Alterations in Hyper-Variable N-Terminal Region of Cardiac Troponin T Result in Diminished Cardiac Myofilament Function

Abstract

Cardiac troponin T (cTnT) plays a central role in Ca2+-dependent regulation of myofilament activation in cardiac muscle. cTnT is characterized by its unique hyper-variable N-terminal extension (T1) that is rich in negative charge when compared to skeletal TnT (sTnT) isoforms, implying that T1 has a cardiac-specific role in Ca2+-based myofilament activation. Studies have shown that alterations in T1 affect the overall confirmation of cTnT and the binding affinity of cTnT to Tropomyosin, Troponin C, and Troponin I. To examine the unique role of T1 on cardiac activation, we created transgenic (TG) mice expressing recombinant chimeric form of TnT, where mouse cTnT1-73 amino acid residues were replaced by mouse fast sTnT1-41 residues resulting in a less acidic isoform of TnT. Papillary muscle fiber bundles isolated from TG and wild-type (WT) mouse hearts were used to measure tension, ATPase activity, myofilament Ca2+-sensitivity (pCa50), rate of tension redevelopment (ktr), and crossbridge recruitment dynamics at sarcomere length (SL) of 1.9 and 2.3 μm. Maximal tension and ATPase activity were unaltered in fiber bundles from TG and WT mouse hearts. However, an interesting finding was a significant increase in myofilament Ca2+-sensitivity in TG mouse hearts (a pCa50 of 5.85 vs. 5.76 in WT mouse hearts). Furthermore, TG mouse hearts exhibited an impaired length-dependent activation, where the length-dependent increase in myofilament Ca2+-sensitivity (ΔpCa50) was 0.04 in TG mouse hearts versus a ΔpCa50 of 0.14 in WT mouse hearts. Measurements of ktr, and crossbridge detachment rate at either SL indicated that the crossbridge kinetics was lower in fibers from TG mouse hearts. Thus, all our results demonstrate that structural alterations in T1 of cTnT lead to diminished cardiac function, implicating a regulatory role for T1 in cardiac myofilament activation.