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Changes in protein kinase C activity and isozyme contents in cardiac hypertrophy due to volume overload in rats
24
Journal of Cardiac Failure Vol. 5 No. 3 Suppl. 1 1999
080
081
Activation of Selective Caspases during Apoptosis in Rat Neonatal Cardiomyocytes Jian Wang, Xiying Wu; Cardiovascular Research Division, Lilly Research Labs, Indianapolis, IN
Changes in Protein Kinase C Activity and Isozyme Contents in Cardiac Hypertrophy Due to Volume Overload in Rats Emmanuelle Sentex, Xi Wang, Xueliang Liu, Naranjan S. Dhalla; Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, & Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, MB, Canada
Caspases have been implicated as key mediators of programmed cell death in a variety of mammalian cell types. Caspase 3 has been shown to be activated during Stanrosporine-induced apoptosis in cardiomyocytes (Yue et al., J. Mol Cell Cardiol 30, 495 (1998)). Here we demonstrate that a selective set of caspase are activated during apoptosis in rat neonatal cardiomyocytes that were treated with either Staurosporine or Puromycin. A substantial high level of caspase 3 activity (DEVD-AFC) was detected in cardiomyocytes that were treated with Staurosporthe or Puromycin. This activity is not detected in untreated cardiomyocytes. Cardiomyocyte apoptosis also results in the activation of caspase 2 (VDVADAFC) and caspase 6 (VEID-AMC), although to a less extend than caspase 3. There was also a minimal but detectable levels of caspase 8 (LIETD-AFC) and caspase 9 (LEHD-AFC) activities in cardiomyocytes treated with Staurosporine and Puromyciu. However, A caspase activity specific for caspase 1 & 4 (YVADAFC) was not detected in these cells. We also investigated the ability of substrate-based caspase inhibitors to inhibit cardiomyocyte apoptosis. At 50uM concentration, inhibitors specific for caspase 3, 2 and 6, as well as a pancaspase inhibitor protected cardiomyocytes from Staurosporine and Puromycin-induced apoptosis. In contrast, caspase inhibitor specific for caspase 1, 4 and 9 are not able to inhibit cardiomyocyte apoptosis. These data suggest that a selective set of caspases are involved in cardiomyocytes apoptosis.
Although protein kinase C (PKC) is largely implicated in modulating cell growth in cardiac hypertrophy, no data are available about its changes in volumeoverload induced heart hypertrophy. The present study evaluated alterations in the PKC activity in the left and right ventricles (LV, RV) at 2 and 4 weeks (w) after the induction of an aorto-caval shunt (AVS). The PKC Ca2+-dependent and Ca2+-independent activities in the homogenate, particulate and cytosolic fractions were determined by measuring the incorporation of 32p in specific substrates. PKC ~-, ]3-, e-, 8- and ~-isozymes were detected by using specific antibodies. The LV wt was increased by 41 and 45% whereas the RV wt was increased by 65 and 70% at 2w and 4w in the AVS group compared to the control, respectively. The PKC Caa+-dependent and PKC Ca2+-independent activities in the LV at 2 and 4w were increased in both the homogenate and particulate fractions but not in the cytosolic fraction. On the other band, the PKC Ca2+dependent and PKC Ca2+-independent activities did not change in the homogenate, particulate and cytosolic fractions of 2 or 4w hypertrophied RV. The Western blots of PKC isozymes in the 2 and 4w hypertrophied LV unlike the RV, showed an increased content of a, 3, and ~ by 25, 10 and 12% respectively. These results suggest differential changes of PKC enzyme in the LV and RV hypertrophy due to volume overload. (Supported by MRC Group in Experimental Cardiology).
082
083
Engrafted Autologous Skeletal Myoblasts Improve Myocardial Performance in Severely Infarcted Rabbit Myocardium B. Zane Atkins, Kelley A. Hutcheson, Matthew T. Hueman, Malsha J. Cottman, Jennifer M. Meuchel, Donald D. Glower, Doris A. Taylor ,; Surgery, Medicine, Biomedical Engineering, Duke University Medical Center, Durham, NC
Gender-Related Differences in Proliferative Response of Cardiac Fibroblasts to Hypoxia: Effect of Estrogen Michael Griffin, Hyeon Lee, John Zhang, Leo Zhao, Mahhoubeh Eghbali-Webb; Yale School of Medicine, New Haven, CT
Myogenic cells have been successfully transplanted into cryoinjured heart by way of cellular cardiomyoplasty (CCM). Yet, the effects of CCM on regional myocardial performance in the setting of ischemic injury, infarct, and failure are unknown. In this study, in vivo regional systolic and diastolic performance was assessed after myocardial infarction (MI) secondary to circumflex marginal coronary ligation and subsequently following intramyocardial injection of autologous skeletal myoblasts (CCM). In 6 rabbits, LV pressure and segment length (SL) were determined over varied preloads following MI and 3 weeks after CCM and compared with control (n=4). Systolic performance was based on the linear relationship between regional stroke work (SW) and end-diastolic (ED) SL. Estimated SW at an extrapolated EDSL (SWEDL) was determined to compare regional contractility. Diastolic performance was assessed by the curvilinear relationship between LVEDP and strain (8). To compare performance, estimated e at standardized LVEDP of 8 mmHg (Ss) was determined. Four of six CCM animals exhibited significantly increased SWEDL (p=0.024) and Ss (P=0.049) demonstrating improved contractility and compliance. In contrast, four control animals showed significant worsening of SWEDL and ~s.
SWEDL (Kerg)-MI 95+2 47±0.1 137-+2 I76±4 186+1 97+3 *p<0.05
SWEDL (Kerg)-CCM
es-MI
es-CCM
124+1* 209±3* 198±4* 417±2" 121-+2 159-+8
0~126 0.136 0.128 0.104 0.082
0.353* 0.645* 0.305* 0.106 0.032
These data suggest that CCM improves regional systolic and diastolic performance in dysfunctional myocardium after coronary ligation. Thus, CCM could be viewed as an alternative intervention after myocardial infarction to prevent the progression to heart failure or potentially as a clinical adjunct to surgical revascularlzation.
Gender-related differences in predisposition to and survival from heart failure are well established. Proliferation of cardiac fibroblasts, the source of matrix proteins, cytokines and growth factors in the heart, is the key event in myocardial remodeling in ischemic heart disease. We tested the hypothesis that male and female cardiac fibroblasts respond differently to hypoxia, a condition of ischemia and examined the proIiferative response to hypoxia (2% oxygen). Fibroblasts were prepared from ventricular tissue of adult age-matched male and female rats. DNA synthesis, as determined by measurement of 3H-thymidine incorporation, decreased under hypoxia in male cardiac fibroblasts, while remaining unchanged in female cells. Consistently, the expression of proliferating cell nuclear antigen (PCNA) reduced in male and increased in female cells under hypoxia. Western analysis with anti-phosphotyrosine antibody showed a different pattern of protein phosphorylation in male and female cells. Estrogen (17g-estradiol, 10 nM) increased DNA synthesis in female cells under normoxia but not hypoxia. Estrogen inhibited DNA synthesis under normoxia and partly reversed the inhibitory effect of hypoxia in male cells. Analysis of total RNA by RT-PCR showed differential hypoxia-induced regulation in estrogen receptor g mRNA, while a receptor mRNA remained unchanged. Western analysis of cell lysate showed coordinate regulation of estrogen receptor g protein in male and female cells under hypoxia. Gel shift analysis of nuclear and cytoplasmic extract with a 32p-Iabelled oligorlucleotide containing the estrogen response element (ERE) showed ERE-binding proteins predominantly in cytoplasmic extract in both genders. Under hypoxia, the ERE-binding increased in male cells but remained unchanged in female cells. These results show differences in proliferative responses of male and female cardiac fibroblasts under hypoxia and provide evidence for a regulatory role of estrogen which may contribute significantly to gender-related differences in heart failure.
Journal of Cardiac Failure Vol. 5 No. 3 Suppl. 1 1999
080
081
Activation of Selective Caspases during Apoptosis in Rat Neonatal Cardiomyocytes Jian Wang, Xiying Wu; Cardiovascular Research Division, Lilly Research Labs, Indianapolis, IN
Changes in Protein Kinase C Activity and Isozyme Contents in Cardiac Hypertrophy Due to Volume Overload in Rats Emmanuelle Sentex, Xi Wang, Xueliang Liu, Naranjan S. Dhalla; Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, & Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, MB, Canada
Caspases have been implicated as key mediators of programmed cell death in a variety of mammalian cell types. Caspase 3 has been shown to be activated during Stanrosporine-induced apoptosis in cardiomyocytes (Yue et al., J. Mol Cell Cardiol 30, 495 (1998)). Here we demonstrate that a selective set of caspase are activated during apoptosis in rat neonatal cardiomyocytes that were treated with either Staurosporine or Puromycin. A substantial high level of caspase 3 activity (DEVD-AFC) was detected in cardiomyocytes that were treated with Staurosporthe or Puromycin. This activity is not detected in untreated cardiomyocytes. Cardiomyocyte apoptosis also results in the activation of caspase 2 (VDVADAFC) and caspase 6 (VEID-AMC), although to a less extend than caspase 3. There was also a minimal but detectable levels of caspase 8 (LIETD-AFC) and caspase 9 (LEHD-AFC) activities in cardiomyocytes treated with Staurosporine and Puromyciu. However, A caspase activity specific for caspase 1 & 4 (YVADAFC) was not detected in these cells. We also investigated the ability of substrate-based caspase inhibitors to inhibit cardiomyocyte apoptosis. At 50uM concentration, inhibitors specific for caspase 3, 2 and 6, as well as a pancaspase inhibitor protected cardiomyocytes from Staurosporine and Puromycin-induced apoptosis. In contrast, caspase inhibitor specific for caspase 1, 4 and 9 are not able to inhibit cardiomyocyte apoptosis. These data suggest that a selective set of caspases are involved in cardiomyocytes apoptosis.
Although protein kinase C (PKC) is largely implicated in modulating cell growth in cardiac hypertrophy, no data are available about its changes in volumeoverload induced heart hypertrophy. The present study evaluated alterations in the PKC activity in the left and right ventricles (LV, RV) at 2 and 4 weeks (w) after the induction of an aorto-caval shunt (AVS). The PKC Ca2+-dependent and Ca2+-independent activities in the homogenate, particulate and cytosolic fractions were determined by measuring the incorporation of 32p in specific substrates. PKC ~-, ]3-, e-, 8- and ~-isozymes were detected by using specific antibodies. The LV wt was increased by 41 and 45% whereas the RV wt was increased by 65 and 70% at 2w and 4w in the AVS group compared to the control, respectively. The PKC Caa+-dependent and PKC Ca2+-independent activities in the LV at 2 and 4w were increased in both the homogenate and particulate fractions but not in the cytosolic fraction. On the other band, the PKC Ca2+dependent and PKC Ca2+-independent activities did not change in the homogenate, particulate and cytosolic fractions of 2 or 4w hypertrophied RV. The Western blots of PKC isozymes in the 2 and 4w hypertrophied LV unlike the RV, showed an increased content of a, 3, and ~ by 25, 10 and 12% respectively. These results suggest differential changes of PKC enzyme in the LV and RV hypertrophy due to volume overload. (Supported by MRC Group in Experimental Cardiology).
082
083
Engrafted Autologous Skeletal Myoblasts Improve Myocardial Performance in Severely Infarcted Rabbit Myocardium B. Zane Atkins, Kelley A. Hutcheson, Matthew T. Hueman, Malsha J. Cottman, Jennifer M. Meuchel, Donald D. Glower, Doris A. Taylor ,; Surgery, Medicine, Biomedical Engineering, Duke University Medical Center, Durham, NC
Gender-Related Differences in Proliferative Response of Cardiac Fibroblasts to Hypoxia: Effect of Estrogen Michael Griffin, Hyeon Lee, John Zhang, Leo Zhao, Mahhoubeh Eghbali-Webb; Yale School of Medicine, New Haven, CT
Myogenic cells have been successfully transplanted into cryoinjured heart by way of cellular cardiomyoplasty (CCM). Yet, the effects of CCM on regional myocardial performance in the setting of ischemic injury, infarct, and failure are unknown. In this study, in vivo regional systolic and diastolic performance was assessed after myocardial infarction (MI) secondary to circumflex marginal coronary ligation and subsequently following intramyocardial injection of autologous skeletal myoblasts (CCM). In 6 rabbits, LV pressure and segment length (SL) were determined over varied preloads following MI and 3 weeks after CCM and compared with control (n=4). Systolic performance was based on the linear relationship between regional stroke work (SW) and end-diastolic (ED) SL. Estimated SW at an extrapolated EDSL (SWEDL) was determined to compare regional contractility. Diastolic performance was assessed by the curvilinear relationship between LVEDP and strain (8). To compare performance, estimated e at standardized LVEDP of 8 mmHg (Ss) was determined. Four of six CCM animals exhibited significantly increased SWEDL (p=0.024) and Ss (P=0.049) demonstrating improved contractility and compliance. In contrast, four control animals showed significant worsening of SWEDL and ~s.
SWEDL (Kerg)-MI 95+2 47±0.1 137-+2 I76±4 186+1 97+3 *p<0.05
SWEDL (Kerg)-CCM
es-MI
es-CCM
124+1* 209±3* 198±4* 417±2" 121-+2 159-+8
0~126 0.136 0.128 0.104 0.082
0.353* 0.645* 0.305* 0.106 0.032
These data suggest that CCM improves regional systolic and diastolic performance in dysfunctional myocardium after coronary ligation. Thus, CCM could be viewed as an alternative intervention after myocardial infarction to prevent the progression to heart failure or potentially as a clinical adjunct to surgical revascularlzation.
Gender-related differences in predisposition to and survival from heart failure are well established. Proliferation of cardiac fibroblasts, the source of matrix proteins, cytokines and growth factors in the heart, is the key event in myocardial remodeling in ischemic heart disease. We tested the hypothesis that male and female cardiac fibroblasts respond differently to hypoxia, a condition of ischemia and examined the proIiferative response to hypoxia (2% oxygen). Fibroblasts were prepared from ventricular tissue of adult age-matched male and female rats. DNA synthesis, as determined by measurement of 3H-thymidine incorporation, decreased under hypoxia in male cardiac fibroblasts, while remaining unchanged in female cells. Consistently, the expression of proliferating cell nuclear antigen (PCNA) reduced in male and increased in female cells under hypoxia. Western analysis with anti-phosphotyrosine antibody showed a different pattern of protein phosphorylation in male and female cells. Estrogen (17g-estradiol, 10 nM) increased DNA synthesis in female cells under normoxia but not hypoxia. Estrogen inhibited DNA synthesis under normoxia and partly reversed the inhibitory effect of hypoxia in male cells. Analysis of total RNA by RT-PCR showed differential hypoxia-induced regulation in estrogen receptor g mRNA, while a receptor mRNA remained unchanged. Western analysis of cell lysate showed coordinate regulation of estrogen receptor g protein in male and female cells under hypoxia. Gel shift analysis of nuclear and cytoplasmic extract with a 32p-Iabelled oligorlucleotide containing the estrogen response element (ERE) showed ERE-binding proteins predominantly in cytoplasmic extract in both genders. Under hypoxia, the ERE-binding increased in male cells but remained unchanged in female cells. These results show differences in proliferative responses of male and female cardiac fibroblasts under hypoxia and provide evidence for a regulatory role of estrogen which may contribute significantly to gender-related differences in heart failure.