experiments by arbitrarily setting the densitometry of control heart samples to 1.0 (shown as mean±SD, n=4, *P<0.05).


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
Meta-analyses of ACE-I trials provide compelling evidence that these drugs attenuate the detrimental effects of Ang II, improve survival, and reduce morbidity in patients with acute MI and heart failure. However, the mechanism for the larger effects of ACE-Is in diabetic patients remains unclear. In the present study, we found that p90RSK activation was increased in diabetic hearts and that PRECE protein and mRNA levels were specifically upregulated in WT-p90RSK-Tg hearts. We detected increased levels of PRECE mRNA expression in the hearts of mice with STZ-induced diabetes. To our knowledge, this is the first report to document the possible role and expression of PRECE in the heart. Interestingly, we found that although an ACE-I did not improve recovery of cardiac function after I/R in NLC hearts, in contrast there was significant improvement in the recovery of cardiac function and alleviation of damage by both an ACE-I and an AT1 receptor blocker in WT-p90RSK-Tg hearts. These data provide a novel mechanism of the RAS in ischemic myocardium and a new paradigm for the treatment of ischemic myocardium in diabetic patients. Previous data have shown controversial results about the effect of AT1 blockers on cardiac damage after I/R among different species. In the mouse, rat, and rabbit, no significant protective effect was shown by an AT1 blocker and in AT1-knockout mice, especially within 1 week of I/R.23–25 In contrast, in the dog and swine model, an AT1 receptor blocker inhibited 40% to 50% of infarct size.30,31 Therefore, it is intriguing to suspect that the previous controversial results with regard to the effect of RAS inhibitors after I/R might have been due to differences in expression of PRECE among different strains and species.

The existence of a local RAS in the heart is still a controversial issue. Supporting evidence for a local RAS comes from the beneficial effect of ACE-Is in heart failure, which are independent, at least partially, of their effect on blood pressure.32,33 Based on previous data, although all RAS components are present in cardiac tissue and both Ang I and Ang II are generated in the heart, the majority of Ang I and Ang II present in cardiac tissue sites originates from the circulation and is therefore, kidney-derived prorenin and renin.32,33 One of the mechanisms by which the heart may regulate its Ang I and Ang II concentrations independent of the circulating levels of these RAS components is the rate of conversion of prorenin to active renin by proteolytic cleavage of 43 amino acids from the prosegment of prorenin. Many enzymes have been proposed to be capable of activating prorenin. These include cathepsin B,34 cathepsin D,35 cathepsin G,36 tissue kallikrein,37 and kallikrein-like PRECE. In the present study, we found that kallikrein-like PRECE expression was increased in hearts of WT-p90RSK-Tg and STZ-injected diabetic mice. The kallikrein-like PRECEs (mouse kallikrein 9 [mKLK9], mKLK13, mKLK22, and mKLK26) cleave prorenin on the COOH side of the Arg residue at the Lys-Arg pair of prorenin.38,39

There are 12 mKLK genes that represent the orthologs of the newly identified human kallikrein genes (KLK4 through KLK15). PRECE-1 (mKLK13) and PRECE-2 (mKLK26) have shown 99% sequence similarity, and it has been suggested that PRECE-1 and PRECE-2 represent allelic variants of the same gene.40,41 Evaluation of the genetic loci in humans and mice has shown that the location of PRECE (KLK13) is conserved between the 2 species, suggesting that human KLK13 is orthologous to the mouse PRECE (mKLK13) gene.40 We also determined the conserved region of mKLK13 in humans from a cross-species comparison by VISTA plot (www-gsd.lbl.gov/vista/index.shtml). We found that exons 2 to 5 in human KLK2 and KLK3 are highly conserved in the mKLK13 and mKLK26 (PRECE) gene (Figure II in online Data Supplement). In addition, the highly conserved regions between humans and mice were localized in the proximate 0.2- to 0.3-kb 5' upstream flanking region of both human KLK2 and KLK3 genes (Figure II in online Data Supplement). Interestingly, Clark et al42 have reported that human KLK3 (prostate-specific antigen) expression is regulated by p90RSK activation. These results suggest that human KLK2 and KLK3 and mKLK13 and mKLK26 (PRECE) may share a similar regulatory mechanism, including p90RSK. Furthermore, it has been reported that plasma prorenin levels are elevated in human subjects with Fletcher trait (prekallikrein deficiency), also suggesting an important role for kallikreins in regulating prorenin levels not only in mice but also in humans.37,43 The biological roles of human KLK2 and KLK3 have been studied only recently,44 and further investigation is required to determine their physiological relevance in regulating RAS activity.

Increasing evidence suggests the importance of circulating prorenin levels and subsequent internalization of prorenin into cardiac cells, which may play a key role in the process of cardiac damage by the RAS.32,45 Therefore, induction of PRECE in WT-p90RSK-Tg and diabetic mice may enhance this process and decrease cardiac function after I/R. To support this notion, Vidotti et al46 have reported that high glucose levels increase intracellular renin activity by increasing the rate of conversion of prorenin to active renin. The strong predictive power of the plasma prorenin level but not of renin for detecting risk of diabetic complications has been reported.47 Because we found p90RSK-dependent PRECE induction in diabetic hearts as well as rapid reduction of Ang levels in WT-p90RSK-Tg mouse hearts after Krebs-Henseleit buffer reperfusion, this increase in cardiac PRECE may explain this phenomenon. In addition, the potential benefits of renin inhibitors for managing diabetic complications has been proposed.48 Our finding of PRECE induction in the diabetic heart may add a novel rationale and therapeutic opportunities for the use of renin inhibitors in preventing cardiac complications in diabetes.


*    Acknowledgments
 
This work was supported by grants from the American Heart Association to Dr Itoh (postdoctoral fellowship 0325769T), Dr Yan (grant in aid 0455847T), and Dr Blaxall (scientist development grant 0435437T) and from the National Institutes of Health to Dr Berk (HL44721) and Dr Abe (HL-66919 and GM-071485-01A1). We thank Sankyo Co, Ltd, for providing olmesartan and Dr Thunder Jalili for critical reading of this manuscript. We also thank Doug Weston, Sarah Mack, and Haodong Xu for technical and constant support.

Disclosures

None.


*    References
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CLINICAL PERSPECTIVE

Diabetes is an independent risk factor for both mortality and morbidity after myocardial infarction (MI). A number of clinical studies have shown that post-MI left ventricular function is significantly worse in diabetic compared with that in nondiabetic patients. In addition, several clinical studies strongly indicate that activation of the renin-angiotensin system (RAS) in diabetic patients is a critical factor for developing heart failure after MI. Although these clinical studies indicate a greater benefit with angiotensin-converting enzyme inhibitor (ACE-I) treatment after MI in diabetic than in nondiabetic patients, the molecular basis for this difference is unclear. In the present study, we found that p90 ribosomal S6 kinase (p90RSK) activation was increased in diabetic hearts and that prorenin-converting enzyme (PRECE) protein and mRNA levels were specifically upregulated in wild-type (WT)-p90RSK-transgenic (Tg)–overexpressing mouse hearts. We detected increased PRECE mRNA expression levels in hearts of mice with streptozotocin (STZ)-induced diabetes. To our knowledge, this is the first report to document the possible role and expression of PRECE in the heart. Interestingly, we found that although an ACE-I did not improve recovery of cardiac function after ischemia/reperfusion in non-Tg littermate control mouse hearts, there was significant improvement in recovery of cardiac function and alleviation of damage by both an ACE-I and an angiotensin II type 1 receptor blocker in WT-p90RSK-Tg hearts. These data provide a novel mechanism of the RAS in ischemic myocardium and a new paradigm for the treatment of ischemic myocardium in diabetic patients.


*    Footnotes
 
The online-only Data Supplement can be found at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.105.578278/DC1.




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