N-terminus Of Troponin Research Articles

Crossbridge Recruitment Dynamics are Divergently Affected by α/β-Myosin Heavy Chain Isoforms in Cardiac Muscle Fibers Containing the Hypertrophic Cardiomyopathy Mutation (A30V) and Protein Kinase C Phosphomimetic (T203E) in Mouse Cardiac Troponin T

The hypertrophic cardiomyopathy (HCM) mutation A30V is located within the N-terminus of troponin T (TnT), a region known to regulate Ca2+-activated maximal tension and length-dependent activation (LDA) in cardiac muscle. Given how the function of the N-terminus is differently altered by α/β-myosin heavy chain (MHC) isoforms, we believe the mutation-mediated effects are also differently altered by α- and β-MHC isoforms. Likewise, the effects of protein kinase C (PKC)-mediated phosphorylation of TnT (residue T203) are also differently altered by MHC isoforms; phosphorylation of TnT is important to consider because PKC isoforms are upregulated in failing human hearts. We tested the effects of TnTA30V (mouse analog of human A28V), PKC phosphomimetic T203E (TnTT203E), and TnTT203E+A30V in α- and β-MHC backgrounds. In α-MHC fibers, Ca2+-activated maximal tension was attenuated by TnTA30V (∼11%) and TnTT203E+A30V (∼59%). However, in β-MHC fibers, maximal tension was augmented by TnTA30V (∼13%), but was attenuated by TnTT203E+A30V (∼29%) to a lesser extent than in α-MHC fibers. Rate constants of tension redevelopment (ktr) and crossbridge (XB) recruitment dynamics (b) were attenuated by TnTT203E+A30V in α-MHC fibers, but were unaffected in β-MHC fibers. The magnitude of muscle length-mediated recruitment of strong XBs (ER) was attenuated by TnTA30V (∼31%) and TnTT203E+A30V (∼43%) in α-MHC fibers. In contrast, ER was augmented by TnTA30V (∼65%) and TnTT203E+A30V (∼65%) in β-MHC fibers. Inferences drawn from dynamic and steady-state contractile measurements suggest that XB-RU cooperativity - a significant player in LDA - is attenuated by TnTA30V and TnTT203E+A30V in α-MHC, but augmented in β-MHC. These observations demonstrate that α/β-MHC isoforms divergently modify contractile dynamics in TnTA30V and TnTT203E+A30V fibers.

Changes in the Myocardial Expression of Tropomyosin Isoforms Modulate Troponin T-Mediated Cardiac Thin Filament Activation

The spatial distribution of the troponin complex (Tn) on the thin filament, and as a result, the functional role of Tn in cardiac thin filament activation depends on structural interactions between the N-terminus of troponin T (T1) and the overlapping ends of tropomyosin (Tm). Thus, T1-Tm interactions have an influential role in regulating cardiac thin filament activation. Structural alterations in T1 have been shown to diminish cardiac activation and in view of the physical interactions between T1 and Tm, it is conceivable that the effect of T1 on cardiac activation can also be modulated by structural alterations in Tm. However, there is a lack of understanding of how structural changes in Tm influence T1-mediation of cardiac activation. To better understand how structural changes in Tm influence T1-mediated cardiac activation, we studied contractile function by reconstituting mouse cardiac troponin T (McTnT) deletion proteins, McTnT 1-44 deletion and McTnT 45-74 deletion, onto detergent-skinned papillary fibers isolated from hearts of transgenic mice expressing β-Tm. Control experiments were performed by reconstituting the McTnT 1-44 deletion and McTnT 45-74 deletion proteins onto detergent-skinned papillary fibers isolated from hearts of wild-type mice containing α-Tm. Our preliminary results show that the T1 deletions induced significant functional alterations in wild-type fibers. Interestingly, the T1-deletion-induced alteration of cardiac function was further modulated by the myocardial expression of β-Tm. For example, the McTnT 1-44 deletion-induced reduction in myofilament Ca2+ sensitivity was abolished by the expression of β-Tm. Furthermore, the McTnT 45-74 deletion-induced reduction in cooperativity of force production was more pronounced under a β-Tm background. Thus, our data shows that changes in the isoform expression of Tm modify T1-dependent cardiac function, indicating that T1-Tm interactions exert a modulatory role in regulating cardiac thin filament activation.

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

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.

Tissue-specific interactions of TNI isoforms with other TN subunits and tropomyosins in C. elegans: The role of the C- and N-terminal extensions

The aim of this study is to investigate the function of the C-terminal extension of three troponin I isoforms, that are unique to the body wall muscles of Caenorhabditis elegans and to understand the molecular interactions within the TN complex between troponin I with troponin C/T, and tropomyosin. We constructed several expression vectors to generate recombinant proteins of three body wall and one pharyngeal troponin I isoforms in Escherichia coli. Protein overlay assays and Western blot analyses were performed using antibodies. We demonstrated that pharyngeal TNI-4 interacted with only the pharyngeal isoforms of troponin C/T and tropomyosin. In contrast, the body wall TNI-2 bound both the body wall and pharyngeal isoforms of these components. Similar to other invertebrates, the N-terminus of troponin I contributes to interactions with troponin C. Full-length troponin I was essential for interactions with tropomyosin isoforms. Deletion of the C-terminal extension had no direct effect on the binding of the body wall troponin I to other muscle thin filament troponin C/T and tropomyosin isoforms.

A mutation in the N-terminus of Troponin I that is associated with hypertrophic cardiomyopathy affects the Ca 2+-sensitivity, phosphorylation kinetics and proteolytic susceptibility of troponin

The first human cardiac troponin I (hcTnI) mutation in the N-terminal 32 residue region, R21C (arginine residue number 21 mutated to cysteine), which has been linked to hypertrophic cardiomyopathy (HCM), has recently been reported. The effect of this mutation on the physiological function of hcTnI was investigated. Human cTnI R21C (in the absence or presence of troponin T and troponin C) was phosphorylated by protein kinase A (PKA) at a significantly slower rate than wild-type hcTnI. In skinned fiber studies, the TnI R21C mutant showed a large increase in Ca 2+-sensitivity of force development when compared to wild-type TnI (Δ pCa 50 = 0.33). Phosphorylation of skinned fibers containing TnI R21C by PKA resulted in a significantly smaller decrease in the Ca 2+-sensitivity of force development when compared to phosphorylation of fibers containing wild-type TnI. The decreased sensitivity of TnI R21C to PKA is most likely due to a decreased ability of PKA to phosphorylate this TnI rather than conformational problems within this TnI. In addition, skinned fibers were found to contain an endogenous kinase that is capable of phosphorylating wild-type TnI. However, the endogenous kinase activity did not affect the Ca 2+-sensitivity of force development, the Hill coefficient or maximal force of these skinned fibers. Actomyosin ATPase assays showed that the R21C mutation did not affect the inhibitory properties of TnI or the maximal ATPase activity. TnI R21C was also found to be more susceptible to proteolysis by calpain II than wild-type TnI. These results suggest that this R21C mutation in TnI affects the Ca 2+-sensitizing effect of Tn, the ability of TnI to be readily phosphorylated by PKA and the stability of TnI to calpain. The results also suggest that the N-terminal region may have important roles such as modulating the Ca 2+-sensitivity of force-development.