Ischemic loss of sarcolemmal dystrophin and spectrin: correlation with myocardial injury.

Sarcolemmal blebbing and rupture are prominent features of irreversible ischemic myocardial injury. Dystrophin and spectrin are sarcolemmal structural proteins. Dystrophin links the transmembrane dystroglycan complex and extracellular laminin receptors to intracellular F-actin. Spectrin forms the backbone of the membrane skeleton conferring an elastic modulus to the sarcolemmal membrane. An ischemic loss of membrane dystrophin and spectrin, in ischemically pelleted rabbit cardiomyocytes or in vivo 30--45 min permanently ischemic, LAD-ligated hearts, was detected by immunofluorescence with monoclonal antibodies. Western blots of light and heavy microsomal vesicles and Triton-extracted membrane fractions from ischemic myocytes demonstrated a rapid loss of dystrophin coincident with sub-sarcolemmal bleb formation, subsequent to a hypotonic challenge. The loss of spectrin from purified sarcolemma of autolysed rabbit heart, and both isolated membrane vesicles and Triton solubilized membrane fractions of ischemic cardiomyocytes correlated linearly with the onset of osmotic fragility as assessed by membrane rupture, subsequent to a hypotonic challenge. In contrast to the ischemic loss of dystrophin and spectrin from the membrane, the dystrophin-associated proteins, alpha-sarcoglycan and beta-dystroglycan and the integral membrane protein, sodium-calcium exchanger, were maintained in the membrane fraction of ischemic cells as compared to oxygenated cells. Preconditioning protected cells, but did not significantly alter ischemic dystrophin or spectrin translocation. This previously unrecognized loss of sarcolemmal dystrophin and spectrin may be the molecular basis for sub-sarcolemmal bleb formation and membrane fragility during the transition from reversible to irreversible ischemic myocardial injury.

Differential translocation or phosphorylation of alpha B crystallin cannot be detected in ischemically preconditioned rabbit cardiomyocytes.

Alpha B Crystallin (alpha BC) is a putative effector protein of ischemic preconditioning (IPC), that is phosphorylated on Ser 45 by ERK1/2 and Ser 59 by the p38 MAPK substrate, MAPKAPK-2. Translocation and phosphorylation of alpha BC was determined in cytosolic and cytoskeletal fractions by 1D SDS-PAGE and IEF, or using Ser 45 and Ser 59 phospho-specific antibodies in: (1) control rabbit cardiomyocytes; (2) cells preconditioned by 10 min in vitro ischemia; or after pre-treatment with specific inhibitors of (3) Ser/Thr protein phosphatase 1/2A (calyculin A); (4) p38 MAPK (SB203580); or (5) ERK 1/2 (PD98059); all prior to 180 min ischemia. Ischemia induced a cytosolic to cytoskeletal translocation of alpha BC, which was similar in all the groups. Highly phosphorylated isoforms (D1/2) of alpha BC were present in cytosolic but not cytoskeletal fractions at 0 min ischemia. By 60-90 min ischemia, D1/2 isoforms had translocated to the cytoskeletal fraction. Calyculin A maintained D1/2 levels throughout prolonged ischemia. SB203580 decreased alpha BC phosphorylation. Neither PD98059 nor IPC altered alpha BC phosphorylation during prolonged ischemia. It is concluded that alpha BC phosphorylation during ischemia is regulated by p38 MAPK but not by ERK 1/2. The inability to detect a correlation between IPC protection and either alpha BC translocation or phosphorylation suggests that the proteins in the highly phosphorylated isoform bands of alpha BC quantitated in this study are not protective end effectors of classical IPC.