The Tropomyosin Binding Region of Cardiac Troponin T Modulates Crossbridge Recruitment Dynamics in Rat Cardiac Muscle Fibers

Abstract

The cardiac muscle comprises dynamically interacting components that use allosteric/cooperative mechanisms to yield unique heart-specific properties. An essential protein in this allosteric/cooperative mechanism is cardiac muscle troponin T (cTnT), the central region (CR) and the T2 region of which differ significantly from those of fast skeletal muscle troponin T (fsTnT). To understand the biological significance of such sequence heterogeneity, we replaced the T1 or T2 domain of rat cTnT (RcT1 or RcT2) with its counterpart from rat fsTnT (RfsT1or RfsT2) to generate RfsT1-RcT2 and RcT1-RfsT2 recombinant proteins. In addition to contractile function measurements, dynamic features of RfsT1-RcT2 and RcT1-RfsT2 reconstituted rat cardiac muscle fibers were captured by fitting the recruitment-distortion model to force response of small amplitude (0.5%) muscle length changes. RfsT1-RcT2 fibers showed a ~40% decrease in tension and ~44% decrease in ATPase activity, but RcT1-RfsT2 fibers were unaffected. The magnitude of length-mediated increase in crossbridge recruitment (E0) decreased by ~33% and the speed of crossbridge recruitment (b) increased by ~100% in RfsT1-RcT2 fibers. Our data suggest the following: (1) the CR of cTnT modulates crossbridge recruitment dynamics; (2) the N-terminal end region of cTnT has a synergistic effect on the ability of CR to modulate crossbridge recruitment dynamics; (3) the T2 region is important for tuning the Ca2+ regulation of cardiac thin filaments. The combined effects of CR-Tm interactions and the modulating effect of the N-terminal end of cTnT on CR-Tm interactions may lead to the emergence of a unique property that tunes contractile dynamics to heart rates.

Interplay between the overlapping ends of tropomyosin and the N terminus of cardiac troponin T affects tropomyosin states on actin

“…It is likely that the N‐terminal end region of cTnT (residues 1‐74 of McTnT) does not interact directly with other thin‐filament proteins (35, 36). However, recent binding affinity studies from our laboratory (37) suggest that the N‐terminal end of cTnT may still influence cardiac thin‐filament activation by synergistically modulating the functional effect of the neighboring region of cTnT (represented by the central region residues 78–193 of cTnT in Fig. 8).…”

The effect of cardiomyopathy mutation (R97L) in mouse cardiac troponin T on the muscle length-mediated recruitment of crossbridges is modified divergently by α- and β-myosin heavy chain

“…Equal amounts (2 µg) of protein samples were loaded and run on an 8% SDS-gel for optimal separation of TnT [8, 25, 28]. Proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane and TnT was probed using a monoclonal anti-TnT primary antibody (M401134, Fitzgerald Industries Int, Concord, MA), followed by HRP-labeled anti-mouse secondary antibody (RPN 2132, Amersham Biosciences, Piscataway, NJ).…”

“…Proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane and TnT was probed using a monoclonal anti-TnT primary antibody (M401134, Fitzgerald Industries Int, Concord, MA), followed by HRP-labeled anti-mouse secondary antibody (RPN 2132, Amersham Biosciences, Piscataway, NJ). Densitometric analysis of the Western blot was performed as described previously [8, 28]. …”

Thick Filament Protein Network, Functions, and Disease Association

“…This is a highly regulated process mediated by thin and thick filament accessory proteins. At rest, binding of tropomyosin (Tm) and troponin-I (TnI) to actin precludes its binding of myosin, while troponin-T (TnT) interlocks the Tn/Tm complex and actin, and contributes to the cooperative activation of thin filaments in response to Ca 2+ , reviewed in (188) (186, 187, 348). In response to depolarization, Ca 2+ released from the sarcoplasmic reticulum (SR) binds to troponin-C (TnC) resulting in displacement of TnI and Tm from the actin filament, and enabling its interaction with the motor head domain of myosin, reviewed in (350).…”