ULTRASTRUCTURAL LOCALIZATION OF CALCIUM IN NORMAL AND ABNORMAL SKELETAL-MUSCLE

Abstract

Ca was demonstrated ultrastructurally as a fine black reaction product with unbuffered 2% saturated potassium pyroantimonate, pH 9.4. In comparison with normal muscle, there was increased precipitate in degenerating skeletal muscle fibers and some degenerating-regenerating fibers occurring in pathologic human muscle, regardless of disease entity, and in experimentally injured rat muscle. The pathologically increased Ca was mainly within the sarcoplasmic reticulum and mitochondria. Both structures were completely blackened. Nuclear Ca was also increased, the precipitates were localized as circular profiles within the nucleoli and heterochromatin and associated with the nuclear envelope. Myofibrillar Ca was only modestly increased. When normal rat muscle was preincubated in 136 mM Ca-enhanced Hanks'' medium, Ca accumulated in the muscle fibers. It was especially heavy in the mitochondria and sarcoplasmic reticulum and appeared identical with the pathologic human and rat muscle fibers. Preincubation of normal rat muscle in 0.1 M acetate buffer (pH 4.65) before Ca loading augmented myofibrillar staining, mainly in the H-zone of the A-bands excluding the M-zone and in broad irregular N1, N2, and N3 lines of the I-bands. EMMA-4 electron probe microanalysis and EGTA (ethylene glycolbis(.beta.-aminoethyl ether)N,N''-tetraacetic acid) chelation prior to staining confirmed that the precipitate in the several loci was calcium antimonate. In skeletal muscle fibers injured by various pathologic processes, a breach of the plasmalemma barrier to Ca occurred as a very early abnormality. Extracellular Ca may then pour into the aqueous sarcoplasm of the muscle fiber, from which it was withdrawn by and accumulated within the still active organelles normally having a great avidity for uptake of this ion, especially the mitochondria and sarcoplasmic reticulum. The resultant organellar calcification may impair function and damage the structure of proteins and phospholipids.