Ryuko Matsuda

Measurement of plasma annexin V by ELISA in the early detection of acute myocardial infarction

Annexin V is a calcium binding protein which is widely present in various cells and tissues. Using annexin V which we isolated and purified from human cardiac muscle, we prepared an anti-human cardiac annexin V monoclonal antibody. Identification of annexin V was made by means of partial amino acid sequences. An enzyme-linked immunosorbent assay (ELISA) was developed using this monoclonal antibody and anti-canine cardiac annexin V polyclonal antibody. With this ELISA, plasma annexin V concentration was measured in 196 normal healthy individuals, 23 acute myocardial infarction (AMI) patients who were hospitalized within 6 h after the onset of chest pain, and 130 patients with other diseases, including lung, liver and kidney disease. The plasma annexin V concentration in normal healthy individuals was 1.7 +/- 0.6 ng/ml (mean +/- S.D.), while that in AMI patients was elevated to 13.2 +/- 6.8 ng/ml (P < 0.0001) at the time of initial blood drawing, 3.2 +/- 1.5 h after onset of pain, and these values were higher than normal in 21 out of 23 cases (91.3%) of AMI. In all cases excepting 3, annexin V concentration immediately decreased after the onset of pain. The annexin V concentration in patients with old myocardial infarction, chest pain syndrome, valvular heart disease, lung disease and kidney disease was 1.8 +/- 0.8, 2.0 +/- 0.7, 1.7 +/- 1.1, 2.3 +/- 1.4 and 2.1 +/- 1.2 ng/ml, respectively, being within normal limits. The values in liver disease patients and trauma patients were 3.7 +/- 2.7 (P < 0.05) and 3.3 +/- 2.4 (P < 0.05) ng/ml, respectively, being slightly higher than that in normal healthy individuals.

Inhibition of annexin V-dependent Ca2+ movement in large unilamellar vesicles by K201, a new 1,4-benzothiazepine derivative

Examination was made of the effect of annexin V on Ca2+ movement into large unilamellar vesicles (LUV) using fura-2, a calcium-sensitive fluorescent dye. To avoid the possible difficulties relating to the addition of annexin V and/or Ca2+ in fura-2-loaded LUV, the burst method was used. LUV, preincubated with rat annexin V in the presence of Ca2+, were collected by centrifugation and resuspended, and then burst with Triton X-100 in the presence of fura-2. Inward Ca2+ movement across the artificial lipid membrane was measured by determination of fura-2 fluorescence due to the leaked Ca2+ from ruptured LUV. The observed Ca2+ signal increased dependent on annexin V and Ca2+ concentrations, whereas bovine serum albumin did not affect this signal up to 1 microM. Thus, annexin V shows Ca2+ channel activity in LUV. K201, a novel 1,4-benzothiazepine, inhibited inward Ca2+ movement into LUV caused by annexin V in a dose-dependent manner. In the presence of 50 nM annexin V and 400 microM Ca2+, 3 microM K201 showed significant inhibition of Ca2+ movement due to annexin V, and 50% inhibition was achieved at 25 microM K201. On the other hand, diltiazem had no such effect even at 30 microM. K201 is thus shown to have inhibitory activity on inward Ca2+ movement due to annexin V in artificial vesicles and may prove useful as a probe for elucidating the functions of annexin V in vivo.

Three-dimensional reconstruction of the human capillary network and the intramyocardial micronecrosis

Three-dimensional reconstruction of the human heart was performed to define the structure of the intramyocardial microvasculature. A total of 200 consecutive serial sections of 6 μm each were prepared from the left ventricular tissue of an autopsied human heart with normal coronary arteries. The corresponding arteriole, venule, and all capillaries were reconstructed using three-dimensional software. The capillary network extended right and left along the cardiomyocyte with major and minor axes of about 130 and 120 μm, respectively. The capillary length from an arteriole to an adjacent venule was about 350 μm. Two types of sack-like structures, the precapillary sinus and the capillary sinus, were present in the capillary network, and many capillaries diverged from these sinuses. The cardiomyocytes were covered with reticular capillaries. In contrast, the precapillary and capillary sinuses were surrounded by many cardiomyocytes. The arterial and venous capillaries were positioned alternately, forming a lattice pattern. Intramyocardial microcirculatory units forming a capillary network from an arteriole to adjacent venules on both sides were present. The sizes of myocardial micronecroses corresponded to that of the intramyocardial microcirculatory unit. These results show that the capillary network is an ordered and anatomically regulated structure and that the microcirculatory unit and the precapillary and capillary sinuses may play an important role in maintaining the intramyocardial microcirculation during contraction and relaxation.

Clinical significance of measurement of plasma annexin V concentration of patients in the emergency room

Annexin V, a calcium-binding protein, is widely present in various organs and tissues. In the present study, plasma annexin V concentration was measured in 158 patients who were brought to the emergency room, including 25 patients suffering from acute myocardial infarction (AMI), 14 with cerebrovascular disease, 11 with trauma of the extremities, 11 with severe trauma associated with visceral damage, and 35 with witnessed cardiac arrest. Annexin V concentration in normal healthy individuals (n=110) was 1.9+/-0.7 ng/ml. Annexin V concentration in AMI and cardiac arrest patients was 11.0+/-4.9 and 15.3+/-7.9 ng/ml, respectively, being significantly higher than that in patients with cerebrovascular disease (5.4+/-2.7 ng/ml). The value in severe trauma patients was 15.9+/-9.4 ng/ml, being significantly higher than that in patients with trauma of the extremities (5.6+/-1.2 ng/ml). Annexin V concentrations in the cardiac arrest and AMI patients who survived more than 24 h after admission were lower than those in patients who died within 24 h after the onset of symptoms. Annexin V content in the lungs and myocardium in normal rats was extremely high in comparison to that in brain and skeletal muscle. These results suggest that the high levels of plasma annexin V in patients with AMI, cardiac arrest and severe trauma reflect the severity of damage of the myocardium and/or other visceral organs, and measurement of plasma annexin V concentration may help to assess the prognosis of patients brought to the emergency room.

Pharmacological Characteristics and Clinical Applications of K201

K201 is a 1,4-benzothiazepine derivative that is a promising new drug with a strong cardioprotective effect. We initially discovered K201 as an effective suppressant of sudden cardiac cell death due to calcium overload. K201 is a nonspecific blocker of sodium, potassium and calcium channels, and its cardioprotective effect is more marked than those of nicorandil, prazosine, propranolol, verapamil and diltiazem. Recently, K201 has also been shown to have activities indicated for treatment of atrial fibrillation, ventricular fibrillation, heart failure and ischemic heart disease, including action as a multiple-channel blocker, inhibition of diastolic Ca2+ release from the sarcoplasmic reticulum, suppression of spontaneous Ca2+ sparks and Ca2+ waves, blockage of annexin V and provision of myocardial protection, and improvement of norepinephrine-induced diastolic dysfunction. Here, we describe the pharmacological characteristics and clinical applications of K201.

Purification of cardiac annexin V from the beagle dog heart and changes in its localization in the ischemic rat heart

SummaryWe isolated and purified 35kDa protein from the myocardium of the beagle dog and identified it to be annexin V from partial amino acid sequence determination. It was confirmed that anticanine cardiac annexin V rabbit polyclonal antibody, which was produced using the 35 kDa protein, cross-reacts with annexin V of the myocardium, lung, liver, kidney, and brain of the rat. The localization of cardiac annexin V and the effect of ischemia for 30–180 min in the rat were immunohistochemically studied with the use of the Langendorff perfusion heart. In the normal myocardium, annexin V, accompanied by cross-striation, was observed throughout the cell. In ischemia of 30 min, extracellular leakage of annexin V was observed with uneven staining in the cytoplasm. When the ischemic time exceeded 60 min, annexin V was observed in the cell membrane with a decrease of annexin V in the cytoplasm. Also, extracellular leakage of annexin V was observed prominently. In ischemia for 180 min, almost all the annexin V in the cytoplasm disappeared. These results suggest that the level of ischemia can be estimated from the changes in localization of annexin V.

Measurement of urinary annexin V by ELISA and its significance as a new urinary-marker of kidney disease.

To confirm the significance of excretion of annexin V into the urine and the change of urinary annexin V concentration in kidney disease, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed using two monoclonal antibodies. Urinary annexin V concentration was measured in healthy individuals and patients with kidney and other diseases. Urinary annexin V did not change over a range of pH between 5.0 and 8.0, and was stable during the course of the study for 24 h at room temperature and for 8 days at 4 degrees C. The mean urinary annexin V concentration in 105 normal healthy individuals was 1.5+/-1.5 ng/ml, while that in patients with nephrotic syndrome and systemic lupus erythematosis (SLE) nephritis was 9.3+/-9.1 and 6.6+/-6.7 ng/ml, respectively, and that in IgA nephropathy and chronic renal failure was 2.6+/-2.1 and 1.3+/-0.7 ng/ml, respectively. Annexin level correlated with urinary protein concentration (r=0. 717), but not the serum creatinine concentration, blood urea nitrogen (BUN) and 24-h creatinine clearance. Mean urinary annexin V concentration in patients with ischemic heart disease, hypertension, and diabetes mellitus was 1.4+/-1.0, 1.4+/-1.1, and 1.7+/-1.3 ng/ml, respectively. In one case of relapsing nephrotic syndrome, the urinary annexin V concentration was markedly increased in the early phase after admission and then decreased. This patient later required hemodialysis. These results suggest that a high urinary annexin V concentration may be an indicator of acute renal injury related to the urinary protein level.

Localization of annexin V in rat normal kidney and experimental glomerulonephritis.

The localization of annexin V, a calcium binding protein, was immunochemically and immunohistologically studied in experimental rat glomerulonephritis using annexin V polyclonal antibody. Plasma and urinary annexin V levels were measured by a sandwich enzyme-linked immunosorbent assay (ELISA). Urinary annexin V level, which was correlated with urinary l-lactate dehydrogenase activity, N-acetyl-β-d-glucosaminidase activity and protein level, increased time-dependently after the injection of nephritogenic antigen (bovine glomerular basement membrane), progressively increasing to attain a peak level at 4 weeks of 51.5±11.3 ng/h. However, plasma annexin V level showed no increase during the study period. Normal kidneys showed strong staining for annexin V in distal tubules, being particularly strong in tubules of the inner stripe of the outer medulla, but could not be detected in proximal tubules. Annexin V was seen in visceral epithelial cells, Bowman’s capsule of the glomerulus, the vascular endothelium of arterioles and interlobular arteries, and vascular smooth muscle. In nephritis, the lumen of distal tubules and the luminal cell membrane were deeply stained, with leakage of annexin V being observed from tubular cells. In the present study, renal annexin V was markedly excreted into urine, and its urinary level reflected the severity of damage of renal tissue and the progression of nephritis. These changes of annexin V in the distal tubule and visceral epithelial cells may be of significance in cell injury of the kidney.

The effect of K201 on isolated working rabbit heart mechanical function during pharmacologically induced Ca2+ overload

Reduced cardiac contractility has been associated with disrupted myocardial Ca2+ signalling. The 1,4 benzothiazepine K201 (JTV‐519) acts on several Ca2+ handling proteins and improves cardiac contractility in vivo in a variety of animal models of myocardial dysfunction. However, it is unclear whether this improvement depends on the systemic effects of K201 or if K201 reverses the effects of Ca2+ dysregulation, regardless of the cause.

Protective Effect of K201 on Isoproterenol-Induced and Ischemic–Reperfusion-Induced Ventricular Arrhythmias in the Rat Comparison With Diltiazem

Aim: Ventricular arrhythmia (VA) is a risk for sudden death. Polymorphic ventricular tachycardia (VT) degenerating to ventricular fibrillation occurs subsequent to the prolongation of the QT interval following administration of catecholamines under Ca2+ loading. Fatal VA also occurs in ischemia and ischemic–reperfusion. We compared the suppressive effect of K201 (JTV519), a multiple-channel blocker and cardiac ryanodine receptor-calcium release channel (RyR2) stabilizer, with that of diltiazem, a Ca2+ channel blocker, in 2 studies of isoproterenol-induced (n = 30) and ischemic–reperfusion-induced VAs (n = 38) in rats. Methods: Adult male Wistar rats were administered 12 mg/kg/min calcium chloride (CaCl2) for 20 minutes and then 6 μg/kg/min isoproterenol was infused with CaCl2 for a further 20 minutes. In other rats, the left coronary artery was ligated for 5 minutes followed by reperfusion for 20 minutes. K201 or diltiazem (both 1 mg/kg) was administered before infusion of the isoproterenol or induction of ischemia. Results: After administration of isoproterenol under Ca2+ loading, fatal VA frequently occurred in the vehicle (9 of 10 animals, 90%) and diltiazem (8 of 10, 80%) groups, and K201 significantly suppressed the incidences of arrhythmia and mortality (2 of 10, 20%). In the reperfusion study, the incidence and the time until occurrence of reperfusion-induced VA and mortality were significantly suppressed in the K201 (2 of 15 animals, 13%) and diltiazem (1 of 9 animals, 11%) groups compared to the vehicle group (8 of 14 animals, 57%). Significance: Induction of VA in an experimental model was achieved with a low dose of isoproterenol under Ca2+ loading. K201 markedly suppressed both the isoproterenol-induced and the reperfusion-induced VAs, whereas diltiazem did not suppress the isoproterenol-induced VA. The results suggest that both VAs are related to early after depolarization (EAD) and indicate that K201 has the potential to suppress EAD by stabilizing RyR2 to mediate Ca2+ release from the sarcoplasmic reticulum and acting as a multiple-channel blocker.