Noboru Takekoshi

Angiotensin II Stimulates c-Jun NH2-Terminal Kinase in Cultured Cardiac Myocytes of Neonatal Rats

Many lines of evidence have suggested that angiotensin II (Ang II)plays an important role in cardiac hypertrophy. Ang II not only increases protein synthesis but also induces the reprogramming of gene expression in cultured cardiac myocytes. In the present study, to elucidate the mechanism by which Ang II regulates gene expression in cardiac myocytes, we examined whether Ang II activates c-Jun NH2-terminal kinase (JNK), which is a member of the mitogen-activated protein kinase family and activates the transcription factor, activator protein-1 (AP-1). The activity of JNK increased 5 minutes after the addition of Ang II, peaked at 20 minutes, and gradually decreased thereafter. Examination of the Ang II dose-response relation revealed detectable JNK activation at 10(-9) mol/L and maximal activation at 10(-6) mol/L. Ang II activated JNK through the AT1 receptor, and the activation was attenuated by the downregulation of protein kinase C or the chelation of intracellular Ca2+. Although the addition of either Ca2+ ionophore or phorbol ester resulted in little or no activation of JNK, simultaneous addition of both Ca2+ ionophore and phorbol ester markedly activated JNK. Slight expressions of the c-jun gene were observed in unstimulated cardiac myocytes, and Ang II increased expressions of the c-jun gene as well as the c-fos gene. Ang II increased transcription of the endothelin-1 gene through the AP-1 binding site. In conclusion, Ang II may activate JNK in cultured cardiac myocytes through an increase in intracellular Ca2+ and activation of protein kinase C, and the activated JNK may regulate gene expression by activating AP-1 during Ang II-induced cardiac hypertrophy.

Mechanical Stretch Induces Hypertrophic Responses in Cardiac Myocytes of Angiotensin II Type 1a Receptor Knockout Mice

Many lines of evidence have suggested that angiotensin II (AngII) plays an important role in the development of cardiac hypertrophy through AngII type 1 receptor (AT1). To determine whether AngII is indispensable for the development of mechanical stress-induced cardiac hypertrophy, we examined the activity of mitogen-activated protein kinase (MAPK) family and the expression of the c-fos gene as hypertrophic responses after stretching cultured cardiac myocytes of AT1a knockout (KO) mice. When cardiac myocytes were stretched by 20% for 10 min, extracellular signal-regulated protein kinases (ERKs) were strongly activated in KO cardiomyocytes as well as wild type (WT) myocytes. Both basal and stimulated levels of ERKs were higher in cardiomyocytes of KO mice than in those of WT mice. Activation of another member of the MAPK family, p38MAPK, and expression of the c-fos gene were also induced by stretching cardiac myocytes of both types of mice. An AT1 antagonist attenuated stretch-induced activation of ERKs in WT cardiomyocytes but not in KO cardiomyocytes. Down-regulation of protein kinase C inhibited stretch-induced ERK activation in WT cardiomyocytes, whereas a broad spectrum tyrosine kinase inhibitor (genistein) and selective inhibitors of epidermal growth factor receptor (tyrphostin, AG1478, and B42) suppressed stretch-induced activation of ERKs in KO cardiac myocytes. Epidermal growth factor receptor was phosphorylated at tyrosine residues by stretching cardiac myocytes of KO mice. These results suggest that mechanical stretch could evoke hypertrophic responses in cardiac myocytes that lack the AT1 signaling pathway possibly through tyrosine kinase activation.

Coronary calcification and coronary atherosclerosis : site by site comparative morphologic study of electron beam computed tomography and coronary angiography

OBJECTIVES We compared, on a site by site basis, the morphologic features of coronary calcifications determined by electron beam computed tomography (EBCT) and angiographically defined coronary atherosclerosis. BACKGROUND Quantification of coronary calcification using EBCT is clinically useful for the prediction of coronary stenosis. However, the relation between calcification and angiographic findings has not been evaluated by site. METHODS We studied 251 consecutive patients who underwent elective coronary angiography for suspected coronary artery disease by EBCT and analyzed findings by site. Coronary calcifications were classified according to their length and width versus the diameter of the coronary artery in which the calcification was observed as: none, spotty, long, wide and diffuse. RESULTS Coronary calcifications were found in 666 (27%) of 2,470 segments. The positive predictive value (PPV) of coronary calcification for significant stenosis (> or = 75% densitometric narrowing) and for all angiographically detectable atherosclerotic lesions in a segment was 0.36 and 0.80, respectively. The PPV for significant stenosis and all atherosclerotic lesions was 0.04 and 0.17 in none, 0.18 and 0.59 in spotty, 0.32 and 0.87 in long, 0.40 and 0.84 in wide and 0.56 and 0.96 in diffuse calcifications, respectively. The PPV for both significant stenosis and all lesions differed significantly (p = 0.001) among the morphologic groups. Of the 105 eccentric significant stenoses, 54 (53%) were classified as long or diffuse calcifications. Of the 95 significant stenoses with multiple irregularities, 61 (64%) showed diffuse calcification. CONCLUSIONS Morphologic evaluation of coronary calcifications using EBCT improved the prediction of coronary stenosis on a site by site basis and provided information related to angiographic morphology.

Noninvasive prediction of coronary atherosclerosis by quantification of coronary artery calcification using electron beam computed tomography: Comparison with electrocardiographic and thallium exercise stress test results

OBJECTIVES This study was designed to compare the usefulness of electron beam computed tomography for prediction of coronary stenosis with that of electrocardiographic (ECG) and thallium exercise tests. BACKGROUND Electron beam computed tomography can quantify coronary calcifications; however, its clinical value has yet to be established. METHODS Using the volume mode of electron beam computed tomography, we studied 251 consecutive patients who underwent elective coronary angiography because of suspected coronary artery disease and compared the results with those of ECG and thallium exercise tests. The total coronary calcification score was calculated by multiplying the area ( > or = 2 pixels) of calcification (peak density > or = 130 Hounsfield units) by an arbitrarily weighted density score (0 to 4) based on its peak density. The mean of two scans was log transformed. RESULTS Calcification was first noted in women in the 4th decade of life, approximately 10 years later than its occurrence in men. Among patients with advanced atherosclerosis (two- and three-vessel disease), calcification scores were uniformly high in women but ranged widely in men. Nine percent of patients with significant stenoses ( > or = 75% by densitometry) had no calcification. The calcification scores of patients with significant stenosis in at least one vessel were significantly higher than those of patients without significant stenosis in the study group as a whole and in most patient subgroups classified according to age and gender. A cutoff calcification score for prediction of significant stenosis, determined by receiver operating characteristic curve analysis, showed high sensitivity (0.77) and specificity (0.86) in all study patients; sensitivity was similarly high even in older patients ( > or = 70 years) and was enhanced in middle-aged patients (40 to < or = 60 years). The difference in specificity between calcification scores and ECG exercise test results had borderline significance (p = 0.058) and that between calcification scores and thallium test results was significant (p = 0.001). The latter difference became small but remained significant (p = 0.01) even after the reevaluation of thallium test results in light of each subjects clinical data. CONCLUSIONS Quantification of coronary artery calcification with electron beam computed tomography noninvasively predicted angiographically confirmed coronary stenosis. Results obtained with this method were at least as useful and potentially better in some patient groups than those obtained with thallium and ECG exercise testing.

Quantification of coronary artery calcification using ultrafast computed tomography: reproducibility of measurements.

BACKGROUND Ultrafast computed tomography (CT) is a non-invasive method of visualizing and quantifying coronary artery calcification; its reproducibility, however, has not been fully elucidated. METHODS To assess intra-observer, inter-observer, and inter-study reproducibility, 75 consecutive patients (51 men and 24 women) were studied. CT images were obtained using the volume mode of ultrafast CT (Imatron C-100). A total coronary calcification score (TCS) was calculated from the lesion area (> or = 2 pixels) and its peak CT density (> or = 130 HU). RESULTS There was no intra-observer variability in two experienced observers. The TCS provided by these observers disagreed in 18 out of 75 (24%) cases, and the differences were -5.1 +/- 53 (mean +/- SD) for TCS and 0.014 +/- 0.13 for In(1 + TCS). They resulted from either 10 incorrect identifications of small coronary branches, or eight variations in determination of the ostial margin. The former was much smaller than the latter in TCS (0.66 +/- 3.0 and -48 +/- 165, respectively), but both were quite similar in In(1 + TCS) (0.082 +/- 0.31, 0.017 +/- 0.22, respectively). Between two scans, 50 out of 75 patients (67%) had different TCS values. The mean differences (95% confidence interval) were 1.8 +/- 106 (-210 to 214) in TCS, and -0.015 +/- 0.46 (-0.94 to 0.91) in In(1 + TCS). Because the differences increased with the mean values, the determination of TCS assumed a constant variance with increasing mean level. A comparison of scan images indicated that partial volume effects were responsible for this constant variance. CONCLUSION Partial volume effects play a key role in producing the variability of TCS determination, and log transformation should be used to interpret TCS values. Thus, for clinical purposes, we recommend that two scans be performed in rapid succession, and that the average of these two scans be used to determine TCS.

Dilated cardiomyopathy defines serum autoantibodies against G-protein-coupled cardiovascular receptors

In order further to identify the prevalence of anti-receptor autoantibodies in the sera of patients with dilated cardiomyopathy (DCM), we attempted to detect autoantibodies against a series of G-protein-coupled cardiovascular receptors in a well-defined population of DCM patients from Japan. Peptides corresponding to the sequences of the second extracellular loops of the human beta 1 and beta 2 adrenoceptors, alpha 1 adrenoceptors, M2 muscarinic acetylcholine receptors and angiotensin II-1 (AT1) receptors were used as antigens in an enzyme immunoassay to screen the sera from patients with DCM (n = 28). Nine sera from patients with DCM (32%) and 2 sera from healthy subjects (9%) recognized the beta 1 adrenoceptor peptide. Ten sera from patients (36%) and 3 sera from healthy subjects (13%) recognized the M2 receptor peptide. Thirty-six per cent of the patients with autoantibody against the beta 1 adrenoceptor peptide. Ten sera from patients (36%) and 3 sera from healthy subjects (13%) recognized the M2 receptor peptide. Thirty-six per cent of the patients with autoantibody against the beta 1 adrenoceptor had autoantibody against the M2 receptor. However, no significantly high frequencies of autoantibodies against the beta 2 adrenoceptor, alpha 1 adrenoceptor and AT1 receptor were found in DCM patients. Our results demonstrate that a subgroup of patients with DCM have a specific spectrum of autoantibodies which are specifically directed against the second extracellular loops of the beta 1 adrenoceptors and M2 muscarinic receptors rather than other cardiovascular receptors.

A novel LIM protein Cal promotes cardiac differentiation by association with CSX/NKX2-5

The cardiac homeobox transcription factor CSX/NKX2-5 plays an important role in vertebrate heart development. Using a yeast two-hybrid screening, we identified a novel LIM domain–containing protein, named CSX-associated LIM protein (Cal), that interacts with CSX/NKX2-5. CSX/NKX2-5 and Cal associate with each other both in vivo and in vitro, and the LIM domains of Cal and the homeodomain of CSX/NKX2-5 were necessary for mutual binding. Cal itself possessed the transcription-promoting activity, and cotransfection of Cal enhanced CSX/NKX2-5–induced activation of atrial natriuretic peptide gene promoter. Cal contained a functional nuclear export signal and shuttled from the cytoplasm into the nucleus in response to calcium. Accumulation of Cal in the nucleus of P19CL6 cells promoted myocardial cell differentiation accompanied by increased expression levels of the target genes of CSX/NKX2-5. These results suggest that a novel LIM protein Cal induces cardiomyocyte differentiation through its dynamic intracellular shuttling and association with CSX/NKX2-5.

Assessment of working skeletal muscle oxygenation in patients with chronic heart failure

Patients with chronic heart failure (CHF) are frequently limited by muscle fatigue resulting from impaired skeletal muscle blood flow. Accordingly, we assessed working skeletal muscle oxygenation in such patients using near-infrared (NIR) spectroscopy. Nine normal subjects (mean age 52 years) and 12 patients with CHF (mean age 60 years) were studied. NIR spectroscopy was used to monitor relative changes in oxygenated hemoglobin (Hb) and myoglobin (Mb) (oxy Hb/Mb), deoxygenated Hb and Mb (deoxy Hb/Mb), and total (oxy + deoxy) Hb and Mb (total Hb/Mb) contents in the vastus lateralis muscle at rest, during warm-up (0 W, 30 cycles/min for 3 min), incremental maximal supine bicycle exercise (ramp protocol, 15 W/min, 50 cycles/min), and recovery. At peak exercise the patients exhibited reduced heart rate, systolic blood pressure, peak exercise oxygen consumption (VO2; 15 +/- 3.0 ml/kg/min vs 32 +/- 8.5 ml/kg/min), and workload (99 +/- 23.4 W vs 183 +/- 68.4 W) as compared with the normal subjects. The respiratory quotient was comparable in both groups. In the normal subjects, oxy Hb/Mb was increased from the warm-up period to the early phase of exercise, followed by a progressive decrease to peak exercise. In the recovery phase, oxy Hb/Mb was increased abruptly. For these patients, change in oxy Hb/Mb followed a pattern similar to that seen in normal subjects, and oxy Hb/Mb was decreased earlier in contrast to that in the normal subjects. There was a significant difference in the change of oxy Hb/Mb during warm-up, early phase exercise, and recovery between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Coronary Disease Morphology and Distribution Determined by Quantitative Angiography and Intravascular Ultrasound

Although previous studies have demonstrated that even quantitative coronary angiography (QCA) can not provide accurate disease morphology, there has not been a systematic comparison of disease morphology determined by QCA and intravascular ultrasound (IVUS), particularly in Japanese patients. Therefore, the present study prospectively examined patients in a multicenter cooperative study. A total of 491 coronary sites from 562 patients (446 men, 116 women; mean age, 64+/-11 years) who underwent coronary interventions were enrolled. The target lesions (>50% diameter stenosis) were evaluated pre-operatively by both QCA and IVUS operating at 30-40 MHz and the percent area stenosis, eccentricity index (EI) and lesion length were determined. The minimal (min) and maximal (max) distances from the center of the stenotic lesion to the outline of the vessel wall were measured, and the EI was calculated by the formula: [(max - min)/max]. By QCA, lesion length was determined by measuring the distance between the proximal and distal shoulders of the lesion. When the lesions were observed by IVUS with a motorized pull-back system, the length was calculated by multiplying the time for observation of the disease and 0.5 or 1 mm/s. Although the severity of the stenosis determined by QCA (86+/-10%, mean +/- SD) did not differ from that by IVUS (83+/-13%), there was no correlation between them (r=0.32, y=0.25x+65) and the correlation did not improve when lesions with remodeling, enlargement (n=176) or shrinkage (n=79) were omitted from the calculation. The EIs by QCA and IVUS were 0.51+/-0.26 and 0.52+/-0.22, respectively (NS), and there was no correlation between them (r=0.30, y=0.36x+33). However, when the lesions with remodeling were excluded, the correlation greatly improved (r=0.80, y=0.84x+10.6, p<0.05). Lesion length determined by QCA (12.4+/-6.1 mm) was significantly shorter than that by IVUS (16.3+/-8.9 mm, p<0.01). These results demonstrate that coronary angiography significantly misinterprets disease morphology in terms of severity, eccentricity and length, in part because of vessel remodeling that can be accurately determined only by IVUS.

Active immunization of combined β1-adrenoceptor and M2-muscarinic receptor peptides induces cardiac hypertrophy in rabbits

BACKGROUND The high prevalence of patients with dilated cardiomyopathy (DCM) with anti-beta1-adrenoceptor and/or anti-M2-muscarinic receptor autoantibodies in their sera has been observed. However, the pathophysiological role of these autoantibodies in the development of cardiomyopathy is unknown. We previously reported an experimental model of early-stage DCM-like cardiomyopathy induced by immunizing rabbits for 1 year with synthetic peptides corresponding to the sequence of the second extracellular loop of either beta1-adrenoceptor or M2-muscarinic receptor. Because approximately half the sera of patients with DCM that recognize one of the two receptor sequences also recognize the second sequence, a model was created in rabbits simultaneously immunized with the synthetic peptides corresponding to the second extracellular loop of the beta1-adrenoceptor and M2-muscarinic receptor. METHODS AND RESULTS All rabbits (n = 8) immunized with both peptides had a high titer of both anti-beta1-adrenoceptor and anti-M2-muscarinic receptor autoantibodies in their sera, whereas none of the sera from control rabbits injected with saline (n = 9) was positive. No significant cross-reaction with peptides other than those used for immunization was found. The weight of the hearts of immunized rabbits increased significantly. The hearts of immunized rabbits showed marked concentric left ventricular hypertrophy with mild inflammatory cell infiltration. In these rabbits, mild or moderate interstitial fibrosis was also observed. In electron micrographs, immunized rabbits showed focal myofibrillar lysis, loss of myofilament, and a marked increase in the number of mitochondria and deposition of dense granules in both sarcoplasm and myofibrils. Conversely, one of the control rabbits showed scant mononuclear cell infiltration. However, in this control rabbit, no significant alteration was found by electron microscopy. CONCLUSION Our results showed the coexistence of both anti-beta1-adrenoceptor and anti-M2-muscarinic receptor autoantibodies in the sera has pathophysiological importance, shown by their ability to induce cardiac hypertrophy in rabbits.