Recombinant Human Cardiac Troponin Research Articles

Slowed Dynamics of Thin Filament Regulatory Units Reduces Ca2+-Sensitivity of Cardiac Biomechanical Function

Actomyosin kinetics in both skinned skeletal muscle fibers at maximum Ca2+-activation and unregulated in vitro motility assays are modulated by solvent microviscosity in a manner consistent with a diffusion limited process. Viscosity might also influence cardiac thin filament Ca2+-regulatory protein dynamics. In vitro motility assays were conducted using thin filaments reconstituted with recombinant human cardiac troponin and tropomyosin; solvent microviscosity was varied by addition of sucrose or glucose. At saturating Ca2+, filament sliding speed (s) was inversely proportional to viscosity. Ca2+-sensitivity (pCa50 ) of s decreased markedly with elevated viscosity (η/η0 ≥ ~1.3). For comparison with unloaded motility assays, steady-state isometric force (F) and kinetics of isometric tension redevelopment (kTR ) were measured in single, permeabilized porcine cardiomyocytes when viscosity surrounding the myofilaments was altered. Maximum Ca2+-activated F changed little for sucrose ≤ 0.3 M (η/η0 ~1.4) or glucose ≤ 0.875 M (η/η0 ~1.66), but decreased at higher concentrations. Sucrose (0.3 M) or glucose (0.875 M) decreased pCa50 for F. kTR at saturating Ca2+ decreased steeply and monotonically with increased viscosity but there was little effect on kTR at sub-maximum Ca2+. Modeling indicates that increased solutes affect dynamics of cardiac muscle Ca2+-regulatory proteins to a much greater extent than actomyosin cross-bridge cycling.

Effects of Elevated Solvent Viscosity on Calcium Dependence of Cardiac Myofilament Contractility

We have previously shown with skinned skeletal muscle fibers at maximum Ca2+ activation and unregulated in vitro motility assays that solvent viscosity modulates actomyosin function in a manner consistent with diffusional limitation of a kinetic process. To determine whether viscosity influences thin filament regulatory protein dynamics, we performed experiments in cardiac muscle preparations with varying [Ca2+]. First, in vitro motility assays were conducted using thin filaments reconstituted with recombinant human cardiac troponin and tropomyosin, and rabbit skeletal HMM and actin; solution viscosity was varied by addition of sucrose.

Ca2+ sensitivity of regulated cardiac thin filament sliding does not depend on myosin isoform

Myosin heavy chain (MHC) isoforms in vertebrate striated muscles are distinguished functionally by differences in chemomechanical kinetics. These kinetic differences may influence the cross-bridge-dependent co-operativity of thin filament Ca(2+) activation. To determine whether Ca(2+) sensitivity of unloaded thin filament sliding depends upon MHC isoform kinetics, we performed in vitro motility assays with rabbit skeletal heavy meromyosin (rsHMM) or porcine cardiac myosin (pcMyosin). Regulated thin filaments were reconstituted with recombinant human cardiac troponin (rhcTn) and alpha-tropomyosin (rhcTm) expressed in Escherichia coli. All three subunits of rhcTn were coexpressed as a functional complex using a novel construct with a glutathione S-transferase (GST) affinity tag at the N-terminus of human cardiac troponin T (hcTnT) and an intervening tobacco etch virus (TEV) protease site that allows purification of rhcTn without denaturation, and removal of the GST tag without proteolysis of rhcTn subunits. Use of this highly purified rhcTn in our motility studies resulted in a clear definition of the regulated motility profile for both fast and slow MHC isoforms. Maximum sliding speed (pCa 5) of regulated thin filaments was roughly fivefold faster with rsHMM compared with pcMyosin, although speed was increased by 1.6- to 1.9-fold for regulated over unregulated actin with both MHC isoforms. The Ca(2+) sensitivity of regulated thin filament sliding speed was unaffected by MHC isoform. Our motility results suggest that the cellular changes in isoform expression that result in regulation of myosin kinetics can occur independently of changes that influence thin filament Ca(2+) sensitivity.

Functional effects of the DCM mutant Gly159Asp Troponin C in skinned muscle fibres

We recently reported a dilated cardiomyopathy (DCM) causing mutation in a novel disease gene, TNNC1, which encodes cardiac troponin C (TnC). We have determined how this mutation, Gly159Asp, affects contractile regulation when incorporated into muscle fibres. Endogenous troponin in rabbit skinned psoas fibres was partially replaced by recombinant human cardiac troponin containing either wild-type or Gly159Asp TnC. We measured both the force-pCa relationship of these fibres and the activation rate using the caged-Ca(2+) compound nitrophenyl-EGTA. Gly159Asp TnC had no significant effect on either the Ca(2+) sensitivity or cooperativity of force generation when compared to wild type. However, the mutation caused a highly significant (ca. 50%) decrease in the rate of activation. This study shows that whilst not affecting the force-pCa relationship, the mutation Gly159Asp causes a significant decrease in the rate of force production and a change in the relationship between the rate of force production and generated force. In vivo, this mutation may cause both a slowing of force generation and reduction in total systolic force. This represents a novel mechanism by which a cardiomyopathy-causing mutation can affect contractility.

Clinical Effect of Recalibration of the Roche Cardiac Troponin T Assay

Objective: To assess the effect of recalibration of the Roche 3rd-generation cTnT assay with recombinant human cardiac troponin T (cTnT) standards on clinical decision limits. Materials and Methods: Serum samples from 77 patients (66 ± 16 years) admitted to the coronary care unit were assayed using the 2nd- and 3rd-generation cTnT assays. Results: There was excellent agreement (r = 0.99 Spearman, 95% CI 0.99–1.0; p ≤ 0.0001, n = 153) between the 2nd- and 3rd-generation cTnT assays across the analytical range, but there was a curvilinear relationship between values. There was concordance between the 2nd- and 3rd-generation cTnT values in the range from 0 to 0.2 µg/l. Above 0.2 µg/l, however, there were increasing but predictable differences. Conclusion: There was no statistical difference between the 2nd- and 3rd-generation cTnT assays, demonstrated by a linear relationship below 0.2 µg/l. This confirms that the 3rd-generation assay was calibrated to that of the 2nd-generation assay in the range of 0–0.2 µg/l. The detection limit and upper reference limit of normal will be unaffected by this change in calibration. A non-linear relationship at higher 2nd-generation concentrations (0.2–25.0 µg/l) was observed. Clinical decision limits up to 0.2 µg/l, associated with increased cardiac risk, are unaffected by the assay calibration, but values greater than 0.2 µg/l are affected.

Prokaryotic expression of human cTnI and preparation of anti-cTnI monoclonal antibody

目的 构建人心肌肌钙蛋白I(cTnI)的表达载体,表达cTnI重组蛋白,并制备其单克隆抗体(mAb).方法以化学方法合成cTnI基因并插入融合表达载体pBV220的多克隆位点,构建重组表达质粒pBV220/ cTnI.以重组质粒转化大肠杆菌DH5α,筛选阳性重组子,经热激诱导目的蛋白的表达,表达产物的免疫学活性用Western blot进行鉴定.以基因重组的cTnI蛋白为抗原,常规方法免疫BALB/ c 小鼠,取其脾细胞与NS-1细胞融合,获得稳定分泌cTnI mAb的杂交瘤细胞株,ELISA 检测mAb的相对亲和力;Western blot检测mAb的特异性.结果酶切鉴定和DNA 测序显示cTnI重组表达载体中含有人cTnI全长编码序列.将该重组载体转化入大肠杆菌DH5α中表达所得蛋白经Western Blot验证为目的蛋白.筛选出2 株能稳定分泌特异性抗cTnI的mAb 杂交瘤细胞株,免疫球蛋白亚类均为IgG类;Western blot结果显示,两株单抗识别相对分子量为24 000的cTnI单一条带;中和抑制试验表明培养上清中的抗体能明显被cTnI中和;cTnI单克隆抗体的亲和常数为Kaff = 1.62 ×109 (mol/ L)-1.结论成功地构建了cTnI表达载体、表达出cTnI重组蛋白,制备出抗cTnI mAb,为进一步用于cTnI的体外免疫学检测奠定了基础。

Alterations in Thin Filament Regulation Induced by a Human Cardiac Troponin T Mutant That Causes Dilated Cardiomyopathy Are Distinct from Those Induced by Troponin T Mutants That Cause Hypertrophic Cardiomyopathy

We have compared the in vitro regulatory properties of recombinant human cardiac troponin reconstituted using wild type troponin T with troponin containing the DeltaLys-210 troponin T mutant that causes dilated cardiomyopathy (DCM) and the R92Q troponin T known to cause hypertrophic cardiomyopathy (HCM). Troponin containing DeltaLys-210 troponin T inhibited actin-tropomyosin-activated myosin subfragment-1 ATPase activity to the same extent as wild type at pCa8.5 (>80%) but produced substantially less enhancement of ATPase at pCa4.5. The Ca(2+) sensitivity of ATPase activation was increased (DeltapCa(50) = +0.2 pCa units) and cooperativity of Ca(2+) activation was virtually abolished. Equimolar mixtures of wild type and DeltaLys-210 troponin T gave a lower Ca(2+) sensitivity than with wild type, while maintaining the diminished ATPase activation at pCa4.5 observed with 100% mutant. In contrast, R92Q troponin gave reduced inhibition at pCa8.5 but greater activation than wild type at pCa4.5; Ca(2+) sensitivity was increased but there was no change in cooperativity. In vitro motility assay of reconstituted thin filaments confirmed the ATPase results and moreover indicated that the predominant effect of the DeltaLys-210 mutation was a reduced sliding speed. The functional consequences of this DCM mutation are qualitatively different from the R92Q or any other studied HCM troponin T mutation, suggesting that DCM and HCM may be triggered by distinct primary stimuli.

Use of recombinant human cardiac Troponin T for standardization of third generation Troponin T methods

The Elecsys® Troponin T third generation assay uses recombinant human cardiac troponin T as standard material. The new assay has a linear calibration curve. Thus, linearity problems observed with the second generation assay have been eliminated. The assay has high precision, especially at the low end of measuring range (inter-assay CV <10 % at 0.1 μg/L). The new standardization does not change the cut-off value of 0.1 μg/L. The use of recombinant human cardiac troponin T as standard material enables a reproducible and reliable standardization of troponin T assays.

Use of recombinant human cardiac Troponin T for standardization of third generation Troponin T methods

Use of recombinant human cardiac Troponin T for standardization of third generation Troponin T methods

Binding of a new Ca2+ sensitizer, levosimendan, to recombinant human cardiac troponin C. A molecular modelling, fluorescence probe, and proton nuclear magnetic resonance study.

The binding of a new calcium sensitizer, levosimendan, to human cardiac troponin C (cTnC) is described. Fluorescence studies done on dansylated recombinant human cTnC and a site-directed mutant showed that levosimendan modulated the calcium-induced conformational change in cTnC, and revealed the role of Asp-88 in the binding of the drug to the NH2-terminal domain of cTnC. Furthermore, NMR studies performed on the NH2-terminal fragment of cTnC showed a spatial proximity between levosimendan and Met81, Met85, and Phe77 in the drug-protein complex. These data were used to build an optimized model of the drug-protein complex, in which levosimendan binds cTnC at the hydrophobic pocket of the NH2-terminal domain. The role of the binding of levosimendan to cTnC in the pharmacological action of this drug in vivo is discussed.