Intracoronary gene transfer of fibroblast growth factor-5 increases blood flow and contractile function in an ischemic region of the heart.

Increased coronary blood vessel development could potentially benefit patients with ischemic heart disease. In a model of stress-induced myocardial ischemia, intracoronary injection of a recombinant adenovirus expressing human fibroblast growth factor-5 (FGF-5) resulted in messenger RNA and protein expression of the transferred gene. Two weeks after gene transfer, regional abnormalities in stress-induced function and blood flow were improved, effects that persisted for 12 weeks. Improved blood flow and function were associated with evidence of angiogenesis. This report documents, for the first time, successful amelioration of abnormalities in myocardial blood flow and function following in vivo gene transfer.

Regional myocardial downregulation of the inhibitory guanosine triphosphate-binding protein (Gi alpha 2) and beta-adrenergic receptors in a porcine model of chronic episodic myocardial ischemia.

Regional myocardial ischemia is associated with increased levels of adenosine and norepinephrine, factors that may alter activation of the beta-adrenergic receptor (beta AR)-G protein-adenylyl cyclase pathway in the heart. We have used the ameroid constrictor model to determine whether alterations in myocardial signal transduction through the beta AR-G protein-adenylyl cyclase pathway occur in the setting of chronic episodes of reversible ischemia. Pigs were instrumented with ameroid occluders placed around the left circumflex coronary artery. 5 wk later, after ameroid closure, flow and function were normal in the ischemic bed, but flow (P = 0.001) and function (P < 0.03) were abnormal when metabolic demands were increased. The ischemic bed showed a reduction in myocardial beta AR number (P < 0.005). Despite regional downregulation of myocardial beta AR number, adenylyl cyclase activity was similar in the ischemic and control beds. Quantitative immunoblotting showed that the cardiac inhibitory GTP-binding protein, Gi alpha 2, was decreased in the ischemic bed (P = 0.02). In contrast, the cardiac stimulatory GTP-binding protein, Gs alpha, was increased in endocardial sections from the ischemic bed (P = < 0.05). Decreased Gi alpha 2 content was associated with decreased inhibition of adenylyl cyclase. Reduced Gi alpha 2 content, in conjunction with increased Gs alpha content in the endocardium, may provide a means by which adrenergic activation is maintained in the setting of chronic episodic myocardial ischemia.

Intracoronary delivery of adenovirus encoding adenylyl cyclase VI increases left ventricular function and cAMP-generating capacity.

BACKGROUND: We tested the hypothesis that intracoronary injection of a recombinant adenovirus encoding adenylyl cyclase type VI (AC(VI)) would increase cardiac function in pigs. METHODS AND RESULTS: Left ventricular (LV) dP/dt and cardiac output in response to isoproterenol and NKH477 stimulation were assessed in normal pigs before and 12 days after intracoronary delivery of histamine followed by intracoronary delivery of an adenovirus encoding lacZ (control) or AC(VI) (1.4x10(12) vp). Animals that had received AC(VI) gene transfer showed increases in peak LV dP/dt (average increase of 1267+/-807 mm Hg/s; P=0.0002) and cardiac output (average increase of 39+/-20 mL. kg(-1). min(-1); P<0.0001); control animals showed no changes. Increased LV dP/dt was evident 6 days after gene transfer and persisted for at least 57 days. Basal heart rate, blood pressure, and LV dP/dt were unchanged, despite changes in cardiac responsiveness to catecholamine stimulation. Twenty-three hour ECG recordings showed no change in mean heart rate or ectopic beats and no arrhythmias. LV homogenates from animals receiving AC(VI) gene transfer showed increased AC(VI) protein content (P=0.0007) and stimulated cAMP production (P=0.0006), confirming transgene expression and function; basal LV AC activity was unchanged. Increased cAMP-generating capacity persisted for at least 18 weeks (P<0.0002). CONCLUSIONS: Intracoronary injection of a recombinant adenovirus encoding AC provides enduring increases in cardiac function.

Effects of dobutamine and arbutamine on regional myocardial function in a porcine model of myocardial ischemia.

OBJECTIVES: The present study was performed to determine the mechanisms for catecholamine-induced wall motion abnormalities and to compare the diagnostic efficacy of two catecholamines: arbutamine and dobutamine. BACKGROUND: Catecholamine stress echocardiography is used to induce regional wall motion abnormalities for the detection of coronary artery disease, but the mechanism by which these abnormalities occur is unknown. METHODS: Ten pigs were instrumented with left circumflex coronary artery ameroid constrictors, sonomicrometers to measure transmural wall thickening in the left circumflex (ischemic) and left anterior descending (control) coronary artery beds and a pressure gauge to measure left ventricular pressure and its first derivative (dP/dt). Myocardial blood flow was measured by microspheres. RESULTS: At 38 +/- 6 days (mean +/- SEM) after surgery, percent wall thickening was normal at rest in both beds but abnormal in the left circumflex coronary artery bed during atrial pacing. These findings were associated with reduced myocardial blood flow in the ischemic bed during atrial pacing. Dobutamine infusion increased percent wall thickening, with no differences between the two beds (p = 0.63). In contrast, arbutamine infusion increased percent wall thickening only in the nonischemic bed, with no effect on percent wall thickening in the ischemic bed (p = 0.03). Although the endocardial/epicardial blood flow ratio tended to be reduced in the left circumflex artery bed during catecholamine infusion (p = 0.07), both agents were similar in this effect. Despite differences in function between the beds, there was no difference in transmural myocardial blood flow between the two beds during catecholamine infusion. When examined at matched metabolic demands, arbutamine elicited greater differences in percent wall thickening than dobutamine between the two beds (p < 0.01). CONCLUSIONS: Arbutamine was able to provoke regional differences in function in a manner superior to dobutamine. This occurred independently of altered transmural myocardial blood flow or differences in hemodynamic effects between the agents. Differences in their inotropic properties may be important in explaining their different effects on ischemic myocardium.

Treadmill performance and cardiac function in selected patients with coronary heart disease.

To investigate the cardiac determinants of treadmill performance in patients able to exercise to volitional fatigue, 88 patients with coronary heart disease free of angina pectoris were tested. The exercise tests included supine bicycle radionuclide ventriculography, thallium scintigraphy and treadmill testing with expired gas analysis. The number of abnormal Q wave locations, ejection fraction, end-diastolic volume, cardiac output, exercise-induced ST segment depression and thallium scar and ischemia scores were the cardiac variables considered. Rest and exercise ejection fractions were highly correlated to thallium scar score (r = -0.72 to -0.75, p less than 0.001), but not to maximal oxygen consumption (r = 0.19 to 0.25, p less than 0.05). Fifty-five percent of the variability in predicting treadmill time or estimated maximal oxygen consumption was explained by treadmill test-induced change in heart rate (39%), thallium ischemia score (12%) and cardiac output at rest (4%). The change in heart rate induced by the treadmill test explained only 27% of the variability in measured maximal oxygen consumption. Myocardial damage predicted ejection fraction at rest and the ability to increase heart rate with treadmill exercise appeared as an essential component of exercise capacity. Exercise capacity was only minimally affected by asymptomatic ischemia and was relatively independent of ventricular function.

Angiogenic gene therapy for heart disease: a review of animal studies and clinical trials.

The current published clinical literature on angiogenic gene therapy for the treatment of myocardial ischemia does not include a single randomized, placebo-controlled trial. Based on current clinical literature, it is an unproven therapy. Successful animal studies combined with published reports of good outcomes in patients enrolled in uncontrolled trials has led to the expectation that angiogenic gene therapy will ultimately become a clinical reality. The next important landmark in the field will be the publication of data showing a favorable effect of angiogenic gene transfer in placebo-controlled, blinded clinical trials.

Improvement in ventricular function during exercise studied with radionuclide ventriculography after cardiac rehabilitation.

A heterogeneous group of 19 consecutive patients with coronary artery disease were studied with radionuclide ventriculography before and after a mean of 6 months of exercise training. Ejection fraction was measured at rest, at matched submaximal supine work loads and during maximal supine bicycle exercise. After training there was no change in mean ejection fraction at rest or during maximal exercise, but a higher maximal mean systolic blood pressure, heart rate and work load were achieved. At equivalent submaximal work loads after training, similar levels of mean heart rate and systolic blood pressure were reached but a statistically greater mean ejection fraction was obtained. These preliminary results suggest that exercise training may improve cardiac function during exercise in selected patients with coronary disease. A randomized study using similar techniques has been initiated.

Hypertonic saline/dextran treatment for severe pressure-driven hemorrhage in dehydrated conscious swine.

Although dehydration impairs the response to a fixed volume hemorrhage, both 7.5% hypertonic saline/6% dextran 70 (HSD: 4 mL/kg) and standard Ringer's lactate (33 mL/kg) are effective resuscitation fluids. However, the efficacy of resuscitation during continuing hemorrhage remains in question. Using a conscious swine model of continuous pressure-driven hemorrhage, we evaluated the effects of dehydration and HSD resuscitation on survival time, hemorrhage volume, regional blood flows, and central hemodynamics. Three groups of pigs were compared: euhydrated control (EC), dehydrated control (48 h water deprived) (DC); and dehydrated and resuscitated with HSD (D + HSD). All pigs were subjected to an initial 37% blood volume hemorrhage for 60 min followed by a continuous hemorrhage proportional to the instantaneous mean arterial pressure. The D + HSD pigs were resuscitated at the end of the 37% blood volume hemorrhage. Dehydration reduced body weight (-6.5 +/- .3%) and increased hematocrit (8.9 +/- 1.8%), serum osmolality (11.6 +/- .9%), serum sodium (11.9 +/- .9%), and serum total protein (9.4 +/- 1.8%). Compared with the EC group, DC had a greater increase in heart rate and arterial base deficit in response to the pressure-driven hemorrhage and a reduced pH and survival time (159 vs. 107 min). In contrast to the DC group, D + HSD had increased mean arterial pressure, cardiac output, oxygen delivery, and regional blood flows to the gut (superior mesenteric artery), kidneys, liver (hepatic artery), and adrenals at 5 min after HSD resuscitation. The HSD did not increase blood loss but tended to prolong survival (+26 min; p = .1079). thus, dehydration compromises survival time (-33%) and the hemodynamic and metabolic responses to pressure-driven hemorrhage, while treatment with HSD improves the hemodynamic responses.

Nitric oxide synthesis inhibition does not improve the hemodynamic response to hemorrhagic shock in dehydrated conscious swine.

Nitric oxide (NO) may influence the hemodynamic response to hemorrhage. To test this hypothesis, the NO synthesis inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) was administered to conscious, dehydrated swine during a 37% blood volume hemorrhage and a 180 min recovery period without fluid resuscitation. L-NAME (.75 mg/kg bolus plus constant infusion of .75 mg/kg/h) was given via a central intravenous catheter during the bleed. The selectivity and specificity of L-NAME as a NO synthesis inhibitor in pigs was validated in pilot studies. The present study shows that inhibition of NO synthesis with L-NAME had no significant effect on the major hemodynamic parameters during and after hemorrhage when compared to dehydrated and euhydrated control groups. Only stroke volume and A-VO2 were significantly different from controls. Mortality was 83% for the L-NAME group and 44% for controls at 180 min of recovery (NS). The results suggest that NO synthesis inhibition provides no hemodynamic benefit during hemorrhage in dehydrated, conscious swine.

Effects of feeding on muscle blood flow during prolonged exercise in miniature swine.

Eight exercise-trained miniature swine were studied during prolonged treadmill runs (100 min) under fasting and preexercise feeding conditions. Each animal ran at identical external work loads that corresponded to 65% of the heart rate reserve (210-220 beats/min) for the two exercise bouts. Cardiac outputs and stroke volumes were higher and heart rates lower for fed than for fasting runs (P less than 0.05). Preexercise feeding did not alter oxygen consumption, core temperature, mean arterial pressure, and arterial-mixed venous oxygen difference during prolonged exercise; however, mixed venous lactate concentration was lower at end exercise than during fasting conditions (1.2 vs. 2.6 mM, P less than 0.05). Microsphere measurements of regional blood flow revealed significantly higher total gastrointestinal flow (23%) for fed than for fasting conditions. Throughout the exercise bout, blood flow to the biceps femoris, semitendinosus, and tibialis anterior muscles was lower in fed than in fasted animals (P less than 0.05). Combined hindlimb muscle blood flow averaged 15 ml.min-1.100 g-1 (18%, P less than 0.05) lower under feeding than fasting run conditions. These findings provide further evidence that cardiovascular reflexes originate in the gut after feeding to increase cardiac output and redistribute a portion of the blood flow away from active muscle to the gastrointestinal tract during prolonged exercise.

Plateau in muscle blood flow during prolonged exercise in miniature swine.

Cardiovascular, metabolic, and thermoregulatory responses were studied in eight male miniature swine during a prolonged treadmill run. Each animal underwent 8-10 wk of exercise training, thoracic surgery, and 3 wk of retraining before the experimental run. This regimen enabled the animals to run at 65% of the heart rate range (210-220 beats/min) for approximately 100 min. Skin wetting and a fan were used to cool the pigs during the run. Regional blood flow was significantly altered with the onset of exercise; however, hindlimb muscle and total gastrointestinal blood flow were unchanged throughout the exercise period. Compared with 5-min values, heart rate and cardiac output were significantly elevated by 17 beats/min and 31 ml.min-1.kg-1 at 60 min and by 20 beats/min and 33 ml.min-1.kg-1 at end exercise, respectively. Core temperatures increased between 5 and 30 min of exercise (39.4 vs. 39.9 degrees C) but then remained unchanged to the end of exercise. Mean arterial pressure, O2 consumption, and blood lactate did not change during the exercise bout. These data indicate that limiting increases in core temperature during prolonged exercise was associated with a plateau in active muscle blood flow.

Exercise-induced cardiac hypertrophy: a correlation of blood flow and microvasculature.

The effects of exercise conditioning on the myocardium were studied in seven instrumented pigs strenuously exercised for 12 wk by treadmill running. Data were compared with eight instrumented untrained pigs. O2 consumption measured during maximum exercise effort was significantly elevated in the trained pigs (71.7 +/- 4.0 vs. 56.3 +/- 3.0 ml X ml-1 X kg-1). Absolute right and left ventricular mass increased by 20 and 13%, respectively, in response to exercise. Myocyte cross-sectional area increased by 21% in the trained hearts compared with the untrained hearts. Transmural left ventricular myocardial blood flow (ml X min-1 X g-1) was not significantly different at rest, during maximum exercise, or during exercise with adenosine infusion. However, training caused an elevation of the regional epicardial blood flow noted during exercise and exercise with adenosine. In the trained pigs mean aortic pressure during maximum exercise with adenosine infusion was not significantly different compared with untrained pigs. Coronary resistance during exercise with adenosine infusion was the same in both animal groups. In the trained group capillary numerical (no./mm2) and length (mm/mm3) densities were reduced, whereas arteriolar numerical and length densities were significantly increased compared with the untrained group. Measurements of capillary luminal surface density (mm2/mm3) in the trained group were unchanged compared with the untrained group. These results suggest that strenuous exercise does not stimulate the production of new capillaries, but this is modified by the ability of existing capillaries to increase their luminal surface area to parallel increases in myocyte growth. The arteriolar data suggest that exercise promotes the formation of new arterioles.(ABSTRACT TRUNCATED AT 250 WORDS)

Validation of a respiratory mask for measuring gas exchange in exercising swine.

A respiratory mask was developed for resting and exercising swine. The lightweight, low-dead-space design fits airtight against the animals' snouts to provide breath-by-breath measurements of respiration and metabolism. Validation of the mask was carried out using the Fick principle with dye-dilution cardiac outputs and arterial and mixed venous O2 content measurements. Linear regression analysis of O2 consumption (VO2) measurements by the two techniques revealed a slope of 1.07 and a Y-intercept of -1.06 ml X kg-1 X min-1. The standard error of the estimate of VO2 was 3.5 ml X kg-1 X min-1. The mask design permits rapid measurements of ventilation and metabolism in response to acute and chronic exercise.

Exercise training in swine promotes growth of arteriolar bed and capillary angiogenesis in heart.

Exercise training induces coronary vascular adaptations. The goal of this study was to contrast the effects of training on capillary and arteriolar growth. Minipigs were trained for 1, 3, 8, and 16 wk and compared with controls. Maximal O2 consumption increased continuously throughout the study. Capillary and arteriolar densities and diameters, and proliferation of vascular cells in these vessels, were determined in perfusion-fixed tissue. The arterioles were subdivided into five groups according to diameter: 10-19.9, 20-30, 31-40, 41-70, and 71-120 microgram. The total vascular bed cross-sectional area increased by 37% at 16 wk, mainly because of an increase in the number of the small arterioles and an increase in the diameter of the larger vessels. Capillary density increased at 3 wk and then returned to control levels by 16 wk; concomitantly, the number of arterioles (20-30 microgram) increased at 16 wk. We speculate that the "extra" capillaries observed at 3 wk were the source of the new arterioles.

Pulmonary gas exchange during exercise in pigs.

Increased ventilation-perfusion (VA/Q) inequality is observed in approximately 50% of humans during heavy exercise and contributes to the widening of the alveolar-arterial O2 difference (A-aDO2). Despite extensive investigation, the cause remains unknown. As a first step to more direct examination of this problem, we developed an animal model. Eight Yucatan miniswine were studied at rest and during treadmill exercise at approximately 30, 50, and 85% of maximal O2 consumption (VO2 max). Multiple inert-gas, blood-gas, and metabolic data were obtained. The A-aDO2 increased from 0 +/- 3 (SE) Torr at rest to 14 +/- 2 Torr during the heaviest exercise level, but arterial PO2 (PaO2) remained at resting levels during exercise. There was normal VA/Q inequality [log SD of the perfusion distribution (log) = 0.42 +/- 0.04] at rest, and moderate increases (log = 0.68 +/- 0.04, P < 0.0001) were observed with exercise. This result was reproducible on a separate day. The VA/Q inequality changes are similar to those reported in highly trained humans. However, in swine, unlike in humans, there was no inert gas evidence for pulmonary end-capillary diffusion limitation during heavy exercise; there was no systematic difference in the measured PaO2 and the PaO2 as predicted from the inert gases. These data suggest that the pig animal model is well suited for studying the mechanism of exercise-induced VA/Q inequality.

Adaptation of the left ventricle to exercise-induced hypertrophy.

Cardiac functional and structural adaptations to exercise-induced hypertrophy were studied in 68 pigs. Pigs were exercise trained on a treadmill for 10 wk. Sequential measurements were made of cardiac dimensions, [left ventricular end-diastolic diameter (EDD), changes in diameter (delta D%), wall thickness (WTh), wall thickening (WTh%), left ventricular pressure (LVP), time derivative of pressure (dP/dt), stroke volume, total body O2 consumption (VO2), blood gases, and systemic hemodynamics] at rest and during moderate and severe exercise. Postmortem studies included morphometric measurements of capillary density, arteriolar density, mitochondria, and myofibrils. All of the exercise-trained pigs showed significant increases in aerobic capacity. Maximum O2 consumption (VO2 max) increased by 37.5% in group 1 (moderate exercise training) and 34% in group 3 (heavy exercise training). Cardiac hypertrophy ranged from less than 15% in a group (n = 8) subjected to moderate exercise training to greater than 30% in a group (n = 11) subjected to heavy exercise training. Before training, exercise was characterized by a decreasing EDD during progressive exercise; this was reversed after exercise training. Stroke volume and end-diastolic volumes during exercise showed a highly significant increase after exercise training and hypertrophy. Morphometric measurements showed that mitochondria and cell membranes increased with increasing myocyte growth in all exercise groups, but there was only a partially compensated adaptation of capillary proliferation. Arteriolar number and length increased in all exercise groups. Intrinsic contractility as measured by delta D%, WTh%, or left ventricular dP/dt did not increase with exercise training and in some instances decreased. Therefore, left ventricular adaptation to strenuous exercise in the pig heart is primarily one of changes in left ventricular dimensions and a compensated hypertrophy. Exercise-induced increases in EDD and stroke volume can be accounted for by decreases in peripheral resistance and increased cardiac dimensions.

Clinical significance of coronary vascular adaptations to exercise training.

Coronary vascular adaptations to exercise training have been extensively studied at the microscopic level in animals and correlated with direct and indirect measurements of myocardial blood flow in patients with coronary artery disease. Animals have permitted more extensive study. These findings have generally supported an increased blood flow to the myocardium with exercise training. However, consistent positive structural and functional adaptations to training have not been observed in large animals. Clinical studies have been limited by methodological problems related to techniques for detecting ischemia and measuring myocardial blood flow and the variability in exercise stimulus. Well-established ischemia and high-intensity, long-duration training were the factors that promoted vascular growth in exercising patients with coronary artery disease. Animals studies also have demonstrated the necessity for myocardial ischemia to be present to induce coronary collateral development with exercise training. Optimal promoters of vascular growth in patients with coronary disease may consist of pharmacological interventions combined with exercise training.

Rest and exercise electrocardiograms and radionuclides in patients presenting for cardiac rehabilitation.

The rest and exercise ECG, 201thallium myocardial scintigram (201T1), and radionuclide ventriculography are noninvasive procedures which can be used to evaluate myocardial damage and ischemia. To compare these procedures and to obtain baseline information, 85 male patients with coronary heart disease were evaluated prior to beginning an exercise program. Findings at rest included Q waves or bundle branch block in 54%; 47% had 201T1 redistribution defects and 33% an abnormal ejection fraction (EF). Of the 39 patients with normal ECGs, 31 had no 201T1 defects and only of these 31 (3%) had an abnormal EF. Abnormal EF or 201T1 redistribution defects did not occur in patients without a history of myocardial infarction. Abnormal resting EF occurred in 63% of patients with abnormal versus 7% of those with normal 201T1 redistribution scans. Exercise test results included an abnormal ST-segment response in 80%, an abnormal EF response in 65%, and a 201T1 ischemic defect in 37%. Twenty patients had exercise-induced ST elevation, and this phenomenon was more related to ventricular aneurysms than to ischemia. 201Thallium imaging, radionuclide ventriculography, and the ECG provide results regarding myocardial damage that agree by more than chance, while the exercise induced ST-segment changes did not agree with the radionuclide indications of exercise-induced ischemia.

Errors in predicting functional capacity for postmyocardial infarction patients using a modified Bruce protocol.

Clinically, the modified Bruce protocol is widely used to predict functional capacity in postmyocardial infarction (post-MI) patients. However, it has been suggested that post-MI patients have lower oxygen uptakes for standard workloads. In order to study this, we measured oxygen uptake (VO2) and venous blood lactic acid concentration in 12 post-MI patients and 12 normal male subjects during a modified Bruce treadmill protocol. During the first four stages of the protocol, mean oxygen uptake was significantly lower (1.0 to 6.2 cc X kg-1 X min-1; p less than 0.001) for the post-MI patients than for the normal male subjects. Venous blood lactic acid concentrations were different only at stage 4. However, a higher respiratory exchange ratio was observed for post-MI patients (p less than 0.001 at stage 3), suggesting an increased anaerobic metabolism and adequate buffering of lactic acid. The post-MI patient's measured VO2 for three stages of the protocol ranged from 1.8 to 7.3 cc X kg-1 X min-1 lower than the Bruce prediction for cardiac patients. In addition, maximal measured VO2 for the post-MI patients ranged from 3.7 to 11.2 cc X kg-1 X min-1 lower than predicted VO2 derived from the normal subjects. These data suggest myocardial damage may slow oxygen uptake kinetics, thus increasing the oxygen deficit at standard workloads during progressive exercise. An increased respiratory exchange ratio secondary to the buffering of lactic acid suggests that anaerobic metabolism may compensate for this oxygen deficit.