A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients'' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.     

Development of an Immunofluorometric, High-Capacity, Cell-Based Assay for the Measurement of Human Type I and Type II Interferons

We have developed a cell-based 96-well microtiter plate, high throughput assay for measuring both type I and type II interferon (IFN) activity on human cells. This assay makes use of a previously described IFN-specific reporter stably expressed in human HT 1080 cells. The induction of the reporter by IFN is determined by measuring the IFN-dependent expression of CD2 on the cell surface. The cytokine-induced expression of CD2 occurs within 48 h and is measured using a time-resolved fluorometric immunoassay. The limit of detection for type I IFN is.0.4 IU/ml. Interassay and intraassay coefficients of variation were 1.1% and 1.3% for the medium control (31 IU IFNb1b/ml), respectively. The limit of detection for type II IFN is.8 IU/ml, and the assay coefficients of variation are similar to those determined for type I IFNs. The level of sensitivity for this assay is comparable to other assays commonly used to measure IFN activity on cells. The current assay has an advantage over antiviral and antiproliferative assays, in that there is no requirement for the use of pathogenic virus or for determining viable cell numbers. The current assay is ideally suited for increasing sample screening and high-capacity automation, making it an excellent tool for drug discovery.     

Elucidation of isoform-dependent pH sensitivity of troponin i by NMR spectroscopy

Myocardial ischemia is characterized by reduced blood flow to cardiomyocytes, which can lead to acidosis. Acidosis decreases the calcium sensitivity and contractile efficiency of cardiac muscle. By contrast, skeletal and neonatal muscles are much less sensitive to changes in pH. The pH sensitivity of cardiac muscle can be reduced by replacing cardiac troponin I with its skeletal or neonatal counterparts. The isoform-specific response of troponin I is dictated by a single histidine, which is replaced by an alanine in cardiac troponin I. The decreased pH sensitivity may stem from the protonation of this histidine at low pH, which would promote the formation of electrostatic interactions with negatively charged residues on troponin C. In this study, we measured acid dissociation constants of glutamate residues on troponin C and of histidine on skeletal troponin I (His-130). The results indicate that Glu-19 comes in close contact with an ionizable group that has a pK(a) of ～6.7 when it is in complex with skeletal troponin I but not when it is bound to cardiac troponin I. The pK(a) of Glu-19 is decreased when troponin C is bound to skeletal troponin I and the pK(a) of His-130 is shifted upward. These results strongly suggest that these residues form an electrostatic interaction. Furthermore, we found that skeletal troponin I bound to troponin C tighter at pH 6.1 than at pH 7.5. The data presented here provide insights into the molecular mechanism for the pH sensitivity of different muscle types.     

Troponin I binds polycystin-L and inhibits its calcium-induced channel activation

Polycystin-L (PCL) is an isoform of polycystin-2, the product of the second gene associated with autosomal dominant polycystic kidney disease, and functions as a Ca(2+)-regulated nonselective cation channel. We recently demonstrated that polycystin-2 interacts with troponin I, an important regulatory component of the actin microfilament complex in striated muscle cells and an angiogenesis inhibitor. In this study, using the two-microelectrode voltage-clamp technique and Xenopus oocyte expression system, we showed that the calcium-induced PCL channel activation is substantially inhibited by the skeletal and cardiac troponin I (60% and 31% reduction, respectively). Reciprocal co-immunoprecipitation experiments demonstrated that PCL physically associates with the skeletal and cardiac troponin I isoforms in overexpressed Xenopus oocytes and mouse fibroblast NIH 3T3 cells. Furthermore, both native PCL and cardiac troponin I were present in human heart tissues where they indeed associate with each other. GST pull-down and microtiter binding assays showed that the C-terminus of PCL interacts with the troponin I proteins. The yeast two-hybrid assay further verified this interaction and defined the corresponding interacting domains of the PCL C-terminus and troponin I. Taken together, this study suggests that troponin I acts as a regulatory subunit of the PCL channel complex and provides the first direct evidence that PCL is associated with the actin cytoskeleton through troponin I.     

Diagnostic and prognostic utility of early measurement with high-sensitivity troponin T assay in patients presenting with chest pain

Background:

High-sensitivity troponin assays are now available for clinical use. We investigated whether early measurement with such an assay is superior to a conventional assay in the evaluation of acute coronary syndromes.

Methods:

Patients presenting to an emergency department with chest pain who did not have ST-segment elevation were prospectively recruited from November 2007 to December 2010. Patients underwent serial testing with a conventional cardiac troponin I assay. Samples were also obtained at presentation and two hours later for measurement of troponin T levels using a high-sensitivity assay. The primary outcome was diagnosis of myocardial infarction on admission; secondary outcomes were death, myocardial infarction and heart failure at one year.

Results:

Of the 939 patients enrolled in the study, 205 (21.8%) had myocardial infarction. By two hours after presentation, the high-sensitivity troponin T assay at the cut-off point of the 99th percentile of the general population (14 ng/L) had a sensitivity of 92.2% (95% confidence interval [CI] 88.1%–95.0%) and a specificity of 79.7% (95% CI 78.6%–80.5%) for the diagnosis of non–ST-segment myocardial infarction. The sensitivity of the assay at presentation was 100% among patients who presented four to six hours after symptom onset. By one year, the high-sensitivity troponin T assay was found to be superior than the conventional assay in predicting death (hazard ratio [HR] 5.4, 95% CI 2.7–10.7) and heart failure (HR 27.8, 95% CI 6.6–116.4), whereas the conventional assay was superior in predicting nonfatal myocardial infarction (HR 4.0, 95% CI 2.4–6.7).

Interpretation:

The high-sensitivity troponin T assay at the cut-off point of the 99th percentile was highly sensitive for the diagnosis of myocardial infarction by two hours after presentation and had prognostic utility beyond that of the conventional assay. To rule out myocardial infarction, the optimal time to test a second sample using the high-sensitivity troponin T level may be four to six hours after symptom onset, but this finding needs verification in future studies before it can become routine practice.For novel cardiac markers to be clinically useful in diagnosing acute coronary syndromes, they need to show their incremental utility beyond that of existing markers, with therapeutic implications designed to improve patient care. Recent improvement in the performance of troponin assays to comply with current guidelines for the diagnosis of acute myocardial infarction¹ has resulted in a new generation of assays with enhanced clinical sensitivity that are now available for use in clinical care. Assays with high sensitivity have been shown to detect myocardial injury earlier^2–8 and identify more patients at risk of future adverse outcomes^8–10 than conventional assays.We conducted a study to assess whether early measurement (at presentation and two hours later) with a high-sensitivity troponin T assay could (a) effectively rule out myocardial infarction without the need for later measurement of troponin levels and (b) identify more patients at risk of adverse cardiac events within one year follow-up compared with a conventional troponin assay.     

Roles of troponin isoforms in pH dependence of contraction in rabbit fast and slow skeletal and cardiac muscles.

Skinned fibers prepared from rabbit fast and slow skeletal and cardiac muscles showed acidotic depression of the Ca2+ sensitivity of force generation, in which the magnitude depends on muscle type in the order of cardiac>fast skeletal>slow skeletal. Using a method that displaces whole troponin-complex in myofibrils with excess troponin T, the roles of Tn subunits in the differential pH dependence of the Ca2+ sensitivity of striated muscle were investigated by exchanging endogenous troponin I and troponin C in rabbit skinned cardiac muscle fibres with all possible combinations of the corresponding isoforms expressed in rabbit fast and slow skeletal and cardiac muscles. In fibers exchanged with fast skeletal or cardiac troponin I, cardiac troponin C confers a higher sensitivity to acidic pH on the Ca2+ sensitive force generation than fast skeletal troponin C independently of the isoform of troponin I present. On the other hand, fibres exchanged with slow skeletal troponin I exhibit the highest resistance to acidic pH in combination with either isoform of troponin C. These results indicate that troponin C is a determinant of the differential pH sensitivity of fast skeletal and cardiac muscles, while troponin I is a determinant of the pH sensitivity of slow skeletal muscle.     

Sensitivity of actomyosin ATPase to calcium and strontium ions. Effect of hybrid troponins

1. Hybrid or reconstituted troponins were prepared from troponin components of rabbit skeletal muscle and porcine cardiac muscle and their effect on the actomyosin ATPase activity was measured at various concentrations of Ca2+ or Sr2+. The Ca2+ concentration required for half-maximum activation of actomyosin ATPase with troponin containing cardiac troponin I was slightly higher than that with troponin containing skeletal troponin I. The Sr2+ concentration required for half-maximum activation of actomyosin ATPase with troponin containing skeletal troponin C was higher than that with troponin containing cardiac troponin C. 2. Reconstituted cardiac troponin was phosphorylated by cyclic AMP-dependent protein kinase. The Ca2+ sensitivity of actomyosin ATPase with cardiac troponin decreased upon phosphorylation of troponin I; maximum ATPase activity was depressed and the Ca2+ concentration at half-maximum activation increased. On the other hand, phosphorylation of troponin I did not change Sr2+ sensitivity. 3. The inhibitory effect of cardiac troponin I on the actomyosin ATPase activity was neutralized by increasing the amount of brain calmodulin at high Ca2+ and Sr2+ concentrations but not at low concentrations. 4. ATPase activity of actomyosin with a mixture of troponin I and calmodulin was assayed at various concentrations of Ca2+ or Sr2+. The Ca2+ or Sr2+ sensitivity of actomyosin ATPase containing skeletal troponin I was approximately the same as that of actomyosin ATPase containing cardiac troponin I. Phosphorylation of cardiac troponin I did not change the Ca2+ sensitivity of the ATPase. 5. The Ca2+ or Sr2+ concentration required for half-maximum activation of actomyosin ATPase with troponin I-T-calmodulin was higher than that of actomyosin ATPase with the mixture of troponin I and calmodulin. Maximum ATPase activity was lower than that with the mixture of troponin I and calmodulin.     

Modulation of cardiac troponin C-cardiac troponin I regulatory interactions by the amino-terminus of cardiac troponin I

Multidimensional heteronuclear magnetic resonance studies of the cardiac troponin C/troponin I(1-80)/troponin I(129-166) complex demonstrated that cardiac troponin I(129-166), corresponding to the adjacent inhibitory and regulatory regions, interacts with and induces an opening of the cardiac troponin C regulatory domain. Chemical shift perturbation mapping and (15)N transverse relaxation rates for intact cardiac troponin C bound to either cardiac troponin I(1-80)/troponin I(129-166) or troponin I(1-167) suggested that troponin I residues 81-128 do not interact strongly with troponin C but likely serve to modulate the interaction of troponin I(129-166) with the cardiac troponin C regulatory domain. Chemical shift perturbations due to troponin I(129-166) binding the cardiac troponin C/troponin I(1-80) complex correlate with partial opening of the cardiac troponin C regulatory domain previously demonstrated by distance measurements using fluorescence methodologies. Fluorescence emission from cardiac troponin C(F20W/N51C)(AEDANS) complexed to cardiac troponin I(1-80) was used to monitor binding of cardiac troponin I(129-166) to the regulatory domain of cardiac troponin C. The apparent K(d) for cardiac troponin I(129-166) binding to cardiac troponin C/troponin I(1-80) was 43.3 +/- 3.2 microM. After bisphosphorylation of cardiac troponin I(1-80) the apparent K(d) increased to 59.1 +/- 1.3 microM. Thus, phosphorylation of the cardiac-specific N-terminus of troponin I reduces the apparent binding affinity of the regulatory domain of cardiac troponin C for cardiac troponin I(129-166) and provides further evidence for beta-adrenergic modulation of troponin Ca(2+) sensitivity through a direct interaction between the cardiac-specific amino-terminus of troponin I and the cardiac troponin C regulatory domain.     

Development and optimization of immunoassays for the detection of botulinum toxins

Monoparametric immunoassay tests for detecting botulinum toxins types A and B and multiparametric assays for simultaneous detection of botulinum toxins type A and B have been developed. It is shown that the sensitivity of assays is affected by the size of nanoparticles of colloidal gold used as a marker of antibodies, load intensity of antibodies of colloidal gold in conjugates, the type of analytical membranes, as well as the chemical composition of buffer solutions used for the storage of conjugates and immunoassay analysis. The detection limit of monoparametric immunoassay tests is 0.5 ng/ml; that of multiparametric assays, 5.0 ng/ml. The developed immunoassay can be used for rapid assay of product quality, for grade control of botulinum toxins in pharmaceuticals, and environmental monitoring.     

Cardiac troponin I is a sensitive, specific biomarker of cardiac injury in laboratory animals

This study directly demonstrates that cardiac troponin I (cTnI) is a sensitive, specific, and persistent biomarker in laboratory animals. Histopathological and pathophysiological cardiac changes in dogs, rats and mice correlated with increased serum cTnI with various cardiac inotropic agents, and cardiotoxic drugs and with cardiac arrhythmias, tachycardia, cardiac effusion with dyspnoea, and ageing. A comparison of six immunoassays for cTnI and cardiac troponin T (cTnT) to detect and monitor cardiac injury in a rodent model indicated that enzyme-linked immunosorbent (Life Diagnostics Inc and TriChem Resources Inc, West Chester, Philadelphia, USA) and Immulite (Diagnostic Products Corporation, Llanberis, UK) assays had low sensitivity and less than 1% of the dynamic range of Centaur (Bayer Healthcare Diagnostics, Newbury, UK) cTnI and Elecsys (Roche Diagnostics, Basel, Switzerland) and M8 (Bioveris Europe, Whitney, UK) cTnT assays. In dogs, however, the Immulite assay was effective and correlated with the Centaur. Serum concentrations were highly correlated but 10-fold lower for cTnT compared with cTnI with cardiac injury. Centaur assay also detected cTnI in myocardium from marmosets, swine, cattle, and guinea pigs, indicating it to be candidate cardiac biomarker for these species as well. Purified rat cTnI was 50% more reactive than purified human cTnI in the Centaur assay. In the rat, an age- and gender-dependent variation in serum cTnI was found. Male rats aged six and eight months had a 10-fold greater serum cTnI than age-matched females and three-month-old rats. These increases correlated with minimal histopathological change. Isoproterenol-induced serum cTnI increased up to 760-fold the minimal detectable concentration of 0.07 microg/L, within 4-6 h and decreased with a half-life of 6 h, with an expected return to baseline of 60 h. Severity of histopathological change correlated with serum cTnI during the ongoing injury.     

Clinical and analytical performance of automated immunochemiluminometric assays for determination of cardiac markers in serum

Three new immunochemiluminometric assays for quantitation of cardiac markers, i.e. creatine kinase isoenzyme MB (CK-MB), myoglobin and cardiac troponin I (cTnI), were evaluated with the Sanofi Access analyser. The complete profile requires 20 min to perform, the method being suitable in true stat situations. In patients with early myocardial infarction (median time of sample collection: 210 min from onset, range 30–450; n = 44), the diagnostic sensitivity of Access cTnI was 66%, compared with 80% for myoglobin, and 43% for CK-MB. For comparison, cTnI, with an automated immunofluorimetric assay was also measured (sensitivity, 45%; p < 0.05 vs. Access cTnI). Our data confirmed myoglobin as the first biochemical marker to appear elevated after infarction. However, cTnI may be a more sensitive marker for early detection of cardiac damage than initially thought, when determined by an ultrasensitive method such as an immunochemiluminometric assay. © 1998 John Wiley & Sons, Ltd.     

Development and optimization of immunoassays for the detection of botulinum toxins

Monoparametric immunoassay tests for detecting botulinum toxins types A and B and multiparametric assays for simultaneous detection of botulinum toxins type A and B have been developed. It is shown that the sensitivity of assays is affected by the size of nanoparticles of colloidal gold used as a marker of antibodies, load intensity of antibodies of colloidal gold in conjugates, the type of analytical membranes, as well as the chemical composition of buffer solutions used for the storage of conjugates and immunoassay analysis. The detection limit of monoparametric immunoassay tests is 0.5 ng/ml; that of multiparametric assays, 5.0 ng/ml. The developed immunoassay can be used for rapid assay of product quality, for grade control of botulinum toxins in pharmaceuticals, and environmental monitoring.     

Development and comparison of high-performance liquid chromatographic methods with tandem mass spectrometric and ultraviolet absorbance detection for the determination of cyclobenzaprine in human plasma and urine

Sensitive assays for the determination of cyclobenzaprine (I) in human plasma and urine were developed utilizing high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS-MS) and ultraviolet (UV) absorbance detections. These two analytical techniques were evaluated for reliability and sensitivity, and applied to support pharmacokinetic studies. Both methods employed a liquid-liquid extraction of the compound from basified biological sample. The organic extract was evaporated to dryness ,the residue was reconstituted in the mobile phase and injected onto the HPLC system. The HPLC assay with MS-MS detection was performed on a PE Sciex API III tandem mass spectrometer using the heated nebulizer interface. Multiple reaction monitoring using the parent → daughter ion combinations of m/z 276 → 215 and 296 → 208 was used to quantitate I and internal standard (II), respectively. The HPLC-MS-MS and HPLC-UV assays were validated in human plasma in the concentration range 0.1–50 ng/ml and 0.5–50 ng/ml, respectively. In urine, both methods were validatedin the concentration range 10–1000 ng/ml. The precision of the assays, as expressed as coefficients of variation (C.V.) was less than 10% over the entire concentration range, with adequate assay specificity and accuracy. In addition to better sensitivity, the HPLC-MS-MS assay was more efficient and allowed analysis of more biological fluid samples in a single working day than the HPLC-UV method.     

High affinity peptides for the recognition of the heart disease biomarker troponin I identified using phage display

Troponin I is a specific and sensitive clinical biomarker for myocardial injury. In this study we have used polyvalent phage display to isolate unique linear peptide motifs which recognize both the human and rat homologs of troponin I. The peptide specific for human troponin I has a sequence of FYSHSFHENWPS and the peptide specific for the rat troponin I has a sequence of FHSSWPVNGSTI. Enzyme‐linked immunosorbent assays (ELISAs) were used to evaluate the binding interactions, and the two phage‐displayed peptides exhibited some cross‐reactivity, but they were both more specific for the troponin I homolog they were selected against. The binding affinities of the phage‐displayed peptides were decreased by the presence of complex tissue culture media (MEM), and the addition of 10% calf serum further interfered with the binding of the target proteins. Kinetic indirect phage ELISAs revealed that both troponin I binding peptides were found to have nanomolar affinities for the troponin proteins while attached to the phage particles. To our knowledge, this is the first example of isolation and characterization of troponin I binders using phage display technology. These new peptides may have potential utility in the development of new clinical assays for cardiac injury as well as in monitoring of cardiac cells grown in culture. Biotechnol. Bioeng. 2010. 105: 678–686. © 2009 Wiley Periodicals, Inc.     

USE OF POLYMYXIN B IN A CAPTURE ENZYME IMMUNOASSAY FOR DETECTION OF SALMONELLAE SPP. LIPOPOLYSACCHARIDE

A capture enzyme immunoassay for detection of salmonellae sp. lipopolysaccharide was developed. The assay made use of polymyxin B sulfate, passively attached to a polystyrene matrix, to capture lipopolysaccharide. Bound lipopolysaccharride was then detected with a monoclonal antibody, specific for salmonellae spp. followed by goat antimouse antibody conjugated with horseradish peroxidase.
The analytical sensitivity of the assay was approximately 1 ng/ml of lipopolysaccharide. The results are comparable to those obtained with a competitive enzyme immunoassay previously developed. The sensitivity of the polymyxin B assay decreased to 4–5 ng/ml when the salmonellae spp. lipopolysaccharide was mixed with 1–100 μg/ml of Escherichia coli lipopolysaccharide, while this level of heterogeneous lipopolysaccharide, did not decrease the sensitivity of the competitive enzyme immunoassay.
The polymyxin B capture assay was advantageous in that polymyxin B is a standardized reagent that is relatively inexpensive and does not require extensive preparation or containment facilities. The assay is robust; however, because of the light sensitivity of polymyxin B, its stickiness to other reagents and interference by other lipopolysaccharides, this assay requires careful attention to detail and may therefore be an unsuitable assay for field use.     

Fluorometric microassays for the determination of cathepsin L and cathepsin S activities in tissue extracts.

We established a continuous semi-microassay, and for large-scale studies both a stopped and a continuous microtiter plate assay for the fluorometric determination of cathepsin L and cathepsin S activities in body fluids, tissues or cell extracts in the presence of cathepsin B. For the detection of enzymatic activities we used the synthetic substrate Z-Phe-Arg-AMC, and for discrimination between cathepsin L, S and cathepsin B the specific inhibitor CA-074 for blocking interfering cathepsin B activities was applied. Furthermore, we took advantage of the stability of cathepsin S at pH 7.5 for further differentiation between cathepsin L and cathepsin S activities. The kinetic assays were characterized in terms of imprecision, analytical sensitivity, accuracy and substrate concentration. The within-run coefficients of variation were found to be 4.9%-7.2% for the continuous semi-microassay, 10.3%-11.7% for the stopped, and 4.5%-11.8% for the continuous microtiter plate assay. The between-days coefficients of variation for the continuous semi-microassay were 8.1%-8.9%, while for the stopped and continuous microtiter plate assays the coefficients were 11.2%-13.5% and 5.8%-12.2%, respectively. Compared to the continuous semi-microassay, the stopped and the continuous microtiter plate assays showed 3-fold and 11-fold higher sensitivity, respectively. Comparison between the continuous enzyme activity assays at substrate concentrations of 40 microM and 200 microM demonstrated a significant correlation of r = 0.97 and r = 0.99, respectively. The newly developed microtiter plate assay will allow efficient, sensitive and high precision determination of cathepsin L and cathepsin S activities in large-scale studies of cysteine-cathepsin dependent diseases.     

Functional consequences of the deletion mutation deltaGlu160 in human cardiac troponin T

To explore the functional consequences of a deletion mutation of troponin T (DeltaGlu160) found in familial hypertrophic cardiomyopathy, the mutant human cardiac troponin T, and wild-type troponins T, I, and C were expressed in Escherichia coli and directly incorporated into isolated porcine cardiac myofibrils using our previously reported troponin exchange technique. The mutant troponin T showed a slightly reduced potency in replacing the endogenous troponin complex in myofibrils and did not affect the inhibitory action of troponin I but potentiated the neutralizing action of troponin C, suggesting that the deletion of a single amino acid, Glu-160, in the strong tropomyosin-binding region affects the tropomyosin binding affinity of the entire troponin T molecule and alters the interaction between troponin I and troponin C within ternary troponin complex in the thin filament. This mutation also increased the Ca(2+) sensitivity of the myofibrillar ATPase activity, as in the case of other mutations in troponin T with clinical phenotypes of poor prognosis similar to that of Glu160. These results provide strong evidence that the increased Ca(2+) sensitivity of cardiac myofilament is a typical functional consequence of the troponin T mutation associated with a malignant form of hypertrophic cardiomyopathy.     

Changes in cardiac troponins with gestational age explain changes in cardiac muscle contractility in the sheep fetus

The development of the adult cardiac troponin complex in conjunction with changes in cardiac function and cardiomyocyte binucleation has not been systematically characterized during fetal life in a species where maturation of the cardiomyocytes occurs prenatally as it does in the human. The aim of this study was to correlate the expression of each of the major adult troponin isoforms (T, I, and C) during late gestation (term of 150 days) to changes in both Ca(2+) sensitivity and maximum Ca(2+)-activated force of the contractile apparatus and the maturation of cardiomyocytes. The percentage of mononucleated cardiomyocytes in the right ventricle decreased with gestational age to 46% by 137-142 days of gestation. The length of binucleated cardiomyocytes did not change with gestational age, but the length of binucleated cardiomyocytes relative to heart weight decreased with gestational age. There was no change in the expression of adult cardiac troponin T with increasing gestation. The contractile apparatus was significantly more sensitive to Ca(2+) at 90 days compared with either 132 or 139 days of gestation, consistent with an ～30% increase in the expression of adult cardiac troponin I between 90 and 110 days of gestation. Maximum Ca(2+)-activated force significantly increased from 90 days compared with 130 days consistent with an increase of ～40% in cardiac troponin C protein expression. These data show that increased adult cardiac troponin I and C protein expression across late gestation is consistent with reduced Ca(2+) sensitivity and increased maximum Ca(2+)-activated force. Furthermore, changes in cardiac troponin C, not I, protein expression track with the timing of cardiomyocyte binucleation.     

An enzyme immunoassay of estrone in swine serum

An enzyme immunoassay for estrone in swine serum was established. For this, beta-galactosidase from E. coli was conjugated through estrone-17 (O-carboxymethyl)oxime using a mixed anhydride reaction. The percentage of immunoreactive estrone-17 (O-carboxymethyl)oxime-beta-galactosidase conjugate was estimated to be about 70%. The recovery rate of estrone (25-500 pg) added to 0.05 ml of swine serum averaged 91.4%. The sensitivity of the present enzyme immunoassay was 5 pg/tube. The coefficients of variation (CV) were 5.9-8.2% (within assays) and 4.1-5.9% (between assays), respectively. Estrone values determined by the present enzyme immunoassay were highly correlated with those determined by radioimmunoassay (r = 0.99, P less than 0.005). This method of enzyme immunoassay was determined to be suitable for the routine assay of serum estrone.     

Engineered troponin C constructs correct disease-related cardiac myofilament calcium sensitivity

Aberrant myofilament Ca(2+) sensitivity is commonly observed with multiple cardiac diseases, especially familial cardiomyopathies. Although the etiology of the cardiomyopathies remains unclear, improving cardiac muscle Ca(2+) sensitivity through either pharmacological or genetic approaches shows promise of alleviating the disease-related symptoms. Due to its central role as the Ca(2+) sensor for cardiac muscle contraction, troponin C (TnC) stands out as an obvious and versatile target to reset disease-associated myofilament Ca(2+) sensitivity back to normal. To test the hypothesis that aberrant myofilament Ca(2+) sensitivity and its related function can be corrected through rationally engineered TnC constructs, three thin filament protein modifications representing different proteins (troponin I or troponin T), modifications (missense mutation, deletion, or truncation), and disease subtypes (familial or acquired) were studied. A fluorescent TnC was utilized to measure Ca(2+) binding to TnC in the physiologically relevant biochemical model system of reconstituted thin filaments. Consistent with the pathophysiology, the restrictive cardiomyopathy mutation, troponin I R192H, and ischemia-induced truncation of troponin I (residues 1-192) increased the Ca(2+) sensitivity of TnC on the thin filament, whereas the dilated cardiomyopathy mutation, troponin T ΔK210, decreased the Ca(2+) sensitivity of TnC on the thin filament. Rationally engineered TnC constructs corrected the abnormal Ca(2+) sensitivities of the thin filament, reconstituted actomyosin ATPase activity, and force generation in skinned trabeculae. Thus, the present study provides a novel and versatile therapeutic strategy to restore diseased cardiac muscle Ca(2+) sensitivity.