Robert M. Lewis

Biochemical and morphologic correlates of cardiac ischemia. I. Membrane systems.

Functions of membrane-linked myocardial systems and morphology of the myocardial cell were examined in normal and acutely and chronically ischemic myocardium. Hemodynamic measurements of ischemic tissue showed depressed force development as well as decreased (and variable) ventricular peak pressures. Mitochondrial respiratory function was reduced, with state 3 (phosphorylating) respiration showing the most marked impairment. Losses in cytochromes c and a 3 were observed. Diminished mitochondrial calcium uptake with subsequent release of the calcium taken up during continued respiratory activity was characteristic of severely ischemic tissue, that is, 7 days after ligation. Defects in the carnitine-mediated oxidation of palmitic acid in isolated mitochondria were severe between 1 and 8 days after ligation. Losses in tissue carnitine also occurred simultaneously with decreased mitochondrial carnitine palmityltransferase activity. During this period the oxidation of hexanoic acid was unaffected. The binding and release cycle of calcium was studied in sarcoplasmic reticulum (cardiac relaxing system) isolated from control tissues and from acutely and chronically ischemic heart. An early impairment in the release phase of calcium from an acutely ischemic preparation occurred at times (12 to 60 minutes after ligation) when the other membrane-associated functions maintained normal integrity. In the chronically ischemic dog, there was marked impairment of calcium-binding variables in the first 2 weeks after ligation. This impairment occurred at times when both mitochondrial and sodium, potassium adenosine triphosphatase (Na + ,K + -ATPase) activity levels were severely impaired. The Na + ,K + -ATPase activity level was consistently lower than the control level by 7 days after ligation. However, there was no change in kinetic indexes or in the ouabainbinding characteristics in the functional enzyme remaining. Morphologic studies of tissue taken from left posterior papillary muscle 3 to 8 days after ligation revealed significant and specific changes in ultrastructure (disrupted Z and I bands, appearance of pseudo-N band and appearance of dense bodies in mitochondria); the greatest occurrence of damaged cells occurred 7 to 8 days after ligation.

Comparative hemodynamic effects of isoproterenol, dopamine, and dobutamine in the newborn dog.

Summary: To compare the hemodynamic effects of isoproterenol, dopamine, and dobutamine in the immature animal, each drug was infused into anesthetized open chest puppies and cardiac output was measured, as were systemic arterial blood pressure, heart rate, and renal artery blood flow.Cardiac output was increased by dopamine and dobutamine. Isoproterenol caused a significantly greater increment of heart rate than either of the other agents to achieve a similar change of cardiac output.Systemic arterial mean blood pressure was increased by dopamine and dobutamine, but decreased by isoproterenol.Dopamine produced a significant increase of renal artery blood flow while renal artery blood flow was unchanged by dobutamine and decreased by isoproterenol.Speculation: Dopamine and dobutamine may have potential advantages over isoproterenol in augmenting cardiovascular function in young animals.

Hemodynamic and biochemical effects of a new positive inotropic agent. Antibiotic ionophore RO 2-2985

The antibiotic ionophores RO 2-2985 (Hoffman-LaRoche; X537A) and A23187 (Lilly) produced an abrupt release of previously bound calcium from isolated sarcoplasmic reticulum. Only RO 2-2985 produced a significant inhibition of sodium, potassium-adenosinetriphosphatase activity. In anesthetized, open-chest dogs, RO 2-2985 caused a marked increase in right and left ventricular contractile force, aortic and coronary flow, and mean central aortic blood pressure with modest changes in heart rate and no change in calculated mean peripheral resistance. A single injection of 1 mg/kg produced a sustained augmentation of mean blood pressure from a control of 45 mm Hg in dogs that were in induced shock to approximately 125 mm Hg for a period up to 7–9 hours. At that time, tyramine produced a release of catecholamines, and norepinephrine and isoproterenol produced a marked increase in force of contraction. Dogs that were treated with reserpine did not respond to the drug. RO 2-2985, but not A23187, produced positive inotropism in isolated rabbit atrial and perfused ventricular preparations. RO 2-2985 still produced an augmentation in blood pressure after β-receptor blockade. It is suggested that RO 2-2985 acts by a mechanism involving a very slow release of humoral substances, by a direct effect on a specific calcium-proton exchange, or by both. It is thought that this agent has potential therapeutic applications.

The effect of ingestion of meat on hepatic extraction of insulin and glucagon and hepatic glucose output in conscious dogs

The effect of ingestion of protein on hepatic extraction of insulin and glucagon and hepatic glucose output were investigated in conscious dogs. The ingestion of meat stimulated both insulin and glucagon secretion but the glucagon response was much more rapid and greater than that of insulin. Secretion of glucagon demonstrated a biphasic pattern while insulin release was monophasic. The fractional hepatic extraction of glucagon increased gradually from the basal value of 15 +/- 3% to a peak of 36 +/- 5% at 90 min, and that of insulin increased from the basal level of 41 +/- 2% to 54 +/- 4% at 45 and 60 min. The increased hepatic extraction of glucagon and insulin after meat ingestion may be explained by neural or hormonal signals from the gut. The blood glucose and hepatic glucose output did not increase significantly despite the significant decrease of the portal vein insulin to glucagon molar ratio as well as the significant decrease of the molar ratio of the hepatic uptake of these hormones. The absence of greater hepatic glucose production despite the augmented glucagon secretion and decreased portal vein insulin to glucagon molar ratio could reflect down regulation by glucagon.

Effects of Portal and Peripheral Venous Insulin Infusion on Glucose Production and Utilization in Depancreatized, Conscious Dogs

The relation between portal vein insulin concentrations and suppression of hepatic glucose production, as well as peripheral venous insulin level and increase of peripheral glucose utilization, was compared in conscious, depancreatized, diabetic dogs after infusion of insulin at 0.25 and 0.5 mU/kg/min into either the portal system or the peripheral circulation. Glucose appearance and clearance was measured using [3-3H]-glucose. Before infusion of insulin, portal vein insulin concentrations were undetectable. The intraportal infusion of insulin at 0.25 mU/kg/min increased portal vein insulin to 16 ± 1 μU/ml, significantly higher than the arterial concentration (9 ± 1 μU/ml). Infusion of the same amount of insulin into a peripheral vein raised peripheral insulin levels to 14 ± 1 μU/ml and portal vein concentrations to 12 ± 1 μU/ml. When 0.5 mU/kg/ min of insulin was infused into the portal system, the portal vein insulin level was 28 ± 2 μU/ml and significantly greater than the arterial concentration (16 ± 1 μU/ml). After the same amount of insulin was infused into a peripheral vein, the arterial insulin level was higher than that of the portal vein (25 ±1 μU/ml versus 20 ± 1 μU/ml, respectively). The ensuing hypoglycemie was greater after the 0.5 mU/kg/min infusion compared with the 0.25 mU/kg/min infusion. At each dose there was no significant difference between the peripheral venous or the portal route. Despite different portal vein insulin levels, suppression of glucose appearance (as measured by the area under the extended basal value) during insulin infusion was similar in all four groups (29 ± 6% at portal vein insulin concentration of 12 ± 1 μU/ml after 0.25 mU/kg/min insulin into the peripheral vein, 36 ± 4% at 16 ± 1 μU/ml after 0.25 mU/kg/min insulin into the portal system, 37 ± 6% at 20 ± 1 μU/ml after 0.5 mU/kg/min insulin into the pe-ripheral vein, and 31 ± 7% at 28 ± 2 μU/ml after 0.5 mU/kg/min insulin into the portal system, respectively). Glucose clearance did not increase after infusion of insulin at 0.25 mU/kg/min into either the portal or the peripheral system despite an increase of peripheral venous insulin levels (9 ± 1 μU/ml and 14 ± 1 μU/ ml, respectively). Glucose clearance (measured as the area above the curve) did increase significantly when 0.5 mU/kg/min was infused. The increase after peripheral infusion (197 ± 27 ml/kg) was significantly greater than after portal infusion (141 ± 22 ml/kg) of insulin. Under these two circumstances, the peripheral insulin concentrations were 25 ± 1 μU/ml and 16 ± 1 μU/ml, respectively. These results demonstrate that the liver in diabetic dogs is more sensitive than peripheral tissue to small changes in the plasma insulin concentration. While the peripheral administration of small amounts of insulin is equally effective as portal insulin infusion in suppressing hepatic production of glucose, glucose clearance is more directly related to peripheral insulin concentrations.

Adaptation of canine saphenous veins to grafting. Correlation of contractility and contractile protein content.

Saphenous veins are used extensively to replace stenotic coronary arteries. However, the contractile and biochemical adaptations of grafted veins are unknown. The three purposes of this work were to characterize the contractile properties of grafted veins, to determine whether altered contractile characteristics were associated with quantitative changes in actin, myosin and collagen, and to determine which changes were associated with the surgical procedure and which with placement in the arterial circulation. Canine saphenous veins were removed and returned to their original location (venous autograft), while others were used to replace a segment of femoral artery (arterial graft). The grafts were removed 1, 4, and 8 weeks later and compared with the contralateral saphenous vein. Both graft types exhibited an increase in sensitivity to norepinephrine but not to potassium chloride. The venous autograft exhibited a reversible reduction in myosin content and in maximum contractile response (force/cross-sectional area) to potassium chloride and norepinephrine. In contrast, the arterial graft exhibited increased wall thickness and content of all measured proteins and decreased maximum contractile response. The latter occurred even though there was an increase in the net production of actin and myosin. Expressing the maximum contractile response in terms of the myosin content did not normalize the contractile response. These results suggest that, except for the elevated sensitivity to norepi nephrine, the vein is capable of recovering from the effects of surgery within 8 weeks; however, placement of the vein in the arterial circulation delays this recovery and initiates a hypertrophic response that includes an attenuation of contractile function.

Effect of chronic myocardial ischemia on the activity of carnitine palmitylcoenzyme A transferase of isolated canine heart mitochondria.

The effect of chronic ischemia on the activity of carnitine palmitylcoenzyme A transferase (PCoAT) was examined. Control mitochondria were isolated from the left ventricle of normal hearts and from the uninvolved areas of ischemic hearts. Mitochondria from ischemic tissue were isolated from the left ventricle of chronically ischemic hearts. Formation of the enzyme product, palmityl-14C-carnitine, was markedly depressed in control mitochondria isolated from ischemic hearts; however, the pH optimums of the enzymes from control and ischemic mitochondria were in the same range. After 1 day of chronic ischemia, the kinetics of the enzyme from ischemic mitochondria (compared with that of the enzyme from control mitochondria) showed marked changes in Vmax. Periods of ischemia longer than 1 day significantly decreased the Km of carnitine PCoAT for carnitine from 2.59 ± 0.476 mM to 0.713 ± 0.148 mM, and Vmax was still depressed (15 to 3 nmoles/min mg−1). The Km for palmityl CoA was not significantly affected. Arrhenius plots for the enzyme from ischemic (32 hours) mitochondria revealed a single phasic response to temperature change. Two nonintersecting slopes were characteristic of the normal 1-day ischemic and the normal sonicated activity. As a result of ischemia, changes in the lipid components in the membrane containing carnitine PCoAT were postulated: alterations in the hydrophobic environment of the enzyme produce interference in the binding of palmityl CoA to the second substrate site and may result in a decrease in the Km of the enzyme for carnitine. The single phasic response of the enzyme to temperature change may be additional evidence for membrane lipid alterations. A defect in the transport of long-chain fatty acids may be an early change associated with myocardial ischemia.

Effects of an inotropic agent, RO 2-2985 (X-537A), on regional blood flow and myocardial function in chronically instrumented conscious dogs and anesthetized dogs.

RO 2-2985 produced a marked positive inotropic effect in unanesthetized, chronically instrumented dogs, measured as an increase in left ventricular dP/dt and an increase in maximum velocity of myocardial fiber shortening. Similar changes produced in dogs in hemorrhagic shock lasted for 2–3 hours. RO 2-2985 increased peripheral blood flow and caused marked increases in both coronary and renal blood flows. The drug altered the response of the renal vascular bed to subsequent norepinephrine administration. After administration of a single dose of RO 2-2985, norepinephrine produced sustained increases in renal blood flow, and this altered responsiveness to norepinephrine persisted for periods ranging from 1 to 3 weeks.

Comparison of the cardiovascular action of isoproterenol, dopamine, and dobutamine in the neonatal and mature dog

SummaryTo determine if there are age-related differences of cardiovascular responses to isoproterenol, dopamine hydrochloride, and dobutamine hydrochloride, we recorded cardiac output, renal artery blood flow, central aortic blood pressure, and heart rate in 11 anesthetized puppies ranging in age from 0 to 10 days and in five adult dogs during incremental infusion of isoproterenol (0.05 to 1.25μg/kg/min), dopamine (2 to 50μg/kg/min), and dobutamine (2 to 50μg/kg/min).Isoproterenol decreased systemic arterial mean blood pressure and renal blood flow more in adult dogs than in puppies. Cardiac output was increased in both age groups, but the increase was more marked in adults than in puppies. Heart rate increased similarly in puppies and adults.Dopamine increased systemic arterial mean blood pressure, heart rate, renal blood flow, and cardiac output in both puppies and adult dogs, but the increase of cardiac output was more marked in adult dogs than in puppies.Dobutamine significantly increased systemic arterial mean blood pressure, cardiac output, and renal blood flow in adult dogs but not in puppies. Heart rate increased in both groups of dogs.This study demonstrates age-related differences in the response of the cardiovascular system of anesthetized dogs to isoproterenol, dopamine, and dobutamine.

Myocardial ischemia: platelet and thromboxane concentrations in cardiac lymph and the effects of ibuprofen and prostacyclin.

Blood platelets have been implicated in several mechanisms leading to and/or modifying myocardial ischemia. Cardiac lymph examination allows insight into the extracellular fluid that is in equilibrium with the capillary blood. In order to obtain an index of platelet activation during coronary artery events in the awake chronic animal, we wished to ascertain whether evaluation of cardiac lymph would detect changes in platelet activation resulting from a vascular occlusion. The study used conscious dogs in which cardiac lymph vessels had been previously cannulated by openchest surgical protocol. The concentrations of immunoreactive thromboxane B2 and platelet counts were assessed in the cardiac lymph during the control period, the 10–60 minute occlusions, and the reperfusion periods. The same protocols were effected on another series of dogs after infusion of ibuprofen or prostacyclin. Initially, immunoreactive thromboxane B2 concentrations in the systemic blood and cardiac lymph were identical. A three-fold increase in immunoreactive thromboxane B2 concentrations occurred in untreated animals and was accompanied by a fall in platelet count in the lymph. The infusion of ibuprofen or prostacyclin, which inhibit platelet aggregation by different mechanisms, prevented both the decrease in platelets and the increase in immunoreactive thromboxane B2. In this study, intravascular events resulting from coronary occlusion invoke a rapid rise of immunoreactive thromboxane B2 in the extravascular fluid. A decrease in platelet escape into the extravascular compartment is interpreted as a result of intravascular aggregation promoting decreased platelet numbers. Thus, examination of continuously flowing cardiac lymph allows rapid detection of intravascular activation of platelets in the awake animal in the absence of surgical trauma.