Up-Regulation of AT1 and AT2 Receptors in Postinfarcted Hypertrophied Myocytes and Stretch-Mediated Apoptotic Cell Death
2000; Elsevier BV; Volume: 156; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)65037-7
ISSN1525-2191
AutoresAnnarosa Leri, Yu Liu, Baosheng Li, Fabio Fiordaliso, Ashwani Malhotra, Roberto Latini, Jan Kajstura, Piero Anversa,
Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoTo determine whether up-regulation of AT1 and AT2 receptors occurred in hypertrophied myocytes after infarction and whether AT2 played a role in stretch-mediated apoptosis, left ventricular myocytes were dissociated from the surviving portion of the wall 8 days after coronary occlusion and cardiac failure in rats. Control cells were obtained from sham-operated animals. Myocytes were stretched in an equibiaxial stretch apparatus and angiotensin II (Ang II) formation and cell death were measured 3 and 12 hours later. AT1 and AT2 proteins were evaluated in freshly isolated myocytes and after stretch. The effects of AT1 and AT2 antagonists on stretch-induced Ang II synthesis and apoptosis were also established. Myocardial infarction increased AT1 and AT2 in myocytes and stretch further up-regulated these receptors. Ang II levels were higher in postinfarcted myocytes and this peptide increased with the duration of stretch in both groups of cells. Similarly, apoptosis increased with time in control and postinfarcted myocytes. Absolute values of Ang II and apoptosis were greater in myocytes from infarcted hearts at 3 and 12 hours after stretch. Addition of AT1 blocker to cultures inhibited stretch-activated apoptosis in both myocyte populations as well as the generation of Ang II in postinfarcted myocytes. In contrast, AT2 antagonists had no impact on these cellular events. In conclusion, Ang II stimulated cell death through AT1 receptor activation, whereas ligand binding to AT2 receptor did not alter Ang II concentration and apoptosis in normal and postinfarcted hypertrophied myocytes. To determine whether up-regulation of AT1 and AT2 receptors occurred in hypertrophied myocytes after infarction and whether AT2 played a role in stretch-mediated apoptosis, left ventricular myocytes were dissociated from the surviving portion of the wall 8 days after coronary occlusion and cardiac failure in rats. Control cells were obtained from sham-operated animals. Myocytes were stretched in an equibiaxial stretch apparatus and angiotensin II (Ang II) formation and cell death were measured 3 and 12 hours later. AT1 and AT2 proteins were evaluated in freshly isolated myocytes and after stretch. The effects of AT1 and AT2 antagonists on stretch-induced Ang II synthesis and apoptosis were also established. Myocardial infarction increased AT1 and AT2 in myocytes and stretch further up-regulated these receptors. Ang II levels were higher in postinfarcted myocytes and this peptide increased with the duration of stretch in both groups of cells. Similarly, apoptosis increased with time in control and postinfarcted myocytes. Absolute values of Ang II and apoptosis were greater in myocytes from infarcted hearts at 3 and 12 hours after stretch. Addition of AT1 blocker to cultures inhibited stretch-activated apoptosis in both myocyte populations as well as the generation of Ang II in postinfarcted myocytes. In contrast, AT2 antagonists had no impact on these cellular events. In conclusion, Ang II stimulated cell death through AT1 receptor activation, whereas ligand binding to AT2 receptor did not alter Ang II concentration and apoptosis in normal and postinfarcted hypertrophied myocytes. 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The initiation and evolution of ischemic cardiomyopathy are characterized hemodynamically by an increase in left ventricular end-diastolic pressure and diastolic wall stress which result in sustained sarcomere stretching.10Vitali-Mazza L Anversa P Tedeschi F Mastandrea R Mavilla V Visioli O Ultrastructural basis of acute left ventricular failure from severe acute aortic stenosis in the rabbit.J Mol Cell Cardiol. 1972; 4: 661-671Abstract Full Text PDF PubMed Scopus (28) Google Scholar, 11Ross J Sonnenblick EH Taylor RR Spotnitz HM Covell JW Diastolic geometry and sarcomere lengths in the chronically dilated canine left ventricle.Circ Res. 1971; 28: 49-61Crossref PubMed Scopus (144) Google Scholar Load-dependent sarcomere elongation in vivo up-regulates the cellular RAS leading to an increase in surface angiotensin II (Ang II) receptors on myocytes and local synthesis of Ang II.12Meggs LG Coupet J Huang H Cheng W Li P Capasso JM Homcy CJ Anversa P Regulation of angiotensin II receptors on ventricular myocytes after myocardial infarction in rats.Circ Res. 1993; 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83: 1182-1191Crossref PubMed Scopus (435) Google Scholar For this purpose, control myocytes and hypertrophied myocytes, surviving an 8-day myocardial infarct, were studied. This was done because Ang II binding sites are doubled in postinfarcted myocytes at this interval.12Meggs LG Coupet J Huang H Cheng W Li P Capasso JM Homcy CJ Anversa P Regulation of angiotensin II receptors on ventricular myocytes after myocardial infarction in rats.Circ Res. 1993; 72: 1149-1162Crossref PubMed Scopus (165) Google Scholar Myocytes were dissociated and stretched in an equibiaxial stretch apparatus for 3 and 12 hours. The expression of AT1 and AT2 receptors was determined at baseline and after stretch. Similarly, Ang II formation, extent of apoptosis, and influence of AT1 and AT2 receptor antagonists, alone and in combination, on Ang II formation and cell death with stretch were established. Under ether anesthesia, MI was induced in 84 male Sprague-Dawley rats (Charles River Breeding Labs, North Wilmington, MA), weighing approximately 250 g.12Meggs LG Coupet J Huang H Cheng W Li P Capasso JM Homcy CJ Anversa P Regulation of angiotensin II receptors on ventricular myocytes after myocardial infarction in rats.Circ Res. 1993; 72: 1149-1162Crossref PubMed Scopus (165) Google Scholar, 13Reiss K Capasso JM Huang H Meggs LG Li P Anversa P Ang II receptors, c-myc, and c-jun in myocytes after myocardial infarction and ventricular failure.Am J Physiol. 1993; 264: H760-H769PubMed Google Scholar, 25Liu Y Leri A Li B Wang X Cheng W Kajstura J Anversa P Angiotensin II stimulation in vitro induces hypertrophy of normal and postinfarcted ventricular myocytes.Circ Res. 1998; 82: 1145-1159Crossref PubMed Scopus (87) Google Scholar To reduce pain, buprenorphine hydrochloride was administered (0.65 mg/kg body weight, intramuscularly; Brufenex, Reckitt and Colman Pharmaceuticals, Richmond, VA). Thirty-seven infarcted rats died shortly after the operation; 47 infarcted rats were used. Thirty-one sham-operated (SO) rats were used as controls. Animals were killed 8 days after coronary occlusion or sham-operation. Before sacrifice, animals were anesthetized with chloral hydrate (300 mg/kg body weight, intraperitoneally), and measurements of left ventricular and right ventricular pressures and rate of pressure rise, + dP/dt, and decay, − dP/dt, were obtained.12Meggs LG Coupet J Huang H Cheng W Li P Capasso JM Homcy CJ Anversa P Regulation of angiotensin II receptors on ventricular myocytes after myocardial infarction in rats.Circ Res. 1993; 72: 1149-1162Crossref PubMed Scopus (165) Google Scholar, 13Reiss K Capasso JM Huang H Meggs LG Li P Anversa P Ang II receptors, c-myc, and c-jun in myocytes after myocardial infarction and ventricular failure.Am J Physiol. 1993; 264: H760-H769PubMed Google Scholar, 14Zhang X Dostal DE Reiss K Cheng W Kajstura J Li P Huang H Sonnenblick EH Meggs LG Baker KM Anversa P Identification and activation of autocrine renin-angiotensin system in adult ventricular myocytes.Am J Physiol. 1995; 269: H1791-H1802PubMed Google Scholar, 25Liu Y Leri A Li B Wang X Cheng W Kajstura J Anversa P Angiotensin II stimulation in vitro induces hypertrophy of normal and postinfarcted ventricular myocytes.Circ Res. 1998; 82: 1145-1159Crossref PubMed Scopus (87) Google Scholar Protocols were approved by the New York Medical College. Hearts were placed on a stainless steel cannula for retrograde perfusion through the aorta and left ventricular myocytes were enzymatically dissociated. The solutions were supplements of modified commercial minimum essential medium (MEM) Joklik (Sigma Chemical Co., St. Louis. MO).12Meggs LG Coupet J Huang H Cheng W Li P Capasso JM Homcy CJ Anversa P Regulation of angiotensin II receptors on ventricular myocytes after myocardial infarction in rats.Circ Res. 1993; 72: 1149-1162Crossref PubMed Scopus (165) Google Scholar, 13Reiss K Capasso JM Huang H Meggs LG Li P Anversa P Ang II receptors, c-myc, and c-jun in myocytes after myocardial infarction and ventricular failure.Am J Physiol. 1993; 264: H760-H769PubMed Google Scholar, 14Zhang X Dostal DE Reiss K Cheng W Kajstura J Li P Huang H Sonnenblick EH Meggs LG Baker KM Anversa P Identification and activation of autocrine renin-angiotensin system in adult ventricular myocytes.Am J Physiol. 1995; 269: H1791-H1802PubMed Google Scholar, 17Leri A Claudio PP Li Q Wang X Reiss K Wang S Malhotra A Kajstura J Anversa P Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin system and decreases the Bcl-2-to-Bax protein ratio in the cell.J Clin Invest. 1998; 101: 1326-1342Crossref PubMed Google Scholar, 18Leri A Liu Y Claudio PP Kajstura J Wang X Wang S Kang P Malhotra A Anversa P Insulin-like growth factor-1 induces Mdm2 and down-regulates p53, attenuating the myocyte renin-angiotensin system and stretch-mediated apoptosis.Am J Pathol. 1999; 154: 567-580Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 22Pierzchalski P Reiss K Cheng W Cirielli C Kajstura J Nitahara JA Rizk M Capogrossi MC Anversa P p53 induces myocyte apoptosis via the activation of the renin-angiotensin system.Exp Cell Res. 1997; 234: 57-65Crossref PubMed Scopus (125) Google Scholar, 23Kajstura J Cigola E Malhotra A Li P Cheng W Meggs LG Anversa P Angiotensin II induces apoptosis of adult ventricular myocytes in vitro.J Mol Cell Cardiol. 1997; 29: 859-870Abstract Full Text PDF PubMed Scopus (381) Google Scholar, 25Liu Y Leri A Li B Wang X Cheng W Kajstura J Anversa P Angiotensin II stimulation in vitro induces hypertrophy of normal and postinfarcted ventricular myocytes.Circ Res. 1998; 82: 1145-1159Crossref PubMed Scopus (87) Google Scholar The cell isolation procedure, used for SO and MI hearts, consisted of three main steps: 1) calcium-free perfusion: blood wash-out and collagenase (selected type II, Worthington Biochemical Corp., Freehold, NJ. perfusion of the heart were carried out at 37°C with HEPES/MEM gassed with 85% O2 and 15% N2. 2) Mechanical tissue dissociation: after the heart was removed from the cannula, the left ventricle, inclusive of the septum, was separated from the right ventricular free wall and minced. Pieces were subsequently shaken in resuspension medium containing collagenase. 3. Separation of intact cells: intact cells were enriched by centrifugation at 30 × g for 3 minutes. Supernatant was discarded. This procedure was repeated four times to remove nonmyocytes, cellular debris, and residual collagenase. Myocytes were resuspended in Percoll solution (final concentration 41%) and centrifuged for 10 minutes at 34 × g. Intact cells were recovered and washed. Smears were made to control the purity of the preparation. Nonmyocytes constituted 1 to 2% of the cells.12Meggs LG Coupet J Huang H Cheng W Li P Capasso JM Homcy CJ Anversa P Regulation of angiotensin II receptors on ventricular myocytes after myocardial infarction in rats.Circ Res. 1993; 72: 1149-1162Crossref PubMed Scopus (165) Google Scholar, 13Reiss K Capasso JM Huang H Meggs LG Li P Anversa P Ang II receptors, c-myc, and c-jun in myocytes after myocardial infarction and ventricular failure.Am J Physiol. 1993; 264: H760-H769PubMed Google Scholar, 14Zhang X Dostal DE Reiss K Cheng W Kajstura J Li P Huang H Sonnenblick EH Meggs LG Baker KM Anversa P Identification and activation of autocrine renin-angiotensin system in adult ventricular myocytes.Am J Physiol. 1995; 269: H1791-H1802PubMed Google Scholar, 25Liu Y Leri A Li B Wang X Cheng W Kajstura J Anversa P Angiotensin II stimulation in vitro induces hypertrophy of normal and postinfarcted ventricular myocytes.Circ Res. 1998; 82: 1145-1159Crossref PubMed Scopus (87) Google Scholar The average number of myocytes obtained from the left ventricle was 6 to 7 × 106 and 3.3 × 106 in SO and MI rats, respectively. Myocytes were plated on a silicon rubber substrate (Figure 1) and stretched in an equibiaxial strain device.17Leri A Claudio PP Li Q Wang X Reiss K Wang S Malhotra A Kajstura J Anversa P Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin system and decreases the Bcl-2-to-Bax protein ratio in the cell.J Clin Invest. 1998; 101: 1326-1342Crossref PubMed Google Scholar, 18Leri A Liu Y Claudio PP Kajstura J Wang X Wang S Kang P Malhotra A Anversa P Insulin-like growth factor-1 induces Mdm2 and down-regulates p53, attenuating the myocyte renin-angiotensin system and stretch-mediated apoptosis.Am J Pathol. 1999; 154: 567-580Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 26Lee AA Delhaas T Waldman LK MacKenna DA Villarreal FJ McCulloch AD An equibiaxial strain system for cultured cells.Am J Physiol. 1996; 271: 1400-1408Google Scholar Stretching effects on cell injury were established by exposing cultures to ethidium monoazide bromide (EMB; Molecular Probes, Eugene, CA). EMB binds to nucleic acids only in cells with membrane breakage.27Riedy MC Muirhead KA Jensen CP Stewart CC Use of a photolabeling technique to identify nonviable cells in fixed homologous or heterologous cell populations.Cytometry. 1991; 12: 133-139Crossref PubMed Scopus (115) Google Scholar Nonstretched and stretched myocytes from noninfarcted and infarcted hearts were evaluated at 3 and 12 hours. In some cultures, the AT1 antagonist, losartan (Merck, Rahway, NJ), and/or the AT2 antagonist, PD123319 (Parke-Davis, Ann Arbor, MI), were added at 10−8 mol/L 30 minutes before stretch, and kept for the duration of the experiment. Cells were collected for histochemical and molecular assays.17Leri A Claudio PP Li Q Wang X Reiss K Wang S Malhotra A Kajstura J Anversa P Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin system and decreases the Bcl-2-to-Bax protein ratio in the cell.J Clin Invest. 1998; 101: 1326-1342Crossref PubMed Google Scholar, 18Leri A Liu Y Claudio PP Kajstura J Wang X Wang S Kang P Malhotra A Anversa P Insulin-like growth factor-1 induces Mdm2 and down-regulates p53, attenuating the myocyte renin-angiotensin system and stretch-mediated apoptosis.Am J Pathol. 1999; 154: 567-580Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar Myocyte dimensions were measured in freshly isolated binucleated myocytes with a computer system: 200 cells from each control and infarcted ventricle were analyzed to collect length, width, and area. Isolated myocytes flatten and the cross-sectional area resembles an ellipse. The minor-to-major axis ratio of the ellipse was obtained by examining 40 cells in each case by confocal microscopy. These values were used to compute myocyte volume.25Liu Y Leri A Li B Wang X Cheng W Kajstura J Anversa P Angiotensin II stimulation in vitro induces hypertrophy of normal and postinfarcted ventricular myocytes.Circ Res. 1998; 82: 1145-1159Crossref PubMed Scopus (87) Google Scholar Cultures were incubated with 5 units of 2.5 mmol/L CoCl2, 0.2 mol/L potassium cacodylate, 25 mmol/L Tris-HCl, 0.25% bovine serum albumin, and 0.5 nmol/L biotin-16-deoxyuridine triphosphate (dUTP). After exposure to fluorescein-labeled Extravidin (Sigma), cytoplasm was stained by α-sarcomeric actin and nuclei by propidium iodide. Double-strand DNA fragments for in situ ligation to 3′ overhangs were prepared by polymerase chain reaction using primers 5′-GTGGCCTGCCCAAGCTCTACCT-3′ and 5′-GGCTGGTCTGCCGTTTTCGACCCTG-3′ complementary to pBluescript-bSDI1 plasmid.28Didenko VV Hornsby PJ Presence of double-stranded breaks with single-base 3′ overhangs in cells undergoing apoptosis but not necrosis.J Cell Biol. 1996; 135: 1369-1376Crossref PubMed Scopus (170) Google Scholar, 29Frustaci A Chimenti C Setoguchi M Guerra S Corsello S Crea F Leri A Kajstura J Anversa P Maseri A Cell death in acromegalic cardiomyopathy.Circulation. 1999; 99: 1426-1434Crossref PubMed Scopus (104) Google Scholar Digoxigenin-dUTP fragments were ligated to DNA using T4 ligase.28Didenko VV Hornsby PJ Presence of double-stranded breaks with single-base 3′ overhangs in cells undergoing apoptosis but not necrosis.J Cell Biol. 1996; 135: 1369-1376Crossref PubMed Scopus (170) Google Scholar, 29Frustaci A Chimenti C Setoguchi M Guerra S Corsello S Crea F Leri A Kajstura J Anversa P Maseri A Cell death in acromegalic cardiomyopathy.Circulation. 1999; 99: 1426-1434Crossref PubMed Scopus (104) Google Scholar The number of myocytes labeled by terminal deoxynucleotidyl transferase or Taq was determined by examining 2000 to 3000 cells in each condition by confocal microscopy.6Olivetti G Abbi R Quaini F Kajstura J Cheng W Nitahara JA Quaini E Di Loreto C Beltrami CA Krajewski S Reed JC Anversa P Apoptosis in the failing human heart.N Engl J Med. 1997; 336: 1131-1141Crossref PubMed Scopus (1487) Google Scholar, 17Leri A Claudio PP Li Q Wang X Reiss K Wang S Malhotra A Kajstura J Anversa P Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin system and decreases the Bcl-2-to-Bax protein ratio in the cell.J Clin Invest. 1998; 101: 1326-1342Crossref PubMed Google Scholar, 18Leri A Liu Y Claudio PP Kajstura J Wang X Wang S Kang P Malhotra A Anversa P Insulin-like growth factor-1 induces Mdm2 and down-regulates p53, attenuating the myocyte renin-angiotensin system and stretch-mediated apoptosis.Am J Pathol. 1999; 154: 567-580Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 29Frustaci A Chimenti C Setoguchi M Guerra S Corsello S Crea F Leri A Kajstura J Anversa P Maseri A Cell death in acromegalic cardiomyopathy.Circulation. 1999; 99: 1426-1434Crossref PubMed Scopus (104) Google Scholar Myocytes were incubated with lysis buffer (50 mmol/L Tris/HCl, pH 7.5, 5 mmol/L EDTA, 250 mmol/L NaCl, 0.1% Triton X-100) containing protease inhibitors (0.2 mmol/L phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 5 mmol/L dithiothreitol, 1 mmol/L Na3VO4) on ice for 30 minutes. Equivalents of 50 μg of myocyte proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose and exposed to rabbit anti-human AT1 (306, Santa Cruz, Santa Cruz, CA) and goat anti-human AT2 (C-18, Santa Cruz) at a concentration of 1 μg/ml in Tris-buffered saline/Tween 20 (TBST). Bound antibodies were detected by peroxidase-conjugated anti-rabbit and anti-goat IgG. AT1 was detected as a 41-kd band and AT2 as a 44-kd band.17Leri A Claudio PP Li Q Wang X Reiss K Wang S Malhotra A Kajstura J Anversa P Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin system and decreases the Bcl-2-to-Bax protein ratio in the cell.J Clin Invest. 1998; 101: 1326-1342Crossref PubMed Google Scholar, 18Leri A Liu Y Claudio PP Kajstura J Wang X Wang S Kang P Malhotra A Anversa P Insulin-like growth factor-1 induces Mdm2 and down-regulates p53, attenuating the myocyte renin-angiotensin system and stretch-mediated apoptosis.Am J Pathol. 1999; 154: 567-580Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 30Leri A Liu Y Wang X Kajstura J Malhotra A Meggs LG Anversa P Overexpression of insulin-like growth factor-1 attenuates the myocyte renin-angiotensin system in transgenic mice.Circ Res. 1999; 84: 752-762Crossref PubMed Scopus (90) Google Scholar Human cloned AT1 receptor (BioSignal, Montreal, Canada) was used as positive control. Ang II in conditioned medium was measured by enzyme-linked immunosorbent assay (Peni
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