Inhibition of caspase-3 improves contractile recovery of stunned myocardium, independent of apoptosis-inhibitory effects
Hartmut Ruetten, MD, PhDa,
Cornel Badorff, MDb,
Christian Ihling, MDc,
Andreas M. Zeiher, MDb and
Stefanie Dimmeler, PhDb,*
a Aventis Pharmaceuticals, Frankfurt, Germany
b Molecular Cardiology, Department of Medicine IV, Goethe-University, Frankfurt, Germany
c Department of Pathology, University of Freiburg, Freiburg, Germany
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Figure 1 The caspase-3 inhibitor Ac-DEVD-CHO improves contractile recovery of isolated working hearts of rats exposed to ischemia and reperfusion. A, Schematic diagram of the experimental set-up. B, Effects of the caspase-3 inhibitor Ac-DEVD-CHO (0.1 to 1 µmol/liter) or vehicle (0.01% DMSO) on cardiac output (CO), external heart power per gram of LV wet weight (EHPLV), left ventricular developing pressure (LVDP) and contractility (dP/dtmax). Data are presented as the mean value ± SEM; there are 8 to 10 animals per group. *p < 0.05 versus vehicle-treated group.
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Figure 2 Ac-DEVD-CHO is effective when given 5 min before reperfusion. The effect of Ac-DEVD-CHO on contractile recovery is shown, calculated as percent recovery compared with baseline (pre-ischemic) values of left ventricular developing pressure (LVDP), contractility (dp/dtmax), aortic flow (AF), coronary flow (CF), cardiac output (CO) and external heart power (EHP). Data are presented as the mean value ± SEM; there are 8 to 10 animals in each group. Ac-DEVD-CHO was started 15 min before ischemia (top) or 5 min before reperfusion (bottom). Note the similar beneficial effect of Ac-DEVD-CHO on the relative recovery. *p < 0.05 versus vehicle-treated group. Open bars = 0.01% DMSO; solid bars = 1 µmol/l of AC-DEVD-CHO.
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Figure 3 Ac-DEVD-CHO blocks ischemia/reperfusion (I/R)-induced caspase-3 activation but not apoptosis. A (left), Immunoblotting of rat hearts with an antibody against the p17 subunit of caspase-3; three independent experiments are shown. Arrowheads indicate the molecular weight of the inactive zymogen (32 kD) and the processed, active subunit (17 kD). A (right), Caspase-3–like proteolytic activity of rat hearts (percent increase relative to the proteolytic activity in control hearts), as measured with a fluorogenic substrate of caspase-3. *p < 0.05. B (left), Representative photomicrographs of myocardial sections stained with the TUNEL technique to detect apoptotic cells in situ in a nonischemic control heart (top), a vehicle-treated heart subjected to ischemia/reperfusion (middle) or an Ac-DEVD-CHO-treated heart subjected to ischemia/reperfusion (bottom). Arrows indicate TUNEL-positive cells. Magnification 200x, before XX% reduction. (B, right) Autoradiogram of radioactively labeled DNA strand breaks, followed by agarose gel electrophoresis; results from four independent samples in each group. Equal loading was verified by ethidium bromide staining. Note the internucleosomal DNA fragmentation ("ladder pattern") induced by ischemia/reperfusion.
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Figure 4 Effects of DEVD-CHO on troponin I degradation and calpain activity. (A) Immunoblots of rat hearts with an antibody against troponin I (upper blots) or actin (lower blots). Arrowheads indicate the molecular weight. The top right arrow indicates full-length troponin I; the lower arrow indicates the major degradation product. (B) Effects of 0.1 µmol/l of calpain inhibitor I (left panel) or Ac-DEVD-CHO (right panel) on the catalytic activity of 5 U of calpain I, as measured in vitro with a fluorogenic calpain I substrate. (C) Quantitative evaluation of the percent activity after addition of the inhibitor, compared with the activity measured before addition of the inhibitor. Data are presented as the mean value ± SEM from three independent measurements for each data point.
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