Age-related abnormalities in regulation of the ryanodine receptor in rat fast-twitch muscle

Abstract

The tibialis anterior (TA) muscles of 6-month-old and 24-month-old male Wistar rats, after being characterized, at the fast motor unit level, for twitch properties, were dissected and processed by a procedure [Margreth A., Damiani E., Tobaldin G. Biochem Biophys Res Commun 1993; 197: 1303–1311] aimed at obtaining a representative total membrane fraction comprising 70–80% of the total muscle content of sarcoplasmic reticulum (SR) and transverse tubule (TT) membranes (about 20 mg protein/g). Skeletal muscle membranes were analyzed for protein composition, and the content and functional properties of specific components of the free and junctional subcompartments of the SR and of junctional TT. Our results, while confirming a twitch prolongation in TA of old rats, do not demonstrate any associated age-related change concerning: (a) the overall number and functional properties of Ca 2+ pumps, as characterized by kinetic parameters, Ca 2+-dependency, and the protein isoform specificity of SR Ca 2+-ATPase; (b) the number of functional junctional SR Ca 2+-release channels, on the basis of B max values for high-affinity binding of [ 3H]-ryanodine to skeletal muscle membranes at optimal Ca 2+; (c) the overall muscle dihydropyridine receptor/ryanodine receptor (RyR) ratio. We conclude from these findings, and the additional negative evidence for changes in membrane density of specific components of junctional SR, including 60 kDa Ca 2+-calmodulin protein kinase, that this membrane domain, like the Ca 2+-pump domain of the SR, are in no way basically altered at early stages of the aging process, as investigated here. Because of that, we allege particular significance to the occurrence of age-related, specific abnormalities in regulation of RyR in rat TA. The main supportive evidence is as follows: (a) an increased sensitivity to Ca 2+ of the RyR of old muscle, and, more importantly; (b) an increased sensitivity to caffeine of [ 3H]ryanodine binding to the RyR at optimal Ca 2+ and also optimal for the activity of the Ca 2+-release channel. The results reported here also demonstrate that there are two classes of caffeine sites in rat TA muscle, as defined by differences in EC 50 values at resting (pCa 7) and at high Ca 2+ (pCa 4–5), that sites involved in stimulation of [ 3H]-ryanodine binding to the RyR are distinguished by a higher affinity (caffeine below mM), and that only these sites undergo age-related changes. Thus, although the underlying age-related abnormality of the RyR remains to be elucidated, it appears to satisfy the requirement for being regarded as a specific change, which in itself might argue for its being fundamentally related to the twitch prolongation of the muscle.