A novel method to study contraction characteristics of a single cardiac myocyte using carbon fibers.

To facilitate cardiac muscle research, we developed a novel method by which the force and length of a single ventricular myocyte can be recorded with a pair of carbon graphite fibers attached firmly to both ends. One fiber was stiff, whereas the other fiber was compliant to allow the recording of force and shortening during twitch contractions. The image of the compliant carbon fiber was projected onto a pair of photodiodes, and their output was fed to a piezoelectric transducer after variable amplifications to alter the effective compliance of the carbon fiber. Thus contraction of the myocyte was induced under virtually isometric conditions as well as under auxotonic conditions. We obtained a bell-shaped relation between the compliance under an auxotonic load and the work output of the myocyte, which was directly related to myocyte performance in the heart. Because it is easy to attach myocytes to the experimental apparatus, the present method would allow us to study cardiac muscle mechanics at the cellular and molecular levels.

Local antithrombotic therapy using a novel porous balloon catheter.

The efficacy of local treatment of thrombosis with low-dose antithrombotic drugs (heparin: 30 U/kg, or argatroban: 0.02 mg/kg) was investigated using a novel porous balloon catheter. This novel balloon catheter can deliver drug into arterial walls without causing vascular trauma. Thrombus formation was significantly inhibited in balloon-injured and locally-treated iliac arteries compared with control balloon-injured arteries in 12 dogs. In the systemic high-dose delivery group (ten times as high as the low dose), thrombus formation in injured arteries was significantly less than that of controls in 7 dogs. Low-dose systemic delivery was not effective at inhibiting this thrombus formation. Thus, local treatment with an antithrombotic drug using this novel porous balloon catheter can prevent thrombosis without influencing systemic coagulability.

Analysis of nitric oxide in the exhaled air of patients with chronic glomerulonephritis.

BACKGROUND: Nitric oxide (NO) plays an important role in renal hemodynamics and function. Although production of NO in the glomeruli has been found to be increased in animal models of glomerulonephritis, it remains unclear whether its endogenous production is enhanced in patients with chronic glomerulonephritis (CGN). SUBJECTS AND METHODS: We measured NO output in exhaled air as an indicator of its local production in the lungs and plasma and urinary nitrite plus nitrate (NO2-/NO3-) levels as indicators of its production in the whole body in 21 patients with CGN in 31 healthy controls. RESULTS: The patients exhaled higher concentrations of NO (29.5 +/- 1.4 vs. 18.7 +/- 1.0 parts per billion (ppb), mean +/- SEM, p < 0.0001) and exhaled NO output was also higher than in controls (166.6 +/- 6.8 vs. 95.5 +/- 5.6 nl/min/m2, p < 0.0001). Plasma NO2-/NO3- concentrations were also significantly greater in the patients than in the controls (81.6 +/- 7.2 vs. 41.1 +/- 4.3 micromol/l, p < 0.001). In patients with CGN, exhaled NO output correlated negatively with creatinine clearance (r = -0.62, p < 0.05). Oral administration of prednisolone (60 mg/day) for two weeks did not significantly affect the exhaled NO output in the patients (160 +/- 7 vs. 200 +/- 30 nl/min/m2, p = NS) despite a decrease in urinary protein excretion (12.0 +/- 2.9 vs. 1.4 +/- 0.6 g/day, p < 0.01). CONCLUSION: These findings suggested that endogenous NO production is increased in patients with CGN. Increased endogenous NO production may play some pathophysiological role in these patients.

Insulin action on heart and skeletal muscle FDG uptake in patients with hypertriglyceridemia.

UNLABELLED: Abnormal heart and skeletal muscle 18F-fluorodeoxyglucose (FDG) uptake in patients with insulin resistance has been demonstrated. Although the existence of whole-body insulin resistance has been reported in hypertriglyceridemics, its specific role in heart and skeletal muscle FDG uptake in hypertriglyceridemics has not been clarified. METHODS: We compared heart and skeletal muscle FDG uptake using PET and the whole-body glucose disposal rate (GDR) during insulin clamping in 17 hypertriglyceridemics and 12 age-matched control subjects to increase our knowledge of whole-body insulin resistance and its relationship to heart and skeletal muscle FDG uptake in hypertriglyceridemics. RESULTS: GDR was significantly reduced in hypertriglyceridemics compared with control subjects (4.50 +/- 1.37 mg/min/kg versus 10.0 +/- 2.97 mg/min/kg, P = 0.00001), as were the skeletal muscle FDG Ki = (k1 x k3)/(k2 + k3) (SFKi: 0.007 +/- 0.003 mL/min/g versus 0.018 +/- 0.01 mL/min/g, P = 0.0001) and skeletal muscle FDG uptake ([SMFU] 0.725 +/- 0.282 mg/min/100 g versus 1.86 +/- 1.06 mg/min/100 g, P = 0.00023). However, myocardial FDG Ki (MFKi) tended to be reduced in hypertriglyceridemics compared with that in control subjects (0.062 +/- 0.017 mL/min/g versus 0.068 +/- 0.015 mL/min/g), but the difference was statistically insignificant (P = 0.3532). Moreover, myocardial FDG uptake (MFU) in hypertriglyceridemics (6.47 +/- 1.72 mg/min/100 g) tended to be reduced compared with that in control subjects (6.97 +/- 1.73 mg/min/100 g), but the difference was statistically insignificant (P = 0.4485). GDR was significantly correlated with SFKi (r = 0.69, P = 0.0022), SMFU (r = 0.612, P = 0.009), MFKi (r = 0.57, P = 0.0174) and MFU (r = 0.505, P = 0.0385) in hypertriglyceridemics. CONCLUSION: Both heart and skeletal muscle glucose utilization were related to insulin resistance in hypertriglyceridemics. However, the less severe reduction in MFU (compared with SMFU) suggests that myocardium may have a mechanism to oppose insulin resistance in hypertriglyceridemics.

Improvement of impaired myocardial vasodilatation due to diffuse coronary atherosclerosis in hypercholesterolemics after lipid-lowering therapy.

BACKGROUND: Diminished myocardial vasodilatation (MVD) in hypercholesterolemics without overt coronary stenosis has been reported. However, whether the diminished MVD of angiographically normal coronary arteries in hypercholesterolemics can be reversed after lipid-lowering therapy is not known. METHODS AND RESULTS: A total of 27 hypercholesterolemics and 16 age-matched controls were studied. All patients had >1 normal coronary artery, and those segments that were perfused by anatomically normal coronary arteries were studied. Myocardial blood flow (MBF) was measured during dipyridamole loading and at baseline using positron emission tomography and 13N-ammonia, after which MVD was calculated before and after lipid-lowering therapy. Total cholesterol was significantly higher in hypercholesterolemics (263+/-33.8) than in controls (195+/-16.6), and it normalized after lipid-lowering therapy (197+/-19.9). Baseline MBF (ml. min-1. 100 g-1) was comparable among hypercholesterolemics (both before and after therapy) and controls. MBF during dipyridamole loading was significantly lower in hypercholesterolemics before therapy (189+/-75.4) than in controls (299+/-162, P<0.01). However, MBF during dipyridamole loading significantly increased after therapy (226+/-84.7; P<0.01). MVD significantly improved after therapy in hypercholesterolemics (2.77+/-1.35 after treatment [P<0.05] versus 2. 02+/-0.68 before treatment [P<0.01]), but it remained significantly higher in controls (3.69+/-1.13, P<0.01). There was a significant relationship between the percent change of total cholesterol and the percent change of MVD before and after lipid-lowering therapy (r=-0. 61, P<0.05). CONCLUSIONS: Diminished MVD of anatomically normal coronary arteries in hypercholesterolemics can be reversed after lipid-lowering therapy.

Role of positron emission tomography using fluorine-18 fluoro-2-deoxyglucose in predicting improvement in left ventricular function in patients with idiopathic dilated cardiomyopathy.

Improvement in left ventricular (LV) function in patients with idiopathic dilated cardiomyopathy (DCM) by medical treatment has been suggested. Thus, it is important to evaluate which patients will respond to medical therapy. Positron emission tomography (PET) with fluorine-18 fluoro-2-deoxyglucose (FDG) and cardiac catheterization were performed in 20 patients with DCM before the initiation of medical therapy. The regional myocardial glucose utilization rate (rMGU) was measured with FDG PET. Subjects were divided into two groups, group 1 (event-free patients, n=10) and group 2 (clinical cardiac events, n=10). Haemodynamic and PET parameters before the initiation of medication were compared between the two groups and between patients with and patients without improvement in LV function. Ejection fraction (EF) was significantly higher in group 1 (35.8%+/-9.0%) than in group 2 (24.8%+/-7.0%) and LV end-diastolic pressure (LVEDP) was significantly lower in group 1 (8.4+/-1.7 mmHg) than in group 2 (11.6+/-3.5 mmHg). Average rMGU (mg min-1 100 g-1) was similar in group 1 (11.2+/-2.5 mg min-1 100 g-1) and group 2 (11.2+/-2.9 mg min-1 100 g-1), while %CV of rMGU was significantly lower in group 1 (11.1%+/-6.3%) than in group 2 (29. 9%+/-13.9%, P<0.01). Furthermore, LV function normalized in seven patients in group 1. In these seven patients, EF (35.1%+/-10.9%), LVEDP (8.2+/-2.0 mmHg) and average rMGU (11.8+/-2.7 mg min-1 100 g-1) were comparable with those in patients without LV functional improvement (EF: 31.6%+/-9.1%; LVEDP: 10.7+/-3.3 mmHg; average rMGU: 10.8+/- 2.7 mg min-1 100 g-1). However,% CV of rMGU in patients with LV functional improvement (9.6%+/-5.6%) was significantly lower than in those without such improvement (26.3%+/-14.1%, P<0.01). %CV of rMGU <13.6% predicted prognosis with a sensitivity of 80%, a specificity of 100% and an accuracy of 90%. %CV of rMGU <13.6% also predicted improvement in LV function, with a sensitivity of 75%, a specificity of 92% and an accuracy of 85%. However, EF failed to predict improvement of LV function. In is concluded that homogeneous myocardial glucose utilization rate can predict both prognosis and improvement in LV function achieved by medical therapy in patients with DCM.

Comparison of unitary displacements and forces between 2 cardiac myosin isoforms by the optical trap technique: molecular basis for cardiac adaptation.

To provide information on the mechanism of cardiac adaptation at the molecular level, we compared the unitary displacements and forces between the 2 rat cardiac myosin isoforms, V1 and V3. A fluorescently labeled actin filament, with a polystyrene bead attached, was caught by an optical trap and brought close to a glass surface sparsely coated with either of the 2 isoforms, so that the actin-myosin interaction took place in the presence of a low concentration of ATP (0.5 micromol/L). Discrete displacement events were recorded with a low trap stiffness (0.03 to 0.06 pN/nm). Frequency distribution of the amplitude of the displacements consisted of 2 gaussian curves with peaks at 9 to 10 and 18 to 20 nm for both V1 and V3, suggesting that 9 to 10 nm is the unitary displacement for both isoforms. The duration of the displacement events was longer for V3 than for V1. On the other hand, discrete force transients were recorded with a high trap stiffness (2.1 pN/nm), and their amplitude showed a broad distribution with mean values between 1 and 2 pN for V1 and V3. The durations of the force transients were also longer for V3 than for V1. These results indicate that both the unitary displacements and forces are similar in amplitude but different in duration between the 2 cardiac myosin isoforms, being consistent with the reports that the tension cost is higher in muscles consisting mainly of V1 than those consisting mainly of V3.

Altered myocardial vasodilatation in patients with hypertriglyceridemia in anatomically normal coronary arteries.

Reduced myocardial vasodilatation (MVD) in hypercholesterolemics without overt coronary stenosis has been reported. However, the status of MVD in hypertriglyceridemics has not yet been clarified. The aim of this study was to investigate whether MVD is impaired in patients with hypertriglyceridemia without overt coronary stenosis. Twenty-three hypertriglyceridemics (10 normocholesterolemic hypertriglyceridemics [HTGs] and 13 mixed combined hyperlipidemics [MCHLs]) and 13 age-matched controls were studied. All patients were proven to have more than one normal coronary artery, as diagnosed by coronary angiography, and those segments that were perfused by anatomically normal coronary arteries were used in the study. Myocardial blood flow (MBF) during dipyridamole (DP) loading and baseline MBF were measured by using positron emission tomography and [13N]ammonia, after which MVD was calculated. Baseline MBF (mL.min(-1).100 g(-1)) was comparable among HTG (76.0+/-26.1), MCHL (77.0+/-26.1), and controls (80.3+/-38.5). However, MBF during DP loading was significantly lower in MCHL (159+/-52.5) than in control subjects (292+/-166, P<.01), while it was comparable in HTG (202+/-104) and controls. MVD was significantly reduced in both HTG (2.70+/-1.09, P<.05) and MCHL (2.07+/-.70, P<.01) compared with controls (3.73+/-1.14). MVD in MCHLs tended to be reduced compared with that in HTGs, but the difference was statistically insignificant (P=.08). There was a significant relationship between MVD and both plasma triglycerides (r=-.47, P<.01) and plasma total cholesterol (r=-.55, P<.01). When controls and HTGs were combined, the relationship between MVD and plasma total triglycerides became more prominent (r=-.55, P<.05), and the significant relationship between cholesterol level and MVD disappeared. Multivariate regression analysis has revealed that the triglyceride level (F=5.2, P<.05) was independently related to MVD (r=.69, P<.01). In conclusion, MVD was reduced in hypertriglyceridemics in anatomically normal coronary arteries. Hypertriglyceridemia is an independent factor for this abnormality.

Distinct kinetic properties of cardiac myosin isoforms revealed by in vitro studies.

To clarify the physiological significance of myosin isoform redistribution in cardiac adaptation process, we compared the kinetic property of the two cardiac myosin isoforms using in vitro motility assay techniques. Cardiac myosin isoforms V1 and V3 were obtained from ventricular muscle of young rats and hypothyroid rats respectively. On each of these myosin isoforms fixed on a glass coverslip, fluorescently labeled actin filaments were made to slide in the presence of ATP. To measure the force generated by actomyosin interaction, a small latex bead was attached to the barbed end of an actin filament and the bead was captured by the laser optical trap installed in a microscope. The force was determined from the distance between the bead and the trap positions under either auxotonic or isometric conditions. The time-averaged force generated by multiple cross-bridges did not differ significantly between the two isoforms. On the other hand, the unitary force measurement revealed the same level of amplitude but a longer duration for V3 isoform. The same level of time-averaged force is in agreement with not only our previous finding but the results of maximum force measurement in muscle preparations. The difference in kinetic characteristics of the two isoforms could account for the difference in economy of force development and the basis for cardiac adaptation mechanism.

Inhibition of carnitine synthesis protects against left ventricular dysfunction in rats with myocardial ischemia.

During myocardial ischemia, inhibition of the carnitine-mediated transportation of fatty acid may be beneficial because it facilitates glucose utilization and prevents an accumulation of fatty acid metabolites. We orally administered 3-(2,2,2-trimethyl hydrazinium) propionate (MET), an inhibitor of carnitine synthesis, for 20 days to rats. Then we evaluated left ventricular (LV) function during brief ischemia by using a buffer-perfused isovolumic heart model. After 15 min of reoxygenation after the transient ischemia, LV peak systolic pressure (PSP) almost completely returned to the baseline level in rats given MET (96 +/- 4%), whereas it was only partially (77 +/- 16%) recovered in the placebo-treated rats. We induced myocardial infarction in other rats by ligating the left anterior descending coronary artery. Then the animals were given MET for 20 days, and LV function was compared. In the placebo-treated rats (with myocardial infarction, but without drug treatment), LVPSP was lower than that in the sham group [108 +/- 19 (n = 10) vs. 136 +/- 15 mm Hg (n = 13); p < 0.05], and the time constant (T) of LV pressure decay was elongated (36 +/- 4 vs. 30 +/- 7 ms; p < 0.05). In MET-treated groups, however, neither PSP nor T differed from those in the sham group. In conclusion, inhibition of the carnitine-mediated transportation of fatty acid by MET protected against left ventricular dysfunction in acute and chronic myocardial ischemia.

Direct characterization of single molecular kinetics of cardiac myosin in vitro.

Distinct crossbridge kinetics among cardiac myosin isoforms have been proposed as the basis of differences in energetic characteristics. However, direct evidence for this hypothesis is lacking because of experimental difficulty. As a preliminary approach to this problem, we applied an in-vitro force measurement technique to directly observe force impulse generated by a single cardiac myosin molecule. The force measurement system was constructed with an inverted microscope coupled with a laser optical trap. With the feedback of the position signal to the driving circuit for a galvanomirror that steers the laser beam, trap stiffness was increased; thus, isometric force measurement was made possible. We measured the force generated by the cardiac myosin V3 isoform purified from hypothyroid rat ventricular muscle. With very low myosin density and low adenosine triphosphate (ATP) concentration of the assay buffer, we successfully observed a single force impulse similar in shape to that of skeletal muscle myosin. With this approach, we will be able to gain a clear view of the molecular basis of cardiac mechanoenergetics.

Different cardiac myosin isoforms exhibit equal force-generating ability in vitro.

We measured forces generated by myosin molecules and a single actin filament using an optical trap system. The force per unit length of actin filament did not differ significantly between cardiac myosin isoforms. V1 and V3. This indicates that the ability to generate force is equal between V1 and V3, despite their difference in the unloaded sliding velocity past actin.