Breakdown and Release of Myofilament Proteins During Ischemia and Ischemia/Reperfusion in Rat Hearts

Identification of Degradation Products and Effects on the pCa-Force Relation

From the Department of Physiology and Biophysics (J.E.V.E., F.P., W.L., R.J.S.), College of Medicine, University of Illinois at Chicago; the Department of Physiology (J.E.V.E.), Queen's University, Kingston, Ontario, Canada; the Department of Surgery (F.P., W.L.), College of Medicine, University of Illinois at Chicago; Sanofi Diagnostics Pasteur (C.L.), Marnes-La-Coquette, France; and the Department of Biochemistry (R.S.H.), University of Alberta, Edmonton, Canada.

Correspondence to Jennifer E. Van Eyk, PhD, Department of Physiology, Queen's University, Kingston, Ontario K7L 3W6, Canada.

Abstract—Our objective in experiments reported here was to identify myofilament proteins of rat hearts either lost or degraded by cardiac ischemia (15- or 60-minute duration) with and without 45 minutes of reperfusion. We correlated these changes with alterations in myofilament sensitivity to Ca²⁺ and maximum force generation. Protein degradation and loss were assessed by high-performance liquid chromatography, SDS-PAGE, Western blotting analysis, and amino acid sequencing. Compared with nonischemic control hearts, bundles of skinned fibers from hearts subjected to ischemia alone demonstrated a decrease in maximum force generation and an increase in sensitivity to Ca²⁺. These changes in function were increased with the duration of the ischemia and with reperfusion. With increasing duration of ischemia, there was an increased loss and degradation of myofibrillar -actinin and troponin I (TnI) at its C-terminus. -Actinin and TnI were most susceptible to ischemia, but with 60 minutes of ischemia/reperfusion, there was also degradation of myosin light chain-1 (MLC1) involving a clip of residues 1 to 19. The MLC1 degradation product was detected in the reperfusion effluent (along with troponin T, tropomyosin, and -actinin) but not in the tissue with 60 minutes of ischemia with no reperfusion. Moreover, with ischemia the following proteins became associated with the myofibrils: GAPDH and proteins of the mitochondrial ATP synthase complex. Our results provide new evidence regarding the mechanism by which ischemia/reperfusion causes myocardial injury and support the hypothesis that an important element in the injury is altered activity and structure of the myofilaments.

Key Words: protein degradation • myocardial ischemia/reperfusion • myofilament • troponin I • -actinin

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