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• W2109585407 abstract "Our previous study has shown that human tissue kallikrein protected against ischemia/reperfusion-induced myocardial injury. In the present study, we investigated the protective role of local kallikrein gene delivery in ischemia/reperfusion-induced cardiomyocyte apoptosis and its signaling mechanisms in promoting cardiomyocyte survival. Adenovirus carrying the human tissue kallikrein gene was delivered locally into the heart using a catheter-based technique. Expression and localization of recombinant human kallikrein in rat myocardium after gene transfer were determined immunohistochemically. Kallikrein gene delivery markedly reduced reperfusion-induced cardiomyocyte apoptosis identified by both in situ nick end-labeling and DNA fragmentation. Delivery of the kallikrein gene increased phosphorylation of Src, Akt, glycogen synthase kinase (GSK)-3β, and Bad(Ser-136) but reduced caspase-3 activation in rat myocardium after reperfusion. The protective effect of kallikrein on apoptosis and its signaling mediators was blocked by icatibant and dominant-negative Akt, indicating a kinin B2 receptor-Akt-mediated event. Similarly, kinin or transduction of kallikrein in cultured cardiomyocytes promoted cell viability and attenuated apoptosis induced by hypoxia/reoxygenation. The effect of kallikrein on cardiomyocyte survival was blocked by dominant-negative Akt and a constitutively active mutant of GSK-3β, but it was facilitated by constitutively active Akt, catalytically inactive GSK-3β, lithium, and caspase-3 inhibitor. Moreover, kallikrein promoted Bad·14-3-3θ complex formation and inhibited Akt-GSK-3β-dependent activation of caspase-3, whereas caspase-3 administration caused reduction of the Bad·14-3-3θ complex, indicating an interaction between Akt-GSK-caspase-3 and Akt-Bad·14-3-3 signaling pathways. In conclusion, kallikrein/kinin protects against cardiomyocyte apoptosis in vivo and in vitro via Akt-Bad·14-3-3 and Akt-GSK-3β-caspase-3 signaling pathways. Our previous study has shown that human tissue kallikrein protected against ischemia/reperfusion-induced myocardial injury. In the present study, we investigated the protective role of local kallikrein gene delivery in ischemia/reperfusion-induced cardiomyocyte apoptosis and its signaling mechanisms in promoting cardiomyocyte survival. Adenovirus carrying the human tissue kallikrein gene was delivered locally into the heart using a catheter-based technique. Expression and localization of recombinant human kallikrein in rat myocardium after gene transfer were determined immunohistochemically. Kallikrein gene delivery markedly reduced reperfusion-induced cardiomyocyte apoptosis identified by both in situ nick end-labeling and DNA fragmentation. Delivery of the kallikrein gene increased phosphorylation of Src, Akt, glycogen synthase kinase (GSK)-3β, and Bad(Ser-136) but reduced caspase-3 activation in rat myocardium after reperfusion. The protective effect of kallikrein on apoptosis and its signaling mediators was blocked by icatibant and dominant-negative Akt, indicating a kinin B2 receptor-Akt-mediated event. Similarly, kinin or transduction of kallikrein in cultured cardiomyocytes promoted cell viability and attenuated apoptosis induced by hypoxia/reoxygenation. The effect of kallikrein on cardiomyocyte survival was blocked by dominant-negative Akt and a constitutively active mutant of GSK-3β, but it was facilitated by constitutively active Akt, catalytically inactive GSK-3β, lithium, and caspase-3 inhibitor. Moreover, kallikrein promoted Bad·14-3-3θ complex formation and inhibited Akt-GSK-3β-dependent activation of caspase-3, whereas caspase-3 administration caused reduction of the Bad·14-3-3θ complex, indicating an interaction between Akt-GSK-caspase-3 and Akt-Bad·14-3-3 signaling pathways. In conclusion, kallikrein/kinin protects against cardiomyocyte apoptosis in vivo and in vitro via Akt-Bad·14-3-3 and Akt-GSK-3β-caspase-3 signaling pathways. It has been widely accepted that apoptosis and necrosis are the major contributors of cardiomyocyte dysfunction associated with acute ischemia and reperfusion (I/R). 1The abbreviations used are: I/R, ischemia/reperfusion; Ad, adenoviral; CMV, cytomegalovirus; DN, dominant-negative; GSK, glycogen synthase kinase; GSK-KM, adenoviral vector expressing kinase mutant GSK; GSK-S9A, adenoviral vector expressing constitutively active GSK; H/R, hypoxia/reoxygenation; KM, catalytically inactive; m.o.i., multiplicity of infection; MOPS, 4-morpholinepropanesulfonic acid; Myr, constitutively inactive; PBS, phosphate-buffered saline; PI 3-kinase, phosphatidylinositol 3-kinase; TK, tissue kallikrein; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling.1The abbreviations used are: I/R, ischemia/reperfusion; Ad, adenoviral; CMV, cytomegalovirus; DN, dominant-negative; GSK, glycogen synthase kinase; GSK-KM, adenoviral vector expressing kinase mutant GSK; GSK-S9A, adenoviral vector expressing constitutively active GSK; H/R, hypoxia/reoxygenation; KM, catalytically inactive; m.o.i., multiplicity of infection; MOPS, 4-morpholinepropanesulfonic acid; Myr, constitutively inactive; PBS, phosphate-buffered saline; PI 3-kinase, phosphatidylinositol 3-kinase; TK, tissue kallikrein; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. Because the capacity of cell proliferation in cardiomyocytes is limited, even a small loss of cardiomyocytes resulting from apoptosis is likely to cause reduced cardiac function and the development of heart failure. Significant increases in myocardial apoptosis have been documented in patients with ischemic cardiomyopathy and terminal stage heart failure (1Di Napoli P. Taccardi A.A. Grilli A. Felaco M. Balbone A. Angelucci D. Gallina S. Calafiore A.M. De Caterina R. Barsotti A. Am. Heart. J. 2003; 146: 1105-1111Crossref PubMed Scopus (46) Google Scholar). Growing evidence from in vitro and in vivo studies indicate that inhibition of cardiomyocyte apoptosis would minimize cardiac injury induced by myocardial I/R (2Suzuki K. Murtuza B. Smolenski R.T. Sammut I.A. Suzuki N. Kaneda Y. Yacoub M.H. Circulation. 2001; 104: I308-I313Crossref PubMed Scopus (585) Google Scholar, 3Condorelli G. Roncarati R. Ross Jr., J. Pisani A. Stassi G. Todaro M. Trocha S. Drusco A. Gu Y. Russo M.A. Frati G. Jones S.P. Lefer D.J. Napoli C. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9977-9982Crossref PubMed Scopus (138) Google Scholar).Akt signaling is an important mediator of survival in response to growth factors. Akt promotes the survival of cardiomyocytes in vitro in addition to protection against acute I/R-induced injury in the mouse heart (4Zou Y. Zhu W. Sakamoto M. Qin Y. Akazawa H. Toko H. Mizukami M. Takeda N. Minamino T. Takano H. Nagai T. Nakai A. Komuro I. Circulation. 2003; 108: 3024-3030Crossref PubMed Scopus (67) Google Scholar, 5Bell R.M. Yellon D.M. J. Mol. Cell. Cardiol. 2003; 35: 185-193Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Similarly, our recent study showed that kallikrein gene delivery protects against cerebral ischemia injury and apoptosis along with activation of Akt signaling (6Xia C.F. Yin H. Borlongan C.V. Chao L. Chao J. Hypertension. 2004; 43: 452-459Crossref PubMed Scopus (115) Google Scholar). Activation of Akt triggers downstream signaling pathways such as GSK-3 phosphorylation in a variety of cell lines (7Roberts M.S. Woods A.J. Dale T.C. Van Der Sluijs P. Norman J.C. Mol. Cell. Biol. 2004; 24: 1505-1515Crossref PubMed Scopus (124) Google Scholar). GSK activity is inactivated by Akt-induced phosphorylation at serine 21 (α isoform) and serine 9 (β isoform), leading to inhibition of apoptosis in insulin-mediated signal transduction (8Stoica B.A. Movsesyan V.A. Lea IV, P.M. Faden A.I. Mol. Cell. Neurosci. 2003; 22: 365-382Crossref PubMed Scopus (138) Google Scholar, 9Loberg R.D. Vesely E. Brosius III, F.C. J. Biol. Chem. 2002; 277: 41667-41673Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). GSK-mediated signaling events in cardiomyocyte apoptosis are not clear. Apoptosis can be induced by overexpression of catalytically active GSK-3 and prevented by dominant-negative GSK-3 (10Kim H.S. Skurk C. Thomas S.R. Bialik A. Suhara T. Kureishi Y. Birnbaum M. Keaney Jr., J.F. Walsh K. J. Biol. Chem. 2002; 277: 41888-41896Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Under ischemic preconditioning, GSK-3β phosphorylation increases through a PI 3-kinase-Akt-dependent pathway, suggesting that inhibition of GSK-3β is protective in myocardial ischemia (11Tong H. Imahashi K. Steenbergen C. Murphy E. Circ. Res. 2002; 90: 377-379Crossref PubMed Scopus (315) Google Scholar). Moreover, several studies reported that GSK-3β exerted its effect via activation of caspase-3 (8Stoica B.A. Movsesyan V.A. Lea IV, P.M. Faden A.I. Mol. Cell. Neurosci. 2003; 22: 365-382Crossref PubMed Scopus (138) Google Scholar, 12Song L. Sarno P.D. Jope R.S. J. Biol. Chem. 2002; 277: 44701-44708Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 13King T.D. Bijur G.N. Jope R.S. Brain Res. 2001; 919: 106-114Crossref PubMed Scopus (166) Google Scholar). Taken together, these studies support a role for GSK-3β in regulating cardiomyocyte apoptosis. Akt has also been shown to play an important role in regulating Bcl-2 family members and thus cell fate. Akt phosphorylates Bad, thereby inhibiting its proapoptotic function, which may partly account for its antiapoptotic effect (8Stoica B.A. Movsesyan V.A. Lea IV, P.M. Faden A.I. Mol. Cell. Neurosci. 2003; 22: 365-382Crossref PubMed Scopus (138) Google Scholar, 14Somervaille T.C. Linch D.C. Khwaja A. Blood. 2001; 98: 1374-13781Crossref PubMed Scopus (106) Google Scholar). Bad is known to bind to Bcl-xL in mitochondria and promote apoptosis, whereas phosphorylation of Bad by Akt results in binding to 14-3-3 proteins and promotes the survival of neurons and fibroblasts (8Stoica B.A. Movsesyan V.A. Lea IV, P.M. Faden A.I. Mol. Cell. Neurosci. 2003; 22: 365-382Crossref PubMed Scopus (138) Google Scholar, 15Shimamura H. Terada Y. Okado T. Tanaka H. Inoshita S. Sasaki S. J. Am. Soc. Nephrol. 2003; 14: 1427-1434Crossref PubMed Scopus (102) Google Scholar, 16Chiang C.W. Kanies C. Kim K.W. Fang W.B. Parkhurst C. Xie M. Henry T. Yang E. Mol. Cell. Biol. 2003; 23: 6350-6362Crossref PubMed Scopus (126) Google Scholar, 17Shinoda S. Schindler C.K. Quan-Lan J. Saugstad J.A. Taki W. Simon R.P. Henshall D.C. J. Neurochem. 2003; 86: 460-469Crossref PubMed Scopus (24) Google Scholar). These studies suggest a potential role of Akt-Bad·14-3-3 signaling in promoting cell survival. However, the relationship between GSK-3β-caspase-3 and Bad·14-3-3 in cardiomyocyte apoptosis has not been examined. We hypothesized that interactions of the proapoptotic members of Bcl-2 family with 14-3-3 may contribute greatly to the regulation of cardiomyocyte fate after I/R injury and that GSK-3β-caspase-3 facilitates these signaling effects.The tissue kallikrein/kinin system has been implicated in protection against cardiac remodeling (18Agata J. Chao L. Chao J. Hypertension. 2002; 40: 653-659Crossref PubMed Scopus (73) Google Scholar, 19Chao J. Zhang J.J. Lin K.F. Chao L. Hum. Gene Ther. 1998; 9: 21-31Crossref PubMed Scopus (91) Google Scholar, 20Silva Jr., J.A. Araujo R.C. Baltatu O. Oliveira S.M. Tschope C. Fink E. Hoffmann S. Plehm R. Chai K.X. Chao L. Chao J. Ganten D. Pesquero J.B. Bader M. FASEB J. 2000; 14: 1858-1860Crossref PubMed Scopus (101) Google Scholar, 21Campbell D.J. Dixon B. Kladis A. Kemme M. Santamaria J.D. Am. J. Physiol. 2001; 281: R1059-R1070Crossref PubMed Google Scholar). Our previous study showed that systemic delivery of the kallikrein gene inhibited I/R-induced myocardial injury, which was accompanied by increased kinin and cGMP levels (22Yoshida H. Zhang J.J. Chao L. Chao J. Hypertension. 2000; 35: 25-31Crossref PubMed Scopus (104) Google Scholar). Similarly, adrenomedullin gene transfer also increased cardiac cGMP levels after I/R (23Kato K. Yin H. Agata J. Chao L. Chao J. Am. J. Physiol. 2003; 285: H1506-H1514PubMed Google Scholar). cGMP is an indicator of nitric oxide formation, and these results indicate that the nitric oxide-cGMP signaling cascade may serve as the common signaling cascade for cardio-protective stimuli in the injured myocardium. In this study, we employed a catheter-based gene delivery technique to investigate the role and signaling mechanisms by which kallikrein/kinin inhibits myocardial apoptosis in an acute I/R rat model and in primary cardiomyocytes. Our results show that kallikrein/kinin protects against cardiomyocyte apoptosis via activation of Akt-Bad·14-3-3 and Akt-GSK-3-caspase-3 signaling pathways. We also observed a novel mechanism in which caspase-3 directly disrupts the Bad·14-3-3 complex in cardiomyocytes, establishing a link between these two pathways.EXPERIMENTAL PROCEDURESMaterials—Monoclonal anti-α-actinin sarcomeric, lithium chloride, and bisbenzimide (Hoechst 33342) were purchased from Sigma. Z-VAD(OMe)-CH2F was purchased from Enzyme System Products (Livermore, CA). Recombinant caspase-3 was from purchased Calbiochem. Hoe140 was a gift from Hoechst Marion Russell Co. (Frankfurt, Germany). LY294002, anti-c-Src, anti-v-Src, anti-Akt, anti-phospho-Akt, anti-Bad, anti-phospho-Bad(Ser-136), anti-phospho-GSK-3β(Ser-9), anti-GSK-3β, anti-cleaved caspase-3, and anti-Bcl-xL were from Cell Signaling Technology (Beverly, MA). Polyclonal anti-14-3-3θ was from Chemicon (Temecula, CA).Preparation of Replication-deficient Adenoviral Vectors—Adenoviral vector harboring the human tissue kallikrein cDNA (Ad.CMV-TK) under the control of the cytomegalovirus (CMV) enhancer/promoter or adenoviral vector alone (Ad.Null) were constructed and prepared as described previously (23Kato K. Yin H. Agata J. Chao L. Chao J. Am. J. Physiol. 2003; 285: H1506-H1514PubMed Google Scholar). Adenoviruses containing the dominant-negative mutant of Akt (Ad.DN-Akt), constitutively active Akt (Ad.Myr-Akt), catalytically inactive GSK-3β (Ad.GSK3β-KM) and constitutively active mutant of GSK-3β (Ad.GSK3β-S9A) were kindly provided by Dr. Kenneth Walsh, St. Elizabeth's Medical Center in Boston.Catheter-based Gene Delivery in Rat Myocardium and Immunological Evidence—Wistar rats (male, 250–280 g, Harlan, Indianapolis, IN) were employed in this study. The study complied with the standards for care and use of animal subjects as stated in the Guide for the Care and Use of Laboratory Animals (National Academy of Sciences, Bethesda, MD). Five days before coronary occlusion, an adenoviral gene was delivered using a catheter-based strategy as described previously (24Del Monte F. Butler K. Boecker W. Gwathmey J.K. Hajjar R.J. Physiol. Genom. 2002; 9: 49-56Crossref PubMed Google Scholar, 25Nozato T. Ito H. Watanabe M. Ono Y. Adachi S. Tanaka H. Hiroe M. Sunamori M. Marum F. J. Mol. Cell. Cardiol. 2001; 33: 1493-1504Abstract Full Text PDF PubMed Scopus (30) Google Scholar). Briefly, rats were anesthetized, intubated, and mechanically ventilated before surgery. The chest was entered via a left intercostals approach. A 24-gauge catheter (BD Biosciences) containing 300 μl of virus solution (2 × 1010 plaque-forming units/ml in PBS) was advanced from the apex of the left ventricle to the aortic root. The aorta and pulmonary trunk were clamped distal to the site of the catheter, and the solution was injected. The clamp was maintained for 20 s. After injection, the exposed heart was monitored for 5 min for resumption of normal sinus rhythm. The chest incision was then closed after removal of air and blood, and the animals were allowed to recover. Expression and localization of TK in rat ventricles after gene delivery were identified immunohistochemically by antibody to TK. Kallikrein levels in rat heart were determined using an enzyme-linked immunosorbent assay specific for human tissue kallikrein for detection of active kallikrein. TK standard ranges from 0.4 to 25 ng/ml.In Vivo Hemodynamics Measurements—To eliminate the hemodynamic effects of TK, we measured hemodynamic parameters before and 1 and 5 days after adenovirus solution was injection. Rats were underwent left ventricular catheterization via left common carotid artery by a 2.5 French micromanometer (Millar Instruments, Houston, TX) advanced into the left ventricle cavity. Heart rate, Mean arterial pressure, and ±left ventricle dP/dt (mm Hg/sec) were recorded and analyzed by a polygraph system (BIOPAC, Santa Barbara, CA). Cardiac output was measured by injection of microspheres for 10 s, collection of blood for 10 s before injection, and continued for a total time of 90 s at 0.68 ml/min. Cardiac output was calculated as described previously (26Dobrzynski E. Montanari D. Agata J. Zhu J. Chao J. Chao L. Am. J. Physiol. 2002; 283: E1291-E1298Crossref PubMed Scopus (58) Google Scholar).Acute Myocardial I/R Model—Acute myocardial I/R models were established 5 days after gene delivery. Rats were anesthetized, intubated, and mechanically ventilated. A thoracotomy was performed via the fourth intercostal space, and the heart was exposed. An electrocardiographic monitor was then connected. A 6-0 polypropylene suture (Ethicon, Somerville, NJ) was passed loosely around the left anterior descending coronary artery near its origin. Once hemodynamics were stabilized, left anterior descending occlusion was performed by tightening the suture loop for 30 min. Acute myocardial ischemia was deemed successful on the basis of regional cyanosis of the myocardial surface distal to the suture, accompanied by elevation of the ST segment on electrocardiogram. The loop was then loosened and reperfusion was identified on the basis of return of the original color, accompanied by obvious ST segment change. At the end of reperfusion period, the ischemic regions were then removed for further analysis.Animals were randomly divided into eight groups. In the sham groups, the chest was opened and injected with saline (n = 5), Ad.Null (n = 7), or Ad.CMV-TK (n = 6). The I/R-injured rats were divided into five groups. Control group (n = 6) was also injected with saline, the second group received Ad.Null (n = 6), and the third group received Ad.CMV-TK (n = 7) delivery. The fourth group received Ad.CMV-TK together with administration of kinin B2 antagonist, icatibant, delivered intraperitoneally by an osmotic minipump (Alza, Palo Alto, CA) at 1 μmol/kg of body weight/day (n = 6). The fifth group was injected with Ad.DN-Akt followed by Ad.CMV-TK via the same catheter (n = 6).Detection of Apoptosis in Situ and DNA Laddering—DNA fragmentation was determined using a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay in 4-μm-thick paraffin-embedded sections as described previously (22Yoshida H. Zhang J.J. Chao L. Chao J. Hypertension. 2000; 35: 25-31Crossref PubMed Scopus (104) Google Scholar). The procedure was performed using an in situ cell death detection kit (Roche Applied Science) according to the manufacturer's instructions. TUNEL-positive cardiomyocytes in the ischemic myocardium were carefully distinguished from TUNEL-positive noncardiomyocytes and evaluated under double-blinded conditions. The ratio of TUNEL-positive cardiomyocytes to the total number of cardiomyocytes was calculated.Fresh ischemic and nonischemic myocardium (100 mg) was minced and homogenized in 600 μl of lysis buffer (50 mm Tris-HCl, 100 mm EDTA, 100 mm NaCl, 1% SDS, pH 7.4). Tissues were digested with 100 μg/ml proteinase K (Invitrogen) at 55 °C overnight followed by centrifugation at 13,000 × g for 15 min. After incubation, tissues were precipitated and centrifuged at 13,000 × g for 5 min. Supernatants containing DNA were precipitated with isopropyl alcohol. After centrifugation at 13,000 × g for 5 min, the resulting DNA pellets were washed with 75% ethanol and dissolved in DNA hydration solution and measured at 260 nm by spectrophotometry. 10 μg of DNA was loaded onto 1.2% agarose gel containing 0.5 μg/ml ethidium bromide. DNA electrophoresis was carried out at 80 V for 1.5 h. DNA ladders, an indicator of tissue apoptotic nucleosomal DNA fragmentation, were visualized under ultraviolet light and photographed.Primary Cardiomyocyte Culture and Hypoxia/Reoxygenation (H/R)— Cardiomyocytes were isolated from the hearts of 2–3-day-old Wistar rats. The ventricles were cut into four equal parts and digested enzymatically through multiple rounds in 0.05% pancreatin (Sigma) and 84 units/ml collagenase (Sigma) in a balanced salt solution. The cells were centrifuged at 800 rpm for 10 min at 4 °C and resuspended in F-12 Nutrient Mixture (Invitrogen). Afterward, the cells were differentially plated for 2 h to remove contaminating nonmyocytes. The enriched cardiomyocyte fractions were then cultured in Dulbecco's modified Eagle's medium and F-12 (Invitrogen) medium with 10% fetal bovine serum. Cardiomyocyte origin was confirmed immunocytochemically using antibody to α-actinin sarcomeric (Sigma).Cultured cells were growth arrested for 18 h at 37 °C before the experiments. Cells were transduced with Ad.CMV-TK, Ad.Null, Ad-.Myr-Akt, Ad.DN-Akt, Ad.GSK3β-KM, or Ad.GSK3β-S9A at m.o.i. 50 for 12 h followed by 12-h hypoxia (95% N2 and 5% CO2) and 24-h reoxygenation (95% O2 and 5% CO2). Prior to H/R, myocytes were also treated with GSK-3β inhibitor 20 mm LiCl for 30 min or caspase-3 inhibitor 100 μm Z-VAD for 60-min. Expression and localization of human kallikrein in cardiomyocytes after Ad.CMV-TK transduction were identified immunocytochemically using a rabbit anti-kallikrein antibody. Apoptotic cardiomyocytes were identified in cells (fixed in 4% paraformaldehyde) by or Hoechst 33342 staining (27Nebigil C.G. Etienne N. Messaddeq N. Maroteaux L. FASEB J. 2003; 17: 1373-1375Crossref PubMed Scopus (118) Google Scholar, 28Craig R. Wagner M. McCardle T. Craig A.G. Glembotski C.C. J. Biol. Chem. 2001; 276: 37621-37629Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The positive cells were determined by counting 500–800 cardiac myocytes in six randomly chosen fields. Cell viability was determined by trypan blue eliminating assay.GSK-3β Kinase and Caspase-3 Activity Assay—Control and injured ventricular tissues were pulverized under liquid nitrogen and homogenized in ice-cold lysis buffer as described previously (29Haq S. Choukroun G. Kang Z.B. Ranu H. Matsui T. Rosenzweig A. Molkentin J.D. Alessandrini A. Woodgett J. Hajjar R. Michael A. Force T. J. Cell Biol. 2000; 151: 117-130Crossref PubMed Scopus (333) Google Scholar). For GSK-3β activity assay, 10 μg of protein from cardiac tissue or cell extracts was incubated at 37 °C for 30 min with the reaction buffer (8 mm MOPS, 0.2 mm EDTA, 10 mm magnesium acetate), 62.5 μm GSK-3β substrate peptide (Upstate Biotechnology, Inc., Lake Placid, NY) and 1 μCi/10 μl [γ-32P]ATP (PerkinElmer Life Sciences). Samples were transferred to P-81 paper and washed three times with 0.75% phosphoric acid followed by a final rinse with acetone. Radioactivity was measured in a scintillation counter. Caspase-3 activity in lysates was determined using a fluorometric caspase-3 assay kit (Oncogene, San Diego, CA) according to the manufacturer's instructions. Reaction was monitored by a blue to green shift in fluorescence upon cleavage of the 7-amino-4-trifluoromethylcoumarin. Samples were read with a fluorescence reader (PerkinElmer Life Sciences).Immunoprecipitation and Western Blot Analysis—Heart tissues were homogenized, and cultured cardiomyocytes were harvested in the extraction buffer (10 mmol/liter Tris, pH 7.4, 100 mmol/liter NaCl, 0.1% SDS, 1% Triton X-100, 5 mmol/liter EDTA, 2 mmol/liter Na3VO4, protease inhibitor mixture including 104 mmol/liter benzenesulfonyl fluoride, 0.08 mmol/liter aprotinin, 2.1 mmol/liter leupeptin, 3.6 mmol/liter bestatin, 1.5 mmol/liter pepstatin A, and 1.4 mmol/liter leucylamido butane (Sigma). The protein concentration was determined by micro-Lowry. Aliquots was separated on SDS-PAGE and transferred to a nitrocellulose membrane (Amersham Biosciences). The membrane was blocked with T-PBS (1× PBS, 0.3% Tween 20) containing 5% dry milk and incubated with primary antibody (1:1,000) overnight at 4 °C. After three washes with T-PBS, the membrane was reblocked and incubated with secondary antibody for 1 h at room temperature. The primary antibodies used were anti-c-Sac, anti-v-Sac, anti-Akt, anti-phospho-Akt(Ser-473) antibody, anti-Bad, anti-phospho-Bad(Ser-136), anti-phospho-GSK-3β(Ser-9), anti-GSK-3β, anti-cleaved caspase-3 at 4 °C overnight. The secondary antibodies were anti-rabbit IgG/horseradish peroxidase conjugate or anti-mouse IgG/horseradish peroxidase conjugate (1:2,500 dilution, Promega). Bound antibodies were visualized by ECL chemiluminescence (PerkinElmer Life Sciences). For determination of Bad·Bcl-xL or Bad·14-3-3, cell lysates were immunoprecipitated with anti-Bad antibody overnight at 4 °C followed by incubation with protein A-agarose Beads (Santa Cruz Biotechnology, Santa Cruz, CA). Immunoprecipitates were then separated by SDS-PAGE and immuoblotted with antibodies to 14-3-3θ (Chemicon, Temecula, CA) or Bcl-xL (Cell Signaling).Statistical Analysis—Data were expressed as the mean ± S.E. and were compared between experimental groups with the use of one-way analysis of variance followed by Fisher's protected least significant difference. Probability values of p < 0.05 were considered statistically significant.RESULTSExpression of Human Tissue Kallikrein and Hemodynamics Parameters—Using a catheter-based strategy, we delivered Ad.CMV-TK locally into the rat left ventricle. Five days after kallikrein gene delivery, expression and localization of recombinant human tissue kallikrein were identified immunohistochemically in the left ventricle (Fig. 1). No specific staining was found in the left ventricle injected with or without control adenovirus, Ad.Null. Immunoreactive human tissue kallikrein levels (2.3 ± 0.2 ng/mg protein, n = 7) in cardiac extracts were measured by enzyme-linked immunosorbent assay. Human kallikrein was not detectable in rats injected with control virus. These results demonstrate that human tissue kallikrein was expressed in rat heart after local gene delivery. Table I shows that the hemodynamic parameters are comparable prior to and at 1 day and 5 days after gene delivery, indicating that local kallikrein gene transfer had no effect on the hemodynamic parameters during the experiment.Table IHemodynamic variables throughout the experimental periodBefore delivery (n = 3)Post gene delivery1 day (n = 4)p5 days (n = 4)pHR (bpm)239.7 ± 47.4228.5 ± 28.60.709244.3 ± 38.50.878MAP (mm Hg)93.5 ± 18.698.9 ± 13.10.65799.9 ± 14.80.595CO (ml/min)65.7 ± 8.559.3 ± 9.70.36962.3 ± 7.90.626+dP/dT (mm Hg/sec)2,345.7 ± 362.52,223.8 ± 238.10.6172,408.3 ± 325.60.796-dP/dT (mm Hg/sec)2,206.7 ± 365.82,045.3 ± 216.90.4762,263.8 ± 265.90.795 Open table in a new tab Kallikrein/Kinin Attenuates Cardiomyocyte Apoptosis after I/R—Apoptotic cardiomyocytes were detected by TUNEL staining in the myocardium of I/R. The ratio of TUNEL-positive cardiomyocytes to total number of cardiomyocytes in the Ad.CMV-TK group was significantly reduced compared with the Ad.Null group (28.4 ± 5.8% versus 41.3 ± 6.9%, n = 7 and 6, p < 0.01). However, the protective effect of kallikrein was blocked by icatibant (28.4 ± 5.8% versus 42.7 ± 6%, n = 7 and 6, p < 0.01) (Fig. 2A). The effect of kallikrein on apoptosis was confirmed further by DNA laddering (Fig. 2B). DNA fragmentation was not visualized in the myocardium of sham-operated rats, whereas I/R markedly increased DNA laddering in rat myocardium with or without Ad.Null delivery. Kallikrein gene transfer abrogated I/R-induced DNA fragmentation, whereas the effect of kallikrein was reversed by both icatibant and dominant-negative Akt (Ad.DN-Akt). These findings indicate that kallikrein/kinin protects against I/R-induced cardiomyocyte apoptosis via the kinin B2 receptor-Akt signaling pathway.Fig. 2Kallikrein gene transfer reduced cardiomyocyte apoptosis after acute I/R injury in rat hearts. A, representative photomicrographs show TUNEL-positive apoptotic cardiomyocytes from I/R-injured ventricular sections of rats injected with Ad.Null, Ad.CMV-TK, and Ad.CMV-TK plus icatibant infusion. TUNEL-positive noncardiomyocytes were excluded. Original magnification is ×200. B, quantitative analysis of apoptotic cardiomyocytes expressed as percentage of TUNEL-positive nuclei in cardiomyocytes (mean ± S.E., n = 6–7). *, p < 0.05 versus other group. C, representative DNA laddering analysis in I/R-injured myocardium from sham, sham receiving Ad.Null or Ad.CMV-TK, I/R control and I/R receiving Ad.Null, Ad.CMV-TK, or Ad.CMV-TK/icatibant or Ad.CMV-TK/DN-Akt. Nucleosomal DNA fragmentation of myocardial extracts was analyzed from three separate experiments.View Large Image Figure ViewerDownload (PPT)Kallikrein/Kinin Activates Akt Signaling Cascade in the Ischemic Myocardium—We next examined the effect of kallikrein gene transfer on the Akt signaling cascade in the ischemic heart by Western blot analysis (Fig. 3A). Kallikrein gene transfer resulted in increased phosphorylation of Src, Akt, GSK-3β, and Bad(Ser-136) in the I/R-injured myocardium compared with the control with or without injection of Ad.Null. However, kallikrein gene transfer had no effect on total Src, Akt, GSK-3β, and Bad levels. Furthermore, kallikrein did not lead to phosphorylation of Bad at Ser-112 or Ser-155 (data not shown). Cleaved caspase-3, which is a downstream proapoptotic signal, was reduced markedly after kallikrein gene delivery. The effects of kallikrein on these signaling effectors were blocked by icatibant and Ad.DN-A" @default.
• W2109585407 created "2016-06-24" @default.
• W2109585407 creator A5025581121 @default.
• W2109585407 creator A5058401832 @default.
• W2109585407 creator A5091050446 @default.
• W2109585407 date "2005-03-01" @default.
• W2109585407 modified "2023-10-15" @default.
• W2109585407 title "Kallikrein/Kinin Protects against Myocardial Apoptosis after Ischemia/Reperfusion via Akt-Glycogen Synthase Kinase-3 and Akt-Bad·14-3-3 Signaling Pathways" @default.
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