Roles for the troponin tail domain in thin filament assembly and regulation. A deletional study of cardiac troponin T.
Striated muscle contraction is regulated by Ca2+ binding to troponin, which has a globular domain and an elongated tail attributable to the NH2-terminal portion of the bovine cardiac troponin T (TnT) subunit. Truncation of the bovine cardiac troponin tail was investigated using recombinant TnT fragments and subunits TnI and TnC. Progressive truncation of the troponin tail caused progressively weaker binding of troponin-tropomyosin to actin and of troponin to actin-tropomyosin. A sharp drop-off in affinity occurred with NH2-terminal deletion of 119 rather than 94 residues. Deletion of 94 residues had no effect on Ca2+-activation of the myosin subfragment 1-thin filament MgATPase rate and did not eliminate cooperative effects of Ca2+ binding. Troponin tail peptide TnT1-153 strongly promoted tropomyosin binding to actin in the absence of TnI or TnC. The results show that the anchoring function of the troponin tail involves interactions with actin as well as with tropomyosin and has comparable importance in the presence or absence of Ca2+. Residues 95-153 are particularly important for anchoring, and residues 95-119 are crucial for function or local folding. Because striated muscle regulation involves switching among the conformational states of the thin filament, regulatory significance for the troponin tail may arise from its prominent contribution to the protein-protein interactions within these conformations. (+info)
Tissue-specific distribution of breast-muscle-type and leg-muscle-type troponin T isoforms in birds.
In order to show the tissue-specific distribution of troponin T (TnT) isoforms in avian skeletal muscles, their expression was examined by electrophoresis of the breast and leg muscles of seven avian species and immunoblotting with the antiserum against fast skeletal muscle TnT. It has been reported in the chicken that breast-muscle-type (B-type) and leg-muscle-type (L-type) TnT isoforms are expressed specifically in the adult breast and leg muscles, respectively. Their differential expression patterns were confirmed in all birds examined in this study. The expression of a segment encoded by the exon x series of TnT was also examined by immunoblotting with the antiserum against a synthetic peptide derived from the exon x3 sequence, because the segment has been shown to be included exclusively in the B-type, but not in the L-type TnT. The expression of the segment was found only in the breast muscle, but not in the leg muscle of all birds examined. TnT cDNA sequences from the duck breast and leg muscles were determined and showed that only B-type TnT had an exon x-related sequence, suggesting that the expression of B-type TnT containing the exon x-derived segment is conserved consistently in the birds. (+info)
Ca2+ sensitization and potentiation of the maximum level of myofibrillar ATPase activity caused by mutations of troponin T found in familial hypertrophic cardiomyopathy.
Human wild-type cardiac troponin T, I, C and five troponin T mutants (I79N, R92Q, F110I, E244D, and R278C) causing familial hypertrophic cardiomyopathy were expressed in Escherichia coli, and then were purified and incorporated into rabbit cardiac myofibrils using a troponin exchange technique. The Ca2+-sensitive ATPase activity of these myofibrillar preparations was measured in order to examine the functional consequences of these troponin mutations. An I79N troponin T mutation was found to cause a definite increase in Ca2+ sensitivity of the myofibrillar ATPase activity without inducing any significant change in the maximum level of ATPase activity. A detailed analysis indicated the inhibitory action of troponin I to be impaired by the I79N troponin T mutation. Two more troponin T mutations (R92Q and R278C) were also found to have a Ca2+-sensitizing effect without inducing any change in maximum ATPase activity. Two other troponin T mutations (F110I and E244D) had no Ca2+-sensitizing effects on the ATPase activity, but remarkably potentiated the maximum level of ATPase activity. These findings indicate that hypertrophic cardiomyopathy-linked troponin T mutations have at least two different effects on the Ca2+-sensitive ATPase activity, Ca2+-sensitization and potentiation of the maximum level of the ATPase activity. (+info)
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. (+info)
Diagnostic marker cooperative study for the diagnosis of myocardial infarction.
BACKGROUND: Millions of patients present annually with chest pain, but only 10% to 15% have myocardial infarction. Lack of diagnostic sensitivity and specificity of clinical and conventional markers prevents or delays treatment and leads to unnecessary costly admissions. Comparative data are lacking on the new markers, yet using all of them is inappropriate and expensive. METHODS AND RESULTS: The Diagnostic Marker Cooperative Study was a prospective, multicenter, double-blind study with consecutive enrollment of patients with chest pain presenting to the emergency department. Diagnostic sensitivity and specificity and frequency of increase in patients with unstable angina were determined for creatine kinase-MB (CK-MB) subforms, myoglobin, total CK-MB (activity and mass), and troponin T and I on the basis of frequent serial sampling for +info)
Effects of the prostanoid EP3-receptor agonists M&B 28767 and GR 63799X on infarct size caused by regional myocardial ischaemia in the anaesthetized rat.
1. This study investigates the effects of two agonists of the prostanoid EP3-receptor (M&B 28767 and GR 63799X) on the infarct size caused by regional myocardial ischaemia and reperfusion in the anaesthetized rat. 2. One hundred and sixty-seven, male Wistar rats were anaesthetized (thiopentone, 120 mg kg(-1) i.p.), ventilated (8-10 ml kg(-1), 70 strokes min(-1), inspiratory oxygen concentration: 30%; PEEP: 1-2 mmHg) and subjected to occlusion of the left anterior descending coronary artery (LAD, for 7.5, 15, 25, 35, 45 or 60 min) followed by reperfusion (2 h). Infarct size was determined by staining of viable myocardium with a tetrazolium stain (NBT), histological evaluation by light and electron microscopy and determination of the plasma levels of cardiac troponin T. 3. M&B 28767 (0.5 microg kg(-1) min(-1), i.v., n=7) or GR 63799X (3 microg kg(-1) min(-1), i.v., n=7) caused significant reductions in infarct size from 60+/-3% (25 min ischaemia and 2 h reperfusion; saline-control, n=8) to 39+/-6 and 38+/-4% of the area at risk, without causing a significant fall in blood pressure. Pretreatment of rats with 5-hydroxydecanoate (5-HD), an inhibitor of ATP-sensitive potassium channels, attenuated the cardioprotective effects of both EP3-receptor agonists. The reduction in infarct size afforded by M&B 28767 was also abolished by glibenclamide and the protein kinase C (PKC) inhibitors staurosporine and chelerythrine. 4. Thus, M&B 28767 and GR 63799X reduce myocardial infarct size in the rat by a mechanism(s) which involves the activation of PKC and the opening of ATP-sensitive potassium channels. (+info)
Intraoperative cardiac troponin T release and lactate metabolism during coronary artery surgery: comparison of beating heart with conventional coronary artery surgery with cardiopulmonary bypass.
OBJECTIVE: To compare cardiac troponin T release and lactate metabolism in coronary sinus and arterial blood during uncomplicated coronary grafting on the beating heart with conventional coronary grafting using cardiopulmonary bypass. DESIGN: A prospective observational study with simultaneous sampling of coronary sinus and arterial blood: before and 1, 4, 10, and 20 minutes after reperfusion for analysis of cardiac troponin T and lactate. Cardiac troponin T was also analysed in venous samples taken 3, 6, 24, 48, and 72 hours after surgery. SETTING: Cardiac surgical unit in a tertiary referral centre. PATIENTS: 18 patients undergoing coronary grafting on the beating heart (10 single vessel and eight two-vessel grafting) and eight undergoing two-vessel grafting with cardiopulmonary bypass. RESULTS: Cardiac troponin T was detected in coronary sinus blood in all patients by 20 minutes after beating heart coronary artery surgery before arterial concentrations were consistently increased. Peak arterial and coronary sinus cardiac troponin T values on the beating heart during single (0.03 (0 to 0. 05) and 0.09 (0.07 to 0.16 microg/l, respectively) and two-vessel grafting (0.1 (0.07 to 0.11) and 0.19 (0.14 to 0.25) microg/l) were lower than the values obtained during cardiopulmonary bypass (0.64 (0.52 to 0.72) and 1.4 (0.9 to 2.0) microg/l) (p < 0.05). The area under the curve of venous cardiac troponin T over 72 hours for two-vessel grafting on the beating heart was less than with cardiopulmonary bypass (13 (10 to 16) v 68 (26 to 102) microg.h/l) (p < 0.001). Lactate extraction began within one minute of snare release during beating heart coronary surgery while lactate was still being produced 20 minutes after cross clamp release following cardiopulmonary bypass. CONCLUSIONS: Lower intraoperative and serial venous cardiac troponin T concentrations suggest a lesser degree of myocyte injury during beating heart coronary artery surgery than during cardiopulmonary bypass. Oxidative metabolism also recovers more rapidly with beating heart coronary artery surgery than with conventional coronary grafting. Coronary sinus cardiac troponin T concentrations increased earlier and were greater than arterial concentrations during beating heart surgery, suggesting that this may be a more sensitive method of intraoperative assessment of myocardial injury. (+info)
Increasing levels of interleukin (IL)-1Ra and IL-6 during the first 2 days of hospitalization in unstable angina are associated with increased risk of in-hospital coronary events.
BACKGROUND: A growing body of evidence suggests a role for inflammation in acute coronary syndromes. The aim of this study was to assess the role of proinflammatory cytokines, their time course, and their association with prognosis in unstable angina. METHODS AND RESULTS: We studied 43 patients aged 62+/-8 years admitted to our coronary care unit for Braunwald class IIIB unstable angina. In each patient, serum levels of interleukin-1 receptor antagonist (IL-1Ra), interleukin-6 (IL-6) (which represent sensitive markers of biologically active IL-1beta and tumor necrosis factor-alpha levels, respectively), and troponin T were measured at entry and 48 hours after admission. Troponin T-positive patients were excluded. Patients were divided a posteriori into 2 groups according to their in-hospital outcome: group 1 comprised 17 patients with an uneventful course, and group 2 comprised 26 patients with a complicated in-hospital course. In group 1, mean IL-1Ra decreased at 48 hours by 12%, and IL-6 diminished at 48 hours by 13%. In group 2, IL-1Ra and IL-6 entry levels were higher than in group 1 and increased respectively by 37% and 57% at 48 hours (P<0.01). CONCLUSIONS: These findings indicate that although they receive the same medical therapy as patients who do not experience an in-hospital event, patients with unstable angina and with complicated in-hospital courses have higher cytokine levels on admission. A fall in IL-1Ra and IL-6 48 hours after admission was associated with an uneventful course and their increase with a complicated hospital course. These findings may suggest novel therapeutic approaches to patients with unstable angina. (+info)