Caspase-dependent and serine protease-dependent DNA fragmentation of myocytes in the ischemia-reperfused rabbit heart: these inhibitors do not reduce infarct size.
Some infarcted myocytes undergo caspase-dependent DNA fragmentation, but serine protease-dependent DNA fragmentation may also be involved. There is controversy regarding whether caspase inhibitors can reduce infarct size, so the present study investigated whether serine protease inhibitor can reduce the DNA fragmentation of infarcted myocytes and whether serine protease or caspase inhibitors attenuates myocardial infarct size in Japanese white rabbits without collateral circulation. Rabbits were subjected to 30-min coronary occlusion followed by 48-h reperfusion. A vehicle (dimethylsulfoxide, control group, n=8) or Z-Val-Ala-Asp(Ome)-CH2F (ZVAD-fmk, a caspase inhibitor, ZVAD group, 0.8 mg/kg iv at 20 min before coronary occlusion and 0.8 mg/kg at 90 min after reperfusion, n=8) or 3,4-dichloroisocoumarin (DCI, a serine protease inhibitor, 2 mg/kg iv at 20 min before coronary occlusion, DCI group, n=8) was administered. Animals were killed at 48h after reperfusion for the detection of myocardial infarct size and at 4h after reperfusion for the detection of dUTP nick end-labeling (TUNEL)-positive myocytes, the electrophoretic pattern of DNA fragmentation and ultrastructural analysis. The left ventricle (LV) was excised and sliced. The myocardial infarct size as a percentage of the area at risk was assessed by triphenyltetrazolium chloride staining. DNA fragmentation was assessed by in situ TUNEL at the light microscopic level. ZVAD and DCI significantly reduced the mean blood pressure during reperfusion without affecting heart rate. There was no significant difference in the % area at risk (AAR) of LV among the 3 groups (control: 26.3+/-3.0%; ZVAD: 25.6+/-2.6%; DCI: 25.6+/-2.0%). The % infarct size as a percentage of the AAR in the ZVAD group (41.3+/-4.5%) and the DCI group (50.4+/-3.8%) was not significantly different from the control group (43.5+/-4.5%). However, the percent DNA fragmentation in the infarcted area in the ZVAD (3.5+/-0.8%) and DCI groups (4.2+/-0.9%) was significantly reduced compared with the control group (10.7+/-1.9%). The DNA ladder pattern observed in the control group was attenuated in both the ZVAD and DCI groups. There was no difference in electron microscopic changes among the 3 groups. Serine protease-dependent DNA fragmentation is present in infarcted myocytes, in addition to caspase-dependent DNA fragmentation, but an infarct-size reducing effect was not observed with either of these inhibitors.
Minatoguchi
S
S
Second Department of Internal Medicine, Gifu University School of Medicine, Japan.
Kariya
T
T
Uno
Y
Y
Arai
M
M
Nishida
Y
Y
Hashimoto
K
K
Wang
N
N
Aoyama
T
T
Takemura
G
G
Fujiwara
T
T
Fujiwara
H
H
D000590:Amino Acid Chloromethyl Ketones / Q000008:administration & dosage* / Q000494:pharmacology; D000818:Animals; D061945:Caspase Inhibitors; D020169:Caspases / Q000502:physiology; D053938:DNA Fragmentation / Q000187:drug effects* / Q000502:physiology; D004195:Disease Models, Animal; D004791:Enzyme Inhibitors / Q000008:administration & dosage / Q000494:pharmacology; D020287:In Situ Nick-End Labeling; D008297:Male; D008854:Microscopy, Electron; D009203:Myocardial Infarction / Q000188:drug therapy / Q000473:pathology / Q000628:therapy; D017202:Myocardial Ischemia / Q000188:drug therapy* / Q000473:pathology* / Q000628:therapy; D015425:Myocardial Reperfusion; D009206:Myocardium / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D011817:Rabbits; D015842:Serine Proteinase Inhibitors / Q000008:administration & dosage* / Q000494:pharmacology
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D015842:Serine Proteinase Inhibitors; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D020169:Caspases