Structural studies of rigor bovine myofibrils using fluorescence microscopy. II. Influence of sarcomere length on the binding of myosin subfragment-1, alpha-actinin and G-actin to rigor myofibrils

Abstract

Sarcomere length influences the textural quality of meat, yet the molecular basis for the mechanism of post-mortem shortening and the toughness associated with shortened muscle remain obscure. Bovine and rabbit myofibrillar structure was investigated over a range of sarcomere lengths (SL), using the high affinity of the myosin head for actin and of alpha-actinin for the Z-line. Myofibrils were incubated with fluorescent conjugates of myosin subfragment-1 (S1), alpha-actinin and actin. When S1 and alpha-actinin were added together, S1 bound in the I-band and A-band but not at the Z-line, while alpha-actinin bound at the Z-line. The pattern of S1 binding was highly influenced by SL, and could be explained using a model with the ratio of myofibrillar actin to myosin heads in the overlap region of 2:1 and thin filament penetration into opposing half sarcomeres. Double staining with S1 and actin demonstrated that, once the tip of the thin filament reached the bare zone, few intrinsic myosin heads were available for fluorescent actin. The patterns observed for both S1 and actin staining suggest that myofibrillar rigor bonds form even at very short SL. These observations lead to the hypothesis that the toughness associated with short sarcomeres is due to thick-filament interactions and not to the density of rigor bonds in the myofibril. Regulation of S1 binding was investigated by incubating myofibrils with low levels of fluorescent S1 in the presence and absence of calcium; S1 binding was in the overlap region when calcium was absent, but in the I-band when it was present. These results suggest that the thin filament can be activated for muscle shortening by the binding of myosin heads, a mechanism that may contribute to post-mortem muscle shortening.