Localization of calcium in skeletal and cardiac muscle

The requirement of calcium (Ca2+) in the excitation-contraction coupling of both skeletal and cardiac muscle is well established. However, the exact location of the intracellular storage sites of Ca2+ is not firmly established. We report here on the ultrastructural ultrastructural distribution of Ca2+ in white and red skeletal muscle and in cardiac muscle of the rat using combined phosphate-pyroantimonate (PPA) and oxalate-pyroantimonate (OPA) procedures. The methods are based on (a) stabilization and/or trapping of Ca2+ during the primary fixation step in glutaraldehyde by potassium phosphate or oxalate; (b) subsequent wash-out of all non-trapped cations such as Na+ and Mg2+ in potassium phosphate or oxalate; (c) conversion of the complexed or trapped Ca2+ into an electron-dense calcium pyroantimonate salt in 100 micron-thick tissue sections; and (d) wash-out of the excess potassium pyroantimonate at alkaline pH. With the OPA procedure, mitochondria of all muscle types showed little precipitate. The junctional sarcoplasmic reticulum was strongly reactive in relaxed white skeletal muscle, negative in contracted white fibres and negative in red skeletal and cardiac muscle, independent of the state of relaxation-contraction. Other organelles were essentially free of deposits. With the PPA method, the precipitate was almost exclusively confined to the sarcolemma and its T-tubular invaginations in cardiac and slow skeletal muscle, and was absent in fast skeletal muscle. Apart from occasional deposits in mitochondria, all other organelles were free of muscle. Apart from occasional deposits in mitochondria, all other organelles were free of precipitate. The sarcolemma-associated deposits were clearly confined to the inner leaflet of the lipid bilayer. The amount of precipitate varied within the contraction cycle, relaxed cells possessing the highest density. Exposure of the tissue to La3+ resulted in the complete absence of sarcolemma-bound precipitate suggesting that the Ca2+ is exchangeable. Furthermore, these cytological data suggest a basic difference in Ca2+ storage between white skeletal muscle on the one hand, and red skeletal and cardiac muscle on the other.