Deletions in the N-Terminus of Troponin T Reveal a Region Important for Ca 2+- and Length-Dependent Cardiac Myofilament Activation

Abstract

Interactions between the N-terminus of cardiac troponin T (cT1), a region rich in negatively charged amino acids, and the head-to-tail overlapping region of tropomyosin are responsible for localizing the troponin complex on the thin filament. Thus, cT1 is uniquely positioned on the thin filament to modulate cardiac contractile activity. Previous studies have demonstrated that truncations of cT1 result in severe inhibition of Ca2+-activated force development and ATPase activity of cardiac myofilaments, indicating that cT1 is essential for cardiac myofilament activation. However, the specific region of cT1 that plays an important role in cardiac contractile activation remains unknown. To determine the specific region of cT1 that plays an important role in cardiac contractile activation, we progressively deleted cT1 in rat cardiac troponin T (RcTnT) to generate four deletion mutants, RcTnT 1-29 deletion, 1-43 deletion, 30-43 deletion, and 44-73 deletion. Cardiac contractile activity and crossbridge recruitment dynamics were studied in detergent-skinned rat cardiac papillary bundles reconstituted with either wild-type (control) or the deletion proteins. Our preliminary studies show that the Ca2+-dependent force development, ATPase activity, myofilament Ca2+ sensitivity, and rate of crossbridge recruitment were significantly depressed in reconstituted fibers containing RcTnT 1-43 deletion. Fiber bundles reconstituted with RcTnT 44-73 deletion exhibited an increase in myofilament Ca2+ sensitivity. Another significant finding is that both RcTnT 1-43 and RcTnT 1-29 deletions resulted in an ablation of length-dependent increase in myofilament Ca2+ sensitivity, an important component of the Frank-Starling mechanism of healthy heart function. Thus, our data indicates that the region of cT1 comprising 1-43 amino acids plays an important role in Ca2+- and length-dependent cardiac myofilament activation. Furthermore, our findings suggest that other regions of cT1 have specific roles in mediating cardiac myofilament activation.