Pressure-induced changes in the connectin/titin localization in the myofibrils revealed by immunoelectron microscopy

Abstract

Changes in the connectin/titin localization in post-mortem and pressurized chicken muscles were investigated by immunoelectron microscopy. The anti-connectin monoclonal antibody, 1D11, strongly labeled the sides of thick filaments near the H-zone and weakly labeled the sides of Z-line in the sarcomere prepared immediately after death. With the development of the muscle contraction, the shortening of the sarcomere and the dispersion of the connectin epitope near the H-zone were observed. With the gradual increase of the sarcomere length during further storage, the apparent increase of the width of the epitope in the A-band region stained by the antibody was observed, but the distance from the epitope to M-line remained almost the same length. In the case of high pressure treatment, significant changes in the labeling pattern of the antibody were observed with the increase of the pressure applied. The increase of the distance from the epitope to M-line and dispersion of the epitope were observed in the fiber pressurized at 100 MPa. These phenomena were accelerated with the increase of the pressure applied. The discontinuous dense materials labeled by the antibody at the thick filament near the H-zone were observed in the fiber bundles pressurized at 200 MPa or more. This is probably due to the accumulation of connectin molecule from ordinary location in the sarcomere, because of the pressure-induced destruction of the thick and connectin filaments. In the fiber bundles pressurized at 300 MPa, a significant increase in the distance from the epitope to M-line accompanied with the increase of the sarcomere length was observed. From the results obtained, it was clear that the changes in the location of the connectin epitope induced by the brief exposure to high pressure were drastic in comparison with that in the sarcomere during post-mortem storage.

Introduction

The presence of connectin (also called titin) filaments within the sarcomere of vertebrate skeletal muscle is now well-established. Connectin, a giant structural protein that was first identified by Maruyama, Natori, and Nonomura (1976), is thought to be elastic in nature and could account for some of the elastic properties of skeletal muscle (Maruyama, 1986, Wang, 1985). The influence of connectin on meat tenderization during post-mortem conditioning has been investigated by many workers.

The gap filaments, which are considered to be composed of connectin, have been shown to be susceptible to protease by Locker and Leet (1976), and the disappearance of insoluble connectin from myofibrils during conditioning of meat has been reported by Takahashi and Saito (1979). Therefore, the degradation of connectin has been implicated as being responsible for the increasing meat tenderness that occurs during conditioning. On the other hand, King, Kurth, and Shorthose (1981) and King (1984) have denied any significance of the degradation of connectin in meat tenderness on the ground that the protein is destroyed by cooking, even though the degradation of connectin has been observed during conditioning of mutton. No disappearance of connectin from chicken muscle and no significant differences in the content and electrophoretic pattern of connectin isolated from chicken muscle during conditioning were reported by Locker (1984) and Suzuki, Sawaki, Hosaka, Ikarashi, and Nonami (1985), respectively. From these observations, connectin is unlikely to be responsible for the meat tenderization caused by conditioning. However, there are still some uncertainties about the fate and role of connectin during conditioning. As suggested by Suzuki et al. (1987), it is important to clarify qualitative change of connectin structure during post-mortem storage if we are to understand the role of connectin in meat tenderness.

In normal skeletal muscle, connectin exists as α-connectin (titin-1) together with a small amount of β-connectin (titin-2) (Maruyama et al., 1984, Wang et al., 1979). Alpha-connectin is easily degraded to β-connectin during post-mortem storage (Maruyama et al., 1977, Seki and Watanabe, 1984), and the extent of degradation is dependent upon time and temperature (Lusby, Ridpath, Parrish, & Robson, 1983). It has been suggested that the amount of titin is less in tender steaks and that the rate of titin degradation during post-mortem storage is accelerated in tender steaks compared to their less-tender counterparts (Paterson & Parrish, 1987). From these observations the amount of connectin and the conversion of α-connectin to β-connectin may be one of the causes of meat tenderization. Nevertheless Fritz, Mitchell, Marsh, and Greaser (1993) reported that titin content (sum of titins-1,-2 and-3), as determined by gel electrophoresis, did not distinguish ‘tough’ from ‘tender’ meat. The changes in the position of titin epitope in the sarcomere during posts mortem storage by using indirect immunofluorescent method have been reported by Ringkob, Marsh, and Greaser (1988) and Fritz and Greaser (1991). However, the changes in the titin epitope obtained by the phase microscopic observation were insufficient to clarify the relationship between the changes of connectinltitin filament and meat tenderization.

In our previous report, we revealed that a brief exposure of muscle to high hydrostatic pressure could induce the conversion of α-connectin to β-connectin, and that calpain was responsible for the pressure-induced conversion of α- to β-connectin (Kim et al., 1993, Kim et al., 1992). The increase in the amount of extractable connectin from the pressurized muscle, i.e. the increase of the conversion of α-connectin to β-connectin, is probably one of the causes of the pressure-induced tenderization of meat (Suzuki, Kim, Tanji, & Ikeuchi, 1998).

This paper describes high pressure effects on the connectin epitope in the sarcomere by using immunoelectron microscopy, in comparison with those naturally observed in the conditioned muscle.