ACTIN REMOVAL FROM CARDIAC MYOCYTES SHOWS THAT NEAR Z-LINE TITIN ATTACHES TO ACTIN WHILE UNDER TENSION

The I band of cardiac sarcomeres contains both actin and titin/connect in filaments. Earlier work has suggested that titin binds to actin in situ. This interaction must be weak in the region of the I band where titin behaves elastically. On the other hand, titin may bind strongly to actin in the similar to 100-nm-wide region adjoining the Z line, wh ere titin has been found to be inelastic. To study the putative intera ction between titin and actin, techniques for selective removal of act in from different regions of the I band are needed. Here we report stu dies with a gelsolin fragment (FX-45) and extract actin from rat cardi ac myocytes. Actin extraction was biphasic: the majority of actin was extracted in similar to 10 min, whereas actin near the Z line (where t itin is inelastic) required a similar to 10-fold longer extraction tim e. Thus, by controlling the extraction time, we could remove either th e full actin filament outside the Z line or just the segment of the ac tin filament that extends beyond the inelastic region of titin that ad joins the Z line. The actin filament-free I band contained titin filam ents, typically with one filament extending from each thick filament. In addition, we observed a dark transverse line (junction line), the l ocation of which in the sarcomere varied linearly with sarcomere lengt h. The position in the sarcomere of the junction line coincided with t he binding site of the anti-titin antibody 9D10. Actin removal signifi cantly affected the slack sarcomere length. Slack sarcomere length was 1.85 +/- 0.04 mu m in control cells and decreased to 1.71 +/- 0.05 mu m after actin near the Z line was extracted. This length reduction ma y be caused by contraction of the titin segment that becomes exposed a fter actin removal near the Z line, indicating that titin is not only attached to the actin filament but is also under tension.