Paper-based cascade cationic isotachophoresis: Multiplex detection of cardiac markers.

Paper-based analytical devices (PADs) are widely used in point-of-care testing (POCT) as they are cost-effective, simple and straightforward. However, poor sensitivity hinders their use in detecting diseases with low abundance biomarkers. The poor detection limit of PADs is mainly attributed to the low concentration of analytes, and the complexity of biological fluid, leading to insufficient interactions between analytes and capture antibodies. This study aims to overcome these difficulties by developing a paper-based cationic isotachophoresis (ITP) approach for simultaneously detecting pico-molar levels of two essential cardiac protein markers: acidic troponin T (cTnT) and basic troponin I (cTnI) spiked into human serum samples. The approach utilizes 3-aminopropyltrimethoxysilane (APTMS) treated glass fiber papers with decreasing cross-sectional area assembled on a 3D printed cartridge device. Our results showed that in the presence of cTnT monoclonal antibody (mAb), fluorescently labeled cTnI and cTnT could be effectively enriched in cationic ITP. Each individual target was captured subsequently by a test line in the detection zone where the capture mAb was immobilized. Detailed analysis suggests that the technology is capable of simultaneous on-board depletion of abundant plasma proteins and enrichment of cTnI/cTnT by ~1300-fold with a sensitivity of 0.6 pmol/L for cTnT and a sensitivity of 1.5 pmol/L for cTnI in less than 6 min. The results demonstrate the potential of this technology for rapid, ultra-sensitive and cost-effective analysis of multiplex protein markers in clinical serum samples at point of care.

Effects of cardiomyopathy-linked mutations K15N and R21H in tropomyosin on thin-filament regulation and pointed-end dynamics.

Missense mutations K15N and R21H in striated muscle tropomyosin are linked to dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), respectively. Tropomyosin, together with the troponin complex, regulates muscle contraction and, along with tropomodulin and leiomodin, controls the uniform thin-filament lengths crucial for normal sarcomere structure and function. We used Förster resonance energy transfer to study effects of the tropomyosin mutations on the structure and kinetics of the cardiac troponin core domain associated with the Ca2+-dependent regulation of cardiac thin filaments. We found that the K15N mutation desensitizes thin filaments to Ca2+ and slows the kinetics of structural changes in troponin induced by Ca2+ dissociation from troponin, while the R21H mutation has almost no effect on these parameters. Expression of the K15N mutant in cardiomyocytes decreases leiomodin's thin-filament pointed-end assembly but does not affect tropomodulin's assembly at the pointed end. Our in vitro assays show that the R21H mutation causes a twofold decrease in tropomyosin's affinity for F-actin and affects leiomodin's function. We suggest that the K15N mutation causes DCM by altering Ca2+-dependent thin-filament regulation and that one of the possible HCM-causing mechanisms by the R21H mutation is through alteration of leiomodin's function.

Dynamic Equilibrium of Cardiac Troponin C's Hydrophobic Cleft and Its Modulation by Ca2+ Sensitizers and a Ca2+ Sensitivity Blunting Phosphomimic, cTnT(T204E).

Several studies have suggested that conformational dynamics are important in the regulation of thin filament activation in cardiac troponin C (cTnC); however, little direct evidence has been offered to support these claims. In this study, a dye homodimerization approach is developed and implemented that allows the determination of the dynamic equilibrium between open and closed conformations in cTnC's hydrophobic cleft. Modulation of this equilibrium by Ca2+, cardiac troponin I (cTnI), cardiac troponin T (cTnT), Ca2+-sensitizers, and a Ca2+-desensitizing phosphomimic of cTnT (cTnT(T204E) is characterized. Isolated cTnC contained a small open conformation population in the absence of Ca2+ that increased significantly upon the addition of saturating levels of Ca2+. This suggests that the Ca2+-induced activation of thin filament arises from an increase in the probability of hydrophobic cleft opening. The inclusion of cTnI increased the population of open cTnC, and the inclusion of cTnT had the opposite effect. Samples containing Ca2+-desensitizing cTnT(T204E) showed a slight but insignificant decrease in open conformation probability compared to samples with cardiac troponin T, wild type [cTnT(wt)], while Ca2+ sensitizer treated samples generally increased open conformation probability. These findings show that an equilibrium between the open and closed conformations of cTnC's hydrophobic cleft play a significant role in tuning the Ca2+ sensitivity of the heart.

Fluorescence Based Characterization of Calcium Sensitizer Action on the Troponin Complex.

Calcium sensitizers enhance the transduction of the Ca(2+) signal into force within the heart and have found use in treating heart failure. However the mechanisms of action for most Ca(2+) sensitizers remain unclear. To address this issue an efficient fluorescence based approach to Ca(2+) sensitizer screening was developed which monitors cardiac troponin C's (cTnC's) hydrophobic cleft. This approach was tested on four common Ca(2+) -sensitizers, EMD 57033, levosimendan, bepridil and pimobendan with the aim of elucidating the mechanisms of action for each as well as proving the efficacy of the new screening method. Ca(2+) -titration experiments were employed to determine the effect on Ca(2+) sensitivity and cooperativity of cTnC opening, while stopped flow experiments were used to investigate the impact on cTnC relaxation kinetics. Bepridil was shown to increase the sensitivity of cTnC for Ca(2+) under all reconstitution conditions, sensitization by the other drugs was context dependent. Levosimendan and pimobendan reduced the rate of cTnC closing consistent with a stabilization of cTnC's open conformation while bepridil increased the rate of relaxation. Experiments were also run on samples containing cTnT(T204E), a known Ca(2+) -desensitizing phosphorylation mimic. Levosimendan, bepridil, and pimobendan were found to elevate the Ca(2+) -sensitivity of cTnT(T204E) containing samples in this context.

FRET study of the structural and kinetic effects of PKC phosphomimetic cardiac troponin T mutants on thin filament regulation.

FRET was used to investigate the structural and kinetic effects that PKC phosphorylations exert on Ca(2+) and myosin subfragment-1 dependent conformational transitions of the cardiac thin filament. PKC phosphorylations of cTnT were mimicked by glutamate substitution. Ca(2+) and S1-induced distance changes between the central linker of cTnC and the switch region of cTnI (cTnI-Sr) were monitored in reconstituted thin filaments using steady state and time resolved FRET, while kinetics of structural transitions were determined using stopped flow. Thin filament Ca(2+) sensitivity was found to be significantly blunted by the presence of the cTnT(T204E) mutant, whereas pseudo-phosphorylation at additional sites increased the Ca(2+)-sensitivity. The rate of Ca(2+)-dissociation induced structural changes was decreased in the C-terminal end of cTnI-Sr in the presence of pseudo-phosphorylations while remaining unchanged at the N-terminal end of this region. Additionally, the distance between cTnI-Sr and cTnC was decreased significantly for the triple and quadruple phosphomimetic mutants cTnT(T195E/S199E/T204E) and cTnT(T195E/S199E/T204E/T285E), which correlated with the Ca(2+)-sensitivity increase seen in these same mutants. We conclude that significant changes in thin filament Ca(2+)-sensitivity, structure and kinetics are brought about through PKC phosphorylation of cTnT. These changes can either decrease or increase Ca(2+)-sensitivity and likely play an important role in cardiac regulation.