A loss of sarcolemmal dystrophin was observed by immuno-fluorescence studies in rabbit hearts subjected to in situ myocardial ischemia and by immuno-blotting of the Triton soluble membrane fraction of isolated rabbit cardiomyocytes subjected to in vitro ischemia. This ischemic loss of dystrophin was a specific event in that no ischemic loss of sarcolemmal α-sarcoglycan, γ-sarcoglycan, αDG, or βDG was observed. The maintenance of sarcolemmal βDG (43 Kd) during ischemia was interesting in that dystrophin binds to the C-terminus of βDG. However, during late in vitro ischemia, a 30 Kd band was observed that was immuno-reactive for βDG. Additionally, this 30 Kd-βDG band was observed in rabbit myocardium subjected to autolysis. Finally, the 30 Kd-βDG was observed in the purified sarcolemmal fraction of rabbit cardiomyocytes subjected to a prolonged period of in vitro ischemia, confirming the sarcolemmal localization of this band. The potential patho-physiologic significance of this band was indicated by the appearance of this band at 120-180 min of in vitro ischemia, directly correlating with the onset of irreversible injury, as manifested by osmotic fragility. Additionally the appearance of this band was significantly reduced by the endogenous cardioprotective mechanism, in vitro ischemic preconditioning, which delays the onset of osmotic fragility. In addition to dystrophin, βDG binds caveolin-3 and Grb-2 at its C-terminus. The presence of Grb-2 and caveolin-3 in the membrane fractions of oxygenated and ischemic cardiomyocytes was determined by Western blotting. An increase in the level of membrane Grb-2 and caveolin-3 was observed following ischemic preconditioning as compared to control cells. The formation of this 30 Kd-βDG degradation product is potentially related to the transition from the reversible to the irreversible phase of myocardial ischemic cell injury and a decrease in 30 Kd-βDG might mediate the cardioprotection provided by ischemic preconditioning.

The hypothesis that irreversible ischemic injury is related to sub-sarcolemmal blebbing and an inherent osmotic fragility of the blebs was tested by subjecting isolated control and ischemically preconditioned (IPC) or calyculin A (CalA)-pretreated (protected) rabbit cardiomyocytes to ischemic pelleting followed by resuspension in 340, 170 or 85 mosmol medium containing trypan blue. At time points from 0-240 min, osmotic fragility was assessed by the percentage of trypan blue permeable cells. Membrane blebs were visualized with India ink preparations. Bleb formation, following acute hypo-osmotic swelling, developed by 75 min and increased with longer periods of ischemia. Osmotic fragility developed only after 75 min. Cells resuspended in 340 mosmol media did not form blebs and largely retained the ability to exclude trypan blue, even after 240 min ischemia. Although the latent tendency for osmotic blebbing preceded the development of osmotic fragility, most osmotically fragile cells became permeable without evident sarcolemmal bleb formation. The onset of osmotic fragility was delayed in protected cells, but protection did not reduce the bleb formation. It is concluded that blebbing and osmotic fragility are independent manifestations of ischemic injury. The principal locus of irreversible ischemic injury and the protection provided by IPC may lie within the sarcolemma rather than at sarcolemmal attachments to underlying adherens junctions.