Keishiro Kawamura

Left ventricular midwall mechanics in systemic arterial hypertension. Myocardial function is depressed in pressure-overload hypertrophy.

BackgroundLeft ventricular (LV) midwall geometry has been described conventionally as the sum of the chamber radius and half of the wall thickness; this convention is based on the assumption of uniform transmural thickening during systole. However, theoretical considerations and experimental data indicate that the inner half (inner shell) of the LV wall thickens more than the outer half (outer shell). Thus, an end-diastolic circumferential midwall fiber exhibits a relative migration toward the epicardium during systole. As a result, the conventional method provides an overestimate of the extent of the midwall fiber shortening. Methods and ResultsWe developed an ellipsoidal model with a concentric two-shell geometry (nonuniform thickening) to assess midwall fiber length transients throughout the cardiac cycle. This modified midwall method was used in the analysis of LV cineangiograms from 15 patients with systemic arterial hypertension and 14 normal subjects. Study groups were classified according to LV mass index (LVMI): 14 normal subjects (group I), eight hypertensive patients with a normal LVMI (group II), and seven hypertensive patients with an increased LVMI (group III). There were no significant differences in LV end-diastolic pressure or volume among the three groups; the ejection fraction was slightly greater in group 11 (70 + 5%) than in groups I (65±8%) and III (664±4%), but this trend did not achieve statistical significance. Values for endocardial and conventional midwall fractional shortening (FS) were also similar in the three groups. By contrast, FS by the concentric two-shell geometry (modified midwall method) in group III (16±2%) was significantly less than that seen in groups I and II (21±4% and 21 + 5%, respectively; both p < 0.05). This difference achieves greater importance when it is recognized that mean systolic circumferential stress was lower in group III (151±22 g/cm2) than in groups I and 11 (244±37 g/cm2 and 213+38 g/cm2, respectively; both p<0.01). The midwall stressshortening coordinates in six of the seven group III patients were outside the 95% confidence limits for the normal (group I) subjects. Thus, despite a normal ejection fraction, systolic function is subnormal in hypertensive patients with LV hypertrophy. ConclusionsChamber dynamics provide an overestimate of myocardial function, especially when LV wall thickness is increased. This is due to a relatively greater contribution of inner shell thickening in pressure-overload hypertrophy.

Serum and urinary human heart fatty acid-binding protein in acute myocardial infarction

A competitive enzyme immunoassay (C-EIA) was developed for the measurement of serum and urinary levels of human heart fatty acid-binding protein (hh-FABP), and the appearance and time-course changes of hh-FABP levels were evaluated in patients with acute myocardial infarction (AMI). Control serum and urinary hh-FABP levels, which were determined in 86 serum and 42 urine samples from 86 patients without AMI, were found to range between 0 and 2.8 ng/mL. Serial determinations performed on 11 patients with AMI demonstrated that hh-FABP levels were significantly elevated in the first serum and urine samples obtained within 14 h of the onset of clinical symptoms. Two serum and 2 urine samples obtained only 1.5 h after the onset of symptoms already showed elevated hh-FABP levels, while in the same serum samples the activity of the myocardial-specific isoenzyme of creatine kinase (CK-MB) was still normal. Maximal serum and urinary hh-FABP levels appeared between 5 and 10 h after symptoms developed, and fell sharply towards normal thereafter. The hh-FABP levels in serum and urine both peaked earlier than the elevation of CK-MB activity in serum. The presence of hh-FABP in serum and/or urine seems to be a marker for myocardial damage and could be used as a useful tool for the early diagnosis of AMI.

Human Heart-Type Cytoplasmic Fatty Acid-Binding Protein (H-FABP) for the Diagnosis of Acute Myocardial Infarction. Clinical Evaluation of H-FABP in Comparison with Myoglobin and Creatine Kinase Isoenzyme MB

Abstract Heart-type fatty acid-binding protein (H-FABP) is a low molecular weight cytoplasmic protein and present abundantly in the myocardium. When the myocardium is injured, as in the case of myocardial infarction, low molecular weight cytoplasmic proteins including H-FABP are released into the circulation and H-FABP is detectable in a blood sample. We have already developed a direct sandwich-ELISA for quantification of human H-FABP using two distinct types of monoclonal antibodies specific for human H-FABP. In this study we investigated the clinical validity of H-FABP as a biochemical diagnostic marker in the early phase of acute myocardial infarction (AMI). To evaluate the diagnostic usefulness of H-FABP in the early phase of AMI, blood samples were obtained from the following patients within 12 hours after the appearance of symptoms, and serum levels of H-FABP were compared with those of conventional diagnostic markers, such as myoglobin and creatine kinase isoenzyme MB (CK-MB). Blood samples were collected from patients with confirmed AMI (n=140), patients with chest pain who were afterwards not classified as AMI by normal CK-MB levels (non-AMI) (n=49) and normal healthy volunteers (n=75). The serum concentration of H-FABP was quantified with our direct sandwich-ELISA. The concentration of myoglobin mass was measured with a commercial RIA kit. The serum CK-MB activity was determined with an immuno-inhibition assay kit. The overall sensitivity of H-FA B P, within 12 hours after the appearance of symptoms, was 92.9%, while it was 88.6% with myoglobin and 18.6% with CK-MB. The overall specificity of H-FABP was 67.3%, while it was 57.1% with myoglobin and 98.0% with CK-MB. The diagnostic efficacy rates with these markers were 86.2% (H-FABP), 80.4% (myoglobin) and 39.2% (CK-MB), respectively. The diagnostic validity of H-FABP was further assessed by receiver operating characteristic (ROC) curve analysis. The area under the curve (AUC) of H-FABP was 0.921, which was significantly greater than with myoglobin (AUC: 0.843) and CK-MB (AUC: 0.654). These parameters, such as sensitivity, specificity, diagnostic efficacy and diagnostic accuracy, obtained for patients with chest pain within 3 hours and/or 6 hours after the onset of symptoms were almost the same as those for patients within 12 hours after symptoms. H-FABP is more sensitive than both myoglobin and CK-MB, more specific than myoglobin for detecting AMI within 12 hours after the onset of symptoms, and shows the highest values for both diagnostic efficacy and ROC curve analysis. Thus, H-FABP has great potential as an excellent biochemical cardiac marker for the diagnosis of AMI in the early phase.

Evaluation of viral infection in the myocardium of patients with idiopathic dilated cardiomyopathy

OBJECTIVES The aim of this study was to evaluate the viral etiology of idiopathic dilated cardiomyopathy (DCM). BACKGROUND The demonstration of enteroviral genome in hearts with DCM has reinforced the importance of enteroviruses in the pathogenesis of DCM. However, there is uncertainty about the character and activity of enteroviruses detected in the myocardium. Recently, the association of hepatitis C virus or adenovirus with DCM has been reported. METHODS Myocardial specimens from 26 patients with idiopathic DCM, which were obtained at partial left ventriculectomy (PLV), were examined virologically. Strand-specific detection of enteroviral RNA was performed to differentiate active viral replication from latent persistence. Polymerase chain reaction was used to detect genomic sequences of hepatitis C virus, adenovirus, cytomegalovirus, influenza viruses, mumps virus, herpes simplex viruses, varicella-zoster virus and Epstein-Barr virus. RESULTS Plus-strand enteroviral RNA was detected in 9 (35%) of the 26 patients. Minus-strand enteroviral RNA was determined in seven (78%) of these nine plus-strand RNA-positive patients. Sequence analysis revealed that the enteroviruses detected were coxsackie B viruses, such as coxsackievirus B3 and B4. However, genetic material from other viruses was not detected. Six (86%) of seven minus-strand enteroviral RNA-positive patients died of cardiac insufficiency within the first six months after PLV. CONCLUSIONS Coxsackie B viruses were seen in hearts with idiopathic DCM. Active viral RNA replication appeared to be present in a significant proportion of these cases. Minus-strand coxsackieviral RNA in the myocardium can be a marker for poor clinical outcome after PLV. There was no evidence of persistent infection by other viruses in hearts with DCM.

Iodine-123 metaiodobenzylguanidine myocardial scintigraphy for prediction of response to β-blocker therapy in patients with dilated cardiomyopathy

This study was performed to evaluate whether iodine-123 metaiodobenzylguanidine (MIBG) myocardial scintigraphy could predict the response to beta-blocker therapy in patients with nonischemic dilated cardiomyopathy (DCM). Beta-Blocker therapy is effective in some patients with DCM. MIBG myocardial scintigraphy has also been suggested to be useful in evaluating the severity of myocardial damage in DCM. However, no data exist on whether MIBG imaging can be used to predict which patients with DCM will respond to beta-blocker therapy. We prospectively evaluated whether MIBG myocardial imaging was useful in predicting responses to beta-blocker therapy in patients with DCM. MIBG imaging was performed in 45 patients with DCM (35 men, 10 women, aged 13 to 68 years) before the start of bisoprolol. The heart to mediastinum (H/M) MIBG uptake ratio was evaluated on initial and delayed images, and the percent washout rate of myocardial MIBG was also obtained from these data. Of the 45 patients, 30 (67%) responded to beta-blocker therapy, whereas 2 were resistant and 13 showed progression of heart failure or died of heart failure. By logistic regression analysis, the H/M uptake ratio on delayed images was seen to be a good predictor of the response to beta-blocker therapy with a threshold of 1.7 (sensitivity = 91%, specificity = 92%, accuracy = 91%, positive and negative predictive value = 97% and 80%, respectively). These results indicate that an H/M ratio > 1.7 on the delayed MIBG myocardial scintigraphic images provides a useful indication of whether patients with DCM will respond to beta-blocker therapy.

Point mutations in mitochondrial DNA in patients with hypertrophic cardiomyopathy

Recent advances suggest that mutations in nuclear DNA are involved in the etiology of autosomal dominant hypertrophic cardiomyopathy. Mitochondria have their own DNA, and mutations in mitochondrial DNA have been shown to contribute to the genesis of various diseases. In this study, we developed rapid sequencing methods with the use of a fluorescence-based sequencing system and analyzed total mitochondrial DNA of seven patients with nonautosomal dominant hypertrophic cardiomyopathy. Multiple point mutations were observed in all patients with hypertrophic cardiomyopathy, although some of them were common among the subjects examined and the others are unique to each subject. Point mutations in transfer RNA genes were observed in five of the seven patients, and point mutations that replaced conserved amino acids were also observed. These mutations may result in the impairment of mitochondrial function. According to these results, mutations in mitochondrial DNA may contribute to the genesis of some cases of nonautosomal dominant hypertrophic cardiomyopathy, and our methods may be useful for the detection of point mutations in mitochondrial DNA.

Development of a sandwich enzyme-linked immunosorbent assay for the determination of human heart type fatty acid-binding protein in plasma and urine by using two different monoclonal antibodies specific for human heart fatty acid-binding protein

We have developed a sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of human heart type fatty acid-binding protein (H-FABP) in human plasma and urine using the combination of two distinct monoclonal antibodies (MAbs) directed against human H-FABP purified from human heart muscle. The total assay time of the ELISA is practically much shorter than that of the competitive enzyme immunoassay (EIA) we previously reported. The immunoreactive mass of human H-FABP was specifically measured using a horseradish peroxidase (HRPO)-labeled anti-human H-FABP MAb as an enzyme-linked MAb, and anti-human H-FABP MAb immobilized on the polystyrene microtiter plate as a solid-phase MAb, and purified human H-FABP as standard materials. The assay range of the ELISA was 0-250 ng/ml of plasma and urine. The ELISA yielded a coefficient of variation of less than 10% in inter- and intra-assays, and the good linearity was obtained in dilution test using clinical samples. Anticoagulants, except sodium fluoride and a high concentration of hemoglobin and bilirubin, did not interfere with the assay of plasma samples. A high concentration of hemoglobin, bilirubin and immunoglobulin, and contamination with seminal plasma did not interfere with the assay of urine samples. The average recovery of purified human H-FABP added to human plasma and urine samples was 98.5% and 97.0%, respectively. Myoglobin and myosin did not crossreact in the ELISA. The minimum detection limit of the ELISA was 1.25 ng/ml. The immunoreactive masses of human H-FABP in plasma and urine samples, obtained from one hundred normal healthy subjects were quantified by the sandwich ELISA. The normal mean (+/- SD) level of human H-FABP mass in plasma was 3.65 +/- 1.81 ng/ml, and that in urine was 3.20 +/- 2.70 ng/ml. In conclusion, this sandwich ELISA is a useful tool for the sensitive and precise determination of human H-FABP in human plasma and urine, and it may be used specifically for clinical investigation and diagnosis of myocardial injury.

Patients with idiopathic cardiomyopathy belong to the same mitochondrial DNA gene family of Parkinson's disease and mitochondrial encephalomyopathy

Comparison of total mitochondrial DNA sequences of patients with idiopathic (deleted or hypertrophic) cardiomyopathy with those of patients with Parkinsons disease and mitochondrial encephalomyopathies revealed distinct clustering of point mutations among patients. Furthermore, an inverse relation was found between the total number of base-substitution and life span of the patients. Among point mutations found in each patient, sequentially diverged six clusters consisting of 14, 10, 7, 1, 2, and 3 mutations, respectively, were detected. Five sub-clusters consisting of 2, 2, 11, 1, and 1 mutations, respectively, were detected. From each cluster, the patients unique mutations were diverged with three types of the mutations specific for the disease. The divergence allowed construction of a phylogenetic tree which clearly indicated that patients with idiopathic cardiomyopathy belong to the same mitochondrial DNA gene family of Parkinsons disease and mitochondrial encephalomyopathies.

CD36 mediates long-chain fatty acid transport in human myocardium: Complete myocardial accumulation defect of radiolabeled long-chain fatty acid analog in subjects with CD36 deficiency

Long-chain fatty acids (LCFA) are the major energy substrate for heart and their oxidation is important for achieving maximal cardiac work. However, the mechanism of uptake of LCFA by myocardium has not been clarified. We previously reported that bovine myocardial LCFA transporter has a sequence homology to human CD36. Clinically, total defect of myocardial uptake of radiolabeled long-chain fatty acid analog [123I-BMIPP: Iodine-123 15-(p-iodophenyl)-(R,S)-methylpentadecanoic acid] has been reported in some restricted cases, but the etiology has not been clarified. In the present study, we analyzed CD36 expression and CD36 gene in subjects who showed total lack of myocardial 123I-BMIPP accumulation, and, vice versa, evaluated myocardial 123I-BMIPP uptake in subjects with CD36 deficiency. Four unrelated subjects were evaluated; Two were found to have negative myocardial LCFA accumulation by 123I-BMIPP scintigraphy, after which the expression of CD36 on their platelets and monocytes was analyzed. Remaining two subjects were identified as CD36 deficiency by screening, then 123I-BMIPP scintigraphy was performed. Expression of CD36 on platelets and monocytes was measured by flow cytometric analysis. The molecular defects responsible for CD36 deficiency was detected by allele-specific restriction enzyme analysis. CD36 expression was totally deficient in all 4 subjects on both platelets and monocytes. Two subjects were homozygous for a 478C→T mutation. One was heterozygous for the dinucleotide deletion of exon V and single nucleotide insertion of exon X, and remaining one was considered to be heterozygous for the dinucleotide deletion of exon V and an unknown gene abnormality. All cases demonstrated a completely negative accumulation of myocardial LCFA despite of normal myocardial perfusion, which was evaluated by thallium scintigraphy. In addition, all cases demonstrated apparently normal hepatic LCFA accumulation Thus, these findings suggested that CD36 acts as a major myocardial specific LCFA transporter in humans.

Usefulness of low-dose dobutamine stress echocardiography for evaluating reversibility of brain death–induced myocardial dysfunction

Many of the myocardial wall motion abnormalities in heart donors are reversible after transplantation, indicating that the presence of wall motion abnormalities should not automatically lead to the exclusion of donor hearts. The present study observes the natural course of brain death-induced myocardial dysfunction, and investigates whether low-dose dobutamine stress echocardiography could identify reversible myocardial dysfunction in brain-dead patients. We prospectively measured the serial changes of left ventricular fractional shortening (FS) using echocardiography and cardiac troponin T from admission to the time of cardiac standstill in 30 brain-dead patients. Patients were divided into 2 groups according to FS at the time of brain death; group I (FS > or =30%) and group II (FS <30%). Dobutamine stress echocardiography was performed in group II. Twenty-three patients were in group I and 7 patients were in group II. Four patients among 7 patients in group II showed dobutamine-nonresponsive wall motion (group IIa) and the remaining 3 patients showed dobutamine-responsive wall motion (group IIb). Troponin T at the time of brain death was markedly higher in group IIa than in groups I and IIb (5.13+/-3.79 vs 0.23+/-0.20, 0.22+/-0.16 ng/ml, p <0.0001, respectively). FS remained normal and troponin T was not increased until cardiac standstill in group I. FS remained decreased and troponin T remained elevated until cardiac standstill in group IIa, whereas FS became normal at 7 days after brain death with no change in troponin T in group IIb. Thus, some brain death-induced myocardial dysfunction is reversible and low-dose dobutamine stress echocardiography may identify reversible myocardial dysfunction.