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Caspase-3 expression in failing human hearts
22
Journal of Cardiac Failure Vol. 4 No. 3 Suppl. 1 1998
079
080
Efficient T r a n s m u r a l Cardiac G e n e Transfer b y Intrapericardial Injection in Neonatal Mice Janet C.L. Zhang, Y. Joseph Woo, Jo-Arm Chert, Judith L.Swain, H. Lee Sweeney, University of Pennsylvania, Philadelphia, PA
Apoptosis During Remodeling of Reperfused Myocardial Infarction in Dogs
An efficient gene transfer technique in murine models would greatly facilitate the elucidation of the pathophysiology of cardiomyopathies and aid in the development of genetic therapies for cardiovascular diseases. Currently employed techniques of myocardial gene delivery in large animals, such as direct injection into the myocardinm or catheterbased approaches, are not easily achieved in mice. Additionally, the transgene expression achieved by those techniques is often limited to either the site of direct injection or to the myocardinm supplied by selective coronary arteries. A replication-defective, recombinant adenovims (Ad5) encoding luciferase (5xl09 pfu) or [3-galactosidase (5x10 ~ to 5xl0 u particles) was injected by a subxyphoid approach into the pericardial cavity of 4-5 day old mice. Chemiluminescence assay for luciferase activity at 3 days post-injection revealed the highest activity in heart (heart=288+l10, lungs=t9+5, liver=l 1+5 ng luciferase/g, n=ll). X-gal staining of cryostat sections of the hearts (n=22) demonstrated lacZ expression in the epicardium, myocardium, and endocardium as well as in the fight ventricle and atrial appendages. This transmural pattern of X-gaI staining was observed from the apex to the base of the left ventricle, with an average fractional area of X-gal staining in the left ventricle of 66 +16% (range 40 - 92%, n=21 sections). In summary, injection of adenoviral vectors into the pericardial space in neonatal mice results in transmural expression of a reporter gene throughout the heart. The degree of transgene expression achieved may be sufficient to allow for the functional evaluation of specific genetic manipulations that may help to elucidate the pathophysiology of cardiovascular diseases.
Different levels of apoptosis or programmed cell death (PCD) in infarct and non-infarct zones might modulate remodeling after myocardial infarction (MI) with or without reperfusiou (RP). We assessed PCD (by in situ nick end-labeling or ISEL) in 255 tissue samples from 51 formalin fixed hearts of dogs in 9 treatment groups after MI: sham; RP; no R.P; enalapril (EL) 4- RP; angiotensin II (AngII) type 1 (AT1) blocker 4- RP; amlodipine (AMLO, an antioxidant) 4- RP. We studied 5 zones (infarct, non-infarct, border, septum, right ventricle) in transverse sections of hearts reperfused for 15 minutes to 6 weeks after MI (30 to 120 minutes of coronary occlusion). We carefully graded the level of PCD using semiquantitative scores (1 to 10) of 4 morphologic features: faint nuclear labeling; strong nuclear labeling; apoptotic bodies; fragmented nuclei. PCD was correlated with remodeling in vivo (2Dechocardiograms) and ex vivo (computerized planimetry), as well as in vivo hemodyrmmics and function. A burst of PCD found after acute RP was attenuated by ACE inhibition (EL) and AT~ blockade. Chronic RP was associated with less PCD than no RP. EL therapy ~: RP was also associated with less PCD. Chronic AMLO was associated with less PCD in RP than no RP hearts. The average PCD score was 2 to 5-fold less in both infarct and non-infarct zones of hearts with chronic EL than AMLO 4- RP. Attenuation of PCD correlated with less ventricular remodeling and improved function. Residual apoptutic bodies found between collagen bundles of infarct at 6 weeks suggest ongoing remodeling. The overall results suggest that i)levels of PCD differ after RP in infarct and non-infarct zones, ii) PCD might modulate functional recovery after acute RP and ongoing remodeling in infarct and non-infarct zones, and iii) AngII and oxidative stress after RP might promote PCD.
081
082
Caspase-3 Expression in Failing Human Hearts Bruce L Goldman, Carol A. Fisher, Elaine Johnston, Kenneth B. Margulies, John Wurzel, Jack L. Martin. Temple University Medical School, Philadelphia PA and John S. Sharpe Foundation of Bryn M a w r Hospital, Bryn Mawr, PA
Primary Pulmonary Hypertension Dennis M. McNamara, Jue J. Wang, Kathleen Zell, T a m m y Tokarczyk, Barry London, Srinivas Murali, Steven E. Reis, University of Pittsburgh, Pittsburgh, PA
Background: Apoptosis (programmed cell death) in non-cardiac tissues has been associated with increased expression of caspases, intracellular cysteine proteases which induce apeptosis via proteolyfic cascade. We charactedzed myocyte expression of caspase-3 in failing human hearts and determined its relationship to myocyte death. Methods: Immunohistochemical staining with a caspase-3 specific monoclonal antibody was performed on formalin fixed, paraffin embedded left ventricular tissue from hearts failing with acute/recent myocardial infarction (MI; n=l 0), chronic atherosclerotic heart disease (ASHD; n=6) and dilated cardiomyopathy (DCM; n=10). Tissue was obtained from cardiectomies removed at orthotopic transplantation (n=20) or from left ventricular cores removed for placement of assist devices (n=6); control tissue was obtained from donor hearts not used for transplantation (n=4). Myocyte caspase-3 immunostaining score reflected maximum number of stained cells/400x microscopic field ( O, no cells; 1+, 1-3 cells; 2+, 4-I 0 cells; 3+, >10 cells). In situ dUTP nick end-labeling (TUNEL assay) was used to identify cell death. Results:Seven of 10 hearts with MI showed 3+ caspase-3 staining of myocytes in peri-infarct zones; no staining was seen away from infarcts. Myocyte staining in cases of DCM and ASHD ranged from 0-1+ only (p=O.003 vs. MI by Fisher's exact test). Numerous myocyte nuclei in the areas of infarcfion were labeled by TUNEL assay, but no caspase-3 positive myocytes were identified as TUNEL positive in sequential sections. All control samples were negative for myocyte caspase-3 staining; 1 of 4 control samples showed myocyte TUNEL positivity. Conclusions: Caspase3 is strongly induced in myocytee neighboring recent infarcts, but is induced to lesser extent in DCM and ASHD. Myocyte caspase-3 expression does not necessadly indicate cell death, but caspase-3 mediated apoptosis could contdbute to infarct extension.
Sorin Musat-Marcu, Darryl O'Brien, Yi Xu, Bodh I. Jugdu~t, University of Alberta, Edmonton, Alberta, C A N A D A
Estrogen Receptor Mutations are not Associated in Women with
Primary pulmonary hypertension (PPH) is a devastating disorder of unknown cause that affects predominantly young premenopausal women. Abnormalities of the estrogen receptor (ER) have been found to be important in other homaonally mediated vascular disorders such as accelerated coronary disease. We hypothesized that ER mutations may affect receptor function and increase the risk of PPH and evaluated this through a case control study. DNA was isolated from peripheral blood in 26 women with non-familial PPH and 40 control women with no history of cardiac or pulmonary disease. Primers which bracketed the eight exnns of the coding region were utilized for PCR. Using primers end labeled with P32, singlestranded contbrmation pnlymorphism (SSCP) analysis was then utilized to identify mutations by differential migration and subsequent sequencing. Mutations were identified in exon 3 only and in each case represented an C to T transition in codon 243, which does not change the amino acid sequence. The frequency of this mutation did not differ in patients with PPH compared to controls (see table below). Conclusion: Although mutations of the estrogen receptor are seen in a minority of women with PPH, these mutations are functionally silent and are seen in an equal frequency in a control population. Though hormonal factors may still be important in the pathogenesis of PPH, mutations of the coding region of the ER do not appear to play a role. PPH
Exon 3 Mutation Wild Type
3(12%) 23 (88%)
Controls 3 (8%) 37 (92%)
Journal of Cardiac Failure Vol. 4 No. 3 Suppl. 1 1998
079
080
Efficient T r a n s m u r a l Cardiac G e n e Transfer b y Intrapericardial Injection in Neonatal Mice Janet C.L. Zhang, Y. Joseph Woo, Jo-Arm Chert, Judith L.Swain, H. Lee Sweeney, University of Pennsylvania, Philadelphia, PA
Apoptosis During Remodeling of Reperfused Myocardial Infarction in Dogs
An efficient gene transfer technique in murine models would greatly facilitate the elucidation of the pathophysiology of cardiomyopathies and aid in the development of genetic therapies for cardiovascular diseases. Currently employed techniques of myocardial gene delivery in large animals, such as direct injection into the myocardinm or catheterbased approaches, are not easily achieved in mice. Additionally, the transgene expression achieved by those techniques is often limited to either the site of direct injection or to the myocardinm supplied by selective coronary arteries. A replication-defective, recombinant adenovims (Ad5) encoding luciferase (5xl09 pfu) or [3-galactosidase (5x10 ~ to 5xl0 u particles) was injected by a subxyphoid approach into the pericardial cavity of 4-5 day old mice. Chemiluminescence assay for luciferase activity at 3 days post-injection revealed the highest activity in heart (heart=288+l10, lungs=t9+5, liver=l 1+5 ng luciferase/g, n=ll). X-gal staining of cryostat sections of the hearts (n=22) demonstrated lacZ expression in the epicardium, myocardium, and endocardium as well as in the fight ventricle and atrial appendages. This transmural pattern of X-gaI staining was observed from the apex to the base of the left ventricle, with an average fractional area of X-gal staining in the left ventricle of 66 +16% (range 40 - 92%, n=21 sections). In summary, injection of adenoviral vectors into the pericardial space in neonatal mice results in transmural expression of a reporter gene throughout the heart. The degree of transgene expression achieved may be sufficient to allow for the functional evaluation of specific genetic manipulations that may help to elucidate the pathophysiology of cardiovascular diseases.
Different levels of apoptosis or programmed cell death (PCD) in infarct and non-infarct zones might modulate remodeling after myocardial infarction (MI) with or without reperfusiou (RP). We assessed PCD (by in situ nick end-labeling or ISEL) in 255 tissue samples from 51 formalin fixed hearts of dogs in 9 treatment groups after MI: sham; RP; no R.P; enalapril (EL) 4- RP; angiotensin II (AngII) type 1 (AT1) blocker 4- RP; amlodipine (AMLO, an antioxidant) 4- RP. We studied 5 zones (infarct, non-infarct, border, septum, right ventricle) in transverse sections of hearts reperfused for 15 minutes to 6 weeks after MI (30 to 120 minutes of coronary occlusion). We carefully graded the level of PCD using semiquantitative scores (1 to 10) of 4 morphologic features: faint nuclear labeling; strong nuclear labeling; apoptotic bodies; fragmented nuclei. PCD was correlated with remodeling in vivo (2Dechocardiograms) and ex vivo (computerized planimetry), as well as in vivo hemodyrmmics and function. A burst of PCD found after acute RP was attenuated by ACE inhibition (EL) and AT~ blockade. Chronic RP was associated with less PCD than no RP. EL therapy ~: RP was also associated with less PCD. Chronic AMLO was associated with less PCD in RP than no RP hearts. The average PCD score was 2 to 5-fold less in both infarct and non-infarct zones of hearts with chronic EL than AMLO 4- RP. Attenuation of PCD correlated with less ventricular remodeling and improved function. Residual apoptutic bodies found between collagen bundles of infarct at 6 weeks suggest ongoing remodeling. The overall results suggest that i)levels of PCD differ after RP in infarct and non-infarct zones, ii) PCD might modulate functional recovery after acute RP and ongoing remodeling in infarct and non-infarct zones, and iii) AngII and oxidative stress after RP might promote PCD.
081
082
Caspase-3 Expression in Failing Human Hearts Bruce L Goldman, Carol A. Fisher, Elaine Johnston, Kenneth B. Margulies, John Wurzel, Jack L. Martin. Temple University Medical School, Philadelphia PA and John S. Sharpe Foundation of Bryn M a w r Hospital, Bryn Mawr, PA
Primary Pulmonary Hypertension Dennis M. McNamara, Jue J. Wang, Kathleen Zell, T a m m y Tokarczyk, Barry London, Srinivas Murali, Steven E. Reis, University of Pittsburgh, Pittsburgh, PA
Background: Apoptosis (programmed cell death) in non-cardiac tissues has been associated with increased expression of caspases, intracellular cysteine proteases which induce apeptosis via proteolyfic cascade. We charactedzed myocyte expression of caspase-3 in failing human hearts and determined its relationship to myocyte death. Methods: Immunohistochemical staining with a caspase-3 specific monoclonal antibody was performed on formalin fixed, paraffin embedded left ventricular tissue from hearts failing with acute/recent myocardial infarction (MI; n=l 0), chronic atherosclerotic heart disease (ASHD; n=6) and dilated cardiomyopathy (DCM; n=10). Tissue was obtained from cardiectomies removed at orthotopic transplantation (n=20) or from left ventricular cores removed for placement of assist devices (n=6); control tissue was obtained from donor hearts not used for transplantation (n=4). Myocyte caspase-3 immunostaining score reflected maximum number of stained cells/400x microscopic field ( O, no cells; 1+, 1-3 cells; 2+, 4-I 0 cells; 3+, >10 cells). In situ dUTP nick end-labeling (TUNEL assay) was used to identify cell death. Results:Seven of 10 hearts with MI showed 3+ caspase-3 staining of myocytes in peri-infarct zones; no staining was seen away from infarcts. Myocyte staining in cases of DCM and ASHD ranged from 0-1+ only (p=O.003 vs. MI by Fisher's exact test). Numerous myocyte nuclei in the areas of infarcfion were labeled by TUNEL assay, but no caspase-3 positive myocytes were identified as TUNEL positive in sequential sections. All control samples were negative for myocyte caspase-3 staining; 1 of 4 control samples showed myocyte TUNEL positivity. Conclusions: Caspase3 is strongly induced in myocytee neighboring recent infarcts, but is induced to lesser extent in DCM and ASHD. Myocyte caspase-3 expression does not necessadly indicate cell death, but caspase-3 mediated apoptosis could contdbute to infarct extension.
Sorin Musat-Marcu, Darryl O'Brien, Yi Xu, Bodh I. Jugdu~t, University of Alberta, Edmonton, Alberta, C A N A D A
Estrogen Receptor Mutations are not Associated in Women with
Primary pulmonary hypertension (PPH) is a devastating disorder of unknown cause that affects predominantly young premenopausal women. Abnormalities of the estrogen receptor (ER) have been found to be important in other homaonally mediated vascular disorders such as accelerated coronary disease. We hypothesized that ER mutations may affect receptor function and increase the risk of PPH and evaluated this through a case control study. DNA was isolated from peripheral blood in 26 women with non-familial PPH and 40 control women with no history of cardiac or pulmonary disease. Primers which bracketed the eight exnns of the coding region were utilized for PCR. Using primers end labeled with P32, singlestranded contbrmation pnlymorphism (SSCP) analysis was then utilized to identify mutations by differential migration and subsequent sequencing. Mutations were identified in exon 3 only and in each case represented an C to T transition in codon 243, which does not change the amino acid sequence. The frequency of this mutation did not differ in patients with PPH compared to controls (see table below). Conclusion: Although mutations of the estrogen receptor are seen in a minority of women with PPH, these mutations are functionally silent and are seen in an equal frequency in a control population. Though hormonal factors may still be important in the pathogenesis of PPH, mutations of the coding region of the ER do not appear to play a role. PPH
Exon 3 Mutation Wild Type
3(12%) 23 (88%)
Controls 3 (8%) 37 (92%)