Calcium depletion in rabbit myocardium. Ultrastructure of the sarcolemma and correlation with the calcium paradox.

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Abstract
The ultrastructure of the Ca-depleted myocardial sarcolemma (via Ca-free and Ca-free plus EGTA perfusion at 28 degree C and 37 degree C) was studied in the vascularly perfused interventricular septum of the rabbit. Thin-section and freeze-fracture electron microscopy was used. Two major structural defects in the sarcolemma were found. (1) Ninety percent of the Ca-depleted cells have between 30 and 40% of their glycocalyx separated from the bilayer. With tannic acid staining, the separation is seen to occur between the external lamina and the surface coat. (2) Freeze-fracture data showed an apparent decrease in intramembrane particles on the P face of unidirectionally shadowed replicas. Quantitation of rotary-shadowed replicas showed no decrease in density of intramembrane particles. It was concluded from this that there was no loss of intramembrane particles, but rather a reorientation in the plane of the bilayer after Ca depletion. Both glycocalyx and bilayer changes were present after perfusion of the heart for only 5 minutes (37 degree C) with Ca-free perfusate. With low temperature and Cd substitution, separation of the glycocalyx occurred in less than 1% of the cells. After Ca depletion at 18 degree C, the density of intramembrane particles on the P face was not significantly different from controls. Cd substitution did not prevent the decrease total intramembrane particles per square micron, but the larger intramembrane particles had similar densities (154/micrometer2) as control (181/micrometer2), and as Ca-depletion with hypothermia (180/micrometer2). These findings indicate that structural changes in the glycocalyx and the bilayer can be totally prevented by hypothermia. Cd, on the other hand, prevents glycocalyx separation and affords protection only to the large intramembrane particles. Upon reperfusion with Ca, the intramembrane particles undergo the further alteration of aggregation, while numerous vesicles can be seen in the fracture plane of the membrane.