Ca 2+ sensitivity of stunned myocardium after skinning using retrograde infusion of detergent

Abstract

Myofilament Ca2+ desensitization contributes to the contractile dysfunction of ischemic/reperfused ("stunned") myocardium. We examined the presence of reduced Ca2+ sensitivity of isometric force in chemically skinned fibers obtained from stunned myocardium using different modes of applying the detergent Triton X-100. Langendorff-perfused rat hearts underwent 20 min ischemia/20 min reperfusion, which caused a 35 +/- 3% decrease in left ventricular developed pressure, compared to continuously perfused control hearts. Stunned and control hearts were randomly allocated to two different permeabilization protocols: In group A, trabeculae were dissected and immersed in skinning solution containing 1% Triton X-100 for 20 min. Group B hearts remained fixed to the aortic cannula and skinning solution was infused retrogradely for 6 min prior to dissection of trabeculae. Extraction of cytosolic marker proteins was more complete in group-B than in group-A preparations. Group-A preparations from stunned hearts exhibited significant Ca2+ desensitization (pCa50 = 5.07 and 5.15 in stunned and control myocardium, respectively). In group B no such difference was observed, all preparations showing higher Ca2+ sensitivity and maximum force than group-A preparations (pCa50 = 5.32 in stunned versus 5.33 in control hearts). Prolonging group-A skinning to 150 min also abolished the difference in Ca2+ sensitivity between stunned and control myocardium. In conclusion, compared to a conventional protocol, skinning by perfusion results in more complete permeabilization and better preservation of myocardial contractile function. Ischemia/reperfusion at this moderate degree of contractile dysfunction induces Ca2+ desensitization at least partially by factors that can be extracted by thorough skinning.