Electrochemical cardiovascular platforms: Current state of the art and beyond.

Cardiovascular diseases (CVD) remain the leading cause of death within industrialized nations as well as an increasing cause of mortality and morbidity in many developing countries. Smoking, alcohol consumption and increased level of blood cholesterol are the main CVD risk factors. Other factors, such as the prevalence of overweight/obesity and diabetes, have increased considerably in recent decades and are indirect causes of CVD. Among CVDs, the acute coronary syndrome (ACS) represents the most common cause of emergency hospital admission. Since the prognosis of ACS is directly associated with timely initiation of revascularization, missed, misdiagnosis or late diagnosis have unfavorable medical implications. Early ACS diagnosis can reduce complications and risk of recurrence, finally decreasing the economic burden posed on the health care system as a whole. To decrease the risk of ACS and related CVDs and to reduce associated costs to healthcare systems, a fast management of patients with chest pain has become crucial and urgent. Despite great efforts, biochemical diagnostic approaches of CVDs remain difficult and controversial medical challenges as cardiac biomarkers should be rapidly released into the blood at the time of ischemia and persistent for a sufficient length of time to allow diagnostics, with tests that should be rapid, easy to perform and relatively inexpensive. Early biomarker assessments have involved testing for the total enzyme activity of aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and creatine kinase (CK), which cardiac troponins being the main accepted biomarkers for diagnosing myocardial injury and acute myocardial infarction (AMI). To allow rapid diagnosis, it is necessary to replace the traditional biochemical assays by cardiac biosensor platforms. Among the numerous of possibilities existing today, electrochemical biosensors are important players as they have many of the required characteristics for point-of-care tests. Electrochemical based cardiac biosensors are highly adapted for monitoring the onset and progress of cardiovascular diseases in a fast and accurate manner, while being cheap and scalable devices. This review outlines the state of the art in the development of cardiac electrochemical sensors for the detection of different cardiac biomarkers ranging from troponin to BNP, N-terminal proBNP, and others.

Isolation and characterization of three skeletal troponin genes and association with growth-related traits in Exopalaemon carinicauda.

Growth is among the most important traits for animal breeding. Muscle growth is controlled by different cellular and molecular pathways and environments, and it also relies heavily on high-quality muscle contractions. The troponin complex, composed of troponin T (TnT), troponin C (TnC) and troponin I (TnI), plays a vital role in the regulation of muscle contraction. In this study, the cDNA of EcTnT, EcTnC and EcTnI of the ridgetail white prawn Exopalaemon carinicauda were cloned and characterized. The full length cDNA of EcTnT, EcTnC and EcTnI were 1 373 bp, 692 bp, and 1 475 bp, encoding a protein of 385, 150 and 193 amino acid residues, respectively. The expression of all genes was predominantly detected in abdominal muscle, while extremely lesser expressed in gill and hepatopancreas. Higher expression level of EcTnI was observed in heavier shrimp of the same age during different developmental stages, excepted for 120 days. Eleven single nucleotide polymorphisms (SNPs) were revealed in the three skeletal troponin genes, and only c.TnI66 A>G from EcTnI was significantly associated with both body weight and body length (P < 0.05). In summary, the result of this study suggested that EcTnI is growth-related gene of the troponin complex gene and the presence of SNP suggests that it could be a candidate gene for shrimp genetic improvement research.

¿Quo vadis, troponina? / Quo vadis, troponin?

No disponible

Troponina elevada en pacientes sin síndrome coronario agudo / Troponin Elevation in patients without acute coronary syndrome

Introducción y objetivos: Las troponinas son biomarcadores específicos de daño miocárdico y tienen implicación en el diagnóstico y el pronóstico de pacientes con síndrome coronario agudo. El objetivo es determinar las características clínicas y el pronóstico en pacientes con elevación de troponina no diagnosticados de síndrome coronario agudo. Métodos: Se estudió retrospectivamente a 1.032 pacientes con determinación de troponinas en un servicio de urgencias, que quedaron distribuidos en tres grupos: 681 pacientes sin elevación de troponina y sin síndrome coronario agudo, 139 con síndrome coronario agudo y 212 con troponina elevada sin diagnóstico de síndrome coronario agudo. Se compararon las características clínicas de estos tres grupos y su mortalidad hospitalaria y a los 12 meses de seguimiento. Conclusiones: La troponina elevada es un importante predictor de mortalidad, independientemente del diagnóstico definitivo del paciente (AU)

Introduction and objectives: Troponins are specific biomarkers of myocardial injury and are implicated in the diagnosis and prognosis of patients with acute coronary syndrome. Our purpose was to determine the clinical characteristics and prognosis of patients with troponin elevation who are not diagnosed with acute coronary syndrome. Methods: A total of 1032 patients with an emergency room troponin measurement were studied retrospectively, dividing them into 3 groups: 681 patients with no troponin elevation and without acute coronary syndrome, 139 with acute coronary syndrome, and 212 with troponin elevation and not diagnosed with acute coronary syndrome. The clinical characteristics and in-hospital and 12-month mortality of these 3 groups were compared. Results: Patients with troponin elevation not diagnosed with acute coronary syndrome were older and had greater comorbidity than patients with acute coronary syndrome or no troponin elevation. The 12-month mortality was 30.2%, compared with 15.1% and 4.7% in the other groups (log rank test, P < .001). In the Cox logistic regression model adjusted for confounding variables, patients with troponin elevation and no diagnosis of acute coronary syndrome had higher mortality compared with patients with negative troponin without acute coronary syndrome (hazard ratio = 3.99; 95% confidence interval, 2.36-6.75; P < .001) and similar prognosis as patients with acute coronary syndrome. Conclusions: Troponin elevation is an important predictor of mortality, regardless of the patient's final diagnosis (AU)

Efficient algorithm for simulation of isoelectric focusing.

IEF simulation is an effective tool to investigate the transport phenomena and separation performance as well as to design IEF microchip. However, multidimensional IEF simulations are computationally intensive as one has to solve a large number of mass conservation equations for ampholytes to simulate a realistic case. In this study, a parallel scheme for a 2D IEF simulation is developed to reduce the computational time. The calculation time for each equation is analyzed to identify which procedure is suitable for parallelization. As expected, simultaneous solution of mass conservation equations of ampholytes is identified as the computational hot spot, and the computational time can be significantly reduced by parallelizing the solution procedure for that. Moreover, to optimize the computing time, electric potential behavior during transient state is investigated. It is found that for a straight channel the transient variation of electric potential along the channel is negligible in a narrow pH range (5∼8) IEF. Thus the charge conservation equation is solved for the first time step only, and the electric potential obtain from that is used for subsequent calculations. IEF simulations are carried out using this algorithm for separation of cardiac troponin I from serum albumin in a pH range of 5-8 using 192 biprotic ampholytes. Significant reduction in simulation time is achieved using the parallel algorithm. We also study the effect of number of ampholytes to form the pH gradient and its effect in the focusing and separation behavior of cardiac troponin I and albumin. Our results show that, at the completion of separation phase, the pH profile is stepwise for lower number of ampholytes, but becomes smooth as the number of ampholytes increases. Numerical results also show that higher protein concentration can be obtained using higher number of ampholytes.

Expression and assembly of active human cardiac troponin in Escherichia coli.

Cardiomyopathy-related mutations in human cardiac troponin subunits, including troponin C (hcTnC), troponin I (hcTnI), and troponin T (hcTnT), are well-documented. Recently, it has been recognised that human cardiac troponin (hcTn) is a sophisticated allosteric system. Therefore, the effect of drugs on this protein complex should be studied with assembled hcTn rather than a short fragment of a subunit or the subunit itself. Here, we describe the expression and assembly of active hcTn in Escherichia coli, a novel method that is rapid and simple, and produces large amounts of functional hcTn.

Purification of tropomyosin, paramyosin, actin, tubulin, troponin and kinases for chemiproteomics and its application to different scientific fields.

BACKGROUND: p-aminobenzamidine (p-ABA) is used as a ligand in the purification of many serine proteases and in their removal from heterogeneous samples. Moreover, p-ABA has a potent ability to bind Ca(2+)-binding proteins. The binding ability and use of p-ABA in purification processes is still not fully understood. METHODOLOGY/PRINCIPAL FINDINGS: A p-Aminobenzamidine (p-ABA) ligand enabled the purification of the panallergenic proteins tropomyosin and paramyosin, as well as actin, tubulin, troponin and several kinases and annexins, with variable specificity depending on the tissue source and slight modifications to the purification process. The high affinity of p-ABA to tropomyosin, paramyosin, actin, troponin and myosin is calcium-dependent, since calcium regulates the function of these proteins. In addition, p-ABA probably simulates phosphorylated serine and therefore purified appropriate kinases. Because p-ABA binds to calcium-dependent proteins, and probably those with binding sites containing serine, it is not a suitable inhibitor of proteolysis during the purification of such proteins. p-ABA is widely used to inhibit proteases during protein purification processes, but it is used in columns here to purify non-protease proteins. Two strategies were applied; the first was the inactivation of proteases that were not of interest using protease inhibitors. The second strategy employed was the use of a Ca(2+) wash solution to remove calcium-dependent proteins. The removal of calcium-dependent proteins from rabbit hind muscle pointed out even more selective purification. It is possible to obtain two purified samples: a) calcium dependent proteins and b) calcium independent proteins. Moreover, p-ABA may be useful as a model to study processes involving the phosphorylation of serine. CONCLUSION: A p-Aminobenzamidine (p-ABA) ligand enabled the purification of non-protease proteins, with variable specificity depending on the tissue source and slight modifications to the purification process. The method is applicable to various scientific branches, but is especially practical for medicinal applications.

Extraction and replacement of the tropomyosin-troponin complex in isolated myofibrils.

Tropomyosin (Tm) is an essential component in the regulation of striated muscle contraction. Questions about Tm functional role have been difficult to study because sarcomere Tm content is not as easily manipulated as Troponin (Tn). Here we describe the method we recently developed to replace Tm-Tn of skeletal and cardiac myofibrils from animals and humans to generate an experimental model of homogeneous Tm composition and giving the possibility to measure a wide range of mechanical parameters of contraction (e.g. maximal force and kinetics of force generation). The success of the exchange was determined by SDS-PAGE and by mechanical measurements of calcium dependent force activation on the reconstituted myofibrils. In skeletal and cardiac myofibrils, the percentage of Tm replacement was higher than 90%. Maximal isometric tension was 30-35% lower in the reconstituted myofibrils than in control myofibrils but the rate of force activation (k(ACT)) and that of force redevelopment (k(TR)) were not significantly changed. Preliminary results show the effectiveness of Tm replacement in human cardiac myofibrils. This approach can be used to test the functional impact of Tm mutations responsible for human cardiomyopathies.

Troponin is a potential regulator for actomyosin interactions.

Troponin extracted from rabbit skeletal muscle directly binds to an actin filament in a molar ratio of 1:1 even in the absence of tropomyosin. An actin filament decorated with troponin did not exhibit significant difference from pure actin filaments in the maximum rate of actomyosin ATP hydrolysis and the sliding velocity of the filament examined by means of an in vitro motility assay. However, the relative number of troponin-bound actin filaments moving in the absence of calcium ions decreased to half that in their presence. The amount of HMM bound to the filaments was less than 4% of actin monomers in the presence of TNs. In addition, actin filaments could not move when Tn molecules were bound in the molar ratio of about 1:1 although they sufficiently bind to myosin heads. These results indicate that troponin can transform an actin monomer within a filament into an Off-state without sterically blocking of the myosin-binding sites with tropomyosin molecules.

Expression and purification of human cardiac troponin subunits and their functional incorporation into isolated cardiac mouse myofibrils.

The three subunits of the human cardiac troponin complex (hcTnC, hcTnI, hcTnT) were overexpressed in E. coli, purified and reconstituted to form the hcTn complex. This complex was then incorporated into subcellular bundles of mouse cardiac myofibrils whereby the native mcTn complex was replaced. On thus exchanged myofibrils, isometric force kinetics following sudden changes in free Ca(2+) concentration were measured using atomic force cantilevers. Following the exchange, the myofibrillar force remained fully Ca(2+) regulated, i.e. myofibrils were completely relaxed at pCa 7.5 and developed the same maximum Ca(2+)-activated isometric force upon increasing the pCa to 4.5 as unexchanged myofibrils. The replacement of endogenous mcTn by wild-type hcTn neither altered the kinetics of Ca(2+)-induced force development of the mouse myofibrils nor the kinetics of force relaxation induced by the sudden, complete removal of Ca(2+). Preparations of functional Tn reconstituted myofibrils provide a promising model to study the role of Tn in kinetic mechanisms of cardiac myofibrillar contraction and relaxation.

Overexpression of human cardiac troponin in Escherichia coli: its purification and characterization.

All three subunits of the human cardiac troponin complex (cTn), namely the major isoform of the tropomyosin binding subunit (hcTnT3), the inhibitory subunit (cTnI), and the calcium binding subunit (cTnC), have been coexpressed in Escherichia coli. The cDNAs of each subunit have been cloned into the pSBET vector and transformed into E. coli. The coexpressed subunits assembled within the bacterial cells to form the hcTn complex (hcTnT3.hcTnI.hcTnC). The complex was isolated and purified by three chromatographic steps. Per 6-L cell culture about 10 mg of a highly purified troponin complex showing the expected 1:1:1 molar ratio of hcTnT3:cTnI:cTnC was obtained. Upon phosphorylation by protein kinase A at Ser22 and Ser23 in cTnI, this recombinant troponin complex shows a nearly identical (31)P NMR spectrum to the native one isolated from bovine heart. By measuring the rate of myosin S1 binding to reconstituted thin filaments it was shown that the dependence of the regulation of S1 binding upon calcium concentration and bisphosphorylation was comparable to the native complex.

An improved method for exchanging troponin subunits in detergent skinned rat cardiac fiber bundles.

We describe a method for the removal of endogenous troponin (Tn) complex from bundles of detergent-treated cardiac fibers. After 70 min treatment with cTnT-cTnI most of the endogenous Tn complex was removed from fiber bundles. Complete reconstitution of the Tn complex was achieved by reconstituting with cardiac troponin C (cTnC) in fully relaxing conditions. Ca(2+)-dependent maximum force of the fibers treated with cTnT-cTnI or cTnT-cTnI(33-211), which was used to aid in the visualization of the troponin exchange, decreased to 85-90% of the force developed by fibers before the treatment. SDS-PAGE analysis of the cTnT-cTnI(33-211) and the cTnT(77-289)-cTnI(33-211) treated fiber bundles demonstrated that 70-80% of the endogenous Tn subunits were removed. After reconstitution with cTnC, approximately 80-85% of the Ca(2+)-regulated force was restored in cTnT-cTnI/cTnI(33-211) treated fibers. Our results demonstrate that by minimizing the prolonged exposure of skinned cardiac fiber bundles to rigor conditions, successful exchange of all three subunits of the Tn complex can be accomplished with minimal loss of function.

The isolation of differentially expressed cDNA clones from the filarial nematode Brugia pahangi.

A cDNA library constructed from 3 day post-infective L3 of the filarial nematode Brugia pahangi was screened by differential hybridization with cDNA probes prepared from different life-cycle stages. Five cDNA clones hybridizing selectively to the mosquito-derived L3 probe were isolated and characterized. Northern blot analysis of 4 of the clones confirmed that each was most highly expressed in the mosquito-derived L3. The expression of each mRNA during parasite development in the mosquito vector was investigated using RT-PCR, and all were shown to be abundant in the immature L3. Four of the 5 cDNAs cloned coded for structural proteins: 2 cuticular collagens, and the muscle proteins tropomyosin and troponin. Further studies on troponin using an antiserum raised to the recombinant protein demonstrated that the protein, unlike the mRNA, was present in all life-cycle stages examined, while immunogold labelling demonstrated that it was localized to the muscle blocks.

Characterization of the cardiac holotroponin complex reconstituted from native cardiac troponin T and recombinant I and C.

Cardiac troponin I (cTnI), the inhibitory subunit of cardiac troponin (cTn), is phosphorylated by the cAMP-dependent protein kinase A at two adjacently located serine residues within the heart-specific N-terminal elongation. Four different phosphorylation states can be formed. To investigate each monophosphorylated form cTnI mutants, in which each of the two serine residues is replaced by an alanine, were generated. These mutants, as well as the wild-type cardiac troponin I (cTnI-WT) have been expressed in Escherichia coli, purified and characterized by isoelectric focusing, MS and CD-spectroscopy. Monophosphorylation induces conformational changes within cTnI that are different from those induced by bisphosphorylation. Functionality was assessed by measuring the calcium dependence of myosin S1 binding to thin filaments containing reconstituted native, wild-type and mutant cTn complexes. In all cases a functional holotroponin complex was obtained. Upon bisphosphorylation of cTnI-WT the pCa curve was shifted to the right to the same extent as that observed with bisphosphosphorylated native cTnI. However, the absolute values for the midpoints were higher when recombinant cTn subunits were used for reconstitution. Reconstitution itself changed the calcium affinity of cTnC: pCa50-values were higher than those obtained with the native cardiac holotroponin complex. Apparently only bisphosphorylation of cTnI influences the calcium sensitivity of the thin filament, thus monophosphorylation has a function different from that of bisphosphorylation; this function has not yet been identified.

Functional analysis of human cardiac troponin by the in vitro motility assay: comparison of adult, foetal and failing hearts.

OBJECTIVE: Human cardiac development and heart failure are associated with altered troponin isoform expression and phosphorylation. As the functional effects of these changes in troponin are unknown, we isolated troponin from human foetal, normal adult and failing adult hearts and investigated their regulatory function. METHODS: Human cardiac troponin was assayed for regulatory function by in vitro motility assay and for protein content by SDS PAGE and immunoblotting. RESULTS: Human cardiac troponin regulated movement of actin-tropomyosin filaments over a bed of immobilised heavy meromyosin. At pCa 9, troponin from foetal and adult hearts reduced the fraction of filaments moving from 90% to less than 15% with a modest (25-30%) decrease in velocity. At pCa 5, troponin from normal adult hearts increased filament velocity by up to 47 +/- 3% with no change in the fraction of filaments moving. Foetal troponin increased velocity by only 4 +/- 6% and the effect of troponin from failing hearts was between these values at 31 +/- 5%. Foetal hearts showed different troponin I and T isoform expression compared with adult hearts. No differences in troponin isoform expression were demonstrated between normal and failing adult hearts. CONCLUSIONS: Functioning troponin and tropomyosin may be isolated from human heart and their properties investigated by in vitro motility assay. Both functional and isoform expression differences exist between foetal and adult cardiac troponin. The regulatory function of troponin from adults with end stage heart failure is different from normal adult troponin. These data suggest a role for altered troponin function in human cardiac development and heart failure.

Isolation of rabbit liver heat shock protein with molecular weight 90 kD (Hsp90) and its interaction with troponin components and calponin.

Using a modified method consisting of chromatography on phenyl-Sepharose, Q-Sepharose, and hydroxyapatite, we isolated a highly purified heat shock protein with molecular weight 90 kD (Hsp90) from rabbit liver. The isolated protein was recognized on immunoblot by commercially available monoclonal anti-Hsp90 antibodies. The chromatographic properties, interaction with actin and calmodulin, phosphorylation in the presence of Mg-ATP, and one-dimensional peptide maps of rabbit liver Hsp90 are similar to the corresponding properties of Hsp90 isolated from other sources. In the presence of soluble carbodiimide and N-hydroxysuccinimide, rabbit liver Hsp90 can be cross-linked with calmodulin, troponin C, troponin I, and calponin. The data obtained indicate that Hsp90 may participate in the assembly of regulatory proteins of the actin filament.

Incorporation of the troponin regulatory complex of post-ischemic stunned porcine myocardium reduces myofilament calcium sensitivity in rabbit psoas skeletal muscle fibers.

Decreased calcium sensitivity of tension in post-ischemic myocardium is thought to be a mechanism of depressed function in stunning. The purpose of this study was to determine if the decrease in calcium sensitivity of tension results from ischemia and/or reperfusion-induced alterations in the thin filament regulatory troponin. The experiments utilized an open-chest porcine model of regional LAD myocardial stunning that has previously been shown to cause a decrease in calcium sensitivity of tension in permeabilized myocytes. Stunning was induced by 45 min of low-flow ischemia to the left anterior descending (LAD) coronary artery perfusion bed, which was followed by 30 min of reperfusion. Regional LAD function after reperfusion was 0.5+/-2.8%, as assessed by systolic wall thickening (v 23.9+/-4.1% thickening in control, P<0.001). Core biopsy samples from control circumflex and stunned LAD myocardium were acquired from each heart (n=9) after LAD reperfusion, and were used to obtain purified troponin complexes. Isometric tension-pCa relationships were measured in permeabilized psoas skeletal fibers before and after partial exchange of cardiac troponin from either control circumflex (n=6) or stunned LAD (n=8) myocardium for endogenous skeletal troponin. Calcium sensitivity of tension as assessed by pCa50 (i.e. pCa for half-maximal tension) was unchanged after exchange of troponin from control circumflex myocardium (pCa50=5. 98+/-0.02 v 5.96+/-0.06), but there was a significant decrease in calcium sensitivity of tension after exchange of troponin from stunned LAD myocardium (pCa50=5.97+/-0.07 v 5.82+/-0.05, P<0.05). We conclude that the decrease in calcium sensitivity of tension in postischemic stunned myocardium is, in part, due to ischemia and/or reperfusion-induced alterations in the cardiac troponin regulatory complex.