Reduction of myocardial infarction by calpain inhibitor A-705253 in isolated perfused rabbit hearts

Ein Anstieg der zytosolischen Kalziumionenkonzentration während Ischämie und Reperfusion führt zur Aktivierung der Cysteinprotease Calpain. die die myokardialen Proteine schädigt und somit ausschlaggebend für den Ischämie-Reperfusionsschaden ist. Untersucht wurde der kardioprotektive Einfluss des spezifischen Calpaininhibitors A-705253 auf die Degeneration des Herzmuskels als alleiniges Pharmakon zu Beginn der Reperfusionsphase und in Kombination mit dem Na+-H+-Antiporter-Inhibitor Cariporide®, der zu einer Reduktion der intrazellulären Kalziumionenkonzentration führt. Am Versuchsmodell des isoliert perfundierten Kaninchenherzen nach Langendorff wurde eine linksventrikuläre Ischämie durch Verschluss eines Koronarastes provoziert. Der Calpaininhibitor A-705253 in den Konzentrationen 10-6M und 10-7M, gegeben zu Beginn der Reperfusion, verkleinerte die Infarktnekrosezone im Vergleich zur Kontrollgruppe um 66,4 %. Da die Reduktion bei präischämischer Gabe von 10-8M einen Wert von 61,8 % erreichte, ist von einer Schädigung auszugehen, die im Wesentlichen in der Reperfusion stattfindet. In der Kombination mit Cariporide® ,dem Na+-H+-Antiporter-Inhibitor, ergab sich kein additiver Effekt. So dass anzunehmen ist, dass die Wirkung von Cariporide® über die Reduktion der Calpainaktivierung zu erklären ist. Desweiteren ist bei fehlendem additivem Effekt in unserem Modell eine kardioprotektive Wirkung durch die Hemmung von Cathepsin B und L, die in den oben genannten Konzentrationen ebenfalls durch A-705253 gehemmt werden, aber bekanntlich keine Ca2+-abhängigen Proteasen sind, unwahrscheinlich. Die postischämische Infarkttherapie ist von höchster klinischer Relevanz. Unser neuartiger Calpaininhibitor A-705253 zeigt zum ersten Mal eine Reduktion der Ausdehnung des Infarktareals verabreicht zu Beginn der Reperfusionsphase und könnte folglich in der Zukunft erheblich zur Reduktion der Infarktletalität beitragenCytosolic Ca2+overload during ischemia and reperfusion (Kihara et al. 1989; Marban et al. 1990) is considered to be mainly responsible for the activation of calcium-dependent calpains, localized primarily in the Z-disk/I-band region (Yoshimura et al. 1986; Kumamoto et al. 1992), and the subsequent breakdown of myocardial proteins. The calpain-dependent degradation of desmin, calspectrin (Yoshida et al. 1995), troponin I and T, and sarcoplasmic reticulum proteins, corresponding to the myofilament disintegration observed by electron microscopy (Gao et al. 1997; Goette et al. 2002), has been reported in ischemia-reperfusion models. Furthermore, calpain activation in ischemia and reperfusion is accompanied by the downregulation of its natural inhibitor calpastatin (Sorimachi et al. 1997; Enns et al. 2002). Besides direct myofilament disintegration, mitochondrial dysfunction is also considered to have an essential contribution to cardiac dysfunction and myocyte injury during ischemia and reperfusion (Lesnefsky et al., 2001). Therefore, the present study seeks to evaluate the cardioprotective properties of a newly developed calpain inhibitor A-705253 (Lubisch et al. 2003) in isolated perfused rabbit hearts following temporary coronary occlusion, when administered alone and in combination with Cariporide®, a Na+/H+ exchange inhibitor. Cariporide® reduces the intracellular Ca2+-concentration and hence a decrease in calpain-activity (Chen et al. 2002). Both inhibitors were studied in isolated perfused rabbit hearts subjected to 60-min occlusion of the ramus interventricularis of the left coronary artery (below the origin of the first diagonal branch), followed by 120 min of reperfusion. The inhibitors were added to the perfusion fluid in various final concentrations five minutes before the tourniquet was reopened and the period of reperfusion began. Myocardial infarct size and the area at risk (transiently non-perfused myocardium) were determined from left ventricular slices after a special staining procedure with Evans blue and 2,3,5- triphenyltetrazolium chloride. The infarcted area (dead myocardium) was 77.9 ± 2.3% of the area at risk in untreated controls. The infarct size was significantly reduced in the presence of A-705253 calpain inhibitors in concentrations of 10-6M and 10-7M to 48,3 ± 2,3 % and 49,2 ± 4,1 % of the area at risk, corresponding to an infarct reduction of 66,4. %. As a result of an infarct size reduction of 61,8% (10-8M), when A-705253 is administered from the beginning (Neuhof et al. 2004), experiments demonstrate that the essential myocyte injury takes place during the time of reperfusion. As the calpain inhibitors used in our study also inhibit the lysosomal cysteine proteases cathepsin B and L, the discussion should be focused whether inhibition of both proteases could account for the cardioprotective effect. So far, very little information is available for both cathepsins in the heart. Thus, activation of cathepsin L was found in the intralysosomal compartment of cardiomyocytes during coronary aortal bypass (Turski and Zaslonka 2000). However, the increase in functional recovery and the decrease in protein degradation, total activity of cathepsin B and L, and in cathepsin B leakage by cysteine proteinase inhibitors in experimental models of myocardial ischemia and infarction (Tsuchida et al 1986; Shibata et al. 1992), cannot be related to both proteinases, as these inhibitors could also inhibit calpains. The results showed that, both calpain inhibitor A- 705253 and the Na+/H+ exchange inhibitor Cariporide® reduced infarct size by the same degree, without an additive effect when they were combined. These findings suggest that cardioprotection in this present study is due to calpain inhibition, because cathepsin B and L are not calcium-activated proteases and their activity would not be inhibited by an Na+/H+ exchange inhibitor. In a nut shell, having a firm understanding of the properties of A-705253 is a valuable tool for further pharmacological studies in coherence with cell damage due to ischemia and reperfusion