The I band of cardiac sarcomeres contains both actin and titin/connectin 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, where titin has been found to be inelastic. To study the putative interaction
between titin and actin, techniques for selective removal of actin from different
regions of the I band are needed. Here we report studies with a gelsolin fragment
(FX-45) and extract actin from rat cardiac myocytes. Actin extraction was biphasic:
the majority of actin was extracted in similar to 10 min, whereas actin near the Z
line (where titin is inelastic) required a similar to 10-fold longer extraction time.
Thus, by controlling the extraction time, we could remove either the full actin filament
outside the Z line or just the segment of the actin filament that extends beyond the
inelastic region of titin that adjoins the Z line. The actin filament-free I band
contained titin filaments, typically with one filament extending from each thick filament.
In addition, we observed a dark transverse line (junction line), the location of which
in the sarcomere varied linearly with sarcomere length. The position in the sarcomere
of the junction line coincided with the binding site of the anti-titin antibody 9D10.
Actin removal significantly 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 may be caused by contraction
of the titin segment that becomes exposed after actin removal near the Z line, indicating
that titin is not only attached to the actin filament but is also under tension.