Human Cardiac Tropomyosin Research Articles

Mutations in troponin T associated with Hypertrophic Cardiomyopathy increase Ca2+-sensitivity and suppress the modulation of Ca2+-sensitivity by troponin I phosphorylation

We investigated the effect of 7 Hypertrophic Cardiomyopathy (HCM)-causing mutations in troponin T (TnT) on troponin function in thin filaments reconstituted with actin and human cardiac tropomyosin. We used the quantitative in vitro motility assay to study Ca2+-regulation of unloaded movement and its modulation by troponin I phosphorylation. Troponin from a patient with the K280N TnT mutation showed no difference in Ca2+-sensitivity when compared with donor heart troponin and the Ca2+-sensitivity was also independent of the troponin I phosphorylation level (uncoupled). The recombinant K280N TnT mutation increased Ca2+-sensitivity 1.7-fold and was also uncoupled. The R92Q TnT mutation in troponin from transgenic mouse increased Ca2+-sensitivity and was also completely uncoupled. Five TnT mutations (Δ14, Δ28 + 7, ΔE160, S179F and K273E) studied in recombinant troponin increased Ca2+-sensitivity and were all fully uncoupled. Thus, for HCM-causing mutations in TnT, Ca2+-sensitisation together with uncoupling in vitro is the usual response and both factors may contribute to the HCM phenotype. We also found that Epigallocatechin-3-gallate (EGCG) can restore coupling to all uncoupled HCM-causing TnT mutations. In fact the combination of Ca2+-desensitisation and re-coupling due to EGCG completely reverses both the abnormalities found in troponin with a TnT HCM mutation suggesting it may have therapeutic potential.

Thermal Activation of Cardiac Thin Filaments Induces Contraction without Intracellular Ca2+ Changes: Studies with Cardiomyocytes and an In Vitro Motility Assay

It is well established that heart beat is triggered by electrical excitation, followed by an increase in [Ca2+]i and ensuing activation of myofilaments in cardiomyocytes. This property is the basis for the regulation of the heart with electric stimulators or chemical compounds that mediate Ca2+ signaling. Recently, we found that microscopic heat pulses induce contractions in isolated rat cardiomyocytes without a rise in [Ca2+]i (Oyama, K. et al., Biochem. Biophys. Res. Commun., 417, 607-612, 2012). Microscopic heat pulses produced by focusing infrared laser beam increase temperature of the extracellular solution near cardiomyocytes. In our experiments, a microscopic heat pulse (ΔT = 5°C for 0.5 sec) induced contractions at a basal temperature of 36°C. When heat pulses were repeatedly applied at 2.5 Hz, oscillatory contractions were observed. In contrast to the electric field stimulation, Ca2+ transients were not detected during the course of contraction. Moreover, we confirmed that skinned cardiomyocytes are thermally activated in the Ca2+-free solution. To elucidate the molecular mechanism of thermal activation, microscopic heat pulses were applied to an in vitro motility assay system utilizing reconstituted thin filaments (rabbit skeletal actin filaments reconstituted with human cardiac tropomyosin and bovine cardiac troponin). Thin filaments started to move during heating in the absence of Ca2+, and most of filaments immediately stopped after switching the laser off, demonstrating that thin filaments can be thermally activated, presumably due to the weakening of the binding affinity of the troponin-tropomyosin complex with actin (Ishiwata, S., Biochim. Biophys. Acta, 534, 350-357, 1978). These results suggest that heat pulses can regulate cardiac contractions by shifting the “on-off” equilibrium in the thin filament toward the “on” state, without accompanying intracellular Ca2+ transients.

Structural Dynamics of Troponin I during Ca2+-Activation of Cardiac Thin Filaments: A Multi-Site Förster Resonance Energy Transfer Study

A multi-site, steady-state Förster resonance energy transfer (FRET) approach was used to quantify Ca2+-induced changes in proximity between donor loci on human cardiac troponin I (cTnI), and acceptor loci on human cardiac tropomyosin (cTm) and F-actin within functional thin filaments. A fluorescent donor probe was introduced to unique and key cysteine residues on the C- and N-termini of cTnI. A FRET acceptor probe was introduced to one of three sites located on the inner or outer domain of F-actin, namely Cys-374 and the phalloidin-binding site on F-actin, and Cys-190 of cTm. Unlike earlier FRET analyses of protein dynamics within the thin filament, this study considered the effects of non-random distribution of dipoles for the donor and acceptor probes. The major conclusion drawn from this study is that Ca2+ and myosin S1-binding to the thin filament results in movement of the C-terminal domain of cTnI from the outer domain of F-actin towards the inner domain, which is associated with the myosin-binding. A hinge-linkage model is used to best-describe the finding of a Ca2+-induced movement of the C-terminus of cTnI with a stationary N-terminus. This dynamic model of the activation of the thin filament is discussed in the context of other structural and biochemical studies on normal and mutant cTnI found in hypertrophic cardiomyopathies.

Mechanical Properties of Human Cardiac Tropomyosin in Familial Hypertrophic Cardiomyopathy (FHC) Probed by Atomic Force Microscopy

α-Tropomyosin (αTm) is a 66kDa alpha-helical coiled-coil protein in thin filaments of cardiac muscle. Together with the Troponin complex (Tn), it is responsible for Ca2+ regulation of striated muscle contraction. When Ca2+ is released from sarcoplasmic reticulum and bound to Tn, the complex undergoes a conformational change that moves Tm away from myosin binding sites on actin, sites that are blocked in the absence of Ca2+. This allows actomyosin cross-bridge cycling, resulting in force generation and/or sarcomere shortening.

Functional Analysis of a Unique Troponin C Mutation, GLY159ASP, that Causes Familial Dilated Cardiomyopathy, Studied in Explanted Heart Muscle

Familial dilated cardiomyopathy can be caused by mutations in the proteins of the muscle thin filament. In vitro, these mutations decrease Ca(2+) sensitivity and cross-bridge turnover rate, but the mutations have not been investigated in human tissue. We studied the Ca(2+)-regulatory properties of myocytes and troponin extracted from the explanted heart of a patient with inherited dilated cardiomyopathy due to the cTnC G159D mutation. Mass spectroscopy showed that the mutant cTnC was expressed approximately equimolar with wild-type cTnC. Contraction was compared in skinned ventricular myocytes from the cTnC G159D patient and nonfailing donor heart. Maximal Ca(2+)-activated force was similar in cTnC G159D and donor myocytes, but the Ca(2+) sensitivity of cTnC G159D myocytes was higher (EC(50) G159D/donor=0.60). Thin filaments reconstituted with skeletal muscle actin and human cardiac tropomyosin and troponin were studied by in vitro motility assay. Thin filaments containing the mutation had a higher Ca(2+) sensitivity (EC(50) G159D/donor=0.55 + or - 0.13), whereas the maximally activated sliding speed was unaltered. In addition, the cTnC G159D mutation blunted the change in Ca(2+) sensitivity when TnI was dephosphorylated. With wild-type troponin, Ca(2+) sensitivity was increased (EC(50) P/unP=4.7 + or - 1.9) but not with cTnC G159D troponin (EC(50) P/unP=1.2 + or - 0.1). We propose that uncoupling of the relationship between phosphorylation and Ca(2+) sensitivity could be the cause of the dilated cardiomyopathy phenotype. The differences between these data and previous in vitro results show that native phosphorylation of troponin I and troponin T and other posttranslational modifications of sarcomeric proteins strongly influence the functional effects of a mutation.

Antibodies in the sera of acute rheumatic fever patients bind to human cardiac tropomyosin.

Previous work from this laboratory has demonstrated the occurrence of heart reactive antibodies (HRA) in the sera of acute rheumatic fever (ARF) patients which bound primarily to the sarcolemmal sheath of cardiac myofibres. While many investigators have reported the presence of antibodies to myosin in the sera of ARF patients, the question of whether these sera also contain antibodies to other cytoskeletal proteins has not been addressed. In this study, crude human sarcolemmal sheaths were extracted with 3 M KCl and partially purified using a DEAE cellulose column and a step gradient of NaCl. Maximum reactivity with ARF sera was seen with a protein fraction eluted with 0.3 M NaCl. Using FPLC, a single polypeptide of 38 kDa reacted in ELISA preferentially with ARF sera when compared to sera from patients with uncomplicated streptococcal infections and acute post-streptococcal glomerulonephritis (APSGN). A comparison of the N-terminal sequence of the purified protein and competitive inhibition assays indicated that the reactive sarcolemmal antigen was human cardiac tropomyosin.

Relation of streptococcal M protein with human and rabbit tropomyosin: The complete amino acid sequence of human cardiac alpha tropomyosin, a highly conserved contractile protein

Partial sequences of group A streptococcal M proteins exhibit up to 50% sequence identity with segments of rabbit skeletal tropomyosin. It is well recognized that rheumatic fever and rheumatic heart disease in humans are sequelae of group A streptococcal infection. To examine whether the human cardiac tropomyosin would exhibit greater homology with the streptococcal M proteins, we have now determined its complete amino acid sequence. The amino acid sequence of human cardiac tropomyosin was established from sequence analyses of its peptides derived by enzymic and chemical cleavages, and comparison of these sequences to the reported sequence of rabbit skeletal tropomyosin. These studies have revealed that the amino acid sequence of human cardiac alpha tropomyosin is identical to that of the rabbit skeletal alpha tropomyosin, but for a single conservative substitution of Arg/Lys at position 220. This observation increases the significance of the previously observed sequence homology between streptococcal M protein and rabbit skeletal tropomyosin and may have relevance to the pathogenesis of rheumatic fever. Furthermore, these results rank tropomyosin as one of the most highly conserved contractile proteins between vertebrate species reported thus far.

Radioimmunoassay of Human Cardiac Tropomyosin in Acute Myocardial Infarction

1. Tropomyosin was prepared from fresh human myocardium and antisera raised in rabbits. A sensitive radioimmunoassay was developed for the detection of human cardiac tropomyosin in human sera down to levels of 1 ng/ml. 2. Values for human cardiac tropomyosin in normal patients ranged from less than 1 to 3 ng/ml. In 18 patients with acute myocardial infarction all had elevated tropomyosin levels ranging from 41 to above 200 ng/ml with a mean peak level of 101 ng/ml. In this study there were no false positive or false negative results. 3. In the initial stages of infarction the time course of appearance and peak levels of cardiac tropomyosin, total creatine kinase and creatine kinase MB isoenzyme were similar. Although total creatine kinase and creatine kinase MB isoenzyme levels were normal after 72 h in patients with single, uncomplicated infarction, cardiac tropomyosin levels were still significantly elevated above normal after this time, being 30-60% of peak values. 4. Radioimmunoassay of human cardiac tropomyosin may prove useful in the diagnosis and in the management of patients with acute myocardial infarction, particularly in the long-term postinfarction period.