Experimental myocardial hypertrophy induced by a minimally invasive ascending aorta coarctation

Ascending aorta coarctation was produced by a minimally invasive technique in rabbits. Animal mortality was 5%. Morphometric and hemodynamic parameters were evaluated. A parabiotically isolated heart model was used to assess the hemodynamic parameters. Left ventricular weight/body weight ratio and muscle area showed clear evidence of hypertrophy when compared to control. The hemodynamic changes in the isolated heart model suggested decreased diastolic and systolic function in the coarcted group. The present model produced hypertrophy with low mortality rates as a result of its less invasive nature.

Head-to-head comparison of dipyridamole, dobutamine and pacing stress echocardiography for the detection of myocardial ischemia in an animal model of coronary artery stenosis

To compare the sensitivity of dipyridamole, dobutamine and pacing stress echocardiography for the detection of myocardial ischemia we produced a physiologically significant stenosis in the left circumflex artery of 14 open-chest dogs (range: 50 to 89% reduction in luminal diameter). In each study, dobutamine (5 to 40 µg kg-1 min-1 in 3-min stages) and pacing (20 bpm increments, each 2 min, up to 260 bpm) were performed randomly, and then followed by dipyridamole (up to 0.84 mg/kg over 10 min). The positivity of stress echocardiography tests was quantitatively determined by a significant (P<0.05) reduction of or failure to increase absolute and percent systolic wall thickening in the stenotic artery supplied wall, as compared to the opposite wall (areas related to the left anterior descending artery). Systolic and diastolic frozen images were analyzed off-line by two blinded observers in the control and stress conditions. The results showed that 1) the sensitivity of dobutamine, dipyridamole and pacing stress tests was 57, 57 and 36%, respectively; 2) in animals with positive tests, the mean percent change of wall thickening in left ventricular ischemic segments was larger in the pacing (-19 ± 11%) and dipyridamole (-18 ± 16%) tests as compared to dobutamine (-9 ± 6%) (P = 0.05), but a similar mean reduction of wall thickening was observed when this variable was normalized to a control left ventricular segment (area related to the left anterior descending artery) (pacing: -16 ± 7%; dipyridamole: -25 ± 16%; dobutamine: -26 ± 10%; not significant), and 3) a significant correlation was observed between magnitude of coronary stenosis and left ventricular segmental dysfunction induced by ischemia in dogs submitted to positive stress tests. We conclude that the dobutamine and dipyridamole stress tests showed identical sensitivities for the detection of myocardial ischemia in this one-vessel disease animal model with a wide range of left circumflex artery stenosis. The pacing stress test was less sensitive, but the difference was not statistically significant. The magnitude of segmental left ventricular dysfunction induced by ischemia was similar in all stress tests evaluated.

Butyrate induces apoptosis in murine macrophages via caspase-3, but independent of autocrine synthesis of tumor necrosis factor and nitric oxide

We demonstrated that 4 mM butyrate induces apoptosis in murine peritoneal macrophages in a dose- and time-dependent manner as indicated by studies of cell viability, flow cytometric analysis of annexin-V binding, DNA ladder pattern and the determination of hypodiploid DNA content. The activity of caspase-3 was enhanced during macrophage apoptosis induced by butyrate and the caspase inhibitor z-VAD-FMK (100 µM) inhibited the butyrate effect, indicating the major role of the caspase cascade in the process. The levels of butyrate-induced apoptosis in macrophages were enhanced by co-treatment with 1 µg/ml bacterial lipopolysaccharide (LPS). However, our data indicate that apoptosis induced by butyrate and LPS involves different mechanisms. Thus, LPS-induced apoptosis was only observed when macrophages were primed with IFN-gamma and was partially dependent on iNOS, TNFR1 and IRF-1 functions as determined in experiments employing macrophages from various knockout mice. In contrast, butyrate-induced macrophage apoptosis was highly independent of IFN-gamma priming and of iNOS, TNFR1 and IRF-1 functions.

Myocardial antioxidant and oxidative stress changes due to sex hormones

The purpose of the present study was to examine myocardial antioxidant and oxidative stress changes in male and female rats in the presence of physiological sex hormone concentrations and after castration. Twenty-four 9-week-old Wistar rats were divided into four groups of 6 animals each: 1) sham-operated females, 2) castrated females, 3) sham-operated males, and 4) castrated males. When testosterone and estrogen levels were measured by radioimmunoassay, significant differences were observed between the castrated and control groups (both males and females), demonstrating the success of castration. Progesterone and catalase levels did not change in any group. Control male rats had higher levels of glutathione peroxidase (50%) and lower levels of superoxide dismutase (SOD, 14%) than females. Control females presented increased levels of SOD as compared to the other groups. After castration, SOD activity decreased by 29% in the female group and by 14% in the male group as compared to their respective controls. Lipid peroxidation (LPO) was assessed to evaluate oxidative damage to cardiac membranes by two different methods, i.e., TBARS and chemiluminescence. LPO was higher in male controls compared to female controls when evaluated by both methods, TBARS (360%) and chemiluminescence (46%). Castration induced a 200% increase in myocardial damage in females as determined by TBARS and a 20% increase as determined by chemiluminescence. In males, castration did not change LPO levels. These data suggest that estrogen may have an antioxidant role in heart muscle, while testosterone does not.

Slaughterhouse blood as a perfusate for studying myocardial function under ischemic conditions

Metabolic studies using the in vitro non-recirculating blood-perfused isolated heart model require large volumes of blood. The present study was designed to determine whether heterologous pig blood collected from a slaughterhouse can be used as perfusate for isolated pig hearts perfused under aerobic and constant reduced flow conditions. Eight isolated working pig hearts perfused for 90 min at a constant flow of 1.5 ml g-1 min-1 with non-recirculated blood diluted with Krebs-Henseleit bicarbonate buffer at a hematocrit of 23% were compared to eight hearts subjected to the same protocol but perfused only with Krebs-Henseleit bicarbonate buffer solution. Hearts were paced at 100 bpm and subjected to aerobic perfusion at 38ºC. Hearts were weighed before perfusion and at the end of the experiment and the results are reported as percent weight gain (mean ± SD). Comparisons between groups were performed by the Student t-test (P<0.05). After 90 min of perfusion with modified Krebs-Henseleit, perfused hearts presented a larger weight gain than blood-perfused hearts (39.34 ± 9.27 vs 23.13 ± 5.42%, P = 0.003). Left ventricular end-diastolic pressure was higher in the modified Krebs-Henseleit-perfused group than in the blood group (2.8 ± 0.4 vs 2.3 ± 0.3 mmHg, respectively, P = 0.01). We conclude that heterologous blood perfusion, by preserving a more physiological myocardial water content, is a better perfusion fluid than modified Krebs-Henseleit solution for quantitative studies of myocardial metabolism and heart function under ischemic conditions.

Protective effects of centrally acting sympathomodulatory drugs on myocardial ischemia induced by sympathetic overactivity in rabbits

It is recognized that an imbalance of the autonomic nervous system is involved in the genesis of ventricular arrhythmia and sudden death during myocardial ischemia. In the present study we investigated the effects of clonidine and rilmenidine, two centrally acting sympathomodulatory drugs, on an experimental model of centrally induced sympathetic hyperactivity in pentobarbital-anesthetized New Zealand albino rabbits of either sex (2-3 kg, N = 89). We also compared the effects of clonidine and rilmenidine with those of propranolol, a ß-blocker, known to induce protective cardiovascular effects in patients with ischemic heart disease. Central sympathetic stimulation was achieved by intracerebroventricular injection of the excitatory amino acid L-glutamate (10 µmol), associated with inhibition of nitric oxide synthesis with L-NAME (40 mg/kg, iv). Glutamate triggered ventricular arrhythmia and persistent ST-segment shifts in the ECG, indicating myocardial ischemia. The intracisternal administration of clonidine (1 µg/kg) and rilmenidine (30 µg/kg) or of a nonhypotensive dose of rilmenidine (3 µg/kg) decreased the incidence of myocardial ischemia (25, 14 and 25%, respectively, versus 60% in controls) and reduced the mortality rate from 40% to 0.0, 0.0 and 12%, respectively. The total number of ventricular premature beats per minute fell from 30 ± 9 in the control group to 7 ± 3, 6 ± 3 and 2 ± 2, respectively. Intravenous administration of clonidine (10 µg/kg), rilmenidine (300 µg/kg) or propranolol (500 µg/kg) elicited similar protective effects. We conclude that clonidine and rilmenidine present cardioprotective effects of central origin, which can be reproduced by propranolol, a lipophilic ß-blocking agent.

Pentoxifylline decreases tumor necrosis factor and interleukin-1 during high tidal volume

Tumor necrosis factor-alpha (TNF-alpha) is one of the most important proinflammatory cytokines which plays a central role in host defense and in the acute inflammatory response related to tissue injury. The major source of TNF-alpha are immune cells such as neutrophils and macrophages. We tested the hypothesis that pentoxifylline, a methylxanthine derivative, down-regulates proinflammatory cytokine expression during acute lung injury in rats. Male Wistar rats weighing 250 to 450 g were anesthetized ip with 50 mg/kg sodium thiopental and randomly divided into three groups: group 1 (N = 7): tidal volume (V T) = 7 ml/kg, respiratory rate (RR) = 50 breaths/min and normal saline infusion; group 2 (N = 7): V T = 42 ml/kg, RR = 9 breaths/min and normal saline infusion; group 3 (N = 7): V T = 42 ml/kg, RR = 9 breaths/min and pentoxifylline infusion. The animals were ventilated with an inspired oxygen fraction of 1.0, a positive end-expiratory pressure of 3 cmH2O, and normal saline or pentoxifylline injected into the left femoral vein. The mRNA of TNF-alpha rapidly increased in the lung tissue within 180 min of ventilation with a higher V T with normal saline infusion. The concentrations of inflammatory mediators were decreased in plasma and bronchoalveolar lavage (BAL) in the presence of higher V T with pentoxifylline infusion (TNF-alpha: plasma, 102.2 ± 90.9 and BAL, 118.2 ± 82.1; IL-1ß: plasma, 45.2 ± 42.7 and BAL, 50.2 ± 34.9, P < 0.05). We conclude that TNF-alpha produced by neutrophil influx may function as an alert signal in host defense to induce production of other inflammatory mediators.

Evaluation of stunned and infarcted canine myocardium by real time myocardial contrast echocardiography

Differentiation between stunned and infarcted myocardium in the setting of acute ischemia is challenging. Real time myocardial contrast echocardiography allows the simultaneous assessment of myocardial perfusion and function. In the present study we evaluated infarcted and stunned myocardium in an experimental model using real time myocardial contrast echocardiography. Sixteen dogs underwent 180 min of coronary occlusion followed by reperfusion (infarct model) and seven other dogs were submitted to 20 min of coronary occlusion followed by reperfusion (stunned model). Wall motion abnormality and perfusional myocardial defect areas were measured by planimetry. Risk and infarct areas were determined by tissue staining. In the infarct model, the wall motion abnormality area during coronary occlusion (5.52 ± 1.14 cm²) was larger than the perfusional myocardial defect area (3.71 ± 1.45 cm²; P < 0.001). Reperfusion resulted in maintenance of wall motion abnormality (5.45 ± 1.41 cm²; P = 0.43 versus occlusion) and reduction of perfusional myocardial defect (1.51 ± 1.29 cm²; P = 0.004 versus occlusion). Infarct size determined by contrast echocardiography correlated with tissue staining (r = 0.71; P = 0.002). In the stunned model, the wall motion abnormality area was 5.49 ± 0.68 cm² during occlusion and remained 5.1 ± 0.63 cm² after reperfusion (P = 0.07). Perfusional defect area was 2.43 ± 0.79 cm² during occlusion and was reduced to 0.2 ± 0.53 cm² after reperfusion (P = 0.04). 2,3,5-Triphenyl tetrazolium chloride staining confirmed the absence of necrotic myocardium in all dogs in the stunned model. Real time myocardial contrast echocardiography is a noninvasive technique capable of distinguishing between stunned and infarcted myocardium after acute ischemia.

Chronic experimental myocardial infarction produces antinatriuresis by a renal nerve-dependent mechanism

The present study focused on the role of sympathetic renal nerve activity, in mediating congestive heart failure-induced sodium retention following experimental chronic myocardial infarction. Groups of male Wistar rats (240-260 g) were studied: sham-operated coronary ligation (CON3W, N = 11), coronary ligation and sham-operated renal denervation (INF3W, N = 19), 3 weeks of coronary ligation and sympathetic renal nerve denervation (INF3WDX, N = 6), sham-operated coronary ligation (N = 7), and 16 weeks of coronary ligation (INF16W, N = 7). An acute experimental protocol was used in which the volume overload (VO; 5% of body weight) was applied for 30 min after the equilibration period of continuous iv infusion of saline. Compared to control levels, VO produced an increase (P < 0.01, ANOVA) in urine flow rate (UFR; 570%) and urinary sodium excretion (USE; 1117%) in CON3W. VO induced a smaller increase (P < 0.01) in USE (684%) in INF3W. A similar response was also observed in INF16W. In INF3WDX, VO produced an immediate and large increase (P < 0.01) in UFR (547%) and USE (1211%). Similarly, in INF3W VO increased (P < 0.01) UFR (394%) and USE (894%). Compared with INF3W, VO induced a higher (P < 0.01) USE in INF3WDX, whose values were similar to those for CON3W. These results suggest that renal sympathetic activity may be involved in sodium retention induced by congestive heart failure. This premise is supported by the observation that in bilaterally renal denervated INF3WDX rats myocardial infarction was unable to reduce volume expansion-induced natriuresis. However, the mechanism involved in urinary volume regulation seems to be insensitive to the factors that alter natriuresis.

Food restriction induces in vivo ventricular dysfunction in spontaneously hypertensive rats without impairment of in vitro myocardial contractility

Cardiac structures, function, and myocardial contractility are affected by food restriction (FR). There are few experiments associating undernutrition with hypertension. The aim of the present study was to analyze the effects of FR on the cardiac response to hypertension in a genetic model of hypertension, the spontaneously hypertensive rat (SHR). Five-month-old SHR were fed a control or a calorie-restricted diet for 90 days. Global left ventricle (LV) systolic function was evaluated in vivo by transthoracic echocardiogram and myocardial contractility and diastolic function were assessed in vitro in an isovolumetrically beating isolated heart (Langendorff preparation). FR reduced LV systolic function (control (mean ± SD): 58.9 ± 8.2; FR: 50.8 ± 4.8%, N = 14, P < 0.05). Myocardial contractility was preserved when assessed by the +dP/dt (control: 3493 ± 379; FR: 3555 ± 211 mmHg/s, P > 0.05), and developed pressure (in vitro) at diastolic pressure of zero (control: 152 ± 16; FR: 149 ± 15 mmHg, N = 9, P > 0.05) and 25 mmHg (control: 155 ± 9; FR: 150 ± 10 mmHg, N = 9, P > 0.05). FR also induced eccentric ventricular remodeling, and reduced myocardial elasticity (control: 10.9 ± 1.6; FR: 9.2 ± 0.9%, N = 9, P < 0.05) and LV compliance (control: 82.6 ± 16.5; FR: 68.2 ± 9.1%, N = 9, P < 0.05). We conclude that FR causes systolic ventricular dysfunction without in vitro change in myocardial contractility and diastolic dysfunction probably due to a reduction in myocardial elasticity.

Cardiac and vascular effects of diltiazem, dobutamine and amrinone, drugs used after myocardial revascularization

Hemodynamic care during postoperative management of myocardial revascularization should include vasorelaxing drugs to insure adequate graft and coronary flow, and stimulation of stroke volume to maintain vascular perfusion pressure. We tested the cardiac (inotropic and lusitropic) and vascular (relaxant) effects of diltiazem (0.1 nM to 0.1 mM), dobutamine (10 µM to 10 mM) and amrinone (10 µM to 1 mM) on isolated rat atria and thoracic aorta, and also on isolated human saphenous vein (HSV) and human mammary artery (HMA). Dobutamine produced a maximal positive inotropic effect (+dF/dt max = 29 ± 7%) at its ED50 for aortic relaxation (88 ± 7 µM). Conversely, at their ED50 for aortic relaxation diltiazem depressed myocardial contractility and amrinone did not exhibit myocardial effects. In HSV and HMA contracted with 80 mM potassium, diltiazem and dobutamine (but not amrinone) had a vasorelaxant activity similar to that in rat aorta. Norepinephrine-contracted human vessels were significantly more sensitive than potassium-contracted vessels to the relaxant effect of amrinone (ED50 HMA = 15 ± 5 µM, ED50 HSV = 72 ± 31 µM, P < 0.05). We conclude that at concentrations still devoid of myocardial effects dobutamine and amrinone are effective dilators in graft segment vessels and rat aorta contracted by membrane depolarization. If the difference between aortic and myocardial tissue still holds in human tissues, at the appropriate concentrations these drugs should be expected to improve cardiac performance while still contributing to the maintenance of graft patency.

Myocardial contractility of the isovolumetrically beating isolated rat heart

Several indexes of myocardial contractility have been proposed to assess ventricular function in the isovolumetrically beating isolated heart. However, the conclusions reached on the basis of these indexes may be influenced by ventricular geometry rather than contractility itself. The objective of the present study was to assess the performance of widely used contractility indexes in the isovolumetrically beating isolated heart in two experimental models of hypertrophy, the spontaneously hypertensive rat (SHR) and infrarenal aortocava fistula. Compared to normotensive controls (N = 8), SHRs with concentric hypertrophy (N = 10) presented increased maximum rate of ventricular pressure rise (3875 ± 526 vs 2555 ± 359 mmHg/s, P < 0.05) and peak of isovolumetric pressure (187 ± 11 vs 152 ± 11 mmHg, P < 0.05), and decreased developed stress (123 ± 20 vs 152 ± 26 g/cm², P < 0.05) and slope of stress-strain relationship (4.9 ± 0.42 vs 6.6 ± 0.77 g/cm²/%). Compared with controls (N = 11), rats with volume overload-induced eccentric hypertrophy (N = 16) presented increased developed stress (157 ± 38 vs 124 ± 22 g/cm², P < 0.05) and slope of stress-strain relationship (9 ± 2 vs 7 ± 1 g/cm²/%, P < 0.05), and decreased maximum rate of ventricular pressure rise(2746 ± 382 vs 3319 ± 352 mmHg, P < 0.05) and peak of isovolumetric pressure (115 ± 14 vs 165 ± 13 mmHg/s, P < 0.05). The results suggested that indexes of myocardial contractility used in experimental studies may present opposite results in the same heart and may be influenced by ventricular geometry. We concluded that several indexes should be taken into account for proper evaluation of contractile state, in the isovolumetrically beating isolated heart.

Temperature, air pollution, and mortality from myocardial infarction in São Paulo, Brazil

An increase in daily mortality from myocardial infarction has been observed in association with meteorological factors and air pollution in several cities in the world, mainly in the northern hemisphere. The objective of the present study was to analyze the independent effects of environmental variables on daily counts of death from myocardial infarction in a subtropical region in South America. We used the robust Poisson regression to investigate associations between weather (temperature, humidity and barometric pressure), air pollution (sulfur dioxide, carbon monoxide, and inhalable particulate), and the daily death counts attributed to myocardial infarction in the city of São Paulo in Brazil, where 12,007 fatal events were observed from 1996 to 1998. The model was adjusted in a linear fashion for relative humidity and day-of-week, while nonparametric smoothing factors were used for seasonal trend and temperature. We found a significant association of daily temperature with deaths due to myocardial infarction (P < 0.001), with the lowest mortality being observed at temperatures between 21.6 and 22.6ºC. Relative humidity appeared to exert a protective effect. Sulfur dioxide concentrations correlated linearly with myocardial infarction deaths, increasing the number of fatal events by 3.4% (relative risk of 1.03; 95% confidence interval = 1.02-1.05) for each 10 µg/m³ increase. In conclusion, this study provides evidence of important associations between daily temperature and air pollution and mortality from myocardial infarction in a subtropical region, even after a comprehensive control for confounding factors.

Increased production of tumor necrosis factor-alpha in whole blood cultures from children with primary malnutrition

Because low tumor necrosis factor-alpha (TNF-alpha) production has been reported in malnourished children, in contrast with high production of TNF-alpha in experimental protein-energy malnutrition, we reevaluated the production of TNF-alpha in whole blood cultures from children with primary malnutrition free from infection, and in healthy sex- and age-matched controls. Mononuclear cells in blood diluted 1:5 in endotoxin-free medium released TNF-alpha for 24 h. Spontaneously released TNF-alpha levels (mean ± SEM), as measured by enzyme immunoassay in the supernatants of unstimulated 24-h cultures, were 10,941 ± 2,591 pg/ml in children with malnutrition (N = 11) and 533 ± 267 pg/ml in controls (N = 18) (P < 0.0001). TNF-alpha production was increased by stimulation with lipopolysaccharide (LPS), with maximal production of 67,341 ± 16,580 pg/ml TNF-alpha in malnourished children and 25,198 ± 2,493 pg/ml in controls (P = 0.002). In control subjects, LPS dose-dependently induced TNF-alpha production, with maximal responses obtained at 2000 ng/ml. In contrast, malnourished patients produced significantly more TNF-alpha with 0.02-200 ng/ml LPS, responded maximally at a 10-fold lower LPS concentration (200 ng/ml), and presented high-dose inhibition at 2000 ng/ml. TNF-alpha production a) was significantly influenced by LPS concentration in control subjects, but not in malnourished children, who responded strongly to very low LPS concentrations, and b) presented a significant, negative correlation (r = -0.703, P = 0.023) between spontaneous release and the LPS concentration that elicited maximal responses in malnourished patients. These findings indicate that malnourished children are not deficient in TNF-alpha production, and suggest that their cells are primed for increased TNF-alpha production.

A routine electrocardiogram cannot be used to determine the size of myocardial infarction in the rat

Nine lead electrocardiograms of non-infarcted (N = 61) and infarcted (N = 71) female Wistar rats (200-250 g) were analyzed in order to distinguish left ventricle myocardial infarction (MI) larger than 40% (LMI) from MI smaller than 40% (SMI). MI larger than 40% clearly caused a deviation of ÂQRS and ÂT from normal values of 270-360 degrees to 90-270 degrees. Infarcted rats showed Q wave in D1 larger than 1 mm with 94% sensitivity and 100% specificity. The sum of QRS positivity in V1, V2 and V6 lower than 10 mm identified MI with 82% sensitivity and 100% specificity. The data showed that MI can be easily and reliably diagnosed by electrocardiogram in the rat. However, contradicting what is frequently believed, when specificity and sensitivity were analyzed focusing on MI size, none of these current electrocardiographic indices of MI size adequately discriminates LMI from SMI.