kb-r7943 and Necrosis

We examined whether KB-R7943 reduced infarct size by attenuating apoptosis during reperfusion and also compared antiapoptotic effects of KB-R7943 and IPost. For this purpose, isolated rat hearts underwent 30-min global ischemia and 120-min reperfusion. Ischemic postconditioning (IPost) (n = 15; three cycles of 10-s reperfusion/10-s ischemia or three cycles of 30-s reperfusion/30-s ischemia) and KB-R7943 (n = 15; 1 μM KB-R at the onset of reperfusion or before ischemia) were compared with controls (n = 12; ischemia-reperfusion only). Myocardial injury was determined by TTC staining, TUNEL assay and caspase-3 activity. AKT and eNOS phosphorylation were measured by immunoblotting. We found that IPost (10 s), Pre KB-R, and Reperf KB-R reduced infarct size (29 ± 4.1, 35 ± 5.0, 28.6 ± 3.4 %, respectively, vs. controls 46 ± 8.7 %; P < 0.05) and attenuated cell apoptosis (TUNEL-positive cardiomyocyte nuclei) in the myocardium (P < 0.01). Moreover, IPost (10 s), Pre KB-R and Reperf KB-R significantly decreased caspase-3 activation caused by myocardial ischemia-reperfusion. However, IPost (30 s) did not show any effect on necrosis and apoptosis. Akt, eNOS phosphorylation, at 30 min of reperfusion/IPost-10 s was significantly higher than other groups. In conclusion, KB-R7943 was as effective as IPost in reducing necrosis and inhibiting apoptosis and it might be an ideal pharmacological agent to provide a more amenable approach to cardioprotection.

Topics: Animals; Anti-Arrhythmia Agents; Apoptosis; Cardiotonic Agents; Caspase 3; Heart; In Situ Nick-End Labeling; Ischemic Postconditioning; Male; Myocardial Reperfusion Injury; Necrosis; Nitric Oxide Synthase Type III; Organ Culture Techniques; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Thiourea

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the regulation of cardiac excitation-contraction coupling (ECC) as well as in apoptotic signaling and adverse remodeling. The goal of the present study is to investigate the role of CaMKII in irreversible ischemia and reperfusion (I/R) injury.. Isovolumic Langendorff perfused rat hearts were subjected to global no-flow I/R (45 min/120 min), and isolated myocytes were subjected to a protocol of simulated I/R (45 min simulated ischemia/60 min reoxygenation) either in the absence or presence of CaMKII inhibition [KN-93 (KN) or the CaMKII inhibitory peptide (AIP)].. In I/R hearts, an increase in CaMKII activity at the beginning of reperfusion was confirmed by the significantly increased phosphorylation of the Thr(17) site of phospholamban. In the presence of KN, contractile recovery at the end of reperfusion was almost double that of I/R hearts. This recovery was associated with a significant decrease in the extent of infarction, lactate dehydrogenase release (necrosis), TUNEL-positive cells, caspase-3 activity, and an increase in the Bcl-2/Bax ratio (apoptosis). In isolated myocytes, both KN and AIP prevented simulated I/R-induced spontaneous contractile activity and cell mortality. Similar results were obtained when inhibiting the reverse mode Na(+)/Ca(2+) exchanger (NCX) with KB-R7943, sarcoplasmic reticulum (SR) function with ryanodine and thapsigargin, or SR Ca(2+) release with tetracaine. In contrast, overexpression of CaMKII decreased cell viability from 52+/-3% to 26+/-2%.. Taken together, the present findings are the first to establish CaMKII as a fundamental component of a cascade of events integrating the NCX, the SR, and mitochondria that promote cellular apoptosis and necrosis in irreversible I/R injury.

Topics: Animals; Apoptosis; Benzylamines; Calcium; Calcium-Binding Proteins; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; L-Lactate Dehydrogenase; Male; Myocardial Reperfusion Injury; Myocardium; Myocytes, Cardiac; Necrosis; Perfusion; Phosphorylation; Rats; Sarcoplasmic Reticulum; Sodium-Calcium Exchanger; Sulfonamides; Thiourea; Time Factors

Reverse-mode activation of the Na+-Ca2+ exchanger (NCX) at the time of reperfusion following ischemia contributes to Ca2+ overload and cardiomyocyte injury. The aim of the present study was to determine whether increased NCX in the myocardium that survived after infarction enhances its vulnerability to ischemia/reperfusion injury. Rabbits were divided into post-MI and sham groups and underwent ligation of the left circumflex coronary artery and sham operation, respectively. Two weeks later, hearts were isolated and perfused with crystalloid in the Langendorff mode with monitoring of left ventricular (LV) pressure. NCX level in the myocardium was determined by Western blotting. Myocardial stunning was induced by 5 episodes of 5-min global ischemia/5-min reperfusion. Using separate groups of hearts, myocardial infarction was induced by 30-min global ischemia/2-h reperfusion with or without treatment with 0.3 microM KB-R7943, a reverse-mode selective blocker of NCX. Heart weight-to-body weight ratio was 20% larger and NCX protein level was 60% higher in the post-MI group than in the sham group. However, there were no significant differences between severities of myocardial stunning after the repetitive ischemia/ reperfusion (18 +/- 7 vs. 25 +/- 2% reduction in LV developed pressure) and between infarct sizes after 30-min ischemia (59.1 +/- 4.1 vs. 63.0 +/- 4.5% of risk area) in the post-MI and sham groups. KB-R7943 limited infarct size in the post-MI group by 53%, and the extent of this protection was not different from that we have reported for hearts without previous infarcts (i.e. 45% reduction of infarct size). These results suggest that enhanced NCX expression does not necessarily increase myocardial vulnerability to myocardial stunning and infarction.

Topics: Animals; Anti-Arrhythmia Agents; Blotting, Western; Heart Ventricles; Ischemia; Myocardial Infarction; Myocardium; Necrosis; Organ Size; Oxygen; Rabbits; Reperfusion Injury; Sodium-Calcium Exchanger; Thiourea; Time Factors