- Email: [email protected]
Differential activation of mitogen-activated protein kinase cascades by protein kinase C (PKC) delta and epsilon
in prior TNF- (r Expression Precedes Expressions for Matrix Metalloproteinase (MMP) and Tissue Inhibitor of MMP in Infarcted Tomoji Hata, Naoki Mekino, Myocardium
A Na+iCaz’ EXCHANGE INHIBITOR, KBR7943, ON DIGITALIS AND ISCHEMIAREPERFUSION ARRHYTHMIA MODELS Keitaro Kazunorl Medical
Hashimoto, Kamlya. University,
Shigeki Miyamoto, Sing-Mel Zhu & Dept of Pharmacology, Yamanashi Yamanashi 4093998, JAPAN.
Sugano. Inst.
Dep. of Bioclimatol~y
of Bioreguiatfon,
Kyushu
& Medicine, Univ.
,BEPPU,
to
Masahiro, Medical
JAPAN
Na+/H+ exchange inhibitors have been shown to suppress ischemia-reperfusion arrhythmias, probably by decreasing Na’ accumulation in the cell, resulting in the inhibition of Ca2+ influx via the Na+/Ca’+ exchanger. KB-R7943 (KB-R) has been used as a relatively selective pharmacological tool to block the CaZ+ influx-mode (reverse-mode) of the Na+/Ca’+ exchanger. We examined whether direct Na+/Ca”+ exchange inhibition is also effective on ischemiain beagles and digitiaiis reperfusion arrhythmias arrhythmias in beagles and guinea-pigs. Lead II ECG and blood pressure (BP) were measured. KB-R or the solvent (10% DMSO) was injected i.v. as a bolus, and 5 min later, the left anterior descending coronary artery was occluded for 30 min followed by reperfusion. Five and 10 mglkg KBR increased BP significantly without changing ECG parameters including the heart rate. Mortality due to ischemia-reperfusion was not suppressed by KB-R (2-10 mg/kg). Five mg/kg KB-R also did not suppress the ouabain-induced arrhythmias in dogs. In guinea-pigs, 5 mglkg KB-R7943 also did not suppress the appearance of and death induced by ouabain, but these were suppressed by 5 mg/kg pilsicainide, a Na+ channel blocking antiarrhythmic drug. These negative results suggest that KB-R7943 may not be a selective Na+/Ca2+ exchange inhibitor and/or may have multiple effects on the heart.
It is not fully known that whether tissue TNF-a is associated with expressions for MMPs and TiMPs of infarkted myocardium in animal model. Myocardiai infarction (MI) was induced by surgical occlusion of the left main coronary artery in rats. Collagen content in infarct zone increased four folds at the first week, while in the non-infarct zone this content increased two folds at the first week. Using RT-PCR, we determined that MMP-9 mRNA was expressed from day 1 to day 7. The mRNA levels for MMP-13 and MMPQ were both enhanced from day 7 to day 14 and thereafter decreased at day 28. MMPB and MMP-14 mRNA expressions were expressed at day 14 and 28 after MI. The levels of TIMP-1 and TIMP-2 mRNA expression were elevated within 1 week and thereafter reduced. TIMP-3 mRNA expression was greater from day 14. TNF-a mRNA in the infarct zone increased from day 2 to day 7 and those levels were still elevated by day 28. The maximal TNF-a protein levels (7-fold increase) Thus, MMP-9 (neutrophii were on day 7 in the infarct zone. geiatinase) was co-expressed with TNF-a at an early phase after Ml. On the other hand, other MMPs and TiMPs were more expressed after one week. We also made an immunohistochemicai study of protein expressions in the infarct zone. Our results suggest that MMP-9 may affect ventricular remodeling through TNF-a expression, which in turn may lead to enhanced expression of other MMPs.
INCREASED DOES NOT SIMULATED CULTURED MYOCYTES.
DiFFERENTiAL ACTIVATION OF YITOGENACTIVATED PROTEIN KINASE CASCADES BY PROTEIN KINASE C (PKC) DELTA AND EPSILON Maria C. Heidkamp, Allison L. Bayer, Jody L. Martin, and Allen M. Samarel, The Cardiovascular institute, Loyola University Medical Center, Maywood, IL USA
EXPRESSION OF PKC-E PROTECT AGAINST ISCHEMIAlREPERFUSION NEONATAL RAT
INJURY VENTRICULAR
IN
Richard S. Vander Heide, Dept. of Pathology, Wayne State University, Detroit, MI, USA Ischemic preconditioning (IP) is a potent endogenous cardioprotective effect against irreversible injury. Current evidence suggests that activation protein kinase C is important in mediating IP. To determine if increased expression of PKC-E is cardioprotective, we subjected neonata1 rat ventricular myocytes expressing increased PKCE levels to simulated ischemitireperfusion (IR) injury. Methods: Neonatal rat ventricular myocytes were isolated using standard techniques. Following isolation, approximately 2-3 x lo6 myocytes were plated in each well of a standard 6 well plate. After 24 hours, cells were infected with recombinant adenovirus expressing native PKC-E, a dominant negative form of PKC-E, and empty adenovirus. After two days, cells were exposed to IR by incubation with buffer containina 3mM iodoacetic acid and 3 mM amvtai for 2 hours followid by reperfusion in hypotonic --s;a&.rd buffer. Cell viability was assayed by measuring lactate dehydrogenase release. m: The -viability of- infected cells did not differ from control cells. Increased expression of both native and dominant negative forms of PKC-E was present in infected cells (avg. 2-3 fold vs. control at 3 days; n=5). Infection with empty virus (control) did not increase PKC-E expression. IR resulted in 50% injury to cells expressing increased native PKC-E, which did not differ from other groups or control. IR did induce translocation of PKCE in all groups. We conclude that following overexpression, IR results in translocation of PKC-E from the cytosol to membrane fraction but does not result in cardioprotection in neonatal rat myocytes.
We and others have demonstrated that endothelin-induced PKCS and PKCE translocation is accompanied bX subsequent activation of the ERK, JNK and ~38~~~ cascades. However, it is currently not known if either or both PKC’s are necessary for their downstream activation. Use of PKC inhibitors to answer this auestion is comolicated bv a lack of isoenzyme specificity, and the fact that many $KC inhibitors stimulate basal JNK and ~138~‘~~ activitv. Therefore, replicationdefective adenovirus& (Adv) encodin$ constitutively active (ca) mutants of PKCG and PKCE were used to examine which, if either of these novel PKC isoenzymes, activate ERKs, JNKs and/or ~38~~~~ in neonatal rat ventricular myocytes. Adv-caPKCG infection (l-25 moi, 448h) increased total PKCG levels in a dose-dependent manner, with maximal expression observed 24h after Adv infection. Adv-caPKCS (5moi; 24h) induced an 18- and 29fold increase in pJNK1 and pJNK2 levels, respecbvely, as compared to a control Adv encoding P-galactosidase iAdvpgal; 5moi; 24h). ~38”~~ was also si nificantly activated (15-fold increase in phosphorylated ~38%“~~ over Adv-pgal infected cells). In contrast, there was no significant increase in pERKI or pERK2. Adv-CaPKCs infection (l-25 moi; 4-48h) increased total PKCE levels in a similar fashion, and induced a 4-fold increase in fERK2, as compared to Adv-pgal (5mor, 8h). JNK and ~38~ PK were only minimally activated. We conclude that the novel HPKCs differentially regulate the MAPK cascades, with isoenzyme specific, and timedependent activation.
A45
A Na+iCaz’ EXCHANGE INHIBITOR, KBR7943, ON DIGITALIS AND ISCHEMIAREPERFUSION ARRHYTHMIA MODELS Keitaro Kazunorl Medical
Hashimoto, Kamlya. University,
Shigeki Miyamoto, Sing-Mel Zhu & Dept of Pharmacology, Yamanashi Yamanashi 4093998, JAPAN.
Sugano. Inst.
Dep. of Bioclimatol~y
of Bioreguiatfon,
Kyushu
& Medicine, Univ.
,BEPPU,
to
Masahiro, Medical
JAPAN
Na+/H+ exchange inhibitors have been shown to suppress ischemia-reperfusion arrhythmias, probably by decreasing Na’ accumulation in the cell, resulting in the inhibition of Ca2+ influx via the Na+/Ca’+ exchanger. KB-R7943 (KB-R) has been used as a relatively selective pharmacological tool to block the CaZ+ influx-mode (reverse-mode) of the Na+/Ca’+ exchanger. We examined whether direct Na+/Ca”+ exchange inhibition is also effective on ischemiain beagles and digitiaiis reperfusion arrhythmias arrhythmias in beagles and guinea-pigs. Lead II ECG and blood pressure (BP) were measured. KB-R or the solvent (10% DMSO) was injected i.v. as a bolus, and 5 min later, the left anterior descending coronary artery was occluded for 30 min followed by reperfusion. Five and 10 mglkg KBR increased BP significantly without changing ECG parameters including the heart rate. Mortality due to ischemia-reperfusion was not suppressed by KB-R (2-10 mg/kg). Five mg/kg KB-R also did not suppress the ouabain-induced arrhythmias in dogs. In guinea-pigs, 5 mglkg KB-R7943 also did not suppress the appearance of and death induced by ouabain, but these were suppressed by 5 mg/kg pilsicainide, a Na+ channel blocking antiarrhythmic drug. These negative results suggest that KB-R7943 may not be a selective Na+/Ca2+ exchange inhibitor and/or may have multiple effects on the heart.
It is not fully known that whether tissue TNF-a is associated with expressions for MMPs and TiMPs of infarkted myocardium in animal model. Myocardiai infarction (MI) was induced by surgical occlusion of the left main coronary artery in rats. Collagen content in infarct zone increased four folds at the first week, while in the non-infarct zone this content increased two folds at the first week. Using RT-PCR, we determined that MMP-9 mRNA was expressed from day 1 to day 7. The mRNA levels for MMP-13 and MMPQ were both enhanced from day 7 to day 14 and thereafter decreased at day 28. MMPB and MMP-14 mRNA expressions were expressed at day 14 and 28 after MI. The levels of TIMP-1 and TIMP-2 mRNA expression were elevated within 1 week and thereafter reduced. TIMP-3 mRNA expression was greater from day 14. TNF-a mRNA in the infarct zone increased from day 2 to day 7 and those levels were still elevated by day 28. The maximal TNF-a protein levels (7-fold increase) Thus, MMP-9 (neutrophii were on day 7 in the infarct zone. geiatinase) was co-expressed with TNF-a at an early phase after Ml. On the other hand, other MMPs and TiMPs were more expressed after one week. We also made an immunohistochemicai study of protein expressions in the infarct zone. Our results suggest that MMP-9 may affect ventricular remodeling through TNF-a expression, which in turn may lead to enhanced expression of other MMPs.
INCREASED DOES NOT SIMULATED CULTURED MYOCYTES.
DiFFERENTiAL ACTIVATION OF YITOGENACTIVATED PROTEIN KINASE CASCADES BY PROTEIN KINASE C (PKC) DELTA AND EPSILON Maria C. Heidkamp, Allison L. Bayer, Jody L. Martin, and Allen M. Samarel, The Cardiovascular institute, Loyola University Medical Center, Maywood, IL USA
EXPRESSION OF PKC-E PROTECT AGAINST ISCHEMIAlREPERFUSION NEONATAL RAT
INJURY VENTRICULAR
IN
Richard S. Vander Heide, Dept. of Pathology, Wayne State University, Detroit, MI, USA Ischemic preconditioning (IP) is a potent endogenous cardioprotective effect against irreversible injury. Current evidence suggests that activation protein kinase C is important in mediating IP. To determine if increased expression of PKC-E is cardioprotective, we subjected neonata1 rat ventricular myocytes expressing increased PKCE levels to simulated ischemitireperfusion (IR) injury. Methods: Neonatal rat ventricular myocytes were isolated using standard techniques. Following isolation, approximately 2-3 x lo6 myocytes were plated in each well of a standard 6 well plate. After 24 hours, cells were infected with recombinant adenovirus expressing native PKC-E, a dominant negative form of PKC-E, and empty adenovirus. After two days, cells were exposed to IR by incubation with buffer containina 3mM iodoacetic acid and 3 mM amvtai for 2 hours followid by reperfusion in hypotonic --s;a&.rd buffer. Cell viability was assayed by measuring lactate dehydrogenase release. m: The -viability of- infected cells did not differ from control cells. Increased expression of both native and dominant negative forms of PKC-E was present in infected cells (avg. 2-3 fold vs. control at 3 days; n=5). Infection with empty virus (control) did not increase PKC-E expression. IR resulted in 50% injury to cells expressing increased native PKC-E, which did not differ from other groups or control. IR did induce translocation of PKCE in all groups. We conclude that following overexpression, IR results in translocation of PKC-E from the cytosol to membrane fraction but does not result in cardioprotection in neonatal rat myocytes.
We and others have demonstrated that endothelin-induced PKCS and PKCE translocation is accompanied bX subsequent activation of the ERK, JNK and ~38~~~ cascades. However, it is currently not known if either or both PKC’s are necessary for their downstream activation. Use of PKC inhibitors to answer this auestion is comolicated bv a lack of isoenzyme specificity, and the fact that many $KC inhibitors stimulate basal JNK and ~138~‘~~ activitv. Therefore, replicationdefective adenovirus& (Adv) encodin$ constitutively active (ca) mutants of PKCG and PKCE were used to examine which, if either of these novel PKC isoenzymes, activate ERKs, JNKs and/or ~38~~~~ in neonatal rat ventricular myocytes. Adv-caPKCG infection (l-25 moi, 448h) increased total PKCG levels in a dose-dependent manner, with maximal expression observed 24h after Adv infection. Adv-caPKCS (5moi; 24h) induced an 18- and 29fold increase in pJNK1 and pJNK2 levels, respecbvely, as compared to a control Adv encoding P-galactosidase iAdvpgal; 5moi; 24h). ~38”~~ was also si nificantly activated (15-fold increase in phosphorylated ~38%“~~ over Adv-pgal infected cells). In contrast, there was no significant increase in pERKI or pERK2. Adv-CaPKCs infection (l-25 moi; 4-48h) increased total PKCE levels in a similar fashion, and induced a 4-fold increase in fERK2, as compared to Adv-pgal (5mor, 8h). JNK and ~38~ PK were only minimally activated. We conclude that the novel HPKCs differentially regulate the MAPK cascades, with isoenzyme specific, and timedependent activation.
A45