Masafumi Koide

Expression pattern of novel chick T-box gene, Tbx20.

Abstract. Little is known about the molecular mechanisms involved with the initial specifications of the cardiac mesoderm. In order to identify potential regulatory factors that play important roles in early heart specification, we attempted to isolate the chick H15-related T-box gene and analyze its expression pattern during early development. The chick Tbx20 gene was found to be highly homologous to human, mouse, and zebrafish hrT/Tbx20. Its expression was initially detected in the posterior lateral mesoderm, after which it expanded to the anterior and was intensively co-expressed with a cardiogenic gene, Nkx2.5, in the anterior lateral mesoderm.

Impaired Force-Frequency Relations in Patients With Hypertensive Left Ventricular Hypertrophy A Possible Physiological Marker of the Transition From Physiological to Pathological Hypertrophy

BACKGROUND The extent to which force-frequency and relaxation-frequency relations (FFR and RFR, respectively) and exercise-induced adrenergic stimulation affect myocardial inotropic and lusitropic reserves has not been established in patients with left ventricular (LV) hypertrophy (LVH). METHODS AND RESULTS We calculated the maximum first derivative of LV pressure (LV dP/dtmax) and the LV pressure half-time (T1/2) during pacing, exercise, and isoproterenol infusion in 17 patients with hypertensive LVH and 9 control subjects to investigate the influence of increases in heart rate (HR) and adrenergic stimulation on inotropic and lusitropic reserves. Group A consisted of 10 LVH patients who showed a progressive increase in the HR-LV dP/dtmax relation. Group B consisted of 7 LVH patients in whom the HR-dP/dtmax relation at physiological pacing rates was biphasic. The LV mass index was larger and the LV ejection fraction was smaller in group B than in group A (244+/-72 g/m2 versus 172+/-22 g/m2 and 55+/-18% versus 72+/-6%, respectively; both P<0.05). The increase in LV dP/dtmax was greater during exercise than pacing alone for similar increases in HR in all groups (P<0.05) (group A, 111+/-22% versus 25+/-14%; group B, 105+/-35% versus 14+/-10%; control, 111+/-24% versus 25+/-12%). T1/2 was shorter (P<0.05) during exercise than with pacing alone in all groups (group A, 41+/-6% versus 11+/-3%; group B, 38+/-9% versus 14+/-4%; control, 44+/-6% versus 12+/-5%). Isoproterenol infusion caused similar increases in LV dP/dtmax and similar decreases in T1/2 in all groups. CONCLUSIONS The FFR was biphasic in patients with severe LVH irrespective of LV function but was preserved in patients with less severe LVH and control subjects. Importantly, the RFR and adrenergic control of both inotropic and lusitropic reserves were well preserved in all LVH patients. A biphasic FFR at physiological pacing rates may be one of the earliest markers of the transition from physiological adaptation to the pathological process in LVH patients.

Mechanism of abnormal postexercise systolic blood pressure response and its diagnostic value in patients with coronary artery disease

To define the factors that affect an abnormal postexercise systolic blood pressure (SBP) response and to verify the diagnostic value of that response in patients with coronary artery disease (CAD), we studied 33 normal subjects, eight patients with hypertension who had no CAD, and 42 patients with documented CAD who underwent supine leg exercise testing. SBP recovery ratios were derived by dividing the values obtained 1 and 3 minutes after exercise by the peak value. The upper normal limit of the SBP ratio was defined by two standard deviations from the mean for 33 normal subjects. The specificity of this criterion for identifying patients with CAD was 97% and the sensitivity was 60%. None of the eight patients with hypertension showed an abnormal postexercise SBP response. At peak exercise the pulmonary artery wedge pressure was significantly greater in the patients with CAD having an abnormal response. In multiple regression analysis the exercise pulmonary artery wedge pressure, the exercise SBP, and the systemic vascular resistance after exercise were determining factors for an abnormal SBP response. Three-vessel disease was more common in patients with an abnormal response. Results of this study indicate that an abnormal postexercise SBP response appears to be determined by (1) the extent of exercise-induced impairment of left ventricular function and (2) peripheral vascular tone during recovery. This study also suggests that this criterion can increase the specificity of supine exercise testing in patients with CAD including those with hypertension and might provide a marker for the severity of impaired left ventricular function by exercise in CAD.

The important role of left ventricular relaxation and left atrial pressure in the left ventricular filling velocity profile

To evaluate the determinants of left ventricular filling, left ventricular filling velocity was measured by pulsed Doppler flowmetry during catheterization of the right and left sides of the heart in 37 patients with cardiac disease before and during leg elevation. During leg elevation, despite no significant change in the time constant of isovolumic relaxation (T), the peak rapid filling velocity (PVRF) increased in association with an increase in pulmonary wedge pressure (PWP), but the peak atrial filling velocity was unchanged. The PVRF correlated with the pulmonary wedge V wave - left ventricular minimum pressure difference (r = 0.68) and in multivariate regression with both T and mean PWP (R = 0.73). These results indicate that left ventricular filling is determined by both left ventricular relaxation and left atrial pressure and that an increase in left atrial pressure changes the left ventricular filling velocity profile in a manner that mimics the pattern with normal diastolic function.

Sex‐specific and left‐right asymmetric expression pattern of Bmp7 in the gonad of normal and sex‐reversed chicken embryos

A genetic switch determines whether the indifferent gonad develops into an ovary or a testis. In adult females of many avian species, the left ovary is functional while the right one regresses. In the embryo, bone morphogenetic proteins (BMP) mediate biological effects in many organ developments but their roles in avian sex determination and gonadal differentiation remains largely unknown. Here, we report the sex‐specific and left‐right (L‐R) asymmetric expression pattern of Bmp7 in the chicken gonadogenesis. Bmp7 was L‐R asymmetrically expressed at the beginning of genital ridge formation. After sexual differentiation occurred, sex‐specific expression pattern of Bmp7 was observed in the ovary mesenchyme. In addition, ovary‐specific Bmp7 expression was reduced in experimentally induced female‐to‐male reversal using the aromatase inhibitor (AI). These dynamic changes of expression pattern of Bmp7 in the gonad with or without AI treatment suggest that BMP may play roles in determination of L‐R asymmetric development and sex‐dependent differentiation in the avian gonadogenesis.

Effects of a new second generation calcium channel blocker, nilvadipine (FR34235), on exercise-induced hemodynamic changes in stable angina pectoris.

The mechanism of the antianginal actions of nilvadipine was investigated in 11 patients with effort angina pectoris. Hemodynamic data were obtained by angina-limited supine multistage bicycle ergometer exercise testing before and after a single 6 mg dose of nilvadipine. Compared with chest pain during control exercise testing, pain at peak exercise disappeared or abated and the ST segment at peak exercise also showed less significant depression after administration of nilvadipine. At rest and at peak exercise, mean blood pressure, pulmonary artery wedge pressure and systemic vascular resistance decreased significantly, whereas heart rate and cardiac index increased significantly after nilvadipine. Rate-pressure product and stroke volume index did not change significantly. Coronary sinus flow at peak exercise increased significantly and total coronary vascular resistance at rest and at peak exercise decreased significantly after nilvadipine. The plasma concentrations of nilvadipine 1.5 hours after administration ranged from 1.15 to 8.23 ng/ml. These data suggest that the principal factors in the antianginal actions of nilvadipine are an increase in myocardial oxygen supply due to increased coronary blood flow and a reduction in myocardial oxygen demand mainly by a decrease in afterload and additionally by a decrease in preload.

Dynamic exercise-induced elevation in plasma levels of atrial natriuretic peptide in patients with effort angina pectoris

We investigated the relationship between plasma atrial natriuretic polypeptide (ANP) levels and hemodynamic indices during dynamic exercise testing in 15 patients with effort angina pectoris. Patients exercised on an angina-limited, supine, multistage bicycle ergometer, and plasma ANP levels and hemodynamic indices were measured at rest, at peak exercise, and 6 minutes after exercise. Plasma ANP levels increased significantly at peak exercise. Pulmonary artery wedge pressure (PAWP) and coronary sinus blood flow (CSBF) were significantly correlated with plasma ANP levels before and at peak exercise (PAWP: r = 0.69, p less than 0.001; CSBF; r = 0.45, p less than 0.05). In six of eight patients whose PAWP exceeded 20 mm Hg at peak exercise, plasma ANP levels were increased at 6 minutes after exercise, whereas PAWP had decreased relative to the values obtained at peak exercise. Plasma ANP concentrations at 6 minutes after exercise were not correlated with PAWP at the same time. However, PAWP at peak exercise was correlated with the plasma ANP levels at 6 minutes after exercise (r = 0.80, p less than 0.001). These results suggest that in patients with effort angina pectoris left ventricular dysfunction resulting from exercise-induced myocardial ischemia may increase preload excessively and may contribute to the excess secretion of ANP after dynamic exercise.

Heart‐selective expression of the chicken FK506‐binding protein (FKBP) 12.6 gene during embryonic development

FKBP12.6, a member of the family of FK506‐binding proteins, selectively associates with the cardiac isoform of the ryanodine receptor and thereby stabilizes this Ca2+ release channel. A chicken FKBP12.6 (chFKBP12.6) cDNA was cloned and shown to encode a protein of 108 amino acids. The deduced amino acid sequence of chFKBP12.6 is 91–92% identical to those of mammalian FKBP12.6 proteins. Northern blot analysis revealed that chFKBP12.6 mRNA is largely restricted to the heart during embryonic development and that the abundance of this mRNA in the heart decreases, and it becomes restricted to the atrium during cardiogenesis. In situ hybridization revealed that chFKBP12.6 mRNA is localized to the precardiac mesoderm before formation of the primitive heart tube. Expression of the chFKBP12.6 gene was initially apparent throughout the developing multichambered heart but became restricted to the atria before hatching. Reverse transcription and polymerase chain reaction analysis demonstrated that chFKBP12.6 mRNA is present in the embryo from early gastrulation and is most abundant immediately after the onset of the heartbeat. These observations suggest that the chFKBP12.6 gene is expressed before heart morphogenesis to play a role in excitation–contraction coupling in cardiomyocytes and that the function of the encoded protein becomes increasingly restricted to the atrium during embryonic development.

INFLUENCE OF CALCIUM ON PROLIFERATION AND PHENOTYPE ALTERATION OF CARDIOMYOCYTE IN VITRO

An accelerated weight gain is noted in the heart of Ca‐deficient, hypertensive chick embryos maintained in a shell‐less culture in vitro. We previously observed that the Ca handling property of cardiomyocytes isolated from the shell‐less embryo is altered, i.e., faster Ca uptake, suggesting a requirement for adequate Ca supply and/or proper Ca handling in embryonic cardiac development. In this study, we have examined the function of Ca on cardiomyocytes by analyzing the effects of (1) various Ca concentration in the culture medium (NCa, 1.8 mmol/L; HCa, 2.8 mmol/L; LCa, 0.9 mmol/L), and (2) various modulators of Ca handling on cell proliferation and phenotype regulation in chick embryonic cardiomyocytes. The analytical parameters included cell number, DNA content, expression of cell cycle–specific and cardiomyocyte‐specific proteins, and creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) enzyme activities. Cell number and total DNA were significantly larger (P < 0.01) in LCa cultures compared with those in NCa. The level of LDH was elevated (P < 0.01), but that of CPK was lowered in LCa. Expression of the G1‐S–specific protein PCNA was raised, but that of the contractile proteins myosin and tropomyosin was substantially suppressed in LCa; in HCa, the cells did not proliferate as well, whereas the level of contractile proteins was higher. Thapsigargin, a sarcoplasmic reticulum (SR)‐specific, Ca‐ATPase inhibitor, simulated the effects of LCa by enhancing cell proliferation and lowering the expression of tropomyosin. These results suggest that culturing in low Ca concentration and inhibition of SR Ca pumping enhance myocardial cell proliferation and suppress sarcomeric protein expression, perhaps by inducing cellular de‐differentiation. The in vitro effects of medium Ca concentration and Ca handling modulators on cardiomyocytes also suggest that the in vivo cardiomegaly of the SL embryos is a direct result of Ca‐deficiency, and that Ca is important in the phenotype regulation of cardiomyocytes. J. Cell. Physiol. 177:289–298, 1998.

Hemodynamic mechanisms of the antianginal action of a novel vasodilator FK409 in dynamic exercise-induced angina.

FK409 is a novel vasodilator with a unique chemical structure. We wished to elucidate the mechanisms of antianginal action of FK409 in dynamic exercise-induced angina. Twelve patients with stable effort angina pectoris were studied before and after a single 40-mg oral dose of FK409. Chest pain was induced in all of 12 patients during the control multistage bicycle ergometer exercise. After FK409 administration, the same workload did not induce chest pain in 6 patients. The ST segment at peak exercise showed less severe depression from 0.15 ± 0.02 to 0.05 ± 0.01 mV (p < 0.001). Left ventricular (LV) filling estimated by the Doppler method was reduced, and pulmonary artery wedge pressure decreased significantly (p < 0.001) throughout exercise testing after FK409. Myocardial oxygen uptake and coronary sinus flow throughout exercise testing decreased significantly (p < 0.05) after FK409 administration. The results of the present study demonstrate that decrease in myocardial oxygen demand may be caused by pre- and afterload reduction and that it could be a major mechanism of the antianginal action of FK409. However, other mechanisms such as redistribution of coronary blood flow to the subendocardium, direct dilatation of the stenotic parts of the epicardial arteries, and an increase in collateral blood flow should be considered additional possible mechanisms of the antianginal action of FK409.