A method for measuring systolic and diastolic microcirculatory red cell flux within the canine myocardium.

BACKGROUND: Knowledge of the patterns of movement of red cells during the cardiac cycle in the microcirculation within the contracting myocardium is largely unknown. We describe a method of making such measurements in the canine myocardium using the technique of laser Doppler velocimetry. METHODS: A lensed 100 microm fiber-optic probe was inserted into the beating myocardium at various sites. Using an ultra-stable laser and achieving measurement stability by heterodyning the laser light and reflected light from the tissue, it was possible to obtain a stable high quality measurement of predominately red cell movement in the microcirculation. RESULTS: Unique regional patterns of red cell movement within the myocardium were observed. Epicardial flux was continuous with peaks while endocardial flux was predominately diastolic. Stopping flow in the epicardial artery for 5-6 s demonstrated that red cell movement continues in the microcirculation with some reduction followed by a delayed reactive hyperemia. Modeling demonstrates an important role for the small coronary veins in control of microcirculatory red cell movement. CONCLUSIONS: It is possible using laser Doppler velocimetry to measure red blood cell flux in the beating canine myocardium. Such measurements demonstrate a high degree of complexity which is not reflected in epicardial coronary arterial or venous flow.

A method for detecting chaos in canine myocardial microcirculatory red cell flux.

OBJECTIVE: To determine whether red cell movement, as measured by laser Doppler velocimetry, in the capillary net of the beating heart is chaotic. METHODS: Using two dog hearts, in situ red blood cell flux was measured at many sites. Simultaneously, epicardial arterial flow and left ventricular pressure were recorded via transit-time flowmeter and catheter manometer, respectively. The presence or absence of chaos was tested by two methods: Lyapunov exponents and correlation dimension. RESULTS: For capillary red cell flux, the Lyapunov was strongly positive at most sites. It was less so for coronary arterial flow and least for left ventricular pressure. Correlation dimension calculation was less able to distinguish the presence or absence of chaos in capillary red cell tissue flux, coronary arterial flow, and left ventricular pressure. CONCLUSIONS: Capillary red cell flux (movement of red cells in capillaries) is nonlinear, (i.e., chaotic). This complexity suggests that the primary control for oxygen delivery to cardiac myocytes by red blood cells resides in the microcirculation. Also, capillary red cell flux is bifractal, suggesting an ordering of control.

Calcified subcutaneous arterioles with infarcts of the subcutis and skin ("calciphylaxis") in chronic renal failure.

Patients with chronic renal failure (CRF) are at increased risk for pathological calcifications because of increased serum calcium-phosphorus products. A minority, including those undergoing dialysis, develop a syndrome of deep skin ulcerations in association with calcification of subcutaneous arterioles. The body distribution of the skin lesions may be proximal (central), distal (peripheral), or both. Since 1968, this syndrome has been called "calciphylaxis" in the belief that it is the human analogue of Selye's experimental models of tissue calcification. Our review emphasizes that this syndrome comprises two separate processes not found in calciphylaxis: calcification of subcutaneous arterioles and infarctions of subcutaneous adipose tissue (panniculus adiposus) and skin. The infarctions are acute and clinically dramatic, whereas the calcific arteriolopathy is preexistent, having developed slowly, sometimes over years, and silently. Separating these two processes facilitates analyses of pathogenetic factors, such as those that target subcutaneous arterioles for calcification and those that interfere with blood flow through the calcified arterioles, sufficient in some patients to cause the infarctions, and of why obesity in CRF is a syndrome risk factor. This approach further helps to provide a much needed standardized definition of the syndrome, thereby facilitating comparisons of the results of such treatments as parathyroidectomy, anticoagulants, and phosphate binders. Finally, the separation shows why the application of such terms as calciphylaxis and calcifying panniculitis to this syndrome is inappropriate.

Pharmacokinetics and hemodynamic effects of diltiazem in healthy volunteers: comparing resting with the effect of exercise.

PURPOSE: To determine the steady-state plasma concentrations of diltiazem (DTZ) and hemodynamic effect in humans at rest and during exercise. METHODS: Healthy volunteers (10 F, mean age 22, and 11 M, mean age 24) were recruited. Prior to receiving DTZ, each volunteer performed two 3-minute stages of treadmill exercise according to the Bruce protocol. Intra-arterial BP and ECG recordings were obtained before, during and immediately post exercise. Each volunteer then received DTZ 60 mg qid for one week. The same exercise protocol was repeated 1 h after the last dose. Steady-state plasma concentrations of DTZ were determined by a previously reported HPLC. RESULTS: DTZ decreased resting DBP from 84 +/- 13 to 79 +/- 10 mmHg (p > 0.05), and HR from 89 +/- 11 to 82 +/- 13 bpm (p < 0.05). During exercise, an average of 32 and 10% increase in SBP and DBP, respectively, and a 47% increase of HR was found (p < 0.05). DTZ limited these increases to 21% for SBP, 5% for DBP, and 44% for HR (p < 0.05 for drug effect). Steady-state plasma DTZ concentrations were 141 +/- 56 ng/ml. CONCLUSION: DTZ significantly decreased resting HR but not BP in health volunteers. It decreased both hemodynamic variables during exercise. Thus, the hemodynamic effects of diltiazem are more profound during exercise, and may be more useful surrogate markers for calcium antagonists and other cardiovascular agents in healthy volunteer studies.

The coronary circulation: quo vadis?

This review examines past and present observations concerning the structure and function of the coronary circulation in health and disease. These observations are considered in the context of how we might proceed to increase understanding of this circulation in the future. The coronary microcirculation with its intimate relationship of capillaries and myocytes and the coronary venous circulation are identified as parts of the coronary circulation in need of further study. Observations using a laser Doppler velocimeter placed within the beating myocardium are presented. Such devices measure the velocity of red cells continuously (red cell flux) and can demonstrate that tissue hematocrit and hence oxygen delivery can be regulated independently of total epicardial arterial volume flow. Implications for the understanding of the pathophysiology of coronary artery disease are presented. As considerations are given to molecular genetic techniques to revascularize the ischemic myocardium we will require a more complete knowledge of coronary circulatory dynamics, myocardial support tissues' responses, and cardiac myocyte interactions to design appropriate interventions. The clinical trial is an appropriate clinical tool to measure effectiveness but a blunt instrument to determine pathophysiology. The purpose of the review is to suggest that advances in measurement of end points are required to permit the right question to be posed.

Brain-derived HIV-1 tat sequences from AIDS patients with dementia show increased molecular heterogeneity.

HIV-1 infection results in a dementing illness affecting 20% of patients with AIDS. Several HIV-1 genes have been implicated in the pathogenesis of HIV-induced neurological disease. To search for distinct HIV-1 sequences associated with the development of dementia, brain-derived tat, env, and pol sequences were examined from AIDS patients defined pre-mortem as demented (HIV-D)[n=5] or non-demented (HIV-ND)[n=5]. Estimations of evolutionary distances and frequency of non-synonymous mutation rates revealed significant differences between brain-derived tat, env, and pol-encoded reverse transcriptase sequences. However, established zidovudine-associated resistance mutations in reverse transcriptase sequences were identified in only one HIV-D and one HIV-ND patient despite prolonged treatment of some patients. Non-synonymous/synonymous substitution rates among the tat sequences derived from patients with HIV-D were significantly higher compared to the HIV-ND group (P < 0.001). The ratios of transversions to transitions were also significantly higher among the HIV-D tat sequences (P< 0.01). Phylogenetic analyses showed clustering of sequences from each clinical group among the brain-derived tat and env sequences. These studies indicated that differing selective forces act on individual HIV-1 genes in the brain which may influence the development of dementia.

Decreasing mortality from acute myocardial infarctions: effect of attack rates and case severity.

Mortality from myocardial infarction (MI) has declined in many countries and the reasons for the decline have not been fully quantified. We used the database of the Halifax County MONICA Project to test the hypothesis that the decline of in-hospital mortality from MI can be explained by a trend toward less severe disease as opposed to improved treatment. During the study period 1984-1993, 14,130 people aged 25-74 had been admitted to hospital with suspected MI. Of these, 3774 were diagnosed as definite MI by standardized criteria (480 fatal). For each patient, clinical history, serial cardiac enzymes, and ECG treatment regimen during hospital stay were extracted from patient charts. Survival status 28 days after onset of symptoms was determined. A severity index predicting 28-day case fatality was derived from health status at admission time. During the study period the rate of definite MI in the MONICA target population showed a general downward trend from 221 to 179 per 100,000/year (p = 0.0002). The severity index increased during the observation time (p < 0.0001), predicting 25% higher mortality. Case fatality fluctuated, but showed a marginally significant decline. We conclude that part of the decreased in-hospital mortality from MI is due to lower attack rates. The remainder occurred despite increased case severity and is possibly due to improved in-hospital treatment.

Red cell flux during the cardiac cycle in the rabbit myocardial microcirculation.

OBJECTIVE: (1) To measure regional phasic myocardial red cell flux during a cardiac cycle using a laser Doppler velocimeter. (2) To test the responses of regional red cell flux to a vasodilator (adenosine), a vasoconstrictor (angiotensin II), and an inotrope (isoproterenol). METHODS: Using an anaesthetised open-chest rabbit with the pericardium intact a 140-micron-tip fibre optic probe was placed in the left ventricular myocardium in various locations. With the fibre in place drugs were given to alter myocardial loading conditions while red cell flux was registered. RESULTS: Phasic red cell flux was similar in the epicardium to endocardium giving an average endo/epi ratio of 1.14 in the rabbit heart. At least two peaks of increased red cell flux within a single cardiac cycle were observed. Some unique patterns for red cell flux were observed in specialised myocardial structures. Adenosine increased red cell flux but minimally changed the pattern of phasic flux throughout the cycle. CONCLUSIONS: Laser Doppler velocimetry permits the recording of phasic red cell flux during the cardiac cycle in the myocardial microcirculation. Its pattern is determined by both coronary arterial inflow and venous outflow. The pattern of red cell flux may be characteristic for a region-probably determined by difference in tissue pressure (attributable to the pattern of muscle fibre shortening and collagen tethering) and changes in capillary length and density.

Effect of diltiazem on plasma concentrations of oxypurines and uric acid.

To determine the clinical effect of diltiazem on the metabolism of adenosine, and its importance in ischemic heart disease, arterial plasma concentrations of the purine metabolites were determined in 21 healthy volunteers (10 female and 11 male) and 19 patients with effort angina (8 female and 11 male) before, during, and immediately after standard treadmill exercise tests conducted before and after they had taken 60 mg diltiazem (Cardizem; Hoechst Marion Roussel, Laval, QC, Canada) four times a day for 1 week. The results showed that the cardiac patients had significantly lower mean plasma concentrations of uric acid (46.82 +/- 25.51 versus 95.47 +/- 35.41 micrograms/ml, p 0.05), inosine (0.25 +/- 0.19 versus 0.84 +/- 0.17 microgram/ml, p < 0.05), and hypoxanthine (0.28 +/- 0.35 versus 0.50 +/- 0.27 microgram/ml, p < 0.05). Diltiazem decreased the mean resting plasma concentrations of uric acid in patients (uric acid 43.47 +/- 22.26 versus 46.82 +/- 25.51 micrograms/ml, p < 0.05) and healthy volunteers (uric acid 85.68 +/- 26.71 versus 95.47 +/- 35.41 micrograms/ml, p < 0.05). There was no statistically significant change in the plasma concentrations of the purine metabolites during exercise (p < 0.05). Female subjects had significantly lower plasma concentrations of uric acid than males (patients, 34.87 +/- 26.93 versus 55.78 +/- 21.25 micrograms/ml; healthy volunteers, 84.79 +/- 32.07 versus 104.22 +/- 37.05 micrograms/ml; p < 0.05 for both). Results of the study suggest that normal therapeutic doses of diltiazem may modulate the metabolism of adenosine and that some of the purine metabolites may be useful markers for specific types of ischemic heart disease.

Effect of diltiazem on intraarterial blood pressure and heart rate during stress testing in patients with angina: a gender comparison study.

The purpose of this study was to measure the blood pressure and electrocardiographic responses of a small, matched group of women (n = 8) and men (n = 9) who experienced typical, effort angina during an exercise on the treadmill (up to the second stage of a Bruce protocol). These responses were measured before and after therapy with diltiazem (60 mg four times daily for 1 week). Reports of previous studies have described significant gender differences in blood pressure responses to diltiazem in healthy volunteers tested with the same protocol. In contrast to the data in healthy individuals, gender differences in blood pressure responses to exercise before and after diltiazem administration were not observed. Results of analysis of pulse pressure responses to exercise were also similar in male and female patients with angina. A significant postexercise drop in blood pressure was observed, which was augmented by diltiazem. These data suggest that gender differences in drug action may be difficult to demonstrate in patients with vascular disease.

Endothelin-induced electrical activity and calcium dynamics in vascular smooth muscle cells: a model study.

A model is proposed to describe the electrical activity and intracellular calcium dynamics of vascular smooth muscle cells (SMC) induced by endothelin (ET1). The conductance of the nonselective channels (NSCs), proportional to the ET1-receptor complex (ET . R), is intracellular calcium dependent. Inositol (1,4,5)-trisphosphate (IP3) produced by ET1 releases Ca2+ from the IP3-sensitive Ca2+ store. The transient increase of intracellular Ca2+ triggers the release of Ca2+ from the Ca(2+)-sensitive store by a Ca(2+)-induced Ca2+ (CICR) mechanism and activates the Ca(2+)-activated K+ current (IK,Ca). The inward current (Iin) via the NSC can depolarize the cell to a level at which the L-type Ca2+ current becomes activated (ICa). The level of depolarization is determined by the relative amplitude of (Iin + ICa + IK,Ca) and the voltage- and time-dependent K+ current. The model simulations show that (a) in cells without a CICR mechanism, short-lasting stimulation by ET1 elicits higher membrane potential and Ca2+ than long-lasting stimulation; (b) in cells with or without a CICR mechanism, a reduction of normal membrane capacitance (1 muf/cm2) results in either significant and sustaining or oscillatory membrane potential and intracellular calcium concentration. The applicability of the model to the study of electrical activity and calcium dynamics associated with hypercholesterolemia is discussed.

Body position, age and mass effect of adiposity on adipose tissue red cell flux in morbid obesity.

OBJECTIVE: To measure red cell flux of adipose tissue in morbidly obese patients' pannus in the upright and supine position to determine factors which would render the lower pannus susceptible to ischemic necrosis. DESIGN: A cohort study of morbidly obese subjects without ischemic necrosis. SETTING: University teaching hospital. PATIENTS: Twenty-three consecutive morbidly obese patients referred for gastroplasty. MEASUREMENTS: Red cell flux, measured as RMS voltage by a laser Doppler velocimeter. An optical fiber with a tip diameter of 250 mu was inserted into the upper and lower pannus and output recorded in the upright and supine positions. Other variables recorded were age, BMI, blood pressure and serum lipids. RESULTS: Adipose tissue red cell flux demonstrates considerable spatial and temporal heterogeneity from subject to subject and in various locations in the pannus. No differences in red cell flux were detected in response to change in position. However, regression analysis demonstrated that the gradient between the upper and lower abdomen in the supine position was increasingly positive with age and in the upright position it was increasingly positive with increasing weight or BMI. CONCLUSIONS: These data suggest that red cell flux is heterogeneously distributed in the abdominal pannus and is not greatly influenced by body position. However, with increasing age and adiposity there is a gradient for decreased red cell flux to the lower portion of the pannus. This may be a factor in rendering this part of the pannus prone to ischemic fat necrosis.

Steady-state plasma concentrations of diltiazem and its metabolites in patients and healthy volunteers.

Diltiazem (DTZ) is a calcium antagonist widely used in the treatment of angina and hypertension. It is extensively metabolized in humans via N-demethylation, O-demethylation, deacetylation, and oxidative deamination, yielding a host of metabolites, some of which have potent pharmacological properties. After our initial identification of O-desmethyl DTZ (Mx) and N,O-didesmethyl DTZ (MB) as major metabolites of DTZ and our subsequent of identification of their chemical synthesis, an improved high-performance liquid chromatography assay was developed to determine the plasma concentrations of DTZ and seven of its major basic metabolites, including the previously unquantitated Mx and MB. The system consisted of a C18 analytical column protected by a C18 cartridge guard column and a variable wavelength ultraviolet detector set at 237 nm. The mobile phase was a mixture of methanol, 0.04 M ammonium acetate, and acetonitrile (38:36:26) containing 0.08% triethylamine, with final pH of the mobile phase adjusted to 7.5. The system was operated at room temperature isocratically at a flow rate of 1.2 ml/min. Using verapamil as an internal standard, DTZ and the basic metabolites in plasma were determined in young healthy volunteers (n = 21) and in patients with ischemic heart disease (n = 19) at steady state after repeated oral doses of 60 mg DTZ four times daily. Preliminary results show that steady-state plasma concentrations of DTZ and its metabolites were higher in the older patients than in young healthy subjects (p < 0.05).

Evidence of type 2 herpes simplex infection in human coronary arteries at the time of coronary artery bypass surgery.

OBJECTIVE: To examine histologically biopsies from the coronary arteries of patients undergoing coronary artery bypass grafting (CABG) for evidence of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) antigen and to correlate the incidence with pathological and clinical data. DESIGN: Sequential patients undergoing CABG in whom adequate tissue could be obtained for histology. SETTING: University teaching hospital. PATIENTS: Forty-six patients were enrolled. Thirty-one provided sufficient tissue and clinical information for the analysis. METHODS: Biopsy material was collected in the operating room and prepared immediately for histology and electron microscopy. Slides were prepared by staining with hematoxylin and eosin, Masson trichrome, avidin biotin complex immunoperoxidase for HSV-1 and HSV-2 protein and specific DNA probes for HSV-1 and HSV-2 by hybridization. Clinical data were obtained in structured interviews. RESULTS: Sixty-one per cent of biopsies demonstrated evidence of inflammation, 45% were positive for antigen to HSV-2 and only one to HSV-1. Significant positive correlations were detected between inflammatory cells in the biopsy and a recent history of cold sores and between the presence of the infiltrate and positivity to HSV-2 antigen. CONCLUSION: A correlation exists between HSV-2 infection and the inflammatory response associated with atherosclerosis.

Gender differences in exercise and recovery blood pressure responses in normal volunteers given diltiazem.

This preliminary phase I study was conducted in healthy volunteers to determine whether gender differences exist in the hemodynamic effects of diltiazem at rest, during exercise, and after exercise. At comparable serum concentrations of the drug, women demonstrated lower systolic and diastolic pressure during exercise and after exercise. ST slope after diltiazem administration in women became less positive during exercise and was gender specific. Heart rate and P-R interval changes were not gender dependent. Results of this study demonstrate that some hemodynamic responses to diltiazem are gender specific while others are not. It indicates that direct comparison studies may be required to detect such differences. In healthy women, hypotension after exercise and the effects of diltiazem are more synergistic than in men. Such a gender difference in response may be an important consideration in determining the correct dosages of this drug for treatment of hypertension.

A model of electrical activity and cytosolic calcium dynamics in vascular endothelial cells in response to fluid shear stress.

A mathematical model is proposed to describe the intracellular Ca2+ (Cai) transient and electrical activity of vascular endothelial cells (VEC) elicited by fluid shear stress (tau). The intracellular Ca2+ store of the model VEC is comprised of a Cai-sensitive (sc) and an inositol (1,4,5)-trisphosphate (IP3)-sensitive compartment (dc). The dc [Ca2+] is refilled by the sc whose [Ca2+] is the same as extracellular [Ca2+]. IP3 produced by the tau-deformed mechanoreceptors discharges the dc Ca2+ into the cytosol. The increase of cytosolic [Ca2+] induces Ca2+ release (CICR) from the sc. The raised Cai activates a Cai-activated K+ current (IK,Ca) and inhibits IP3 production. The cell membrane potential is determined by IK,Ca, voltage-dependent Na+ and K+ currents. Steady tau > 0.1 dyne/cm2 elicits a Cai transient which reaches peak value at 19-54 sec. The peak Cai varies sigmoidally with Log10(tau) with a maximal peak Cai of 150 nM at tau = 4 dynes/cm2. Step increases of tau fail to elicit a Ca2+ response in cells previously stimulated by a lower shear. The Ca2+ response gradually decreases with repetitive tau stimuli. Pulsatile shear elicits two to three times higher Cai and hyperpolarizes the cell more than steady shear of the same magnitude. The simulated Ca2+ responses to tau are quantitatively and qualitatively similar to those observed in cultured VEC. The model provides a possible explanation of why the vasodilating stimulus is greater for pulsatile flow than for nonpulsatile flow.

Functional anatomy of the epicardial coronary veins.

OBJECTIVE: To describe the functional anatomy of the epicardial veins, including their dimensional changes in systole and diastole, and to relate these observations to the pattern of bloodflow in these veins. DESIGN: Using quantitative angiography and casts of epicardial veins in dogs, measurements of dimensions and anatomical patterns were observed. INTERVENTIONS: Venous angiograms were performed on 21 mongrel dogs. The diameters of epicardial veins in systole and diastole were measured using ECG gating. The dynamic anatomy of epicardial veins observed from video tapes was compared with static images and casts. RESULTS: The transverse diameter of the epicardial veins was similar in systole and diastole. The accuracy of measurement was limited by definition of the edges of the veins. An area of reduced contrast was observed in the posterior lateral section of the great cardiac vein and the appearance of gaps in the column of contrast material within the epicardial veins was consistently seen. CONCLUSIONS: These observations suggest that flow in epicardial coronary veins occurs as a bolus during each cardiac cycle.

Erythrocyte adenosine transport. A rapid screening test for cardiovascular drugs.

An erythrocyte (RBC) model based on whole blood was used to investigate the effect of cardiovascular drugs on the uptake of adenosine in vitro. Fresh whole blood obtained from healthy volunteers was allowed to equilibrate with various concentrations (5-1000 microM) of a tested agent. (2-3H)-Adenosine was used as a substrate, and the reaction was terminated after 2 sec of incubation at room temperature by rapid addition of a "Stopping Solution" which was a mixture of erythro-9-(2-hydroxy-3-nonyl)adenine, dipyridamole, and EDTA. The mixture was centrifuged (1760 g, 4 degrees C, 10 min), and the radioactivity of an aliquot of the supernatant was determined by a scintillation counter. The results showed that dipyridamole was the most potent agent tested (IC50 = 0.2 microM). Amongst the calcium antagonists studied, isradipine was most potent, followed by verapamil, clentiazem, diltiazem, and then nifedipine. The racemates of two metabolites of diltiazem, MX and MB, were more potent than the parent drug. The antiarrhythmic agents, amiodarone and sotalol, the two new lipid peroxidation inhibitors, U-74389F and U-78517F, and the anxiolytic agent, alprazolam, were as active as verapamil. The beta-receptor antagonist propranolol and the angiotensin converting enzyme (ACE) inhibitor, enalapril, were practically inactive. In addition, the model was stereoselective such that the S(-)-enantiomer of verapamil was considerably more potent than the R(+)-antipote, whereas d(+)-sotalol was practically inactive compared to racemic sotalol.

Pharmacokinetics and metabolism of diltiazem in healthy males and females following a single oral dose.

Plasma concentrations and urinary excretion of DTZ and its metabolites were determined in 20 healthy volunteers (10 males and 10 females) after they had each been given a single oral 90 mg dose of DTZ. DTZ and six of its metabolites which included N-monodesmethyl DTZ (MA), deacetyl DTZ (M1), deacetyl N-monodesmethyl DTZ (M2), deacetyl O-desmethyl DTZ (M4) and deacetyl DTZ N-oxide (M1NO) and deacetyl N,O-didesmethyl DTZ (M6), were determined by a sensitive and specific HPLC assay. The major metabolites measurable in the plasma of all the volunteers were MA, M1, and M2. The terminal half-lives (t1/2) of M1 and M2 were considerably longer than those of DTZ and MA. Less than 5% of the dose was excreted as unchanged DTZ in the urine over the 24 h period. The major urinary metabolite was MA, followed by M6, M2, and then M1. Except for the urinary excretion of M4 there were no statistically significant differences in any of the pharmacokinetic parameters between the males and the females. The mean 24 h urinary recovery of M4 was higher in the males than in the females (P < 0.05). However there were large inter-individual variations in the plasma concentrations and urinary excretion of DTZ and its metabolites with some parameters differing by more than 20-fold. In addition, O-desmethyl DTZ (Mx) and N,O-didesmethyl DTZ (MB) were identified as two other major urinary metabolites.