Z Papp1, J van der Velden, G J Stienen. 1. Department of Physiology, University Medical School of Debrecen, Hungary. pz@phys.dote.hu
Abstract
OBJECTIVE: The involvement of Calpain-I mediated proteolysis has been implicated in myofibrillar dysfunction of reperfused myocardium following ischemia (stunning). This study addresses the question whether ultrastructural alterations might be responsible for the depressed contractility. METHODS: Mechanical properties and protein composition of isolated myocytes after Calpain-I exposure (1.25 U/ml; 10 min; 15 degrees C; pCa 5.0) and of ischemic rat hearts following reperfusion were characterized. RESULTS: Maximal isometric force (44 +/- 5 kN/m2) at pCa 4.5 (pCa = -log[Ca2+]) decreased by 42.5% in Triton permeabilized myocytes (n = 11) after Calpain-I treatment. Force (and consequent myofilament disarrangement) during Calpain-I treatment was prevented by 40 mM BDM. The contractile force of Calpain-I exposed myocytes was significantly higher at submaximal levels of activation (pCa 5.5, 5.4 and 5.3) before maximal force development (pCa 4.5) than after maximal force development. The pCa50 value (5.40 +/- 0.02) determined from these initial test contractures did not differ significantly from that of untreated controls (5.44 +/- 0.03). However, after full activation Ca(2+)-sensitivity of force production in Calpain-I treated myocytes was significantly reduced (pCa50 5.34 +/- 0.02). This change in pCa50 was positively correlated with the reduction in maximal isometric force and was accompanied by sarcomere disorder. These findings imply that at least part of the Calpain-I induced mechanical alterations are dependent on force history. Measurements of the rate of force redevelopment after unloaded shortening suggested that Calpain-I did not affect cross-bridge kinetics. SDS gel electrophoresis and Western immunoblotting of Calpain-I treated myocytes revealed desmin degradation. The desmin content of postischemic myocardium was also reduced. CONCLUSION: Our results indicate that ultrastructural alterations may play an important role in the Calpain-I mediated cardiac dysfunction.
OBJECTIVE: The involvement of Calpain-I mediated proteolysis has been implicated in myofibrillar dysfunction of reperfused myocardium following ischemia (stunning). This study addresses the question whether ultrastructural alterations might be responsible for the depressed contractility. METHODS: Mechanical properties and protein composition of isolated myocytes after Calpain-I exposure (1.25 U/ml; 10 min; 15 degrees C; pCa 5.0) and of ischemicrat hearts following reperfusion were characterized. RESULTS: Maximal isometric force (44 +/- 5 kN/m2) at pCa 4.5 (pCa = -log[Ca2+]) decreased by 42.5% in Triton permeabilized myocytes (n = 11) after Calpain-I treatment. Force (and consequent myofilament disarrangement) during Calpain-I treatment was prevented by 40 mM BDM. The contractile force of Calpain-I exposed myocytes was significantly higher at submaximal levels of activation (pCa 5.5, 5.4 and 5.3) before maximal force development (pCa 4.5) than after maximal force development. The pCa50 value (5.40 +/- 0.02) determined from these initial test contractures did not differ significantly from that of untreated controls (5.44 +/- 0.03). However, after full activation Ca(2+)-sensitivity of force production in Calpain-I treated myocytes was significantly reduced (pCa50 5.34 +/- 0.02). This change in pCa50 was positively correlated with the reduction in maximal isometric force and was accompanied by sarcomere disorder. These findings imply that at least part of the Calpain-I induced mechanical alterations are dependent on force history. Measurements of the rate of force redevelopment after unloaded shortening suggested that Calpain-I did not affect cross-bridge kinetics. SDS gel electrophoresis and Western immunoblotting of Calpain-I treated myocytes revealed desmin degradation. The desmin content of postischemic myocardium was also reduced. CONCLUSION: Our results indicate that ultrastructural alterations may play an important role in the Calpain-I mediated cardiac dysfunction.
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