Real-time myocardial contrast echocardiography for the detection of stress-induced myocardial ischemia. Comparison with 99mTc-sestamibi single photon emission computed tomography.

BACKGROUND: Real-time contrast echocardiography (MCE) is a new promising technique for assessing myocardial perfusion. The purpose of this study was to test whether realtime MCE can be used to detect functionally significant coronary artery stenosis in patients with known or suspected coronary artery disease. Myocardial contrast echocardiographic studies were compared with nearly simultaneous 99mTc-sestamibi single photon emission computed tomography (SPECT) as a clinical standard reference to evaluate regional myocardial perfusion defects. METHODS: Real-time MCE based on continuous infusion of Optison (8-10 ml/h) was performed in 66 patients during standard 99mTc-SPECT dipyridamole (0.56 mg/kg x 4 min) stress testing. Images were obtained in apical 4- and 2-chamber views, each divided into 6 segments. Tracer uptake and myocardial opacification were visually analyzed for each segment by two pairs of blinded observers and graded as normal, mildly reduced, severely reduced, or absent. In 792 myocardial segments, myocardial opacification by MCE was uninterpretable in 143 (18%) segments and tracer uptake by SPECT was not clearly defined in 92 (12%) segments. Interobserver variability for MCE was good with concordance rates of 83% (kappa=0.72) for rest- and 86% (kappa=0.76) for stress images. Overall concordance between MCE and SPECT was good (83%, kappa=0.63) at a segmental level. In the diagnosis of fixed and reversible defects, and of normal perfusion, concordance rates were 73, 65 and 83%, respectively. When analysis was performed at the regional level, we found comparable levels of concordance rates for LAD (83%, kappa=0.59), LCX (86%, kappa=0.64) and RCA (80%, kappa=0.68) perfusion territories. CONCLUSIONS: These findings suggest that realtime MCE is a clinically acceptable method to evaluate myocardial perfusion defects during dipyridamole stress testing.

Identification and characterisation of a novel KCNQ1 mutation in a family with Romano-Ward syndrome.

Romano-Ward syndrome (RWS), the autosomal dominant form of the congenital long QT syndrome, is characterised by prolongation of the cardiac repolarisation process associated with ventricular tachyarrhythmias of the torsades de pointes type. Genetic studies have identified mutations in six ion channel genes, KCNQ1, KCNH2, SCN5A, KCNE1 and KCNE2 and the accessory protein Ankyrin-B gene, to be responsible for this disorder. Single-strand conformation polymorphism (SSCP) analysis and subsequent DNA sequence analysis have identified a KCNQ1 mutation in a family that were clinically conspicuous due to several syncopes and prolonged QTc intervals in the ECG. The mutant subunit was expressed and functionally characterised in the Xenopus oocyte expression system. A novel heterozygous missense mutation with a C to T transition at the first position of codon 343 (CCA) of the KCNQ1 gene was identified in three concerned family members (QTc intervals: 500, 510 and 530 ms, respectively). As a result, proline 343 localised within the highly conserved transmembrane segment S6 of the KCNQ1 channel is replaced by a serine. Co-expression of mutant (KCNQ1-P343S) and wild-type (KCNQ1) cRNA in Xenopus oocytes produced potassium currents reduced by approximately 92%, while IKs reconstitution experiments with a combination of KCNQ1 mutant, wild-type and KCNE1 subunits yielded currents reduced by approximately 60%. A novel mutation (P343S) identified in the KCNQ1 subunit gene of three members of a RWS family showed a dominant-negative effect on native IKs currents leading to prolongation of the heart repolarisation and possibly increases the risk of malign arrhythmias with sudden cardiac death.

Expression of the IKr components KCNH2 (rERG) and KCNE2 (rMiRP1) during late rat heart development.

To understand molecular mechanisms that regulate formation and maintenance of cardiac IKr (rapidly activating component of the delayed rectifier K+ current), we have investigated the spatiotemporal expression pattern of two rat potassium voltage-gated channels, namely subfamily H (eag-related), member2 (KCNH2) (alias name: rERG) and Isk-related family, member2 (KCNE2) (alias name: rMiRP1) during late embryonic development by means of the in situ hybridization technique. KCNE2 is transcribed predominantly in atrial und ventricular myocardium at stages E14.5-E18.5dpc and only a minor signal emerged in the tongue at E16.5dpc. In contrast, KCNH2 transcripts appeared in a less confined pattern with intense signals in atrial and ventricular myocardium, somites, spinal cord, bowel system, central nervous system and thymus at stages E14.5-E18.5dpc. Non-cardiac expression even exceeds the intensity of the cardiac signal, indicating that KCNH2 contributes to K+ currents in non-cardiac tissue as well. Transcription of the rat b-subunit KCNE2 is present in all regions of the fetal myocardium and co-distributes perfectly with transcription of the pore forming a-subunit KCNH2. It seems likely that KCNH2 and KCNE2 are linked to form cardiac IKr channels, associated to cardiogenesis and cardiomyocyte excitability.

Troponin T concentrations 72 hours after myocardial infarction as a serological estimate of infarct size.

BACKGROUND: After acute myocardial infarction, the structural protein T is released considerably longer than cytosolic creatine kinase (CK), CK MB isoenzyme (CK-MB), or lactate dehydrogenase (LDH) and late troponin T release (> 48 hours after onset of chest pain) appears to be less affected by early coronary reperfusion. OBJECTIVE: To investigate the precision of a single measurement of circulating troponin T concentrations 72 hours after onset of chest pain compared with standard scintigraphic and enzymatic estimates of myocardial infarct size. METHODS: Quantitative single photon emission computed tomography thallium-201 scintigraphy at rest was performed in 37 patients 2-3 weeks after myocardial infarction (group 1: 14 patients without early coronary reperfusion; group 2: 23 patients with early reperfusion achieved by thrombolytic therapy, by percutaneous transluminal coronary angioplasty, or by both). RESULTS: In both groups, the number of myocardial segments with abnormal thallium-201 uptake indicating the individual extent of irreversible myocardial damage correlated significantly with the troponin T concentrations 72 hours after infarction as well as with peak concentrations of CK, CK-MB, and LDH. CONCLUSION: The data show that a single measurement of circulating troponin T 72 hours after onset of chest pain--independent of reperfusion--is superior for the estimation of myocardial infarct size to measurement of peak CK, CK-MB, or LDH, which require serial determinations and depend on coronary reperfusion.

Molecular cloning and expression of cERG, the ether à go-go-related gene from canine myocardium.

Given the anatomical and physiological similarities to the human heart, canine in vivo heart models may facilitate the analysis of molecular mechanisms underlying cardiac repolarization abnormalities. The development of such models depends, however, on information about canine K+ channels responsible for the establishment of IK currents. In this context, we isolated and sequenced the reverse transcript of the canine ether à go-go-related gene (cERG). The complementary deoxyribonucleic acid (cDNA-derived cERG polypeptide consists of 1,158 amino acids, the sequence of which shows striking homology to human, rat and mouse ERG subunits (97%, 94% and 95% identity respectively). In highly conserved peptide domains like the PAS domain, the membrane-spanning segments S1, S3-S6 and the pore-forming region, there was 100% identity. Analysis of cERG transcription revealed abundant expression of cERG messenger ribonucleic acid (mRNA) in heart and brain and low expression in liver, spleen and kidney. Membrane currents recorded in Xenopus oocytes expressing cERG channels showed functional properties very similar to the human K+ channel hERG, which encodes the alpha-subunit of the cardiac rapidly activating, delayed rectifier (IKr) channel.

Elevated serum concentrations of cardiac troponin T in acute allograft rejection after human heart transplantation.

OBJECTIVES: This study evaluates the concept and diagnostic efficacy of using serum troponin T for the detection of cardiac graft rejection. BACKGROUND: Cardiac troponin T is a cardiospecific myofibrillar protein, which is only detectable in the circulation after cardiac myocyte damage. It might be expected to be released during acute heart allograft rejection, allowing noninvasive rejection diagnosis. METHODS: In 35 control subjects and in 422 samples from 95 clinically unremarkable heart allograft recipients more than 3 months postoperatively, troponin T serum concentrations were compared to the histological grade of acute graft rejection in concurrent endomyocardial biopsies. RESULTS: Mean troponin T serum concentrations were identical in control subjects (23.2 +/- 1.4 ng/liter) and in heart transplant recipients without graft rejection (International Society for Heart and Lung Transplantation [ISHLT] grade 0; 22.4 +/- 1.7 ng/liter). Mean troponin T concentrations increased in parallel with the severity of graft rejection (ISHLT grade 1: 27.8 +/- 1.8 ng/liter; grade 2: 33.2 +/- 2.7 ng/liter; grade 3A: 54.6 +/- 6.5 ng/liter; grade 3B and 4: 105.4 +/- 53.7 ng/liter; p < 0.001 for grades 3 and 4 vs. grades 0 and 1). The proportion of positive samples also increased in parallel with rejection severity, reaching 100% in rejections of grade 3B and 4. Sensitivity and specificity for the detection of significant graft rejection (ISHLT grade 3/4) were 80.4% and 61.8%, respectively. The negative predictive value was most remarkable with 96.2%. Intraindividual longitudinal analysis of troponin T levels and biopsy results in 15 patients during long-term follow-up confirmed these findings. CONCLUSIONS: The present data demonstrate that acute allograft rejection after human heart transplantation is often associated with increased serum concentrations of troponin T. All cases of serious forms of graft rejection would have been detected before the development of clinical symptoms. Measurement of troponin T levels may become a useful ancillary parameter for noninvasive rejection diagnosis, being most valuable in the exclusion of severe cardiac graft rejection.

Cardiac troponin T in patients with end-stage renal disease: absence of expression in truncal skeletal muscle.

In patients with end-stage renal disease (ESRD), the serum concentration of cardiac troponin T (cTnT) may be increased without cardiac ischemia. One reason for this unexplained increase could be the extracardiac expression of cTnT. However, truncal skeletal muscle biopsies of five patients with ESRD showed no evidence of the expression of either cTnT mRNA (reverse transcription-PCR) or protein (immunoblot, immunofluorescence). We also measured the serum concentration of cTnT in 97 patients with ESRD. The serum cTnT concentration determined in both first and second generation cTnT assays was significantly lower P <0.01 in patients with a low cardiac risk than in patients with positive indicators of coronary artery disease. The correlation between cTnT and indicators of coronary artery disease is consistent with the hypothesis that cTnT in the serum of patients with ESRD originates from the heart.

Novel splice donor site mutation in the cardiac myosin-binding protein-C gene in familial hypertrophic cardiomyopathy. Characterization Of cardiac transcript and protein.

Familial hypertrophic cardiomyopathy is a disease generally believed to be caused by mutations in sarcomeric proteins. In a family with hypertrophic cardiomyopathy linked to polymorphic markers on chromosome 11, we found a new mutation of a splice donor site of the cardiac myosin-binding protein-C gene. This mutation causes the skipping of the associated exon in mRNA from lymphocytes and myocardium. Skipping of the exon with a consecutive reading frame shift leads to premature termination of translation and is thus expected to produce a truncated cardiac myosin-binding protein-C with loss of the myosin- and titin-binding COOH terminus. However, Western blot analysis of endomyocardial biopsies from histologically affected left ventricular myocardium failed to show the expected truncated protein. These data show for the first time that a splice donor site mutation in the myosin-binding protein-C gene is transcribed to cardiac mRNA. Truncated cardiac myosin-binding protein-C does not act as a "poison polypeptide," since it seems not to be incorporated into the sarcomere in significant amounts. The absence of mutant protein and of significantly reduced amounts of wild-type protein in the presence of the mutated mRNA argues against the "poison protein" and the "null allele" hypotheses and suggests yet unknown mechanisms relevant to the genesis of chromosome-11- associated familial hypertrophic cardiomyopathy.

Improved troponin T ELISA specific for cardiac troponin T isoform: assay development and analytical and clinical validation.

The first generation of troponin T ELISA (TnT 1) can yield false-positive results in patients with severe skeletal muscle injury. Therefore, a cardiac-specific second-generation troponin T ELISA (TnT 2) was developed, in which the cross-reactive antibody 1B10 has been replaced by a high-affinity cardiac-specific antibody M11.7. No cross-reactivity of TnT 2 was observed with purified skeletal muscle troponin T (1000 micrograms/L) or in test samples from 43 marathon runners and 24 patients with rhabdomyolysis and highly increased creatine kinase. TnT 2 was increased > 0.2 microgram/L in 5 of 40 patients with renal failure and in 4 of 20 muscular dystrophy patients. The detection limit is 0.012 microgram/L. Day-to-day imprecision (CV) within the range 0.19-14.89 micrograms/L was < 5.8%. In 4955 patients without myocardial damage, 99.6% had TnT < 0.10 microgram/L. Assay comparison (TnT 1 vs TnT 2) over the whole concentration range (i.e., in 323 samples from AMI-suspected patients) showed a slope, intercept, and standard error of estimate (Sey) of 1.18, 0.01 micrograms/L, and 0.81 microgram/L, respectively.

Trapping and metabolism of radioiodinated PHIPA 3-10 in the rat myocardium.

UNLABELLED: PHIPA 3-10 [13-(4'-iodophenyl)-3-(p-phenylene)tridecanoic acid] is a p-phenylene-bridged, radioiodinated omega-phenyl fatty acid that has recently been developed to study coronary artery disease or cardiomyopathies. Here, we demonstrate that PHIPA 3-10 exhibits the characteristics of a long-chain fatty acid, including its ability to be efficiently taken up by myocytes and to function as a substrate for beta-oxidation before it is trapped. METHODS: Myocardial metabolism of carrier-added and carrier-free 131I-PHIPA 3-10 preparations were investigated in rats in vivo and in isolated Langendorff rat hearts. Heart extracts were analyzed by high-performance liquid chromatography, negative-ion electrospray mass spectrometry and investigation of intracellular distribution using density-gradient centrifugation. RESULTS: A single, rapidly formed metabolite was found in the heart extract and also, surprisingly, in the hydrolyzed lipids. The total amount of metabolite increased from 43% to 51% between 15 and 60 min postinjection. By high-performance liquid chromatography comparison with synthetic potential catabolites, the metabolite was assigned the name PHIPA 1-10 [11-(4'-iodophenyl)-1-(p-phenylene)undecanoic acid] and was the product of one beta-oxidation cycle. Additional proof was obtained from the mass spectrometric analysis of the metabolite formed in vivo. The formation of this metabolite could be suppressed by Etomoxir, a carnitine palmitoyl transferase I inhibitor, indicating beta-oxidation of 131I-PHIPA 3-10 in mitochondria. Final evidence for the involvement of mitochondria in the degradation of 131I-PHIPA 3-10 was obtained by density-gradient centrifugation of homogenized rat heart tissue. The position of the labeled free PHIPA 3-10 and free metabolite peaked within the fraction containing mainly mitochondria. CONCLUSION: In spite of its bulky structure, 131I-PHIPA 3-10 is extracted by the myocardium in a manner similar to the extraction of the unmodified fatty acid analog, IPPA. The retention of PHIPA 3-10 in heart muscle results from the presence of the p-phenylene group, which prevents more than one beta-oxidation cycle. Intracellular free PHIPA 3-10 and free PHIPA 1-10 are present in the mitochondria, whereas most of the esterified metabolite was found in the cytosolic lipid pool. Hence, the rapid appearance of PHIPA 1-10 in the lipid pool must be accounted for by mitochondrial leakage or by an unknown in-out transport system.

Troponin T: a diagnostic marker for myocardial infarction and minor cardiac cell damage.

The diagnosis of acute myocardial infarction is straightforward when anginal pain is accompanied by typical ECG changes and in these patients measurements of cardiac markers are unnecessary in deciding whether thrombolytic therapy is appropriate. Cardiac markers in patients with acute ischaemic coronary syndromes, however, may serve to identify a high risk subgroup of patients with small acute infarctions or minor myocardial damage. In many patients with chest pain a valid diagnosis of myocardial cell injury depends on the result of biochemical assays. In 30% of patients with unstable angina, troponin T is elevated although myocardial infarction was ruled out by cardiac enzymes and ECG recordings. The outcome of these patients at 4 weeks and 6 months follow-up is not different from that of patients with definite myocardial infarction. To guide therapeutic decisions on these patients a troponin T test result needs to be available rapidly. The rapid troponin T test strip assay, which allows the determination of troponin T levels in whole blood at the patient's bedside, can be performed conveniently in the emergency room or in laboratories with less sophisticated equipment and has the potential to aid in the triage of chest pain patients and the selection of therapeutic strategies.

Residual 201Tl activity in irreversible defects as a marker of myocardial viability. Clinicopathological study.

BACKGROUND: The objective of the present study was to characterize the relation between the residual 201Tl activity in irreversible perfusion defects and the extent of irreversible myocardial damage indicated by the volume fraction of myocardial interstitial fibrosis in patients with chronic coronary artery disease. METHODS AND RESULTS: Stress planar 201Tl scintigraphy with tracer reinjection at rest was performed in 37 patients with > or = 75% stenosis of the left anterior descending coronary artery, and anteroseptal 201Tl activity was quantified by computer-assisted placement of regions of interest from the serial myocardial images. During coronary artery bypass grafting (performed within 6 +/- 3 weeks after scintigraphy), two transmural biopsy specimens were taken from the anterior wall of the left ventricle and the amount of interstitial fibrosis was assessed by use of light microscopic morphometry. A wide spectrum of interstitial fibrosis was obtained, ranging from 15 vol% to 60 vol%. Interstitial fibrosis was similar in patients with reversible (n = 11) or irreversible (n = 15) tracer defects in conventional stress-redistribution images. However, interstitial fibrosis was significantly lower in patients who had enhanced regional 201Tl activity after tracer reinjection compared with those who did not have enhancement of tracer activity after reinjection (28 +/- 8 vol%, n = 7, versus 41 +/- 12 vol%, n = 8; P = .031). The correlation between relative poststenotic 201Tl activity and interstitial fibrosis after tracer reinjection was significantly improved compared with conventional redistribution images (r = -.622 versus r = -.851, n = 15; P < .01). CONCLUSIONS: The present data demonstrate that the level of regional 201Tl activity in redistribution and, in particular, reinjection images is significantly related to the mass of preserved viable myocytes in poststenotic left ventricular myocardium. Therefore, the residual 201Tl activity provides information about viability within irreversible perfusion defects and may itself serve as marker of myocardial viability.

[Clinical aspects and follow-up in dilated cardiomyopathy]. / Klinik und Verlauf der dilatativen Kardiomyopathie.

Dilative cardiomyopathy is a heterogeneous myocardial disease characterized by a depressed contractile function and ventricular dilation. The exercise intolerance of patients with dilative cardiomyopathy is partly explained by changes of metabolism and composition of skeletal muscle, whereas the physical findings result from progressive heart failure and cardiac remodeling. The clinical course of the disease is highly variable and prognostic indicators of progressive heart failure or cardiac arrhythmias are of little value in the assessment of cardiac risk in an individual patient. During recent years the mortality of patients with dilative cardiomyopathy was 5-15%, which appears to be lower than previously reported. The differences in mortality today and in previous years may be explained by a more precise and earlier diagnosis of the disease and possibly better treatment strategies. Patients with a severely depressed left ventricular function but a stable clinical course, a good hemodynamic response to therapy, and an oxygen uptake of > 12 ml/kg min can be followed without heart transplantation. It can be expected that the definition of the molecular causes of dilative cardiomyopathy will lead to a better classification of the patients and the development of more efficient treatment strategies.

Assessment of reperfusion of the infarct zone after acute myocardial infarction by serial cardiac troponin T measurements in serum.

BACKGROUND: The purpose of this study was to derive indices of reperfusion and non-reperfusion after acute myocardial infarction (AMI) from changes in serum concentrations of cardiac troponin T and to test the predictive value of these indices. METHODS: The indices were derived from a retrospective analysis of changes in serum troponin T concentration in 71 patients given thrombolytic treatment who had immediate and late angiography (group 1). These troponin T indices were first tested in a blinded and prospective study of 53 consecutive patients eligible for thrombolytic therapy (group 2). They were then used for the non-invasive assessment of reperfusion of AMI in 48 patients (group 3). RESULTS: In group 1 troponin T serum concentration curves were biphasic in patients who had reperfusion < or = 5.8 h after the onset of symptoms. Release of the cytosolic troponin T pool resulted in a peak at 14 h and ended at 38 h. The probability of reperfusion was > 95% when the ratio of peak cytosolic troponin T concentration to concentration at 38 h (PV1/38) exceeded 1.42 or the ratio of troponin T concentration at 14 h to that at 38 hours (14/38) exceeded 1.09. The probability of the presence of non-reperfused AMI was < 5% when troponin T PV1/38 and 14/38 ratios were < 0.99 and < 0.84 respectively. These discriminatory values of troponin T indices correctly classified (efficiency 96%) 48 of the 53 group 2 patients in whom immediate and late angiography were performed. When troponin T indices were used to classify 48 group 3 patients who were not studied by immediate angiography, thrombolytic therapy was deemed to have been successful in 82% of the treated patients, with spontaneous recanalisation in 11% and 23% of the non-treated patients assessed by PV1/38 and 14/38 respectively. CONCLUSION: The PV1/38 or 14/38 ratios of serum troponin T concentration indicated the effectiveness of thrombolytic therapy in achieving reperfusion of AMI.

[Monitoring ischemia with new markers]. / Ischämiemonitoring mit neuen Markern.

The measurement of cardiac enzymes is critical for the diagnosis of acute myocardial infarction. Cardiac enzymes, however, are by no means ideal marker molecules, primarily due to their non-specific tissue distribution and low concentration in cardiomyocytes. Many limitations of cardiac enzymes can be overcome by the measurement of cardiospecific troponin T or I with immunological techniques. In the evaluation of new diagnostic methods it is important to define the purpose of marker molecule measurement, i.e., monitoring of definite myocardial infarction or establishing the proper diagnosis in patients with suspected myocardial infarction. For monitoring of success of reperfusion therapy and for the detection of reocclusion short-lived perfusion markers with rapid appearance in circulation such as myoglobin, fatty acid binding protein or glycogenisophosphorylase BB are preferable. For proper diagnosis in patients with suspected acute myocardial infarction test systems with high sensitivity and specificity are needed due to the low prevalence of disease in the patients tested. Troponin T determinations are particularly useful in this group of patients. With troponin T determinations it could be shown that some patients so far classified as having unstable angina do in fact have microinfarction. These data indicate the need for re-definition of diagnostic criteria of acute myocardial infarction.