TITIN ELASTICITY AND MECHANISM OF PASSIVE FORCE DEVELOPMENT IN RAT CARDIAC MYOCYTES PROBED BY THIN-FILAMENT EXTRACTION

Titin (also known as connectin) is a giant filamentous protein whose e lastic properties greatly contribute to the passive force in muscle. I n the sarcomere, the elastic I-band segment of titin may interact with the thin filaments, possibly affecting the molecule's elastic behavio r. Indeed, several studies have indicated that interactions between ti tin and actin occur in vitro and may occur in the sarcomere as well. T o explore the properties of titin alone, one must first eliminate the modulating effect of the thin filaments by selectively removing them. In the present work, thin filaments were selectively removed from the cardiac myocyte by using a gelsolin fragment. Partial extraction left behind similar to 100-nm-long thin filaments protruding from the Z-lin e, whereas the rest of the I-band became devoid of thin filaments, exp osing titin. By applying a much more extensive gelsolin treatment, we also removed the remaining short thin filaments near the Z-line. After extraction, the extensibility of titin was studied by using immunoele ctron microscopy, and the passive force-sarcomere length relation was determined by using mechanical techniques. Titin's regional extensibil ity was not detectably affected by partial thin-filament extraction. P assive force, on the other hand, was reduced at sarcomere lengths long er than similar to 2.1 mu m, with a 33 +/- 9% reduction at 2.6 mu m. A fter a complete extraction, the slack sarcomere length was reduced to similar to 1.7 mu m. The segment of titin near the Z-line, which is ot herwise inextensible, collapsed toward the Z-line in sarcomeres shorte r than similar to 2.0 mu m, but it was extended in sarcomeres longer t han similar to 2.3 mu m. Passive force became elevated at sarcomere le ngths between similar to 1.7 and similar to 2.1 mu m, but was reduced at sarcomere lengths of >2.3 mu m. These changes can be accounted for by modeling titin as two wormlike chains in series, one of which incre ases its contour length by recruitment of the titin segment near the Z -line into the elastic pool.