title: Staurosporine-induced apoptosis in cardiomyocytes: A potential role of caspase-3.
abstract: Cardiomyocyte apoptosis has been demonstrated in animal models of cardiac injury as well as in patients with congestive heart failure or acute myocardial infarction. Therefore, apoptosis has been proposed as an important process in cardiac remodeling and progression of myocardial dysfunction. However, the mechanisms underlying cardiac apoptosis are poorly understood. The present study was designed to determine whether the family of caspase proteases and stress-activated protein kinase (SAPK/JNK) are involved in cardiac apoptosis. Cultured rat neonatal cardiac myocytes were treated with staurosporine to induce apoptosis as evidenced by the morphological (including ultrastructural) characteristics of cell shrinkage, cytoplasmic and nuclear condensation, and fragmentation. Nucleosomal DNA fragmentation in myocytes was further identified by agarose gel electrophoresis (DNA ladder) as well as in situ nick end-labeling (TUNEL). Staurosporine-induced apoptosis in myocytes was a time- and concentration-(0.25-1 micro M)-dependent process. Staurosporine-induced apoptosis in myocytes was reduced by a cell-permeable, irreversible tripeptide inhibitor of caspases, ZVAD-fmk, but not by the ICE-specific inhibitor, Ac-YVAD-CHO. At 10, 50 and 100 muM of ZVAD-fmk, staurosporine-induced myocyte apoptosis was reduced by 5.8, 39.1 (P<0.01) and 53.8% (P<0.01), respectively. Staurosporine, at 0.25-1 micro M, increased caspase activity in cardiomyocytes by five- to eight-fold, peaking at 4-8 h after stimulation. Based on substrate specificity analysis, the major component of caspases activated in myocytes was consistent with caspase-3 (CPP32). Moreover, the appearance of the 17-kD subunit of active caspase-3 in staurosporine-treated myocytes was demonstrated by immunocytochemical analysis. In contrast, staurosporine induced a rapid and transient inhibition of SAPK/JNK in myocytes. The SAPK activity in myocytes was reduced by 68.3 and 58.3% (P<0.01 v basal) at 10 and 30 min after treatment with 1 micro M of staurosporine, respectively. Our results suggest that staurosporine-induced cardiac myocyte apoptosis involves activation of caspases, mainly caspase-3, but not activation of the SAPK signaling pathway.
authors.affiliation: Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406-0939, USA.
authors.forename: T L
authors.initials: TL
authors.lastname: Yue
authors.forename: C
authors.initials: C
authors.lastname: Wang
authors.forename: A M
authors.initials: AM
authors.lastname: Romanic
authors.forename: K
authors.initials: K
authors.lastname: Kikly
authors.forename: P
authors.initials: P
authors.lastname: Keller
authors.forename: W E
authors.initials: WE
authors.lastname: DeWolf
authors.forename: T K
authors.initials: TK
authors.lastname: Hart
authors.forename: H C
authors.initials: HC
authors.lastname: Thomas
authors.forename: B
authors.initials: B
authors.lastname: Storer
authors.forename: J L
authors.initials: JL
authors.lastname: Gu
authors.forename: X
authors.initials: X
authors.lastname: Wang
authors.forename: G Z
authors.initials: GZ
authors.lastname: Feuerstein
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006321:Heart / Q000187:drug effects; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D009206:Myocardium / Q000166:cytology* / Q000201:enzymology*; D009842:Oligopeptides / Q000737:chemistry; D051381:Rats; D015398:Signal Transduction; D019311:Staurosporine / Q000494:pharmacology*; D013379:Substrate Specificity
chemical_list: D009842:Oligopeptides; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D019311:Staurosporine