Calpain-I induced alterations in the cytoskeletal structure and impaired mechanical properties of single myocytes of rat heart

Objective: The involvement of Calpain-I mediated proteolysis has been impli cated in myofibrillar dysfunction of reperfused myocardium following ischem ia (stunning). This study addresses the question whether ultrastructural al terations might be responsible for the depressed contractility. Methods: Me chanical properties and protein composition of isolated myocytes after Calp ain-I exposure (1.25 U/ml; 10 min; 15 degrees C; pCa 5.0) and of ischemic r at hearts following reperfusion were characterized. Results: Maximal isomet ric force (44 +/- 5 kN/m(2)) at pCa 4.5 (pCa= -log[Ca2+]) decreased by 42.5 % in Triton permeabilized myocytes (n=11) after Calpain-I treatment. Force land 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 forc e development. The pCa,, value (5.40 +/- 0.02) determined from these initia l test contractures did not differ significantly from that of untreated con trols (5.44 +/- 0.03). However, after full activation Ca2+-sensitivity of f orce production in Calpain-I treated myocytes was significantly reduced (pC a(50) 5.34 +/- 0.02). This change in pCa(50) 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 Calpai n-I did not affect cross-bridge kinetics. SDS gel electrophoresis and Weste rn immunoblotting of Calpain-I treated myocytes revealed desmin degradation . The desmin content of postischemic myocardium was also reduced. Conclusio n: Our results indicate that ultrastructural alterations may play an import ant role in the Calpain-I mediated cardiac dysfunction. (C) 2000 Elsevier S cience BN. All rights reserved.